DNA fastening and ripping actions of novel Knoevenagel condensed dicarboxylic acid complexes in antitumor journey

Article abstract of DOI:10.1016/j.ejmech.2014.04.032

Few novel Cu(II) and Zn(II) oxali-platin type complexes of stoichiometry [ML(ox)] where, L is a Knoevenagel ligand and ox is oxalic acid, have been explored. They are well characterized by spectroanalytical methods. The binding and cleavage propensity of these complexes on DNA and their cytotoxic effect in tumor cells have been investigated. They bind to DNA preferentially by intercalation and cleave the strands under mild reaction conditions even in the absence of external cofactors. However, in H₂O₂ medium they exhibit better efficacy in the nuclease reaction process by initiating DNA cleavage in an oxidative pattern. Complex 1 shows higher in vitro cytotoxic property against HeLa/EAC cells comparing to other complexes and the standards (cisplatin/5-FU). Moreover, the in vivo antitumor efficacy of copper complexes against EAC tumor model reveals that they are non-toxic to normal cells (lymphocytes). Among the copper complexes, complex 1 reveals excellent antitumor activity.

Full text of DOI:10.1016/j.ejmech.2014.04.032

European Journal of Medicinal Chemistry 80 (2014) 57e70
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
DNA fastening and ripping actions of novel Knoevenagel condensed
dicarboxylic acid complexes in antitumor journey
*
Natarajan Raman , Narayanaperumal Pravin
Research Department of Chemistry, VHNSN College, Virudhunagar 626 001, Tamil Nadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Few novel Cu(II) and Zn(II) oxali-platin type complexes of stoichiometry [ML(ox)] where, L is a Knoe-
venagel ligand and ox is oxalic acid, have been explored. They are well characterized by spectroanalytical
methods. The binding and cleavage propensity of these complexes on DNA and their cytotoxic effect in
tumor cells have been investigated. They bind to DNA preferentially by intercalation and cleave the
strands under mild reaction conditions even in the absence of external cofactors. However, in H2O2
medium they exhibit better efficacy in the nuclease reaction process by initiating DNA cleavage in an
oxidative pattern. Complex 1 shows higher in vitro cytotoxic property against HeLa/EAC cells comparing
to other complexes and the standards (cisplatin/5-FU). Moreover, the in vivo antitumor efficacy of copper
complexes against EAC tumor model reveals that they are non-toxic to normal cells (lymphocytes).
Among the copper complexes, complex 1 reveals excellent antitumor activity.
Received 18 November 2013
Received in revised form
18 March 2014
Accepted 8 April 2014
Available online 13 April 2014
Keywords:
Oxali-platin type complexes
Intercalative mode
DNA cleavage
Ó 2014 Elsevier Masson SAS. All rights reserved.
Cytotoxicity
Antitumor effect
1. Introduction
pharmacological profile different than those of platinum drugs
[5,6]. Many authentic reports are available in the literature which
The major focus of research in chemotherapy for cancer in
recent times includes the identification, characterization and
development of new and safe cancer chemopreventive agents [1].
Over the past 30 years platinum compounds have played a very
important and well documented role in treating cancer [2,3].
Especially, oxali-platin, as the prototype of the third-generation
platinum drugs which are characterized by substitution of both
the amine carrier ligands and the labile chlorido leaving groups of
cisplatin with 1,2-diaminocyclohexane (1,2-DACH) and carboxylato
groups, possesses rare cross-resistance with cisplatin, appropriate
water-solubility and reduced toxicity. Also, it is the first platinum-
based chemotherapeutics in the treatment of metastatic colorectal
cancer [4]. As it possesses inherent limitations such as lesser side
effects, general toxicity, and acquired drug resistance, attempts are
being made to replace the platinum-based drugs with suitable al-
ternatives, and numerous metal based complexes are synthesized
and screened for their anticancer activities. Further, the non-
platinum antitumor compounds show various geometries and co-
ordination numbers, various oxidation states, better solubility,
feasible substitution kinetic pathways and factors influencing the
reveals the anticancer activity of the non-platinum transition metal
complexes containing oxygen and nitrogen donors [7]. Large
number of studies has been carried out on non-platinum transition
metal complexes for their ability to mediate oxidative damage to
nucleobases and/or to the 2-deoxyribose moiety in presence
of oxidizing or reducing agent, light, or redox-active metal center
[8e11].
Recently, few of the mono-,di- and multinuclear metal com-
plexes of Cu(II), Fe(III), Zn(II), Ru(II), Co(III), Ln(III)/(IV) with aza-
macrocyclic, aminocarboxylic, pyridyl, benzimidazolyl, ferrocenyl
and others are reported to show strong chemical nucleases activity
under physiological conditions [12]. Compared with other transi-
tion metals, Cu(II) ion has relatively strong Lewis acidity and high
nucleobase affinity to induce efficient DNA cleavage activity.
Generally, copper is a biometal and its complexes are less toxic and
some of them have even important cellular effects such as neuro-
transmission, cellular respiration and others. Recent studies on new
kinds of chemotherapeutic Schiff bases are now attracting the
attention of biochemists. The coordination behavior of b-diketone
derivatives also has significant influences on the relative stabilities
of the mixedeligand complexes as well as their use in biomedicine
[13,14]. Moreover, the emerging Knoevenagel condensate Schiff
base metal complexes have been a wide variety of applications in
various fields, like agriculture, industry, pharmaceutical etc.,
[15,16]. In this regard, N2O2 mixed-ligand metal complexes are
* Corresponding author.
E-mail
addresses:
ramchem1964@gmail.com,
drn_raman@yahoo.co.in
(N. Raman), pravinknp2012@gmail.com (N. Pravin).
http://dx.doi.org/10.1016/j.ejmech.2014.04.032
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

