Journal of Inorganic Biochemistry 128 (2013) 85–96

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Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Antioxidant capacity and DNA-interaction studies of zinc complexes

with a non-steroidal anti-inflammatory drug, mefenamic acid
Alketa Tarushi a, Zoi Karaflou a, Jakob Kljun b, Iztok Turel b, George Psomas a,
Athanasios N. Papadopoulos c,⁎, Dimitris P. Kessissoglou a,⁎⁎
a
Laboratory of Inorganic Chemistry, Department of General and Inorganic Chemistry, Faculty of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
b
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva 5, 1000 Ljubljana, Slovenia
c
Department of Nutrition and Dietetics, Faculty of Food Technology and Nutrition, Alexandrion Technological Educational Institution, Sindos, Thessaloniki, Greece

a r t i c l e i n f o a b s t r a c t

Article history: Zinc(II) complexes of a non-steroidal anti-inflammatory drug, mefenamic acid(= Hmef) in the absence or
Received 17 March 2013 presence of the nitrogen donor heterocyclic ligands 2,2′-bipyridine(= bipy), 2,2′-bipyridylamine(= bipyam),
Received in revised form 1 July 2013 2,2′-dipyridylketone oxime(= Hpko) or 1,10-phenanthroline(= phen) have been synthesized and charac-
Accepted 8 July 2013
terized. The crystal structures of [Zn(mef-O,O′)2(bipy)], 2, [Zn(mef-O)2(Hpko-N,N′)2]·EtOH, 4 and
Available online 15 July 2013
[Zn(mef-O)(mef-O,O′)(phen)(H2O)], 5, have been determined by X-ray crystallography showing distinct
Keywords:
binding modes of mefenamato carboxylato group, bidentate in 2, monodentate in 4 or both in 5. Interaction
Zinc complexes studies of the complexes with calf-thymus DNA (CT DNA) have shown that complexes can bind to CT DNA
Mefenamic acid with [Zn(mef-O)2(Hpko)2] exhibiting the highest binding constant to CT DNA (Kb = 1.93(±0.04) × 107 M−1).
Interaction with calf-thymus DNA The complexes can bind to CT DNA via intercalation as concluded by DNA solution viscosity measurements.
Interaction with serum albumins Competitive studies with ethidium bromide (EB) have shown that the complexes can displace the
Antioxidant capacity DNA-bound EB. The complexes exhibit good binding affinity to serum albumin proteins with [Zn(mef-O)2(H2O)4],
1 exhibiting the highest quenching ability (kq = 1.46 × 1015 M−1 s−1 for human and 5.55 × 1015 M−1 s−1 for
bovine serum albumin). 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. The scavenging activity is low to
moderate against 1,1-diphenyl-picrylhydrazyl (DPPH) radicals and high against hydroxyl and 2,2′-azinobis-
(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+•) radicals, with [Zn(mef-O)2(H2O)4], 1 (ABTS%, 0.1 mM:
94.75(±1.06)%; •OH%, 0.1 mM: 96.69(±0.27)%; LOX: IC50 = 27.34(±0.90) μM) exhibiting the highest scavenging
activity of the ABTS radical cation among the complexes. Additionally, the complexes exhibit higher scavenging and
LOX inhibitory activity than free mefenamic acid (ABTS%, 0.1 mM: 66.32(±0.38)%; •OH%,0.1 mM: 92.51(±0.44)%;
LOX: IC50 = 48.52(±0.88) μM).
© 2013 Elsevier Inc. All rights reserved.

1. Introduction worth to note the unique role of zinc in nucleic acid chemistry in
many aspects; it is quite interesting that only zinc(II) ions from all
Zinc is the second most abundant trace element in the human metal ions are able to facilitate the rewinding of molten DNA [12].
body and a major regulatory ion in the metabolism of cells [1,2]. Non-steroidal anti-inflammatory drugs (NSAIDs) are the most
Zinc has many beneficial effects in human health but changes in its frequently administrated analgesic, anti-inflammatory and antipyret-
metabolism or trafficking may be related to some diseases [3,4]. The ic drugs with known (mainly gastrointestinal (e.g. gastric ulceration,
extensive use of zinc to treat children suffering from deadly diarrhea nausea, diarrhea, dyspepsia) and renal (e.g. salt and fluid retention,
has resulted in significant reduction of child mortality [5]. Zinc hypertension, and in rare cases interstitial nephritis, acute renal
complexes with drugs have been tested for the treatment of failure)) side-effects [13]. The action of NSAIDs is mainly through
Alzheimer disease [6] with others showing noteworthy antibacterial inhibition of the cyclo-oxygenase (COX)-mediated production of
[7], anticonvulsant [8], antidiabetic [9] antiinflammatory [10], antimi- prostaglandins [14]. NSAIDs have a synergistic role on the activity
crobial [11] and antiproliferative-antitumor [10,11] activity. It is also of certain antitumor drugs [15] and can lead to cell death of a series
of cancer cell lines via apoptosis [16]. The antitumor activity of the
NSAIDs may be explained either with COX-independent mechanisms
⁎ Corresponding author. Tel.: +30 2310013925.
⁎⁎ Corresponding author. Tel.: +30 2310997723; fax: +30 2310997738.
by modulating cell proliferation and cell death in cancer cells lacking
E-mail addresses: papadnas@nutr.teithe.gr (A.N. Papadopoulos), COX [17], or with apoptosis via an activation of caspases [18] or via an
kessisog@chem.auth.gr (D.P. Kessissoglou). unknown molecular mechanism where free radicals may also be

0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2013.07.013
86 A. Tarushi et al. / Journal of Inorganic Biochemistry 128 (2013) 85–96

