Investigation of in vitro anticancer and DNA strap interactions in live cells using carboplatin type Cu(II) and Zn(II) metalloinsertors

Article abstract of DOI:10.1016/j.ejmech.2014.08.036

A series of carboplatin type Cu(II) and Zn(II) metalloinsertors (1-8) having β-diketone analogues and biologically significant cyclobutane-1,1-dicarboxylic acid have been synthesized and characterized by spectral and analytical methods. The binding and cleavage propensity of these metalloinsertors on DNA and their cytotoxic effects in live cells have been explored. From the gel electrophoresis study, it is observed that the complexes 1-8 cleave pBR322 DNA via a hydrolytic mechanism induced by hydroxyl radical scavengers, DMSO and EtOH as the reactive oxygen species (ROS). In vivo antitumor efficacy has been studied on EAC tumor bearing mice which is assessed by mean survival time, effect on hematological parameters and solid tumor volume. The results strongly support that complex 1 shows potent antitumor effect against EAC and higher than the standard drug carboplatin. Moreover, the cytotoxicity of the complexes, screened on a panel of human cancerous cell lines viz., human cervical cancer cells (HeLa), human breast adenocarcinoma cells (MCF-7), human laryngeal epithelial carcinoma cells (HEp-2), human liver carcinoma cells (Hep G2) and non-cancerous NIH 3T3 mouse embryonic fibroblasts cell lines, reveals that complex 1 exhibits a better anticancer activity than other complexes and standards.

Full text of DOI:10.1016/j.ejmech.2014.08.036

European Journal of Medicinal Chemistry 85 (2014) 675e687
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Investigation of in vitro anticancer and DNA strap interactions in live
cells using carboplatin type Cu(II) and Zn(II) metalloinsertors
Narayanaperumal Pravin, Natarajan Raman*
Research Department of Chemistry, VHNSN College, Virudhunagar 626 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of carboplatin type Cu(II) and Zn(II) metalloinsertors (1e8) having b-diketone analogues and
Received 28 June 2014
Received in revised form
8 August 2014
Accepted 9 August 2014
Available online 12 August 2014
biologically significant cyclobutane-1,1-dicarboxylic acid have been synthesized and characterized by
spectral and analytical methods. The binding and cleavage propensity of these metalloinsertors on DNA
and their cytotoxic effects in live cells have been explored. From the gel electrophoresis study, it is
observed that the complexes 1e8 cleave pBR322 DNA via a hydrolytic mechanism induced by hydroxyl
radical scavengers, DMSO and EtOH as the reactive oxygen species (ROS). In vivo antitumor efficacy has
been studied on EAC tumor bearing mice which is assessed by mean survival time, effect on hemato-
logical parameters and solid tumor volume. The results strongly support that complex 1 shows potent
antitumor effect against EAC and higher than the standard drug carboplatin. Moreover, the cytotoxicity of
the complexes, screened on a panel of human cancerous cell lines viz., human cervical cancer cells (HeLa),
human breast adenocarcinoma cells (MCF-7), human laryngeal epithelial carcinoma cells (HEp-2), hu-
man liver carcinoma cells (Hep G2) and non-cancerous NIH 3T3 mouse embryonic fibroblasts cell lines,
reveals that complex 1 exhibits a better anticancer activity than other complexes and standards.
© 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Cu(II) and Zn(II) metalloinsertors
Carboplatin
Cyclobutane-1,1-dicarboxylic acid
Reactive oxygen species
Antitumor activity
1. Introduction
towards normal cells which are tremendously valuable in biological
systems [5e9].