58
N. Raman, N. Pravin / European Journal of Medicinal Chemistry 80 (2014) 57e70
found to be particularly useful because of their potential to bind
DNA via a multitude of interactions and to cleave the duplex by
virtue of their intrinsic chemical, electrochemical, and photo-
chemical activities [17,18].
4.1. Infrared spectra
The tentative assignments of the IR bands are useful tools for
determining the coordination mode of the ligands with metal
ions. There are some elevated peaks in the spectra of the ligands
which are good in accomplishing this goal. The position and/or
the intensities of these peaks are expected to be changed upon
chelation. The bands observed in the range of 1638e1631 cmꢀ1
Over the last few years, dicarboxylic acids [19e21] and the ox-
alate containing compounds hold an active field of research in the
systematic construction of mixed ligand metal complexes with
interesting properties in biological systems towards applications in
biological chemistry, catalysis, photochemistry and magneto-
chemistry [22]. Also, this is widely used in crystal engineering that
may serve as “templates” for new functional materials [23,24].
Based on the above facts, we are tempted to develop new non-
platinum anticancer agents. In continuation of our journey in
designing and exploring the newer anticancer agents [25] in order
to improve the anticancer efficacy, we hereby report the synthesis
and characterization of few new oxali-platin type copper(II) and
zinc(II) complexes. Herein, the in vitro and in vivo antitumor ac-
tivities against two human cancer cell lines (HeLa and MCF-7) and
animal tumor cell line (EAC) have been evaluated.
for the azomethine group
n(C]N) authenticating an effective
Schiff base condensation between 3-benzylidene-pentane-2,4-
dione and p-substituted anilines, have undergone a negative
shift in the complexes in the range of 1615e1601 cmꢀ1 ca.
30 cmꢀ1 on comparing with the free ligands. This may be
explained on the basis of a drift of lone pair density of azome-
thine nitrogen towards the metal ions suggesting that the co-
ordination takes place through nitrogen of n(eC]N) [26]. Be-
sides, the free eOH group of the ligand L3 vibrated at ca.
3430 cmꢀ1 does not show any significant shift on complex for-
mation, which confirms that the phenolic group is not partici-
pated on complex formation.
2. Chemistry
The additional bands in complexes at 1686, 1347 and 816 cmꢀ1
could be ascribed to the vibrations of the oxalic acid moiety. This
suggests the presence of dicarboxylic acid as a bidentate ligand in
the metal(II) centers via two monodentate carboxylate groups.
The mixed oxalato complexes show the stretching vibration of
The novel Knoevenagel condensate Schiff bases L1eL4 and their
mixed-ligand (i.e., oxalic acid) Cu(II) and Zn(II) complexes were
synthesized and characterized by spectral and elemental analysis
data. The complexes were found to be air stable. The ligands are
soluble in common organic solvents but their complexes are solu-
ble only in DMF and DMSO. The results of elemental analysis for the
metal complexes are in good agreement with the calculated values
showing that the complexes have 1:1:1 (Knoevenagel condensate
Schiff base: metal: oxalic acid) stoichiometry of the type [ML(ox)]
wherein L acts as a bidentate ligand. The metal(II) complexes were
dissolved in DMF and the molar conductivities of 10ꢀ3 M of their
solution at room temperature were measured. The lower conduc-
n
C]O of the oxalate groups at 1689e1681 cmꢀ1. The relatively
high value for this group indicates the unsharing of the C]O
group in coordination to metal ion and thus the oxalato group
acts as a dianionic bidentate ligand. This is further confirmed by
the formation of metaleoxygen bond in the complexes in the
region 505e533 cmꢀ1. The new band observed in the complexes
in the range 448e430 cmꢀ1 indicates the formation of MeN
bond.
tance values (8.3e15.2
U
ꢀ1 cmꢀ2 molꢀ1) of the complexes support
4.2. Elemental analysis and molar conductivity measurements
their non-electrolytic nature.
The results of elemental analysis for the metal complexes are in
good agreement with the calculated values showing that the
complexes have 1:1:1 metaleligand stoichiometry of the type
[ML(ox)] wherein L and (ox) act as bidentate ligands (Scheme 1).
3. Pharmacology
The DNA binding study of the synthesized complexes with CT
DNA was carried out by electronic absorption spectroscopy, vis-
cosity measurements and cyclic voltammetry techniques. The
extent of pBR322 DNA cleavage, in the absence and presence of an
activating agent H2O2 and various radical scavengers like sodium
azide (singlet oxygen), SOD (superoxide), DMSO (hydroxyl radical
scavenger), was monitored using agarose gel electrophoresis. A
meticulous sympathetic of the structural and electronic properties
of drug-DNA complexes and their mechanism of binding is the key
step in elucidating the principles of their anti-cancer activity.
In vitro and in vivo anti-tumor functions of synthesized complexes
against Ehrlich ascites carcinoma (EAC) tumor model were inves-
tigated. The antitumor activity was assessed by hematological pa-
rameters, median survival time and cell viability with trypan blue
dye exclusion assay. In vitro cytotoxicity was performed by MTT
assay against human cervical cancer cell lines (HeLa) and Human
breast cancer lines (MCF-7). The minimum inhibitory concentration
(MIC) values of the complexes were determined.
The complexes are found to be non-electrolytic nature in 10ꢀ3
M
DMF solution, implying the replace of chloride anions by bidentate
oxalate to the central metal ion. The absence of counter (chloride)
ion is conformed from Volhard’s test. The molar conductance values
(8.3e15.2 Uꢀ1 cmꢀ2 molꢀ1) of the complexes support their non-
electrolytic nature.
4.3. Magnetic moments and electronic spectra
The electronic absorption spectra are often very helpful in the
evaluation of results furnished by other methods of structural
investigation. The geometry of the metal complexes has been
deduced from electronic spectra and magnetic data of the com-
plexes, which were recorded in DMF solution. The free ligands
exhibit two intense bands in 45,857e41,656 and 28,361e
27,138 cmꢀ1 region due to
respectively. In all the metal complexes, the absorption bands at
40,475e43,836 and 29,561e35,225 cmꢀ1 are due to
* and
p / p* and n / p* transitions [27],
p
/ p
4. Results and discussion
n / p* transitions that are observed in the spectra of the free li-
gands L1, L2, L3 and L4. These transitions are shifted to blue or red
frequencies due to the coordination of the ligand with metal ions.
The electronic spectra of Cu(II) complexes (1e4) show broad bands
The formation of the investigated Schiff bases and their mixed
ligand complexes is represented in Scheme 1. The ligands and their
complexes are found to be stable in air. The ligands are soluble in
common organic solvents but their complexes are soluble only in
DMF and DMSO.
at around 22,883e23,364 cmꢀ1
2B1g
planar environment around the copper(II) ion. The observed
,
which are assigned to the
/
2A1g transition which is characteristic of distorted square

N. Raman, N. Pravin / European Journal of Medicinal Chemistry 80 (2014) 57e70
59
Scheme 1. Synthesis of Schiff base ligands and their metal complexes.
magnetic moment of the Cu(II) complexes (1e4) (1.84e1.88 B.M) at
room temperature indicates the non-coupled mononuclear com-
plexes of magnetically diluted d9 system with S ¼ 1/2 spin-state.
The monomeric nature of the complexes is further supported by
the microanalytical and ESI mass spectral data. The electronic ab-
sorption spectra of the diamagnetic Zn(II) complexes show the
bands at 40,561e42,359 and 29,198e33,853 cmꢀ1 which are
assigned to intra-ligand charge transfer transitions [28].

60
N. Raman, N. Pravin / European Journal of Medicinal Chemistry 80 (2014) 57e70
Table 1
4.4. NMR spectra of Zn(II) complexes
The spin Hamiltonian parameters of the Cu(II) complexes in DMSO solution at 77 K.
In 1H NMR, the aromatic region is a set of multiplets in the range
of 6.8e7.4 ppm for all the ligands and their Zn(II) complexes. The
phenolic eOH proton for L3 ligand and its Zn(II) complex is
observed as a singlet at ca.10.3 ppm. It is suggesting that phenolic e
OH group is not taking part in the complexation. 1H NMR spectra of
aliphatic methyl protons exhibit at 2.1e2.4 ppm for all the Schiff
base ligands and their Zn(II) complexes. There is no appreciable
change in all other signals of the complexes. The spectra of ligand L4
and its complex 8 are given in Figs. S1a and S1b (Supplementary
file).
Complexes
g-tensor
A ꢁ 10ꢀ4 (cmꢀ1
)
g
jj/A
G
jj
g
jj
gt
giso
A
jj
At
Aiso
1
2
3
4
2.15
2.19
2.22
2.21
2.04
2.03
2.05
2.04
2.08
2.09
2.11
2.10
149
145
142
151
25
23
27
24
66
64
65
66
144
151
156
146
4.12
5.02
4.41
5.63
ꢀ
ꢁ.
G ¼ g ꢀ 2 ðgt ꢀ 2Þ
jj
The 13C NMR spectra of the ligands show aromatic carbons at
119e129 ppm. The ligands also show the C]N carbons at 172.8e
175.4 ppm, which are shifted to downfield at 168.2e170.4 ppm,
upon coordination indicating that the C]N groups participate in
complex formation. The peak at 164.3e168.7 ppm of Zn(II) com-
which measures the exchange interaction between the copper
centers in polycrystalline solid. The G values lie within the range
4.12e5.63 for all the Cu(II) complexes indicating negligible ex-
change interaction of CueCu in the complexes according to Hath-
plexes indicates the presence of oxalic acid carbon moiety. The 13
C
away [29]. The empirical factor f ¼ g /A cmꢀ1 is an index of
jj jj
NMR spectra of ligand L4 and its complex 8 are given in Figs. S2a
and S2b. Comparison of all carbon peaks of the ligands with
those of Zn(II) complexes show some upfield and downfield shifts,
but these shifts are not so large. This indicates the coordination of
the ligand to the metal ion.
tetragonal distortion. Values of this factor may vary from 105 to 135
for small to extreme distortions in square planar complexes and it
depends on the nature of the coordinated atoms. The f values of
copper complexes (1e4) are in the range from 144e156, indicating
significant distortion from planarity. The EPR studies of the Cu(II)
complexes have provided supportive evidence to the conclusion
obtained on the basis of electronic and magnetic moment values.
4.5. Mass spectra
The ESI-mass spectra of synthesized ligands and their com-
plexes were recorded and the obtained molecular ion peaks
confirm the proposed formulas. The mass spectrum of L1 ligand
shows [M þ 1] peak at m/z 429 (86.4%) corresponding to
[C24H20N4O4]þ ion. Also, the spectrum exhibits the fragments at m/
z 184, 102 and 77 corresponding to [C12H12N2]þ, [C8H5]þ and
[C5H6]þ respectively. The mass spectrum of complex 1 shows
peaks at m/z 581 with 65.2% abundances, respectively. The stron-
gest peaks (base beak) at m/z 429 represent the stable species
4.7. DNA binding studies
4.7.1. Absorption titration measurement
Most of the antitumor drugs act by targeting DNA and therefore,
DNA binding is one of the most critical steps for the action of a large
number of metal based anticancer drugs. Electronic absorption
spectroscopy is used as a distinctive characterization tool for
examining the binding mode of metal complexes with DNA [30].
DNA is known to offer several binding modes (outer-sphere non-
covalent binding, metal coordination to nucleobases and phos-
phate backbone interactions) to the anticancer drugs [31]. The
binding of intercalative ligand to DNA has been well characterized
classically through absorption titration, following the hypochrom-
ism and red shift associated with the binding of the colored com-
plex to the helix, due to the intercalative mode involving a strong
stacking interaction between an aromatic chromophore and the
DNA base pairs. The magnitudes of the hypochromism and red shift
are commonly found to depend on the strength of the intercalative
interaction [32].
C
24H20N4O4. Moreover, the spectrum exhibits the fragments at m/z
184, 102 and 77 corresponding to [C12H12N2]þ, [C8H5]þ and [C5H6]þ
respectively. The m/z of all the fragments of ligands and their
complexes confirm the stoichiometry of the complexes as
[ML1(ox)]. The observed peaks are in good agreement with their
formulas as expressed from microanalytical data. Thus, the mass
spectral data reinforce the conclusion drawn from the analytical
and conductance values.
4.6. EPR spectra
The absorption spectra of the mixed oxalato complexes 1 and 5
in the presence or absence of DNA were mutually compared, which
are shown in Fig. 1 and Fig. 2. In the UV region of the spectra, all the
Cu(II) complexes exhibited an intense absorption around 331.0e
386.4 nm and Zn(II) complexes showed bands in the region 322.5e
The EPR spectra of Cu(II) complexes (1e4) provide information
about hyperfine and super hyperfine structures. It is very
important to understand the metal ion environment in the
complexes, i.e., the geometry, nature of the donating atoms from
the ligands and degree of covalency of the Cu(II)eligand bonds.
The X-band EPR spectra of all Cu(II) complexes (1e4) have been
recorded in DMSO at liquid nitrogen temperature and at room
temperature. The spectra of the Cu(II) complexes at RT show one
intense absorption band in the high field and are isotropic due to
the tumbling motion of the molecules. However, these complexes
at LNT show three-well resolved peaks with low field region. The
spin Hamiltonian parameters of the complexes were calculated
and are summarized in Table 1. From this spectral data, it is found
339.3 nm (due to nep* transition). With increasing concentration
of DNA, both the ligands and their complexes showed hypo-
chromicity and a red-shifted charge transfer peak maxima in the
absorption spectra. The hypochromicity values of all the complexes
observed in the presence of DNA were in the range 6.0e16.2%, and
their red shifts in the region 2.0e6.2 nm. This feature might be
ascribed to the fact that all the complexes could untwist the helix
structure of DNA and made more bases embedding in DNA exposed
[33,34]. The change in the absorbance values with increasing
amount of DNA was used to evaluate the intrinsic binding constant
Kb for the present complexes, the values of which are given in
Table 2. The change in hypochromicity may be attributed to the
nature of the binding of the complexes with DNA, which is signif-
that A (142e151) > At (23e27); g (2.22e2.15) > gt(2.05e
jj
jj
2.03) > ge (2.0023), which support the dx2ꢀy2 as the ground state,
characteristic of square-planar geometry and axially symmetric.
Further, in an axial symmetry, the G-values are related by the
expression,
icant due to p-stacking or hydrophobic interactions of the aromatic