involved [19,20]. Within this context and in an attempt to investigate and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+•)
the potential anticancer as well as the anti-inflammatory activity of radicals as well as their in vitro inhibitory activity against soybean
the NSAIDs and their complexes, the interaction with DNA as well lipoxygenase (LOX), because the use of NSAIDs in medicine as anti-
as the antioxidant activity should be considered of great importance inflammatory agents may be also related to free radical scavenging.
and further evaluated; it is noteworthy that only few relevant reports
on the interaction of NSAIDs and their complexes with DNA have 2. Experimental
been published so far [21,22]. Furthermore, the number of structurally
characterized zinc complexes with NSAIDs is quite limited; an 2.1. Materials–instrumentation–physical measurements
aspirinate [23], a flufenamato complex [24] and a series of indometh-
acin [11] and tolfenamic acid [25] as ligands [26]. Mefenamic acid, ZnCl2, Zn(NO3)2⋅6H2O, bipy, bipyam, Hpko, phen,
Phenylalkanoic acids, anthranilic acids, oxicams, salicylate de- KOH, trisodium citrate, NaCl, CT DNA, BSA, HSA and EB were purchased
rivatives, sulfonamides and furanones constitute the chemical from Sigma-Aldrich Co and all solvents were purchased from Merck. All
classes of NSAIDs [27]. The NSAID mefenamic acid (= Hmef) is a the chemicals and solvents were reagent grade and were used as
N-phenylanthranilic acid and resembles chemically to tolfenamic purchased.
and flufenamic acids and other fenamates in clinical use (Scheme 1). DNA stock solution was prepared by dilution of CT DNA to buffer
Mefenamic acid is an effective analgesic and antipyretic agent with (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0)
quite mild side-effects including headaches, diarrhea, vomiting and followed by exhaustive stirring at 4 °C for three days, and kept at
nervousness. Structures of tin(IV) [28], copper(II) [29–31] and 4 °C for no longer than a week. The stock solution of CT DNA gave a
cobalt(II) [32] mefenamato complexes have been recently reported. ratio of UV absorbance at 260 and 280 nm (A260/A280) of 1.89, indi-
Taking into consideration the importance of NSAIDs in medicine, the cating that the DNA was sufficiently free of protein contamination.
enhanced activity of their metal complexes and the application of zinc The DNA concentration was determined by the UV absorbance at
in drugs, we report the synthesis, the structural and spectroscopic 260 nm after 1:20 dilution using ε = 6600 M−1 cm−1 [31,32].
characterization and the biological properties of the neutral zinc(II) Infrared (IR) spectra (400–4000 cm−1) were recorded on a Nico-
complexes with the NSAID mefenamic acid in the presence of methanol let FT-IR 6700 spectrometer with samples prepared as KBr pellets.
or a N,N′-donor heterocyclic ligand such as 2,2′-bipyridine (=bipy), UV–visible (UV–vis) spectra were recorded as nujol mulls and in
2,2′-bipyridylamine (=bipyam), 2,2′-dipyridylketone oxime (=Hpko) solution at concentrations in the range of 10−5–10−3 M on a Hitachi
or 1,10-phenanthroline (=phen). Interaction of Zn(II) with mefenamic U-2001 dual beam spectrophotometer. 1H NMR spectra were measured
acid leads to the formation of the complex [Zn(mef-O,O′)2(H2O)4], at room temperature on a Bruker AM300 NMR spectrometer using
(= 1), while in the presence of bipy, bipyam, Hpko or phen the dmso-d6 as solvent. C, H and N elemental analysis was performed on
mononuclear complexes [Zn(mef-O,O′)2(bipy)], (=2), [Zn(mef-O,O′)2 a Perkin-Elmer 240B elemental analyzer. Molar conductivity measure-
(bipyam)], (=3), [Zn(mef-O)2(Hpko-N,N′)2]·EtOH, (=4 · EtOH) and ments were carried out in 1 mM dmso solution of the complexes with
[Zn(mef-O)(mef-O,O′)(phen)(H2O)], (=5) have been isolated, respec- a Crison Basic 30 conductometer. Fluorescence spectra were recorded
tively. The crystal structures of [Zn(mef-O,O′)2(bipy)], 2, [Zn(mef-O)2 in solution on a Hitachi F-7000 fluorescence spectrophotometer. Viscos-
(Hpko-N,N′)2]·EtOH, 4·EtOH and [Zn(mef-O)(mef-O,O′)(phen)(H2O)], ity experiments were carried out using an ALPHA L Fungilab rotational
5 have been determined by X-ray crystallography. As to investigate the viscometer equipped with an 18 mL LCP spindle.
existence of potential anticancer and/or anti-inflammatory activity of
complexes 1–5, the study of the biological properties of the complexes 2.2. Synthesis of the complexes
has been focused on (i) their binding properties with calf-thymus (CT)
DNA investigated by UV spectroscopy and viscosity measurements, 2.2.1. [Zn(mefenamato-O)2(H2O)4] or [Zn(mef-O)2(H2O)4], 1
(ii) their ability to displace ethidium bromide (EB) from the EB–DNA A methanolic solution (15 mL) containing mefenamic acid
complex in order to clarify the existence of a potential intercalation (0.4 mmol, 97 mg) and KOH (0.4 mmol, 22 mg) was stirred for 1 h.
of the complexes to CT DNA in competition to the classical DNA- The solution was added dropwise to a methanolic solution (10 mL)
intercalator EB performed by fluorescence spectroscopy, (iii) the affinity of ZnCl2 (0.2 mmol, 27 mg) and the reaction mixture was stirred for
of the complexes to bovine (BSA) and human serum albumin 1 h. The reaction solution was filtered and left for slow evaporation.
(HSA); binding to such proteins that are involved in the transport of After a few days colorless microcrystalline product of [Zn(mef)2(H2O)4],
metal ions and metal–drug complexes through the blood stream may 1 (90 mg, 74%) was deposited and collected by filtration (found C,
result in lower or enhanced biological properties of the original drug, or 58.43; H, 6.17; N, 4.78; C30H36N2O8Zn (MW = 618.01) requires C
new paths for drug transportation [33] investigated by fluorescence 58.31; H, 5.87; N, 4.53%). IR: νmax/cm− 1; νasym(CO2): 1614 (vs);
spectroscopy and (iv) their antioxidant capacity by investigating their νsym(CO2): 1399 (vs); Δ = νasym(CO2) − νsym(CO2): 215 cm− 1
scavenge ability of 1,1-diphenyl-picrylhydrazyl (DPPH), hydroxyl (•OH) (KBr disk); UV–vis: λ/nm (ε/M− 1 cm− 1) as nujol mull: 343, 298;
in dmso: 344 (14,600), 295 (16,400). 1H NMR in dmso-d6 (δ/ppm):
2.08 (6H, s, H11-mef), 2.23 (6H, s, H10-mef), 6.65 (2H, d(J = 7.4 Hz),
H5-mef), 6.77 (2H, m(J = 8.3 Hz), H3-mef), 6.92 (2H, m(J = 7 Hz),
H9-mef), 7.05 (2H, m(J = 7.5 Hz), H8-mef), 7.11 (2H, d(J =
8.3 Hz), H7-mef), 7.20 (2H, m(J = 7.6 Hz), H4-mef), 7.96 (2H, d
(J = 7.6 Hz), H2-mef), 9.98 (2H, s, H6-mef). The complex is soluble
in dmf and dmso (ΛM = 15 mho cm2 mol− 1, 1 mM in dmso).

2.2.2. [Zn(mefenamato-O,O′)2(2,2′-bipyridine)] or [Zn(mef-O,O′)2(bipy)], 2

A methanolic solution (10 mL) of mefenamic acid (0.4 mmol, 97 mg)
and KOH (0.4 mmol, 22 mg) after 1 h stirring was added dropwise
slowly and simultaneously with a methanolic solution (10 mL) of
bipy (0.2 mmol, 31 mg) to a methanolic solution (10 mL) of ZnCl2
(0.2 mmol, 27 mg). The resultant solution was left for slow evaporation.
Scheme 1. Mefenamic acid (Hmef) with H atom numbering in accordance to 1H NMR Colorless crystals of [Zn(mef)2(bipy)], 2 (100 mg, 71%) suitable for X-ray
proton's assignment. structure determination, were collected after a few days (found C, 68.15;
 A. Tarushi et al. / Journal of Inorganic Biochemistry 128 (2013) 85–96 87

H, 5.25; N, 8.24; C40H36N4O4Zn (MW = 702.10) requires C, 68.43; H, stirring, the mixture was left for slow evaporation. Colorless crystals
5.17; N, 7.98%). IR: νmax/cm−1; νasym(CO2): 1581 (vs); νsym(CO2): 1393 of [Zn(mef)2(phen)(H2O)], 5 (90 mg, 60%) suitable for X-ray structure
(vs); Δ = 188 cm−1 (KBr disk); UV–vis: λ/nm (ε/M−1 cm−1) as nujol determination, were deposited after a week (found C, 67.53; H, 4.89;
mull: 353, 286; in dmso: 351 (10,400), 284 (20,500). 1H NMR in N, 7.35; C42H38N4O5Zn (MW = 744.16) requires C, 67.79; H, 5.15; N,
dmso-d6 (δ/ppm): 2.03 (6H, s, H11-mef), 2.21 (6H, s, H10-mef), 6.60 7.53%). IR: νmax/cm−1; νasym(CO2): 1574 (vs); νsym(CO2): 1385 (vs);
(2H, m(J = 7.5 Hz), H3-mef), 6.75 (2H, d(J = 8.0 Hz), H5-mef), Δ = 189 cm−1 (KBr disk); UV–vis: λ/nm (ε/M−1 cm−1) as nujol
6.88 (2H, m(J = 7.2 Hz), H9-mef), 7.03 (2H, m(J = 7.6 Hz), mull: 339, 291 (sh); in dmso: 344 (4600), 286 (19,500). 1H NMR in
H8-mef), 7.08 (2H, d(J = 8.2 Hz), H7-mef), 7.16 (2H, m(J = dmso-d6 (δ/ppm): 1.97 (6H, s, H11-mef), 2.20 (6H, s, H10-mef), 6.57
7.7 Hz), H4-mef), 7.65 (2H, m(J = 6.5 Hz), H4- and H4′-bipy), 7.92 (2H, d(J = 7.3 Hz), H5-mef), 6.73 (2H, m(J = 8.2 Hz), H3-mef),
(2H, d (J = 7.8 Hz), H2-mef), 8.15 (2H, d(J = 6.5 Hz), H6- and 6.87 (2H, m(J = 6.9 Hz), H9-mef), 7.01 (2H, m(J = 7.5 Hz), H8-mef),
H6′-bipy), 8.56 (2H, d(J = 7.3 Hz), H5- and H5′-bipy), 8.82 (2H, d(J = 7.04 (2H, d(J = 7.6 Hz), H7-mef), 7.13 (2H, m(J = 7.7 Hz), H4-mef),
3.1 Hz), H3- and H3′-bipy), 10.11 (2H, s, H6-mef). The complex is soluble 7.86 (2H, d(J = 7.7 Hz), H2-mef), 8.06 (2H, d(J = 8.1 Hz), H3-
in dmso (ΛM = 12 mho cm2 mol−1, 1 mM in dmso). and H8-phen), 8.25 (2H, d(J = 4.1 Hz), H5- and H6-phen), 8.86
(4H, m(J = 8.1 Hz), H2-, H4-, H7- and H9-phen), 10.17 (2H, s, H6-mef).
2.2.3. [Zn(mefenamato-O,O′)2(2,2′-bipyridylamine)] or The complex is soluble in dmf and dmso (ΛM = 7 mho cm2 mol−1,
[Zn(mef-O, O′)2(bipyam)], 3 1 mM in dmso).
In a similar way, with the use of bipyam (0.2 mmol, 34 mg) instead
of bipy, colorless microcrystalline product of [Zn(mef)2(bipyam)], 3 2.3. X-ray crystallography
(95 mg, 65%) was deposited after ten days and collected by filtration
(found C, 67.25; H, 5.28; N, 10.15; C40H37N5O4Zn (MW = 717.15) X-ray diffraction data for compounds 2 and 4 (Table S1) were col-
requires C, 66.99; H, 5.20; N, 9.77%). IR: νmax/cm−1; νasym(CO2): 1581 lected on a Nonius Kappa CCD diffractometer equipped with a Mo
(vs); νsym(CO2): 1402 (vs); Δ = 179 cm−1 (KBr disk); UV–vis: λ/nm anode (Kα radiation, λ = 0.71073 Å) and a graphite monochromator
(ε/M−1 cm−1) as nujol mull: 342, 301 (sh); in dmso: 345 (4200), 298 at 293(2) and 150(2) K, respectively, while the X-ray diffraction data
(14,600). 1H NMR in dmso-d6 (δ/ppm): 2.06 (6H, s, H11-mef), 2.24 for 5 were collected on an Oxford Diffraction SuperNova diffractome-
(6H, s, H10-mef), 6.64 (2H, m(J = 7.2 Hz), H3-mef), 6.79 (2H, d(J = ter with Mo microfocus X-ray source (Kα radiation, λ = 0.71073 Å)
8.3 Hz), H5-mef), 6.91 (2H, m(J = 7.2 Hz), H9-mef), 7.02 (2H, d(J = with mirror optics and an Atlas detector at 150(2) K. The structures
7.6 Hz), H4- and H4′-bipyam), 7.05 (2H, m(J = 7.8 Hz), H8-mef), 7.10 were solved by direct methods implemented in SIR92 [34] and
(2H, d(J = 8.3 Hz), H7-mef), 7.23 (2H, m(J = 7.7 Hz), H4-mef), 7.57 refined by a full-matrix least-squares procedure based on F2 using
(2H, d(J = 7.7 Hz), H6- and H6′-bipyam), 7.79 (2H, d(J = 7.4 Hz), H5- SHELXL-97 [35]. All non-hydrogen atoms were refined anisotropically.
and H5′-bipyam), 7.95 (2H, d(J = 7.8 Hz), H2-mef), 8.35 (2H, d(J = The hydrogen atoms were placed at calculated positions and treated
4.6 Hz), H3- and H3′-bipyam), 10.11 (2H, s, H6-mef). The complex is sol- using appropriate riding models. The programs Mercury, ORTEP and
uble in dmso (ΛM = 13 mho cm2 mol−1, 1 mM in dmso). Platon were used for data analysis and figures preparation [36–38].

