Non-steroidal antiinflammatory drug-copper(II) complexes: Structure and biological perspectives

Article abstract of DOI:10.1039/c1dt10714c

Copper(ii) complexes with the non-steroidal antiinflammatory drug mefenamic acid in the presence of aqua or nitrogen donor heterocyclic ligands (2,2′-bipyridine, 1,10-phenanthroline, 2,2′-bipyridylamine or pyridine) have been synthesized and characterized

Full text of DOI:10.1039/c1dt10714c

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PAPER
Non-steroidal antiinflammatory drug–copper(II) complexes: Structure and
biological perspectives†
Filitsa Dimiza,a Stella Fountoulaki,a Athanasios N. Papadopoulos,b Christos A. Kontogiorgis,b
Vassilis Tangoulis,a Catherine P. Raptopoulou,c Vassilis Psycharis,c Aris Terzis,c Dimitris P. Kessissogloua and
George Psomas*a
Received 19th April 2011, Accepted 9th June 2011
DOI: 10.1039/c1dt10714c
Copper(II) complexes with the non-steroidal antiinflammatory drug mefenamic acid in the presence of
aqua or nitrogen donor heterocyclic ligands (2,2¢-bipyridine, 1,10-phenanthroline, 2,2¢-bipyridylamine
or pyridine) have been synthesized and characterized. The crystal structures of [(2,2¢-bipyridine)-
bis(mefenamato)copper(II)], 2, [(2,2¢-bipyridylamine)bis(mefenamato)copper(II)], 4, and
[bis(pyridine)bis(methanol)bis(mefenamato)copper(II)], 5, have been determined by X-ray
crystallography. 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(aqua)tetrakis(mefenamato)dicopper(II)]
exhibits the highest binding constant to CT DNA. The cyclic voltammograms of the complexes in the
presence of CT DNA solution have shown that the complexes can bind to CT DNA by the intercalative
binding mode verified also by DNA solution viscosity measurements. Competitive studies with
ethidium bromide (EB) indicate that the complexes can displace the DNA-bound EB suggesting strong
competition with EB. Mefenamic acid and its complexes exhibit good binding propensity to human or
bovine serum albumin protein having relatively high binding constant values. All the compounds have
been tested for their antioxidant and free radical scavenging activity as well as for their in vitro
inhibitory activity against soybean lipoxygenase showing significant activity.
antitumor drugs.3c Although their interaction with DNA should
be of great interest in order to explain the anticancer and
1. Introduction
the antiinflammatory activity of the NSAIDs,4 few reports on
the interaction of NSAIDs and their complexes with DNA
have been published.5 Additionally, metal complexes have also
exhibited synergetic activity when administered in conjunction
with NSAIDs, as they have been shown to be more active drugs
than their parent compounds.6
Non-steroidal antiinflammatory drugs (NSAIDs) are common
medicinal drugs mainly used as analgesic, antiinflammatory and
antipyretic agents since their side-effects have been well studied.1
They may work through inhibition of the cyclo-oxygenase (COX)-
mediated production of prostaglandins or via COX-independent
mechanisms.2 NSAIDs have exhibited significant chemopreventive
and antitumor activity by modulating cell proliferation and
cell death in cultured colon cancer cells and by reducing the
number and size of carcinogen-induced colon tumors.3a,b They
have also exhibited a synergistic role on the activity of certain
Copper(II) complexes with diverse drugs have been the subject
of a large number of research studies,6a,7 since they exhibit
potential synergetic activity with drugs8 and numerous biological
activities such as antitumor,9 antibacterial10 and antifungal.11
A
thorough survey of the literature has revealed reports on the crystal
structures of copper(II) complexes with the NSAIDs aspirin,
diclofenac, flufenamic acid, ibuprofen, indomethacin, naproxen,
niflumic acid, piroxicam, suprofen and tolmentin as ligands.6a,12–14
The NSAID mefenamic acid (Hmef) belongs to the derivatives
of N-phenylanthranilic acid and resembles chemically tolfenamic
and flufenamic acids and other fenamates in clinical use (Fig. 1).
To the best of our knowledge, the crystal structures of two tin(IV)
complexes15a and two copper(II) complexes15b,c have been reported,
as well as the crystal structures and the biological properties of two
Co(II)-mefenamato complexes that have recently been reported by
our group.16
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: Experimental
data for compounds 1–5. CCDC reference numbers 797033 and 797034.
For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c1dt10714c
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All complexes are soluble in DMSO and are non-electrolytes in
1 mM DMSO solution (KM = 6–14 mho cm2 mol-1).
IR spectroscopy has been used in order to confirm the de-
protonation and binding mode of mefenamic acid. In the IR
spectrum of Hmef, the observed absorption bands at 3370(br,
m) cm-1, attributed to the n(H–O) stretching vibration has
disappeared upon binding to the metal ion. The bands at
1655(s) cm-1 and 1255(s) cm-1 attributed to the stretching vibra-
tions n(C O)carboxylic and n(C–O)carboxylic of the carboxylic moiety
(–COOH) respectively, have shifted in the IR spectra of complexes
2–5, in the range 1613–1620 cm-1 and 1377–1393 cm-1 as-
signed to antisymmetric, 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 227–246 cm-1 for 2–5
indicative of an asymmetrically binding mode of the mefenamato
ligand, while in the case of 1, the nasym(C O) and nsym(C O)
stretching vibrations were observed at 1595 cm-1 and 1425 cm-1,
respectively, resulting in a D value of 170 cm-1, which is indicative
of a bidentate bridging coordination mode.19c
Fig. 1 Mefenamic acid (Hmef = 2-[(2,3-dimethylphenyl)-amino]benzoic
acid or N-(2,3-xylyl)anthranilic acid).
Our studies have been focused on the co-ordination chem-
istry of carboxylate-containing herbicides,17 antimicrobial9,18 or
antiinflammatory12,16,19 agents with metal ions in an attempt to
explore their mode of binding and possible biological perspectives.
We have reported studies on the interaction of metal complexes
with nucleic acids and their biological activity.
In this context, we report the synthesis, structural character-
ization and the electrochemical and biological properties of the
neutral copper(II) complexes with NSAID mefenamic acid in
the absence or presence of a nitrogen-donor heterocyclic ligand
such as 2,2¢-bipyridine (bipy), 1,10-phenanthroline (phen), 2,2¢-
bipyridylamine (bipyam) or pyridine (py). Interaction of Cu(II)
with mefenamic acid leads to the formation of the dinuclear
complex [Cu2(mef)4(H2O)2], 1. While in the presence of bipy,
phen, bipyam or py the mononuclear complexes [Cu(mef)2(bipy)],
2, [Cu(mef)2(phen)], 3, [Cu(mef)2(bipyam)]·MeOH, 4·MeOH,
and [Cu(mef)2(py)2(MeOH)2], 5‡ have been isolated, re-
spectively. The crystal structures of [Cu(mef)2(bipy)] and
[Cu(mef)2(bipyam)]·MeOH and [Cu(mef)2(py)2(MeOH)2] have
been determined by X-ray crystallography. The study of the
biological properties of the complexes has been focused on
(i) their binding properties with CT DNA investigated by UV
spectroscopy, cyclic voltammetry, viscosity measurements and
competitive binding studies with EB, (ii) their affinity for bovine
serum albumin (BSA) and human serum albumin (HSA) inves-
tigated by fluorescence spectroscopy and (iii) their capacity to
scavenge 1,1-diphenyl-picrylhydrazyl (DPPH), hydroxyl and 2,2¢-
azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals
and their inhibitory activity against soybean lipoxygenase.
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,
one low-intensity band in the region 650–695 nm is observed
assigned to a 2Eg
→
2T2g d–d transition,20 typical for Cu(II)
complexes with octahedral local symmetry while an absorption
band at around 430 nm may be attributed to MLCT transition.
The fact that the complexes 1–5 are non-electrolytes in DMSO
solution and they have the same UV-vis spectral pattern in nujol
and in DMSO solution as well as in the presence of the buffer
solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0)
used in the biological experiments suggests that the compounds
keep their integrity in solution. Nevertheless, some dissociation of
mefenamato ligand might occur in solution.
The observed values of meff (1.80–1.95 BM) for complexes 2–
5 with d9 configuration of the central atom are higher than the
spin-only value (1.73 BM) at room temperature and typical for
mononuclear Cu(II) complexes with d9 configuration (S = 1/2).20
The meff value for 1 (1.40 BM) is typical for a dinuclear copper(II)
complex with a paddle-wheel arrangement and is in agreement
with values of similar and isostructural Cu(II) complexes of the
NSAIDs naproxen19b and diclofenac.14d
Results and discussion
Synthesis and spectroscopic study of the complexes
X-Band EPR measurements were carried out in powder samples
as well as in frozen solutions of 2 and 5 in DMSO and are shown
in Fig. 2. The EPR signals of powder samples of the complexes
exhibit a main derivative at g^ = 2.06 and a broad peak at gꢀ = 2.22.
From solution EPR measurements for complex 2 it was possible
to resolve the hyperfine pattern (Fig. 2(B)) and the results are: Aꢀ =
580(20) MHz, A^ = 40(10) MHz, g^ = 2.07(1), gꢀ = 2.28(1).
