Mixed-ligand Cu(II)-vanillin Schiff base complexes; Effect of coligands on their DNA binding, DNA cleavage, SOD mimetic and anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2012.08.019

SOD mimics with varying coligand are momentous in developing potential chemotherapeutic drugs. Cu(II) based SOD mimics 1-4 [CuLH(OAc)(H ₂O)Y)] (LH = 2-((E)-(1,3-dihydroxy-2-methylpropan-2-ylimino)methyl)- 6-methoxyphenol, OAc = CH₃CO

Full text of DOI:10.1016/j.ejmech.2012.08.019

European Journal of Medicinal Chemistry 60 (2013) 216e232
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Mixed-ligand Cu(II)evanillin Schiff base complexes; effect of coligands on their
DNA binding, DNA cleavage, SOD mimetic and anticancer activity
,
a
a
b,
c
Sartaj Tabassum a , Samira Amir , Farukh Arjmand , Claudio Pettinari
, Fabio Marchetti ,
*
**
Norberto Masciocchi d, Giulio Lupidi b, Riccardo Pettinari b
a Department of Chemistry, Aligarh Muslim University (AMU), UP 202002, India
b School of Pharmacy, Via S. Agostino 1, University of Camerino, I-62032 Camerino (MC), Italy
c School of Science and Technology, Via S. Agostino 1, University of Camerino, I-62032 Camerino (MC), Italy
d Department of Chemical and Environmental Sciences, University of Insubria, Via Valleggio 11, 22100 Como, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
SOD mimics with varying coligand are momentous in developing potential chemotherapeutic drugs.
Cu(II) based SOD mimics 1e4 [CuLH(OAc)(H2O)Y)] (LH ¼ 2-((E)-(1,3-dihydroxy-2-methylpropan-2-
ylimino)methyl)-6-methoxyphenol, OAc ¼ CH3COO, 1: Y ¼ H2O; 2: Y ¼ phen (1,10-phenanthroline), 3:
Y ¼ tpimH (2,4,5-triphenylimidazole); 4: Y ¼ tfbimH (2-(trifluoromethyl)benzimidazole) were synthe-
sized and thoroughly characterized. Their interaction with CT-DNA showed different non-covalent
binding behaviour. SOD activity of 2 was highest among 1e4 which was further validated by gel elec-
Received 20 November 2011
Received in revised form
26 July 2012
Accepted 13 August 2012
Available online 23 August 2012
$ꢀ
trophoresis. The pBR322 plasmid strand break offered by 2 þ O2 system reveals oxidative cleavage
Keywords:
Coligands
DNA binding
SOD activity
Antitumor activity
mechanism. In vitro antimicrobial activity of 1e4 was shown by percent inhibition data while in vitro
anticancer activity of 1e4 was screened using 16 human carcinoma cell lines of different histological
origin. Complex 2 showed higher efficacy towards 14 cell lines.
Ó 2012 Published by Elsevier Masson SAS.
1. Introduction
therapeutic strategies and elicit antitumor effect by inducing
cancer cell apoptosis by generating large amount of noxious radi-
In recent years there has been a rapid expansion in the devel-
opment of metal complexes as extensive diagnostic agents/
chemotherapeutic drugs [1]. In this regard therapeutic agents that
pertains to less toxic and more effective metallic component like
cals into the cancer cells [6,7]. SOD can putatively participate in
such apoptotic events leading to tumor reduction and cell
proliferation.
Cu(II)containing SOD enzyme, Cu2, Zn2SOD is the most efficient
catalytic species found in the mammalian cell plasma and extra-
cellular spaces. It catalyzes the dismutation of superoxide radical
(O2ꢀ) and converts it into molecular oxygen and hydrogen peroxide
via one electron redox cycle involving its Cu(II) centre. Since cancer
cells have been investigated for rates of metabolism and generate
Cu(II) are of particular interest and include
a plethora of
compounds: antitumor, antioxidant, antimicrobial and anti-
flammatory agents [2]. The appropriate redox property of Cu(II) is
essential for various metabolic pathways like mitochondrial
respiration, free radical scavenging, and iron absorption where it
acts as a catalytic cofactor [3]. The Cu(II) complexes follows
different mode of action towards DNA (non-covalent) as compared
to cisplatin (covalent). Therefore, Cu(II)-based generic complexes
exhibit higher antineoplastic potency towards human ovarian
carcinoma (CH1), murine leukaemia (L1210), and various cervico-
uterine carcinomas as compared to cisplatin [4,5]. A large number
of chemotherapeutic agents and cytokines imply potentially useful
$ꢀ
large amount of intracellular O2 as compared to normal cells [8],
the SOD activity in cancer cell is lower than the normal cell [9]. SOD
mimic enzyme affects tumor cell proliferation, due to the genera-
tion of increased amount of H2O2 and its metabolite OHꢁ radical
(formed via Fenton’s reaction), which crucially causes cytotoxicity
in affected cell lines [10]. Thus synthetic SOD mimics can be
considered as a perfect tool in mediating apoptosis by implying
oxidative stress induced by OHꢁ radical.
Lippard et al. [11] have described a number of imidazolate-
bridged homo and heterodinuclear SOD mimics and much of the
attention has been laid towards their structure activity relationship.
Such model complexes are often constructed by self-assembly of
one or two complex units by the incorporation of imidazole bridge
* Corresponding author. Tel.: þ91 9358255791.
** Corresponding author. Tel.: þ39 0737 402234; fax: þ39 0737 637345.
E-mail addresses: tsartaj62@yahoomail.com (S. Tabassum), claudio.pettinari@
unicam.it (C. Pettinari).
0223-5234/$ e see front matter Ó 2012 Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2012.08.019

S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
217
between both the units. However, the labile nature of bimetallic
Cu(II) and Zn(II) complexes led the dissociation of self-assembled
structures and made it difficult to study in detail their solution
studies and SOD functions [12]. This is a prerequisite for the
synthesis of monometallic SOD mimic complexes. The crystal
structure of native Cu, ZneSOD reported by J. A. Tainer et al. have
also revealed Cu(II) as the active site of the enzyme [13]. The penta
coordinated geometry around Cu(II) centre with a water molecule
attached to the Arg 141 residue through hydrogen bonding is
believed to facilitate the transportation of superoxide anion to the
active site of the enzyme. The present work embodies the design
and application of mononuclear penta coordinated Cu(II)
complexes that have low molecular weight and provides models for
metalloprotein (SOD) active sites by facilitating hydrogen bondinꢀg
in the outer coordination sphere of the redox active centre for O2
stabilization [14e16].
base complexes derived from o-vanillin and pro-ligand: 2-amino-2-
methylpropane 1,3-diol with varying coligands. As illustrated in
Chart 1, complexes exhibit novelty in structure due to presence of
several pharmacophore-active sites that mimics native SOD enzyme
viz., i) two free hydroxyl group of the ligand has been introduced as
hydrogen bonding site pertained to guanidinium group in the Arg
residue of native bovine Cu, ZneSOD; ii) nitrogen containing
heterocyclic units have been considered as a structural model of His
residues; iii) distorted structure around Cu(II) provides feasible
$ꢀ
condition for the attack of O2 [22]. Beside determining the SOD
activity of these Cu(II) ternary systems with varying coligands, other
parameters viz., DNA binding, nuclease, antimicrobial and anti-
tumor activities were also evaluated thoroughly.
2. Chemistry
The presence of different substituents in the intercalative ligand
could affect changes in space configuration and in the electron
density distribution around transition metal complexes, which not
only influence their spectral properties but renders a clear under-
standing in evaluating the changes in SOD-like activity, as well as in
the binding mechanism of transition metal complexes to DNA. The
presence of coordination sites belonging to nitrogen heteroatomic
rings such as imidazole, pyrazole, 1,10-phenanthroline, 2,20-bipyr-
idine, pyridine etc are considered important for high SOD activity
and posses remarkable DNA binding propensity [17,18]. Among
these coligands, phen (1,10-phenanthroline), is an important moiety
which has attracted considerable attention for its versatility in
exhibiting electronic properties and high cleavage efficiency [19];
tpimH, (2,4,5-triphenylimidazole), the imidazole derivative is an
important domainpresent in histidyl residues of metalloprotein and
possess several metal binding sites and tfbimH, (2-trifluoromethyl-
benzimidazole), a member of the benzimidazole family which can
yield complexes that could serve as model compounds for purine
nucleobases [20] and selectively inhibit endothelial cell growth and
suppress angiogenesis in vitro and in vivo [21].
The Cu(II) complex [Cu(LH)(OAc)(H2O)2] was synthesized by
mixing stoichiometric amounts of Cu(OAc)2$H2O with Schiff base
LH in methanol (Scheme 1). The final Rietveld refinement plot of
the ligand LH, is shown in Fig. 1. The preformed complex [Cu(L-
H)(OAc)(H2O)2] was further reacted with coligands: phen, tpimH
and tfbimH to yield the ternary complexes [Cu(LH)(phen)(OAc)],
[Cu(LH)(tpimH)(OAc)(H2O)] and [Cu(LH)(tfbimH)(OAc)(H2O)],
respectively by removal of water molecule from the coordination
sphere of Cu(II) ion. The proposed structures of complexes (Fig. 2)
were established by elemental analysis and spectroscopic studies
(UVevisible, IR, conductivity, mass spectra, EPR and magnetic
susceptibility measurements). All the complexes are soluble in
common organic solvents. The molar conductivity (L ) data of all
M
complexes is consistent with their non-electrolytic nature.
3. Pharmacology
3.1. Proposed mechanistic pathway of reactive oxygen species
responsible for DNA cleavage
To ascertain the therapeutic potential of Cu(II)-based SOD
mimics, we have carried out a systematic study on the Cu(II) Schiff
DNA cleavage mediated by complex [Cu(LH)(Y)(OAc)] is similar
to that proposed for other multinuclear Cu(II) complexes [23,24].
Scheme 1. Proposed scheme for the synthesis of complexes 1e4.