N. Raman, N. Pravin / European Journal of Medicinal Chemistry 80 (2014) 57e70
61
Table 2
Electronic absorption spectral properties of Cu(II) and Zn(II) mixed ligand
complexes.
Complexes
lmax (nm)
Dl (nm)
aH%
bKb (Mꢀ1
)
Free
Bound
1
331.0
384.0
351.5
341.8
386.4
322.5
320.6
339.3
329.5
329.0
379.0
354.5
335.6
384.3
319.5
315.4
337.6
331.5
2.0
5.0
3.0
6.2
2.1
3.0
5.2
1.7
2.0
14.8
12.3
8.5
8.7
6.0
10.1
16.2
9.4
2.3 ꢃ 0.05 ꢁ 105
2.0 ꢃ 0.07 ꢁ 105
1.9 ꢃ 0.13 ꢁ 104
1.2 ꢃ 0.04 ꢁ 104
1.5 ꢃ 0.15 ꢁ 104
1.8 ꢃ 0.03 ꢁ 105
1.5 ꢃ 0.08 ꢁ 104
1.3 ꢃ 0.14 ꢁ 104
1.2 ꢃ 0.11 ꢁ 104
2
3
4
5
6
7
8
6.1
a
H% ¼ [(AfreeꢀAbound)/Afree] ꢁ 100%.
b
Kb ¼ Intrinsic DNA binding constant determined from the UVeVis absorption
spectral titration.
hand, in the existence of a partial and/or non-classic intercalation of
a compound to DNA grooves, a bend or kink in the DNA helix should
appear resulting in a slight reduce of its effective length with the
DNA viscosity showing a slight decrease or remaining unchanged.
The viscosity of the DNA solution increased with increasing ratio of
the complexes to DNA. As expected, the known DNA-intercalator EB
increased the relative viscosity of DNA due to its strong intercala-
tion (Fig. 3). Compared with EB, complexes exhibit minor increase
in the relative viscosity of CT-DNA, suggesting an intercalation
mode between the complexes and DNA. This result further suggests
an intercalating binding mode of the complexes with DNA and also
parallels the above spectroscopic results, such as hypochromism
and red shift of the complexes in the presence of DNA. The viscosity
studies provide a strong evidence for intercalation. The information
obtained from this study could be helpful to understand the
mechanism of the interaction of small molecules with nucleic acids,
and should be useful in the development of potential probes of DNA
structure and conformation.
Fig. 1. Absorption spectrum of complex 1 in buffer pH ¼ 7.2 at 25 ꢂC in presence of
increasing amount of DNA.
phenyl rings [35]. However, the metal ions play decisive role in DNA
binding by these complexes. The binding strength of the synthe-
sized complexes with DNA is shown as in the following order:
1 z 5 > 2 z 6 > 4 z 8 > 3 z 7. The strong binding affinity of the
metal complexes is due to additional pep interaction through the
aromatic phenyl rings and central metal ions which act as strong
intercalators can greatly promote the DNA-binding ability of their
complexes.
4.7.2. Viscosity measurements
To further confirm the interaction mode of the complexes with
DNA, a viscosity study was carried out. As known, the viscosity of
DNA is sensitive to changes occurring on the length of DNA helix
mainly upon interaction of DNA with a compound. In the case of
classic intercalation such as ethidium bromide [EB], the compound
inserts in between the DNA base pairs leading to an increase in the
separation of base pairs at intercalation sites in order to host the
bound compound [36] and the length of the DNA helix and, sub-
sequently, the DNA viscosity will exhibit an increase, which is
usually proportional to the strength of the interaction. On the other
4.7.3. Electrochemical parameters
Cyclic voltammetry is one of the important and most popular
electrochemical techniques for DNA binding studies with a fact that
compound bound to DNA is redox active [37]. Electrochemical
behavior of the synthesized complexes was investigated using cy-
clic voltammetric technique. The cyclic voltammetric experiment
Fig. 2. Absorption spectrum of complex 5 in buffer pH ¼ 7.2 at 25 ꢂC in presence of
Fig. 3. Effect of increasing amounts of [EB] (-), 1 ( ), 2 ( ), 3 ( ), 4 ( ), 5 ( ), 6 ( ), 7
increasing amount of DNA.
(
) and 8 ( ) on the relative viscosity of DNA.1/R ¼ [Complex]/[DNA] or [EB]/[DNA].

