Biological evaluation of non-steroidal anti-inflammatory drugs-cobalt(ii) complexes

Article abstract of DOI:10.1039/b927472c

Cobalt(ii) complexes with the non-steroidal anti-inflammatory drug mefenamic acid in the presence or absence of nitrogen donor heterocyclic ligands (2,2′-bipyridine, 1,10-phenanthroline or pyridine) have been synthesized and characterized with physicochemical and spectroscopic techniques. The experimental data suggest that mefenamic acid acts as deprotonated monodentate ligand coordinated to Co(ii) ion through a carboxylato oxygen. The crystal structures of tetrakis(methanol)bis(mefenamato)cobalt(ii), 1 and (2,2′-bipyridine)bis(methanol)bis(mefenamato)cobalt(ii), 2 have been determined by X-ray crystallography. The EPR spectra of complexes 1 and 2 in frozen solution reveal that they retain their structures. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that the complexes can bind to CT DNA and bis(methanol)bis(pyridine)bis(mefenamato) cobalt(ii) exhibits the highest binding constant. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. The cyclic voltammograms of the complexes recorded in dmso solution and in the presence of CT DNA in 1:2 dmso:buffer (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0) solution have shown that they can bind to CT DNA by the intercalative binding mode. Mefenamic acid and its cobalt(ii) complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values. The antioxidant activity of the compounds has been evaluated indicating their high scavenging activity against hydroxyl free radicals and superoxide radicals.

Full text of DOI:10.1039/b927472c

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/dalton | Dalton Transactions
Biological evaluation of non-steroidal anti-inflammatory drugs-cobalt(II)
complexes†
Filitsa Dimiza,a Athanasios N. Papadopoulos,b Vassilis Tangoulis,a Vassilis Psycharis,c
Catherine P. Raptopoulou,c Dimitris P. Kessissogloua and George Psomas*a
Received 5th January 2010, Accepted 15th March 2010
First published as an Advance Article on the web 6th April 2010
DOI: 10.1039/b927472c
Cobalt(II) complexes with the non-steroidal anti-inflammatory drug mefenamic acid in the presence or
absence of nitrogen donor heterocyclic ligands (2,2¢-bipyridine, 1,10-phenanthroline or pyridine) have
been synthesized and characterized with physicochemical and spectroscopic techniques. The
experimental data suggest that mefenamic acid acts as deprotonated monodentate ligand coordinated
to Co(II) ion through a carboxylato oxygen. The crystal structures of tetrakis(methanol)bis-
(mefenamato)cobalt(II), 1 and (2,2¢-bipyridine)bis(methanol)bis(mefenamato)cobalt(II), 2 have been
determined by X-ray crystallography. The EPR spectra of complexes 1 and 2 in frozen solution reveal
that they retain their structures. UV study of the interaction of the complexes with calf-thymus DNA
(CT DNA) has shown that the complexes can bind to CT DNA and bis(methanol)bis(pyridine)bis-
(mefenamato)cobalt(II) exhibits the highest binding constant. Competitive study with ethidium
bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind
to DNA in strong competition with EB. The cyclic voltammograms of the complexes recorded in dmso
solution and in the presence of CT DNA in 1 : 2 dmso : buffer (containing 150 mM NaCl and 15 mM
trisodium citrate at pH 7.0) solution have shown that they can bind to CT DNA by the intercalative
binding mode. Mefenamic acid and its cobalt(II) complexes exhibit good binding propensity to human
or bovine serum albumin protein having relatively high binding constant values. The antioxidant
activity of the compounds has been evaluated indicating their high scavenging activity against hydroxyl
free radicals and superoxide radicals.
cultured colon cancer cells lacking COX.2 NSAIDs have presented
Introduction
chemopreventive and antitumorigenic activity by reducing the
Non-steroidal anti-inflammatory drugs (NSAIDs‡) are among
number and size of carcinogen-induced colon tumors and exhibit-
ing a synergistic role on the activity of certain antitumor drugs.3
In an attempt to explain the tentative anticancer as well as the
anti-inflammatory activity of the NSAIDs, their interaction with
DNA should be of great interest.4 Nevertheless, the anionic form
they present at physiological pH is an obstacle to their approach
to the poly-anionic DNA backbone resulting in few reports on
the interaction of NSAIDs and their complexes with DNA.5
Additionally, metal complexes have also exhibited synergistic
activity when administered in conjunction with NSAIDs.6 It has
been found that the copper complexes of some antiarthritic drugs
are more active as anti-inflammatory agents than their parent
compounds.7
Mefenamic acid (Hmef = 2-[(2,3-dimethylphenyl)-amino]-
benzoic acid or N-(2,3-xylyl)anthranilic acid), (Fig. 1), is a NSAID
that belongs to the derivatives of N-phenylanthranilic acid. It
chemically resembles tolfenamic and flufenamic acids and other
fenamates in clinical use. In the literature, the crystal structures of
two tin(IV), a mononuclear and a dinuclear Cu(II) complexes of
mefenamic acid have been reported.8
the most frequently used medicinal drugs and they are utilized
primarily as analgesic, anti-inflammatory and antipyretic agents
since their side-effects have been well studied.1 Their mode of
action is either through inhibition of the cyclo-oxygenase(COX)-
mediated production of prostaglandins or via COX-independent
mechanisms by modulating cell proliferation and cell death in
aDepartment of General and Inorganic Chemistry, Faculty of Chemistry,
Aristotle University of Thessaloniki, P.O. Box 135, GR-54124, Thessa-
loniki, Greece. E-mail: gepsomas@chem.auth.gr; Fax: +30+2310997738;
Tel: +30+2310997790
bDepartment of Nutrition and Dietetics, Faculty of Food Technology
and Nutrition, Alexandrion Technological Educational Institution, Sindos,
Thessaloniki, Greece
cInstitute of Materials Science, NCSR “Demokritos”, GR-15310, Aghia
Paraskevi Attikis, Greece
† Electronic supplementary information (ESI) available: Additional data.
CCDC reference numbers 759764 and 759765. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/b927472c
‡ Abbereviations: ABTS
=
2,2¢-azinobis-(3-ethylbenzothiazoline-6-
sulfonic acid) radical cation, BHT = butylated hydroxytoluene, bipy = 2,2¢-
bipyridine, BSA = bovine serum albumin, COX = cyclo-oxygenase, CT =
calf-thymus, DPPH = 1,1-diphenyl-picrylhydrazyl, EB = ethidium bro-
mide, Hmef = mefenamic acid, HSA = human serum albumin, NDGA =
nordihydroguairetic acid, NSAID = Non-steroidal anti-inflammatory
drug, phen = 1,10-phenanthroline, py = pyridine, TEAP = Tetraethylam-
monium perchlorate, Trolox = 6-hydroxy-2,5,7,8-tetramethylchromane-2-
carboxylic acid
Cobalt is an element of biological interest. Its biological
role is mainly focused on its presence in the active center of
vitamin B12, which regulates indirectly the synthesis of DNA.
Additionally, there are at least eight cobalt-dependent proteins.9
Cobalt is involved in the co-enzyme of vitamin B12 used as a
This journal is
The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 4517–4528 | 4517
©

View Article Online
CoCl2·6H2O + 2 C14H14N-COOH + 2 KOH + 4 CH3OH →
[Co(C14H14N-COO)2(CH3OH)4] + 2 KCl + 8 H2O
(1)
(2)
CoCl2·6H2O + 2 C14H14N-COOH + 2 KOH + C10H8N2 + 2
CH3OH → [Co(C14H14N-COO)2(C10H8N2)(CH3OH)2] + 2
KCl + 8 H2O
The complexes are soluble in dmso and dmf, stable in air and no
electrolytes in dmso.
In the IR spectrum, the observed absorption bands at 3370
(br,m) cm-1, 1655 (s) cm-1 and 1255 (s) cm-1 attributed to the
=
Fig. 1 Mefenamic acid (Hmef = 2-[(2,3-dimethylphenyl)-amino]benzoic
acid).
stretching vibrations n(H–O), n(C O)carboxylic and n(C–O)carboxylic
,
respectively, of the carboxylic moiety (–COOH) of the mefenamic
acid, have been replaced in the IR spectra of the complexes by
two very strong characteristic bands in the range 1609–1615 cm-1
supplement of the vitamin.10 Since the first reported studies into
the biological activity of Co complexes in 1952,11 many cobalt
complexes of biological interest have been reported with the most
structurally characterized showing antitumor, antiproliferative,12
antimicrobial,13 antifungal14 and antiviral15 activity. To the best
of our knowledge, no cobalt(II) complexes of the NSAID group
of N-phenylanthranilic acids have been structurally characterized
yet.
and 1382–1389 cm-1 assigned as anti-symmetric, nasym(C O), and
=
=
symmetric, nsym(C O), stretching vibrations of the carboxylato
=
=
group, respectively. The difference D [nasym(C O) - nsym(C O)], a
useful characteristic tool for determining the coordination mode
of the carboxylato ligands, gives a value falling in the range 224–
230 cm-1 indicative of a monodentate coordination mode for the
mefenamato ligand.18c
Our studies have been focused on the coordination chemistry
of carboxylate-containing herbicides,16 antimicrobial17 or anti-
inflammatory18 agents with metal ions in an attempt to examine
their mode of binding and possible biological relevance. In
addition, we have reported studies on the interaction of metal
complexes with nucleic acids and their biological activity. Taking
into consideration the reported biological role and activity of
cobalt and its complexes as well as the significance of the
NSAIDs in medicine, we have initiated the investigation of the
interaction of cobalt(II) with ligands that belong to the NSAID
group. In this context, we report the synthesis, the structural
characterization, the electrochemical and the biological properties
of the neutral mononuclear cobalt(II) complexes with the NSAID
mefenamic acid in the absence ([Co(mef)2(MeOH)4]·2MeOH,
1·2MeOH) or presence of a nitrogen-donor heterocyclic ligand
such as bipy, phen or pyridine (py) ([Co(mef)2(bipy)(MeOH)2] 2,
[Co(mef)2(phen)(MeOH)2] 3 and [Co(mef)2(py)2(MeOH)2] 4). The
crystal structures of 1·2MeOH and 2 have been determined by
X-ray crystallography. The study of the biological properties of
the compounds has been focused on (i) the binding properties with
CT DNA investigated with UV spectroscopy, cyclic voltammetry
and competitive binding studies with ethidium bromide (EB),
(ii) the affinity for bovine (BSA) and human serum albumin
(HSA), proteins involved in the transport of metal ions and metal
complexes with drugs through the blood stream, investigated with
fluorescence spectroscopy and (iii) the antioxidant capacity, since
the use of NSAIDs in medicine as anagelsics and antiinflammato-
ries may be related to free radicals scavenging.