The solution EPR spectrum was interpreted using the spin
Hamiltonian:
The synthesis of the complexes in high yield was achieved via the
aerobic reaction of mefenamic acid (C14H14N–COOH) and KOH
with CuCl2·2H2O 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:
2CuCl2·2H2O + 4C14H14N–COOH + 4KOH →
(1)
[Cu2(C14H14N–COO)4(H2O)2] + 4KCl + 6H2O
CuCl2·2H2O + 2C14H14N–COOH + 2KOH + C10H8N2 →
H = bB·g·S + S·A·I
(3)
(2)
[Cu(C14H14N–COO)2(C10H8N2)] + 2KCl + 4H2O
where b stands for Bohr magneton and g and A are the g and
hyperfine splitting tensors, respectively.21 When there is a large shift
of one g-tensor from ge = 2.023, an extra component arises in the
hyperfine coupling due to induced orbital motion of the electron
which must be allowed for before orbital population calculations
‡ Compound 5 has been reported earlier in ref. 15b. In the present study
only some structural features related to the biological evaluation of this
compound are being reported.
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Fig. 2 (A) X-Band powder EPR spectra of complexes 2 and 5 at 4 K. (B) X-Band frozen solution EPR spectrum of complex 2 at 4 K (dotted line) along
with its theoretical spectrum (solid line). See text for details.
can be performed. Symons22 has shown that the isotropic, Aiso,
and dipolar, 2B, contributions to the hyperfine interaction can be
related to Aꢀ and A^ through equations (4) and (5):
(4)
(5)
With Aꢀ taken to be negative and A^ positive, the values of Aiso
and 2B were obtained and their values are 308 and 550 MHz,
respectively. These were then used to estimate the s and d orbital
populations on the Cu centre (for more details see ref. 23) and the
2
2
values obtained are as = 0.050, ad = 0.84. The remaining spin
density is delocalized onto the ligands.
The EPR spectrum is typical of a (near) axial specie with gꢀ >
g^. In general, the perpendicular feature of the spectrum consisted
of a series of closely spaced, overlapping lines arising as a result
of coupling of the unpaired electron with the copper nucleus and
the nitrogen nuclei. Simulations of the lineshape were in good
agreement with the experimental spectrum and therefore all spin
Hamiltonian parameters used in calculations are taken from the
simulation. The spin Hamiltonian parameters are typical of a
tetragonally distorted octahedral in which elongation along the
Fig. 3 The molecular structure and partial labeling of 2.
Table 1. The complex is mononuclear and the mefenamato ligands
behave as deprotonated ligands coordinated to the copper atom
via the carboxylato group, which adopts the asymmetric chelating
mode.
The copper atom is six-coordinate and is surrounded by
two mefenamato ligands and a bidentate 2,2¢-bipyridine ligand
showing a distorted octahedral geometry with the six vertices
occupied by two nitrogen and four oxygen atoms, giving a CuN2O4
chromophore. More specifically, the equatorial positions of the
distorted octahedron are occupied by the two nitrogen atoms of
bipy and two oxygen atoms from the mefenamato ligands, whereas
the axial Jahn–Teller positions are occupied by the carboxylato
2
2
z-axis has occurred with the unpaired electron in the dx
orbital.
-y
Symons has shown that for many Cu(II) complexes the variation
in hyperfine values arises directly as a consequence of variations
in g.22 Octahedral/square planar complexes lie on a line defined
by |Aꢀ| = 570 MHz (upper limit) and 480 MHz (lower limit)
with |2B| = 521 MHz, while for tetrahedral complexes the line is
defined by |Aꢀ| = 111 MHz and |2B| = 456 MHz. Peisach and
Blumberg have also found similar variations between the |Aꢀ| and
gꢀ for type I and type II copper complexes in biological systems.24
The present samples fall in the area of octahedral/square planar
complexes, something confirmed also by crystallographic results.
˚
oxygens O(1) and O(2) at 2.413(2) and 2.534(2) A, respectively.
The bond distances around the copper atom in the equatorial
plane are not equal, with the coordinated carboxylate oxygen
˚
˚
atoms (Cu–O(21) = 1.944(1) A and Cu–O(2) = 1.980(2) A) being
˚
closer to Cu than the bipy nitrogens (Cu–N(41) = 1.997(2) A and
Crystal structures of the complexes
˚
Cu–N(42) = 2.006(2) A). The two oxygen atoms are displaced at
˚
Crystal structure of [Cu(mef)2(bipy)], 2. A diagram of 2 is
shown in Fig. 3, and selected bond distances and angles are listed in
~0.13 A from the mean CuN2O2 plane. The N(41)–Cu–N(42) angle
observed is 80.80(8)◦ and is similar to reported values of other
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Table 1 Selected bond distances and angles for complexes 2 and 4·MeOH
˚
˚
Bond distances
(A)
Bond distances
(A)
2
4·MeOH
2.004(1)
2.500(1)
1.951(1)
2.587(1)
1.988(1)
2.003(1)
1.279(2)
1.257(2)
1.289(2)
1.250(2)
Cu–O(1)
Cu–O(2)
2.413(2)
1.980(2)
1.944(1)
2.534(2)
1.997(2)
2.006(2)
1.245(3)
1.296(3)
1.282(3)
1.238(3)
Cu–O(11)
Cu–O(12)
Cu–O(31)
Cu–O(32)
Cu–N(1)
Cu–O(21)
Cu–O(22)
Cu–N(41)
Cu–N(42)
C(1)–O(1)
C(1)–O(2)
C(21)–O(21)
C(21)–O(22)
Cu–N(3)
C(11)–O(11)
C(11)–O(12)
C(31)–O(31)
C(31)–O(32)
Bond angles
(◦)
Bond angles
(◦)
2
4·MeOH
131.42(4)
168.95(5)
161.20(5)
112.92(5)
110.02(5)
103.88(5)
91.96(6)
O(1)–Cu–O(22)
O(21)–Cu–N(42)
O(2)–Cu–N(41)
O(22)–Cu–N(42)
O(1)–Cu–N(41)
O(2)–Cu–O(22)
N(41)–Cu–N(42)
144.03(5)
O(12)–Cu–O(32)
O(31)–Cu–N(1)
O(11)–Cu–N(3)
O(32)–Cu–N(1)
O(32)–Cu–N(3)
O(12)–Cu–N(3)
N(1)–Cu–N(3)
171.03(7)
168.69(7)
116.22(7)
112.14(6)
99.31(6)
80.80(8)
Fig. 4 The molecular structure and partial labeling of 4.
˚
0.39 A, respectively, from the mean basal CuN2O2 plane, while
the carboxylate oxygens in the axial positions O(12) and O(32)
˚
are lying below and above the mean CuN2O2 plane at 2.24 A and
˚
2.22 A, respectively. Additionally, the tetrahedrality of the basal
plane is equal to 21.28◦ showing a distortion from the regular
square planar geometry.25b
chelating polycyclic diimines.25a The bipyridine ligand is planar
with the copper atom lying in this plane.
The trans atom system of the CuN2O4 chromophore gives
angles of O(11)–Cu–N(3) = 161.20(5)◦ and O(31)–Cu–N(1) =
168.95(5)◦ for the basal plane atoms and an angle of O(12)–
Cu–O(32) = 131.42(4) for the Jahn–Teller apical positions. The
2,2¢-bipyridylamine ligand is not planar as observed in the crystal
structures of other bipyam copper(II) complexes.17a
The carboxylate oxygens in the axial positions O(1) and O(22)
are lying below and above the mean CuN2O2 plane at distances
˚
˚
2.145 A and 2.217 A. The trans atom system of the CuN2O2 plane
gives angles of O(2)–Cu–N(41) = 168.69(7) and O(21)–Cu–N(42) =
171.03(7)◦. If we assume a four-coordinate copper complex, the
tetrahedrality calculated for complex 2 gives a dihedral angle of
10.70◦ (the tetrahedrality for a four-coordinate copper complex
can be determined from the angle subtended by two planes, each
encompassing the copper and two adjacent atoms; for strictly
square planar complexes with D4h symmetry, the tetrahedrality
is 0◦; for tetrahedral complexes with D2d symmetry, the tetra-
hedrality equals 90◦), supporting a square planar geometry for
the basal plane CuN(1)2O(1)2.25b A similar arrangement of the
carboxylato group of the NSAID naproxen has been observed in
the crystal structures of the complexes [Cu(naproxenato)2(bipy)]
and [Cu(naproxenato)2(phen)] recently reported by our group.12
The NH group of the mefenamato ligands participates in
intra-molecular hydrogen bonding interactions to the coordinated
The NH group of the mefenamato ligands participates in
intra-molecular hydrogen bonding interactions to the coordinated
˚
carboxylato oxygen atoms [N11 ◊ ◊ ◊ O11 = 2.660 A, H11N ◊ ◊ ◊ O11 =
◦
˚
˚
2.051 A, N11–H11N ◊ ◊ ◊ O11 = 135.4 ; N31 ◊ ◊ ◊ O32 = 2.680 A,
◦
˚
H31N ◊ ◊ ◊ O32 = 1.966 A, N31–H31N ◊ ◊ ◊ O32 = 136.5 ], whereas
the NH group of bipyam and the solvate methanol molecule
participate in inter-molecular hydrogen bonds to the coordi-
˚
nated carboxylato atoms [N2 ◊ ◊ ◊ O12 (1-x,-y,1-z) = 2.762 A,
◦
˚
H2N ◊ ◊ ◊ O12 = 1.985 A, N2–H2N ◊ ◊ ◊ O12 = 172.3 ; Om ◊ ◊ ◊ O31◦=
˚
˚
2.969 A, H1Om ◊ ◊ ◊ O31 = 2.161 A, Om–H1Om ◊ ◊ ◊ O31 = 162.9 ]
thus forming dimers of 4.