62
N. Raman, N. Pravin / European Journal of Medicinal Chemistry 80 (2014) 57e70
was performed at room temperature in DMF using a glassy carbon
working electrode a platinum counter electrode and an Ag/Agþ
reference electrode.
The voltammetric parameters obtained for all the complexes
without and in the presence of DNA are given in Table 3. The cyclic
voltammogram of the complex 1 in the absence and presence of
different amounts of DNA are shown in Fig. 4. In the absence of CT
DNA, the first redox cathodic peak appeared at 0.597 V for
Cu(II) / Cu(I) (Ep ¼ 0.544 V, Ep ¼ 0.597 V,
D
Ep ¼ ꢀ0.053 V, and E1/
a
c
¼ 0.571 V) and in the second redox couple, the cathodic peak
2
appeared at 0.067 V for Cu(I) / Cu(0) (Ep ¼ 0.018 V, Ep ¼ 0.067 V,
a
c
D
Ep ¼ ꢀ0.049 V, and E1/2 ¼ 0.043 V). The Ip /Ip ratios for these two
a
c
redox couples are 1.43, and 0.78, respectively, which indicate that
reaction of the complex on the glassy carbon electrode surface is a
quasi-reversible redox process assignable to the Cu(II)/Cu(I) and
Cu(I)/Cu(0) couple, as evidenced by the following criteria: (i) the
peak-to-peak separation DEp is greater than 49 mV, (ii) the current
ratios Ip /Ip are constantly nearly equal to 1, and E1/2 ¼ [Ep þ Ep /2]
c
c
a
is found toabe 0.571 V [38]. The voltammogram of the Zn(II) com-
plex also shows quasi reversible process. The reason for the quasi-
reversible electron transfer process may be due to slow electron
transfer or the adsorption of the complex onto the electrode surface
[39].
Fig. 4. Cyclic voltammogram of complex 1 in buffer pH ¼ 7.2 at 25 ꢂC in presence of
increasing amount of DNA.
withdrawing nitro group on the aromatic ring increases the anti-
microbial activities of the tested metal complexes compared to
complexes having no substituent [43]. The nature of metal ion also
plays a decisive role in determining antimicrobial properties. In the
present study, the order of the antimicrobial activity of the syn-
thesized compounds (based on the substituent present in the
phenyl ring) is as follows: 1 z 5 > 4 z 8 > 3 z 7 > 2 z 6. It is
inferred from the results that electron-withdrawing nitro group has
effective and direct impact on selective antimicrobial activities
against both bacteria and fungi [44]. The mode of action of the
compounds may involve the formation of a hydrogen bond through
the azomethine group with the active centers of cell constituents,
resulting in interferences with the normal cell process [45].
A quasi-reversible transfer process with the redox couple
[Zn(II) / Zn(0)] was observed for the complexes 5e8.The cathodic
peak appeared at 1.057 V in the absence of DNA (Ep ¼ ꢀ0.461 V,
a
Ep ¼ 1.057 V,
D
Ep ¼ ꢀ1.517 V, and E1/2 ¼ 0.301 V). The Ip /Ip ratio is
c
a
c
0.93. This indicates the quasi-reversible redox process of the metal
complexes. Incremental addition of DNA to the complex 5 resulted
in a slight decrease in the current intensity and less negative shift of
the oxidation peak potential. The resulting minor changes in the
current and potential are indicative of diffusion of the metal com-
plexes bound to the large, slowly diffusing DNA molecule [40].
4.8. Antimicrobial activity
4.9. Interaction with pBR322 plasmid DNA
The minimal inhibitory concentrations of tested compounds
against certain bacteria and fungi are shown in Tables 4and 5. The
ligands (L1eL4) and their metal complexes were prepared and
tested for their in vitro antimicrobial activity against the five strains
of bacteria (Gram negative and Gram positive), and five strains of
The intact double-stranded plasmid DNA exists in a compact
circular closed conformation, the supercoiled DNA. If only one DNA
strand is cleaved, the supercoiled DNA form assumes a more
relaxed conformation known as “open circular DNA”, which may be
converted into linear DNA, following a cleavage event on the
complementary strand near the first cleavage site. These three
fungi. Few metal complexes of the functionalized
b-diketimines
showed high in vitro antimicrobial activity. All the mixed ligand
metal complexes 1e8 showed significant antibacterial and anti-
fungal activities compared to free ligands, but the activity was
lesser than the standard drugs. Such increased activity of the
complexes can be explained on the basis of Overtone’s concept [41]
and the Tweedy’s Chelation theory [42]. The presence of electron-
Table 4
Minimum inhibitory concentration of the synthesized compounds against growth of
bacteria (mM).
Compound Minimum inhibitory concentration (MIC) (ꢁ104
m
M) SEM ¼ ꢃ2
Table 3
Staphylococcus Bacillus
Escherichia Klebsiella
Salmonella
typhi
Redox potential profiles for interaction of DNA with Cu(II) and Zn(II) complexes.
aureus
subtilis
coli
pneumoniae
Complexes
aDEp (V)
bE1/2 (V)
Ip /Ip
L1
L2
L3
L4
1
2
3
4
5
16.7
17.0
19.3
18.5
9.6
10.6
10.3
10.0
11.1
13.6
12.7
12.3
16.1
18.2
10.9
19.6
10.1
11.9
11.4
10.1
11.4
13.3
11.9
11.7
2.8
15.3
16.3
18.7
17.0
8.7
10.9
10.3
9.4
12.4
13.6
13.3
12.9
1.4
16.7
16.4
18.3
17.6
8.3
10.2
9.6
9.1
11.5
13.3
12.1
12.3
2.3
16.3
16.8
17.1
17.9
9.8
11.3
11.0
10.4
13.3
14.3
14.3
14.1
2.6
a
c
Free
Bound
Free
Bound
1
2
3
4
5
6
7
8
ꢀ0.053
ꢀ0.064
ꢀ0.112
ꢀ0.122
ꢀ1.517
ꢀ0.121
0.119
ꢀ0.046
ꢀ0.042
ꢀ0.106
ꢀ0.071
ꢀ1.479
0.254
0.571
0.052
ꢀ0.173
ꢀ0.231
0.301
ꢀ0.144
ꢀ0.852
ꢀ0.042
0.582
0.64
ꢀ0.181
ꢀ0.108
0.330
ꢀ0.131
ꢀ0.942
ꢀ0.051
1.43
0.81
0.71
0.68
0.32
3.28
6.18
0.79
0.199
0.168
6
7
8
0.174
Data from cyclic voltammetric measurements.
aKanamycin 1.6
a
D
Ep ¼ Ep dEp
.
a
c
b
a
E1/2 is calculated as the average of anodic (Epa) and cathodic (Ep ) peak poten-
Kanamycin is used as the standard. The results obtained were the average of
three replicates.
c
tials; E1/2 ¼ Ep þ Ep /2.
a
c

N. Raman, N. Pravin / European Journal of Medicinal Chemistry 80 (2014) 57e70
63
Table 5
cleavage. Based on the above observations we speculate that the
complexes generated singlet oxygen and/or singlet oxygen like
reactive oxygen species (ROS) which were responsible for nuclease
activity. Considering that these types of active species mostly occur
in oxidative cleavage, it is most likely that complexes 1e8 cleaved
the DNA through a self-activating oxidative pathway. The possible
redox mechanism between the metal complex and oxidant may be
explained as follows:
Minimum inhibitory concentration of the synthesized compounds against the
growth of fungi (mM).
Compound Minimum inhibitory concentration (MIC) (ꢁ104
m
M) SEM ¼ ꢃ2
Aspergillus Fusarium Curvularia Rhizoctonia
Candida
albicans
niger
solani
lunata
bataticola
L1
L2
L3
L4
1
2
3
4
5
16.2
18.9
20.3
19.2
10.1
14.9
14.1
12.0
13.7
18.4
15.2
14.1
17.4
18.5
21.6
19.0
12.1
16.7
16.1
14.4
15.1
17.1
16.9
16.3
1.7
15.5
17.6
19.5
18.1
12.3
14.9
14.1
13.4
13.4
16.9
15.4
14.1
1.2
14.9
15.8
17.5
16.9
13.6
16.9
15.1
14.3
16.3
17.4
17.1
16.3
1.5
16.4
17.6
18.4
17.9
14.2
16.1
14.9
14.4
16.9
12.6
16.5
16.1
1.8
½MðIIÞLðoxÞꢄ þ eꢀ/½MðIÞLðoxÞꢄ
½MðIÞLðoxÞꢄ þ O2/½MðIIÞLðoxÞꢄ þ O2ꢀ
2Oꢀ2 þ 2Hþ/H2O2 þ O2
6
7
8
Oꢀ2 þ H2O2/O2 þ OHꢀ þ OH$
aFluconazole 1.4
a
To further test the involvement of H2O2 in the cleavage reaction,
the analysis has been carried out under redox reaction conditions.
The complexes 1e8 were found to form the same ROS involved in
the reaction, and H2O2 was the crucial active intermediate involved
in the DNA cleavage (Fig. 5(d)). Further, these complexes 1e8, are
capable of promoting DNA cleavage through an oxidative DNA
damage pathway, in which giving active oxygen species such as
singlet oxygen or singlet oxygen-like entity, probably a copper-
peroxide, which cleaves DNA [47e49]. The resulting nuclease ac-
tivity and mechanism under redox conditions strongly indicate a
“self-activating” mechanism, which is most likely initiated by both
the complexes through an oxidative pathway [50e52]. Further
studies are currently underway to clarify the cleavage mechanism.
Fluconazole is used as the standard. The results obtained were the average of
three replicates.
plasmid DNA conformations are distinguishable when subjected to
agarose gel electrophoresis. The cleavage of plasmid pBR322 DNA
induced by Cu(II) and Zn(II) complexes was performed in the
absence of external cofactor agents (either oxidant or reductant).
The Cu(II) complexes were found to promote the cleavage of
plasmid pBR322 DNA from the supercoiled form (I) to the nicked
form (II) and linear form (III) (Fig. 5(a)). No DNA cleavage was
observed for the control in which the metal complex was absent
(lane 1) and in presence of free CuCl2 (100 mM). When the con-
centrations of the Cu(II) and Zn(II) complexes were increased (lanes
3e8), the amount of form I of pBR322 DNA diminished gradually,
whereas the amounts of form II and III increased. These show, both
the complexes are able to induce significant cleavage of the plasmid
4.10. Cytotoxicity
DNA at the concentration of 25 mM. At the concentration of 50 mM,
Cu(II) and Zn(II) almost promote the complete conversion of DNA
from form I to forms II and III.
Cytotoxicity is a common limitation in terms of the introduction
of new compounds into the pharmaceutical industry. Schiff base
complexes of copper have recently been investigated for their
therapeutic potential [53] and the positive results obtained from
DNA binding, cleavage and efficient microbial activity of the novel
mixed-ligand complexes encouraged us to test their cytotoxicity
against a panel of human cancer cell lines namely, human cervical
cancer cell lines (HeLa) and human breast cancer cell lines (MCF-7)
by colorimetric (MTT) assay in which mitochondrial dehydrogenize
activity was measured as an indication of cell viability.
The IC50 values have been calculated after 72 h of incubation
with complexes and are listed in Table 6. As shown in Table 6, HeLa
cells are more sensitive to complex 1 than MCF-7 cells, and the
cytotoxicity of complex 1 against HeLa cells are higher compared
with other complexes and cis-Pt(NH3)2Cl2. Copper complexes
inhibit the growth of HeLa cells in a dose-dependent manner
In the presence of an oxidant (100
conditions (Fig. 5(b)), only 5 M of Cu(II) and Zn(II) complexes can
significantly cleave DNA. At highest concentration 50 M the form I
mM H2O2) under aerobic
m
m
DNA has been fully depleted. This observation indicates that the
chemical nuclease activity of the Cu(II) complex is significantly
promoted with the addition of oxidants compared to Zn(II) com-
plex. These observations support the hypothesis that Cu(II) and
Zn(II) mixed ligand complexes most likely cleaved the plasmid DNA
by a self-activating oxidative mechanism, which is similar to the
manner in which bleomycin cooperates with a redox metal (Fe(II))
to trigger DNA cleavage without exogenous agents [46].
4.9.1. A mechanistic investigation of the DNA cleavage reaction
To explore the cleavage mechanism, the copper-mediated DNA
cleavage was investigated with several different potential radical
scavengers to identify the intermediate reactive oxygen species
(ROS) in the absence of external cofactors (Fig. 5(c)). DMSO was
used as hydroxyl radical (HO_) scavenger; sodium azide was used as
singlet oxygen (1O2) scavenger; and superoxide dismutase (SOD)
was used as an O$2ꢀ radical scavenger. The DNA breaks mediated by
complexes 1e8 were not affected by the presence of the hydroxyl
radical inhibitor DMSO, suggesting that diffusible hydroxyl radicals
were not involved in the DNA damage mechanism. Addition of
singlet oxygen scavenger NaN3 revealed complete inhibition of
nuclease (Fig. 5(c); lanes 4 and 5). These results suggested that 1O2
or any other singlet oxygenelike entity may participate in the DNA
strand scission. On the other hand addition of SOD (Fig. 5(c); lanes 8
and 9) also showed comparatively little inhibitory effect on the DNA
(IC50 ¼ 0.50
mM). Although higher complex concentration reduces
the percentages of cell survival, there is a significant difference in
susceptibility between the complexes and cis-Pt(NH3)2Cl2. Based
on the results, we speculate that the better cytotoxic activity of the
complexes 1e4 may be attributed to their stronger ability to cleave
DNA. All the complexes are remarkable in displaying effective
cytotoxicity against the HeLa human cervical cancer cell line and
they are more potent than the widely used drug cisplatin.
The in vitro cytotoxicity against the animal tumor cell line (EAC)
is also performed by trypan blue dye exclusion method. The IC50
values of all the synthesized complexes are given in Table 6. This
table shows that the complexes demonstrate different anti-tumor
activities. Moreover, the results show excellent potential of com-
plexes 1 and 4 towards EAC cell lines with IC50 values (107.21 and
109.32 mM respectively), very sensitive with the value obtained for