2.2.4. cis,cis,trans-[Zn(mefenamato-O)2(2,2′-dipyridylketonoxime-N, 2.4. Antioxidant biological assay

N′)2]·EtOH or [Zn(mef-O)2(Hpko-N,N′)2]·EtOH, 4·EtOH
Mefenamic acid (0.4 mmol, 97 mg) was dissolved in ethanol In the in vitro assays each experiment was performed at least in
(15 mL) followed by the addition of KOH (0.4 mmol, 22 mg). After triplicate and the standard deviation of absorbance was less than
1 h stirring, the resultant solution was added slowly and simulta- 10% of the mean.
neously with an ethanolic solution of Hpko (0.4 mmol, 80 mg), to
an ethanolic solution (10 mL) of ZnCl2 (0.2 mmol, 27 mg) and was 2.4.1. Determination of the reducing activity of the stable radical DPPH
stirred for 30 min. The solution was left for slow evaporation. Pale To a solution of DPPH (0.1 mM) in absolute ethanol an equal volume
yellow crystals of [Zn(mef)2(Hpko)2]·EtOH, 4·EtOH (130 mg, 65%) of the compounds dissolved in ethanol was added. As control solution
suitable for X-ray structure determination were deposited after a ethanol was used. The concentration of the solution of the compounds
few days (found C, 65.81; H, 5.34; N, 11.45; C54H52N8O7Zn (MW = was 0.1 mM. The absorbance at 517 nm was recorded at room temper-
990.41) requires C, 65.49; H, 5.29; N, 11.31%). IR: νmax/cm− 1; ature, after 20 and 60 min in order to examine the time-dependence
νasym(CO2): 1574 (vs); νsym(CO2): 1383 (vs); Δ = 191 cm− 1 (KBr of the radical scavenging activity [31,39]. The radical scavenging
disk); UV–vis: λ/nm (ε/M− 1 cm− 1) as nujol mull: 339, 291(sh); in activity of the compounds was expressed as the percentage reduction
dmso: 342 (5700), 288 (18,500). 1H NMR in dmso-d6 (δ/ppm): of the absorbance values of the initial DPPH solution (RA%). NDGA
2.05 (6H, s, H11-mef), 2.22 (6H, s, H10-mef), 6.63 (2H, m(J = (nordihydroguaiaretic acid) and BHT (butylated hydroxytoluene)
7.4 Hz), H3-mef), 6.75 (2H, d(J = 8.3 Hz), H5-mef), 6.90 (2H, were used as reference compounds.
m(J = 7.1 Hz), H9-mef), 7.04 (2H, m(J = 7.6 Hz), H8-mef), 7.09
(2H, d(J = 7.4 Hz), H7-mef), 7.18 (2H, m(J = 7.0 Hz), H4-mef), 7.38 2.4.2. Competition of the tested compounds with dmso for hydroxyl
(4H, m(J = 5.7 Hz), H4- and H4′-Hpko), 7.52 (2H, d(J = 7.7 Hz), radicals
H6′-Hpko), 7.83 (2H, d(J = 7.6 Hz), H6-Hpko), 7.87 (4H, m(J = The hydroxyl radicals generated by the Fe3+/ascorbic acid system,
7.8 Hz), H5- and H5′-Hpko), 7.94 (2H, d(J = 6.5 Hz), H2-mef), 8.45 were detected according to Nash [31,39], by the determination
(2H, d(J = 7.0 Hz), H3′-Hpko), 8.60 (2H, d(J = 4.7 Hz), H3-Hpko), of formaldehyde produced from the oxidation of dmso. The reaction
10.00 (2H, s, H6-mef), 11.94 (2H, s, H1-Hpko). The complex is soluble mixture contained EDTA (0.1 mM), Fe3+ (167 μM), dmso (33 mM)
in dmf and dmso (ΛM = 9 mho cm2 mol−1, 1 mM in dmso). in phosphate buffer (50 mM, pH 7.4), the tested compounds
(concentration 0.1 mM) and ascorbic acid (10 mM). After 30 min
2.2.5. [Zn(mefenamato-O)(mefenamato-O,O′)(1,10-phenanthroline)(H2O)] of incubation (37 °C) the reaction was stopped with CCl3COOH
or [Zn(mef-O)(mef-O,O′)(phen)(H2O)], 5 (17% w/v) and the absorbance at λ = 412 nm was measured. Trolox
The complex was prepared by the simultaneous addition of a was used as an appropriate standard. The competition of the com-
methanolic solution (5 mL) of phen (0.2 mmol, 36 mg) and a pounds with dmso for •OH, generated by the Fe3+/ascorbic acid sys-
methanolic solution (20 mL) of Hmef (0.4 mmol, 97 mg) and KOH tem, expressed as percent inhibition of formaldehyde production,
(0.4 mmol, 22 mg), stirred for 1 h, to a methanolic solution was used for the evaluation of their hydroxyl radical scavenging ac-
(10 mL) of Zn(NO3)2⋅6H2O (0.2 mmol, 59 mg) and, after 15 min of tivity (•OH%).
88 A. Tarushi et al. / Journal of Inorganic Biochemistry 128 (2013) 85–96