The crystal structure of [Cu(mef)2(py)2(MeOH)2], 5, was found
to be similar to a structure recently reported15b with slight
differences on the bond distances and angles. Therefore only
the geometrical characteristics and the bond connectivities will
be discussed. A diagram of 5 is shown in Fig. 5 and selected
bond distances and angles are listed in Table S1.†The complex is
mononuclear and the mefenamato ligands behave as monodentate
deprotonated ligands coordinated to the copper atom via a
carboxylate oxygen.
˚
carboxylato oxygen atoms [N1 ◊ ◊ ◊ O2 = 2.662 A, H1N ◊ ◊ ◊ O2 =
◦
˚
˚
2.005 A, N1–H1N ◊ ◊ ◊ O2 = 137.7 ; N11 ◊ ◊ ◊ O22 = 2.666 A,
H11N ◊ ◊ ◊ O22 = 1.985 A, N11–H11N ◊ ◊ ◊ O22 = 137.1 ].
◦
˚
Crystal structure of [Cu(mef)2(bipyam)]·MeOH, 4·MeOH.
A
diagram of 4 is shown in Fig. 4, and selected bond distances and
angles are listed in Table 1. The complex is mononuclear and the
mefenamato ligands behave as a deprotonated ligand coordinated
to the copper atom through carboxylato oxygens.
The structure of the complex is centrosymmetric, the copper(II)
ion is sitting on a center of symmetry and is coordinated to two
mefenamato, two pyridine and two methanol ligands. Thus, the
copper atom displays an octahedral geometry. In the equatorial
plane of the octahedron, the bond distances Cu–Npy (Cu–N(1) =
The complex is isostructural with 2. The copper atom is
six-coordinate and is surrounded by two mefenamato ligands
and a bidentate 2,2¢-bipyridylamine ligand showing a distorted
octahedral geometry with the six vertices occupied by two nitrogen
and four oxygen atoms, giving a CuN2O4 chromophore. The
oxygen atoms O(11) and O(31) and the nitrogen atoms N(1)
˚
˚
1.992(3) A) are longer than the Cu–Ocarb (Cu–O(11) = 1.972(2) A).
The uncoordinated carboxylate oxygens are lying out of the
˚
˚
˚
˚
and N(3) are displaced at ca. 0.24 A, 0.16 A, 0.21 A and
CuN2O2 plane at distances Cu ◊ ◊ ◊ O(1) = 3.242 A. The axial
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Fig. 5 The molecular structure and partial labeling of 5.
Jahn–Teller positions of the octahedron are occupied by the
1, 5% for 2, 4% for 3, 1% for 4 and 4% for 5) 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.12,16
The Stern–Volmer and Scatchard equations and plots are
usually used in order to study the interaction of a quencher in
the presence of SAs and calculate the corresponding quenching
and binding constants. According to the Stern–Volmer quenching
equation:27a
˚
methanol oxygen atoms at 2.463(3) A distance.
Binding of serum albumins
It is important to consider the interactions of drugs with plasma
proteins particularly with serum albumin (SA), 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.26a Bovine serum
albumin (BSA) is the most extensively studied serum albumin, due
to its structural homology with human serum albumin (HSA).26b
BSA (containing two tryptophans, Trp-134 and Trp-212) and HSA
(one Trp-214) solutions exhibit a strong fluorescence emission with
peaks at 343 nm and 351 nm, respectively, due to the tryptophan
residues, when excited at 295 nm. The interaction of Hmef
and complexes 1–5 with serum albumins has been studied from
tryptophan emission-quenching experiments. 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,16 since Hmef and complexes 1–5 in buffer
solutions do not exhibit any emission spectra under the same
experimental conditions.
(6)
where I0 = the initial tryptophan fluorescence intensity of SA, I =
the tryptophan fluorescence intensity of SA after the addition of
the quencher, kq = the quenching rate constants for SA, KSV = the
dynamic quenching constant, to = the average lifetime of the SA
without the quencher, [Q] = the concentration of the quencher, the
dynamic quenching constant (KSV, M-1) can be obtained by the
slope of the diagram
vs. [Q] (Fig. S1†). Taking a fluorescence
lifetime (to) of tryptophan in SA at around 10-8 s and eqn. (7)
(7)
KSV = kqto
Addition of Hmef or complexes 1–5 to BSA results in a
significant decrease of the fluorescence signal (up to 10% of the
initial fluorescence intensity of BSA for Hmef (Fig. 6(A)), 2% for
the approximate quenching constant (kq, M-1 s-1) is calculated.27a
The values of Ksv and kq for the interaction of Hmef and
complexes 1–5 with BSA (Table 2) indicate good BSA binding
Fig. 6 (A) Plot of % relative fluorescence intensity at lem = 343 nm (I/I0 %) vs r (r = [complex]/[BSA]) for Hmef and complexes 1–5 in buffer solution
(150 mM NaCl and 15 mM trisodium citrate at pH 7.0). (B) Plot of % relative fluorescence intensity at lem = 351 nm (I/I0 %) vs r (r = [complex]/[HSA])
for Hmef and complexes 1–5 in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0).
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Table 2 The BSA and HSA binding constants and parameters (Ksv, kq, K, n) derived for Hmef and complexes 1–5
Albumin
BSA
Compound
KSV (M-1)
kq (M-1 s-1)
K (M-1)
n
Hmef16
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6.12( 0.30) ¥ 105
2.88( 0.10) ¥ 106
1.31( 0.05) ¥ 106
2.78( 0.20) ¥ 1013
9.86( 0.12) ¥ 1013
3.76( 0.25) ¥ 1013
6.12( 0.30) ¥ 1013
2.88( 0.10) ¥ 1014
1.31( 0.05) ¥ 1014
1.35 ¥ 105
2.47 ¥ 105
1.58 ¥ 105
2.59 ¥ 105
1.28 ¥ 106
2.33 ¥ 105
1.20
1.22
1.24
1.15
1.13
1.29
[Cu2(mef)4(H2O)2], 1
[Cu(mef)2(bipy)], 2
[Cu(mef)2(phen)], 3
[Cu(mef)2(bipyam)], 4
[Cu(mef)2(py)2(MeOH)2], 5
HSA
Hmef16
7.13( 0.34) ¥ 104
1.15( 0.04) ¥ 105
2.44( 0.14) ¥ 105
2.08( 0.09) ¥ 105
3.34( 0.20) ¥ 105
1.26( 0.09) ¥ 105
7.13( 0.34) ¥ 1012
1.15( 0.04) ¥ 1013
2.44( 0.14) ¥ 1013
2.08( 0.09) ¥ 1013
3.34( 0.20) ¥ 1013
1.26( 0.09) ¥ 1013
1.32 ¥ 105
1.69 ¥ 105
1.37 ¥ 105
1.61 ¥ 105
2.52 ¥ 105
2.21 ¥ 105
0.82
0.89
1.05
1.04
1.04
0.94
[Cu2(mef)4(H2O)2], 1
[Cu(mef)2(bipy)], 2
[Cu(mef)2(phen)], 3
[Cu(mef)2(bipyam)], 4
[Cu(mef)2(py)2(MeOH)2], 5
propensity of the complexes with 4 exhibiting the strongest
protein-binding ability (kq = 1.31( 0.05) ¥ 1014 M-1 s-1). The kq
values (>1013 M-1 s-1) are higher than diverse kinds of quenchers
for biopolymer fluorescence (2.0 ¥ 1010 M-1 s-1) indicating the
existence of a static quenching mechanism.27b
The binding affinity for SA of complexes 1–5 is similar to
that of the previously reported Co(II) mefenamato complexes.16
Complexes 1–5 present higher K values, especially for BSA, than
the Cu(II) complexes with the NSAIDs naproxen and diclofenac
as ligands,12 which is mainly due to the existence of mefenamato
ligands. We may conclude that the interaction between metal
complexes and SA is affected only by the nature of the NSAID
ligand. Mefenamic acid may be considered a stronger binding
ligand to SA than naproxen and diclofenac.
From the Scatchard equation:27a
(8)
where n is the number of binding sites per albumin and K is the
association binding constant, K (M-1) is calculated by the slope
Interaction with DNA
Transition metal complexes can bind to DNA via both covalent
and/or noncovalent 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 noncovalent DNA in-
teractions include intercalative, electrostatic and groove (surface)
binding of metal complexes outside of DNA helix, along major or
minor groove.28a To the best of our knowledge, metal complexes
of the oxicam NSAIDs have been found to bind to DNA via
the intercalative mode,5b while from the group of the anthranilic
acid NSAIDs only the interaction of DNA with a series of Co(II)
mefenamato complexes has been reported by our lab.16
in plots
versus
(Fig. S2†) and n is given by the ratio of
the y intercept to the slope.27a The K values of the compounds
(Table 2) are relatively high with 4 having the highest K value.
Hmef exhibits the lowest value, indicating that its coordination to
Cu2+ leads to enhanced stability of the corresponding compound
with BSA. The n values of all compounds are of similar magnitude
(1.15–1.29) with 5 showing the highest value.