The UV-vis spectra of the complexes have been recorded as nujol
mull and in dmso solution and are similar, suggesting that the
complexes retain their structure in solution. In the visible region,
three low-intensity bands are observed and can be assigned to
d–d transitions. More specifically, for local Oh symmetry, band I
observed in the region 733–742 nm may be attributed to a 4T1g(F)
4
→ T2g transition, band II in the region 535–565 nm to a 4T2g(F)
4
4
→ A2g transition and band III at 440–475 nm to a T1g(F) →
4T1g(P) transition and are typical for distorted octahedral high-
spin Co2+ complexes.9 Additionally, an absorption band assigned
to charge transfer transition for the mefenamato ligand exists at
around 395 nm.
The observed values of meff (4.35–4.50 BM) for the complexes at
room temperature are higher than the spin-only value (3.87 MB)
2
showing spin–orbit coupling due to t2g5eg electron configuration.
The values are within the range reported for mononuclear high-
spin Co(II) complexes (S = 3/2).9
X-Band EPR measurements were carried out in powder samples
as well as in frozen solutions of 1 and 2 in dmso and are shown
in Fig. S1.† As a consequence of the fast spin–lattice relaxation
time of high-spin Co(II), signals were observed only below 70 K.
For the powder spectra, at temperatures T < 25 K, 1 exhibits a
broad derivative and an axial broad peak with g1 = 3.9(1) and g2 =
2.04(1) while 2 exhibits a rhombic broad signal with three g values
g1 = 5.5(1), g2 = 3.5(1) and g3 = 3.0(1), (giso = 4.0) and a weak
signal centred also at g = 2.04(1). The frozen solutions of both
systems are more isotropic as it is expected with axial g-values:
g1 = 4.0(1), g2 = 2.02(1) revealing that the systems do retain their
structures.
Results and discussion
The dominant broadening effect emerges when the g-strain is
Synthesis and characterization of the complexes
⎛
⎜
⎜
⎜
⎞
⎛
⎞
hn ⎟ Dg
⎟
⎟
⎟
⎟
⎟
⎠
⎜
⎟
⎜
converted into B-strain through the equation
,
DB = −
⎟
⎜
⎟
2
⎜
⎜
⎝
⎜
m
⎟⎜
The synthesis of the complexes in high yield was achieved via the
aerobic reaction of mefenamic acid (C14H14N-COOH) and KOH
with CoCl2·6H2O in the absence, (eqn (1)) for 1, or presence of the
corresponding N-donor heterocyclic ligand, e.g. (C10H8N2, eqn
(2)) for 2, according to the equations:
g
⎝
⎠
B
where the parameters have their usual meaning. Thus, the largest
and smallest g-values of the powder and solution spectra have field
widths that differ by an order of magnitude, thereby rationalizing
the broad high-field features of the spectrum.19
4518 | Dalton Trans., 2010, 39, 4517–4528
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
Table 1 Selected bond distances and angles for complex 1
Table 2 Selected bond distances and angles for complex 2
˚
˚
˚
˚
Bond distances/A
Bond distances/A
Bond distances/A
Bond distances/A
Co–O(1)
Co–O(21)
Co–O(22)
C(1)–O(2)
2.060(2)
2.084(3)
2.063(3)
1.254(4)
C(21)–O(21)
C(22)–O(22)
C(1)–O(1)
1.377(6)
1.305(5)
1.267(4)
Co–O(2)
Co–N(21)
C(1)–O(2)
2.063(2)
2.115(3)
1.257(4)
Co–O(31)
C(1)–O(1)
2.072(3)
1.244(4)
Bond angles/◦
O(2)–Co–O(2)¢
O(2)–Co–O(31)
O(2)–Co–O(31)¢
O(31)–Co–N(21)¢
N(21)–Co–N(21)¢
Bond angles/◦
Bond angles/◦
O(1)–Co–O(22)
O(1)–Co–O(22)¢
O(21)–Co–O(22)
O(1)–Co–O(1)¢
O(22)–Co–O(22)¢
Bond angles/◦
179.6(2)
88.0(2)
92.3(1)
171.5 (2)
77.0(2)
O(2)–Co–N(21)
O(2)–Co–N(21)¢
O(31)–Co–N(21)
O(31)–Co–O(31)¢
92.7(1)
87.0(1)
96.4(2)
90.6(3)
90.2(1)
89.8(1)
88.2(2)
180.0
O(1)–Co–O(21)
O(1)¢–Co–O(21)
O(21)–Co–O(22)¢
O(21)–Co–O(21)¢
90.6(1)
89.4(1)
91.8(2)
180.0
180.0
Primed atoms are generated by symmetry: (¢) -x, y, -z.
Primed atoms are generated by symmetry: (¢) -x, 1 - y, -z.
Crystal structure of [Co(mef)2(MeOH)4]·2MeOH, 1·2MeOH
A diagram of 1 is shown in Fig. 2, and selected bond distances
and angles are listed in Table 1. The complex is mononuclear with
the mefenamato ligand behaving as a monodentate deprotonated
ligand coordinated to cobalt atom via a carboxylate oxygen.
Fig. 3 The molecular structure and partial labeling of 2 with only the
heteroatoms labeled.
molecules form the basal plane of the octahedron with the two
oxygen atoms from the mefenamato ligands lying at the apical.
The bond distances around the cobalt atom are not equal, with
the coordinated carboxylate oxygen atoms (Co–O(2) = 2.064(2)
Fig. 2 The molecular structure and partial labeling of 1 with only the
heteroatoms labeled.
˚
A) lying closer to Co than the methanol oxygen atoms (Co–
˚
O(31) = 2.072(3) A) and the bipy nitrogen atoms (Co–N(21))
˚
The structure of the complex is centrosymmetric, the cobalt(II)
ion is sitting on a center of symmetry and is coordinated to two
mefenamato ligands and four methanol molecules related by the
inversion center. Thus, the cobalt atom is six-coordinate and it
displays an octahedral geometry. All the Co–O distances are of the
same magnitude with Co–Ocarboxylate being the shortest (Co–O(1) =
being at a distance 2.115(3) A. The methanol molecules are lying
at cis positions (O(31)–Co–O(31)¢ = 90.6(3)◦) and the mefenamato
oxygen atoms are trans (O(2)–Co–O(2)¢ = 179.6(2)◦) to each other.
The carboxylate group is asymmetrically bound to cobalt (C(1)–
˚
˚
O(2) = 1.257(4) A and C(1)–O(1) = 1.244(4) A).
The N(21)–Co–N(21)¢ angle observed is 77.0(2)◦ and is similar
to reported values of other chelating polycyclic diimines.20 The
2,2¢-bipyridine ligand is planar with the cobalt atom lying in this
plane.
˚
2.060(2) A) and the inequivalent Co–Omethanol (Co–O(21) 2.084(3),
˚
Co–O(22) = 2.063(3) A) being the longest. Taking into account
the small differences found in the Co–O distances in combination
with the angles around cobalt (O(1)–Co–O(22) = 90.2(1)◦, O(1)–
Co–O(21) = 90.6(1)◦ and O(21)–Co–O(22) = 88.2(2)◦), the
octahedron displays a slight distortion. The carboxylate group
The crystal structures of 1 and 2 are the only structures of
Co(II)-anthranilic acid complexes reported. Generally structurally
characterized metal complexes of mefenamic acid or flufenamic,
tolfenamic, niflumic and meclofenamic acid, members of the
NSAID group of anthranilic acids are rare, with only copper(II)
and tin(II) complexes reported so far (Table 3).21
˚
is asymmetrically bound to cobalt (C(1)–O(1) = 1.267(4) A and
˚
C(1)–O(2) = 1.254(4) A).
Crystal structure of [Co(mef)2(bipy)(MeOH)2], 2
Interaction with DNA
A diagram of 2 is shown in Fig. 3, and selected bond distances
and angles are listed in Table 2. The complex is mononuclear and
the mefenamato ligand behaves as a monodentate deprotonated
ligand coordinated to cobalt atom via a carboxylate oxygen.
