Antioxidant activity and interaction with DNA and albumins of zinc-tolfenamato complexes. Crystal structure of [Zn(tolfenamato) 2(2,2′-dipyridylketoneoxime)2]

Article abstract of DOI:10.1016/j.ejmech.2013.12.019

The zinc(II) complex of the non-steroidal anti-inflammatory drug tolfenamic acid (=Htolf) in the presence of 2,2′-dipyridylketone oxime (=Hpko) as a N,N′-donor heterocyclic ligand, [Zn(tolf-O)₂(Hpko-N,N′) ₂]·MeOH (=1·MeOH), has been synthesized and characterized by physicochemical techniques including X-ray crystallography. The complex exhibits good binding affinity to human or bovine serum albumin with high binding constant values. Complex 1 and previously reported Zn-tolfenamato complexes were tested for their free radical scavenging activity and in vitro inhibitory activity against soybean lipoxygenase and exhibited significant activity with [Zn(tolf)₂(1,10-phenantroline)] being the most active compound. The complexes interact with calf-thymus (CT) DNA via intercalation, and can displace the DNA-bound ethidium bromide with 1 exhibiting the highest binding constant to CT DNA.

Full text of DOI:10.1016/j.ejmech.2013.12.019

European Journal of Medicinal Chemistry 74 (2014) 187e198
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antioxidant activity and interaction with DNA and albumins of
zincetolfenamato complexes. Crystal structure of
[Zn(tolfenamato)2(2,20-dipyridylketoneoxime)2]
,
d
Alketa Tarushi a, Xanthippi Totta a, Athanasios Papadopoulos b, Jakob Kljun c
,
,
Iztok Turel d, Dimitris P. Kessissoglou a, George Psomas a
*
a Laboratory of Inorganic Chemistry, Department of General and Inorganic Chemistry, Faculty of Chemistry, Aristotle University of Thessaloniki, GR-54124
Thessaloniki, Greece
b Department of Nutrition and Dietetics, Faculty of Food Technology and Nutrition, Alexandrion Technological Educational Institution, Sindos, Thessaloniki,
Greece
c EN-FIST Centre of Excellence, Dunajska 156, 1000 Ljubljana, Slovenia
d Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva 5, 1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
The zinc(II) complex of the non-steroidal anti-inflammatory drug tolfenamic acid (¼Htolf) in the pres-
ence of 2,20-dipyridylketone oxime (¼Hpko) as a N,N0-donor heterocyclic ligand, [Zn(tolf-O)2(Hpko-
N,N0)2]$MeOH (¼1$MeOH), has been synthesized and characterized by physicochemical techniques
including X-ray crystallography. The complex exhibits good binding affinity to human or bovine serum
albumin with high binding constant values. Complex 1 and previously reported Zn-tolfenamato com-
plexes were tested for their free radical scavenging activity and in vitro inhibitory activity against soybean
lipoxygenase and exhibited significant activity with [Zn(tolf)2(1,10-phenantroline)] being the most active
compound. The complexes interact with calf-thymus (CT) DNA via intercalation, and can displace the
DNA-bound ethidium bromide with 1 exhibiting the highest binding constant to CT DNA.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Received 5 November 2013
Received in revised form
20 December 2013
Accepted 21 December 2013
Available online 3 January 2014
Keywords:
Zinc(II) complexes
Tolfenamic acid
Interaction with calf-thymus DNA
Interaction with serum albumins
Antioxidant activity
1. Introduction
many countries of Asia and Africa [4]. The role of zinc in nucleic
acid chemistry is noteworthy since zinc(II) ions are the only metal
Zinc is the second most abundant trace element in the human
body, has a major regulatory role in the metabolism of cells [1] and
presents many beneficial effects to human health, while changes in
its metabolism or trafficking are related to some diseases [2,3].
Zinc is extensively used to treat children suffering from deadly
diarrhea resulting in significant reduction of child mortality in
ions able to facilitate the rewinding of molten DNA [5]. In the
literature diverse zinc compounds have exhibited a potential bio-
logical activity with zinc complexes with drugs being tested for the
treatment of Alzheimer disease [6] and others showing antibac-
terial [7,8], anticonvulsant [9], antidiabetic [10], anti-inflammatory
[11], antioxidant [12] and antiproliferative-antitumor [8,11]
activity.
Non-steroidal anti-inflammatory drugs (NSAIDs)y are among the
most frequently used drugs as analgesics, anti-inflammatories and
antipyretics with known side-effects (mainly gastrointestinal such
as gastric ulceration, nausea, diarrhea and renal including salt and
fluid retention, hypertension, and in rare cases interstitial nephritis,
acute renal failure) [13]. The inhibition of the cyclo-oxygenase
(COX)-mediated production of prostaglandins is the main mode
of action of the NSAIDs [14]. They have also shown a synergistic role
on the activity of certain antitumor drugs [15] and have presented
antitumor activity leading to cell death of a series of cancer
cell lines via apoptotic pathways [16] or via diverse molecular
Abbreviations: ABTS, 2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)
radical cation; BHT, butylated hydroxytoluene; bipy, 2,20-bipyridine; BSA, bovine
serum albumin; COX, cyclo-oxygenase; CT, calf-thymus; DMF, N,N-dime-
thylformamide; DMSO, dimethylsulfoxide; DPPH, 1,1-diphenyl-picrylhydrazyl; EB,
ethidium bromide, 3,8-diamino-5-ethyl-6-phenyl-phenanthridinium bromide;
Hpko, 2,20-dipyridylketone oxime; Htolf, tolfenamic acid, 2-[(3-chloro-2-
methylphenyl)amino]benzoic acid; HSA, human serum albumin; LOX, soybean
lipoxygenase; NDGA, nordihydroguaiaretic acid; NSAID, non-steroidal anti-
inflammatory drug; phen, 1,10-phenanthroline; RA, DPPH radical scavenging ac-
tivity; SA, serum albumin; tolf, tolfenamato, anion of tolfenamic acid.
* Corresponding author. Tel.: þ30 2310997790; fax: þ30 2310997738.
E-mail address: gepsomas@chem.auth.gr (G. Psomas).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.019

188
A. Tarushi et al. / European Journal of Medicinal Chemistry 74 (2014) 187e198
mechanisms [17] where free radicals have been also involved [18].
In an attempt to investigate potential mechanisms of the anticancer
as well as the anti-inflammatory activity of the NSAIDs and their
complexes, the interaction with DNA as well as the antioxidant
activity are considered of great importance and should be further
evaluated; it is noteworthy that only few relevant reports on the
interaction of NSAIDs and their complexes with DNA have been
published so far [19,20]. Furthermore, the number of structurally
characterized zinc complexes with NSAIDs [21] is still expanding
including an aspirinate complex [22], a flufenamato complex [23]
and a series of complexes with indomethacin [8], mefenamic acid
[12] and tolfenamic acid [24] as ligands.
binding to these proteins which are involved in the transport of
metal ions and metal-drug complexes through the blood stream
may result in lower or enhanced biological properties of the orig-
inal drug, or may propose new paths for drug transportation [31],
(iii) their binding properties with calf-thymus (CT) DNA investi-
gated by UV spectroscopy and viscosity measurements and
ethidium bromide (EB) displacement ability from the EB-DNA
compound performed by fluorescence spectroscopy (EB is a
typical DNA-intercalator and the competition with it for the DNA
intercalation sites may serve as an indirect evidence of potential
intercalation).
Tolfenamic acid (¼Htolf, Scheme 1) is a NSAID belonging to the
N-phenylanthranilic acid derivatives with similar properties to
mefenamic acid and flufenamic acid and other fenamates in clinical
use [25]. Htolf is found in diverse analgesic, anti-inflammatory,
antipyretic and antirheumatoid drugs and is also used for veteri-
nary purposes [26]. The crystal structures of a series of tin(IV) [27],
copper(II) [28,29], cobalt(II) [30], and zinc(II) [24] complexes with
tolfenamato ligands have been reported in the literature.