218
S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
Complexes 1e4, fulfils every criterion to behave as potent SOD
mimic by various reasons. Firstly, Cu(II) center are initially reduced
to Cu(I) which further reacts with dioxygen to form a perox-
odicopper(II) derivative, and could generate active oxygen species
required for cleavage. The reaction proceeds through preliminary
steps. Step 1: formation of dioxygen by superoxide causing redox
changes at the metal centre either by i) superoxide coordination to
the metal ion (inner sphere) prior to electron transfer or ii) direct
electron transfer to the outer sphere of metal ion without coordi-
nation as shown in Fig. 3 [25]. Step 2: formation of H2O2 by the
$ꢀ
oxidation of O2 in presence of redox active metal centre leads to
proton-coupled electron transfer. For this purpose, metal ion
should possess “vacant” coordination site that can directly bind to
$ꢀ
O2 in its coordination sphere. In complexes 1e4, the free ‘vacant
site’ is facilitated by square pyramidal geometry of these complexes
$ꢀ
favouring direct O2 binding.
Another aspect for a complex to show SOD activity depends
upon the availability of labile ligand in complex structure. It has
been cited in the literature that the axial site of Cu(II) complex that
consists of solvent molecules with little steric hindrance, undergoes
$ꢀ
a fast attachment of O2 at the Cu(II) centre [26]. Complexes 1, 3,
and 4 possesses labile water molecules coordinated to the Cu(II)
$ꢀ
centre and could provides excellent locus for O2
attachment.
$ꢀ
Third possibility of O2 binding to the complex is based upon the
investigation of A. Klanicova et al. [27]. Their proposed mechanism
involves the substitution of the ligand (other than N donar ligands
$ꢀ
i.e. coordinated perchlorate) by O2
with the formation of
a superoxo-complex. Moreover, the reduction of Cu(II) to Cu(I)
occurs, with the concomitant release of O2 forming tetracoordi-
$ꢀ
nated Cu(I) species. This, Cu(I) species interact with another O2
and re-oxidizes to Cu(II) species forming H2O2 molecule. Again, this
particular condition could be applied to the complexes 1e4 and is
likely that the coordinated acetate ligand in these complexes could
be easily substituted by O2$ꢀ. Thus, to a greater extent, our
complexes represent the (mechanism) active center of native Cu,
ZneSOD.
$ꢀ
The general O2 attack could be described as:
½CuðLHÞðYÞðOAcÞꢂþþþO2$ꢀ/½CuðLHÞðYÞðOAcÞꢂ$O2
þ
½CuðLHÞðYÞðOAcÞꢂ$O2þ þ O2$ꢀ/½CuðLHÞðYÞðOAcÞꢂ$O2 þ O2
½CuðLHÞðYÞðOAcÞꢂ$O2 þ 2H þ /½CuðLHÞðYÞðOAcÞꢂþþþH2O2
½CuðLHÞðYÞðOAcÞꢂ$O2þþH2O2/½CuðLHÞðYÞðOAcÞꢂþþþOH$
þ OHꢀ
The OHꢁ formation leads to the oxidative cleavage of pBR322
DNA.

S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
219
Fig. 2. Cylindrical bonded three dimensional model of metal complex [Cu(LH)(OAc)(H2O)2](1), [Cu(LH)(Phen)(OAc)](2), [Cu(LH)(tpimH)(OAc)(H2O)](3) and [Cu(LH)(tfbimH)(OA-
c)(H2O)] (4). Colour scheme: N dark blue; O red; C grey; F light green and Cu(II) dark grey; H atoms are omitted for clarity. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
Fig. 3. Diagramatic representation of dioxygen formation by superoxide anion via inner and outer coordination sphere (A), and oxidation by superoxide to give hydrogen peroxide (B).

220
S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
4. Results and discussion
crystalline, the orientation of their spins in the material are not
truly random. Thus, powder samples can sometimes give
misleading g values due to the presence of significant bipolar
interactions between molecules in the sample. The X-band EPR
spectra of the Cu(II) complexes [Cu(LH)(OAc)(H2O)2], [Cu(LH)(-
phen)(OAc)], [Cu(LH)(tpimH)(OAc)(H2O)] and [Cu(LH)(tfbim-
H)(OAc)(H2O)], were recorded at 133 K with a magnetic field
strength of 3000 ꢄ 1000 G. All the complexes showed anisotropic
4.1. IR spectroscopy
The IR spectrum of the ligand LH exhibited characteristic azome-
thine band
bonded phenolic eOH and
n
(eHC]Ne) at 1637 cmꢀ1 while intermolecular H-
n
(CeO) were observed at 2602 cmꢀ1
and 1466 cmꢀ1, respectively [28]. The IR spectra of complexes
[Cu(LH)(OAc)(H2O)2], [Cu(LH)(phen)(OAc)], [Cu(LH)(tpimH)(OA-
spectra with g ¼ 2.26, gt ¼ 2.02 and A ¼ 176 for complex
jj
jj
c)(H2O)] and [Cu(LH)(tfbimH)(OAc)(H2O)] undergoes
decrease in
(eHC]Ne) frequency in the range 1621e1626 cmꢀ1
supportive of the coordination of imine nitrogen to Cu(II) ion [29].
a
relative
[Cu(LH)(OAc)(H2O)2]. Complex [Cu(LH)(phen)(OAc)] exhibits
n
g ¼ 2.25, gt ¼ 2.01 and A ¼ 183 (Fig. 4). For complex [Cu(LH)(t-
jj
jj
pimH)(OAc)(H2O)] the EPR spectrum reveals g ¼ 2.22, gt ¼ 2.01
jj
The shift of phenolic
n
(CeO) from 1466 cmꢀ1 to lower wavenumber
and A ¼ 183 while complex [Cu(LH)(tfbimH)(OAc)(H2O)] have
jj
1438e1440 cmꢀ1 in the studied complexes has been attributed to
the coordination of phenolic oxygen to the Cu(II) ion [30]. The
extent of the separation between carboxylate stretches
g
jj
¼ 2.22, gt ¼ 2.01 and A ¼ 183, respectively. In solution, indi-
jj
vidual molecule persist true random orientations that are well
separated from one another. Therefore, EPR spectra remains unaf-
(
D
¼
nas(COOꢀ) ꢀ ns(COOꢀ)) of the acetate, determines binding mode
fected. Since g > gt > 2.0023 reveals that the unpaired electron is
jj
of carboxylate [31]. The following criteria can be used for
carboxylate complexes of divalent metal cations.
D
values of
present in the dx2 ꢀ y2 orbital and is consistent with Cu(II) ion in
a square pyramidal geometry [39a]. The values of g lie in the range
jj
2.22e2.28, suggesting the presence of a CuN2O2 chromophore [40]
D
(monodentate) >
D(ionic) >
D(bridging bidentate) >
D(chelating
in solution and also g < 2.3 indicates an appreciable metal-ligand
covalent character [41,42].
jj
bidentate).
Usually
group, while the
D
value of w170 cmꢀ1 corresponds to ionic carboxylate
w187e200 cmꢀ1 indicates monodentate coor-
4.4. Electronic absorption spectra
D
dination [32]. Upon complexation, change in the force field around
the metal atom causes redistribution of the electron density which
shifts nas(COOꢀ) to higher wave number with respect to ionic
The UVevis spectra of complexes 1e4 was measured at room
temperature in the region 190e1100 nm showing distinct regions.
The electronic intra-ligand
pep* and LMCT transitions were
acetates (D
w170 cmꢀ1) and leads to monodentate coordination of
observed in the region 202e278 nm and 368e381 nm, respectively.
However, ded transitions observed in the region 633e675 nm are
acetates. However, the shifting of nas(COOꢀ) to lower wavenumbers
with respect to the ionic group leads to bidentate acetates
typical for penta coordinated Cu(II) complexes having a distorted
D
<170 cmꢀ1). The nas(COOꢀ) for ‘bridging’ and ionic acetates
22
(
square pyramidal geometry (dxz, dyz / d ) [43].
xꢀy
shows slight difference in their frequencies [33].
The
D
values of all the studied complexes lie in the range 201e
4.5. Crystal structure of LH
287 cmꢀ1 suggestive of monodentate coordination of the acetate to
the Cu(II) ion. The presence of coordinated water molecules in
complexes [Cu(LH)(OAc)(H2O)2], [Cu(LH)(tpimH)(OAc)(H2O)] and
[Cu(LH)(tfbimH)(OAc)(H2O)] were ascertained by rocking mode in
the region 840e860 cmꢀ1 [34]. In complex [Cu(LH)(phen)(OAc)],
The ligand LH crystallizes in the triclinic space group with one
crystallographically independent molecule, shown in Fig. 5. As
described in the experimental section, within the molecule, only
torsional angles were free, which resulted in the following
conformational parameters: C5eC6eC8]N1 176.7(4)ꢅ (maintain-
the aromatic (Phen) stretching vibrations
n(C]C) and n(C]N)
were observed at 1429 and 1516 cmꢀ1, respectively. The complex
ing the iminic group nearly coplanar with the
p-system of the
[Cu(LH)(tpimH)(OAc)(H2O)] displays a strong band at 695 cmꢀ1
phenyl residue) C6eC8]N1eC9 176.4(2)ꢅ (in agreement with the
trans-configuration of the iminic moiety) and C1eO1eC2eC7
175.3(4)ꢅ, indicating the anti-disposition of the OMe group with
respect to the other (adjacent) substituents. Interestingly, the
ligand LH shows a strong intramolecular H-bond between O2 and
N1 (O1/N1 2.50 Å), along with several intramolecular bonds
due to out of plane mode of tpimH ring. Moreover, the n(C]N)
vibrations of the imidazole ring in complexes [Cu(LH)(tpim-
H)(OAc)(H2O)] and [Cu(LH)(tfbimH)(OAc)(H2O)] lie in the range
1400e1500 cmꢀ1 [35]. A strong band at 1128 cmꢀ1 observed in the
complex [Cu(LH)(tfbimH)(OAc)(H2O)] corresponds to CeF vibra-
tion [36]. The far IR spectra of the complexes exhibited absorption
bands, 500e600 cmꢀ1, 410e480 cmꢀ1 assigned to CueO and CueN
stretching vibrations [37].
4.2. Magnetic susceptibility studies
The magnetic susceptibility values for complexes [Cu(LH)(OA-
c)(H2O)2], [Cu(LH)(phen)(OAc)], [Cu(LH)(tpimH)(OAc)(H2O)] and
[Cu(LH)(tfbimH)(OAc)(H2O)], at room temperature lies in the
range of 1.8e1.9 BM which correspond to one unpaired electron
and are consistent with the d9 configuration around Cu(II) ion
[38].
4.3. EPR spectra
In the absence of single crystal EPR analysis, reliable parameters
for Cu(II) complex can be obtained by running the solution EPR
spectrum as a frozen glass. Since powdered samples are sufficiently
Fig. 4. EPR spectra of complex [Cu(L)(phen)(OAc)(H2O)](2) in methanol at 133 K.