The quenching provoked by Hmef or complexes 1–5 to the HSA
fluorescence at l = 351 nm (Fig. 6(B)) is moderate for Hmef and
significant for the complexes (up to 40% of the initial fluorescence
intensity for Hmef, 28% for 1, 14% for 2, 18% for 3, 4% for 4 and
20% for 5) without any other spectroscopic changes indicating
that the binding of Hmef or each complex to HSA quenches the
intrinsic fluorescence of the single tryptophan in HSA.27b
The calculated values of KSV and kq, as obtained by the slope of
the Stern–Volmer plot (Fig. S3†), for Hmef and 1–5 (Table 2)
indicate their good HSA binding propensity, with complex 4
exhibiting the strongest protein-binding ability and provoking the
highest quenching. The kq values (>1013 M-1 s-1) are higher than
diverse kinds of quenchers for biopolymers fluorescence (2.0 ¥
1010 M-1 s-1) suggesting a static quenching mechanism.26b
From the Scatchard plot (Fig. S4†) and eqn. (8),27a the K and
n values of each compound have been calculated (Table 2) with
complex 4 exhibiting the highest association binding constant to
HSA and 2–4 the highest n values (1.04–1.05).
Study of the DNA-interaction with UV spectroscopy
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. 7(A). Hmef
and the complexes exhibit similar behaviour upon their addition to
CT DNA solution. The decrease of the intensity at lmax = 258 nm
is accompanied by a red-shift of the lmax up to 266 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.28a The observed hypochromism could be attributed to a
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.28b
Comparing the affinity of Hmef and complexes 1–5 for BSA and
HSA (K values), it is obvious that 1, 3 and 4 show higher affinity
for BSA than HSA, while for complexes 2 and 5 and mefenamic
acid the corresponding values of the constants K are of the same
magnitude.
In the UV spectra of the complexes, the intense absorption
bands observed are attributed to the intra-ligand transition of
the coordinated groups of mefenamato ligands. Any interaction
8560 | Dalton Trans., 2011, 40, 8555–8568
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Fig. 7 (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 [Cu(mef)2(bipy)]
2. The arrows show the changes upon increasing amounts of the complex. (B) UV spectra of DMSO solution (1 ¥ 10-5 M) of [Cu(mef)2(bipy)] 2 in the
presence of increasing amounts of CT DNA. The arrows show the changes upon increasing amounts of CT DNA. Inset in (B): plot of
[DNA].
vs.
Table 3 The DNA binding constants (Kb) of complexes 1–5
(9)
Hyperchromism/
Hypochromism (%)
Compound
Kb (M-1)
where [DNA] is the concentration of DNA in base pairs, eA
=
Hmef16
Hyperchromism
1.05( 0.02) ¥ 105
Aobsd/[complex], ef = the extinction coefficient for the free complex
[Cu2(mef)4(H2O)2], 1
[Cu(mef)2(bipy)], 2
[Cu(mef)2(phen)], 3
[Cu(mef)2(bipyam)], 4
Hypochromism, 25% 7.59( 0.22) ¥ 105
Hypochromism, 40% 9.03( 0.04) ¥ 104
Hypochromism, 33% 6.95( 0.15) ¥ 105
Hypochromism, 19% 2.20( 0.17) ¥ 105
and eb = the extinction coefficient for the complex in the fully
bound form. The calculated Kb values for Hmef and complexes
1–5 (Table 3) suggest a relatively strong binding of Hmef and
complexes 1–5 to CT DNA18b–e and increase in the order 5 <
2 < Hmef < 4 < 3 < 1 with complex 1 exhibiting the highest
Kb value (7.59( 0.22) ¥ 105 M-1). It is obvious (Table 3) that the
coordination of Hmef to Cu(II) results in a significant increase
of the Kb value as calculated for 1, while the co-existence of the
N-donor heterocyclic ligands leads to lower Kb values, even lower
than that of free Hmef (= 1.05( 0.02) ¥ 105 M-1) as for 2 and 5,
indicating that the existence of a nitrogen-donor ligand may not
enhance the affinity for DNA. The Kb values of all complexes are
of the same magnitude as that of the classical intercalator EB (Kb =
1.23( 0.07) ¥ 105 M-1).17c,18b
In comparison to the previously reported Co(II) mefenamato
complexes,16 Cu(II) mefanamato complexes, especially 1–3, exhibit
much higher affinity for DNA than their corresponding Co(II)
complexes. This could be clearly attributed as a synergetic effect
between copper and mefenamic acid. Additionally, complexes 1–5
present much higher Kb values than the Cu(II) complexes with the
NSAIDs naproxen and diclofenac as ligands,12 which is mainly due
to the existence of mefenamato ligands in 1–5; free Hmef presents
much higher Kb than free naproxen and sodium diclofenac. The
interaction between metal complexes and CT DNA is affected
either by the metal or by the nature of the NSAID ligand. Cu(II)
should be considered a better binder than Co(II) and mefenamato
ligand a stronger binding ligand than naproxen and diclofenac.
[Cu(mef)2(py)2(MeOH)2], 5 Hypochromism, 26% 6.93( 0.50) ¥ 104
between each complex and CT DNA that could perturb its
intra-ligand centred spectral transitions during the titration upon
addition of CT DNA in diverse r values can be observed.29a In
the UV spectrum of 2, the band centered at 341 nm exhibits
initially a hypochromism of 40% (Fig. 7(B)) suggesting tight
binding to CT DNA probably by intercalation. Further addition
of DNA results in a blue-shift with a gradual elimination of this
band. Additionally, the band at 291 nm presents a hyperchromism
accompanied by a red-shift of 9 nm (up to 300 nm), suggesting
tight binding and stabilization. A distinct isosbestic point at 334
nm appears upon addition of CT DNA. The behavior of complexes
1, 3–5 upon addition of CT DNA is quite similar to 2 while the
observed hypochromism varies from 19% for 4 up to 40% for 2
(Table 3). Additionally, for Hmef the band centred at 324 nm has
shown a hyperchromism in the presence of increasing amounts of
CT DNA.16
The results derived from the UV titration experiments suggest
that all the complexes can bind to CT DNA29b although the exact
mode of binding cannot be merely proposed by UV spectroscopic
titration studies. Nevertheless, the existence of hypochromism
could be considered as evidence that the binding of the complexes
involving intercalation between the base pairs of CT DNA cannot
be ruled out.18b–e The binding constant of the complexes to CT
DNA, Kb, is calculated by the ratio of the slope to the y intercept
Cyclic voltammetry and DNA-binding study
The cyclic voltammograms of 3 in DMSO solution (Fig S5†)
exhibit two cathodic waves at -550 mV (Epc1) and at -1170 mV
(Epc2) followed by two anodic waves at +200 mV (Epa1) and at
+600 mV (Epa2). The two one-electron waves at Epc1 and Epc2
in plots
versus [DNA] (Inset in Fig. 7(B)), according to
the equation:28b
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Table 4 Cathodic and anodic potentials (mV) for the redox couples in
means of partial and/or non-classic intercalation causes a bend or
kink in the DNA helix reducing its effective length and, as a result,
DNA solution viscosity is decreased or remains unchanged.30
Viscosity measurements were carried out on CT DNA solutions
upon addition of increasing amounts of the Hmef or complexes
1–5. The addition of Hmef or the complexes results in an increase
in the relative viscosity of DNA (Fig. 8). This may be explained
by the insertion of the compounds in between the DNA base
pairs, leading to an increase in the separation of base pairs at
intercalation sites and, thus, an increase in overall DNA length.12
DMSO solution of the complexes
Complex
Epc1
Epc2
Epa1
Epa2
[Cu2(mef)4(H2O)2], 1
[Cu(mef)2(bipy)], 2
-590
-480
-550
-530
-530
-1240
-1225
-1170
-1080
-1260
+175
+310
+200
+240
+240
+675
+770
+660
+695
+740
[Cu(mef)2(phen)], 3
[Cu(mef)2(bipyam)], 4
[Cu(mef)2(py)2(MeOH)2], 5
suggest reduction of the mononuclear species in two steps and
they can be assigned to the process [Cu(II)] → [Cu(I)], and the
formation of metallic copper, respectively.12 Additionally, the two
anodic waves at Epa1 and Epa2 can be attributed to the oxidation
of metallic copper and of [Cu(I)], respectively. The remaining
complexes 1, 2, 4 and 5 have similar cyclic voltammograms in
DMSO solution and the corresponding potentials are given in
Table 4.
The electrochemical investigations of metal–DNA interactions
can provide a useful supplement to spectroscopic methods, e.g., for
nonabsorbing species, and yield information about interactions
with both the reduced and oxidized form of the metal. In
general, the electrochemical potential of a small molecule will shift
positively when it intercalates into a DNA double helix, and it will
shift to a negative direction in the case of electrostatic interaction
with DNA.12,16
The quasi-reversible redox couple Cu(II)/Cu(I) for each complex
in 1/2 DMSO/buffer solution has been studied upon addition of
CT DNA (Fig. S6†) 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
explained in terms of an equilibrium mixture of free and DNA-
bound complexes to the electrode surface.12,16
Fig. 8 Relative viscosity of CT DNA (h/ho)1/3 in buffer solution (150 mM
NaCl and 15 mM trisodium citrate at pH 7.0) in the presence of Hmef and
complexes 1–5 at increasing amounts (r).