2. Results and discussion
2.1. Synthesis and spectroscopic characterization
The synthesis of complex 1 was achieved in high yield via the
aerobic reaction of tolfenamic acid, deprotonated by KOH, with
ZnCl2 in the presence of the N,N0-donor heterocyclic ligand Hpko in
a ratio Zn2þ:tolf-:Hpko of 1:2:2. The complex is stable in air, soluble
in DMSO and DMF and non-electrolyte in DMSO (for 1 mM solution,
Taking into consideration the significance of NSAIDs in medicine
and the presence of zinc in diverse drugs, we have synthesized and
characterized the structure and the spectroscopic (IR, UV and 1H
NMR) properties of the neutral zinc(II) complexes with the NSAID
tolfenamic acid in the presence of the N,N0-donor heterocyclic
ligand 2,20-dipyridylketone oxime (¼Hpko). The crystal structure of
the resultant complex [Zn(tolf-O)2(Hpko-N,N0)2]$MeOH, 1$MeOH
has been determined by X-ray crystallography. Furthermore, the 1H
NMR spectra of the recently reported Zn-tolfenamato complexes
[Zn(tolf)(phen)Cl] (2), [Zn(tolf)(bipy)Cl], (3), [Zn(tolf)2(phen)], (4),
[Zn(tolf)2(bipy)], (5) and [Zn3(tolf)6(MeOH)2] (6) (where phen and
bipy are the N,N0-donor heterocyclic ligands 1,10-phenanthroline
and 2,20-bipyridine, respectively) [24] have been recorded and
evaluated. In an attempt to further investigate the existence of
potential anti-inflammatory activity of complexes 1e6, the study of
their biological properties has been focused on (i) their antioxidant
capacity by investigating their scavenging ability of 1,1-diphenyl-
picrylhydrazyl (DPPH) and 2,20-azinobis-(3-ethylbenzothiazoline-
6-sulfonic acid) (ABTSþꢀ) radicals, their ability to antagonize DMSO
in hydroxyl radicals (ꢀOH) binding, as well as their in vitro inhibitory
activity against soybean lipoxygenase (LOX), because the use of
NSAIDs in medicine as anti-inflammatories is also related to free
radicals scavenging, (ii) the affinity of the complexes to bovine
(BSA) and human serum albumin (HSA), as characteristic serum
proteins, investigated by fluorescence spectroscopy since the
15 mS/cm). Complex 1 was characterized by elemental analysis, IR,
UV and 1H NMR spectroscopic techniques and by X-ray crystal-
lography. Additionally, the 1H NMR spectra of compounds 2e6
were recorded and evaluated.
IR spectroscopy is a useful tool to confirm the deprotonation and
the binding mode of tolfenamic acid. In the IR spectrum of Htolf, the
absorption band at 3355(br,m) cmꢁ1, attributed to the
n(HeO)
stretching vibration has disappeared upon binding to the zinc ion.
The strong bands appearing at 1661 cmꢁ1 and 1265 cmꢁ1 which
were attributed to n(C]O)carboxylic and n(CeO)carboxylic stretching
vibrations of the carboxylic moiety (-COOH) of Htolf, respectively,
have shifted, in the IR spectra of complex 1, at 1584 cmꢁ1 and
1387 cmꢁ1 assigned to antisymmetric, nasym(C]O), and symmetric,
nsym(C]O), stretching vibrations of the coordinated carboxylato
group, respectively. The parameter
D
[¼nasym(C]O) e nsym(C]O)]
which is a useful characteristic tool for determining the coordina-
tion mode of the carboxylato ligands, has a value of 197 cmꢁ1 that is
indicative of asymmetrically monodentate binding mode of the
tolfenamato ligands [24,30].
1H NMR spectroscopy can be considered a valuable tool to study
the behavior of complexes in solution. The recorded 1H NMR
spectra of compounds 1e6 in DMSO-d6 solution confirm the
formulae and the purity of the prepared compounds as well as the
integrity of each complex in solution. More specifically, in the 1H
NMR spectra of complexes 1e6, the absence of the signal at
13.10 ppm assigned to carboxylic hydrogen of the free tolfenamic
acid [32] confirms the deprotonated mode of the bound drug, while
the rest signals of the tolfenamato ligand are slightly shifted
downfield or upfield as expected upon binding to zinc metal ion
[12,23,33,34]. Furthermore, all sets of signals related to the pres-
ence of the N-donor ligands are present and the ratios of integrated
peaks confirm the ratio of ligands in the solid state, e.g. represen-
tatively for 1, 2 and 4 in Fig. 1: (i) four signals for bipy or phen li-
gands and (ii) seven signals for Hpko ligands; six of them are
attributed to aromatic hydrogen atoms and the seventh signal at
12.21 ppm is assigned to the oxime hydroxyl proton confirming
that the Hpko ligand is bound in its non-deprotonated state. The
absence of any additional sets of signals related to dissociated li-
gands is in agreement with the molecular conductance measure-
ments and suggests that all complexes remain intact in solution
[35e37].
The UVevis spectra of complex 1 were recorded as nujol mull
and in DMSO solution and were similar suggesting that it retains its
structure in solution. Additionally, in order to explore the stability
Scheme 1. Tolfenamic acid (¼Htolf) with H atom numbering in accordance to 1H NMR
proton’s assignment.

A. Tarushi et al. / European Journal of Medicinal Chemistry 74 (2014) 187e198
189
370e390 nm, they exhibit an intense emission band with lem,max
lying in the region 438e445 nm and a second band (as a peak or as
a shoulder) of lower intensity at 483e488 nm. An additional pair of
excitation and emission bands at 330 nm and 395 nm, respectively,
has been observed for complex 3 (Table S1).
The fact that the complexes are non-electrolytes in DMSO so-
lution (L ꢂ 15
mS/cm, in 1 mM DMSO solution), they have the
M
same UVeVis spectral pattern in nujol and in DMSO solution, as
well as in buffer solution, and all 1H NMR spectra confirm that the
complexes do not dissociate, suggests that the compounds are
stable and keep their integrity in solution. We have reported earlier
that Co(II) tolfenamato complexes [30] as well as the Zn(II) mefe-
namato complexes [12] are also stable in solution.
2.2. Structure of cis, cis, trans-[Zn(tolf-O)2(Hpko-N,N0)2]$CH3OH,
1$CH3OH
A drawing of the molecular structure of [Zn(tolf-O)2(Hpko-
N,N0)2], 1 is shown in Fig. 3 and selected bond distances and angles
are listed in Table 1.
In complex 1, the zinc atom is six-coordinate surrounded by two
tolfenamato ligands and two Hpko showing a distorted octahedral
environment with a ZnN4O2 metal center. The bond distances
around Zn atom are not equal (Table 1) with ZneO bond distances
ꢀ
ꢀ
(Zn(1)eO(8) ¼ 2.0384(14) A and Zn(1)eO(9) ¼ 2.0768(13) A) being
ꢀ
shorter than the ZneN bond distances (Zn(1)eN(10) ¼ 2.1748(17) A,
ꢀ
ꢀ
Zn(1)eN(14) ¼ 2.1602(16) A, Zn(1)eN(15) ¼ 2.1777(16) A and
ꢀ
Zn(1)eN(17) ¼ 2.1316(17) A). These distances are consistent with
those of previously reported structurally related zinc complexes
with fenamic acid NSAIDs and bidentate nitrogen ligands,
[Zn(tolf)2(bipy)], [Zn(tolf)2(phen)] [24], [Zn(mefenamato)2(bipy)],
[Zn(mefenamato)2(bipyam)], [Zn(mefenamato)2(phen)(H2O)] [12],
[Zn(bipy)2(ONO)](NO2) [38], [Zn(phen)2(MeCO2)](ClO4) [39], and
[Zn(pipdtc)2(bipy)] (pipdtcꢁ ¼ piperidineecarbodithioato anion)
[40] and is isostructural with its mefenamato analogue cis,cis,trans-
[Zn(mefenamato-O)2(Hpko-N,N0)2]$EtOH recently published by our
group [12].
The tolfenamato ligands behave as deprotonated monodentate
ligands coordinated to zinc ion via a carboxylato-oxygen atom. The
carboxylate groups of the tolfenamato ligands are monodentately
ꢀ
bound to zinc in asymmetric fashion (C(56)eO(6) ¼ 1.265(2) A and
ꢀ
ꢀ
C(56)eO(9) ¼ 1.273(2) A, C(52)eO(7) ¼ 1.260(2) A and C(52)e
ꢀ
O(8) ¼ 1.274(2) A). Weak intraligand interactions through hydrogen
bonds between the amine hydrogen atoms and the coordinated
ꢀ
ꢀ
oxygen atoms (H(12)/O(8) ¼ 1.961 A and H(13)/O(9) ¼ 2.016 A)
contribute to the stabilization of the complex (Table S2).