S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
221
On contrary, complexes [Cu(LH)(tpimH)(OAc)(H2O)] and
[Cu(LH)(tfbimH)(OAc)(H2O)] exhibited both ‘hyperchromic’ and
‘hypochromic effect’ at intra-ligand and LMCT regions. Upon addi-
tion of increasing concentration of CT-DNA to the complex
[Cu(LH)(tpimH)(OAc)(H2O)] and [Cu(LH)(tfbimH)(OAc)(H2O)],
‘hyperchromism’ was observed in the intra-ligand region (277e
278 nm) with a blue shift of 12 nm and 10 nm, respectively.
Appreciable changes in the position of the LMCT band in the region
371e372 nm were observed with the decreasing order of the band
intensities, resulting into ‘hypochromism’ along with the red shift
of 4e6 nm, respectively (Fig. 6C, D and C0, D0). A distinct isosbestic
point was observed at 310 nm for complex [Cu(LH)(tpimH)(OA-
c)(H2O)] and 315 nm for complex [Cu(LH)(tfbimH)(OAc)(H2O)]
indicating intercalative mode of binding of these complexes to the
double helix structure of DNA. The presence of aromatic chromo-
phore viz., tpimH and tfbimH facilitates the stacking interaction of
the complex with the base pair of DNA. The
intercalated ligand couple with the orbital of the DNA base,
decreasing of the * transitions energy which ultimately leads
p* orbital of the
Fig. 5. Schematic drawing of the molecule of LH (with partial labelling scheme),
showing the conformation derived from our X-ray powder diffraction study. Colour
code: carbon (grey); nitrogen (blue); oxygen (red); hydrogen (white). The fragmented
line indicates the intramolecular H-bond interaction. (For interpretation of the refer-
ences to colour in this figure legend, the reader is referred to the web version of this
article.)
p
pep
into the red shift [48]. To quantify the extent of DNA binding, the
intrinsic binding constant Kb of the studied complexes with varying
coligands were determined and the data were presented in Table 1
that shows either electrostatic and/or intercalative binding trends
of the complexes 1, 3 and 4, respectively with CT-DNA. Complex 2,
however possesses a planar aromatic moiety (phen) and the
probability of binding of this complex to CT-DNA via partial inter-
calation cannot be ruled out (Kb ¼ 2.70 ꢃ 104), nonetheless
‘hyperchromism’ supports electrostatic interaction.
(O2/O3 and O2/O4 of 2.71 and 2.70 Å, respectively) granting an
efficient packing and a high melting point (165 ꢅC).
Accordingly, O2 is tetraconnected (with three H-bond interac-
tions in nearly tetrahedral geometry), while the O3 and O4 atoms,
belonging to a more peripheral section of the molecule, and show
a single H-bond interaction. Inter alia, this conformational analysis,
as well as the determination of the main crystal packing features,
witness the high quality of our powder diffraction study, which,
although coping with idealized bond distances and angles (derived
from simple molecular mechanics optimization), was nevertheless
able to rescue important, otherwise inaccessible, structural features
(no single-crystal of suitable quality being available).
5.2. Steady state emission titrations
In absence of CT-DNA, all the complexes when excited between
265 and 268 nm, emitted luminescence in TriseHCl buffer/pH 7.2,
with emission bands observed in the range of 282e290 nm. Fixed
amount of all the metal complexes were titrated with increasing
amount of CT-DNA (R ¼ 40), and as a consequence an increase in
the emission intensity was observed for complexes 1e4 (Table 2).
The hydrophobic molecular structure of CT-DNA could be respon-
sible for enhancing the fluorescence quantum yield of complexes
which in turn leads to the higher fluorescence intensity
with increasing concentration of CT-DNA. The binding constant
of the complexes were obtained from SterneVolmer relationship
[49] and followed the order, [Cu(LH)(phen)(OAc)] > [Cu(LH)
(tfbimH)(OAc)(H2O)] > [Cu(LH)(tpimH)(OAc)(H2O)] > [Cu(LH)
(OAc)(H2O)2]. The interaction of [Cu(LH)(phen)(OAc)] with CT-DNA
displays a higher binding propensity ca. three times greater in
magnitude as compared to other complexes (Fig. 7).
To evaluate the affinity of complexes towards CT-DNA,
quenching experiments were performed using [Fe(CN)6]4ꢀ
(Table 3) which reveal the binding strength of complexes with CT-
DNA. The emission spectra with increasing concentration of the
quencher are depicted in Fig. 8. The accessibility of DNA-bound
exciplex to the quencher depends upon the strength of binding of
complex to DNA. Stronger the binding, lesser the chances for
availability of DNA-bound exciplex to the quencher and vice versa.
The quenching efficiency is evaluated by using SterneVolmer
constant Ksv which follows the order [Cu(LH)(tfbimH)(OAc)
(H2O)] > [Cu(LH)(tpimH)(OAc)(H2O)] > [Cu(LH)(phen)(OAc)] > [Cu
(LH)(OAc)(H2O)2].
5. DNA-binding mode and affinity
5.1. Absorption spectral studies
Electronic absorption spectroscopy is employed to examine the
binding mode of CT-DNA with small molecules [44]. The absorption
spectra of complexes [Cu(LH)(OAc)(H2O)2], [Cu(LH)(phen)(OAc)],
[Cu(LH)(tpimH)(OAc)(H2O)] and [Cu(LH)(tfbimH)(OAc)(H2O)] with
CT-DNA were depicted in Fig. 6. A fixed concentration of the
complexes were titrated with increasing concentration of CT-DNA
(R ¼ [DNA]/[Cu] ¼ 1e40). It was observed that the complex
[Cu(LH)(OAc)(H2O)2] exhibited ‘hyperchromism’ in the intra-ligand
region (at 278 nm) with a noticeable blue shift of 10 nm (Table 1).
This indicates strong binding propensity of the complex towards
CT-DNA (non-covalent interactions) leading to the damaged DNA
double helix structure. Complex [Cu(LH)(phen)(OAc)] also dis-
played ‘hyperchromism’ with the blue shift of 8 nm in the same
region (at 273 nm) (Fig. 6B). In general, ‘hyperchromism’ and blue
shift suggests the corresponding (conformational and structural)
changes after the complexes are bound to CT-DNA electrostatically
via external contact (surface binding) with the DNA duplex. While
‘hypochromism’ and a red shift (bathochromism) of absorption
band implicates intercalative mode of binding and is likely that the
metal complexes with aromatic chromophore stabilizes the DNA
duplex [45,46]. However, ‘hyperchromism’ obtained for complex
[Cu(LH)(phen)(OAc)] is characteristic of the complex bound to DNA
through non-covalent interactions or the complex could uncoil the
helix and made more bases embedded in the DNA explored [47].
5.3. Viscosity measurements
Viscosity is considered as least ambiguous and most critical test
in predicting the nature of binding of the complexes to CT-DNA, in
absence of crystallographic data [50]. A classical intercalator causes

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S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
Fig. 6. Absorption spectral traces of complexes [Cu(L)(CH3COO)(H2O)2](1), [Cu(L)(Phen)(CH3COO)](2), [Cu(L)(tpimH)(CH3COO)(H2O)](3); C & C0, [Cu(L)(tfbimH)(CH3COO)(H2O)](4);
D & D0 ([complex 1e4] ¼ 1 ꢃ 10ꢀ5 M) in MeOH solution in the absence and presence of increasing amounts of CT-DNA ([CT-DNA] ¼ 0e1.35 ꢃ 10ꢀ4 M). The arrows show the changes
upon increasing amounts of CT-DNA. Inset: Plots of [DNA]/εa ꢀ εf (m2 cm) vs [DNA] for the titration of CT-DNA with complexes 1e4;- experimental data points; full lines, linear
fitting of the data. C0 and D0 represent the spectral changes of absorbance of complexes 3, and 4 in the range 315e475 nm (‘Hypochromic effect’).
significant increase in the viscosity of DNA solution due to the
increase in the separation in overall DNA contour length [51]. A
partial/or non-classical intercalation of metal complexes 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
[52,53]. In presence of increasing amount of complexes [Cu(L-
H)(OAc)(H2O)2], [Cu(LH)(phen)(OAc)], [Cu(LH)(tpimH)(OAc)(H2O)]
and [Cu(LH)(tfbimH)(OAc)(H2O)] the relative viscosity of CT-DNA
Table 1
Electronic spectral properties of Cu(II) complexes [Cu(LH)(OAc)(H2O)2](1), [Cu(LH)(phen)(OAc)](2), [Cu(LH)(tpimH)(OAc)(H2O)](3) and [Cu(LH)(tfbimH)(OAc)(H2O)](4) in
presence of CT-DNA with mean standard deviation values.
Complex
Ligand based
l
max (nm)
CT
Ra
Changes in absorbance (
Dε%)
Shifts (nm)
Blue
Kb (Mꢀ1
)
pe
p*
Red
1
2
3
278
273
278
e
e
e
27
40
32
e
Hyperchromism (84.46)
Hyperchromism (78.88)
Hyperchromism (56)
Hypochromism (66)
Hyperchromism (84)
Hypochromism (55)
10
8
12
e
e
e
e
4
8.10 ꢃ 103(ꢄ0.04)
2.70 ꢃ 104(ꢄ0.02)
5.79 ꢃ 104(ꢄ0.04),
8.30 ꢃ 104(ꢄ0.02)
6.10 ꢃ 104(ꢄ0.06),
4.06 ꢃ 105(ꢄ0.04)
372
e
4
5
277
e
9
e
10
e
e
371
6
a
R ¼ [DNA]/[Cu complex] where concentration of Cu(II) solutions ¼ 1 ꢃ 10ꢀ5 M.