Over the past decades, metallopharmaceuticals are known to act
on DNA by inhibiting DNA replication and transcription. Thus,
studies on the interaction of metal complexes with DNA may reveal
useful information on the rational drug design and development of
perceptive chemical probe for DNA [1,2]. To date, cisplatin is
considered to be one of the most successful and extensively used as
anticancer drugs. Second generation platinum drugs including
cisplatin, oxaliplatin and carboplatin have been developed for
clinical application [3]. Among them, carboplatin displays a more
tolerable toxicological profile due to the higher stability of the
chelating 1,1-cyclobutanedicarboxylato ligand when compared to
the chlorido ligands in cisplatin. This in turn affects the adminis-
tration of the drugs [4]. These limitations have aroused curiosity
towards the design and evaluation of metal complexes other than
platinum derivatives for therapeutic use. In this esteem, non plat-
inum metal complexes predominantly low molecular weight
metals such as copper and zinc are being used at the forefront of
many of these efforts due to their stable, inert and low toxicity
In recent times, dicarboxylic acids have been widely used as a
polydentate ligand involving in diverse reactions to complexes
which have interesting properties in biological systems. Many
different bridging ligands such as pyridine (di)carboxylic acids and
imidazole (di)carboxylic acids [10,11] have effectively been familiar
with the construction of coordination compounds. These ligands
have diversiform coordination modes, and they contain two
carboxyl groups. This trait could increase the water-solubility of the
compounds for the cell membrane and thus facilitate the entrance
into the cell [12]. It is prominent that Schiff bases belong to a class
of organic compounds which have potential biological profile,
indeed the activity of these compounds augment when they
complex with metal ions [13]. This factor defends the drug against
enzymatic degradations because of the inertness of certain metal-
eligand linkages which have better hydrophobicity/hydrophilicity
properties than the free ligand and through this it can improve the
transport process in the tissues. Moreover, the complexation can
liberate the active drug(s) in a specific organ, and its action can be
reinforced by the combination of effects from the ligands and from
the metal residue. The application of these principles has previ-
ously resulted in the design of victorious metal based drugs [14,15].
Many studies suggest that DNA is the primary intracellular target of
* Corresponding author.
E-mail addresses: pravinknp2012@gmail.com (N. Pravin), drn_raman@yahoo.co.
in, ramchem1964@gmail.com (N. Raman).
http://dx.doi.org/10.1016/j.ejmech.2014.08.036
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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N. Pravin, N. Raman / European Journal of Medicinal Chemistry 85 (2014) 675e687
Scheme 1. Synthesis of Schiff base ligands and their metal complexes.
antitumor drugs because the interaction between small molecules
and DNA can cause DNA damage in cancer cells [16e18]. Thus, to
facilitate the development of new potential DNA targeting anti-
tumor drugs, the binding mechanisms of metal complexes to DNA
should be premeditated.
Our group has continuously been interesting in DNA in-
teractions of mixed-ligand complexes and has reported the syn-
thesis, DNA binding and cleavage activity of various transition
metal complexes [19,20]. In continuation of our journey in
designing and exploring the newer anticancer agents [21,22] in
order to improve the anticancer efficacy, we hereby report the
synthesis and characterization of few novel carboplatin type Cu(II)
and Zn(II) complexes. Furthermore, the in vitro, in vivo (EAC tumor
model) and DNA strap interactions and antitumor activities against
four human cancer cell lines along with one normal mouse em-
bryonic fibroblasts cell line have been probed.
2. Chemistry
The analytical, physicochemical and electronic spectral param-
eters of mixed ligand (i.e., cyclobutane-1,1-dicarboxylic acid)
complexes are compiled in the experimental section. The com-
plexes are found to be air stable. The ligands are soluble in common
organic solvents but their complexes are soluble 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:
cyclobutane-1,1-dicarboxylic acid) stoichiometry of the type
[ML(CBDCA)] wherein L acts as a bidentate ligand. The metal(II)
complexes are dissolved in DMF and the molar conductivities of
10ꢀ3 M of their solution at room temperature are measured. The
lower conductance values (9.6e16.3 Uꢀ1 cm2 molꢀ1) of the com-
plexes support their non-electrolytic nature.

N. Pravin, N. Raman / European Journal of Medicinal Chemistry 85 (2014) 675e687
677
3. Pharmacology
(9.6e16.3 Uꢀ1 cm2 molꢀ1) of the complexes support their non-
electrolytic nature [24].