The Hpko ligands act as bidentate neutral chelators and are
coordinated to zinc via one pyridine nitrogen and the oxime ni-
trogen with the ketonoxime oxygen remaining unbound. This co-
ordination mode of Hpko ligand (1.0110 according to the
Harris notation [41]) has been observed in a series of mononuclear
metal complexes [42], [Zn(mefenamato)2(Hpko)2] [12] and in
the trinuclear Ni(II) complex [Ni3(shi)2(Hpko)2(py)2] (where
H3shi ¼ salicylhydroxamic acid and py ¼ pyridine) [43]. The
ketoxime groups also form hydrogen bonds with the uncoordi-
nated carboxylic oxygens of the adjacent tolfenamato ligands
Fig. 1. 1H NMR spectra of (A) 1, (B) 2 and (A) 4 in DMSO-d6.
of the complex in buffer solution, the UVeVis spectra in the series
of pH (pH range 6e8, since the biological experiments are per-
formed at pH ¼ 7) with the use of diverse buffer solutions (150 mM
NaCl and 15 mM trisodium citrate at pH values regulated by HCl
solution) have also been recorded. No significant changes (shift of
the lmax or new peaks) have been observed indicating that the
complex keeps its integrity in the pH range 6e8. The same behavior
in solution has been reported for complexes 2e6 [24].
ꢀ
ꢀ
(H(5)/O(6) 1.706 A and H(4)/O(7) 1.709 A; Table S2). The lattice is
further stabilized by the formation of a hydrogen bond between the
solvate methanol molecule and one of the uncoordinated pyridine
nitrogen atoms of a dipyridylketoxime ligand (N(18)).
The fluorescence emission spectra of the complexes have been
recorded in DMSO solution. As an example, the fluorescence exci-
tation and emission spectra for complexes 1 and 4 are shown
in Fig. 2. When all complexes are excited in the range of lex,max
2.3. Antioxidant capacity of the compounds
It is known that free radicals play an important role in the in-
flammatory process. Compounds with antioxidant properties could

190
A. Tarushi et al. / European Journal of Medicinal Chemistry 74 (2014) 187e198
4000
2000
1500
1000
500
0
(A)
(B)
excitation
emission
excitation
emission
3000
2000
1000
0
350
400
450
500
550
600
650
350
400
450
500
550
600
650
λ
(nm)
λ
(nm)
Fig. 2. Fluorescence spectra (emission in red, excitation in black) of complex (A) 1 (lex ¼ 390 nm, lem ¼ 438 nm) and (B) 4 (lex ¼ 384 nm, lem ¼ 438 and 488 nm) in DMSO solution.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
potentially have a crucial role against inflammation and, thus, lead
to potentially effective drugs. NSAIDs are drugs capable to act either
as inhibitors of free radical production or as radical scavengers [44]
and the antioxidant activity shown by NSAIDs and their complexes
could be considered as first evidence of a potential anticancer and
anti-inflammatory activity. Therefore, the potential antioxidant
ability of complexes 1e6 has been evaluated in regard to DPPH,
ABTS and hydroxyl radical scavenging ability as well as the in vitro
soybean lipoxygenase inhibition and has been compared to that of
well-known antioxidant agents (e.g. nordihydroguaiaretic acid
(NDGA), butylated hydroxytoluene (BHT), 6-hydroxy-2,5,7,8-
tetramethylchromane-2-carboxylic acid (trolox) and caffeic acid)
used as reference compounds.
Compounds that can scavenge DPPH radicals also present anti-
ageing, anticancer and anti-inflammatory activity [45]. Therefore,
the compounds exhibiting DPPH radical scavenging activity are of
increasing interest because of their potential protection against
rheumatoid arthritis and inflammation which may lead to poten-
tially effective drugs. DPPH is a stable free radical presenting a
strong absorption band at 517 nm, which decreases, when antiox-
idants donate protons to DPPH radical. The scavenging activity of
almost all complexes with exception of 5 and 6 was time-
independent since no significant changes were observed after 20-
min and 60-min measurements (Table S3). The complexes pre-
sent low to moderate reducing ability of DPPH radical compared to
the reference compound NDGA while their DPPH scavenging ac-
tivity is similar to BHT. All complexes but 3 show higher DPPH
radical scavenging activity than free tolfenamic acid with
[Zn(tolf)2(phen)] being the best DPPH scavenger (Fig. 4(A)) among
complexes 1e6. The complexes present similar scavenging activity
of the DPPH radical to that found for a series of zinc(II), copper(II)
and cobalt(II) complexes with mefenamic acid as ligand [12,46,47].
Hydroxyl radicals are among the most reactive oxygen species.
Compounds capable to act as hydroxyl radical scavengers may also
serve as protectors activating the prostaglandin synthesis since the
superoxide anion radicals are generated by phagocytes at the
inflamed site during the inflammatory process and are connected
to other oxidizing species such as ꢀOH [46]. Complexes 1e6 exhibit
higher competition with DMSO (33 mM) at 0.1 mM for hydroxyl
free radicals than free tolfenamic acid and the reference compound
trolox (Table S4) with complex 6 being the most active compound
(ꢀOH% ¼ 96.40(ꢃ0.57) %) and complexes 1 and 3 presenting similar
hydroxyl scavenging ability (Fig. 4(B)). The hydroxyl radical scav-
enging actility of complexes 1e6 is of the same magnitude to that
found for zinc(II), copper(II), and cobalt(II) mefenamato complexes
[12,46,47].
The ability of the compounds to scavenge the cationic radical of
ABTS (ABTSþꢀ) is related to the magnitude of the total antioxidant
activity of the compounds [46]. Complexes 1e6 show significant
ABTS radical scavenging activity which is higher than free Htolf, but
lower than that of the reference compound trolox except complex 4
which is the most active ABTS scavenger among the complexes
(ABTS% ¼ 93.62(ꢃ0.75)%) and even more active than trolox
(Table S4). The results are in agreement to DPPH and hydroxyl
Table 1
Selected bond distances and angles for complex 1.
ꢀ
ꢀ
Bond
Distance (A)
Bond
Distance (A)
Zn(1)eO(8)
Zn(1)eO(9)
Zn(1)eN(10)
O(6)eC(56)
O(9)eC(56)
O(4)eN(14)
2.0384(14)
2.0768(13)
2.1748(17)
1.265(2)
1.273(2)
1.367(2)
Zn(1)eN(14)
Zn(1)eN(17)
Zn(1)eN(15)
O(7)eC(52)
O(8)eC(52)
O(5)eN(15)
2.1602(16)
2.1316(17)
2.1777(16)
1.260(2)
1.274(2)
1.370(2)
Bond angle
(ꢄ)
Bond angle
(ꢄ)
O(8)eZn(1)eO(9)
O(8)eZn(1)eN(10)
O(8)eZn(1)eN(14)
O(8)eZn(1)eN(15)
O(8)eZn(1)eN(17)
N(10)eZn(1)eN(14)
N(10)eZn(1)eN(15)
N(10)eZn(1)eN(17)
85.57(6)
89.84(6)
97.38(6)
95.23(6)
171.05(6)
95.62(6)
74.88(6)
96.47(6)
O(9)eZn(1)eN(10)
O(9)eZn(1)eN(14)
O(9)eZn(1)eN(15)
O(9)eZn(1)eN(17)
N(14)eZn(1)eN(15)
N(14)eZn(1)eN(17)
N(15)eZn(1)eN(17)
169.76(6)
94.04(6)
96.41(6)
89.17(6)
164.19(6)
75.75(6)
92.54(6)
Fig. 3. A drawing of the molecular structure of 1 with only the heteroatoms labeling.