S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
223
Table 2
Table 3
Emission properties of complexes [Cu(LH)(OAc)(H2O)2](1), [Cu(LH)(phen)(OAc)](2),
[Cu(LH)(tpimH)(OAc)(H2O)](3) and [Cu(LH)(tfbimH)(OAc)(H2O)](4) bound to CT-
DNA.
Binding constant values for the quenching of [Fe(CN)6]4ꢀ with complexes [Cu(L-
H)(OAc)(H2O)2](1), [Cu(LH)(phen)(OAc)](2), [Cu(LH)(tpimH)(OAc)(H2O)](3) and
[Cu(LH)(tfbimH)(OAc)(H2O)](4), in absence (A) and in the presence (B) of CT-DNA
with mean standard deviation value.
Complex
Excitation (nm)
Emission (nm)
K (Mꢀ1
)
Complex
Ksv (Mꢀ1) (A)
Ksv (Mꢀ1) (B)
1
2
3
4
260
260
258
265
282
284
285
290
171 ꢃ 104
6.39 ꢃ 104
1.85 ꢃ 104
2.95 ꢃ 104
1
2
3
4
3.28(ꢄ0.6) ꢃ 104
4.21(ꢄ0.3) ꢃ 104
5.14(ꢄ0.4) ꢃ 104
8.17(ꢄ0.5) ꢃ 104
2.4(ꢄ0.5) ꢃ 104
2.21(ꢄ0.2) ꢃ 104
2.78(ꢄ0.3) ꢃ 104
2.82(ꢄ0.3) ꢃ 104
increases steadily (Fig. 9). In case of complex [Cu(LH)(OAc)(H2O)2]
the increase in viscosity is less as compared to complexes
[Cu(LH)(phen)(OAc)], [Cu(LH)(tpimH)(OAc)(H2O)] and [Cu(LH)(tf-
bimH)(OAc)(H2O)] in the same concentration ranges of CT-DNA.
Thus, it is concluded that complexes [Cu(LH)(phen)(OAc)],
5.4. Cyclic voltammetry
Electrochemical techniques are complementary to other
related biophysical techniques that are applied to study the
interaction between the redox active molecules and biomolecules
[55]. Important criteria for a complex to act as SOD mimic is its
metal-centred redox potential that facilitates catalytic dispropor-
tionation of O2$ꢀ. The cyclic voltammogram of complexes [Cu(L-
H)(OAc)(H2O)2], [Cu(LH)(phen)(OAc)], [Cu(LH)(tpimH)(OAc)(H2O)]
and [Cu(LH)(tfbimH)(OAc)(H2O)] in absence and in the presence of
CT-DNA were recorded in aqueous solutions with 0.4 M KNO3 as
[Cu(LH)(tpimH)(OAc)(H2O)]
and
[Cu(LH)(tfbimH)(OAc)(H2O)]
exhibit higher affinity for the intercalative site on DNA as compared
to complex [Cu(LH)(OAc)(H2O)2] that bring conformational changes
due to partial intercalation of these complexes within the hydro-
phobic DNA pockets [54]. The increasing degree of viscosity follows
the order [Cu(LH)(tfbimH)(OAc)(H2O)]
> [Cu(LH)(tpimH)(OAc)
(H2O)] > [Cu(LH)(phen)(OAc)] > [Cu(LH)(OAc)(H2O)2].
Fig. 7. Emission spectra of [Cu(L)(OAc)(H2O)2](1); A, [Cu(L)(Phen)(OAc)](2); B, [Cu(L)(tpimH)(OAc)(H2O)](3); C and [Cu(L)(tfbimH)(OAc)(H2O)](4); D in TriseHCl buffer in the
presence of CT-DNA. [CT-DNA] (0e1.35 ꢃ 10ꢀ4 M). Arrows show the intensity changes upon addition of the complexes (1 ꢃ 10ꢀ5 M).

224
S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
Fig. 8. Emission quenching spectra of [Cu(L)(OAc)(H2O)2](1); A, [Cu(L)(Phen)(OAc)](2); B, [Cu(L)(tpimH)(OAc)(H2O)](3); C, [Cu(L)(tfbimH)(OAc)(H2O)](4); D in presence of [CT-DNA]
(1.89 ꢃ 10ꢀ5 M) with increasing concentration of quencher [Fe(CN)6]4ꢀ (0e1.91 ꢃ 10ꢀ4 M). Arrows show the intensity changes upon addition of the complexes (1 ꢃ 10ꢀ5 M).
a supporting electrolyte (Fig. 10). The cyclic voltammograms of all
the studied complexes exhibited lower peak current ratios (less
than 1) attributed to quasi-reversible one electron redox process
involving Mn/Mnꢀ1 couple. All the values corresponding to EPC (V),
EPA (V), E1/2 (V),
DEP and IPA/IPC are listed in Table 4. We also
investigated the electrochemical responses of all the four
complexes in presence of CT-DNA (Table 4B). A significant reduc-
tion in their respective peak potentials, cathodic and anodic peak
currents can be attributed to slow diffusion of the equilibrium
mixtures of these complexes (free and DNA bound) at the elec-
trode surface [52]. The E1/2 shifts these complexes towards
lower negative potential confirms their binding to CT-DNA
[56]. However, the positive shift in the EPC or EPA value is indica-
tive of intercalation of the complex into the DNA double helix
while negative shift reveals the involvement of electrostatic
interactions [57].
5.5. SOD activity
$ꢀ
The O2
scavenging activity (IC50) of the complexes [Cu(L-
H)(OAc)(H2O)2], [Cu(LH)(phen)(OAc)], [Cu(LH)(tpimH)(OAc)(H2O)]
and [Cu(LH)(tfbimH)(OAc)(H2O)] was determined by NBT assay
using UVevis spectroscopy. All Cu(II) complexes demonstrated SOD
Fig. 9. Effect of increasing amount of complexes 1 (-A-), 2 (- -), 3 (-:-) and 4 (-C-)
;
(prepared in MeOH) on the relative viscosities (h/ho) of CT-DNA in TriseHCl buffer (pH
7.2).

S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
225
Fig. 10. Cyclic voltammogram (5:95 MeOH/H2O, 25 ꢅC) of [Cu(L)(OAc)(H2O)2](1); A, [Cu(L)(Phen)(OAc)](2); B, [Cu(L)(tpimH)(OAc)(H2O)](3); C, [Cu(L)(tfbimH)(OAc)(H2O)](4); D, at
(a) unbound and (b) in presence of CT-DNA, [complex 1e4] ¼ 1 ꢃ 10ꢀ3 M, [DNA] ¼ 6 ꢃ 10ꢀ3 M.
activity in the micromolar range (IC50) varying from 0.58 to
11.40 M as shown in Table 5. Complex [Cu(LH)(phen)(OAc)]
5.6. Gel electrophoresis studies
m
possesses higher SOD mimetic activity among all the studied
complexes. Although, the IC50 values of the Cu(II) complexes are
higher than those reported for native bovine erythrocyte SOD
5.6.1. DNA cleavage demonstrating SOD mimetic activity
The higher SOD activity of complex [Cu(LH)(phen)(OAc)] as
ascertained by NBT assay, was further confirmed by employing DNA
cleavage studies. Our experiment is based on the mechanism of
SOD activity of native bovine erythrocyte SOD (Cu/Zn), which
converts the O2$ꢀ into H2O2 and O2. However, this H2O2was further
converted into H2O by catalase. Native SOD, a homodimer metal-
loenzyme, undergoes glycation in vivo [60] resulting into the
release of free Cu(II) ion, which further participates in the genera-
tion of OHꢁ by Fenton-type reaction. To assess the DNA cleavage
(0.04
mM), they are in good agreement with the IC50 values of
previously reported synthetic SOD mimics [58,59].
Table 4
Cyclic voltammetric results of complexes [Cu(LH)(OAc)(H2O)2](1), [Cu(LH)(phe-
n)(OAc)](2), [Cu(LH)(tpimH)(OAc)(H2O)](3) and [Cu(LH)(tfbimH)(OAc)(H2O)](4), (A)
in the absence of CT-DNA, (B) in the presence of CT-DNA.
Complexes
(A)
EPC (V)
EPA (V)
E1/2 (V)
DEP
IPA/IPC
Table 5
SOD activity profile of complexes [Cu(LH)(OAc)(H2O)2](1),
[Cu(LH)(phen)(OAc)](2), [Cu(LH)(tpimH)(OAc)(H2O)](3) and
[Cu(LH)(tfbimH)(OAc)(H2O)](4).
1
2
3
4
ꢀ0.4896
ꢀ0.4167
ꢀ0.5049
ꢀ0.4459
ꢀ0.2963
ꢀ0.3212
ꢀ0.2842
ꢀ0.3432
ꢀ0.3929
ꢀ0.3689
ꢀ0.3945
ꢀ0.3945
0.1933
0.0955
0.2207
0.1027
0.3150
0.6030
0.5880
0.4632
Complexes
Concentrationa
(mM)
(B)
1
2
3
4
1
2
3
4
11.40 (ꢄ0.08)
0.58 (ꢄ0.06)
3.20 (ꢄ0.04)
0.67 (ꢄ0.06)
ꢀ0.4474
ꢀ0.3947
ꢀ0.5194
ꢀ0.4388
ꢀ0.3215
ꢀ0.3232
ꢀ0.2622
ꢀ0.3435
ꢀ0.3844
ꢀ0.3589
ꢀ0.3908
ꢀ0.3911
0.1259
0.0515
0.2572
0.0885
0.1755
0.6355
0.8303
0.4906
a
Equivalent to 1 U SOD (0.04 mM).