Competitive study with ethidium bromide
Ethidium bromide (EB
=
3,8-diamino-5-ethyl-6-phenyl-
phenanthridinium bromide) is a phenanthridine fluorescent
dye and is a typical indicator of intercalation, forming soluble
complexes with nucleic acids and emitting intense fluorescence in
the presence of CT DNA due to the intercalation of the planar
phenanthridinium ring between adjacent base pairs on the double
helix. The changes observed in the spectra of EB on its binding to
CT DNA are often used for the interaction study between DNA
and metal complexes.31a,b
Hmef and complexes 1–5 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 the
emission spectra. Hence competitive EB binding studies may be
undertaken in order to examine the binding of each compound
with DNA since the fluorescence intensity is highly enhanced
For increasing amounts of CT DNA, both the cathodic (Epc) and
the anodic (Epa) potentials of all complexes exhibit a positive shift
(DEpc = (+15) - (+65) mV) (Table 5) suggesting an intercalative
mode of binding.18b,d,e
DNA-binding study with viscosity measurements
DNA viscosity is sensitive to DNA length change.30 In the case
of classic intercalation, DNA base pairs are separated in order to
host the bound compound resulting in the lengthening of the DNA
helix and subsequently increased DNA viscosity. On the other
hand, the binding of a compound exclusively in DNA grooves by
Table 5 Cathodic and anodic potentials (mV) for the redox couple Cu(II)/Cu(I) in 1/2 DMSO/buffer solution of the complexes in the absence and
presence of CT DNA
a
b
c
a
b
c
Complex
Epc(f)
Epc(b)
DEpc
Epa(f)
Epa(b)
DEpa
[Cu2(mef)4(H2O)2], 1
[Cu(mef)2(bipy)], 2
-686
-738
-830
-765
-710
-670
-720
-815
-730
-695
+16
+18
+15
+35
+15
-555
-400
+170
-535
-540
-540
-335
+185
-520
-485
+15
+65
+15
+15
+55
[Cu(mef)2(phen)], 3
[Cu(mef)2(bipyam)], 4
[Cu(mef)2(py)2(MeOH)2], 5
a Epc/a in DMSO/buffer solution in the absence of CT DNA (Epc/a(f)
- Epc/a(f)
) )
b Epc/a in DMSO/buffer solution in the presence of CT DNA (Epc/a(b) c DEpc/a = Epc/a(b)
8562 | Dalton Trans., 2011, 40, 8555–8568
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Table 6 EB-DNA fluorescence (%) and Stern–Volmer constants (KSV) of
complexes 1–5
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 in a decrease in the
DNA-induced EB emission due to the replacement of EB, and/or
electron transfer.12,16
% of EB fluorescence
(% I/I0)
Compound
KSV (M-1)
Hmef16
20
29
17
27
18
12
1.58( 0.06) ¥ 105
1.99( 0.07) ¥ 105
4.72( 0.37) ¥ 105
1.04( 0.03) ¥ 105
2.54( 0.08) ¥ 106
1.04( 0.05) ¥ 106
The emission spectra of EB bound to CT DNA in the absence
and presence of each compound have been recorded for [EB] =
20 mM, [DNA] = 26 mM for increasing amounts of each compound.
The addition of Hmef or each complex 1–5 at diverse r values
(Fig. 9) 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, 29% for
1, 17% for 2, 27% for 3, 18% for 4 and 12% for 5) indicating
the competition of the complexes with EB in binding to DNA.
The observed significant quenching of DNA–EB fluorescence for
Hmef and 1–5 suggests that they displace EB from the DNA–EB
complex and they can probably interact with CT DNA by the
intercalative mode.12,16,18
[Cu2(mef)4(H2O)2], 1
[Cu(mef)2(bipy)], 2
[Cu(mef)2(phen)], 3
[Cu(mef)2(bipyam)], 4
[Cu(mef)2(py)2(MeOH)2], 5
much higher quenching and Ksv values and subsequently they
are more able to replace EB, a fact that may be attributed to the
existence of mefenamato ligands.
Antioxidant capacity of the complexes
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.14a Therefore, compounds with possible
antioxidant properties could potentially play a crucial role against
inflammation and lead to potentially effective drugs. The new
compounds were tested in comparison to well-known antioxidant
agents e.g. NDGA, BHT and trolox. In this context, Hmef and
complexes 1–5 have been tested with regard to their antioxidant
ability and the results are shown in Tables 7 and 8.
Antioxidants that exhibit DPPH radical scavenging activity
are receiving increased attention since they present interesting
anticancer, antiageing and antiinflammatory activities. Therefore,
compounds with antioxidant properties may offer protection in
rheumatoid arthritis and inflammation and lead to potentially
effective drugs. DPPH is a stable free radical and presents a
strong absorption band at 517 nm. The interaction of Hmef
and complexes 1–5 with the stable free radical DPPH (Table 7)
has revealed their reducing activity. All the compounds have
been tested in two concentrations (0.05 and 0.1 mM). The
measurements were taken after 20 and 60 min. In general, the
tested compounds present low to moderate interaction values. The
order of the reducing activity of the studied compounds increases
in the order 2 < 3 < 4 ~ Hmef < 5 < 1. Complexes 2–5 which bear
a lipophilic moiety (N-donor ligands) have shown low reducing
activity. Complex 1 was found to be the most active and its activity
is time and concentration dependent. In general, complexes 1–5
exhibit more pronounced DPPH radical scavenging activity than
the corresponding Co(II) mefenamato complexes.16
Fig. 9 Plot of EB relative fluorescence intensity at lem = 592 nm (%) vs
r (r = [complex]/[DNA]) for Hmef and complexes 1–5 in buffer solution
(150 mM NaCl and 15 mM trisodium citrate at pH 7.0).
The Stern–Volmer constant KSV is used to evaluate the quench-
ing efficiency for each compound according to eqn. (10):
(10)
where I0 and I are the emission intensities in the absence and the
presence of the quencher, respectively, [Q] is the concentration of
the quencher (complexes 1–5) and KSV is obtained by the slope
of the diagram
vs. [Q]. The Stern–Volmer plots of DNA–EB
Hydroxyl radicals are characterized among 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∑. They
are considered to be responsible for some of the tissue damage
occurring in inflammation. It has been 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, was
used for the evaluation of their hydroxyl radical scavenging
activity. The competition of Hmef and complexes 1–5 with DMSO
for ∑OH generated by the Fe3+/ascorbic acid system expressed as
for the compounds (Fig. S7†) illustrate that the quenching of EB
bound to DNA by the compounds are in good agreement (R =
0.99) with the linear Stern–Volmer equation (eqn. (10)), which
proves that the partial replacement of EB bound to DNA by each
compound results in a decrease in the fluorescence intensity. The
high KSV (Table 6) values of the compounds show that they can
bind tightly to the DNA.12,16,18
A comparison with the previously reported Co(II) mefenamato
complexes16 shows that Co(II) and Cu(II) complexes 1–5 exhibit
similar behaviour and ability to displace EB from the EB–DNA
complex while comparison of the Cu(II) complexes with the
naproxen and diclofenac12 shows that complexes 1–5 presents
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Table 7 Interaction % with DPPH (RA%)a
Compound
20 min, 0.05 mM
20 min, 0.1 mM
60 min, 0.05 mM
60 min, 0.1 mM
Hmef16
15.5
35.9
4.3
5.7
52.4
6.0
13.5
38.4
7.1
13.5
13.7
23.2
nt
11.7
54.1
7.9
[Cu2(mef)4(H2O)2], 1
[Cu(mef)2(bipy)], 2
[Cu(mef)2(phen)], 3
[Cu(mef)2(bipyam)], 4
[Cu(mef)2(py)2(MeOH)2], 5
NDGA
7.5
3.3
7.9
14.8
19.7
nt
17.3
11.8
81
18.5
21.5
82.6
60
BHT
nt
31.3
nt
a Each experiment was performed at least in triplicate SD < 10%; nt = not tested.
Table 8 Competition % with DMSO for hydroxyl radical (·OH%);% superoxide radical scavenging activity (ABTS%); in vitro inhibition of soybean
lipoxygenase (LOX) IC50 (mM)a
Compound
·OH% 0.1 mM
ABTS% 0.1 mM
LOX IC50 (mM)
Hmef16
92.5
67.6
74.6
99.7
77.4
75.3
88.2
—
66.3
75.4
90.3
89.4
80.0
98.2
91.8
—
48.5
4.8
40
67.5
54.5
50
[Cu2(mef)4(H2O)2], 1
[Cu(mef)2(bipy)], 2
[Cu(mef)2(phen)], 3
[Cu(mef)2(bipyam)], 4
[Cu(mef)2(py)2(MeOH)2], 5
Trolox
—
600
Caffeic acid
a Each experiment was performed at least in triplicate SD < 10%.
percent inhibition of formaldehyde production was used for the
evaluation of their hydroxyl radical scavenging activity and the
results are cited in Table 8. All the compounds were found to
present strong competition with DMSO (33 mM) at 0.1 mM for
hydroxyl free radicals. Hmef showed significant scavenging activity
(92.5%). The hydroxyl radical scavenging activity increases in the
order 1 < 2 < 5 < 4 < Hmef < 3. The coordination of mefenamato
ligand to copper leads to lower scavenging activity, while only
complex 3 presents even higher scavenging activity (99.7%) than
Hmef showing that the co-existence of the mefenamato ligand with
Cu(II) and phen in the molecule may lead to increased activity.