A. Tarushi et al. / European Journal of Medicinal Chemistry 74 (2014) 187e198
191
100
80
60
40
20
0
(A)
100
(B)
80
60
40
20
0
Htolf
1
2
3
4
5
6
NDGABHT
Htolf
1
2
3
4
5
6
Trolox
100
80
60
40
20
0
600
550
(C)
(D)
50
40
30
20
10
0
Htolf
1
2
3
4
5
6
Trolox
Htolf
1
2
3
4
5
6Caffeic acid
Fig. 4. (A) Interaction % with DPPH (RA%), (B) competition% with DMSO for hydroxyl radical (ꢀOH%), (C) % superoxide radical scavenging activity (ABTS%) and (D) in vitro inhibition of
soybean lipoxygenase (LOX) (IC50, in M) for Htolf and complexes 1e6.
m
radical scavenging studies with the complexes being more active
scavengers than free Htolf (Fig. 4(C)(C)). Similar scavenging activity
of the ABTS radical has been recorded for Zn(II), Cu(II), and Co(II)
mefenamato complexes [12,46,47].
hydroxyl and ABTS radicals. In the literature, there are examples of
ꢀ
complexes being more active against DPPH radicals than OH or
ABTSþ [55,56] and others with zinc complexes among them
ꢀ
exhibiting much more significant hydroxyl and ABTS scavenging
activity than DPPH [50e54,57,58]. The zincetolfenamato com-
plexes 1e6 exhibit similar or better free radical scavenging activity
(RA% ¼ 12.5e36.7%, ꢀOH% ¼ 85.4e96.4%, ABTS% ¼ 66.5e93.6%,
Lipoxygenases (LOXs) are a family of non-heme iron-containing
dioxygenases and are the key enzymes of the transformation of
arachidonic acid to leukotrienes, compounds playing an important
role in the pathophysiology of several inflammatory and allergic
diseases. The products of the oxygenation catalysed by LOXs are
also involved in the development of rheumatoid arthritis, psoriasis
and asthmatic responses [12]. Most LOX inhibitors could be
considered as potential antioxidants or free radical scavengers
since lipoxygenation occurs via a carbon centered radical [48]. The
“non-heme” iron per molecule of LOXs in the enzyme active site is a
high-spin Fe(III) in the activated state and a high-spin Fe(II) in the
inactive state. The ability of LOX inhibitors, which are considered
excellent ligands for Fe(III), to reduce the Fe(III) at the active site to
the catalytically inactive Fe(II) has been closely related to LOX in-
hibition [49].All complexes present significant inhibition against
soybean lipoxygenase (Table S4), especially in relation to the
LOX ¼ 8.7e32.7
mM) when compared to that of the corresponding
zincemefenamato complexes (RA% ¼ 16.7e41.6%, ꢀOH% ¼ 83e
98.5%, ABTS% ¼ 74e94.5%, LOX ¼ 22.9e51.9
mM) recently reported
[12]. Furthermore, complexes 1e6 can be considered significantly
active when compared to other Zn complexes reported in the
literature [56,57].
2.4. Interaction with serum albumins
Serum albumins (SAs) are the most abundant proteins in plasma
and have the vital role to transport metal ions, drugs and their
metal complexes through the blood stream [31]. It is important to
examine the interaction of potential drugs with SA since differen-
tiated biological properties of the drug or novel transport pathways
may arise. The solutions of human serum albumin (HSA, bearing
one tryptophan, Trp-214) and its extensively studied homologue
bovine serum albumin (BSA, with two tryptophans, Trp-134 and
Trp-212) exhibit an intense fluorescence emission when excited at
295 nm, with lem,max ¼ 351 nm and 343 nm, respectively [59].
Complex 1 in buffer solution exhibits a maximum emission at
w330 nm under the same experimental conditions (as also previ-
ously reported the Co(II)-tolfenamato and Zn(II)-tolfenamato
complexes 2e6) [24,30] and the SA fluorescence spectra have
been corrected before the experimental data processing.
reference compound caffeic acid (IC50 ¼ 600
mM). The complexes
present slightly better LOX inhibition activity than free Htolf and 4
is the most active compound against LOX (IC50 ¼ 8.71(ꢃ0.76)
mM)
(Fig. 4(D)).
In conclusion, the complexes exhibit higher radical scavenging
and LOX inhibitory activity than free tolfenamic acid with complex
4 considered the most active scavenger among the complexes
examined. A series of Cu(II), Co(II), Ni(II), Zn(II), and Pd(II) com-
plexes with diverse ligands exhibiting enhanced antioxidant ac-
tivity in comparison to corresponding free ligands have been found
in the literature [12,45,46,50e54]. In general all complexes present
low to moderate activity against DPPH and high activity against
hydroxyl and ABTS radicals, a fact that could be considered evi-
dence of selective scavenging activity of the complexes against
The quenching observed in the fluorescence emission spectra of
SA solutions upon addition of complex 1 (Fig. 5) may be attributed
to changes in protein conformation, subunit association, substrate

192
A. Tarushi et al. / European Journal of Medicinal Chemistry 74 (2014) 187e198
100
80
100
80
60
40
20
0
1400
(A)
(B)
2500
2000
1500
1000
500
60
1200
40
20
1000
0
800
600
400
200
0
0
2
r = [
4
6
0
2
4
6
1
]/[BSA]
r = [
1
]/[HSA]
0
300 320 340 360 380 400 420 440
300 320 340 360 380 400 420 440
λ
(nm)
λ
(nm)
Fig. 5. Emission spectra (lexit ¼ 295 nm) for (A) BSA ([BSA] ¼ 3
m
M) and (B) HSA ([HSA] ¼ 3
mM) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the
absence and presence of increasing amounts of complex 1 (up to the value of r ¼ 7.3). The arrow shows the changes of intensity upon increasing amounts of 1. Insets: Plot of %
relative fluorescence intensity at (A) lem ¼ 342 nm (%) vs r (r ¼ [complex]/[BSA]) for 1 (1% of the initial fluorescence intensity) and (B) lem ¼ 351 nm (%) vs r (r ¼ [complex]/[HSA])
for 1 (15% of the initial fluorescence intensity).
binding or denaturation [60]. The fluorescence quenching provoked
by complex 1 to the BSA fluorescence signal at 342 nm as well as to
the HSA fluorescence maximum at 351 nm is significant (Insets in
Fig. 5) and is due to possible changes in protein secondary structure
leading to changes in tryptophan environment of SA, and thus
indicating the binding of the complex to SA [61].
among Zn(II)-tolfenamato complexes 1e6 [24]. Comparing the af-
finity of complex 1 for BSA and HSA (K values), it is obvious that it
shows higher affinity for BSA than HSA.
In general, the binding constant of a compound to a protein such
as serum albumin should be high enough to allow its binding and
possible transfer but also not too high so that it can get released
upon arrival at its target. The K values of all complexes 1e6 are
within such an optimum range; high enough (6.20 ꢅ 104e
The SterneVolmer and Scatchard graphs may be used in order to
study the interaction of a quencher with serum albumins. The
values (Table 2) of the quenching constant, kq (in Mꢁ1
s
ꢁ1), as
2.40 ꢅ 106
M
ꢁ1) to allow the binding of the complexes to SAs and
calculated by the SterneVolmer equation (Eq. S(1)) [60] and plots
also quite below the value of K z 1015 ꢁ1, which is the association
M
(data not shown), are higher (>1013
M
ꢁ1 sꢁ1) than diverse kinds of
constant of the one of strongest known non-covalent bonds for the
interaction between avidin and ligands, suggesting a possible
release from the serum albumin to the target cells [61].
In relation to previously reported Co(II) tolfenamato complexes
[30], the quenching constants (kq) and binding constants (K) for
complexes 1e6 are more pronounced than for their Co(II) ana-
logues for both albumins.
quenchers for biopolymers fluorescence (2 ꢅ 1010 Mꢁ1
s
ꢁ1) indi-
cating the existence of static quenching mechanism [60]. These
values indicate good SA quenching ability and the kq values of the
complex are higher than the corresponding values of free Htolf.
Furthermore, complex 1 exhibits the highest quenching ability for
BSA (kq ¼ 7.07(ꢃ0.07) ꢅ 1014 Mꢁ1
complexes 1e6 [24].
s
ꢁ1) among Zn-tolfenamato
The values of the association binding constant, K (in Mꢁ1), and
the number of binding sites per albumin, n, for complex 1 have
been obtained from the corresponding Scatchard equation (Eq.