226
S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
ability of complex [Cu(LH)(phen)(OAc)] a fixed concentration
(200 M) of the complex and pBR322 DNA was taken and incubated
m
in a mixture (1:10) containing xanthine oxidase (prepared with
50 mM TriseHCl, pH 8.0 sample buffer) and hypoxanthine
(prepared in 50 mM TriseHCl, pH 8.0 assay buffer þ 0.1 mM
diethylenetriaminepentaacetic acid (DTPA) þ 0.1 mM hypoxan-
thine). Change in the electrophoretic mobility of plasmid DNA was
$ꢀ
observed on agarose gel. As depicted in Fig. 11 (lane L2), O2
generated by the reaction of xanthine oxidase and hypoxanthine
did not cause loss of the supercoiled form (SC form; Form I) [61]. In
presence of native SOD, an inhibition of the cleavage was observed
(Fig. 11, lane L3), which reveals that OHꢁ is not generated due to the
absence of glycation process in the experimental conditions.
However, OHꢁ is easily produced by mixing complex [Cu(LH)(phe-
n)(OAc)] with O2$ꢀ. As a consequence the supercoiled form (SC
form; Form I) was converted into the nicked circular form (NC form;
Form II) and linear circular form (LC form; Form III) (Fig. 11, lane L4)
which confirms the involvement of complex [Cu(LH)(phen)(OAc)]
in double strand DNA cleavage. Furthermore, the mixture con-
taining complex [Cu(LH)(phen)(OAc)] and O2 reveals minor
conversion of Form I into the Form II as displayed in Fig.11 (lane L5).
To further ascertain the role of OHꢁ in the cleavage process, the
electrophoretic pattern of plasmid pBR322 DNA was observed in
the presence of radical scavenger DMSO (hydroxyl radical) and
NaN3 (singlet oxygen). Upon addition of DMSO to complex
Fig. 12. Gel electrophoresis pattern showing cleavage of pBR322 supercoiled DNA
(300 ng) by complex [Cu(L)(Phen)(OAc)]. Lane L1: DNA; lane L2: 25
[Cu(L)(Phen)(OAc)] þ DNA; lane L3: 50 M [Cu(L)(Phen)(OAc)] þ DNA; lane L4: 150
[Cu(L)(Phen)(OAc)] þ DNA; lane L5: 200 M [Cu(L)(Phen)(OAc)] þ DNA.
m
m
M
M
m
m
electrophoresis. The conversion of supercoiled (SC form; Form I)
into the nicked circular form (NC form; Form II) was observed with
the increase in concentration of complex [Cu(LH)(phen)(OAc)] from
25 to 50
mM while the linear circular form (LC form; Form III) was
observed at 150e200
mM indicating that complex [Cu(LH)(phe-
n)(OAc)] is involved in double strand DNA cleavage. These result
confirms the efficient cleavage activity of complex [Cu(LH)(phe-
n)(OAc)] which corroborates with the results of binding studies.
$ꢀ
[Cu(LH)(phen)(OAc)] þ O2 system, inhibition of the cleavage was
observed (Fig. 11, lane L6) suggestive of the involvement of freely
diffusible OHꢁ in the cleavage mechanism. However, no such
quenching in the cleavage of the pBR322 DNA was observed in
presence of NaN3 revealing the non-participation of singlet oxygen
in cleavage mechanism (Fig. 11, lane L7).
5.6.3. DNA cleavage with added reductant
Cleavage efficiency of Cu(II) complexes was also monitored in
the presence of activators [62,63]. To examine whether reducing
agents present in the reaction mixture could account for the higher
rates of pBR322 DNA degradation by complex [Cu(LH)(phen)(OAc)]
in 5 mM TriseHCl/50 mM NaCl buffer at pH 7.2, cleavage reaction
was performed in presence of various reducing agent viz., H2O2,
ascorbate (Asc), 3-mercaptopropionic acid (MPA), and glutathione
(GSH) (Fig.13). In presence of H2O2, a significant conversion of Form
I into Form II was observed (Fig. 13, lane 4), as compared to other
activators (Fig. 13, lane 2, 3 and 5). And follows the order
H2O2 > Asc ¼ MPA > GSH, which is similar to the nuclease activity
of various mononuclear Cu(II) complexes [64,65].
Besides, Form II was slightly inhibited in presence of catalase
(Fig. 11, lane L8) which reveals slow catalyzation of H2O2 by the
enzyme. Complex [Cu(LH)(phen)(OAc)], however undergoes rapid
interaction with a major part of H2O2 as compared to catalase and
generates OHꢁ as ascertained by the conversion of Form I into Form
II. These findings fulfil the prerequisite for complex [Cu(LH)(phe-
n)(OAc)] to act as a potent SOD mimic.
5.6.2. DNA cleavage without added reductant
To assess the DNA cleavage ability of complex [Cu(LH)(phe-
n)(OAc)] it was incubated with pBR322 DNA at different concen-
trations (25e200 mM) in 15% H2O/0.01 mM HCl/10 mM NaCl buffer
at pH 7.2 (Fig. 12) for 1 h. A concentration dependent DNA cleavage
was observed after the reaction mixture was subjected to gel
5.6.4. DNA cleavage in the presence of recognition elements (groove
binding)
In order to probe the potential interacting site of the complex
[Cu(LH)(phen)(OAc)] with plasmid pBR322 DNA, the cleavage
Fig. 11. Gel electrophoresis pattern showing cleavage of pBR322 supercoiled DNA
$ꢀ
(300 ng) by complex [Cu(L)(Phen)(OAc)](200
m
M). O2
generated by incubation of
Fig. 13. Agarose gel electrophoresis showing the cleavage pattern of pBR322 plasmid
XOD and hypoxanthine (1:10) as described in the experimental procedure. Lane L1:
DNA (300 ng) by complex [Cu(L)(Phen)(OAc)](200
vating agent at 312 K after incubation for 30 min. Lane L1, DNA control; lane L2,
mM) in presence of different acti-
$ꢀ
$ꢀ
DNA; lane L2: O2
;
O2
lane L3: O2
þ
SOD (15 units); lane L4:
$ꢀ
[Cu(L)(Phen)(OAc)]
[Cu(L)(Phen)(OAc)]
[Cu(L)(Phen)(OAc)]
þ
;
lane L5: [Cu(L)(Phen)(OAc)]
þ
O2; lane L6:
DNA
DNA
þ
[Cu(L)(Phen)(OAc)]
[Cu(L)(Phen)(OAc)]
þ
glutathione
MPA
M); lane L5, DNA þ [Cu(L)(Phen)(OAc)] þ Asc
(400
(400
m
M);
M);
lane
lane
L3,
L4,
$ꢀ
$ꢀ
þ
þ
O2
O2
þ
DMSO
NaN3
(400
(400
m
M);
lane
lane
L7:
L8:
þ
þ
m
þ
mM);
DNA þ [Cu(L)(Phen)(OAc)] þ H2O2 (400
m
$ꢀ
[Cu(L)(Phen)(OAc)] þ O2 þ catalase (15 units).
(400 M).
m