Among complexes 1–5, only 3 exhibits higher hydroxyl radical
scavenging activity than the corresponding Co(II) mefenamato
complexes.16
Generation of the ABTS radical cation forms the basis of one
of the spectrophotometric methods that have been applied to the
measurement of the total antioxidant activity of solutions of Hmef
and 1–5. The compounds were found to have high activity as
shown in Table 8. Hmef presents a rather high activity (66.3%)
while complexes 1–5 present a much higher activity that increases
in the order Hmef < 1 < 4 < 3 < 2 < 5. All the complexes are
more potent than mefenamic acid. The activity of mefenamic acid
is enhanced when coordinated to Cu(II) in 1 (75.4%) in the presence
of aqua ligand, while, in the presence of the N,N¢-donor ligands
bipyam, bipy and phen, the activity is much more enhanced (up to
90%). The highest activity has been found in the presence of py in
5 (98%). The radical cation scavenging activity of complexes 1–5
may be mainly attributed to the mefenamato ligands, depending
on the existence and the nature of the nitrogen-donor ligand and
is similar to that of their Co(II) analogues.16
inhibitors are antioxidants or free radical scavengers,32a since
lipoxygenation occurs via a carbon centred radical. Some studies
suggest a relationship between LOX inhibition and the ability
of the inhibitors to reduce the Fe(III) at the active site to the
catalytically inactive Fe(II).32b LOXs contain a ‘non-heme’ iron
per molecule in the enzyme active site as high-spin Fe(III) in
the native state and the high-spin in the activated state Fe(III).
Several LOX inhibitors are excellent ligands for Fe(III). It has
been demonstrated that their mechanism of action is presumably
related to their coordination with a catalytically crucial Fe(III).
Caffeic acid was used as a reference compound (IC50 = 600 mM).
All the compounds were found to present significant inhibition
against soybean lipoxygenase (Table 8) which increases in the
order 3 < 4 < 5 < Hmef < 2 < 1. Complex 1 is the most
active compound (IC50 = 4.8 mM) which could be attributed to a
synergy between copper and mefenamato ligands (Hmef presents
significant inhibition with IC50 = 48.5 mM).
In conclusion, the complexes exhibit higher inhibition than
mefenamic acid and their Co(II) analogues against the DPPH
and ABTS radicals. The inhibition of all the compounds against
hydroxyl radicals is very high with only complex 3 showing better
inhibition than mefenamic acid. Complexes 1 and 2 exhibit better
in vitro inhibitory activity against soybean lipoxygenase than
mefenamic 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.33
Conclusions
The compounds were tested for their inhibitory activity against
soybean lipoxygenase. The majority of lipoxygenase (LOX)
The synthesis and characterization of the neutral mononu-
clear copper(II) complex with the non-steroidal antiinflammatory
8564 | Dalton Trans., 2011, 40, 8555–8568
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drug mefenamic acid in the absence or presence of a nitrogen
donor heterocyclic ligand 2,2¢-bipyridine, 1,10-phenanthroline
2,2¢-bipyridylamine or pyridine has been achieved. The crystal
structures of [Cu(mef)2(bipy)], [Cu(mef)2(bipyam)]·MeOH and
[Cu(mef)2(py)2(MeOH)2] have been determined by X-ray crystal-
lography presenting a similar arrangement of the atoms around
copper to the previously reported Cu(II)–NSAID complexes
[Cu(naproxenato)2(bipy)] and [Cu(naproxenato)2(phen)].
UV spectroscopy, viscosity measurements and cyclic voltamme-
try studies have revealed the ability of the complexes to bind to
DNA as well as the binding mode. The binding constants of the
complexes for CT DNA have been calculated by UV spectroscopic
titrations with [Cu2(mef)4(H2O)2] exhibiting the highest Kb 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 voltammetric studies
as well as viscosity measurements have confirmed intercalation
as the most possible binding mode to DNA. The complexes
show good binding affinity to BSA and HSA proteins giving
high binding constants. All the compounds have been tested
in vitro for their antioxidant, free radical scavenging activity
and inhibitory activity on soybean lipoxygenase. They exhibit
significantly high scavenging activity against hydroxyl free radicals
and superoxide radicals. Complex 1 is the most active one. The
DPPH radical scavenging activity of complex 1 accompanies the
potent lipoxygenase inhibitory activity.
UV absorbance at 260 nm after 1 : 20 dilution using e = 6600 M-1
cm-1.12,16
Infrared (IR) spectra (400–4000 cm-1) were recorded on a
Nicolet FT-IR 6700 spectrometer with samples prepared as KBr
disks. 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 temper-
ature magnetic measurements were carried out on a magnetic
susceptibility balance of Sherwood Scientific (Cambridge, UK) by
the Faraday method using mercury tetrathiocyanatocobaltate(II)
as a calibrant. C, H and N elemental analysis were performed
on a Perkin-Elmer 240B elemental analyzer. Molar conductivity
measurements of 1 mM DMSO solution of the complexes were
carried out with a Crison Basic 30 conductometer. Fluorescence
spectra were recorded in solution on a Hitachi F-7000 fluorescence
spectrophotometer. 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 Cryo-
stat, operating at 9.412 GHz, 10db. Viscosity experiments were
carried out using an ALPHA L Fungilab rotational viscometer
equipped with an 18 mL LCP spindle.
Cyclic voltammetry studies were performed on an Eco chemie
Autolab Electrochemical analyzer. Cyclic voltammetric experi-
ments were carried out in a 30 mL three-electrode electrolytic cell.
The working electrode was a platinum disk, a separate Pt single-
sheet electrode was used as the counter electrode and an 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 vapors. All electrochemical
measurements were performed at 25.0 0.2 ◦C.
The examined Cu(II)-mefenamato complexes exhibit higher
binding constants for CT DNA and serum albumin proteins
than the corresponding Cu(II) complexes with the NSAIDs
naproxen and diclofenac of the same formula. Cu(II)-mefenamato
complexes 1–5 exhibit higher binding constants for CT DNA
and higher inhibition against DPPH and ABTS radicals than the
corresponding Co(II)-mefenamato while their ability to displace
EB and their binding towards serum albumin proteins presents
similar constant values.
Synthesis of the complexes
[Cu2(mef)4(H2O)2], 1. A methanolic solution (15 mL) contain-
ing mefenamic acid (0.4 mmol, 97 mg) and KOH (0.4 mmol,
22 mg) was stirred for 1 h. The solution was added dropwise to a
methanolic solution (10 mL) of CuCl2·2H2O (0.2 mmol, 34 mg)
and the reaction mixture was stirred for 1 h. The reaction solution
was filtered and left for slow evaporation. After a few days an
olive-green microcrystalline product (90 mg, 78%) was deposited
and collected by filtration. (Found C, 63.90; H, 5.56; N, 4.65%;
C60H60Cu2N4O10 (MW = 1124.25) requires C, 64.10; H, 5.38; N,
4.98%). IR: nmax/cm-1 n(O–H)w, 3343 (m); nasym(CO2), 1595 (vs);
Experimental
Materials, instrumentation and physical measurements
Mefenamic acid, CT DNA, BSA, HSA, EB, DPPH, soybean
lipoxygenase, linoleic acid sodium salt, ABTS, 6-hydroxy-2,5,7,8-
tetramethylchromane-2-carboxylic acid (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 CuCl2·2H2O, bipy, phen, bipyam, py, KOH and Nordihy-
droguairetic acid (NDGA) were purchased from Aldrich Co. All
the chemicals and solvents were reagent grade and were used as
purchased without any further purification. Tetraethylammonium
perchlorate (TEAP) was purchased from Carlo Erba and, prior to
its use, it was recrystallized twice from ethanol and dried under
vacuum.
n
sym(CO2), 1425 (vs); D = nasym(CO2) - nsym(CO2): 170 cm-1 (KBr
disk); UV-vis: l nm-1 (e/M-1 cm-1) as nujol mull: 675, 445 (sh),
324, 281; in DMSO: 665 (60), 440 (150), 335 (7800), 292 (14200).
meff = 1.40 BM. The complex is soluble in DMSO and DMF and
is non-electrolyte (KM = 14 mho cm2 mol-1).
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
[Cu(mef)2(bipy)], 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
CuCl2·2H2O (0.2 mmol, 34 mg) and stirred for 30 min. The
solution was left for slow evaporation. Dark green crystals of
◦
4 C for no longer than a week. The stock solution of CT DNA
gave a ratio of UV absorbance at 260 and 280 nm (A260/A280
)
of 1.89, indicating that the DNA was sufficiently free of protein
contamination. The DNA concentration was determined by the
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[Cu(mef)2(bipy)] 2 (120 mg, 85%) suitable for X-ray structure
determination were deposited after a few days. (Found C, 68.41; H,
5.14; N, 7.87%; C40H36CuN4O4 (MW = 700.30) requires C, 68.61;
H, 5.18; N, 8.00%). IR: nmax/cm-1; nasym(CO2): 1613 (vs); nsym(CO2):
1377 (vs); D = 246 cm-1 (KBr disk); UV-vis: l nm-1 (e/M-1 cm-1) as
nujol mull: 650, 440 (sh), 341, 307; in DMSO: 655 (110), 435 (sh)
(120), 346 (11800), 303 (17000). meff = 1.83 BM. The complex is
soluble in DMSO and DMF and is non-electrolyte (KM = 13 mho
cm2 mol-1).
competitive studies with EB by fluorescence spectroscopy have
been thoroughly described in ref. 12 and references therein.
The determination of the reducing activity of the stable radical
DPPH provoked by the complexes, their competition with DMSO
for hydroxyl radicals and their radical cation scavenging activity
have been determined according to the procedures described in ref.