64
N. Raman, N. Pravin / European Journal of Medicinal Chemistry 80 (2014) 57e70
Fig. 5. (a) The gel electrophoresis of pBR322 DNA cleavage catalyzed by complex 1 and 5 in TriseHCl buffer at 37 ꢂC for 1.5 h with histogram of % cleavage forms. Lane 1: DNA control
(10 M); lanes 3e5: DNA þ 5 (5, 25, 50 M); lanes 6e8: DNA þ 1 (5, 25, 50 M). (b) The agarose gel electrophoresis of complexes 1 and 5 showing the
M); lane 2: DNA þ CuCl2 (100
oxidative cleavage of DNA (10 M) in the presence of H2O2 (100 M) with histogram of % cleavage forms. Lane 1: DNA control (10
M); lane 2: DNA þ H2O2; lane 3: DNA þ CuCl2
(500 M) þ H2O2; lanes 7e9: DNA þ 5 (5, 25, 50 M) þ H2O2. (c) The agarose gel electrophoresis of complexes (50 M) cleaving DNA
M) þ H2O2; lanes 4e6: DNA þ 1 (5, 25, 50
(10
M) in the presence of various radical scavengers under redox conditions for 1.5 h at 37 ꢂC with histogram of % cleavage forms. Lane 1: DNA control; lanes 2 and 3: no inhibitor;
complexes 1 and 5 (each 50
M); lanes 4 and 5: complexes 1 and 5 þ NaN3 (100 mM); lanes 6 and 7: complexes 1 and 5 þ DMSO (100 mM); lanes 8 and 9: complexes 1 and 5 þ SOD
(1U). (d) The agarose gel electrophoresis of complexes 1 and 5 (50 M) cleaving DNA (10
M) in the presence of various radical scavengers for 1.5 h at 37 ꢂC with histogram of %
M) þ H2O2; lane 3 and 7: 1 and 5 þ NaN3 (100 mM) þ H2O2; lane 4 and 8: 1 and
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
cleavage forms. Lane 1: DNA control; lane 2 and 6: no inhibitor; complexes 1 and 5 (each 50
5 þ DMSO (100 mM) þ H2O2; lane 5 and 9: complexes 1 and 5 þ SOD (1U) þ H2O2.
m
the standard drug 5-Flurouracil (5-FU), which is taken as positive
control. It suggests that the -diketimine moiety, bio-ligand and its
metal ions have important effect on cytotoxicity. Amongst all the
complexes tested, complex 1 has lowest IC50 value of 107.21 M.
These results indicate that the complexes exert cytotoxic effects
against tested carcinoma cell lines.
4.10.1. In vivo approach
b
Based on the above investigations, it is clearly demonstrated
that the copper(II) complexes (1e4) exhibit superior in vitro bio-
logical responses. The in vitro cytotoxicity of EAC tumor cell lines
show that the complexes 1 and 4 have lower IC50 values which
stimulated us to do the in vivo antitumor activity of EAC tumor
m