S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
227
investigated by studying comparative reactions in presence of
various radical scavengers like DMSO, EtOH (hydroxyl radical),
sodium azide (singlet oxygen) and SOD (superoxide). In presence of
DMSO and EtOH (Fig. 14, lane 4, 5) inhibition of the cleavage was
observed while the presence of sodium azide and superoxide dis-
mutase did not affect the cleavage mechanism (Fig. 14, lane 6, 7).
Thus, freely diffusible OH$ is considered as the active species
responsible for pBR322 DNA cleavage.
6. Antimicrobial screening
Furthermore, the complexes [Cu(LH)(OAc)(H2O)2], [Cu(LH)(-
phen)(OAc)], [Cu(LH)(tpimH)(OAc)(H2O)] and [Cu(LH)(tfbim-
H)(OAc)(H2O)] were screened for their antimicrobial activity
against certain pathogenic bacterial and fungal species using agar
well dilution method and the results of antimicrobial activities
were summarized in Table 6. All the complexes were found to
exhibit significant activity against Gram (þve) and Gram (ꢀve)
bacteria. We observed excellent activity of complex [Cu(LH)(phe-
n)(OAc)] against Candida albicans and Aspergillus niger.
Fig. 14. Agarose gel electrophoresis pattern showing the cleavage of pBR322 plasmid
DNA (300 ng) by complex [Cu(L)(Phen)(OAc)](200 M) in the presence of DNA minor
binding agent DAPI, major binding agent MG and standard radical scavengers at 310 K
after incubation for 30 min. Lane L1, DNA control; lane L2,
DNA þ [Cu(L)(Phen)(OAc)] þ DAPI (8 M); lane L3, DNA þ [Cu(L)(Phen)(OAc)] þ methyl
green (2.5
L of a 0.01 mg/ml solution); lane L4, DNA þ [Cu(L)(Phen)(OAc)] þ DMSO
(400 M); lane L5, DNA [Cu(L)(Phen)(OAc)] EtOH (400 M); lane L6,
M); lane L7, [Cu(L)(Phen)(OAc)] þ SOD (15
m
m
m
m
þ
þ
m
DNA þ [Cu(L)(Phen)(OAc)] þ NaN3 (400
m
units).
7. Antitumor activity
experiment was carried out in presence of minor groove binding
agent, DAPI [66] and major groove binding agent, methyl green
(MG) [67]. The results demonstrate that (Fig. 14, lane 2, 3) the DNA
cleavage was neither affected by DAPI nor methyl green, impli-
cating non affinity of the complex towards the DNA major/minor
groove.
In vitro antitumor activity of complexes [Cu(LH)(OAc)(H2O)2],
[Cu(LH)(phen)(OAc)] and [Cu(LH)(tfbimH)(OAc)(H2O)] was
screened against 16 different human carcinoma cell lines of
different histological origin; K562 (Leukaemia), ZR-75-1, MCF7,
(Breast), Colo205, HT29, HCT15, SW620 (Colon), SiHa (Cervix), T24
(bladder), A498,786-O (kidney), MIAPACA2 (Pancreas), Hop-62
(Lung), PC3 (Prostrate), DWD (Oral) and A549 (Alveolar) and GI50
values were listed in Table 7. However it must be noted that the
test stock solutions of the complexes 1e4 (1 mg/mL) were
5.6.5. DNA cleavage in the presence of reactive oxygen species
To probe the DNA cleavage mechanism by complex [Cu(LH)(-
phen)(OAc)], some standard radical scavengers were used prior to
the addition of complex [Cu(LH)(phen)(OAc)] to plasmid pBR322
DNA. The involvement of ROS in the cleavage mechanism was
prepared by dissolving the substance in 100
mL of DMSO and
completed with 900 L of tissue culture medium. DMSO is
m
Chart 1. Synthetic model representing SOD mimic.

228
S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
Quenching experiments were performed using [Fe(CN)6]4ꢀ
which reveal the binding strength of complexes with CT-DNA. The
quenching efficiency is evaluated by using SterneVolmer constant
Ksv which follows the same order of binding affinity of complexes
towards CT-DNA as observed for Kb: [Cu(LH)(tfbimH)(OAc)
(H2O)] > [Cu(LH)(tpimH)(OAc)(H2O)] > [Cu(LH)(phen)(OAc)] > [Cu
(LH)(OAc)(H2O)2]. SOD activity of complex 2 was measured by
employing NBT assay that showed excellent IC50 value of 0.58
Table 6
Antimicrobial activity of complexes [Cu(LH)(OAc)(H2O)2](1), [Cu(LH)(phe-
n)(OAc)](2), [Cu(LH)(tpimH)(OAc)(H2O)](3) and [Cu(LH)(tfbimH)(OAc)(H2O)](4) by
agar well dilution method.
Cmpa
S. ab
B. sc
S. td
E. ce
C. af
A. ng
1
2
3
4
15
30
e
e
e
12
16
e
20
10
31
10
Under investigation
18
e
e
11
13
e
(ꢄ0.06)
mM for the complex. Gel electrophoretic pattern of complex
‘_’
Activity profile <11 mm, poor activity (þ); <15 mm, moderate activity (þþ);
2 displayed remarkable double strand scission of pBR322 DNA via
oxidative pathway. Also, SOD activity of complex 2 was analyzed by
gel electrophoresis and is consistent with the participation of OHꢁ in
the cleavage process. In vitro antitumor activity results reveal
excellent potential of complex 2 towards 14 different human
carcinoma cell lines of different histological origin. The GI50 values
of complex 2 are comparable to those obtained for standard drug
Adriamycin (ADR) while complex 1 and 4 were showed activity
towards 786-O kidney cell line.
<20 mm, very good activity (þþþ).
a
Complex (1 mg/mL).
S. aureus (SA-22).
B. subtilis (MTCC 121).
S. typhimurium (MTCC 98).
E. coli (K-12).
C. albicans (Clinical isolate).
b
c
d
e
f
g
A. niger (Clinical isolate).
a biocompatible solvent used in several biological test at lower
concentration. Afterwards, the tested complexes were further
diluted in culture medium to reach the final concentrations of 10,
9. Experimental section
20, 40 and 80 ng/
triplicates with each well receiving 900
10 L of the drug solution. But if higher concentration of the
m
L, respectively. Now, test were performed in
9.1. Materials
mL of cell suspension and
m
All the solvents used were of analytical grade and used as
received (SigmaeAldrich). Analytical grade reagents o-vanillin, 2-
amino-2-methylpropane-1,3-diol, 1,10-phenanthroline (phen),
2,4,5-triphenylimidazole (tpimH), 2-trifluoromethyl-benzimid-
azole (tfbimH), Cu(OAc)2.H2O (Sigma Aldrich, Steinheim, Germany)
were used as received. SOD activity was determined by using
superoxide dismutase activity kit II Cat.No. 574601 (Calbiochem)
according to the manufacturer’s protocol. The kit utilizes a tetra-
zolium salt for the detection of superoxide radicals generated by
xanthine oxidase and hypoxanthine. The supercoiled pBR322 DNA
was purchased from Genei, Bangalore (India). Calf thymus DNA (CT-
DNA) was purchased from SigmaeAldrich. Deionized water was
prepared with Bhanu Scientific Instruments co., Bangalore and is
used for the preparation of the TriseHCl buffer.
solvent is used it does not reveal any cytotoxicity [68]. The results
of antitumor activity of the studied complexes show the negligible
effect of DMSO in the complex solutions at a given range of
concentrations. The result showed excellent potential of complex
[Cu(LH)(phen)(OAc)] towards 14 different human carcinoma cell
lines viz., K562 (Leukaemia), MCF7, (Breast), HT29, HCT15, SW620
(Colon), SiHa (Cervix), T24 (bladder), A498,786-O (kidney), MIA-
PACA2 (Pancreas), Hop-62 (Lung), PC3 (Prostrate), DWD (Oral) and
A549 (Alveolar) with a GI50 value comparable with the values
obtained for standard drug Adriamycin (ADR), which is taken as
positive control while complexes [Cu(LH)(OAc)(H2O)2] and
[Cu(LH)(tfbimH)(OAc)(H2O)] were found active towards 786-O
(kidney) cell line. Further in vivo investigations are in progress in
order to understand the mechanism of the antiproliferative
activity for these complexes.
9.2. Synthesis
8. Conclusion
9.2.1. Synthesis of the proligand 2-((E)-(1,3-dihydroxy-2-
methylpropan-2-ylimino)methyl)-6-methoxyphenol (LH)
The proligand LH was synthesized from the reaction of
o-vanillin (100 mg, 0.65 mmol) and 2-amino-2-methylpropane-1,3-
diol (70 mg, 0.65 mmol) according to the procedure reported earlier
[69].
Synthesis and characterization of four Cu(II) o-vanillin Schiff
base complexes [Cu(LH)(OAc)(H2O)2], [Cu(LH)(phen)(OAc)],
[Cu(LH)(tpimH)(OAc)(H2O)] and [Cu(LH)(tfbimH)(OAc)(H2O)] were
carried out using different coligands viz., phen, tpimH and tfbimH.
The effect of varying coligands on the DNA binding and other
biological activities: SOD mimetic, antimicrobic and antitumor
were studied in detail. The results of electronic absorption titra-
tions, fluorescence, viscosity and cyclic voltammetry studies of
Cu(II) complexes carried out using CT-DNA indicated the non-
covalent mode of binding of these complexes with CT-DNA i.e.
electrostatic interactions for complex 1 and intercalation for
complex 2e4.
Yield: 82% (0.012 g, 0.53 mmol). M.p. 165 ꢅC. Anal. (%) Calcd. for
C12H17NO4: C, 60.24; H, 7.16; N, 5.85. Found: C, 60.33; H, 7.24; N,
5.92. FT-IR (cmꢀ1): 3177s br
n
(OH), 1637s
n(C]N of imine), 1494s,
1442s, 1393w, 1369m, 1339m, 1284w, 1216vs, 1160s, 1063vs, 956w,
925s, 888w, 850m, 793w, 746s, 720s
n
(C]O þ C]C). UVevisible
[MeOH, lmax/nm (ε/Mꢀ1 cmꢀ1)]: 216 (46,296), 265 (37,735), 348
(28,735), 417sh (23,980). ESI-MS (MeCN) (þ) (m/z, relative intensity
Table 7
Cytotoxicity against different tumor cell lines in terms of GI50 (mg/ml) value.
Cmp
K562
Zr-75-1
Colo205
MCF7
SiHa
28.6
<10
T24
37.9
<10
A498
MIAPACA2
Hop-62
PC3
23.8
<10
HT29
HCT15
DWD
A549
SW620
786-O
1
2
41.7
<10
>80
33.3
61.0
26.9
29.4
<10
49.5
<10
31.2
<10
55.5
<10
35.4
<10
29.3
10.2
47.1
<10
36.0
<10
42.4
<10
6.9
<10
3
Under investigation
4
39.1
<10
71.4
<10
44.1
<10
27.3
<10
27.5
<10
39.1
<10
38.0
<10
32.0
<10
55.8
<10
24.1
<10
33.7
<10
31.3
<10
33.7
<10
32.9
<10
42.7
<10
10.0
<10
ADRa
‘_’GI50 ꢆ 10 is considered to demonstrate activity.
a
Adriamycin (taken as positive control).