The cobalt atom is six-coordinate and is surrounded by two
mefenamato ligands, two methanol molecules and a bidentate 2,2¢-
bipyridine ligand showing a distorted octahedral geometry. The
two nitrogen atoms of bipy and two oxygen atoms of the methanol
Transition metal complexes can bind to DNA via both covalent
and/or non-covalent interactions. In the case of covalent binding,
the labile ligand of the complexes can be replaced by a nitrogen
base of DNA such as guanine N7, while the non-covalent
DNA interactions include intercalative, electrostatic and groove
(surface) binding of metal complexes outside of DNA helix, along
major or minor groove.22 To the best of our knowledge, metal
This journal is
The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 4517–4528 | 4519
©

View Article Online
Table 3 X-Ray structurally characterized complexes of NSAID group of
stacking interaction between the aromatic chromophore (either
from mefenamato and/or the N-donor ligands) of the complexes
and DNA base pairs consistent with the intercalative binding
mode, while the red-shift is an evidence of the stabilization of
the CT DNA duplex.23
anthranilic acids
Metal Complex
Geometry
Reference
Cu
Cu
Cu
Cu
Cu
Cu
Cu
Cu
Sn
[Cu2(fluf)4(caffeine)(H2O)]
Paddlewheel
Paddlewheel
Paddlewheel
Paddlewheel
Paddlewheel
21a
7a
21b
21c
8c
21d
21e
8b
The changes occurring in the spectrum of a 10-5 M solution of
Hmef, 1 and 4 upon addition of CT DNA in diverse r values can be
observed in Fig. 5. In the UV region, the intense absorption bands
observed in the spectra of the complexes are attributed to the
intraligand transition of the coordinated groups of mefenamato
ligands. Any interaction between each complex and CT DNA
could perturb its intraligand centred spectral transitions.24
In the UV spectrum of Hmef, no significant changes were
observed in the position of the p → p* transition bands upon
addition of CT DNA (Fig. 5(A)). On the other hand, the intensity
of the bands centred at 324 nm is increased upon addition of
CT DNA, suggesting tight binding to CT DNA. The observed
hyperchromic effect suggests binding to CT DNA ascribed either
to external contact or to the fact that mefenamic acid could uncoil
the helix structure of DNA.25
[Cu(nifl)2(DMSO)]2
[Cu2(nifl)4(H2O)2]
[Cu(tolf)2(DMF)]2
[Cu(mef)2(DMSO)]2
[Cu(nifl)2(3-pyridylmethanol)2] Square-planar
[Cu(fluf)2(Et2nia)2(H2O)2]
[Cu(mef)2(MeOH)2(py)2]
[Me2(fluf)SnOSn(fluf)Me2]2
[Bu2Sn(tolf)O(tolf)SnBu2]2
Octahedral
Octahedral
Octahedral
Distorted trigonal 21g
bipyramidal
21f
Sn
Sn
Sn
[Me2Sn(mef)O(mef)SnMe2]2
[Bu2Sn(mef)O(mef)SnBu2]2
Distorted trigonal 8a
bipyramidal
Distorted trigonal 8a
bipyramidal
Co
Co
[Co(mef)2(MeOH)4]·MeOH
Octahedral
Octahedral
This work
This work
[Co(mef)2(bipy)(MeOH)2]
Hfluf = flufenamic acid, Hnifl = niflumic acid, Htolf = tolfenamic acid,
Hmef = mefenamic acid, Et2nia = N,N-diethylnicotinamide.
In the UV spectrum of 1 (Fig. 5(B)), the band centred at
340 nm exhibits a hyperchromism accompanied by a red-shift
of 3 nm (up to 343 nm) suggesting probable external binding to
DNA and stabilization. Additionally, the band at 300 nm presents
a hyperchromism accompanied by a blue-shift of 4 nm (up to
296 nm).
complexes of the oxicam NSAIDs have been found to bind to
DNA via the intercalative mode,5b while the interaction of DNA
with metal complexes of the anthralinic acid NSAIDs has not been
investigated yet.
In the UV spectrum of 4, the band centred at 340 nm exhibits
initially a slight hypochromism (Fig. 5(C)) suggesting tight binding
to CT DNA probably by intercalation. Further addition of
DNA results in a hyperchromism accompanied by a red-shift of
4 nm (up to 343 nm) suggesting tight binding and stabilization.
Additionally, the band at 300 nm presents a hyperchromism
accompanied by a red-shift of 5 nm (up to 305 nm). A distinct
isosbestic point at 331 nm appears upon addition of CT DNA.
The behaviour of complexes 2 and 3 is quite similar to that of 4.
The results derived from the UV titration experiments suggest
that all compounds can bind to CT DNA although the exact
mode of binding cannot be merely proposed by UV spectroscopic
titration studies.26 Nevertheless, the existence of hypochromism for
complexes 2–4 could be considered as evidence that the binding
of the complexes involving intercalation between the base pairs of
CT DNA cannot be ruled out.17b-f The different behaviour between
complexes 2–4 and 1 may be attributed either to the steric effect
due to pyridine ligands in complex 4 or to a combination of steric
and chelate effects provided by the bipy or phen ligands in 2 and
3, respectively.
Study of the DNA-binding with UV spectroscopy
The changes observed in the UV spectra upon titration may give
evidence of the existing interaction mode, since a hypochromism
due to p → p* stacking interactions may appear in the case of
the intercalative binding mode, while a red-shift (bathochromism)
may be observed when the DNA duplex is stabilized.23
The UV spectra have been recorded for a constant CT DNA
concentration in different [compound]/[DNA] mixing ratios (r).
UV spectra of CT DNA in the presence of a compound derived for
diverse r values are shown representatively for 2 in Fig. 4. Hmef and
the complexes exhibit similar behaviour upon their addition on
CT DNA solution. The decrease of the intensity at lmax = 258 nm
is accompanied by a red-shift of the lmax up to 265 nm for all
compounds, indicating that the interaction with CT DNA results
in the direct formation of a new complex with double-helical
CT DNA.22 The observed hypochromism could be attributed to
The binding constant of the compounds to CT DNA, Kb, can
be obtained by the ratio of the slope to the y intercept in plots
[DNA]
versus [DNA] (insets in Fig. 5(B) and (C)), according
(e −e )
A
f
to the equation:23b
[DNA]
[DNA]
1
=
+
(3)
(e −e ) (e −e ) K (e −e )
A
f
b
f
b
b
f
Fig. 4 (A) UV spectra of CT DNA in buffer solution (150 mM NaCl
and 15 mM trisodium citrate at pH 7.0) in the absence or presence of
[Co(mef)2(bipy)(MeOH)2] 2. The arrows show the changes upon increasing
amounts of complex.
where [DNA] is the concentration of DNA in base pairs, eA
=
Aobsd/[compound], ef = the extinction coefficient for the free
compound and eb = the extinction coefficient for the compound in
4520 | Dalton Trans., 2010, 39, 4517–4528
This journal is The Royal Society of Chemistry 2010
©

View Article Online
Table 4 The DNA binding constants (Kb) and the Stern–Volmer con-
stants (KSV) of complexes 1–4
Complex
Kb/M-1
K
SV/M-1
Hmef
1.05 ( 0.02) ¥ 105
5.82 ( 0.25) ¥ 104
4.59 ( 0.24) ¥ 104
3.02 ( 0.45) ¥ 104
3.22 ( 0.04) ¥ 105
1.58 ( 0.06) ¥ 105
7.63 ( 0.42) ¥ 105
1.09 ( 0.03) ¥ 106
4.10 ( 0.22) ¥ 105
2.17 ( 0.09) ¥ 105
[Co(mef)2(MeOH)4], 1
[Co(mef)2(bipy)(MeOH)2], 2
[Co(mef)2(phen)(MeOH)2], 3
[Co(mef)2(py)2(MeOH)2], 4
and 3, indicating that the existence of a N,N¢-donor ligand does
not enhance the affinity for DNA. On the other hand, the presence
of the N-donor ligand pyridine results to increased affinity for
DNA. Finally, the Kb values of all compounds are of the same
magnitude to that of the classical intercalator EB (Kb = 1.23 (
0.07) ¥ 105 M-1).16c,17d
Competitive studies with ethidium bromide
Ethidium bromide (EB
=
3,8-diamino-5-ethyl-6-phenyl-
phenanthridinium bromide) is a typical indicator of intercalation
since it can form soluble complexes with nucleic acids emitting
intense fluorescence in the presence of CT DNA due to the
intercalation of the planar phenenthridinium ring between
adjacent base pairs on the double helix. The changes observed
in the fluorescence spectra of EB on its binding to CT DNA are
often used for the interaction study between DNA and other
compounds, such as metal complexes.27
Hmef and complexes 1–4 show no fluorescence at room
temperature in solution or in the presence of CT DNA, and their
binding to DNA cannot be directly predicted through emission
spectra. Hence, competitive EB binding studies may be undertaken
in order to examine the binding of each compound with DNA.
EB does not show any appreciable emission in buffer solution due
to fluorescence quenching of the free EB by the solvent molecules
and the fluorescence intensity is highly enhanced upon addition
of CT DNA, due to its strong intercalation with DNA base pairs.
Addition of a second molecule, which may bind to DNA more
strongly than EB results to a decrease the DNA-induced EB
emission. Two mechanisms have been proposed to account for
this reduction in the emission intensity: the replacement of EB,
and/or electron transfer.28
The emission spectra of EB bound to CT DNA in the absence
and presence of each compound have been recorded for [EB] = 20
mM and [DNA] = 26 mM upon addition of increasing amounts
of each compound. The addition of Hmef or each complex 1–4
at diverse r values (Fig. 6(A)) results in a significant decrease
of the intensity of the emission band of the DNA-EB system at
592 nm (up to 20% of the initial EB-DNA fluorescence intensity for
Hmef, 14% for 1, 19% for 2, 26% for 3 and 23% for 4) indicating
the competition of the complexes with EB in binding to DNA.
The observed significant quenching of DNA-EB fluorescence for
Hmef and 1–4 suggests that they displace EB from the DNA-
EB complex and they probably interact with CT DNA by the
intercalative mode.17b-f,29
Fig. 5 UV spectra of ([compound] = 1 ¥ 10-5 M) (A) Hmef, (B)
[Co(mef)2(MeOH)4] 1 and (C) [Co(mef)2(py)2(MeOH)2] 4 in dmso solution
in the presence of CT DNA at increasing amounts. The arrows show the
[DNA]
changes upon increasing amounts of CT DNA. Inset: plot of
versus [DNA].