16 and ref. 33a,b. In order to perform the soybean lipoxygenase
inhibition study in vitro, the tested compounds dissolved in
ethanol were incubated at room temperature with sodium linoleate
(0.1 mM) and 0.2 cm3 of enzyme solution (1/9 ¥ 10-4 w/v in saline)
and the conversion of sodium linoleate to 13-hydroperoxylinoleic
acid at 234 nm was recorded and compared with the appropriate
standard inhibitor.33a 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.
[Cu(mef)2(phen)], 3. Complex 3 (110 mg, 75%) 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 CuCl2·2H2O (0.2 mmol, 34 mg).
The dark green microcrystalline product was collected after a
few days. (Found C, 69.87; H, 4.93; N, 7.65%; C42H36CuN4O4
(MW = 724.32) requires C, 69.65; H, 5.01; N 7.74%). IR: nmax/cm-1;
X-ray determination
n
asym(CO2): 1615 (vs); nsym(CO2): 1380 (vs); D = 235 cm-1 (KBr
Slow crystallization from methanol yielded green prismatic crys-
tals of 2, green prismatic crystals of 4 and pink prismatic
crystals of 5. A green crystal of 2 with approximate dimensions
0.10 ¥ 0.13 ¥ 0.24 mm, a green crystal of 4 with approximate
dimensions 0.19 ¥ 0.22 ¥ 0.58 mm and a pink crystal of 5
with approximate dimensions 0.06 ¥ 0.15 ¥ 0.29 mm were taken
directly from the mother liquor and immediately cooled to
-93 ◦C (2, 5) or -113 ◦C (4). Diffraction measurements were
made on a Rigaku R-AXIS SPIDER Image Plate diffractome-
ter using graphite monochromated Cu-Ka radiation (Table 9
and Table S2†). Data collection (w-scans) and processing (cell
refinement, data reduction and Empirical absorption correction)
were performed using the CrystalClear program package.34a The
structures were solved by direct methods using SHELXS-97
and refined by full-matrix least-squares techniques on F2 with
SHELXL-97.34b,c Further experimental crystallographic details for
2: 2qmax = 130◦; reflections collected/unique/used, 33244/5823
[Rint = 0.0221]/5823; 564 parameters refined; (D/s)max = 0.000;
disk); UV-vis: l nm-1 (e/M-1 cm-1) as nujol mull: 675, 440 (sh),
328, 287; in DMSO: 660 (70), 445 (90), 341 (7500), 288 (15800).
meff = 1.92 BM. The complex is soluble in DMSO and DMF and
is non-electrolyte (KM = 11 mho cm2 mol-1).
[Cu(mef)2(bipyam)]·MeOH, 4·MeOH. 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
bipyam (0.2 mmol, 34 mg) to a methanolic solution (10 mL) of
CuCl2·2H2O (0.2 mmol, 34 mg). The resultant solution was left for
slow evaporation. Green crystals of [Cu(mef)2(bipyam)]·MeOH,
4·MeOH (100 mg, 70%) suitable for X-ray structure determina-
tion, were collected after a few days. (Found C, 65.78; H, 5.43; N,
9.56%; C41H41CuN5O5 (MW = 747.33) requires C, 65.89; H, 5.53;
N, 9.37%). IR: nmax/cm-1; nasym(CO2): 1614 (vs); nsym(CO2): 1378
(vs); D = 236 cm-1 (KBr disk); UV-vis: l nm-1 (e/M-1 cm-1) as nujol
mull: 670, 440 (sh), 330, 295; in DMSO: 680 (100), 442 (sh) (130),
326 (6200), 298 (10100). meff = 1.85 BM. The complex is soluble in
DMSO and DMF and is non-electrolyte (KM = 8 mho cm2 mol-1).
-3
˚
(Dr)max/(Dr)min = 0.612/-0.302 e/A ; R1/wR2 (for all data),
0.0469/0.1117. Further experimental crystallographic details for
Table 9 Crystallographic data for complexes 2 and 4·MeOH
[Cu(mef)2(py)2(MeOH)2], 5. 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 CuCl2·2H2O (0.2 mmol,
34 mg) followed by the addition of 1.5 mL of pyridine. Pink
crystals of [Cu(mef)2(py)2(MeOH)2] 5 (90 mg, 65%) suitable for
X-ray structure determination, were deposited after a few days.
(Found C, 65.58; H, 6.18; N, 7.29%; C42H46CuN4O6 (MW = 766.40)
requires C, 65.82; H, 6.05; N, 7.31%). IR: nmax/cm-1; nasym(CO2):
1614 (vs); nsym(CO2): 1382 (vs); D = 232 cm-1 (KBr disk); UV-vis: l
nm-1 (e/M-1 cm-1) as nujol mull: 690, 440 (sh), 327, 285; in DMSO:
695 (80), 435 (sh) (95), 338 (6900), 294 (18500). meff = 1.95 BM.
The complex is soluble in DMSO and is non-electrolyte (KM = 6
mho cm2 mol-1).
2
4·MeOH
Formula
FW
T (K)
C40 H36 Cu N4 O4
700.27
180 (2)
C41 H41 Cu N5 O5
747.33
160 (2)
Crystal system
Space group
Monoclinic
P21/c
7.6941(1)
26.6993(5)
16.9080(3)
90.00
97.756(1)
90.00
3441.6(1)
4
Monoclinic
P21/n
11.1512(2)
14.0929(2)
23.7121(4)
90.00
101.795(1)
90.00
3647.7(1)
4
˚
a (A)
˚
b (A)
˚
c (A)
a (◦)
b (◦)
g (◦)
3
˚
Volume (A )
Z
D(calc), Mg m-3
Abs. coef., m/mm-1
F(000)
1.351
1.278
1460
1.361
1.268
1564
Biological studies
GOF on F2
R1
1.075
1.048
0.0402a
0.1076a
0.0340b
0.0910b
The procedures relating to the study of the interaction of com-
plexes 1–5 with the albumin binding by tryptophan fluorescence
quenching experiments and with CT DNA by UV spectroscopy,
viscosity experiments and cyclic voltammetry as well as their
wR2
a 4970 reflections with I > 2s(I). b 5774 reflections with I > 2s(I).
8566 | Dalton Trans., 2011, 40, 8555–8568
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4: 2qmax = 130◦; reflections collected/unique/used, 41459/6180
[Rint = 0.0302]/6180; 633 parameters refined; (D/s)max = 0.001;
(Dr)max/(Dr)min = 0.510/-0.428 e/A ; R1/wR2 (for all data),
10 (a) M. Ruiz, L. Perello, J. Servercarrio, R. Ortiz, S. Garciagranda, M.
R. Diaz and E. Canton, J. Inorg. Biochem., 1998, 69, 231–239; (b) F.
Hueso-Urena, M. N. Moreno-Carretero, M. A. Romero-Molina, J.
M. Salas-Peregrin, M. P. Sanchez-Sanchez, G. Alvarez de Cienfuegos-
Lopez and R. Faure, J. Inorg. Biochem., 1993, 51, 613–632; (c) D. K.
Saha, U. Sandbhor, K. Shirisha, S. Padhye, D. Deobagkar, C. E. Anson
and A. K. Powell, Bioorg. Med. Chem. Lett., 2004, 14, 3027–3032.
11 A. M. Ramadan, J. Inorg. Biochem., 1997, 65, 183–189.
12 F. Dimiza, F. Perdih, V. Tangoulis, I. Turel, D. P. Kessissoglou and G.
Psomas, J. Inorg. Biochem., 2011, 105, 476–489.
13 (a) R. G. Bhirud and T. S. Srivastava, Inorg. Chim. Acta, 1990,
173, 121–128; (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) A. L. Abuhijleh, J.
Inorg. Biochem., 1994, 55, 255–262; (d) M. Melnik, I. Potocnak, L.
Macaskova, D. Miklos and C. E. Holloway, Polyhedron, 1996, 15, 2159–
2164.
-3
˚
0.0359/0.0925. Further experimental crystallographic details for
5: 2qmax = 126◦; reflections collected/unique/used, 11019/2843
[Rint = 0.0469]/2843; 299 parameters refined; (D/s)max = 0.000;
-3
˚
(Dr)max/(Dr)min = 0.324/-0.584 e/A ; R1/wR2 (for all data),
0.0593/0.1161. Hydrogen atoms were located by difference maps
and were refined isotropically, except those of the methyl groups
which were introduced at calculated positions as riding on bonded
atoms. All non-hydrogen atoms were refined anisotropically.
Abbreviations
14 (a) R. Cini, G. Giorgi, A. Cinquantini, C. Rossi and M. Sabat, Inorg.
Chem., 1990, 29, 5197–5200; (b) A. E. Underhill, S. A. Bougourd, M.
L. Flugge, S. E. Gale and P. S. Gomm, J. Inorg. Biochem., 1993, 52,
139–144; (c) J. E. Weder, T. W. Hambley, B. J. Kennedy, P. A. Lay, D.
MacLachlan, R. Bramley, C. D. Delfs, K. S. Murray, B. Moubaraki,
B. Warwick, J. R. Biffin and H. L. Regtop, Inorg. Chem., 1999, 38,
1736–1744; (d) D. Kovala-Demertzi, A. Theodorou, M. A. Demertzis,
C. P. Raptopoulou and A. Terzis, J. Inorg. Biochem., 1997, 65, 151–157.
15 (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.