S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
229
%): 240 (100) [C12H17NO4 þ H]þ. 1H NMR (400 MHz, CDCl3):
d
, 14.4
þ H]þ. TGA-DTA (mg% vs. ꢅC): heating from 30 to 600 ꢅC with a speed
of 10 ꢅC/min; from 80 to 100 ꢅC loss of one water molecule (weight
loss found 3.1%, calcd. 2.7%), exothermal event with an onset at 182 ꢅC
(s br, 1H, PheOH), 8.43 (s, 1H, N]CHimine), 6.89 (d, 2H, CHPh), 6.74
(t,1H, CHPh), 3.88e3.80 (m, 4H, CH2OH), 3.70 (s, 3H, OCH3), 3.5 (s br,
2H, CH2OH), 1.32 (s, 3H, CCH3). 13C NMR (100 MHz, CDCl3):
d
, 163.9
(D
H ¼ ꢀ6.7 kJ/mol), then progressive decomposition until to 600 ꢅC,
(N]CHimine), 129.2, 126.7, 123.7, 117.1, 114.4, 113.5 (Caromatics), 68.2,
67.1 (CH2OH), 64.0 (tert-C), 56.2 (OCH3).
with residue of 12.2% weight corresponding to CuO (calcd. 11.8%).
9.2.5. Synthesis of [Cu(LH)(tfbimH)(OAc)(H2O)] (4)
9.2.2. Synthesis of [Cu(LH)(OAc)(H2O)2] (1)
This complex was synthesized according to the procedure
described for complex 2 by using complex 1 (500 mg, 1.26 mmol)
with tfbimH (0.234 g, 1.26 mmol).
To a methanolic solution (10 mL) of the Schiff base ligand LH
(0.5 g, 2.08 mmol) was added a solution of Cu(OAc)2$H2O (0.379 g,
2.089 mmol) in methanol (5 mL). The mixture was allowed to reflux
at 60 ꢅC for 1 h to ensure completion of reaction. The reaction
mixture yielded a solid product (green coloured) which was iso-
lated, washed with hexane and dried in vacuo.
Yield: 80% (0.569 g, 1.007 mmol). M.p. 158 ꢅC. Anal. (%) Calcd. for
C22H26CuF3N3O7: C, 46.77; H, 4.64; N, 7.44. Found: C, 46.82; H, 4.66;
N, 7.48. FT-IR (cmꢀ1) 1626
ns(COO), 1545
(CueN), 560
n
(HC]N of imine), 1602 nas(COO), 1316
(C]C), 3213 (NeH), 1128 (CeF), 465
n(CueO). meff (298 K): 1.99 BM. UVevisible [MeOH,
n
(C]N), 1469
n
n
n
Yield: 80% (0.663 g, 1.671 mmol). M.p. 127 ꢅC. Anal. (%) Calcd. for
C14H23CuNO8: C, 42.37; H, 5.84; N, 3.53. Found: C, 42.50; H, 5.95; N,
n
lmax/nm (ε/Mꢀ1 cmꢀ1)]: 205 (48,780), 223 (44,843), 277 (36,101),
3.59. FT-IR (cmꢀ1): 3234
COO), 1401 ns(COO), 466
1.88 BM. UVevisible [MeOH, lmax/nm (ε/Mꢀ1 cmꢀ1)]: 231 (43,290),
n
(OH), 1621
n
(HC]N of imine), 1602 nas(-
(CueO). meff (298 K):
371 (26,954), 643 (15,552). LM (MeOH, 10ꢀ3 M) ¼ 8.0 S cm2 molꢀ1
.
n
(CueN), 528
n
ESI-MS (MeCN) (þ) (m/z, relative intensity %): 525 (20) [Cu(L)
(tfbim)(H2O)2 ꢀ H]þ, 491 (25) [Cu(L)(tfbim) þ 3H]þ, 304 (15) [Cu
(L) þ 2H]þ. TGA-DTA (mg% vs. ꢅC): heating from 30 to 600 ꢅC with
a speed of 10 ꢅC/min; from 100 to 150 ꢅC loss of one water molecule
(weight loss found 3.4%, calcd. 3.2%), melting at 158 ꢅC, then
progressive decomposition until to 600 ꢅC, with residue of 14.5%
weight corresponding to CuO (calcd. 14.1%).
278 (35,866), 368 (27,106), 675 (14,814).
L
(MeOH,
M
10ꢀ3 M) ¼ 8.6 S cm2 molꢀ1. ESI-MS (MeCN) (þ) (m/z, relative
intensity %): 399 (20) [CuC12H17NO4(CH3COO)(H2O)2 þ 4Hþ], 383.3
(80) [CuC12H17NO4(CH3COO)(H2O)
þ
4Hþ], 319.4
(15)
[CuC12H17NO4(H2O)]þ, 301.4 (50) [CuC12H17NO4]þ, 304.6 (100)
[CuC12H17NO4 þ 3H]þ. TGA-DTA (mg % vs. ꢅC): heating from 30 to
600 ꢅC with a speed of 10 ꢅC/min; from 80 to 100 ꢅC loss of two
9.3. Methods
water molecules (weight loss found 9.4%, calcd. 9.0%,
D
H ¼ 13.3 kJ/
mol), melting at 127 ꢅC, exothermal event with an onset at 264 ꢅ
C
9.3.1. Physical measurements
(D
H ¼ ꢀ7.9 kJ/mol), then progressive decomposition till 600 ꢅC,
Elemental analyses were carried out with a Fison instrument
1108 CHNSeO elemental analyzer. Melting points were obtained
using SMP3 stuart scientific melting point apparatus. IR reflectance
spectra were recorded from 4000 to 380 cmꢀ1 with a PerkineElmer
Spectrum 100 FT-IR instrument. Molar conductance measurements
were measured at room temperature on a Digsun Electronic
conductivity Bridge. 1H and 13C {1H} NMR spectra were recorded on
a 400 Mercury Plus instrument operating at room temperature
(400 MHz for 1H, 100 MHz for 13C) and with a Varian (200 MHz for
1H, 50 MHz for 13C). TGA spectra were obtained with a STA 6000
Simultaneous Thermal Analyzer PerkineElmer by conducting the
thermal studies under an inert atmosphere of dry nitrogen. Elec-
tron paramagnetic resonance (EPR) spectra of the Cu(II) complexes
were obtained on a Varian E 112 EPR spectrometer at a frequency of
9.1 GHz under the magnetic field strength 3000 ꢄ 1000 Gauss using
tetracyanoethylene (TCNE) as field marker at LNT. H and C chemical
with residue of 20.3% weight corresponding to CuO (calcd. 20.0%).
9.2.3. Synthesis of [Cu(LH)(phen)(OAc)] (2)
To a methanolic solution (5 mL) of complex 1 (0.5 g, 1.259 mmol)
was added dropwise methanolic solution (5 mL) of phen (0.250 g,
1.26 mmol) and the reaction mixture was refluxed at 60 ꢅC for 5 h.
The reaction mixture yielded a solid product (green coloured)
which was isolated, washed with diethyl ether and methanol in
portion and dried in vacuum.
Yield: 82% (0.559 g, 1.03 mmol). M.p. 147 ꢅC. Anal. (%) Calcd. for
C26H27CuN3O6: C, 57.72; H, 5.03; N, 7.77. Found: C, 57.89; H, 5.12; N,
7.82. FT-IR (cmꢀ1): 3234
n
(H2O þ OH), 1625
nas(COO), 1374 ns(COO), 1516 (C]N), 1429
472 (CueN). meff (298 K): 1.95 BM. UVevisible [MeOH, lmax/nm
n
(HC]N of imine), 1601
n
n
(C]C), 536 (CueO),
n
n
(ε/Mꢀ1 cmꢀ1)]: 218 (45,806), 273 (36,541), 381 (26,246), 633
(15,797). LM (MeOH,10ꢀ3 M) ¼ 5.8 S cm2 molꢀ1. ESI-MS (MeCN) (þ)
(m/z, relative intensity %): 481 (100) [CuC12H17NO4(phen)]þ, 302.4
(65) [CuC12H17NO4 þ 2H]þ. TGA-DTA (mg% vs. ꢅC): heating from 30
to 600 ꢅC with a speed of 10 ꢅC/min; melting at 147 ꢅC, exothermal
shifts (d
) are reported in parts per million (ppm) from SiMe4 (1H
and 13C calibration by internal deuterium solvent lock). Peak
multiplicities are abbreviated: singlet, s; doublet, d; triplet, t;
quartet, q; multiplet, m.
event with an onset at 269 ꢅC (
DH ¼ ꢀ5.3 kJ/mol), then progressive
Absorption spectral titration experiments were performed with
UV-1700 PharmaSpec UVevisible spectrophotometer (Shimadzu)
spectrophotometer. UVevisible data were reported as lmax/nm.
Emission intensity measurements were carried out using Shimadzu
RF-5301PC spectrofluorophotometer at room temperature. DNA
binding experiments including absorption spectral traces, emission
spectroscopy viscosity and cyclic voltammetry were performed in
accordance with the standard methods and practices previously
adopted by our laboratory [70]. A solution of calf thymus DNA in the
decomposition until to 600 ꢅC, with residue of 15.2% weight cor-
responding to CuO (calcd. 14.7%).
9.2.4. Synthesis of [Cu(LH)(tpimH)(OAc)(H2O)] (3)
This complex was synthesized according to the procedure
described for complex 2 by using complex 1 (500 mg, 1.26 mmol)
with tpimH (0.373 g, 1.26 mmol).
Yield: 72% (0.613 g, 0.72 mmol). M.p. >300 ꢅC. Anal. (%) Calcd. for
C35H37CuN3O7: C, 62.26; H, 5.52; N, 6.22. Found: C, 62.38; H, 5.61; N,
Tris-hydroxymethylaminomethane buffer gave
a ratio of UV
6.39. FT-IR (cmꢀ1): 3234
n
(H2O þ OH), 3202
imine), 1602 nas(COO), 1315 ns(COO), 1544 (C]N), 1469
(CueN), 530 (CueO). meff (298 K): 1.97 mB. UVevisible [MeOH, lmax/
n(NeH),1624
n(HC]N of
absorbance at 260 and 280 nm of about 1.90 indicating that the
DNA was sufficiently free of protein [71]. The DNA concentration
per nucleotide was determined by absorption spectroscopy using
the molar absorption coefficient (6600Mꢀ1 cmꢀ1) at 260 nm [72].
Magnetic susceptibilities were determined at 297 K with Sherwood
scientific magnetic susceptibility balance. Diamagnetic corrections
were carried out with Pascal’s increments [73]. Cyclic voltammetry
n
n(C]C), 468
n
n
nm (ε/Mꢀ1 cmꢀ1)]: 202 (49,504), 229 (43,668), 278 (35,971), 372
(26,881), 650 (15,384). LM (MeOH,10ꢀ3 M) ¼ 9.9 S cm2 molꢀ1. ESI-MS
(MeCN) (þ) (m/z, relative intensity %): 676 (15) [C12H17CuNO4
(tpimH)(CH3COO)(H2O) þ 2Hþ], 338 (100) [C12H17CuNO4 þ 2H2O