2.4.3. Assay of radical cation scavenging activity 2.5. DNA binding studies
ABTS was dissolved in water to a 2 mM concentration. ABTS radi-
cal cation (ABTS+•) was produced by reacting ABTS stock solution The interaction of complexes 1–5 with CT DNA has been studied by
with 0.17 mM potassium persulfate and allowing the mixture to UV spectroscopy in order to investigate the possible binding modes to
stand in the dark at room temperature for 12–16 h before use. CT DNA and to calculate the binding constants to CT DNA (Kb). In UV ti-
Because ABTS and potassium persulfate react stoichiometrically at a tration experiments, the spectra of CT DNA in the presence of each com-
ratio of 1:0.5, this will result in incomplete oxidation of the ABTS. pound have been recorded for a constant CT DNA concentration in
Oxidation of the ABTS commenced immediately, but the absorbance diverse [complex] / [CT DNA] mixing ratios (r). The binding constants,
was not maximal and stable until more than 6 h had elapsed. The Kb, of the compounds with CT DNA have been determined using the
radical was stable in this form for more than 2 days when stored in UV spectra of the complexes recorded for a constant concentration in
the dark at room temperature. The ABTS+• solution was diluted with the absence or presence of CT DNA for diverse r values. Control experi-
ethanol to an absorbance of 0.70 at 734 nm. After addition of 10 μL of ments with dmso were performed and no changes in the spectra of CT
diluted compounds or standards (0.1 mM) in dmso, the absorbance DNA were observed [42].
reading was taken exactly 1 min after initial mixing [40,41]. The radical Viscosity experiments were carried out using an ALPHA L Fungilab
scavenging activity of the complexes was expressed as the percentage rotational viscometer equipped with an 18 mL LCP spindle and the
inhibition of the absorbance of the initial ABTS solution (ABTS%). Trolox measurements were performed at 100 rpm. The viscosity of a DNA so-
was used as an appropriate standard. lution has been measured in the presence of increasing amounts of
complexes 1–5. The relation between the relative solution viscosity
(η / η0) and DNA length (L / L0) is given by the equation L / L0 =
2.4.4. Soybean lipoxygenase inhibition study in vitro (η / η0)1 / 3, where L0 denotes the apparent molecular length in the
The in vitro study was evaluated as reported previously [39]. The absence of the compound [31,43]. The obtained data are presented
tested compounds dissolved in ethanol were incubated at room as (η / η0)1 / 3 versus r, where η is the viscosity of DNA in the presence
temperature with sodium linoleate (0.1 mM) and 0.2 mL of enzyme of complex, and η0 is the viscosity of DNA alone in buffer solution.
solution (1/9 × 10−4 w/v in saline). The conversion of sodium linoleate The competitive studies of each complex with EB have been inves-
to 13-hydroperoxylinoleic acid at 234 nm was recorded and compared tigated by fluorescence spectroscopy in order to examine whether the
with the appropriate standard inhibitor caffeic acid. complex 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 EB-displacing ability of complexes 1–5 from the DNA -EB com-
plex was studied by adding a certain amount of a solution of the com-
plex step by step into the solution of the DNA–EB complex. The
influence of the addition of each complex to the DNA–EB complex so-
lution has been obtained by recording the variation of fluorescence
emission spectra.

2.6. Albumin binding experiments

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 342 nm or HSA at 351 nm was moni-
tored using complexes 1–5 as quenchers with increasing concentration
since 1–5 in buffer solutions do not exhibit any emission spectra under
the same experimental conditions [31,32]. Fluorescence spectra were
recorded from 300 to 500 nm at an excitation wavelength of 296 nm.
The Stern–Volmer (Eq. (S3)) and Scatchard equations (Eq. (S4)) and
graphs have been used in order to study the interaction of each quencher
with serum albumins.

Table 1
Selected bond distances and angles for complex 2.

Bond distances (Å) Bond distances (Å)

Zn(1)\O(2) 2.109(2) Zn(1)\O(5) 2.000(2)

Zn(1)\O(3) 2.195(2) Zn(1)\N(6) 2.090(2)
Zn(1)\O(4) 2.462(2) Zn(1)\N(7) 2.109(2)
O(2)\C(10) 1.260(3) O(4)\C(25) 1.256(4)
O(3)\C(10) 1.266(3) O(5)\C(25) 1.261(4)

Bond angles (°)

O(2)\Zn(1)\O(5) 129.11(8) O(3)\Zn(1)\O(4) 116.50(8)

O(4)\Zn(1)\N(7) 142.91(7) O(2)\Zn(1)\N(7) 118.09(8)
O(3)\Zn(1)\N(6) 152.48(9)
Fig. 1. 1H NMR spectra of complexes (A) 1 and (B) 4 in dmso-d6.
 A. Tarushi et al. / Journal of Inorganic Biochemistry 128 (2013) 85–96 89

Table 2 3.2. Spectroscopic study of the complexes

Selected bond distances and angles for complex 4.

Bond distances (Å) Bond distances (Å) The spectroscopic characterization of the resultant complexes 1–5
has been performed by IR, 1H NMR and electronic spectra techniques.
Zn(1)\O(3) 2.069(3) Zn(1)\N(16) 2.159(5)
Zn(1)\O(6) 2.038(3) Zn(1)\N(17) 2.165(4) IR spectroscopy confirms the deprotonation and binding mode of
Zn(1)\N(9) 2.149(4) Zn(1)\N(25) 2.131(4) mefenamic acid. In Hmef's IR spectrum, the absorption band at
O(5)\C(30) 1.253(6) O(3)\C(31) 1.267(6) 3370(br,m) cm−1, attributed to the ν(H\O) stretching vibration dis-
O(6)\C(30) 1.277(6) O(7)\C(31) 1.257(6)
appears upon binding to the metal ion. The bands at 1655(s) cm−1
Bond angles (°) Bond angles (°) and 1255(s) cm−1 attributed to ν(C_O)carboxylic and ν(C\O)carboxylic
stretching vibrations of the carboxylic moiety (\COOH) respec-
O(3)\Zn(1)\N(16) 170.50(15) N(9)\Zn(1)\N(17) 164.95(16)
O(6)\Zn(1)\N(25) 172.41(15)
tively, shift, at the IR spectra of complexes 1–5, in the range of
1574–1614 cm−1 and 1383–1402 cm−1 assigned to antisymmetric,
νasym(C_O), and symmetric, νsym(C_O), stretching vibrations of the
carboxylato group, respectively. The difference Δ [=νasym(C_O) −
Table 3 νsym(C_O)], a useful characteristic tool for determining the coordi-
Selected bond distances and angles for complex 5. nation mode of the carboxylato ligands, gives a value falling in the
Bond distances (Å) Bond distances (Å) range of 179–215 cm− 1 indicative for asymmetrically monodentate
Zn(1)\O(2) 2.1580(14) Zn(1)\O(5) 2.0748(16)
binding modes for values above 190 cm−1 and/or asymmetrically
Zn(1)\O(3) 2.0418(15) Zn(1)\N(7) 2.1496(17) bidentate binding modes for values between 172 and 190 cm−1 for
Zn(1)\O(4) 2.2108(14) Zn(1)\N(8) 2.1689(19) the mefenamato ligands [28,29].
O(2)\C(13) 1.276(2) O(3)\C(22) 1.282(2) 1
H NMR spectroscopy is considered a valuable tool to explore the
O(4)\C(13) 1.267(2) O(6)\C(22) 1.252(2)
behavior of complexes in solution. The collected data of compounds
Bond angles (°) Bond angles (°) 1–5 in dmso-d6 solution confirm the formulae and the purity of the
prepared compounds as well as the integrity of each compound in
O(3)\Zn(1)\N(8) 167.68(6) O(2)\Zn(1)\N(7) 154.79(6)
O(4)\Zn(1)\O(5) 158.52(6)
solution. In complexes 1–5, the signal at 12.95 ppm assigned to
carboxylic hydrogen of the free mefenamic acid (Figs. 1 and S1–S5)
is absent confirming the deprotonated mode of the bound drug. The
3. Results and discussion other signals of the mefenamato ligand are slightly shifted downfield
or upfield as expected upon binding to zinc metal ion [24,44,45].
3.1. Synthesis of the complexes Additionally, all sets of signals related to the presence of the
N-donor ligands are present while the ratios of integrated peaks
The synthesis of the complexes in high yield was achieved via confirm the ratio of ligands in the solid state e.g. Fig. 1(A) for 1; four
the aerobic reaction of mefenamic acid and KOH with ZnCl2 or signals for bipy, bipyam or phen ligands and seven for Hpko ligand for
Zn(NO3)2⋅6H2O in the presence of O-donor ligand, (H2O) for 1, or which six signals are attributed to aromatic hydrogen atoms and the
N,N′-donor heterocyclic ligand (L = bipy, bipyam, Hpko or phen) signal at 11.94 ppm is assigned to the oxime hydroxyl proton thus
for 2–5 according to Eq. (1): confirming that the ligand is bound in its non-deprotonated state,
Fig. 1(B). The absence of any additional sets of signals related to dissoci-
ated ligands is in agreement with the molecular conductance measure-
ZnCl2 or ZnðNO3 Þ2 þ 2 Hmef þ 2 KOH þ ð1–4ÞL→½ZnðmefÞ2 ðLÞ1–4 
ments and suggests that all complexes remain intact in solution [46,47].
þ2 KCl þ 2 H2 O; ðL ¼ bipy; bipyam; phen or H2 OÞ: The UV–vis spectra of the complexes have been recorded as nujol
ð1Þ mull and in dmso solution and are similar suggesting that the com-
plexes retain their structure in solution. In order to explore the stability
The complexes are stable in air, are soluble in dmso and non- of complexes in buffer solution, the UV–vis spectra in the series of pH
electrolytes in dmso (for 1 mM solutions, ≤15 mho cm2 mol−1). (pH range 6–8, since the biological experiments are performed at