S(3)) [60] and plots (data not shown) and are cited in Table 2. It is
obvious that complex 1 exhibits higher K and n values than free
Htolf; indeed, complex 1 presents the highest SA binding constants
2.5. Interaction with calf-thymus DNA
Metal complexes can bind to double-stranded DNA via covalent
and/or noncovalent interactions; upon covalent binding, the
replacement of a labile ligand of the complex by a nitrogen base of
DNA occurs, while the noncovalent DNA interactions include
intercalative (via
p / p* stacking interaction of the complex and
DNA nucleobases), external electrostatic (as a result of Coulomb
forces of metal complexes and the phosphate groups of DNA) and
groove (through van der Waals interaction or hydrogen-bonding or
hydrophobic bonding along major or minor groove of DNA helix)
binding of metal complexes inside the DNA helix, along major or
minor groove [12,30,45,46,62]. The possibility of the existence of
anticancer and/or anti-inflammatory activity of the NSAIDs and
their complexes could be also related to their ability to bind to DNA
[19,20]; within this context, DNA-binding studies should be of
significant interest, although the number of such studies involving
NSAIDs and their complexes so far is rather limited; e.g. complexes
of oxicam NSAIDs bind to DNA via intercalation [20] and the
interaction of DNA with a series of zinc(II), cobalt(II) and copper(II)
complexes of NSAIDs complexes has been studied and reported
quite recently [12,21,30,45,46].
Table 2
The BSA and HSA binding constants and parameters (Ksv, kq, K, n) derived for the
Htolf and complexes 1e6.
Compound
kq (Mꢁ1 sꢁ1
)
K (Mꢁ1
)
n
BSA
Htolf [30]
2.18(ꢃ0.12) ꢅ 1013
7.07(ꢃ0.07) ꢅ 1014
9.99(ꢃ0.60) ꢅ 1013
1.15(ꢃ0.04) ꢅ 1013
1.00(ꢃ0.01) ꢅ 1014
3.67(ꢃ0.15) ꢅ 1013
1.87(ꢃ0.06) ꢅ 1014
1.60 ꢅ 105
2.40 ꢅ 106
5.70 ꢅ 104
1.81 ꢅ 105
1.88 ꢅ 105
6.20 ꢅ 104
2.07 ꢅ 105
1.11
1.01
2.09
0.84
1.31
1.70
1.44
[Zn(tolf)2(Hpko)2], 1
[Zn(tolf)(phen)Cl], 2 [24]
[Zn(tolf)(bipy)Cl], 3 [24]
[Zn(tolf)2(phen)], 4 [24]
[Zn(tolf)2(bipy)], 5 [24]
[Zn3(tolf)6(CH3OH)2], 6 [24]
HSA
Htolf [30]
0.61(ꢃ0.04) ꢅ 1013
1.81(ꢃ0.14) ꢅ 1013
1.41(ꢃ0.07) ꢅ 1013
0.86(ꢃ0.03) ꢅ 1013
4.27(ꢃ0.28) ꢅ 1013
2.84(ꢃ0.12) ꢅ 1013
1.39(ꢃ0.05) ꢅ 1013
3.12 ꢅ 105
4.74 ꢅ 105
4.37 ꢅ 105
1.43 ꢅ 105
1.36 ꢅ 105
1.49 ꢅ 105
4.12 ꢅ 105
0.63
0.79
0.65
0.86
1.20
1.16
0.79
[Zn(tolf)2(Hpko)2], 1
[Zn(tolf)(phen)Cl], 2 [24]
[Zn(tolf)(bipy)Cl], 3 [24]
[Zn(tolf)2(phen)], 4 [24]
[Zn(tolf)2(bipy)], 5 [24]
[Zn3(tolf)6(CH3OH)2], 6 [24]
2.5.1. Study of the DNA-binding with UV spectroscopy
The changes observed in the UV spectra upon titration may
provide evidence of the existing interaction mode, since
a

A. Tarushi et al. / European Journal of Medicinal Chemistry 74 (2014) 187e198
193
3
-0.2
-0.4
-0.6
-0.8
-1.0
-1.2
1.0
0.8
0.6
0.4
0.2
0.0
(A)1.2
(B)
2
1
0
1.0
0.8
0.6
0.4
0.2
0.0
1
2
3
4
5
0
2
4
6
8
[DNA]x10 , (M)
[DNA]x10 , (M)
280
300
320
340
360
380
400
280
300
320
340
360
380
400
λ
(nm)
λ
(nm)
Fig. 6. UV spectra of DMSO solution (1 ꢅ 10ꢁ5 M) of (A) [Zn(tolf)2(Hpko)2], 1 and (B) [Zn3(tolf)6(CH3OH)2], 6 in the presence of increasing amounts of CT DNA. The arrows show the
changes upon increasing amounts of CT DNA. Insets: plot of ½DNAꢆ=ð A ꢁ 3 Þ vs [DNA].
3
f
hypochromism due to
p
/
p
* stacking interactions may appear in
significant hypochromism of 50% (Fig. 6(A)) resulting in an elimi-
nation of the band. These predominant spectra features may sug-
gest tight binding to CT DNA probably by intercalation and
stabilization. In the UV spectrum of 6, the band at 297 nm (band I)
presents a hyperchromism of up to 15% accompanied by a red-shift
of 11 nm (up to 308 nm), suggesting tight binding and stabilization.
Additionally, the band centred at 346 nm (band II) exhibits a sig-
nificant hypochromism of 30% (Fig. 6(B)) suggesting tight binding
to CT DNA probably by intercalation. Further addition of DNA re-
sults in a blue-shift with a gradual elimination of this band. A
distinct isosbestic point at 334 nm appears upon addition of CT
DNA. The behavior of Htolf and complexes 2e5 upon addition of CT
DNA is quite similar to 6; the hypochromism of band I varies from
15% for 2 up to 60% for Htolf and the observed hyperchromism of
band II is between 9% for 5 and 40% for Htolf (Table 3).
In any case, the exact mode of DNA-binding cannot be merely
concluded by UV spectroscopic titration studies and the results
collected from the UV titration experiments suggest that all com-
pounds can bind to CT DNA. The observed hypochromic effect for all
complexes could be considered evidence of tight binding to CT DNA
probably by intercalation; nevertheless, further experiments are
necessary to carry out in order to clarify the binding mode and
possibly verify the existence of intercalation [65].
the case of the intercalative binding mode, while red-shift (bath-
ochromism) is an evidence of stabilization of the DNA duplex [63].
The UV spectra of a CT DNA solution have been recorded for a
constant CT DNA concentration (1.4 ꢅ 10ꢁ4e1.6 ꢅ 10ꢁ4 M) in
different [complex]/[DNA] mixing ratios (r) (complex ¼ 1e6) (up to
r ¼ 0.3). UV spectra of CT DNA in the presence of complex 6 derived
for diverse r values are shown as an example in Fig. S1. The pre-
dominant features observed upon the addition of the complexes is
the decrease of the intensity at lmax ¼ 258 nm and a red-shift of the
lmax up to 266 nm for all complexes, indicating that the interaction
with CT DNA results in the direct formation of a new complex with
double-helical CT DNA [64]. The observed hypochromism may be
attributed to
p / p* stacking interaction between the aromatic
chromophore (either from tolfenamato and/or the N-donor li-
gands) 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 [12,30,45,46,62].
In the UV spectra of the complexes, the intense absorption
bands observed are attributed to the intraligand transitions of the
coordinated groups of tolfenamato ligands. Any interaction be-
tween each complex and CT DNA could perturb the intra-ligand
transitions during the titration upon addition of CT DNA in
diverse r values. In general, the changes observed in the UV spectra
upon titration may give evidence of the existing interaction mode,
The magnitude of the binding strength of complexes with CT
DNA may be estimated through the binding constant Kb, which can
be obtained by the WolfeeShimer equation (Eq. S(4)) [66] and plots
since a hypochromism due to
p / p* stacking interactions may
appear in the case of the intercalative binding mode, while red-shift
(bathochromism) may be observed when the DNA duplex is
stabilized.
In the UV spectra of 1, the band centred at 290 nm (band I)
presents a hypochromism of 20% accompanied by a red-shift of
16 nm (up to 306 nm) and the band at 341 nm (band II) exhibits a
½DNAꢆ=ð
3
A ꢁ f Þ versus [DNA] (e.g. insets in Fig. 6). The Kb values
3
calculated for Htolf and complexes 1e6 (Table 3) are relatively high
and in most cases are higher than that of free tolfenamic acid
suggesting that its coordination to Zn(II) results in a increase of
the Kb value. The Kb values suggest a strong binding of the com-
plexes to CT DNA, with complex 1 having the highest Kb value
Table 3
The DNA binding constants (Kb) of Htolf and complexes 1e6.