The DNA strap interaction of the synthesized complexes with CT
DNA was carried out by electronic absorption spectroscopy, vis-
cosity measurements, cyclic voltammetry and differential pulse
voltammetry techniques. The extent of pBR322 DNA damage, in the
absence and presence of an activators viz., H2O2, AH2, MPA and GSH
and various radical scavengers like sodium azide NaN3 (singlet
oxygen), SOD (superoxide), DMSO, EtOH (hydroxyl radical scav-
enger) was monitored using agarose gel electrophoresis. A scru-
pulous 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), human breast
cancer (MCF-7), human laryngeal epithelial carcinoma (HEp-2)
cells, human liver carcinoma (Hep G2) and normal NIH 3T3 (mouse
embryonic fibroblasts). The minimum inhibitory concentration
(MIC) values of the complexes were determined.
4.3. Magnetic moments and electronic spectra
The geometry of the metal complexes has been deduced from
electronic spectra and magnetic data of the complexes which are
recorded in DMF solution. The free ligands exhibit two intense
bands in 45,857e41,656 and 28,361e27,138 cmꢀ1 region due to
p
/
p* and n /
p* transitions [25], respectively. In all the metal
complexes, the absorption bands at 43,759e41,424 and
29,302e27,659 cmꢀ1 are due to
p / p* and n / p* transitions that
are observed in the spectra of the free ligands L1, L2, L3 and L4. These
transitions are shifted to blue or red frequencies due to the coor-
dination of the ligand with metal ions. The electronic spectra of
Cu(II) complexes 1e4 reveal
a broad band in the region
21,786e21,413
cmꢀ1
with
high
molar
/
intensities
(ε ¼ 1058e1113 L Mꢀ1 cmꢀ1) assigned to B1g
2A1g transition
2
suggesting distorted square planar environment around the Cu(II)
ion. The observed magnetic moment of the Cu(II) complexes 1e4
(1.85e1.89 B.M) at room temperature indicates the non-coupled
mononuclear complexes 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 absorption spectra of the diamagnetic Zn(II)
complexes show the bands in the region 40,762e42,451 and
29,395e33,169 cmꢀ1 which are assigned to intra-ligand charge
transfer transitions [26].
4. Results and discussion
The synthetic pathways of the formation of Schiff bases and
their mixed ligand complexes are sketched 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 solu-
ble only in DMF and DMSO.
4.4. NMR spectra of zinc complexes
In 1H NMR, a set of multiplets appeared in the range of 6.8e7.4
d
for all the ligands and their Zn(II) complexes are assigned to the
aromatic region. The phenolic eOH proton for L3 ligand and its zinc
complex is observed as a singlet at ca. 10.3 ppm. It is suggesting that
phenolic eOH group is not taking part in the complexation. 1H NMR
spectra of aliphatic methyl protons exhibit peaks at 2.1e2.3 ppm for
all the Schiff base ligands and their Zn(II) complexes. There is no
appreciable change in all other signals of the complexes 5e8.
The 13C NMR spectra of the ligands show aromatic carbons at
119e129 ppm. The ligands also show the C]N carbons at
4.1. Infrared spectra
In IR spectra, the n(C]N) band presented in the ligands is shifted
to lower frequency by ca.30 cmꢀ1on complexation [23]. The free
eOH group of the ligand L3 vibrated at ca. 3435 cmꢀ1 does not show
any significant shift on complex formation. The additional bands
observed in complexes at 1683, 1349 and 817 cmꢀ1 are ascribed to
the vibrations of the carboxylate moiety. Therefore, the cyclo-
butane-1,1-dicarboxylic acid acts as bidentate coordinated to the
metal(II) centers via two monodentate carboxylate groups. The
172.8e175.4 ppm, which are shifted to downfield at 170.1e170.6 d,
upon coordination indicating the participation of C]N groups in
complex formation. 13C NMR spectra of ligand L4 and its Zn(II)
complex 8 are given in Fig. S1. The appearance of signals for the
mixed ligand complexes show the stretching vibration of n(C]O) of
the cyclobutanedicarboxylato groups at 1677e1689 cmꢀ1. The
relatively high value for this group shows the unsharing of the C]O
group in coordination to metal ion and thus the cyclo-
butanedicarboxylato group acts as a dianionic bidentate ligand.