Fig. 2. A drawing of the molecular structure of 2 with only the heteroatom labeling. The molecules of complex 2 link themselves into dimers about the center of inversion by for-
mation of π-stacking interactions between the N7-containing ring of bipy and the secondary aromatic ring of ligand A.
90 A. Tarushi et al. / Journal of Inorganic Biochemistry 128 (2013) 85–96

Fig. 3. A drawing of the molecular structure of 4 with only the heteroatom labeling.

pH = 7) with the use of diverse buffer solutions (150 mM NaCl and nitrogen of bipy (O(2), O(4) and N(6) in base 1 and O(3), O(5) and
15 mM trisodium citrate at pH values regulated by HCl solution) have N(7) in base 2) with two almost parallel trigonal basal planes forming
also been recorded. No significant changes (shift of the λmax or new an angle of 6.41° and lying at a (centroid to centroid) distance of 2.272 Å.
peaks) have been observed indicating that the complexes keep their The molecules of complex 2 link themselves into dimers about the
integrity in the pH range of 6–8. center of inversion by formation of π-stacking interactions between
The fact that the complexes are non-electrolytes in dmso solution the N7-containing ring of bipy and the secondary aromatic ring of
(ΛM = 7–15 mho cm2 mol−1, in 1 mM dmso solution), they have ligand A. In both cases, the secondary amine group forms an intramo-
the same UV–vis spectral pattern in nujol and in dmso solution, as lecular hydrogen bond with the neighboring carboxylic oxygen which
well as in the presence of the buffer solution, and all 1H NMR spectra causes the elongation of the Zn\O bond. The interesting fact is, that
confirm no dissociation of the complexes suggests that the compounds the elongation is substantially different in the case of ligands A and
are stable and keep their integrity in solution. We have reported earlier B (Zn(1)\O(3) = 2.195(2) Å; Zn(1)\O(4) = 2.462(2) Å) which
that Cu(II) and Co(II) mefenamato complexes are also stable in solution might be related to the different nature of the two secondary amino
[31,32]. groups in the mefenamato ligands. In ligand A, the angle between
planes of the primary and secondary aromatic groups is considerably
3.3. Crystal structure of the complexes higher (77.1(1)° vs. 44.9(2)°) which gives the bridging nitrogen atom
a more sp3 character as in ligand B.
3.3.1. General structural features of 2, 4 and 5
In these complexes, the zinc atom is six-coordinate surrounded by
two mefenamato ligands and N,N′-donor ligands (bipy in 2, Hpko in
4, phen in 5) showing a distorted trigonal prismatic geometry for
2 and distorted octahedral environment for 4 and 5, giving a
ZnN2O4 metal center.
The bond distances around Zn atom are not equal (Tables 1–3) with
bond distances (Zn\O and Zn\N) consistent with those of previously
reported structurally related compounds, [Zn(tolfenamato)2(bipy)],
[Zn(tolfenamato)2(phen)] [25], [Zn(bipy)2(ONO)](NO2) (2.122(9) Å)
[48], [Zn(phen)2(MeCO2)](ClO4) (2.100(5)–2.160(5) Å) [49] and
[Zn(pipdtc)2(bipy)] (2.196(3) Å, pipdtc− = piperidine-carbodithioato
anion) [50] compounds.

3.3.2. Structure of [Zn(mefenamato-O,O′)2(2,2′-bipyridine)], or

[Zn(mef-O,O′)2(bipy)], 2
A drawing of the molecular structure of [Zn(mef-O,O′)2(bipy)], 2 is
shown in Fig. 2, and selected bond distances and angles are listed in
Table 1.
The complex is mononuclear and the mefenamato ligands behave
as bidentate ligands in deprotonated mode coordinated to the zinc
ion via the carboxylato groups in an asymmetric chelating mode
(C(10)\O(2) = 1.260(3) Å and C(10)\O(3) = 1.266(3) Å, C(25)\
O(4) = 1.256(4) Å and C(25)\O(5) = 1.261(4) Å). The trigonal prism
in 2 is formed by two oxygens from the two mefenamato ligands and a Fig. 4. A drawing of the molecular structure of 5 with only the heteroatom labeling.
 A. Tarushi et al. / Journal of Inorganic Biochemistry 128 (2013) 85–96 91