Compound
Band I (290e297 nm)
Band II (346 nm)
Kb (Mꢁ1
)
% of hyperchromism or hypochromism
% of hypochromism
Htolf [30]
Hyperchromism, 40%
Hypochromism, 20%
Hyperchromism, 20%
Hyperchromism, 10%
Hyperchromism, 10%
Hyperchromism, 9%
Hyperchromism, 15%
60%
50%
15%
30%
18%
25%
30%
5.00(ꢃ0.10) ꢅ 104
2.80(ꢃ0.18) ꢅ 105
9.30(ꢃ0.20) ꢅ 104
3.56(ꢃ0.11) ꢅ 104
1.77(ꢃ0.09) ꢅ 105
2.36(ꢃ0.11) ꢅ 104
5.26(ꢃ0.13) ꢅ 104
[Zn(tolf)2(Hpko)2], 1
[Zn(tolf)(phen)Cl], 2
[Zn(tolf)(bipy)Cl], 3
[Zn(tolf)2(phen)], 4
[Zn(tolf)2(bipy)], 5
[Zn3(tolf)6(CH3OH)2], 6

194
A. Tarushi et al. / European Journal of Medicinal Chemistry 74 (2014) 187e198
Viscosity measurements were carried out on CT DNA solutions
1.4
1.3
1.2
1.1
1.0
Htolf
(0.1 mM) upon addition of increasing amounts of Htolf or com-
plexes 1e6 (up to the value of r ¼ 0.35). The addition of the com-
pounds results in a moderate or significant (for 1 and 5) increase in
the relative viscosity of DNA (Fig. 7). This may be explained by the
insertion of the complexes 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. In conclusion, the
observed increase of the DNA viscosity in the presence of increasing
amounts of the complexes may be considered as an evidence of the
existence of an intercalative binding mode to DNA; a conclusion
which completely enforces the preliminary findings from UV
spectroscopic studies.
1
2
3
4
5
6
0.0
0.1
0.2
0.3
0.4
r = [Compound]/[DNA]
2.5.3. Competitive study with ethidium bromide
The intercalation of ethidium bromide (EB ¼ 3,8-diamino-5-
ethyl-6-phenyl-phenanthridinium bromide) to CT DNA occurs
via the insertion of the planar EB phenanthridine ring in between
adjacent base pairs on the DNA double helix. This intercalation
results in the appearance of intense fluorescence emission which
is due to the formation of the EB-DNA complex. Thus, EB is
considered a typical indicator of intercalation since upon the
addition of a compound, capable to act as a DNA-intercalator
equally or more strongly than EB, into a solution of the EB-DNA
compound, a quenching of the DNA-induced EB fluorescence
emission may appear [69]. Htolf and complexes 1e6 do not show
any significant fluorescence at room temperature in solution or in
the presence of CT DNA, when excited at 540 nm. Furthermore, the
addition of the compounds to a solution containing EB does not
provoke quenching of free EB fluorescence and new peaks do not
appear in the spectra. Within this context, the changes observed in
the fluorescence emission spectra of a solution containing the EB-
DNA compound upon addition of the complexes can be used to
study the ability of the compounds to displace EB from the EB-
DNA complex. The emission spectra of pre-treated EB-CT DNA in
the absence and presence of each complex have been recorded for
1/3
Fig. 7. Relative viscosity of CT DNA (
h/
ho)
in buffer solution (150 mM NaCl and
15 mM trisodium citrate at pH 7.0) in the presence of Htolf and complexes 1e6 at
increasing amounts vs r (r ¼ [Compound]/[DNA]).
(¼2.80(ꢃ0.18) ꢅ 105 M-1) among the compounds, and close to that
of the classical intercalator EB (¼1.23(ꢃ0.07) ꢅ 105
M
ꢁ1) as calcu-
lated in Ref. [67]. The Kb values of complexes 1e6 are slightly lower
than the corresponding Co(II)-tolfenamato [30] and Zn(II)-
mefenamato [12] complexes.
2.5.2. DNA-binding study with viscosity measurements
The information derived by the measurement of the viscosity of
DNA solution upon addition of a compound is useful in an attempt
to clarify the interaction mode of a compound with DNA, since it is
sensitive to DNA length changes; the relation between relative
solution viscosity (
h
/
h0) and DNA length (L/L0) is given by the
equation L/L0 ¼ ( h0
h/ )
1/3, where L0 denotes the apparent molecular
length in the absence of the compound [12,21,30,45,46,62]. When a
compound binds to DNA grooves via a partial or non-classic inter-
calation (i.e. electrostatic interaction or external groove-binding), a
bend or kink in the DNA helix may be provoked resulting in a slight
shortening of the effective length of DNA; in such a case, the vis-
cosity of DNA solution shows a slight decrease or remains un-
changed [12,21,30,45,46,68] In the case of classic intercalation, the
insertion of the compound in between the DNA base pairs with a
subsequent increase in the separation of base pairs at intercalation
sites and subsequently its hosting results in an increase of the
length of the DNA helix leading to an increase of DNA viscosity, the
magnitude of which is in accordance to the strength of the
interaction.
[EB] ¼ 20
m
M and [DNA] ¼ 26
mM for increasing amounts of the
complex up to the value of r ¼ 0.05 (Fig. 8(A)). The addition of
complexes 1e6 at diverse r values results in a significant decrease
of the intensity of the emission band of the DNA-EB system at
592 nm (the final fluorescence is up to 18e32% of the initial EB-
DNA fluorescence intensity in the presence of the complexes)
indicating the competition of the complexes with EB in binding
to DNA (Fig. 8(B)). The observed quenching of DNA-EB fluores-
cence by the complexes suggests that the complexes have signif-
icant ability to displace EB from the DNA-EB compound, thus
(B)
100
80
60
40
20
0
(A)
7000
Htolf
6000
5000
4000
3000
2000
1000
1
2
3
4
5
6
0
0.00
0.01
0.02
0.03
0.04
0.05
560 580 600 620 640 660 680 700 720
r = [Compound] / [DNA]
λ
(nm)
Fig. 8. (A) Fluorescence emission spectra (lexit ¼ 540 nm) for EB-DNA ([EB] ¼ 20
m
M, [DNA] ¼ 26
mM) in buffer solution in the absence and presence of increasing amounts of
complex 1 (up to the value of r ¼ 0.05). The arrow shows the changes of intensity upon increasing amounts of 1. (B) Plot of EB relative fluorescence emission intensity at
lem ¼ 592 nm (%) vs r (r ¼ [complex]/[DNA]) in the presence of Htolf and complexes 1e6 (up to 26% of the initial EB-DNA fluorescence intensity for Htolf, 19% for 1, 32% for 2, 31% for
3, 29% for 4, 26% for 5 and 32% for 6).

A. Tarushi et al. / European Journal of Medicinal Chemistry 74 (2014) 187e198
195
Table 4
the complexes to displace the typical intercalator EB from the EB-CT
DNA complex indicating intercalation as a possible mode of their
interaction with CT DNA. The intercalative binding mode has been
also confirmed by viscosity measurements of CT DNA solutions in
the presence of the complexes.
Percentage of EB-DNA fluorescence emission quenching (
constants (KSV) for Htolf and complexes 1e6.
D
I/Io, %) and SterneVolmer
Compound
EB-DNA fluorescence
quenching (%)
Ksv (Mꢁ1
)
Htolf [30]
74
81
68
69
71
74
68
1.15(ꢃ0.04) ꢅ 106
1.15(ꢃ0.06) ꢅ 106
5.04(ꢃ0.18) ꢅ 105
1.11(ꢃ0.05) ꢅ 106
1.04(ꢃ0.02) ꢅ 106
1.38(ꢃ0.05) ꢅ 106
8.44(ꢃ0.22) ꢅ 105
The reported albumin binding studies is a first attempt to
evaluate the ability of the complexes to bind to serum proteins and
such studies could be expanded to more serum proteins. The results
of the antioxidant activity of the complexes are promising since the
complexes are more active than the reference compounds such as
trolox and caffeic acid and the ability of the reported compounds to
act as DNA intercalators may open a new window for their use as
potential metallodrugs.