(Δ −Δ )
A
f
the fully bound form. The calculated Kb values (Table 4) suggest
a relatively strong binding of Hmef and complexes 1–4 to CT
DNA17b-f and increase in the order 3 < 2 < 1 < Hmef < 4 with
complex 4 exhibiting the highest Kb value (3.22 ( 0.04) ¥ 105 M-1).
The coordination of Hmef to Co(II) results in a decrease of the Kb
value as calculated for 1 (Table 4), while the co-existence of the
N,N¢-donor heterocyclic ligands leads to lower Kb values as for 2
The Stern–Volmer constant, KSV, can be obtained by the slope
Io
of the diagram
versus [Q] and is used to evaluate the quenching
I
efficiency for each compound according to the equation (eqn (4)):
This journal is
The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 4517–4528 | 4521
©

View Article Online
more cathodic wave appears at -270 mV (Epc2). The one-electron
cathodic wave at Epc1 can be attributed to the reduction of [Co(II)]
to [Co(I)], while the two anodic waves at Epa1 and Epa2 can be
attributed to the oxidation processes [Co(I)] → [Co(II)] and [Co(II)]
→ [Co(III)], respectively, with the second cathodic wave at Epc2 to
the reduction of [Co(III)] to [Co(II)] species.17b,c Complexes 1–3
present similar cyclic voltammograms in dmso solution and the
corresponding potentials are given in Table 5.
The electrochemical investigations of metal–DNA interactions
can provide a useful supplement to spectroscopic methods and
yield information about interactions with both the reduced and
oxidized form of the metal. In general, the electrochemical poten-
tial of a small molecule will shift positively when it intercalates
into DNA double helix, and it will shift to a negative direction in
the case of electrostatic interaction with DNA.30
Fig. 6 Plot of EB relative fluorescence intensity at lem = 592 nm (%)
versus r (r = [complex]/[DNA]) for Hmef and complexes 1–4 in buffer
solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0).
The quasi-reversible redox couple Co(II)/Co(I) for each complex
in 1 : 2 dmso : buffer solution has been studied upon addition of CT
DNA and the corresponding potentials as well as their shifts are
given in Table 5. No new redox peaks appeared after the addition of
CT DNA to each complex, but the current intensity of all the peaks
decreased significantly, suggesting the existence of an interaction
between each complex and CT DNA, and can be explained in
terms of an equilibrium mixture of free and DNA-bound complex
to the electrode surface.17b
For increasing amounts of CT DNA, the cathodic potential Epc
for all complexes shows a positive shift (DEpc = 0 - (+50) mV)
(Table 5) while the anodic potential Epa shifts slightly to more
negative or positive values (DEpa = (-15) - (+60) mV) suggesting
an intercalative mode of binding while the co-existence of external
(possibly electrostatic) interaction in the case of 3 (negative shift
of Epa) cannot be ruled out.17b,e,f,30b
Io
(4)
=1+ K [Q]
SV
I
where Io and I are the emission intensities in the absence and the
presence of the quencher, respectively, [Q] is the concentration of
the quencher (Hmef or complexes 1–4). The Stern–Volmer plots
of DNA-EB (Fig. S2†) 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 (eqn (4)), which proves that the
partial replacement of EB bound to DNA by each compound
results in a decrease of the fluorescence intensity. The high KSV
values of the compounds (Table 4) show that they bind tightly to
the DNA.17b-f
Study of the DNA-binding with cyclic voltammetry
The cyclic voltammograms of 4 in dmso solution (Fig. 7) exhibit
one cathodic wave at -920 mV (Epc1) followed by two anodic waves
at -10 mV (Epa1) and at +620 mV (Epa2). In the reverse scan, one
Binding of serum albumins
It is important to consider the interactions of drugs with plasma
proteins particularly with serum albumin, which is the most
abundant protein in plasma. Binding to these proteins may lead to
loss or enhancement of the biological properties of the original
drug, or provide paths for drug transportation. Bovine serum
albumin (BSA) is the most extensively studied serum albumin,
due to its structural homology with human serum albumin
(HSA). BSA with its two tryptophans, Trp-134 and Trp-212, and
HSA with one Trp-214, can bind reversibly to a large number
of endogenous and exogenous compounds.31 The interaction of
Hmef and complexes 1–4 with serum albumins has been studied
from tryptophan emission-quenching experiments. BSA and HSA
solutions exhibit a strong fluorescence emission with a peak at
Fig. 7 Cyclic voltammogram of 0.4 mM dmso solution of 4. Scan rate =
100 mV s-1. Supporting electrolyte = TEAP, 0.1 M.
Table 5 Cathodic and anodic potentials (in mV) for the redox couples Co(II)/Co(I) and Co(III)/Co(II) in dmso solution and for the redox couple
Co(II)/Co(I) in 1 : 2 dmso : buffer solution of the complexes in the absence and presence of CT DNA
a
a
a
a
b
c
d
b
c
d
Complex
Epc1
Epa1
Epa2
Epc2
Epc(f)
Epc(b)
DEpc
Epa(f)
Epa(b)
DEpa
[Co(mef)2(MeOH)4], 1
-1010
-1205
-960
-45
-25
+600
+620
+590
+620
-303
-215
-315
-270
-675
-750
-665
-685
-675
-700
-665
-680
0
+50
0
-547
-550
-525
-560
-527
-490
-540
-535
+20
+60
-15
+25
[Co(mef)2(bipy)(MeOH)2], 2
[Co(mef)2(phen)(MeOH)2], 3
[Co(mef)2(py)2(MeOH)2], 4
+180
-920
-10
+5
a Epc/a in dmso solution. b Epc/a in dmso/buffer in the absence of CT DNA (Epc/a(f)). c Epc/a in dmso/buffer in the presence of CT DNA (Epc/a(b)). d DEpc/a
Epc/a(b) - Epc/a(f)
=
.
4522 | Dalton Trans., 2010, 39, 4517–4528
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
Table 6 The BSA binding constants and parameters (Ksv, kq, K, n) derived for Hmef and complexes 1–4
Compound
K
SV/M-1
kq/M-1 s-1
K/M-1
n
Hmef
2.78 ( 0.20) ¥ 105
2.11 ( 0.22) ¥ 106
2.86 ( 0.23) ¥ 106
6.04 ( 0.25) ¥ 105
6.32 ( 0.37) ¥ 105
2.78 ( 0.20) ¥ 1013
2.11 ( 0.22) ¥ 1014
2.86 ( 0.23) ¥ 1014
6.04 ( 0.25) ¥ 1013
6.32 ( 0.37) ¥ 1013
1.35 ¥ 105
2.22 ¥ 105
2.38 ¥ 105
3.66 ¥ 105
2.37 ¥ 105
1.20
1.27
1.31
1.06
1.12
[Co(mef)2(MeOH)4], 1
[Co(mef)2(bipy)(MeOH)2], 2
[Co(mef)2(phen)(MeOH)2], 3
[Co(mef)2(py)2(MeOH)2], 4
343 nm and 351 nm, respectively, due to their tryptophan residues,
when excited at 295 nm. The changes in the emission spectra
of tryptophan in BSA or HSA are primarily due to change in
protein conformation, subunit association, substrate binding or
denaturation,31b since Hmef and complexes 1–4 in buffer solutions
do not exhibit any emission spectra under the same experimental
conditions.
Addition of Hmef or complexes 1–4 to BSA results in a
significant decrease of the fluorescence (up to 10% of the initial
fluorescence intensity of BSA for Hmef (Fig. 8), 2% for 1, 1%
for 2, 6% for 3 and 7% for 4) at l = 343 nm without any
other spectroscopic changes due to possible changes in protein
secondary structure of BSA indicating the binding of Hmef or
each complex to BSA.
strongest protein-binding ability (kq = 2.86 ( 0.23) ¥ 1014) since
kq increases in the order Hmef < 3 < 4 < 1 < 2 with values (>1013
M-1 s-1) higher than the diverse kinds of quenchers for biopolymer
fluorescence (2.0 ¥ 1010 M-1 s-1) indicating the existence of static
quenching mechanism.31c Similar kq values (1012–1013 M-1 s-1) have
been recently reported for a series of Ni(II) and Zn(II) complexes
with quinolone antibacterial agents, where the complexes have
exhibited higher affinity to human or bovine serum albumin than
the free drug.17e-h
Using the Scatchard equation:33
DI
Io
DI
(6)
= nK − K
[Q]
Io
where n is the number of binding sites per albumin and K is the
association binding constant, K (M-1) may be calculated from the
DI
DI
Io
slope in plots
versus
(Fig. S4†) and n is given by the ratio of
Io
[Q]
the y intercept to the slope.33 The K and n values are cited in Table
6 with 3 having the highest K value and 2 the highest n value. The
K values are relatively high and Hmef exhibits the lowest value,
indicating that its coordination to Co2+ leads to enhanced stability
of the corresponding compound with BSA. The n values of all
compounds are of similar magnitude (1.06–1.30) with 2 showing
the highest value.
The quenching provoked by Hmef or complexes 1–4 to the HSA
fluorescence at l = 351 nm (Fig. 9) is significant (up to 40% of
the initial fluorescence intensity for Hmef, 17% for 1, 13% for 2,
12% for 3 and 28% for 4) without any other spectroscopic changes
indicating that the binding of the compounds to HSA quenches
the intrinsic fluorescence of the single tryptophan in HSA.34
Fig. 8 Plot of % relative fluorescence intensity at lem = 342 nm (%) versus
r (r = [compound]/[BSA]) for Hmef and complexes 1–4 in buffer solution
(150 mM NaCl and 15 mM trisodium citrate at pH 7.0).