ABTS = 2,2¢-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)
radical cation, BHT = butylated hydroxytoluene, bipy = 2,2¢-
bipyridine, bipyam = 2,2¢-bipyridylamine, BSA = bovine serum
albumin, COX = cyclo-oxygenase, CT = calf-thymus, DPPH = 1,1-
diphenyl-picrylhydrazyl, EB = ethidium bromide = 3,8-diamino-
5-ethyl-6-phenyl-phenanthridinium bromide, Hmef = mefenamic
acid = 2-[(2,3-dimethylphenyl)-amino]benzoic acid, HSA = hu-
man serum albumin, LOX = lipoxygenase, NDGA = nordihy-
droguairetic acid, NSAID = non-steroidal antiinflammatory drug,
phen = 1,10-phenanthroline, py = pyridine, r = [compound]/[CT
DNA] mixing ratio, SA = serum albumin, sh = shoulder,
TEAP = tetraethylammonium perchlorate, trolox = 6-hydroxy-
2,5,7,8-tetramethylchromane-2-carboxylic acid, D = nasym(CO2) -
16 F. Dimiza, A. N. Papadopoulos, V. Tangoulis, V. Psycharis, C. P.
Raptopoulou, D. P. Kessissoglou and G. Psomas, Dalton Trans., 2010,
39, 4517–4528.
17 (a) C. Dendrinou-Samara, G. Psomas, K. Christophorou, V. Tangoulis,
V. P. Raptopoulou, A. Terzis and D. P. Kessissoglou, J. Chem. Soc.,
Dalton Trans., 1996, 3737–3743; (b) G. Psomas, C. P. Raptopoulou, L.
Iordanidis, C. Dendrinou-Samara, V. Tangoulis and D. P. Kessissoglou,
Inorg. Chem., 2000, 39, 3042–3048; (c) A. Dimitrakopoulou, C.
Dendrinou-Samara, A. A. Pantazaki, M. Alexiou, E. Nordlander and
D. P. Kessissoglou, J. Inorg. Biochem., 2008, 102, 618–628.
18 (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) A.
Tarushi, G. Psomas, C. P. Raptopoulou and D. P. Kessissoglou, J.
Inorg. Biochem., 2009, 103, 898–905; (d) K. C. Skyrianou, F. Perdih,
I. Turel, D. P. Kessissoglou and G. Psomas, J. Inorg. Biochem., 2010,
104, 740–749; (e) K. C. Skyrianou, V. Psycharis, C. P. Raptopoulou,
D. P. Kessissoglou and G. Psomas, J. Inorg. Biochem., 2011, 105, 63–
74.
19 (a) P. Christofis, M. Katsarou, A. Papakyriakou, Y. Sanakis, N.
Katsaros and G. Psomas, J. Inorg. Biochem., 2005, 99, 2197–2210;
(b) C. Dendrinou-Samara, D. P. Kessissoglou, G. E. Manoussakis, D.
Mentzafos and A. Terzis, J. Chem. Soc., Dalton Trans., 1990, 959–965;
(c) C. Dendrinou-Samara, G. Tsotsou, C. P. Raptopoulou, A. Kortsaris,
D. Kyriakidis and D. P. Kessissoglou, J. Inorg. Biochem., 1998, 71, 171–
179.
20 B. J. Hathaway, in Comprehensive Coordination Chemistry, ed. G.
Wilkinson, Pergamon Press, Oxford, 1987, vol. 5, pp. 533–773.
21 S. Stoll and A. Schweiger, J. Magn. Reson., 2006, 178, 42–55.
22 M. C. R. Symons, D. X. West and G. J. Wilkinson, J. Chem. Soc., Dalton
Trans., 1975, 1696–1700.
23 J. R. Morton and K. F. Preston, J. Magn. Reson., 1978, 30, 577–582.
24 J. Peisach and W. E. Blumberg, Arch. Biochem. Biophys., 1974, 165,
691–708.
25 (a) A. Grirrane, A. Pastor, A. Ienco, C. Mealli and A. Galindo, J. Chem.
Soc., Dalton Trans., 2002, 3771–3777; (b) J. Casanova, G. Alzuet, S.
Ferrer, J. Latorre, J. A. Ramirez and J. Borras, Inorg. Chim. Acta, 2000,
304, 170–177.
26 (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.
n
sym(CO2).
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, 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) W. J. Wechter, E. D. Murray, D. Kantoci, D.
D. Quiggle, D. D. Leipold, K. M. Gibson and J. D. McCracken, Life
Sci., 2000, 66, 745–753.
3 (a) K. Kim, J. Yoon, J. K. Kim, S. J. Baek, T. E. Eling, W. J. Lee, J. Ryu,
J. G. Lee, J. Lee and J. Yoo, Biochem. Biophys. Res. Commun., 2004,
325, 1298–1303; (b) M. Smith, G. Hawcroft and M. A. Hull, Eur. J.
Cancer, 2000, 36, 664–674; (c) B. A. Teicher, T. T. Korbut, K. Menon,
S. A. Holden and G. Ara, Cancer Chemother. Pharmacol., 1994, 33,
515–552.
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 (a) 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; (b) 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, 19–27; (c) 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, 375–385.
7 (a) G. Crisponi, V. M. Nurchi, D. Fanni, C. Gerosa, S. Nemolato and
G. Faa, Coord. Chem. Rev., 2010, 254, 876–889; (b) J. A. Drewry and
P. T. Gunning, Coord. Chem. Rev., 2011, 255, 459–472; (c) B. M. Rode
and Y. Suwannachot, Coord. Chem. Rev., 1999, 190–192, 1085–1099.
8 J. R. J. Sorenson, Prog. Med. Chem., 1989, 26, 437–568.
9 (a) E. K. Efthimiadou, H. Thomadaki, Y. Sanakis, C. P. Raptopoulou,
N. Katsaros, A. Scorilas, A. Karaliota and G. Psomas, J. Inorg.
Biochem., 2007, 101, 64–73; (b) M. E. Katsarou, E. K. Efthimiadou,
G. Psomas, A. Karaliota and D. Vourloumis, J. Med. Chem., 2008, 51,
470–478.
27 (a) S. Wu, W. Yuan, H. Wang, Q. Zhang, M. Liu and K. Yu, J. Inorg.
Biochem., 2008, 102, 2026–2034; (b) 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.
This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 8555–8568 | 8567
©

View Article Online
28 (a) Q. Zhang, J. Liu, H. Chao, G. Xue and L. Ji, J. Inorg. Biochem.,
2001, 83, 49–55; (b) 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.
29 (a) Y. Song, Q. Wu, P. Yang, N. Luan, L. Wang and Y. Liu, J.
Inorg. Biochem., 2006, 100, 1685–1691; (b) A. Jancso, L. Nagy, E.
Moldrheim and E. Sletten, J. Chem. Soc., Dalton Trans., 1999, 1587–
1594.
30 (a) D. Li, J. Tian, W. Gu, X. Liu and S. Yan, J. Inorg. Biochem., 2010,
104, 171–179; (b) M. Jiang, Y. Li, Z. Wu, Z. Liu and C. Yan, J. Inorg.
Biochem., 2009, 103, 833–844; (c) J. L. Garcia-Gimenez, M. Gonzalez-
Alvarez, M. Liu-Gonzalez, B. Macias, J. Borras and G. Alzuet, J. Inorg.
Biochem., 2009, 103, 923–934.
31 (a) T. Afrati, A. A. Pantazaki, C. Dendrinou-Samara, C. Raptopoulou,
A. Terzis and D. P. Kessissoglou, Dalton Trans., 2010, 39, 765–775;
(b) G. Zhao, H. Lin, S. Zhu, H. Sun and Y. Chen, J. Inorg. Biochem.,
1998, 70, 219–226.
33 (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; (d) Z. D. Petrovic, D. Hadjipavlou-
Litina and V. P. Petrovic, J. Mol. Liq., 2009, 144, 55–58; (e) D.
Kovala-Demertzi, D. Hadjipavlou-Litina, A. Primikiri, M. Staninska,
C. Kotoglou and M. A. Demertzis, Chem. Biodiversity, 2009, 6, 948–
960; (f) Z. Liu, B. Wang, Z. Yang, Y. Li, D. Qin and T. Li, Eur. J. Med.
Chem., 2009, 44, 4477–4484; (g) S. B. Bukhari, S. Memon, M. M. Tahir
and M. I. Bhanger, J. Mol. Struct., 2008, 892, 39–46; (h) E. Pontiki,
D. Hadjipavlou-Litina, A. T. Chaviara and C. A. Bolos, Bioorg. Med.
Chem. Lett., 2006, 16, 2234–2237.
34 (a) Rigaku/MSC, CrystalClear. Rigaku/MSC Inc., The Woodlands,
Texas, USA, 2005; (b) G. M. Sheldrick, SHELXS-97, Program for
solution of crystal structures, University of Go¨ttingen, Germany, 1997;
(c) G. M. Sheldrick, SHELXL-97, Program for refinement of crystal
structures, University of Go¨ttingen, Germany, 1997.
32 (a) K. Muller, Arch. Pharm., 1994, 327, 1–19; (b) J. Van der Zee, T. E.
Eling and R. P. Mason, Biochemistry, 1989, 28, 8363–8367.
8568 | Dalton Trans., 2011, 40, 8555–8568
This journal is
The Royal Society of Chemistry 2011
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