This is further confirmed by the formation of metaleoxygen bond
in the complexes in the region 511e529 cmꢀ1. The new band
observed in the complexes in the range 430e448 cmꢀ1 indicates
the formation of metalenitrogen bond.
group COOꢀ at 61.2 and 164.3e168.7
d indicates the presence of
CBDCA carbon moiety. Further, in all the zinc complexes (5e8) the
particular signals of CBDCA carbon moiety have been appeared at
the regions 175.9, 25.7, 136.7 and 129.6 ppm. This indicates the
coordination of the ligand to the metal ion.
4.5. Mass spectra
4.2. Elemental analysis and molar conductivity measurements
The ESI-mass spectra of synthesized ligands and their com-
plexes have been recorded and the obtained molecular ion peaks
authenticate the proposed formulae. The mass spectrum of L1
ligand shows [Mþ1] peak at m/z 429 (86.4%) corresponding to
[C24H20N4O4]þ ion. Also, the spectrum exhibits the fragments at m/z
184 (35.2%), 102 (40.3%) and 77 (20.4%) corresponding to
[C12H12N2]þ, [C8H6]þ and [C6H5]þ respectively. ESI mass spectra of
ligand L1 and its Cu(II) complex 1 are shown in Fig. S2. The mass
spectrum of [CuL1(CBDCA)] shows peaks at m/z 635 with 54.5%
abundance. The strongest peak (base beak) at m/z 429 (66.8%)
represents the stable species C24H20N4O4. Moreover, the spectrum
exhibits the fragments at m/z 184, 102 and 77 corresponding to
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(CBDCA)] wherein both L and CBDCA act as bidentate ligands
(L ¼ Knoevenagel condensed Schiff bases; CBDCA ¼ cyclobutane-
1,1-dicarboxylic acid) (Scheme 1). The complexes are found to be
non-electrolytic nature in 10ꢀ3 M DMF solution, implying the
replacement of chloride anions by bidentate carboxylato group to
the central metal ion. The absence of counter (chloride) ions is
conformed from Volhard's test. The molar conductance values

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N. Pravin, N. Raman / European Journal of Medicinal Chemistry 85 (2014) 675e687
[C12H12N2]þ, [C8H6]þ and [C6H5]þ respectively. The m/z of all the
fragments of ligands and their complexes confirm the stoichiom-
etry of the complexes as [CuL1(CBDCA)]. The observed peaks are in
good agreement with their formulae as expressed from microan-
alytical data. Thus, the mass spectral data reinforce the conclusion
drawn from the analytical and conductance values.
complexes indicating negligible exchange interaction of CueCu in
the complexes according to Hathaway [27].
In order to quantify the degree of distortion of the Cu(II) com-
plexes, the G factor (g /A ) is obtained from the EPR spectra, where
jj jj
the G factor is regarded as an empirical index of tetrahedral
distortion. Its value ranges between 105 and 135 for square planar
complexes, depending on the nature of the coordinated atoms.
Values of this factor may vary from 157 to 166 for extreme distor-
tions in square planar complexes and it depends on the nature of
the coordinated atoms. The f values of Cu(II) complexes are indi-
cating significant distortion from planarity. In addition, the mo-
4.6. EPR spectra
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 EPR spectra of Cu(II) complexes 1 and 4 both at
room temperature (273 K) and liquid nitrogen temperature (77 K)
are depicted in Fig. 1. The spectrum of the copper complex 1 at RT
shows one intense absorption band in the high field and is isotropic
due to the tumbling motion of the molecules. However, this com-
plex at LNT shows three-well resolved peaks with low field region.
The spin Hamiltonian parameters of the complexes have been
calculated and are summarized in Table 1. From this spectral data, it
lecular orbital coefficients
(covalent in-plane -bonding) have been calculated from the
following equations [28]:
a s
2 (covalent in-plane -bonding) and b2
p
ꢀ
.