The Stern–Volmer and Scatchard graphs may be used in order to
study the interaction of a quencher in presence of serum albumins.
According to Stern–Volmer quenching equation:32
Io
(5)
=1+ k t [Q]=1+ K [Q]
q
0
SV
I
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 constant SA, KSV
=
the dynamic quenching constant, to = the average lifetime of SA
without the quencher, [Q] = the concentration of the quencher
respectively and KSV = kqto, and taking the fluorescence lifetime
(to) of tryptophan in SA at around 10-8 s,32 the dynamic quenching
Fig. 9 Plot of % relative fluorescence intensity at lem = 352 nm (%) versus
r (r = [compound]/[HSA]) for Hmef and complexes 1–4 in buffer solution
(150 mM NaCl and 15 mM trisodium citrate at pH 7.0).
constant (KSV, M-1) can be obtained by the slope of the diagram
Io
versus [Q] (Fig. S3†), and subsequently the approximate
I
quenching constant (kq, M-1 s-1) may be calculated. The calculated
values of Ksv and kq (to ~ 10-8 s) for the interaction of Hmef and
complexes 1–4 with BSA are given in Table 6 and indicate good
BSA binding propensity of the complexes. Complex 2 exhibits the
The calculated values of KSV and kq, as obtained by the slope of
the Stern–Volmer plot (Fig. S5†), for Hmef and 1–4 are given in
Table 7 and indicate their good HSA binding propensity. The kq
This journal is
The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 4517–4528 | 4523
©

View Article Online
Table 7 The HSA binding constants (Ksv, kq, K, n) derived for Hmef and complexes 1–4
Compound
K
SV/M-1
kq/M-1 s-1
K/M-1
n
Hmef
7.13 ( 0.34) ¥ 104
1.96 ( 0.06) ¥ 105
1.88 ( 0.08) ¥ 105
2.29 ( 0.20) ¥ 105
1.03 ( 0.06) ¥ 105
7.13 ( 0.34) ¥ 1012
1.96 ( 0.06) ¥ 1013
1.88 ( 0.08) ¥ 1013
2.29 ( 0.20) ¥ 1013
1.03 ( 0.06) ¥ 1013
1.32 ¥ 105
1.46 ¥ 105
1.34 ¥ 105
1.49 ¥ 105
2.43 ¥ 105
0.82
1.07
1.13
1.13
0.79
[Co(mef)2(MeOH)4], 1
[Co(mef)2(bipy)(MeOH)2], 2
[Co(mef)2(phen)(MeOH)2], 3
[Co(mef)2(py)2(MeOH)2], 4
Table 9 Competition % with DMSO for hydroxyl radical (∑OH%); %
values increase in the order Hmef < 4 < 2 < 1 < 3, with complex
3 exhibiting the strongest protein-binding ability and provoking
the highest quenching. The kq values (>1013 M-1 s-1) are similar to
those observed for Ni(II) and Zn(II) quinolone complexes17e-h and
are higher than diverse quenchers for biopolymers fluorescence
(2.0 ¥ 1010 M-1 s-1) suggesting a static quenching mechanism.
From the Scatchard plot (Fig. S6†) and eqn (6),34 the K and n
values of each compound have been calculated (Table 7). All the
complexes have similar K values to that of Hmef except 4 which
exhibits the highest association binding constant to HSA (2.43 ¥
105 M-1), while the n values of complexes 1–3 are higher than that
of Hmef with 2 and 3 having the highest n value (~1.13).
Comparing the affinity of Hmef and complexes 1–4 for BSA
and HSA (K values), it is obvious that 1–3 show higher affinity for
BSA than HSA, while mefenamic acid and 1, the corresponding
KA constants present similar values. Additionally, the affinity of
mefenamic acid is enhanced when coordinated in all complexes
1–4.
superoxide radical scavenging activity (ABTS%)
∑ OH% 0.1 mM
ABTS% 0.1 mM
Hmef
92.5
95.7
96.4
89.3
96.7
88.2
66.3
78.3
92.4
90.4
97.0
91.8
[Co(mef)2(MeOH)4], 1
[Co(mef)2(bipy)(MeOH)2], 2
[Co(mef)2(phen)(MeOH)2], 3
[Co(mef)2(py)2(MeOH)2], 4
Trolox
Each experiment was performed at least in triplicate SD < 10%.
and inflammation. DPPH is a stable free radical and presents
strong absorption band at 517 nm. The interaction of Hmef
and complexes 1–4 with the stable free radical DPPH (Table
8) has revealed their reducing activity. The measurements were
performed after 20 and 60 min. In general, the tested compounds
present low to moderate interaction values. The order of the
reducing activity of the compounds increases in the order Hmef <
2 < 4 < 1 < 3. Complexes 2, 3 and 4 containing a lipophilic moiety
(bipy, phen and py respectively) showed low reducing activity. 1
and 3 seem to be the most active and their activity is not time
dependent.
Hydroxyl radicals are characterized aiming the most reactive
oxygen species. During the inflammatory process, phagocytes
generate the superoxide anion radical at the inflamed site, and
this is connected to other oxidizing species such as OH∑.36b They
are considered to be responsible for some of the tissue damage
occurring in inflammation. It is claimed that hydroxyl radical
scavengers could serve as protectors, thus increasing prostaglandin
synthesis. The competition of the new complexes with DMSO for
HO∑, 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 cited in Table 9. All the compounds are found to
present strong competition with DMSO (33 mM) at 0.1 mM for
hydroxyl free radicals. Hmef shows significant scavenging activity
(92.5%) and complexes 1, 2 and 4 present even higher scavenging
activity. The hydroxyl radical scavenging activity increases in the
order 3 < Hmef <1 < 2 < 4.
Antioxidant capacity
Free radicals play an important role in the inflammatory process
as it is known. Many NSAIDs with a broad spectrum of
effects have been reported to act either as inhibitors of free
radical production or as radical scavengers.35 Thus, compounds
with possible antioxidant properties could play a crucial role
against inflammation and lead to potentially effective drugs. The
compounds were tested in comparison to well-known antioxidant
agents e.g. nordihydroguairetic acid (NDGA), butylated hydrox-
ytoluene (BHT) and 6-hydroxy-2,5,7,8-tetramethylchromane-2-
carboxylic acid (Trolox). In this context, Hmef and complexes
1–4 have been tested with regard to their antioxidant ability and
the results are shown in Tables 8 and 9.
Antioxidants that exhibit 1,1-diphenyl-picrylhydrazyl (DPPH)
radical scavenging activity are receiving increased attention
since they present interesting anticancer, anti-ageing and anti-
inflammatory activities.36a Therefore, compounds with antiox-
idant properties may offer protection in rheumatoid arthritis
Table 8 Interaction % with DPPH (RA%)
20 min, 0.1 mM
60 min, 0.1 mM
Generation of the 2,2¢-azinobis-(3-ethylbenzothiazoline-6-
sulfonic acid) (ABTS) radical cation forms the basis of one of
the spectrophotometric methods applied to the measurement of
the total antioxidant activity of solutions of Hmef and 1–4.36c
Hmef presents a rather high activity (66.3%) while complexes
1–4 present a much higher activity increasing in the order Hmef
< 1 < 3 < 2 < 4 (Table 9). All the complexes are more potent
than mefenamic acid. The activity of mefenamic acid is enhanced
when coordinated to Co(II) in 1 (78.3%), while in the presence of
Hmef
5.7
11.7
30.1
17.9
36,8
29.5
82.6
60
[Co(mef)2(MeOH)4], 1
[Co(mef)2(bipy)(MeOH)2], 2
[Co(mef)2(phen)(MeOH)2], 3
[Co(mef)2(py)2(MeOH)2], 4
NDGA
29.9
20.4
32.5
28.6
81
BHT
31.3
Each experiment was performed at least in triplicate SD < 10%.
4524 | Dalton Trans., 2010, 39, 4517–4528
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
the N,N¢-donor ligands phen and bipy the activity increases even
more (90.4% and 92.4% respectively) and especially in the presence
of py in 4 (97.0%) which presents the highest activity.
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.38 The DNA concentration was determined by the
UV absorbance at 260 nm after 1 : 20 dilution using e = 6600
M-1cm-1.39
In conclusion, all the complexes exhibit higher inhibition than
mefenamic acid against the DPPH and ABTS radicals. The
inhibition of all the compounds against hydroxyl radicals is very
high, with complexes 1, 2 and 4 showing better inhibition than
mefanamic acid. In the literature, diverse Cu(II), Co(II), Ni(II),
Zn(II) and Pd(II) complexes with drugs or Schiff bases as ligands
have presented enhanced antioxidant activity in relation to the free
ligands.36b,37
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 solution at concentrations in the range 10-5–10-3 M on a
Hitachi U-2001 dual beam spectrophotometer. Room temperature
magnetic measurements were carried out by the Faraday method
using mercury tetrathiocyanatocobaltate(II) as a calibrant. C, H
and N elemental analysis were performed on a Perkin-Elmer 240B
elemental analyzer. Molecular conductivity measurements were
carried out with a Crison Basic 30 conductometer. Fluorescence
spectra were recorded in solution on a Hitachi F-7000 fluorescence
spectrophotometer. Solid state and solution EPR measurements
were taken in the temperature range 4-300 K on a Bruker 200D-
SRC X-Band spectrometer, equipped with an Oxford ESR 9
Cryostat, operating at 9.412 GHz, 10 db.
Cyclic voltammetry studies were performed on an Eco chemie
Autolab Electrochemical analyzer. Cyclic voltammetry experi-
ments were carried out in a 30 mL three-electrode electrolytic cell.
The working electrode was platinum disk, a separate Pt single-
sheet electrode was used as the counter electrode and 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 n = 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 vapours. All electrochemical
measurements were performed at 25.0 0.2 ◦C.