230
S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
measurements were carried out using a CH Instrument Electro-
chemical analyzer in a single compartmental cell. A conventional
three electrode arrangement consisting of platinum wire as auxil-
iary electrode, glassy-carbon as working electrode and Ag(s)/AgCl
electrode as reference electrode, was used in the presence of 0.4 M
KNO3 as supporting.
buffer. The gels were stained with ethidium bromide (EB) and
visualized under ultraviolet light.
9.3.6. Measurement of SOD-like activity
Superoxide dismutase (SOD)-like activity was investigated
using Beauchamp and Fridovich’s method e as improved by Ima-
nari et al. [74]. This method is based on the inhibitory effect of SOD
$ꢀ
9.3.2. X-ray powder diffraction analysis
on the reduction of nitrobluetetrazolium (NBT) by the O2
A strictly monophasic sample of the proligand LH was gently
grinded in an agate mortar, and then deposited in the cavity of
a 0.2 mm deep aluminium sample holder, equipped with a quartz
monocrystal zero background plate (supplied by The Gem Dugout,
generated by the xanthine/xanthine oxidase system. The assay was
carried out in the assay buffer containing 50 mM Tris HCl, pH 8.0,
0.1 mM DTPA and 0.1 mM hypoxanthine. Radical detector consists
of a tetrazolium salt and is diluted by assay buffer. Similarly, the
solutions of SOD standards and xanthine oxidase were prepared in
sample buffer consisting of 50 mM TriseHCl, pH 8.0. All the studied
complexes were dissolved in ethanol and absorbance was reported
for each set of concentrations after 15 min interval. Results were
graphed as % inhibition of NBT reduction taken at various concen-
State College, PA). Diffraction data were collected in the 5e105ꢅ 2
q
range, sampling at 0.02ꢅ, on a
q:q vertical scan Bruker AXS D8
Advance diffractometer, equipped with a linear Lynxeye position
sensitive detector, set at 300 mm from the sample. Ni-filtered Cu-
K
a1,2 radiation,
l
¼ 1.5418 Å. Standard peak search methods, fol-
lowed by indexing by TOPAS-R (V 3.0, 2005, Bruker AXS, Karls-
ruhe, Germany), allowed the determination of approximate cell
parameters (GOF(26) ¼ 17.5, space group P ꢀ 1, later confirmed by
successful structure solution and refinement). Structure solution
was initiated by employing a semirigid molecular fragment, flex-
ible about a few torsional angles, build by molecular modelling
and optimized by molecular mechanics (in vacuo) using the
freeware programs distributed by ACD package (ChemSketch
version 11.01, 2007). Simulated annealing allowed the location,
orientation and conformation of the molecule, later refined by the
Rietveld method. The fundamental parameter approach in
describing the peak shapes was employed, the background
contribution was modelled by a polynomial fit, and preferred
orientation effects corrected by the MarcheDollase formalism
(001 pole, g ¼ 0.91) A single isotropic thermal parameter was
adopted for all atoms. Fig. 1 shows the final Rietveld refinement
plots.
trations (0
m
M-20
m
M) for each of the studied complexes. IC50 values
M) to 1U bovine eryth-
are reported equivalent concentrations (
m
rocyte superoxide dismutase (native SOD).
9.3.7. Antimicrobic activity
9.3.7.1. Antifungal screening. All the studied complexes were tested
for their in vitro growth inhibition activity against pathogenic
fungus viz., C. albicans and A. niger cultured on potato dextrose agar
(PDA) medium and incubated at 25 ꢄ 2 for 72 h by using poison
food technique [75]. The PDA medium was sterilized in autoclave in
15 kg/cm2 at 121 ꢅC which keeps the medium to retain its normal
temperature. Solutions of each of the complex was added in the
PDA medium at different concentrations in the range of (10e
50) ꢃ 10ꢀ6 M. The inhibition of the fungal growth, expressed as
percent, was determined on the growth in test plates compared to
respective control plates (untreated), as given by Vincent equation
(2) [76].
9.3.3. Crystal data for LH
Inhibition % ¼ ðC ꢀ TÞ=C ꢃ 100
(2)
LH: C12H17NO4, fw
¼
239.27
g
molꢀ1
;
triclinic,
a
P
ꢀ
1,
a ¼ 7.2777(3) Å, b ¼ 8.1109(2) Å, c ¼ 10.6231(3) Å;
¼ 88.380(2)ꢅ,
Where C is the diameter of fungal growth of the control plate and T
is the diameter of the fungal on the test plate.
b
¼ 87.383(2)ꢅ,
g
¼ 102.853(2)ꢅ; V ¼ 610.24(3) Å3; Z ¼ 2;
rcalc ¼ 1.302 g cmꢀ3; F(000) ¼ 256;
m(Cu-K
a
) ¼ 8.17 cmꢀ1. Rp, Rwp
,
and RBragg, 0.045, 0.063 and 0.037, respectively for 38 parameters.
The CIF file is supplied in the (Electronic Supporting Information)
CCDC No. 829822.
9.3.7.2. Antibacterial screening. Solutions of the studied complexes
(10e50) ꢃ 10ꢀ6 M in DMSO were applied on a paper disc prepared
from sterilized paper (3 mm diameter) with a micropipette. The
discs were left in an incubator for 30 h at 37 ꢅC and then applied on
the bacterial growth viz., Staphylococcus aureus, Bacillus subtilis,
Salmonella typhimurium and Escherichia coli on potato dextrose agar
(PDA) plates. Minimal agar was used for growth of specific bacterial
species. Agar (50 g) was suspended in freshly distilled water (1 L),
allowed to soak for 15 min and then boiled on water bath until the
agar was completely dissolved. The mixture was autoclaved for
15 min at 120 ꢅC and then poured into previously washed and
sterilized petri dishes and stored at 40 ꢅC for inoculation.
9.3.4. Absorption titration of Cu(II)complexes binding to DNA
The binding strength of all the studied complexes with CT-DNA
was determined by intrinsic binding constant Kb employing equa-
tion (1) and monitoring the changes in the absorbance of the
and LMCT bands with increasing concentration of CT-DNA.
pep*
ꢀ
ꢁ
½DNAꢂ=εa ꢀ εf ¼ ½DNAꢂ=εb ꢀ εf þ 1=Kb εb ꢀ εf
(1)
Where εa, εf and εb corresponds to (Aobsd/[Cu]), the extinction
coefficient of free Cu(II) complex and the extinction coefficient for
the Cu(II) complex in the fully bound forms, respectively. A plot of
[DNA]/εa ꢀ εf vs [DNA], where [DNA] is the concentration of DNA in
the base pair, gives Kb as the ratio of slope to the intercept.
9.3.8. Antitumor activity
The cell lines used for in vitro antitumor screening activity were,
K562, ZR-75-1, Colo205, MCF7, SiHa, T24, A498, MIAPACA2, Hop-62,
PC3, HT29, HCT15, DWD, A549, SW620 and 786-O. These human
cancer cell lines were procured and grown in RPMI-1640 medium
supplemented with 10% foetal bovine serum (FBS) and antibiotics to
study growth pattern of these cells. The proliferation of the cells
upon treatment with the studied complexes was determined using
the sulphorhodamine-B (SRB) semi automated assay. Cells were
seeded in 96 well plates at an appropriate cell density to give optical
density in the linear range (from 0.5 to 1.8) and were incubated at
37 ꢅC in CO2 incubator for 24 h. Stock solutions of the studied
9.3.5. DNA strand break analysis
The pBR322 DNA in 50 mm TriseHCl, pH 8.0, containing 0.1 mm
DTPA was incubated with complex [Cu(LH)(phen)(OAc)] which
$ꢀ
exhibit higher SOD activity among complexes 1e4. O2
was
generated from XOD and hypoxanthine for 1 h. The samples were
analyzed by electrophoresis at 100 V in 0.7% agarose gel in Tris
acetate EDTA buffer at 25 ꢅC by adding 4
mL volume of loading

S. Tabassum et al. / European Journal of Medicinal Chemistry 60 (2013) 216e232
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We thank, University of Camerino, Italy, for providing financial
support with useful suggestions throughout this work and facili-
tating NMR, elemental analysis, magnetic susceptibility and IR
facility, Department of Biotechnology, New Delhi for generous
finical support (Scheme No. BT/PR6345/Med/14/784/2005), RSIC,
Punjab University, Chandigarh for spectroscopy facility, Dr. Iqbal
Ahmad, Interdisciplinary agricultural microbiology, Aligarh Muslim
university, for antimicrobial analysis and Dr. (Mrs.) Aarti Juvekar,
(ACTREC), Kharghar, Navi Mumbai, for assessing cellular prolifera-
tion through (SRB) assay against 16 different human carcinoma cell
lines.
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