N. Raman, N. Pravin / European Journal of Medicinal Chemistry 80 (2014) 57e70
65
Table 6
IC50 M) values of complexes and cis-platin against various cancer cell lines.
significant increase in the life span of the tumor bearing mice which
were almost comparable with that of 5-FU.
(
m
Compounds
IC50
(m
M) SEM ¼ ꢃ0.5
MCF-7
HeLa
EAC
5. Conclusion
1
2
3
4
5
6
7
8
0.50
0.56
0.72
0.64
0.54
0.60
0.82
0.74
0.53
e
0.74
0.79
0.86
0.83
0.75
0.86
1.04
0.91
0.72
e
107.21
115.95
113.18
109.32
108.81
118.35
115.41
110.53
e
Few novel mononuclear copper(II) and zinc(II) complexes (1e8)
have been synthesized and characterized by physicochemical and
spectroscopic methods. Characterizations of the new complexes
have shown that, Cu(II) formed square planar complexes with 1:1:1
(metal:ligand) stoichiometry. The study of the interaction of com-
plexes with CT-DNA has been investigated by absorption spectro-
photometry, cyclic voltammetry and viscosity measurements. From
the binding studies, it is evident that 1 z5 > 2 z 6 > 4 z 8 > 3 z 7
according to CT-DNA binding ability. In cyclic voltammetry, it is
observed that there is positive shift of both the Ep and Ep along
Cis-platin
5-FU
114.24
The results obtained were the average of three replicates from three determinations.
IC50, Drug concentration inhibiting 50% cellular growth following 72 h of drug
exposure.
c
a
with the decrease of peak currents. The DNA interaction studies
exhibit that all the complexes strongly interact with CT DNA by
intercalative binding mode. Furthermore, the novel complexes
exhibit good antimicrobial activity against the microbial strains,
which may be due to the strong chelation and increased electron
delocalization in increasing the lipophilic character of the metal ion
into the cells in comparison to free ligands. These complexes could
induce scission of pBR322 supercoiled DNA effectively without
addition of external agents at pH ¼ 7.2 and 37 ꢂC. These results
suggested that 1O2 or any other singlet oxygenelike entity may
participate in the DNA strand scission. The strong DNA binding
ability coupled with the potent DNA cleavage activity shows that all
the complexes might be capable of promoting DNA cleavage
through an oxidative DNA damage pathway. In cytotoxicity exper-
iment, complex 1 shows high in vitro cytotoxic property against
HeLa/EAC cells compared to other complexes and cisplatin/5-FU.
The above investigations clearly demonstrate that the complexes
1e4 exhibit superior in vitro biological responses. Further, the
in vivo antitumor activity of complexes 1e4 against EAC tumor
model in Adult Swiss female albino mice reveals that the complexes
are non-toxic to normal cells (lymphocytes). Among them, complex
1 shows potential antitumor activity. Further studies are warranted
to assess precise molecular mechanism of cytotoxicity and the
pharmacological properties to elucidate the actual mechanism of
the biological activity.
model. On the day 12, the biochemical and hematological param-
eters as regard to hemoglobin level, neutrophils and monocytes
counts, were compared for the group treated with the standard
drug 5-FU [54] and the same with the newly synthesized com-
pound 1. This shows the highest % of increase in life span over
control on comparing the values obtained from normal and control
groups. Hematological parameters of tumor-bearing mice on day
12 showed significant changes when compared to the normal mice
(Table 7). From Table 7, differential count of WBC showed that the
percentage of neutrophils increased (P < 0.001) while that of
lymphocytes decreased (P < 0.001). At the same time interval,
complexes (100 mg/kg/day, p.o.) treatment could change these
altered parameters to almost normal.
The analysis of the hematological parameters showed minimum
toxic effect in mice treated with complexes as compared with 5-FU
[54]. After 12 days of transplantation, complexes were able to
reverse the changes in the hematological parameters consequent to
tumor inoculation. The present study revealed that the complexes
were cytotoxic towards EAC. Fig. 6 shows smear showing matured
EAC control cells (for 24 h) with definite cell wall and structure
without degeneration. After EAC tumor cells treated with 5-FU,
complex 4 for 24 h showing degenerative changes like membrane
blebbing, vacuolated cytoplasm and low staining intensity for
complex 1 with complete destruction of cells have efficient
response than the complex 4 and compared to 5-FU. Interestingly,
all the live normal cells upon treatment with the complexes 1e4
(Table 7) reveal that the complexes are non-toxic to normal cells
(lymphocytes).
6. Experimental protocols
6.1. Reagents and instruments
The effect of complexes 1 and 4 on the survival of tumor-bearing
All reagents, benzaldehyde, acetylacetone, para substituted an-
ilines, oxalic acid and metal(II) chlorides were of Merck products
and they were used as supplied. Commercial solvents were distilled
mice showed at dose of 100
mg/mL life span values 114.98 and
112.64% respectively (Table 8). The other complexes also showed
Table 7
Effect of Cu(II) complexes on hematological parameters of EAC tumor bearing mice.
Design of treatment Hb (gm %)
RBC 106 Cells/CU.MM WBC 103 Cells/CU.MM Total protein mg% PCV (%)
Differential count (%)
Lymphocytes Neutrophils
Monocytes
Normal
Tumor control
1
2
3
4
12.92 ꢃ 0.15
5.78 ꢃ 0.4a
4.9 ꢃ 0.12
6.78 ꢃ 0.14
5.73 ꢃ 0.26
12.23 ꢃ 0.34a
7.19 ꢃ 0.17b,d
7.63 ꢃ 0.27a,d
8.56 ꢃ 0.17a,d
7.96 ꢃ 0.41a,d
16.86 ꢃ 0.55
25.98 ꢃ 0.56a
17.14 ꢃ 0.26d
16.98 ꢃ 0.51d
17.18 ꢃ 0.45d
66.46 ꢃ 1.36
26.23 ꢃ 1.16a
32.6 ꢃ 1.6
1.25 ꢃ 0.45
2.46 ꢃ 0.14a
18.74 ꢃ 0.47a
15.58 ꢃ 0.3a,d
14.95 ꢃ 0.54a,d
12.16 ꢃ 0.63a,d
11.47 ꢃ 0.42a,d
72.8 ꢃ 1.07a 1.20 ꢃ 0.45
12.13 ꢃ 0.21d 4.18 ꢃ 0.19d
11.39 ꢃ 0.13d 3.45 ꢃ 0.15a,e
11.04 ꢃ 0.16a,d 3.94 ꢃ 0.13b,d
13.07 ꢃ 0.14d 4.04 ꢃ 0.15d
65.91 ꢃ 0.86d 35.12 ꢃ 1.24d
0.7 ꢃ 0.45
0.4 ꢃ 0.24
0.6 ꢃ 0.42
0.6 ꢃ 0.27
64.43 ꢃ 1.0d
31.60 ꢃ 1.03d
63.22 ꢃ 1.28d 36.27 ꢃ 1.46d
19.98 ꢃ 0.54b,d 65.41 ꢃ 1.21d 38.54 ꢃ 1.23d
aP<0.001.
bP < 0.01.
cP < 0.05 versus Normal.
dP < 0.001.
eP < 0.01 versus Tumor control. Data were analyzed by using one way ANOVA followed by TukeyeKramer multiple comparison test.

66
N. Raman, N. Pravin / European Journal of Medicinal Chemistry 80 (2014) 57e70
Fig. 6. (a) Smear showing matured EAC control cells (for 24 h) with definite cell wall and structure without degeneration. (b) EAC tumor cells treated with 5-FU for 24 h. (c) EAC
tumor cells treated with complex 4 for 24 h. (d) complex 1 (for 24 h) treated with EAC cells showing degenerative changes like membrane blebbing, vacuolated cytoplasm and low
staining intensity with complete destruction of cells.
and then used for the preparation of ligands and their complexes.
DNA was purchased from Bangalore Genei (India). Microanalyses
(C, H and N) were performed in Carlo Erba 1108 analyzer at So-
phisticated Analytical Instrument Facility (SAIF), Central Drug
Research Institute (CDRI), Lucknow, India. Molar conductivities in
DMF (10ꢀ3 M) at room temperature were measured by using
Systronic model-304 digital conductivity meter. Magnetic suscep-
tibility measurement of the complexes was carried out by Gouy
balance using copper sulfate pentahydrate as the calibrant. Infrared
spectra (4000e400 cmꢀ1 KBr disc) of the samples were recorded on
an IR Affinity-1 FT-IR Shimadzu spectrophotometer. NMR spectra
were recorded on a Bruker Avance Dry 300 FT-NMR spectrometer
in DMSO-d6, using TMS as the internal reference. EPR spectra were
recorded on a Varian E 112 EPR spectrometer in DMSO solution
both at room temperature (300 K) and liquid nitrogen temperature
(77 K) using TCNE (tetracyanoethylene) as the g-marker. The ab-
sorption spectra were recorded by using Shimadzu model UV-1601
spectrophotometer at room temperature.
the above Schiff base(s) (5 mmol), a solution of copper(II)/zinc(II)
chloride (5 mmol) in ethanol was added dropwise. After the reac-
tion for 1 h at 60 ꢂC, a solution of oxalic acid (5 mmol) in ethanol
was added. The reaction solution was refluxed for 2 h. After cooling
the reaction mixture to an ambient temperature, the formed solid
was filtered, washed with diethyl ether and finally dried in vacuum.
[CuL1(ox)] (1) Yield: 64%. Anal. Calc. for C26H20N4O8Cu: Cu, 11.0;
C, 54.0; H, 3.5; N, 9.7; Found: Cu, 10.8; C, 53.6; H, 3.5; N, 9.5 (%). IR
(KBr pellet, cmꢀ1): 1601
n(eC]N); 1501 n(eHC]C); 1471,1304, 845
n
(eCeN str; eNO2); 1322 n(eCeO); 1689 n(non-coordinated C]O
in ox); 433
n(MꢀN), 508
n
(MꢀO). MS m/z (%): 581 [M þ 1]þ. LM
10ꢀ3(ohmꢀ1 cm2 molꢀ1) ¼ 8.6. lmax in DMF, 43,836, 35,225, 22,883.
meff (BM): 1.86.
[CuL2(ox)] (2) Yield: 62%. Anal. Calc. for C26H22N2O4Cu: Cu, 13.0;
C, 64.0; H, 4.5; N, 5.7; Found: Cu, 12.9; C, 63.7; H, 4.5; N, 5.5 (%). IR
(KBr pellet, cmꢀ1): 1605
1686
n
(C]N); 1505
n
(eHC]C); 1324
n
(eCeO);
n
(non-coordinated C]O in ox); 431
n
(MꢀN), 505
n
(MꢀO). MS
m/z (%): 491 [M þ 1]þ. LM 10ꢀ3 (ohmꢀ1 cm2 molꢀ1) ¼ 8.9. lmax
(cmꢀ1) in DMF, 40,475, 29,561, 23,041. meff (BM): 1.88.
[CuL3(ox)] (3) Yield: 57%. Anal. Calc. for C26H22N2O6Cu: Cu, 12.2;
C, 60.0; H, 4.2; N, 5.7; Found: Cu, 12.1; C, 59.8; H, 4.2; N, 5.6 (%). IR
6.2. Synthesis of metal complexes
(KBr pellet, cmꢀ1): 1610
1323
526
n
(eC]N); 1528
n
(eHC]C); 3428
n
(eOH);
(MꢀN),
1]þ. LM 10ꢀ3
The Schiff base ligands were synthesized by the method re-
ported in the literature by us [25]. To a stirred ethanolic solution of
n
(eCeO); 1685
n
(non-coordinated C]O in ox); 439 n
(MꢀO). MS m/z (%): 523 [M
n
þ
(ohmꢀ1 cm2 molꢀ1) ¼ 8.3. lmax (cmꢀ1) in DMF, 41,326, 33,398,
23,201. meff (BM): 1.87.
Table 8
Effect of Cu(II) complexes treatment on the survival of tumor-bearing mice.
[CuL4(ox)] (4). Yield: 52%. Anal. Calc. for C28H26N2O6Cu: Cu, 11.5;
C, 61.1; H, 4.8; N, 5.1; Found: Cu, 11.3; C, 61.0; H, 4.8; N, 5.0 (%). IR
Treatment
MST
Increase in life span
(KBr pellet, cmꢀ1): 1607
CeOeCe); 1321
445
n(eC]N); 1537 n(eHC]C); 1270, 1083,n(e
Tumor control
5- FU
1
2
3
4
16.26 ꢃ 0.52
34.71 ꢃ 0.81*
30.34 ꢃ 0.62*
28.29 ꢃ 0.52*
27.75 ꢃ 0.75*
27.82 ꢃ 0.58*
__
140.67
114.98
111.85
108.14
112.64
n
(eCeO); 1689 n(non-coordinated C]O in ox);
n
n(MꢀN), 531
(MꢀO). MS m/z (%): 551 [M þ 1]þ. LM 10ꢀ3
(ohmꢀ1 cm2 molꢀ1) ¼ 9.1. lmax (cmꢀ1) in DMF, 41,689, 31,626,
23,364. meff (BM): 1.84.
[ZnL1(ox)] (5) Yield: 61%. Anal. Calc. for C26H20N4O8Zn: Zn, 12.0;
C, 56.4; H, 3.6; N, 5.1; Found: Zn, 11.8; C, 56.3; H, 3.6; N, 5.0 (%). IR
N ¼ 6; d of drug treatment ¼ 9, *p < 0.01 versus tumor control.
Data were analyzed by one-way ANOVA followed by Dunnett’s test.