[Zn(tolf)2(Hpko)2], 1
[Zn(tolf)(phen)Cl], 2
[Zn(tolf)(bipy)Cl], 3
[Zn(tolf)2(phen)], 4
[Zn(tolf)2(bipy)], 5
[Zn3(tolf)6(CH3OH)2], 6
subsequently interacting with CT DNA by the intercalative mode
[12,21,30,45,46,62].
4. Experimental
The SterneVolmer constant, KSV, is used to evaluate the
quenching efficiency for each compound according to the Sterne
Volmer equation (Eq. S(5)) and is obtained from the diagram Io=I vs
[Q]. The SterneVolmer plots of DNA-EB for the compounds (e.g. for
complex 1 in Fig. S2) illustrate that the quenching of EB bound to
DNA by the compounds is in good agreement (R ¼ 0.99) with the
linear SterneVolmer equation (Eq. (S5)), which proves that the
replacement of EB bound to DNA by each compound results in a
decrease in the fluorescence intensity [32,33,46,47,50]. The ob-
tained values of KSV (Table 4) are high and show that the complexes
can bind tightly to DNA with complex 5 exhibiting the highest Ksv
4.1. Materials and instrumentation
Tolfenamic acid, ZnCl2, Hpko, KOH, trisodium citrate, NaCl, CT
DNA, BSA, HSA and EB were purchased from SigmaeAldrich Co and
all solvents were purchased from Merck. All the chemicals and
solvents were reagent grade and were used as purchased.
DNA stock solution was prepared by dilution of CT DNA to buffer
(containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0)
followed by exhaustive stirring at 4 ꢄC for three days, and kept at
4 ꢄC 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 1.85,
indicating that the DNA was sufficiently free of protein contami-
nation. The DNA concentration was determined by the UV absor-
value (¼1.38(ꢃ0.05) ꢅ 106
M
ꢁ1) among the complexes. The Ksv
values of complexes 1e6 are lower than those of Zn(II)-
mefenamato complexes [12] and of the same magnitude to their
Co(II)-tolfenamato analogues [30].
bance at 260 nm after 1:20 dilution using
3
¼ 6600 Mꢁ1 cmꢁ1
.
Infrared (IR) spectra (400e4000 cmꢁ1) were recorded on a
Nicolet FT-IR 6700 spectrometer with samples prepared as KBr
pellets. UVevisible (UVevis) spectra were recorded as nujol mulls
and in solution at concentrations in the range 10ꢁ5e10ꢁ3 M on a
3. Conclusions
The synthesis and characterization of the neutral zinc(II) com-
plex with the non-steroidal anti-inflammatory drug tolfenamic acid
in the presence of the nitrogen-donor heterocyclic ligand 2,20-
dipyridylketone oxime has been achieved. The crystal structure of
the complex [Zn(tolf)2(Hpko)2] has been determined by X-ray
crystallography revealing the coordination of the deprotonated
monodentate tolfenamato ligands to zinc atom via a carboxylato
oxygen and of neutral bidentate Hpko ligands via the ketoxime
nitrogen and a pyridyl nitrogen.
Complex 1 shows high quenching ability of the BSA and HSA
fluorescence and tight binding affinity to these proteins providing
relatively high binding constants which are in the optimum range
to allow the binding, the transfer and the release upon arrival at the
targets. Complex 1 exhibits higher SA-binding constants than a
series previously reported Zn(II)-tolfenamato complexes 2e6.
All Zn(II)-tolfenamato complexes 1e6 have been tested in vitro
for their free radical scavenging activity and inhibitory activity on
soybean lipoxygenase and were found to be more active than free
tolfenamic acid. All complexes exhibit low to moderate DPPH
radical scavenging activity and significantly high scavenging ac-
tivity against hydroxyl and superoxide radicals; they also present
significant inhibition against soybean lipoxygenase. Complex
[Zn(tolf)2(phen)], 4 is the most active scavenger of all radicals
tested and the most potent LOX inhibitor.
Hitachi U-2001 dual beam spectrophotometer. C, H and N
elemental analysis were performed on a PerkineElmer 240B
elemental analyzer. Molar conductivity measurements were car-
ried out in 1 mM DMSO solution of the complexes with a Crison
Basic 30 conductometer. Fluorescence spectra were recorded in
solution on a Hitachi F-7000 fluorescence spectrophotometer.
Viscosity experiments were carried out using an ALPHA L Fungilab
rotational viscometer equipped with an 18 mL LCP spindle.
4.2. Synthesis of complex [Zn(tolf-O)2(Hpko-N,N0)2]$MeOH, 1$MeOH
Tolfenamic acid (0.4 mmol, 104 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 simulta-
neously with a methanolic solution of Hpko (0.4 mmol, 80 mg), to a
methanolic solution (10 mL) of ZnCl2 (0.2 mmol, 27 mg) and was
stirred for 30 min. The solution was left for slow evaporation. Pale
yellow crystals of [Zn(tolf)2(Hpko)2]$MeOH, 4$MeOH (110 mg, 55%)
suitable for X-ray structure determination were deposited after two
weeks (found C, 59.87; H, 4.40; N, 10.85;
C51H44Cl2N8O7Zn
(MW ¼ 1017.21) requires C, 60.22; H, 4.36; N, 11.01%). IR: nmax
/
cmꢁ1
(KBr disk); UVevis:
;
nasym(CO2): 1584 (vs); nsym(CO2): 1387 (vs);
D
¼ 197 cmꢁ1
l/nm (
3
/Mꢁ1 cmꢁ1) as nujol mull: 342(sh),
297(sh); in DMSO: 345(sh) (1600), 302 (11,300). 1H NMR in DMSO-
UV spectroscopic titration studies and viscosity measurements
have revealed the ability of complexes 1e6 to bind to CT DNA. The
binding strength of the complexes with CT DNA calculated with UV
spectroscopic titrations have shown that 1 exhibits the highest Kb
d6 (d/ppm), (numbering of the ligands in Schemes 1 and S1): 2.21
(6H, s, H10-tolf), 6.72 (2H, t (J ¼ 7.5 Hz), H3-tolf), 6.92 (2H,
d (J ¼ 8.1 Hz), H5-tolf), 7.12 (4H, m (J ¼ 8.0 Hz), H8-and H9-tolf), 7.25
(4H, t (J ¼ 7.4 Hz), H4- and H7-tolf), 7.40 (4H, t (J ¼ 6.2 Hz), H4- and
0
0
(Kb ¼ 2.80(ꢃ0.18) ꢅ 105
M
ꢁ1) value among the complexes exam-
H4 -Hpko), 7.54 (2H,
d
(J
¼
7.5 Hz), H6 -Hpko), 7.79 (2H,
0
ined, which is of the same order to the Kb value of EB. Competitive
DNA-binding studies with EB have revealed a moderate ability of
d (J ¼ 7.0 Hz), H6-Hpko), 7.88 (4H, m (J ¼ 7.5 Hz), H5- and H5 -Hpko),
0
7.98 (2H, d (J ¼ 7.8 Hz), H2-tolf), 8.47 (2H, d (J ¼ 4.0 Hz), H3 -Hpko),

196
A. Tarushi et al. / European Journal of Medicinal Chemistry 74 (2014) 187e198
8.62 (2H, d (J ¼ 4.0 Hz), H3-Hpko), 10.20 (2H, s, H6-tolf), 12.21 (2H, s,
5. Biological assays
H1-Hpko). The complex is soluble in DMF and DMSO (LM ¼ 13
mS/
cm, 1 mM in DMSO).
5.1. Antioxidant biological assay
4.3. Synthesis of complexes 2e6
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.
The synthesis, the physicochemical and IR spectroscopic data for
complexes 2e6 have been thoroughly presented in Ref. [24]. Herein
the 1H NMR spectral features of the complexes are presented
(numbering of the ligands is presented in Schemes 1 and S1).
5.1.1. 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. Ethanol
was used as control solution. The concentration of the solution of
the compounds was 0.1 mM. The absorbance at 517 nm was
recorded at room temperature, after 20 and 60 min in order to
examine the time-dependence of the radical scavenging activity
[45,46]. The radical scavenging activity of the compounds was
expressed as the percentage reduction of the absorbance values of
the initial DPPH solution (RA%). NDGA and BHT were used as
reference compounds.