3.3.3. Structure of cis,cis,trans-[Zn(mefenamato-O)2(2,2′-dipyridylketone anti-inflammatory activity since the NSAIDs can also act either as
oxime-N,N′)2]·EtOH or [Zn(mef-O)2(Hpko-N,N′)2]·EtOH, 4·EtOH inhibitors of free radical production or as radical scavengers [55].
A drawing of the molecular structure of [Zn(mef-O)2(Hpko-N,N′)2]· Compounds with antioxidant properties could potentially have a cru-
EtOH, 4·EtOH is shown in Fig. 3 and selected bond distances and angles cial role against inflammation and, thus, lead to potentially effective
are listed in Table 2. drugs. In this context, the potential antioxidant ability of complexes
The mefenamato ligands behave as deprotonated monodentate li- 1–5 has been evaluated in regard to DPPH, ABTS and hydroxyl radical
gands coordinated to zinc ion via a carboxylato-oxygen atom. The scavenging ability as well as the in vitro soybean lipoxygenase inhibi-
carboxylate groups of the mefenamato ligands are monodentately tion and has been compared to that of well-known antioxidant agents
bound to zinc in asymmetric fashion (C(30)\O(5) = 1.253(6) Å e.g. NDGA, BHT and trolox used as reference compounds.
and C(30)\O(6) = 1.277(6) Å, C(31)\O(3) = 1.267(6) Å and DPPH scavenging activity is closely related to antiageing, antican-
C(31)\O(7) = 1.257(6) Å). Monodentate coordination mode has cer and anti-inflammatory activity [39]. Therefore, the compounds
also been reported for [Cu(mef)2(py)2(MeOH)2] [29,31], [Co(mef)2 exhibiting DPPH radical scavenging activity are of increasing interest
(MeOH)4] and [Co(mef)2(bipy)(MeOH)2] [32] complexes. Weak since they may offer protection in rheumatoid arthritis and inflam-
intraligand interaction through hydrogen bonds between the amine mation and lead to potentially effective drugs. DPPH is a stable free
hydrogen atoms and the coordinated oxygen atoms (H(13)⋯O(3) = radical presenting a strong absorption band at 517 nm. The initial
2.003 Å and H(12)⋯O(6) = 1.958 Å) contribute to the stabilization absorbance of DPPH solution decreases, when antioxidants donate
of the complex (Table S2). protons to DPPH radical. The scavenging activity of all complexes
The Hpko ligands act as bidentate neutral chelators and are coor- was not found to be time-dependent since no significant changes
dinated to zinc via a pyridine nitrogen and the oxime nitrogen with were observed after 20-min and 60-min measurements (Fig. 5(A)
ketonoxime oxygen remaining unbound. This coordination mode and Table S3). The complexes present low to moderate reducing abil-
of Hpko ligand (1.0110 according to the Harris notation [51]) has ity of DPPH radical when compared to the reference compounds and
been observed in a series of mononuclear metal complexes [52] show higher DPPH radical scavenging activity than free mefenamic
and in the trinuclear Ni(II) complex [Ni3(shi)2(Hpko)2(py)2] (where acid with 1 being the best DPPH scavenger among complexes 1–5.
H3shi = salicylhydroxamic acid and py = pyridine) [53]. The The complexes present similar scavenging activity of the DPPH radical
ketonoxime groups also form hydrogen bonds with the uncoordinated to that found for a series of copper(II) and cobalt(II) complexes with
carboxylic oxygen of the adjacent mefenamato ligands (H(2)⋯O(5) mefenamic acid as ligand [31,32].
1.723 Å and H(4)⋯O(7) 1.451 Å; Table S2). The lattice is further stabi- The ABTS radical cation (ABTS+•) scavenging activity may be related
lized by the formation of a hydrogen bond between the solvate ethanol to the magnitude of the total antioxidant activity of the compounds
molecule and one of the uncoordinated pyridine nitrogen atoms [32]. All complexes show higher ABTS radical scavenging activity than
of a 2,2′-dipyridylketonoxime ligand and partial π-stacking of one free mefenamic acid, and, in some cases, it is even higher than that of
of the mefenamato secondary aromatic rings with a secondary pyridyl the reference compound trolox. These results (Fig. 5(B) and Table S4)
group of the Hpko ligand. are in agreement to DPPH and hydroxyl radical scavenging studies
revealing that complex 1 exhibits the highest scavenging activity of the
3.3.4. Structure of [Zn(mefenamato-O)(mefenamato-O,O′) ABTS radical cation among the complexes. Similar scavenging activity
(phenanthroline)(H2O)] or [Zn(mef-O)(mef-O,O′)(phen)(H2O)], 5 of the ABTS radical has been recorded for Cu(II) and Co(II) mefenamato
A drawing of the molecular structure of [Zn(mef-O)(mef-O,O′) complexes [31,32].
(phen)(H2O)] is shown in Fig. 4 and selected bond distances and an- Hydroxyl radicals are among the most reactive oxygen species. On
gles are listed in Table 3. the other hand, hydroxyl radical scavengers could serve as protectors
Complex 5 is mononuclear and the two mefenamato ligands are activating the prostaglandin synthesis since, during inflammatory
deprotonated and behave in different binding mode; one mefenamato process, the superoxide anion radicals are generated by phagocytes
ligand is bound in asymmetric bidentate mode while the other is at the inflamed site and are connected to other oxidizing species
coordinated in a monodentate fashion with the uncoordinated oxygen such as •OH [32]. All complexes present higher competition with
atom forming a strong hydrogen bond with the coordinated water dmso (33 mM) at 0.1 mM for hydroxyl free radicals than the refer-
molecule. This binding mode is not unprecedented since the CCDC data- ence compound trolox (Fig. 5(C) and Table S4) which is similar to
base contains 24 crystal structures of zinc complexes bearing one previously reported Cu(II) and Co(II) complexes with mefenamic
phenanthroline (or derivative), one aqua ligand and two carboxylates acid [31,32]. Additionally, most complexes present higher competi-
bound in the previously described way [54]; yet it is rare when it tion with dmso for hydroxyl radical than free mefenamic acid, with
comes to mononuclear complexes as only two compounds (one of complexes 1 and 4 being the most active compounds.
them has two different solvates) are known (with p-hydroxy and Lipoxygenases (LOXs) comprise a family of non-heme iron-
p-dimethylamino benzoate), the others being di-, tri-nuclear or poly- containing dioxygenases and are the key enzymes of the transformation
meric in nature. of arachidonic acid to leukotrienes, which play an important role in the
The structure is stabilized by the intraligand hydrogen bonds be- pathophysiology of several inflammatory and allergic diseases. The
tween the secondary amino groups and the bound carboxylic oxygens products of the oxygenation catalyzed by LOXs are also involved in
as in the previous two cases, by the abovementioned intramolecular the development of rheumatoid arthritis, psoriasis and asthmatic
hydrogen bond between the aqua ligand and the free oxygen atom responses [56]. Lipoxygenation occurs via a carbon centered radical;
of the monodentate mefenamato ligand (O(5)\H(5b)⋯O(6)) and therefore, most LOX inhibitors are antioxidants or free radical scaven-
also by the intermolecular hydrogen bond between the aqua ligand gers [57,58]. The ‘non-heme’ iron per molecule of LOXs in the enzyme
and the bidentate mefenamato ligand of the neighboring complex active site is a high-spin Fe(II) in the inactive state and a high-spin
(O(5)\H(5a)⋯O(2)), thus forming supramolecular dimers about a Fe(III) in the activated state. Therefore, a relationship between LOX
center of inversion (Fig. S6). inhibition and the ability of the inhibitors to reduce the Fe(III) at the
active site to the catalytically inactive Fe(II) has been reported and
3.4. Antioxidant capacity several LOX inhibitors are excellent ligands for Fe(III) [59]. Within this
context, the in vitro inhibitory activity of the compounds against soy-
It is known that free radicals play an important role in the inflam- bean lipoxygenase has been also tested. All complexes present signifi-
matory process. Therefore, the antioxidant activity of the NSAIDs and cant inhibition against soybean lipoxygenase (Fig. 5(D) and Table S4),
their complexes may be related to their potential anticancer and especially in relation to the reference compound caffeic acid (IC50 =
92 A. Tarushi et al. / Journal of Inorganic Biochemistry 128 (2013) 85–96

600 μM). The complexes present better LOX inhibition activity than free to [Zn(mef)2(H2O)4], 1, since there is a nuclearity difference)—
Hmef and 5 is the most active compound against LOX (IC50 =22.87(± or cobalt(II) (RA% = 18–37%, •OH% = 89–96.5% and ABTS% =
1.62) μM). Complexes 1–5 present similar or better in vitro inhibition of 78–92.4%)—mefenamato complexes reported [31,32]. Furthermore,
soybean lipoxygenase than the corresponding Cu(II) mefenamato com- complexes 1–5 can be considered significantly active when compared
plexes reported [31]. to other Zn complexes reported in the literature [67,69].
In conclusion, the complexes exhibit higher scavenging and LOX
inhibitory activity than free mefenamic acid. This is in agreement with 3.5. Interaction with CT DNA
the literature data of a series of Cu(II), Co(II), Ni(II), Zn(II) and Pd(II)
complexes with drugs or Schiff bases as ligands that also exhibited en- It is known that transition metal complexes can bind to DNA via
hanced antioxidant activity compared to free corresponding ligands both covalent (via replacement of a labile ligand of the complex by a ni-
[32,60–65]. Additionally, the scavenging activity of all complexes is trogen base of DNA, e.g. guanine N7) and/or noncovalent (intercalation,
low to moderate against DPPH radicals and high against hydroxyl and electrostatic or groove binding) interactions [31,32]. The potential anti-
ABTS radicals; this may be considered as a first evidence of selective cancer and probably the anti-inflammatory activity of the NSAIDs and
scavenging activity of the complexes against hydroxyl and ABTS radi- their complexes may be also related to their ability to bind to DNA
cals. In the literature, there are examples of complexes being more [21,22]. Within this context, DNA-binding studies can be of significant
active against DPPH radicals than •OH or ABTS+• [66,67] and others interest, although their number so far is quite limited; complexes of
exhibiting much more significant hydroxyl and ABTS scavenging activ- oxicam NSAIDs bind to DNA via intercalation [22]. Interaction of DNA
ity than DPPH [61–65,68]. The existence of a series of zinc complexes with the copper(II) complexes of the NSAIDs naproxen, diclofenac,
being active especially against hydroxyl and superoxide radicals is mefenamic acid and diflunisal as well as of cobalt(II) naproxenato or
also noteworthy [69,70]. mefenamato complexes has been recently thoroughly studied by our
The zinc-mefenamato complexes 1–5 exhibit similar or better free lab [26,31,32,43,71–73].
radical scavenging activity (RA% = 16.7–41.6%, •OH% = 83–98.5%, Complexes 1–5 exhibit similar behavior upon their addition on CT
ABTS% = 74–94.5%, LOX = 22.9–51.9 μM) when compared to that DNA solution. The UV spectra of a CT DNA solution with standard
of the copper(II) (RA% = 8–54%, •OH% = 67–99%, ABTS% = 75–90%, concentration (1.1 × 10− 4 M) have been recorded in the presence
LOX = 4.8–67.5 μM; the most active Cu(II)–mefenamato complex is of a compound at diverse different [complex] / [DNA] mixing ratios
the dinuclear [Cu2(mef)4(H2O)2] which cannot be directly compared (r) and representatively for 1 are shown in Fig. S7. The band at

(A) (B)
100 100

80 80
ABTS%

60 60
RA%

40 40

20 20

0 0
ef ], 1 ], 2 ], 3 ], 4 )], 5 DGA BHT ef ], 1 ], 2 ], 3 ], 4 )], 5 rolox
Hm O) 2 ipy) am) ko) 2 O N Hm O) 2 ipy) am) ko) 2 O T
(H 2 f) 2(b (bipy (Hp en)(H 2 (H 2 ( b ip y p )(H 2
f )
e (m 2 e f )
) 2 ef 2 (ph e f) 2 mef) 2 f) 2(b ef) 2(H phen
( m e (m Zn( (me n(m (
[Zn [Zn Zn(m [Zn(m mef) 2 [Zn [ [Z n(me
f) 2
[ ( [Zn
[Zn [Z