ꢁ
ꢀ
ꢁ
ꢀ
ꢁꢀ
ꢁ
a2 ¼
A
0:036
þ
g ꢀ 2:0027
k
þ
3=7 g⊥ ꢀ 2:0027
k
þ 0:04
ꢀ
ꢁ
.ꢀ
ꢁ
jj
jj
22
x-y
b2 ¼ g ꢀ 2:0027 E
ꢀ8la2
is
found
that
A
(131e138)
>
A⊥
(24e29);
g
k
dꢀd
(2.16e2.21) > g⊥(2.03e2.05) > ge(2.0023), which support the d
as the ground state, characteristic of square planar geometry and
axially symmetric. Further, in an axial symmetry, the g-values are
The calculated values of a2 (0.69e0.77) and b2 (0.87e0.96) for
the complexes 1e4 indicate that the in plane
plane -bonding are appreciably covalent and are consistent with
very strong in-plane -bonding in this complex. Furthermore, the
nature of bonding in the Cu(II) complexes can be obtained from the
s-bonding and in-
related by the expression, G ¼ (g ꢀ2)/(g⊥ꢀ2), which measures the
p
jj
exchange interaction between the copper centers in polycrystalline
p
solid. The G values lie within the range 4.2e5.3 for all the copper
Fig. 1. X-Band EPR spectra of Cu(II) complexes 1 and 4 at room temperature (a) (273 K) and at liquid nitrogen temperature (b) (77 K).

N. Pravin, N. Raman / European Journal of Medicinal Chemistry 85 (2014) 675e687
679
Table 1
The spin Hamiltonian parameters of the Cu(II) complexes in DMSO solution at 77 K.
Complexes
g-tensor
A ꢁ 10ꢀ4 (cmꢀ1
)
f
G
a2
b2
K
jj
K⊥
g
g⊥
giso
A
A⊥
Aiso
jj
jj
1
2
3
4
2.17
2.16
2.18
2.21
2.04
2.03
2.04
2.05
2.11
2.07
2.09
2.10
138
134
131
136
24
28
26
29
62
63
61
65
157
161
166
163
4.25
5.33
4.51
4.23
0.72
0.69
0.77
0.74
0.92
0.96
0.87
0.93
0.61
0.63
0.59
0.64
0.83
0.81
0.89
0.85
magnitude of the orbital reduction factors like K and K⊥ by using
jj
the following equations [29]:
ꢀ
ꢀ
ꢁ
Kk2
K⊥2
¼
g ꢀ 2:0027 Edꢀd=8l
k
ꢁ
¼
g⊥ ꢀ 2:0027 Edꢀd=2l
In the present study of the above mentioned complexes, it is
observed that K (0.59e0.64) < K⊥ (0.81e0.89) which indicates the
jj
presence of significant in-plane bonding. Based on the above ob-
servations, a square planar geometry is proposed for the complexes.
In the absence of crystallographic data, the orbital reduction and
bonding parameters of the Cu(II) complexes have provided sup-
portive evidence to the conclusion obtained on the basis of elec-
tronic and magnetic moment values.
4.7. DNA fastening efficacy
4.7.1. Absorption titration measurement
Transition metal complexes can bind to DNA through covalent
or non-covalent interactions [30]. The study of non-covalent in-
teractions of transition metal complexes with DNA is an area of
extreme modern curiosity. Since DNA is an important cellular re-
ceptor, many compounds wield their anticancer effects through
binding to DNA, thereby altering the replication of DNA and
inhibiting the growth of the tumor cells, which is the foundation for
designing new and more efficient anticancer drugs, where their
effectiveness depends on the mode and affinity of the binding [31].
Therefore, the binding studies of metal complexes to DNA are
considered to be highly imperative in the development of new
anticancer drugs. It is well known that the electronic absorption
spectroscopy is widely employed to determine the propensity for
binding of the complexes to DNA (CT DNA). In general, hyper-
chromism and hypochromism are the spectral features of DNA
concerning changes of its double helix structure; hyperchromism
means the breakage of the secondary structure of DNA and hypo-
chromism shows that binding of complex to DNA can be due to
electrostatic effect or intercalation which may stabilize the DNA
duplex. Moreover, the existence of a red-shift is the indicative of
stabilization of DNA duplex [32].