4.3.1. [Zn(tolf)(phen)Cl], 2
1H NMR in DMSO-d6 /ppm): 2.11 (3H, s, H10-tolf), 6.66 (1H, t
(d
(J ¼ 7.1 Hz), H3-tolf), 6.89 (1H, d (J ¼ 8.0 Hz), H5-tolf), 7.09 (2H, m
(J ¼ 7.5 Hz), H8- and H9-tolf), 7.20 (2H, m (J ¼ 6.5 Hz), H4- and H7-
tolf), 7.88 (1H, d (J ¼ 7.6 Hz), H2-tolf), 8.06 (2H, m (J ¼ 6.0 Hz), H3-
and H8-phen), 8.24 (2H, s, H5- and H6-phen), 8.84 (2H, d (J ¼ 8.1 Hz),
H4- and H7-phen), 9.15 (2H, s, H2- and H9-phen), 10.51 (1H, s, H6-
tolf).
4.3.2. [Zn(tolf)(bipy)Cl], 3
1H NMR in DMSO-d6 /ppm): 2.19 (3H, s, H10-tolf), 6.71 (1H, t
(d
(J ¼ 7.4 Hz), H3-tolf), 6.94 (1H, d (J ¼ 8.1 Hz), H5-tolf), 7.13 (2H, m
5.1.2. Competition of the tested compounds with DMSO for hydroxyl
radicals
(J ¼ 7.1 Hz), H8- and H9-tolf), 7.26 (2H, m (J ¼ 7.2 Hz), H4- and H7-
0
tolf), 7.63 (2H, t (J ¼ 6.0 Hz), H4- and H4 -bipy), 7.904 (1H,
d (J ¼ 6.7 Hz), H2-tolf), 8.14 (2H, t 0(J ¼ 7.0 Hz), H6- and H6 -bipy),
8.54 (2H, d0 (J ¼ 7.8 Hz), H5- and H5 -bipy), 8.80 (2H, d (J ¼ 4.5 Hz),
H3- and H3 -bipy), 10.36 (1H, s, H6-tolf).
The hydroxyl radicals generated by the Fe3þ/ascorbic acid sys-
tem, were detected according to Nash [45,46], by the determination
of formaldehyde produced from the oxidation of DMSO. The reac-
tion mixture contained EDTA (0.1 mM), Fe3þ (167
mM), DMSO
(33 mM) in phosphate buffer (50 mM, pH 7.4), the tested com-
pounds (0.1 mM) and ascorbic acid (10 mM). After 30 min of in-
cubation (37 ꢄC) the reaction was stopped with CCl3COOH (17% w/
4.3.3. [Zn(tolf)2(phen)], 4
1H NMR in DMSO-d6 /ppm): 2.11 (6H, s, H10-tolf), 6.66 (2H, t
(d
(J ¼ 7.2 Hz), H3-tolf), 6.89 (2H, d (J ¼ 8.1 Hz), H5-tolf), 7.08 (4H, m
(J ¼ 6.8 Hz), H8- and H9-tolf), 7.20 (4H, m (J ¼ 8.0 Hz), H4- and H7-
tolf), 7.88 (2H, d (J ¼ 7.1 Hz), H2-tolf), 8.06 (2H, m (J ¼ 6.0 Hz), H3-
and H8-phen), 8.24 (2H, s, H5- and H6-phen), 8.85 (2H, d (J ¼ 7.2 Hz),
H4- and H7-phen), 9.15 (2H, s, H2- and H9-phen), 10.45 (2H, s, H6-
tolf).
v) and the absorbance at
l
¼ 412 nm was measured. Trolox was
used as an appropriate standard. The competition of the com-
pounds with DMSO for OH, generated by the Fe3þ/ascorbic acid
ꢀ
system, expressed as percent inhibition of formaldehyde produc-
tion, was used for the evaluation of their hydroxyl radical scav-
enging activity (ꢀOH%).
4.3.4. [Zn(tolf)2(bipy)], 5
5.1.3. Assay of radical cation scavenging activity
1H NMR in DMSO-d6 /ppm): 2.19 (6H, s, H10-tolf), 6.71 (2H, t
(d
ABTS was dissolved in water to a 2 mM concentration. ABTS
radical cation (ABTSþꢀ) was produced by reacting ABTS stock so-
lution with 0.17 mM potassium persulfate and allowing the mixture
to stand in the dark at room temperature for 12e16 h before use.
Because ABTS and potassium persulfate react 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 absor-
bance 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
(J ¼ 7.3 Hz), H3-tolf), 6.92 (2H, d (J ¼ 8.1 Hz), H5-tolf), 7.12 (4H, m
(J ¼ 7.5 Hz), H8- and H9-tolf), 7.25 (4H, m (J ¼ 6.5 Hz), H4- and H7-
0
tolf), 7.63 (2H, m (J ¼ 6.5 Hz), H4- and H4 -bipy), 7.94 (2H,
0
d (J ¼ 7.7 Hz), H2-tolf), 8.13 (2H, t (J ¼ 6.3 Hz), H6- and H6 -bipy),
0
8.54 (2H, d0 (J ¼ 7.5 Hz), H5- and H5 -bipy), 8.80 (2H, d (J ¼ 4.1 Hz),
H3- and H3 -bipy), 10.36 (2H, s, H6-tolf).
4.3.5. [Zn3(tolf)6(MeOH)2], 6
1H NMR in DMSO-d6 ( /ppm): 2.24 (18H, s, H10-tolf), 6.74 (6H, m
d
stored in the dark at room temperature. The ABTSþ solution was
diluted with ethanol to an absorbance of 0.70 at 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 [74]. The radical scavenging activity of the complexes was
expressed as the percentage inhibition of the absorbance of the
initial ABTS solution (ABTS%). Trolox was used as an appropriate
standard.
ꢀ
(J ¼ 7.2 Hz), H3-tolf), 6.94 (6H, d (J ¼ 8.2 Hz), H5-tolf), 7.10 (12H, m
(J ¼ 2.2 Hz), H8- and H9-tolf), 7.35 (12H, m (J ¼ 6.5 Hz), H4- and H7-
tolf), 7.97 (6H, d (J ¼ 7.2 Hz), H2-tolf), 10.38 (6H, s, H6-tolf).
4.4. X-ray crystallography
X-ray diffraction data for 1 were collected on an Oxford
Diffraction SuperNova diffractometer with Mo microfocus X-ray
ꢀ
source (K
a
radiation,
l
¼ 0.71073 A) with mirror optics and an Atlas
detector. The structure was solved by direct methods implemented
in SIR92 [70] and refined by a full-matrix least-squares procedure
based on F2 using SHELXL-97 [71]. All non-hydrogen atoms were
refined anisotropically. The hydrogen atoms were placed at calcu-
lated positions and treated using appropriate riding models. The
programs Mercury [72], and Platon [73] were used for data analysis
(Table S5).
5.1.4. Soybean lipoxygenase inhibition study in vitro
The in vitro study was evaluated as reported in Ref. [45]. The
tested compounds dissolved in ethanol were incubated at room
temperature with sodium linoleate (0.1 mM) and 0.2 mL of enzyme
solution (1/9 ꢅ 10ꢁ4 w/v in saline). The conversion of sodium
linoleate to 13-hydroperoxylinoleic acid at 234 nm was recorded
and compared with the appropriate standard inhibitor caffeic acid.

A. Tarushi et al. / European Journal of Medicinal Chemistry 74 (2014) 187e198
197
5.2. Albumin binding experiments
Appendix B. Supplementary data
The protein binding study was performed by tryptophan fluo-
rescence emission quenching experiments using bovine (BSA,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.12.019.
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 342 nm or HSA at 351 nm was monitored using complex 1 as
quencher with increasing concentration since 1 in buffer solutions
does not exhibit any emission spectra under the same experimental
conditions [46,47]. Fluorescence spectra were recorded from 300 to
500 nm at an excitation wavelength of 295 nm. The SterneVolmer
(Eq. S(1)) and Scatchard equations (Eq. S(3)) and graphs have been
used in order to study the interaction of each quencher with serum
albumins.
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Acknowledgements
This research has been co-financed by European Social Fund
(ESF) and Greek National Funds (National Strategic Reference
Framework (NSRF): Heracleitus II and Archimides III. Financial
support from the Slovenian Research Agency (ARRS) through
project P1-0175 is gratefully acknowledged. This project was also
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