(C) (D)
600
100
LOX, IC50 (μM)

80
550
60
OH %

50
40

20

0 0
f ], 1 )], 2 )], 3 ) ], 4 )], 5 rolox ef ], 1
me ], 2 ], 3 , 4 ], 5 cid
H O) 2 py m o 2 O T Hm O) 2 ipy) am) ko) 2] O) eic a
( H 2 f) 2(bi bipya (Hpk n)(H 2 b
(H 2 f) 2( (bip y p H 2 f f
e f) 2 me ) ( f ) 2 phe ef) 2 e (H hen)( Ca
(m Zn( f 2 e ( ) f) 2
e
(m Zn(m ef) 2 (m Zn(m (mef 2 (me f) 2(p
[Zn [
[Zn [Z n [ [Zn n(me
[ n (m [Zn
[Z [Z

Fig. 5. (A) Interaction % with DPPH (RA%), (B) % superoxide radical scavenging activity (ABTS%), (C) competition % with dmso for hydroxyl radical (•OH%) and (D) in vitro inhibition
of soybean lipoxygenase (LOX) (IC50, in μM) for Hmef and complexes 1–5.
 A. Tarushi et al. / Journal of Inorganic Biochemistry 128 (2013) 85–96 93

λmax =259 nm exhibits a slight red-shift of 3 nm (up to 262 nm) for The measurement of the viscosity of DNA solution upon addition
all complexes, indicating that the interaction with CT DNA results in of a compound may provide significant aid to clarify the interaction
the direct formation of a new complex with double-helical CT DNA mode of a compound with DNA, since it is sensitive to DNA length
[74] that results in a stabilization of CT DNA duplex [75]. changes [43,72,73]. Viscosity measurements were carried out on CT
In the UV spectrum of 2 (Fig. 6), the band centered at 351 nm (band DNA solutions (0.1 mM) upon addition of increasing amounts of the
I) exhibits a significant hypochromism of ~50% suggesting tight binding complexes. Fig. 7(A) shows that the addition of the complexes results
to CT DNA probably by intercalation. Further addition of DNA results in in an increase in the relative viscosity of DNA. The binding of a com-
a gradual elimination of this band. Additionally, the band at 284 nm pound to DNA grooves via a partial or non-classic intercalation (i.e.
(band II) presents a hyperchromism of up to 20% accompanied by a electrostatic interaction or external groove-binding) may provoke a
red-shift of 7 nm (up to 291 nm), suggesting tight binding and stabili- bend or kink in the DNA helix and subsequently shorten its effective
zation. A distinct isosbestic point at 335 nm appears upon addition of length and, as a result, the viscosity DNA solution may show a slight
CT DNA. The behavior of all complexes upon addition of increasing decrease or may remain unchanged [80]. In the case of classic interca-
amounts of CT DNA is similar; i.e. 35% hypochromism of band I at lation, the insertion of the compound in between the DNA base pairs
344 nm and 55% hyperchromism of band II at 295 nm accompanied provokes an increase in the separation of base pairs at intercalation
by a bathochromism of 11 nm for 1, 25% hypochromism of band I at sites in order to host the bound compound and the increase of the
345 nm and 20% hyperchromism of band II at 298 nm with an 8-nm length of the DNA helix will be obvious via an increase of DNA viscos-
bathochromism for 3, 15% hypochromism of band I at 342 nm and 7% ity, the magnitude of which is usually in accordance to the strength of
hyperchromism of band II at 288 nm with a 7-nm red-shift for 4, 30% the interaction. In conclusion, the increase of the DNA viscosity ob-
hypochromism of band I at 344 nm and 4% hyperchromism of band II served upon addition of complexes may be considered an evidence of
at 286 nm accompanied by a red-shift of 3 nm for 5. Moreover, similar the existence of an intercalative binding mode between DNA and each
behavior upon addition of CT DNA solution has been observed in the complex [43,72,73].
spectra of the corresponding Cu(II) mefenamato complexes [31]. Ethidium bromide (EB = 3,8-diamino-5-ethyl-6-phenyl-
It should be noted that the exact mode of binding cannot be merely phenanthridinium bromide) emits intense fluorescence in the
proposed by UV spectroscopic titration studies and the results collected presence of CT DNA as a result of strong intercalation of the planar
from the UV titration experiments suggest that all compounds can bind EB phenanthridine ring between adjacent base pairs on the double
to CT DNA [76]. The observed hypochromic effect may be considered as helix; therefore, EB is considered a typical indicator of intercalation
first evidence of tight binding to CT DNA probably by intercalation while [81]. The changes observed in the fluorescence emission spectra of
the stabilization of the DNA duplex may be concluded as a result of the a solution containing EB bound to DNA may be used to study the
existence of a red-shift [77]. interaction between DNA and other compounds, such as metal com-
The values of the binding constant, Kb, of the compounds with CT plexes, since the addition of a compound, capable to intercalate to
DNA, as calculated by Eq. (S1) [78] and the plots in Fig. S8, are given in DNA equally or more strongly than EB, could result in a quenching of
Table 4. The Kb values are high and in most cases are higher than that the EB–DNA fluorescence emission [82]. The emission spectra of EB
of free mefenamic acid suggesting that its coordination to Zn(II) results bound to CT DNA in the absence and presence of each complex have
in an increase of the Kb value. The Kb values suggest a strong binding been recorded for [EB] = 20 μM, [DNA] = 26 μM for increasing
of the complexes to CT DNA, with complex 4 having the highest Kb amounts of the complex. The addition of complexes 1–5 at diverse r
value (=1.93(±0.04) × 107 M−1) among the compounds, and are of values results in a moderate decrease of the intensity of the emission
the same magnitude to that of the classical intercalator EB (Kb = band of the DNA–EB system at 592 nm (the final fluorescence is up to
1.23(±0.07) × 105 M− 1) [79]. 33–49% of the initial EB–DNA fluorescence intensity for all complexes)
The Kb values of complexes 1–5 are among the highest ones report- indicating the existence of a competition of the complexes with EB in
ed for metal complexes of NSAIDs (e.g. naproxen, sodium diclofenac, binding to DNA (Fig. 7(B)). The observed moderate quenching of
mefenamic acid and tolfenamic acid) studied by our group so far DNA–EB fluorescence from the complexes suggests that they may
[31,32,43,71–73]. A comparison to the corresponding Co(II) and Cu(II) displace EB from the DNA–EB compound, thus probably interacting
mefenamato complexes will reveal that, in most cases, 1–5 exhibit with CT DNA by the intercalative mode [31,43].
higher Kb values than their Cu(II) and Co(II) analogs [31,32]. The Stern–Volmer plots of EB–DNA (Fig. S9) illustrate that the
quenching of EB bound to DNA by the compounds is in good agreement
(R = 0.99) with the linear Stern–Volmer equation (Eq. (S2)) [31,32].
The values (Table 4) of the Stern–Volmer constant (KSV) as obtained
1.6 by Stern–Volmer plots of DNA–EB (Fig. S9) show that the complexes
can bind tightly to DNA [31].
In general, although the quenching of EB–DNA fluorescence
1.2 provoked by complexes 1–5 is not as pronounced as in the case of the
corresponding cobalt(II)- and copper(II)-mefenamato complexes, the
KSV values of 1–5 are higher than their Cu(II) and Co(II) analogs
A

0.8

0.4 Table 4
The DNA binding constants (Kb) and Stern–Volmer constants (KSV) of EB–DNA fluores-
cence for complexes 1–5.

0.0 Compound Kb (M−1) KSV (M−1)

280 300 320 340 360 380 400 Hmef [32] 1.05(±0.02) × 105 1.58(±0.06) × 105
λ (nm) [Zn(mef)2(H2O)4], 1 6.81(±0.10) × 105 1.97(±0.07) × 106
[Zn(mef)2(bipy)], 2 5.37(±0.20) × 105 1.23(±0.04) × 106
[Zn(mef)2(bipyam)], 3 5.82(±0.15) × 105 1.21(±0.03) × 106
Fig. 6. UV spectra of a dmso solution (10−5 M) of [Zn(mef)2(bipy)], 2 in the presence [Zn(mef)2(Hpko)2], 4 1.93(±0.04) × 107 8.08(±0.18) × 105
of CT DNA at increasing amounts. The arrows show the changes upon increasing [Zn(mef)2(phen)(H2O)], 5 3.91(±0.15) × 104 6.27(±0.14) × 105
amounts of CT DNA.
94 A. Tarushi et al. / Journal of Inorganic Biochemistry 128 (2013) 85–96

(A) (B)
1.6 100 1
1 2

EB-DNA fluorescence (%)

2 3
1.5 3 80 4
4 5
1.4 5
60
(η/η0)1/3

1.3

1.2 40

1.1 20
1.0
0
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.00 0.01 0.02 0.03 0.04 0.05 0.06
r = [Complex]/[DNA] r = [Complex]/[DNA]

Fig. 7. (A) Relative viscosity (η / ηo)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 complexes 1–5 at increas-
ing amounts (r). (B) 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 complexes 1–5 (quenching up to 36% of the initial EB–DNA fluorescence for 1, 33% for 2, 48% for 3, 44% for 4 and 49% for 5).