Conclusions
The synthesis and characterization of neutral mononuclear
cobalt(II) complexes with the non-steroidal anti-inflammatory
drug mefenamic acid in the absence or presence of a nitrogen
donor heterocyclic ligand 2,2¢-bipyridine, 1,10-phenanthroline
or pyridine has been achieved. In these complexes, the
mefenamato ligand is bound to cobalt(II) via a carboxy-
lato oxygen. [Co(mef)2(bipy)(MeOH)2] and centrosymmetric
[Co(mef)2(MeOH)4] present a distorted octahedral geometry
around the cobalt atom. EPR signals of 1 and 2 reveal an
octahedral geometry of the Co(II) ion. Complexes 1 and 2 are
the first cobalt(II) complexes of the NSAID anthranilic acids that
have been structurally characterized.
UV spectroscopy and cyclic voltammetry studies have revealed
the ability of the complexes to bind to DNA. The binding strength
of the complexes with CT DNA calculated with UV spectroscopic
titrations have shown that [Co(mef)2(MeOH)4] exhibits the highest
Kb value among the compounds examined. Competitive binding
studies with EB have revealed the ability of the complexes to
displace EB from the EB-DNA complex and cyclic voltammetry
studies have confirmed the intercalation as the most possible
binding mode to DNA.
Synthesis of the complexes
The complexes show good binding affinity to BSA and HSA
proteins giving relatively high binding constants. All the com-
pounds were tested in vitro for their antioxidant and free radical
scavenging activity. They present significantly high scavenging
activity against hydroxyl free radicals and superoxide radicals with
4 being the most active one.
[Co(mef)2(MeOH)4]·2MeOH, 1·2MeOH. A methanolic so-
lution (15 mL) containing mefenamic acid (0.4 mmol, 97 mg)
and KOH (0.4 mmol, 22 mg) was stirred for 1 h. The solution
was added to a methanolic solution (10 mL) of CoCl2·6H2O
(0.2 mmol, 48 mg) and the reaction mixture was stirred for 1 h.
The reaction solution was filtered and left for slow evaporation.
Rose-colored crystals of [Co(mef)2(MeOH)4]·2MeOH, 1·2MeOH,
(105 mg, 75%) suitable for X-ray structure determination, were
deposited after a few days. (Found: C, 59.09; H, 7.16; N, 3.83.
C36H52CoN2O10 (MW = 731.75) requires C, 59.75; H, 6.92;
N, 4.00%). IR: nmax/cm-1; nasym(CO2), 1615 (vs (very strong));
Experimental
Materials and instrumentation
Mefenamic acid, CT DNA, BSA, HSA, EB, DPPH, ABTS, Trolox
and caffeic acid were purchased from Sigma, NaCl and all solvents
were purchased from Merck, trisodium citrate was purchased
from Riedel-de Haen and CoCl2·6H2O, bipy, phen, py, KOH
and NDGA were purchased from Aldrich Co. All the chemicals
and solvents were reagent grade and were used as purchased.
Tetraethylammonium perchlorate (TEAP) was purchased from
Carlo Erba and, prior to its use, it was recrystallized twice from
ethanol and dried under vacuum.
n
sym(CO2), 1389 (vs); D = nasym(CO2) - nsym(CO2): 226 cm-1 (KBr
disk); UV-vis: l/nm (e/M-1 cm-1) as nujol mull: 720, 541, 454,
396, 350, 289; in dmso: 742 (53), 558 (40), 453 (sh (shoulder)) (54),
394 (230), 340 (2300), 292 (5300). meff = 4.40 BM. The complex
is soluble in dmso, dmf, CH2Cl2, CHCl3 and ethanol and is non-
electrolyte.
[Co(mef)2(bipy)(MeOH)2], 2. Mefenamic acid (0.4 mmol,
97 mg) was dissolved in methanol (15 mL) followed by the addition
of KOH (0.4 mmol, 22 mg). After 1 h stirring, the resultant
solution was added slowly, and simultaneously with a methanolic
solution of bipy (0.2 mmol, 31 mg), to a methanolic solution
(10 mL) of CoCl2·6H2O (0.2 mmol, 48 mg) and stirred for 30 min.
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
This journal is
The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 4517–4528 | 4525
©

View Article Online
The solution was left for slow evaporation. Orange crystals of
[Co(mef)2(bipy)(MeOH)2] 2 (105 mg, 70%) suitable for X-ray
structure determination, were deposited after a few days. (Found:
C, 66.65; H, 5.91; N, 7.53. C42H44CoN4O6 (MW = 759.77) requires
C, 66.40; H, 5.84; N, 7.37%). IR: nmax/cm-1; nasym(CO2): 1609 (vs);
trisodium citrate at pH 7.0). The intercalating effect of Hmef and
complexes 1–4 with the DNA-EB complex was studied by adding
a certain amount of a solution of the compound step by step
into the 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.
The interaction of complexes 1–4 with CT DNA has been
also investigated by monitoring the changes observed in the
cyclic voltammogram of a 0.40 mM 1 : 2 dmso : buffer solution
of complex upon addition of CT DNA at diverse r values. The
buffer was also used as the supporting electrolyte and the cyclic
voltammograms were recorded at n = 100 mV s-1.
n
sym(CO2): 1382 (vs); D = nasym(CO2) - nsym(CO2): 227 cm-1 (KBr
disk); UV-vis: l/nm (e/M-1 cm-1) as nujol mull: 745, 550, 480 (sh),
394, 356, 297; in dmso: 740 (50), 565 (25), 475 (40), 395 (sh) (290),
355 (9800), 295 (11 000). meff = 4.46 BM. The complex is soluble
in dmso and dmf and is non-electrolyte.
[Co(mef)2(phen)(MeOH)2], 3. Complex 3 (100 mg, 65%) was
prepared by the addition of a methanolic solution (15 mL)
of Hmef (0.4 mmol, 97 mg) and KOH (0.4 mmol, 22 mg),
which was stirred for 30 min, and of a methanolic solution of
phen (0.2 mmol, 36 mg) to a methanolic solution (10 mL) of
CoCl2·6H2O (0.2 mmol, 48 mg). The brownish microcrystalline
product was collected after a few days. (Found C, 67.75; H, 5.51;
N, 7.32; C44H44CoN4O6 (MW = 783.79) requires C, 67.43; H, 5.66;
N, 7.15%). IR: nmax/cm-1; nasym(CO2): 1615 (vs); nsym(CO2): 1385
(vs); D = nasym(CO2) - nsym(CO2): 230 cm-1 (KBr disk); UV-vis:
l/nm (e/M-1 cm-1) as nujol mull: 695, 520, 445 (sh), 405, 355, 295;
in dmso: 735 (40), 550 (25), 460 (50), 398 (110), 352 (2800), 292
(5300). meff = 4.35 BM. The complex is soluble in dmso and dmf
and is non-electrolyte.
Albumin binding studies
The protein binding study was performed by tryptophan fluo-
rescence quenching experiments using bovine (BSA, 3 mM) or
human serum albumin (HSA, 3 mM) in buffer (containing 15 mM
trisodium citrate and 150 mM NaCl at pH 7.0). The quenching of
the emission intensity of tryptophan residues of BSA at 343 nm or
HSA at 351 nm was monitored using Hoxo or complexes 1–4 as
quenchers with increasing concentration.32 Fluorescence spectra
were recorded from 300 to 500 nm at an excitation wavelength of
296 nm.
[Co(mef)2(py)2(MeOH)2], 4. The complex was prepared by the
addition of a methanolic solution (15 mL) of Hmef (0.4 mmol,
97 mg) and KOH (0.4 mmol, 22 mg), after 30 min of stirring, to
a methanolic solution (10 mL) of CoCl2·6H2O (0.2 mmol, 48 mg)
followed by the addition of 1.5 mL of pyridine. The rose-colored
microcrystalline product (105 mg, 70%) was collected after a few
days. (Found C, 65.97; H, 6.21; N, 7.31; C42H46CoN4O6 (MW =
761.76) requires C, 66.22; H, 6.09; N, 7.36%). IR: nmax/cm-1;
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.
Determination of the reducing activity of the stable radical 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 concentrations of the
solutions of the compounds were 0.05 mM and 0.1 mM. After 20
and 60 min at room temperature, the absorbance at l = 517 nm was
recorded.36a NDGA and BHT were used as reference compounds.
n
n
asym(CO2): 1612 (vs); nsym(CO2): 1388 (vs); D = nasym(CO2) -
sym(CO2): 224 cm-1 (KBr disk); UV-vis: l/nm (e/M-1 cm-1) as
nujol mull: 690, 510, 445 (sh), 390, 342, 295; in dmso: 733 (53),
535 (25), 440 (sh) (30), 395 (320), 340 (3200), 293(6750). meff = 4.50
BM. The complex is soluble in dmso, CH2Cl2 and CHCl3 and is
non-electrolyte.
Competition of the tested compounds with DMSO for hydroxyl
radicals
DNA-binding studies
The interaction of Hmef and complexes 1–4 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). In UV titration experiments, the
spectra of CT DNA in the presence of each compound have
been recorded for a constant CT DNA concentration in diverse
[compound]/[CT DNA] mixing ratios (r). The binding constants,
Kb, of the compounds with CT DNA have been determined
using the UV spectra of the compound recorded for a constant
concentration 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 hydroxyl radicals generated by the Fe3+/ascorbic acid sys-
tem, were detected according to Nash, by the determination
of formaldehyde produced from the oxidation of DMSO. The
reaction mixture contained EDTA (0.1 mM), Fe3+ (167 mM),
DMSO (33 mM) in phosphate 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 l = 412 nm was
measured.36b Trolox was used as an appropriate standard.