N. Raman, N. Pravin / European Journal of Medicinal Chemistry 80 (2014) 57e70
67
(KBr pellet, cmꢀ1): 1612
n
(eC]N); 1503
n
(eHC]C); 1467,1307, 835
.ꢀ
ꢁ
.ꢀ
ꢁ
h
ꢀ
ꢁiꢀ1
½DNAꢄ εa ꢀ εf ¼ ½DNAꢄ εb ꢀ εf þ Kb εb ꢀ εf
(1)
n
(eCeN str; eNO2); 1323 n(eCeO); 1681 n(non-coordinated C]O
in ox). 448
n
(MꢀN), 509
n
(MꢀO). 1H NMR (
d): (aromatic) 6.9e7.4
(m); (eCH3, 6H), 2.1 (s). 13C NMR (
d): 124.6e126.4 (C1 to C3), 134.3
where [DNA] is the concentration of CT DNA in base pairs. The
apparent absorption coefficients εa, εf and εb correspond to Aobs./
[M], the extinction coefficient for the free metal(II) complex and
extinction coefficient for the metal(II) complex in the fully bound
form, respectively [56]. Kb is given by the ratio of slope to the
intercept.
Cyclic voltammetry studies were performed on a CHI 620C
electrochemical analyzer with three electrode system of glassy
carbon as the working electrode, a platinum wire as auxiliary
electrode and Ag/AgCl as the reference electrode. Solutions were
deoxygenated by purging with N2 prior to measurements. Viscosity
experiments were carried on an Ostwald viscometer, immersed in a
thermostated water-bath maintained at a constant temperature at
30.0 ꢃ 0.1 ꢂC. CT DNA samples of approximately 0.5 mM were
prepared by sonicating in order to minimize complexities arising
from CT DNA flexibility [57]. Flow time was measured with a digital
stopwatch three times for each sample and an average flow time
was calculated. Data were presented as (
centration of the metal(II) complexes, where
DNA solution in the presence of complex, and h0 is the viscosity of
CT DNA solution in the absence of complex. Viscosity values were
calculated after correcting the flow time of buffer alone (t0),
t0)/t0 [58].
(C4),136.1 (C5),114.3 (C6),170.4 (C7), 20.4 (C8),153.1 (C9),123.3 (C10),
124.6 (C11) 146.6 (C12), 164.5 (C13). MS m/z (%): 582 [M þ 1]þ. LM
10ꢀ3 (ohmꢀ1 cm2 molꢀ1) ¼ 13.6. lmax (cmꢀ1) in DMF, 42,359, 32,161.
meff (BM): diamagnetic.
[ZnL2(ox)] (6) Yield: 59%. Anal. Calc. for C26H22N2O4Zn: Zn, 13.3;
C, 63.5; H, 4.5; N, 5.7; Found: Zn, 13.1; C, 63.3; H, 4.4; N, 5.4 (%). IR
(KBr pellet, cmꢀ1): 1606
1685
NMR (
n
(eC]N); 1526
n(eHC]C); 1320 n(eCeO);
n
(non-coordinated C]O in ox). 441
n
(MꢀN), 517
n
(MꢀO). 1H
d
, ppm): (aromatic) 6.9e7.3 (m); (eCH3, 6H), 2.2 (s). 13C NMR
(d
, ppm):125.3e128.6 (C1 to C3), 133.1 (C4), 136.4 (C5), 112.3 (C6),
166.5 (C7), 20.4 (C8), 136.9 (C9), 119.4 (C10), 131.6 (C11), 128.3 (C12),
168.7 (C13), MS m/z (%): 492 [M
þ
1]þ. LM 10ꢀ3
(ohmꢀ1 cm2 molꢀ1) ¼ 12.1. lmax (cmꢀ1) in DMF, 40,561, 29,198. meff
(BM): diamagnetic.
[ZnL3(ox)] (7). Yield: 53%. Anal. Calc. for C26H22N2O6Zn: Zn, 12.5;
C, 59.6; H, 4.2; N, 5.4; Found: Zn, 12.2; C, 59.4; H, 4.2; N, 5.3 (%). IR
1/3
(KBr pellet, cmꢀ1): 1615
n
(eC]N); 1524
n
(eHC]C); 3428
n
(eOH);
(MꢀN),
, ppm): (aromatic) 6.8e7.2 (m); (eOH, 1H)
, ppm): 125.0e127.9 (C1 to
h
/
h0)
versus the con-
h
is the viscosity of CT
1322
513
n
(eCeO); 1684
n
(non-coordinated C]O in ox). 430 n
d
n
(MꢀO). 1H NMR (
10.3 (s); (eCH3, 6H), 2.4 (s). 13C NMR (
d
h
¼ (te
C3), 134.4 (C4), 136.3 (C5), 114.5 (C6), 169.3 (C7), 20.3 (C8), 147.2 (C9),
122.9 (C10), 118.4 (C11), 154.6 (C12), 164.7 (C13), MS m/z (%): 524
[M þ 1]þ. LM 10ꢀ3 (ohmꢀ1 cm2 molꢀ1) ¼ 15.2. lmax (cmꢀ1) in DMF,
41,741, 33,853. meff (BM): diamagnetic.
6.4. Interaction with pBR322 plasmid DNA
[ZnL4(ox)] (8) Yield: 51%. Anal. Calc. for C28H26N2O6Zn: Zn, 12.0;
C, 61.0; H, 5.0; N, 5.1; Found: Zn, 11.8; C, 60.8; H, 5.0; N, 4.9 (%). IR
The extent of pBR322 DNA cleavage in the absence and presence
of an activating agents H2O2 and various radical scavengers like
sodium azide (singlet oxygen), SOD (superoxide) DMSO (Hydroxyl
radical scavenger) was monitored using agarose gel electropho-
resis. In reactions using supercoiled pBR322 plasmid DNA form I
(KBr pellet, cmꢀ1): 1611
n(eC]N); 1543
n
(eHC]C); 1264, 1081,
(non-coordinated C]O in ox). 436
): (aromatic) 6.8e7.3 (m); (eCH3,
):124.9e128.6 (C1 to C3),
n(e
CeOeCe); 1326
(MꢀN), 533
n(eCeO); 1683 n
n
n
(MꢀO). 1H NMR (
d
6H), 2.2 (s); 3.7 (eOCH3, 6H). 13C NMR (
d
(2
which was treated with the metal complex (5e50
vating agents (100 M) followed by dilution with the TriseHCl
buffer to a total volume of 20 L. The samples were incubated for
1 h at 37 ꢂC. A loading buffer containing 25% bromophenol blue,
0.35% xylene cyanol, 30% glycerol (3 M) was added and electro-
phoresis was performed at 40 V for each hour in TriseAcetatee
EDTA (TAE) buffer using 1% agarose gel containing 1 M ethidium
m
M,10
m
M) in TriseHCl buffer (50 mM) with 50 mM NaCl (pH 7.2)
132.5 (C4), 137.5 (C5), 112.8 (C6), 168.2 (C7), 20.3 (C8), 142.1 (C9),
mM) and acti-
122.2 (C10), 116.1 (C11), 158.4 (C12), 58.3 (C13), 164.3 (C14), MS m/z
(%): 552 [M þ 1]þ. LM 10ꢀ3 (ohmꢀ1 cm2 molꢀ1) ¼ 12.8 lmax (cmꢀ1
in DMF, 42,359, 31,833. meff (BM): diamagnetic.
)
m
m
m
6.3. DNA binding experiments
m
The interaction between metal complexes and DNA was studied
using electronic absorption, viscosity and electrochemical methods.
Disodium salt of calf thymus DNA was stored at 4 ꢂC. All the ex-
periments involving the interaction of the complexes with calf
thymus (CT) DNA were carried out in TriseHCl buffer (50 mM Trise
HCl, pH 7.2) containing 5% DMF at room temperature. A solution of
CT DNA in the buffer gave a ratio of UV absorbance at 260 and
280 nm of about 1.8e1.9, indicating that the CT DNA was suffi-
ciently free from protein [55]. The concentration of DNA was
measured by using its extinction coefficient at 260 nm
(6600 Mꢀ1 cmꢀ1) after 1: 100 dilution. Stock solutions were stored
at 4 ꢂC and used not more than 4 days. Doubly distilled water was
used to prepare solutions. Concentrated stock solutions of the
complexes were prepared by dissolving the complexes in DMF and
diluting properly with the corresponding buffer to the required
concentration for all the experiments.
Absorption titration experiment was performed by keeping the
concentration of the metal complex as constant at 50
varying the concentration of the CT DNA within 40e400
measuring the absorption spectrum, equal quantity of CT DNA was
added to both the complex solution and the reference solution to
eliminate the absorbance of CT DNA itself. From the absorption
data, the intrinsic binding constant (Kb) was determined from the
plot of [DNA]/(εaeεf) versus [DNA] using the following equation (1):
bromide. The cleavage efficiency was measured by determining the
ability of the complex to convert the supercoiled form I DNA to
nicked circular form II and linear form III. After staining with an EB
solution (1 mM), the bands visualized and photographed. The extent
of DNA form I cleavage was measured from the intensities of the
bands using UVITEC Gel Documentation System. The observed er-
ror in measuring the band intensities was w4%. Inhibition reactions
were carried out by prior incubation of the SC pBR322 DNA (10
with DMSO (2 M).
mM)
m
6.5. Evaluation of antimicrobial activity
Qualitative determination of antimicrobial activity was done
using the disc diffusion method. The biological activities of syn-
thesized Schiff bases and their metal complexes were studied for
their antibacterial and antifungal activities in DMF solvent against
bacterial and fungal species. Suspensions in sterile peptone water
from 24 h cultures of microorganisms were adjusted to 0.5
McFarland. MullereHinton petri discs of 90 mm were inoculated
using these suspensions. Paper discs (6 mm in diameter) containing
m
M while
mM. While
10 mL of the substance to be tested were placed in a circular pattern
in each inoculated plate. DMF impregnated discs were used as
negative controls. Toxicity tests of the solvent, DMF, showed that