Fig. 2. Absorption spectra of complexes 1 (a) and 8 (b) in buffer pH ¼ 7.2 at 25 ꢂC in
presence of increasing amount of DNA.
Table 2
Electronic absorption spectral properties of Cu(II) and Zn(II) mixed ligand
complexes.
We have registered the spectra of solutions of the complexes 1
and 8 in the absence and in the presence of increasing concentra-
tions of CT DNA which are exemplified in Fig. 2(a) and (b). In the UV
region of the spectra, all the Cu(II) complexes exhibit an intense
absorption around 338.2e386.0 nm and zinc complexes show
c
Complexes
lmax (nm)
Dl (nm)
aH%
bKb (Mꢀ1
)
Free
Bound
1
2
3
4
5
6
7
8
386.0
342.5
340.6
338.2
380.3
351.4
353.3
324.5
381.0
339.3
337.8
336.5
376.5
348.1
350.2
320.5
5.0
3.2
2.8
1.7
3.8
3.3
3.1
4.0
20.8
8.5
10.7
14.5
18.2
9.2
3.8 ꢁ 106
2.6 ꢁ 105
1.3 ꢁ 106
1.6 ꢁ 106
2.7 ꢁ 106
2.1 ꢁ 105
1.2 ꢁ 106
1.5 ꢁ 106
bands in the region 324.5e380.3 nm (due to nep* transition). With
increase in DNA to complex ratio, hypochromism and red shift are
observed in the range 8.5e20.8%, and their red shifts in the region
1.7e5.0 nm. The extent of binding strength of complexes is quan-
titatively determined by measuring an intrinsic binding constants
Kb (Table 2). The intrinsic binding constant values of these com-
plexes (1e8) are compared to cisplatin (standard drug) and
ethidium bromide (classical intercalator) which reveal that Kb
11.2
12.3
a
H% ¼ [(Afree ꢀ Abound)/Afree] ꢁ 100%.
b
Kb ¼ Intrinsic DNA binding constant determined from the UVeVis absorption
spectral titration.
values of 1e8 are greater in magnitude (1.2e3.8 ꢁ 106
M
ꢀ1) than
Error limit 3%.
c

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N. Pravin, N. Raman / European Journal of Medicinal Chemistry 85 (2014) 675e687
that of cisplatin (5.73
0.45 ꢁ 104
M
ꢀ1) and ethidium bromide
the complexes 1 and 7 in the absence and presence of different
amounts of DNA are shown in Fig. 4(a) and (b), exhibited significant
shifts in the anodic and cathodic peak potentials followed by
decrease in both peak currents, indicating the interaction existing
among these complexes and CT DNA.
The voltammetric parameters obtained for all the complexes
without and in the presence of DNA are given in Table 3. In the
absence of CT DNA, the first redox cathodic peak appears at 0.048 V
(1.4 ꢁ 106
M
ꢀ1) as reported in the literature [33,34] and relatively
lower
than
the
metallointercalator
[Ru(bpy)2(HBT)]2þ
(5.71 ꢁ107 Mꢀ1) [35]. However, the metal ions play a crucial role in
DNA binding by these complexes. The binding strength of the
synthesized complexes with DNA is shown as in the following or-
der: 1 z 5 > 2 z 6 > 4 z 8 > 3 z 7. The highest binding constant of
complex 1 is due to electron withdrawing group present on the
ligand moiety [36].
for Cu(II)/Cu(I) (Epa ¼ 0.066 V, Epc ¼ 0.597 V,
E1/2 ¼ ꢀ0.009 V) and in the second redox couple, the cathodic peak
appears at ꢀ0.103 for Cu(I)/Cu(0) (Epa ꢀ0.416 V,
Epc ¼ ꢀ0.103 V, Ep ¼ ꢀ0.358 V, and E1/2 ¼ ꢀ0.282 V). The Ipa/Ipc
DEp ¼ ꢀ0.114 V and
V
¼
4.7.2. Hydrodynamic volume measurement
D
Though photophysical experiments afford necessary informa-
tion about binding modes of metal complexes with DNA, but not
provide conclusive evidences for the precise mode of binding.