Assay of radical cation scavenging activity
The competitive studies of each compound with EB have been
investigated with fluorescence spectroscopy in order to examine
whether the compound can displace EB from its CT DNA-EB
complex. The CT DNA-EB complex was prepared by adding 20
mM EB and 26 mM CT DNA in buffer (150 mM NaCl and 15 mM
ABTS was dissolved in water to a 2 mM concentration. ABTS
radical cation (ABTS∑+) was produced by reacting ABTS stock
solution with 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 potassium persulfate react
4526 | Dalton Trans., 2010, 39, 4517–4528
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
Table 10 Crystallographic data for complexes 1·2MeOH and 2
R. NicAmhlaoibh and M. Clynes, Eur. J. Cancer, 1998, 34, 1250–
1259.
2 (a) A. R. Amin, P. Vyas, M. Attur, J. Leszczynskapiziak, I. R. Patel,
G. Weissmann and S. B. Abramson, Proc. Natl. Acad. Sci. U. S. A.,
1995, 92, 7926–7930; (b) M. Smith, G. Hawcroft and M. A. Hull, Eur.
J. Cancer, 2000, 36, 664–674.
3 (a) K. Kim, J. Yoon, J. Kim, S. J. Baek, T. E. Eling, W. Lee, J. Ryu, J. Lee,
J. Lee and J. Yoo, Biochem. Biophys. Res. Commun., 2004, 325, 1298–
1303; (b) B. Reddy, C. Rao, A. Rivenson and G. Kelloff, Carcinogenesis,
1993, 14, 1493–1497.
4 T. Zhang, T. Otevrel, Z. Gao, Z. Gao, S. M. Ehrlich, J. Z. Fields and B.
M. Boman, Cancer Res., 2001, 61, 8664–8667.
5 (a) J. F. Neault, M. Naoui, M. Manfait and H. A. Tajmir-Riahi, FEBS
Lett., 1996, 382, 26–30; (b) S. Roy, R. Banerjee and M. Sarkar, J. Inorg.
Biochem., 2006, 100, 1320–1331.
6 J. E. Weder, C. T. Dillon, T. W. Hambley, B. J. Kennedy, P. A. Lay, J. R.
Biffin, H. L. Regtop and N. M. Davies, Coord. Chem. Rev., 2002, 232,
95–126 and references therein.
7 (a) F. T. Greenaway, E. Riviere, J. J. Girerd, X. Labouze, G. Morgant, B.
Viossat, J. C. Daran, M. Roch Arveiller and N. Dung, J. Inorg. Biochem.,
1999, 76, 19–27and references therein; (b) B. Viossat, J. Daran, G.
Savouret, G. Morgant, F. T. Greenaway, N. Dung, V. A. Pham-Tran and
J. R. J. Sorenson, J. Inorg. Biochem., 2003, 96, 375–385and references
therein.
8 (a) V. Dokorou, Z. Ciunik, U. Russo and D. Kovala-Demertzi, J.
Organomet. Chem., 2001, 630, 205–214; (b) S. Lorinc, M. Koman, M.
Melnik and M. Jaroslava, Acta Crystallogr., Sect. E: Struct. Rep. Online,
2006, 62, m1938–m1939; (c) G. Facchin, M. H. Torre and E. J. Baran,
Z. Naturforsch. Teil B, 1998, 53, 871–874.
9 P. V. Bernhardt and G. A. Lawrance, in Comprehensive Coordination
Chemistry II, ed. J. A. McCleverty and T. J. Meyer, 2003, vol. 6, ch. 1,
pp. 1-45.
1·2MeOH
2
Formula
Fw
C36H52CoN2O10
731.73
¯
P1
C42H44CoN4O6
759.74
Ic2a
7.21870(10)
17.6361(3)
29.6060(5)
90
Space group
˚
a/A
7.66020(10)
7.88870(10)
15.7633(3)
90.2790(10)
99.2940(10)
92.7690(10)
938.88(2)
1
˚
b/A
˚
c/A
a/◦
b/◦
g /◦
90
90
3
˚
V/A
3769.13(10)
4
Z
T/◦C
-93
-93
Radiation
rc/g cm-3
m/mm-1
Cu Ka 1.54178
1.294
Cu Ka 1.54178
1.339
4.053
4.003
a
R1, wR2
0.0523/0.1344b
0.0351/0.0689c
a w = 1/[s (Fo2) + (aP)2 + bP] and P = (max(Fo2,0) + 2Fc2)/3, R1 =
2
ꢀ
ꢀ
ꢀ
ꢀ
(|Fo| - |Fc|)/ (|Fo|) and wR2 = { [w(Fo - Fc2)2]/ [w(Fo2)2]}
.
2
1/2
b For 2743 reflections with I > 2s(I). c For 2295 reflections with I > 2s(I).
stoichiometrically at a ratio of 1 : 0.5, this will result in incomplete
oxidation of the ABTS. Oxidation of the ABTS commenced
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 l = 734 nm. After addition of 10 mL of diluted compounds
or standards (0.1 mM) in DMSO, the absorbance reading was
taken exactly 1 min after initial mixing.36c
10 P. J. Sadler, Adv. Inorg. Chem., 1991, 36, 1–48.
11 (a) M. D. Hall, T. W. Failes, N. Yamamoto and T. W. Hambley, Dalton
Trans., 2007, 3983–3990; (b) F. P. Dwyer, E. C. Gyarfas, W. P. Rogers
and J. H. Koch, Nature, 1952, 170, 190–191.
12 (a) H. Lopez-Sandoval, M. E. Londono-Lemos, R. Garza-Velasco,
I. Poblano-Melendez, P. Granada-Macias, I. Gracia-Mora and N.
Barba-Behrens, J. Inorg. Biochem., 2008, 102, 1267–1276; (b) I. Ott,
A. Abraham, P. Schumacher, H. Shorafa, G. Gastl, R. Gust and
B. Kircher, J. Inorg. Biochem., 2006, 100, 1903–1906; (c) I. Ott, K.
Schmidt, B. Kircher, P. Schumacher, T. Wiglenda and R. Gust, J. Med.
Chem., 2005, 48, 622–629.
13 (a) D. U. Miodragovic, G. A. Bogdanovic, Z. M. Miodragovic, M. D.
Radulovic, S. B. Novakovic, G. N. Kaludjerovic and H. Kozlowski, J.
Inorg. Biochem., 2006, 100, 1568–1574; (b) K. Nomiya, A. Yoshizawa,
K. Tsukagoshi, N. C. Kasuga, S. Hirakawa and J. Watanabe, J. Inorg.
Biochem., 2004, 98, 46–60.
14 (a) J. Lv, T. Liu, S. Cai, X. Wang, L. Liu and Y. Wang, J.
Inorg. Biochem., 2006, 100, 1888–1896; (b) Z. Weiqun, Y. Wen,
X. Liqun and C. Xianchen, J. Inorg. Biochem., 2005, 99, 1314–
1319.
15 (a) A. Bottcher, T. Takeuchi, K. I. Hardcastle, T. J. Meade and H. B.
Gray, Inorg. Chem., 1997, 36, 2498–2504; (b) T. Takeuchi, A. Bottcher,
C. M. Quezada, T. J. Meade and H. B. Gray, Bioorg. Med. Chem., 1999,
7, 815–819.
16 (a) C. Dendrinou-Samara, G. Psomas, K. Christophorou, V. Tangoulis,
C. P. Raptopoulou, A. Terzis and D. P. Kessissoglou, J. Chem.
Soc., Dalton Trans., 1996, 3737–3743; (b) M. Alexiou, I. Tsivikas,
C. Dendrinou-Samara, A. Pantazaki, P. Trikalitis, N. Lalioti, D. A.
Kyriakidis and D. P. Kessissoglou, J. Inorg. Biochem., 2003, 93, 256–
264; (c) A. Dimitrakopoulou, C. Dendrinou-Samara, A. A. Pantazaki,
M. Alexiou, E. Nordlander and D. P. Kessissoglou, J. Inorg. Biochem.,
2008, 102, 618–628.
17 (a) G. Psomas, A. Tarushi, E. K. Efthimiadou, Y. Sanakis, C. P.
Raptopoulou and N. Katsaros, J. Inorg. Biochem., 2006, 100, 1764–
1773; (b) G. Psomas, J. Inorg. Biochem., 2008, 102, 1798–1811; (c) E. K.
Efthimiadou, A. Karaliota and G. Psomas, Bioorg. Med. Chem. Lett.,
2008, 18, 4033–4037; (d) A. Tarushi, G. Psomas, C. P. Raptopoulou and
D. P. Kessissoglou, J. Inorg. Biochem., 2009, 103, 898–905; (e) K. C.
Skyrianou, E. Efthimiadou, V. Psycharis, A. Terzis, D. P. Kessissoglou
and G. Psomas, J. Inorg. Biochem., 2009, 103, 1617–1625; (f) K. C.
Skyrianou, F. Peridh, I. Turel, D. P. Kessissoglou and G. Psomas,
J. Inorg. Biochem., 2010, 104, 161–170; (g) E. K. Efthimiadou, A.
Karaliota and G. Psomas, J. Inorg. Biochem., 2010, 104, 455–466; (h) A.
X-Ray determination
A pink crystal of 1·2MeOH (0.31 ¥ 0.67 ¥ 0.75 mm) and a pink
crystal of 2 (0.10 ¥ 0.27 ¥ 0.31 mm) were taken from the mother
liquor and immediately cooled to -93 ◦C. Diffraction measure-
ments were made on a Rigaku R-AXIS SPIDER Image Plate
diffractometer using graphite monochromated Cu Ka radiation.