68
N. Raman, N. Pravin / European Journal of Medicinal Chemistry 80 (2014) 57e70
the concentration used in antibacterial activity assays did not
interfere with the growth of the microorganisms.
6.6.4. Cytotoxicity
Human cervical cancer cell lines (HeLa) and Human breast
cancer (MCF-7) cells were obtained from National Centre for Cell
Science (Pune, India). Stock cells of HeLa and MCF-7 cell lines were
cultured in RPMI-1640 or DMEM supplemented with 10% in acti-
vated new born calf serum, penicillin (100 IU/mL), streptomycin
6.5.1. Determination of MIC
The in vitro antimicrobial activity was performed against Gram-
positive bacteria: Staphylococcus aureus, Bacillus subtilis and sal-
monella typhi Gram-negative bacteria: Escherichia coli, Pseudo-
monas aeruginosa and fungal strains: Rhizoctonia bataticola,
Fusarium Solani, Candida albicans, Culvularia lunata and Aspergillus
niger. The standard and test samples were dissolved in DMF to give
(100 mM), and amphotericin-B (5 mM) under a humidified atmo-
sphere of 5% CO2 at 37 ꢂC until confluent. The cells were dissociated
in 0.2% trypsin, 0.02% EDTA in phosphate buffer saline solution. The
stock culture was grown in 25 cm2 tissue-culture flasks, and cyto-
toxicity experiments were carried out in 96-well microtiter plates
(Tarsons India, Kolkata, India).
a concentration of 100 mM. The minimum inhibitory concentration
(MIC) was determined by broth microdilution method [59]. Di-
lutions of test and standard compounds were prepared in nutrient
broth (bacteria) or Sabouraud dextrose broth (fungi) [60]. The
samples were incubated at 37 ꢂC for 24 h (bacteria) and at 25 ꢂC for
48 h (fungi), respectively, and the results were recorded in terms of
MIC (the lowest concentration of test substance which inhibited the
growth of microorganisms).
Cell lines in the exponential growth phase were washed, tryp-
sinized and resuspended in complete culture media. Cells were
plated at 10,000 cells/well in 96-well microtiter plates and incu-
bated for 24 h, during which a partial monolayer formed. They were
then exposed to various concentrations of the complexes (0.1e
100
mM) and cisplatin. Control wells received only maintenance
medium. The plates were incubated at 37 ꢂC in a humidified
incubator with 5% CO2 for a period of 72 h at the end of 72 h,
viability was determined by MTT assay.
6.6. Antitumor studies
Adult Swiss female albino mice (20e25 g) were procured from
Animal house, Vivekananda College of Pharmacy, Tiruchengode,
Tamil Nadu, India and used throughout the study. They were
housed in microlon boxes in controlled environment (temperature
25 ꢃ 2 ꢂC and 12 h dark/light cycle) with standard laboratory diet
and water ad libitum. The experiments were performed in accor-
dance with guidelines established by the European community for
the care and use of laboratory animals, and were approved by the
Institutional Animal Ethics Committee (IAEC) of Vivekananda Col-
lege of Pharmacy, Tamil Nadu, India.
6.6.5. Effect of Cu(II) and Zn(II) complexes on in vitro cytotoxicity
Short-term cytotoxicity was assessed by incubating 1 ꢁ 106 EAC
cells in 1 mL phosphate buffer saline with varying concentrations of
the complexes at 37 ꢂC for 3 h in CO2 atmosphere ensured using a
McIntosh field jar. The viability of the cells was determined by the
trypan blue exclusion method [66].
Acknowledgments
The authors express their heartfelt thanks to the Science & En-
gineering Research Board (SERB), DST, New Delhi (File No.SR/S1/IC-
27/2012) for financial Support. They also express their gratitude to
the College Managing Board, Principal and Head of the Department
of Chemistry, VHNSN College, Virudhunagar for providing research
facilities. They thank Prof. R. Senthil Kumar, Department of Phar-
maceutical Chemistry, Swami Vivekananda College of Pharmacy,
Elayampalayam, Tiruchengode, for in vitro and in vivo anti-cancer
studies.
6.6.1. Cells
Ehrlich Ascites Carcinoma (EAC) cells were obtained through the
courtesy of Amala Cancer Research Centre, Trissur, India. They were
maintained by weekly intraperitoneal inoculation of 106 cells/
mouse [61].
6.6.2. Mean survival time [62]
Animals were inoculated with 1 ꢁ106 cells/mouse on day ‘0’ and
treatment with synthesized complexes started 24 h after inocula-
tion, at a dose of 100 mg/kg/day, p.o. The control group was treated
with the same volume of 0.9% sodium chloride solution. All the
treatments were given for nine days. The median survival time
(MST) of each group, consisting of 10 mice was noted. The anti-
tumor efficacy of complexes was compared with that of 5-
fluorouracil (Dabur Pharmaceutical Ltd, India; 5-FU, 20 mg/kg/
day, i.p. for 9 days).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.032.
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