Hydrodynamic measurements are perceptive to DNA length change
and considered to be the most decisive tests in assessing binding
modes in solution in the absence of crystallographic structural data
[37]. Classical intercalative mode causes a significant increase in
viscosity of DNA solution due to separation of base pairs at inter-
calation sites and increase in overall DNA length. In contrast,
complexes those bind exclusively in the DNA grooves typically
cause less positive or negative or no change in DNA viscosity [38].
The viscosity of the DNA solution increases with increasing ratio of
the complexes to DNA. As expected, the known DNA-intercalator,
ethidium bromide (EB) increases the relative viscosity of DNA due
to its strong intercalation. The effects of complexes together with
the viscosity of rod-like DNA are shown in Fig. 3. Compared with EB,
complexes demonstrate minor increase in the relative viscosity of
CT DNA, suggesting an intercalation mode among the complexes
and DNA. This result further suggests an intercalating binding
mode of the complexes with DNA and also parallels the above
photophysical results, such as hypochromism and red shift of the
complexes in the presence of DNA. The experimental results sug-
gest that complexes bind to DNA through a classical intercalation
mode.
ratios for these two redox couples are 1.1 and 0.86 respectively. The
voltammogram of the Zn(II) complex also shows quasi-reversible
process. The reason for the quasi-reversible electron transfer pro-
cess may be due to slow electron transfer or the adsorption of the
complex onto the electrode surface [40]. A quasi-reversible transfer
process with the redox couple [Zn(II)/Zn(0)] is observed for the
complexes 5e8. In complex 7, the cathodic peak appeares
at ꢀ0.172 V in the absence of DNA (Epa ¼ ꢀ0.867 V, Epc ¼ ꢀ0.172 V,
D
Ep ¼ ꢀ0.695 V, and E1/2 ¼ ꢀ0.520 V). The Ipa/Ipc ratio is 1.08. This
indicates the quasi-reversible redox process of the metal com-
plexes. Incremental addition of DNA to the complex 7 results in a
slight decrease in the current intensity and less negative shift of the
oxidation peak potential. The resulting minor changes can be
explained in terms of the slow diffusion of the complexes bound to
the large DNA molecule.
In the differential pulse voltammogram of the complexes 1 and
7 obtained in the absence and the presence of varying amounts of
DNA [in which a significant decrease in current intensity with
4.7.3. Electrochemical profiles
CV can afford useful information on the binding of metal com-
plexes to DNA, because of the resemblance between the electro-
chemical and biological reactions. The application of the cyclic
voltammetric technique to the study of interaction between the
metal complexes and DNA provides a useful complement to the
previously used spectral studies [39]. The cyclic voltammograms of
Fig. 3. Effect of increasing amount of [EB] ( ), 1 ( ), 2 ( ), 3 ( ), 4 ( ), 5 ( ), 6 ( ), 7
Fig. 4. Cyclic voltammograms of complexes 1 (a) and 7 (b) in buffer pH ¼ 7.2 at 25 ꢂ
C
(
) and 8 ( ) on the relative viscosity of DNA.1/R ¼ [Complex]/[DNA] or [EB]/[DNA].
in presence of increasing amount of DNA.

N. Pravin, N. Raman / European Journal of Medicinal Chemistry 85 (2014) 675e687
681
Table 3
Table 4
Redox potential profiles for interaction of DNA with Cu(II) and Zn(II) complexes.