[31,32] and are among the highest ones reported for metal complexes of changes in protein secondary structure leading to changes in tryptophan
NSAIDs (e.g. naproxen, sodium diclofenac, mefenamic acid and environment of HSA, and thus indicating the binding of each complex to
tolfenamic acid) [31,43,71–73]. the albumins [85].
The quenching constant values (kq) for complexes 1–5 interacting
3.6. Interaction with serum albumins with albumins have been calculated with Stern–Volmer quenching
equation (Eq. (S3)) and Stern–Volmer plots (Figs. S10 and S11) and
The most abundant protein in plasma is serum albumin (SA), which are given in Table 5. These values indicate good SA quenching ability
role is mainly the transport of metal ions, drugs and their metal com- and the kq values of the complexes are higher than the corresponding
plexes through the blood stream [33]. HSA has a tryptophan at position values of free Hmef, with 1 exhibiting the highest quenching ability
214, while its most extensively studied structural homolog BSA has two (kq = 1.46 × 1015 M−1 s−1 for HSA and 5.55 × 1015 M−1 s−1 for
tryptophans, Trp-134 and Trp-212 [83]. HSA and BSA solutions exhibit, BSA). The kq values (N 1012 M−1 s−1) are higher than diverse kinds
when excited at 295 nm, an intense fluorescence emission with λem, of quenchers for biopolymer fluorescence (2.0 × 1010 M−1 s−1) indi-
max = 351 nm and 343 nm, respectively, which is attributed to the cating the existence of static quenching mechanism [83].
tryptophans [84]. Complexes 1–5 do not emit any significant fluores- The values of the association binding constant (K) and the number
cence under the same experimental conditions and the quenching of binding sites per albumin (n), as calculated from the Scatchard equa-
occurring in the BSA or HSA fluorescence emission spectra upon addi- tion (Eq. (S4)) [83] and Scatchard plots (Figs. S12 and S13), for the
tion of 1–5 is primarily due to changes in protein conformation, subunit complexes are given in Tables 5 and S5. The relatively high K values
association, substrate binding or denaturation [83]. of all complexes are higher than of free Hmef with 1 having the
Addition of complexes 1–5 to a SA solution results in a signifi- highest K values for both albumins used (K(HSA) = 5.29 × 106 M−1
cant quenching of the HSA fluorescence at λ = 351 nm (Fig. 8(A)) and K(BSA) = 5.05 × 107 M−1). Comparing the affinity of the com-
(e.g. quenching up to 99.9% of the initial fluorescence intensity pro- plexes for BSA and HSA (K values), it is obvious that all complexes
voked by complex 1) and to a much more enhanced quenching of the show higher affinity for BSA than HSA.
BSA fluorescence at λ = 343 nm (Fig. 8(B)) (e.g. quenching higher In conclusion, the quenching of SA fluorescence provoked by com-
than 99.9% of the initial fluorescence intensity in the case of 1). The plexes 1–5 is more pronounced than their Cu(II) and Co(II) analogs
observed quenching provoked by all complexes may be due to possible [31,32] and, subsequently, the corresponding quenching constants

(A) (B)
100 100
1 1
2 2
80 3 80 3
4 4
5 5
I/Io (%)

I/Io (%)

60 60

40 40

20 20

0 0
0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
r = [Complex]/[HSA] r = [Complex]/[BSA]
Fig. 8. (A) Plot of % relative fluorescence intensity at λem = 351 nm (%) vs r (r = [complex] / [HSA]) for complexes 1–5 (b0.1% of the initial fluorescence intensity for 1, 12% for 2,
2% for 3, 29% for 4 and 26% 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 complexes 1–5 (b0.1% of the initial fluorescence intensity for 1, 5% for 2, b0.5% for 3, 14% for 4 and 16% for 5) in buffer solution (150 mM NaCl and
15 mM trisodium citrate at pH 7.0).
 A. Tarushi et al. / Journal of Inorganic Biochemistry 128 (2013) 85–96 95

Table 5
The SA quenching constant (kq) and the SA association binding constant (K) for Hmef and complexes 1–5.

Compound kq(HSA) (M−1 s−1) K(HSA) (M−1) kq(BSA) (M−1 s−1) K(BSA) (M−1)

Hmef [32] 7.13(±0.34) × 1012 1.32(±0.15) × 105 2.78(±0.20) × 1013 1.35(±0.22) × 105
[Zn(mef)2(H2O)4], 1 1.46(±0.48) × 1015 5.29(±0.23) × 106 5.55(±0.14) × 1015 5.05(±0.33) × 107
[Zn(mef)2(bipy)], 2 3.40(±0.23) × 1013 3.43(±0.30) × 105 1.00(±0.05) × 1014 1.05(±0.05) × 106
[Zn(mef)2(bipyam)], 3 7.60(±0.22) × 1013 6.19(±0.34) × 105 2.05(±0.16) × 1014 8.76(±0.45) × 105
[Zn(mef)2(Hpko)2], 4 1.28(±0.03) × 1013 1.59(±0.13) × 105 3.20(±0.14) × 1013 4.78(±0.16) × 105
[Zn(mef)2(phen)(H2O)], 5 1.35(±0.10) × 1013 1.51(±0.25) × 105 2.58(±0.13) × 1013 4.80(±0.40) × 105

(kq) of 1–5, especially of 1, are higher than those reported for Cu(II) and py pyridine
Co(II) mefenamato complexes for both albumins. Additionally, the RA DPPH radical scavenging activity
Zn(II) mefenamato complexes exhibit the highest association binding SA serum albumin
constant values among the metal mefenamato complexes reported. A
comparison of the kq and K values of the Zn complexes with the
NSAID tolfenamic acid [25] reveals that the Zn-mefenamato complexes Acknowledgments
1–5 present higher constants.
In general, the binding constant of a compound to a protein should This research has been co-financed by the European Social Fund
be high enough to allow its binding and possible transfer, but also not (ESF) and the Greek national funds (National Strategic Reference
too high so that it can get released upon arrival at its target. It is quite Framework (NSRF)): Heracleitus II and Archimides III.
noteworthy that the K values of all complexes 1–5 are within such an Financial support from the Slovenian Research Agency (ARRS)
optimum range; high enough (1.3 × 105–5.05 × 107 M−1) to allow through project P1-0175 is gratefully acknowledged. We thank EN-FIST
the binding of the complexes to SAs and also quite below the association Centre of Excellence, Dunajska 156, 1000 Ljubljana, Slovenia for the use
constant of one of the strongest known non-covalent bonds for the of the SuperNova diffractometer. This project was also supported by EU
interaction between avidin and ligands (K ≈ 1015 M−1), suggesting a COST Action CM1105.
possible release from the serum albumin to the target cells [85].
Appendix A. Supplementary data
4. Conclusions
CCDC 910549–910551 contain the supplementary crystallograph-
In summary, characterization of neutral mononuclear zinc(II) com-
ic data for this paper. These data can be obtained free of charge via
plexes with the non-steroidal anti-inflammatory drug mefenamic acid
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge
in the absence or presence of the N,N′-donor heterocyclic ligands
Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ,
reveals several different binding modes for the mefenamato ligand
UK; fax: (+44) 1223-336-033; or deposit@ccde.cam.ac.uk).
that could be monitored through the use of the different bases. The syn-
Supplementary data to this article can be found online at http://
thesized complexes were shown to bind to CT DNA in an intercalative
dx.doi.org/10.1016/j.jinorgbio.2013.07.013.
binding mode that was more favored by the presence of Hpko base
compared to other similar zinc, copper or cobalt metal ion reported
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