Data collection (w-scans) and processing (cell refinement, data
reduction and Empirical absorption correction) were performed
using the CrystalClear program package.40 The structures were
solved by direct methods using41 SHELXS-97 and refined by
full-matrix least-squares techniques on F2 with SHELXL-97.42
Further experimental crystallographic details for 1·2MeOH (Table
10): 2qmax = 130◦; reflections collected/unique/used, 11 946/2968
[Rint = 0.0294]/2968; 270 parameters refined; (D/s)max = 0.002;
3
˚
(Dr)max/(Dr)min = 0.725/-0.673 e/A ; R1/wR2 (for all data),
0.0550/0.1365. Further experimental crystallographic details for
2 (Table 10): 2qmax = 126◦; reflections collected/unique/used,
20 358/2940 [Rint = 0.0399]/2940; 295 parameters refined;
3
˚
(D/s)max = 0.002; (Dr)max/(Dr)min = 0.234/-0.229 e/A ; R1/wR2
(for all data), 0.0566/0.0818. All hydrogen atoms in both struc-
tures either were located by difference maps and were refined
isotropically or were introduced at calculated positions as riding
on bonded atoms. All non-hydrogen atoms in 1 and 2 were refined
anisotropically.
References
1 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,
This journal is
The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 4517–4528 | 4527
©

View Article Online
Tarushi, C. P. Raptopoulou, V. Psycharis, A. Terzis, G. Psomas and D.
P. Kessissoglou, Bioorg. Med. Chem., 2010, 18, 2678–2685.
18 (a) C. Dendrinou-Samara, D. P. Kessissoglou, G. E. Manoussakis, D.
Mentzafos and A. Terzis, J. Chem. Soc., Dalton Trans., 1990, 959–965;
(b) C. Dendrinou-Samara, P. D. Jannakoudakis, D. P. Kessissoglou, G.
E. Manoussakis, D. Mentzafos and A. Terzis, J. Chem. Soc., Dalton
Trans., 1992, 3259–3264; (c) C. Dendrinou-Samara, G. Tsotsou, C.
P. Raptopoulou, A. Kortsaris, D. Kyriakidis and D. P. Kessissoglou,
J. Inorg. Biochem., 1998, 71, 171–179; (d) P. Christofis, M. Katsarou,
A. Papakyriakou, Y. Sanakis, N. Katsaros and G. Psomas, J. Inorg.
Biochem., 2005, 99, 2197–2210.
19 (a) I. Bertini and C. Luchinat, in Advances in Inorganic Biochemistry,
ed. G. L. Eichorn and L. G. Marzilli, Elsevier, New York, 1984, vol.
6, pp. 78-80; (b) J. Zarembowitch and O. Kahn, Inorg. Chem., 1984,
23, 589–593; (c) A. Bencini, C. Benelli, D. Gatteschi and C. Zanchini,
Inorg. Chem., 1979, 18, 2526–2528.
Dendrinou-Samara, C. Raptopoulou, A. Terzis and D. P. Kessissoglou,
Dalton Trans., 2010, 39, 765–775.
28 R. F. Pasternack, M. Cacca, B. Keogh, T. A. Stephenson, A. P. Williams
and F. J. Gibbs, J. Am. Chem. Soc., 1991, 113, 6835–6840.
29 O. Novakova, H. Chen, O. Vrana, A. Rodger, P. J. Sadler and V. Brabec,
Biochemistry, 2003, 42, 11544–11554.
30 (a) M. T. Carter and A. J. Bard, J. Am. Chem. Soc., 1987, 109, 7528–
7530; (b) M. T. Carter, M. Rodriguez and A. J. Bard, J. Am. Chem.
Soc., 1989, 111, 8901–8911.
31 (a) C. Tan, J. Liu, H. Li, W. Zheng, S. Shi, L. Chen and L. Ji, J. Inorg.
Biochem., 2008, 102, 347–358; (b) Y. Wang, H. Zhang, G. Zhang, W.
Tao and S. Tang, J. Lumin., 2007, 126, 211–218; (c) S. Deepa and A.
K. Mishra, J. Pharm. Biomed. Anal., 2005, 38, 556–563.
32 J. R. Lakowicz and G. Weber, Biochemistry, 1973, 12, 4161–4170.
33 S. Wu, W. Yuan, H. Wang, Q. Zhang, M. Liu and K. Yu, J. Inorg.
Biochem., 2008, 102, 2026–2034.
20 A. Grirrane, A. Pastor, A. Ienco, C. Mealli and A. Galindo, J. Chem.
Soc., Dalton Trans., 2002, 3771–3777.
34 V. Rajendiran, R. Karthik, M. Palaniandavar, H. Stoeckli-Evans, V. S.
Periasamy, M. A. Akbarsha, B. S. Srinag and H. Krishnamurthy, Inorg.
Chem., 2007, 46, 8208–8221.
35 R. Cini, G. Giorgi, A. Cinquantini, C. Rossi and M. Sabat, Inorg.
Chem., 1990, 29, 5197–5200.
21 (a) M. Melnik, M. Koman and T. Glowiak, Polyhedron, 1998, 17, 1767–
1771; (b) B. Viossat, F. T. Greenaway, G. Morgant, J. C. Daran, N.
H. Dung and J. R. J. Sorenson, J. Inorg. Biochem., 2005, 99, 355–
367; (c) D. Kovala-Demertzi, A. Galani, M. A. Demertzis, S. Skoulika
and C. Kotoglou, J. Inorg. Biochem., 2004, 98, 358–364; (d) F. Valach,
M. Tokarcik, P. Kubinec, M. Melnik and L. Macaskova, Polyhedron,
1997, 16, 1461–1464; (e) M. Melnik, I. Potocnak, l. Macaskova and D.
Miklos, Polyhedron, 1996, 15, 2159–2164; (f) D. Kovala-Demertzi, V.
N. Dokorou, J. P. Jasinski, A. Opolski, J. Wiecek, M. Zervou and M. A.
Demertzis, J. Organomet. Chem., 2005, 690, 1800–1806; (g) D. Kovala-
Demertzi, N. Kourkoumelis, A. Koutsodimou, A. Moukarika, E. Horn
and E. R. T. Tiekink, J. Organomet. Chem., 2001, 620, 194–201.
22 Q. Zhang, J. Liu, H. Chao, G. Xue and L. Ji, J. Inorg. Biochem., 2001,
83, 49–55.
23 (a) A. M. Pyle, J. P. Rehmann, R. Meshoyrer, C. V. Kumar, N. J. Turro
and J. K. Barton, J. Am. Chem. Soc., 1989, 111, 3053–3063; (b) H. Chao,
W. Mei, Q. Huang and L. Ji, J. Inorg. Biochem., 2002, 92, 165–170; (c) E.
C. Long and J. K. Barton, Acc. Chem. Res., 1990, 23, 271–273.
24 Y. Song, Q. Wu, P. Yang, N. Luan, L. Wang and Y. Liu, J. Inorg.
Biochem., 2006, 100, 1685–1691.
36 (a) C. Kontogiorgis and D. Hadjipavlou-Litina, J. Enzyme Inhib. Med.
Chem., 2003, 18, 63–69; (b) D. Panagoulis, E. Pontiki, E. Skeva, C.
Raptopoulou, S. Girousi, D. Hadjipavlou-Litina and C. Dendrinou-
Samara, J. Inorg. Biochem., 2007, 101, 623–634; (c) R. Re, N. Pellegrini,
A. Proteggente, A. Pannala, M. Yang and C. Rice-Evans, Free Radical
Biol. Med., 1999, 26, 1231–1237.
37 (a) Z. D. Petrovic, D. Hadjipavlou-Litina and V. P. Petrovic, J. Mol. Liq.,
2009, 144, 55–58; (b) D. Kovala-Demertzi, D. Hadjipavlou-Litina, A.
Primikiri, M. Staninska, C. Kotoglou and M. A. Demertzis, Chem.
Biodiversity, 2009, 6, 948–960; (c) Z. Liu, B. Wang, Z. Yang, Y. Li,
D. Qin and T. Li, Eur. J. Med. Chem., 2009, 44, 4477–4484; (d) S.
B. Bukhari, S. Memon, M. M. Tahir and M. I. Bhanger, J. Mol.
Struct., 2008, 892, 39–46; (e) E. Pontiki, D. Hadjipavlou-Litina, A. T.
Chaviara and C. A. Bolos, Bioorg. Med. Chem. Lett., 2006, 16, 2234–
2237.
38 J. Marmur, J. Mol. Biol., 1961, 3, 208–211.
39 M. F. Reichmann, S. A. Rice, C. A. Thomas and P. Doty, J. Am. Chem.
Soc., 1954, 76, 3047–3053.
40 Rigaku/MSC (2005). CrystalClear, Rigaku/MSC Inc., The Wood-
lands, Texas, USA.
25 (a) R. F. Pasternack, E. J. Gibbs and J. J. Villafranca, Biochemistry,
1983, 22, 2406–2414; (b) G. Pratviel, J. Bernadou and B. Meunier, Adv.
Inorg. Chem., 1998, 45, 251–262.
26 A. Jancso, L. Nagy, E. Moldrheim and E. Sletten, J. Chem. Soc., Dalton
Trans., 1999, 1587–1594.
41 G. M. Sheldrick, SHELXS-97: Structure Solving Program, University
of Go¨ttingen, Germany, 1997.
27 (a) W. D. Wilson, L. Ratmeyer, M. Zhao, L. Strekowski and D. Boykin,
Biochemistry, 1993, 32, 4098–4104; (b) T. Afrati, A. A. Pantazaki, C.
42 G. M. Sheldrick, SHELXL-97: Crystal Structure Refinement Program,
University of Go¨ttingen, Germany, 1997.
4528 | Dalton Trans., 2010, 39, 4517–4528
This journal is
The Royal Society of Chemistry 2010
©