Minimum inhibitory concentration of the synthesized compounds against growth of
bacteria ( M).
m
Complexes
aDEp (V)
bE1/2 (V)
Free
Ipa/Ipc
K[red]/K[ox]
Compound Minimum inhibitory concentration (MIC) (ꢁ104
m
M) SEM ¼
2
Free
Bound
Bound
Staphylococcus Bacillus
Escherichia Klebsiella
Salmonella
typhi
1
2
3
4
5
6
7
8
ꢀ0.204
0.462
ꢀ0.114
0.534
ꢀ0.013
0.526
ꢀ0.009
0.564
1.2
0.76
0.43
0.59
0.82
0.68
0.79
0.54
0.93
aureus
subtilis
coli
pneumoniae
0.78
0.58
0.67
0.43
3.21
1.08
0.84
ꢀ0.128
ꢀ0.107
ꢀ0.186
ꢀ0.175
L1
L2
L3
L4
1
2
3
4
5
16.7
17.0
19.3
18.5
9.6
16.1
18.2
10.9
19.6
9.1
15.3
16.3
18.7
17.0
8.6
16.7
16.4
18.3
17.6
8.2
16.3
16.8
17.1
17.9
9.3
10.3
11.1
10.4
9.9
11.6
11.9
13.1
2.6
0.525
0.549
0.378
0.563
0.812
0.823
0.248
0.354
0.381
ꢀ0.119
ꢀ0.695
0.167
ꢀ0.142
ꢀ0.520
ꢀ0.053
ꢀ0.128
ꢀ0.492
ꢀ0.045
ꢀ0.677
0.178
10.7
10.7
10.9
10.1
11.2
12.6
12.4
12.1
11.9
11.3
11.2
13.1
12.1
11.8
2.8
10.9
11.4
12.4
9.1
9.9
9.4
9.3
9.1
Data from cyclic voltammetric measurements: a
D
Ep ¼ Epa ꢀ Epc; bE1/2 is calculated
as the average of anodic (Epa) and cathodic (Epc) peak potentials; bE1/2 ¼ Epa þ Epc/
2.
6
7
8
11.6
13.1
12.8
1.4
10.3
11.1
12.2
2.3
aKanamycin 1.6
increasing DNA concentration is observed (Fig. 5(a) and (b))], the
shift in potential is related to the ratio of the binding constants by
the following equation:
Average of 3 determinations, 3 replicates.
a
Kanamycin is used as the standard.
ꢀ
.
ꢁ
Eb ꢀ Ef ¼ 0:0591 log K
K
½oxꢃ
½redꢃ
4.8. Antimicrobial screening
where Eb and Ef are the peak potentials of the complex in its bound
and free forms, respectively. In the present study, the novel mixed
ligand complexes exhibit one-electron transfer during the redox
process, and the Ipc/Ipa value is less than unity, indicating that the
reaction of the complex on the glassy carbon electrode surface is
quasi-reversible.
As the antimicrobial screening contribution to the field of bio-
inorganic chemistry is important, the synthesized compounds have
been evaluated for their antimicrobial actions. The results of the
bactericidal and fungal study of the synthesized compounds are
reported in Tables 4 and 5. Further, the antimicrobial action of li-
gands may be significantly enhanced on chelation with metal ions,
but the activity is lesser than the standard drugs. Such enhanced
activity of the complexes can also be explained on the basis of
activation of enzymes, fineness of the particles, and size of the
metal ion and the presence of substituted and highly conjugated b-
diketimine analogous. The biological activity involves inhibition of
DNA synthesis by creating lesions in DNA strands by oxidative
rupture and by binding the nitrogen bases of DNA or RNA, hin-
dering or blocking base replication. Due to the complexity of bio-
logical systems, it is rather difficult to stipulate the exact
mechanism for such activities. However, the increased biocidal
properties after complexation can be very well explained by
Overtone's concept [41] and the Tweedy's Chelation theory [42].
The MIC values for the complexes are related to the nature of the p-
substituent as they increase according to the following order p-
(OCH3 < OH < H < NO2) [43]. This can be attributed to the fact that
the effective nuclear charge experienced on d-electrons increases
due to the presence of electron withdrawing p-substituents and
decreases by the electron donating p-substituents.
4.9. DNA ripping actions
4.9.1. Interaction with pBR322 plasmid DNA
The potential of the cleavage DNA of the present complexes is
studied by gel electrophoresis using supercoiled plasmid pBR322
DNA in TAE buffer (pH 7.4). When circular plasmid DNA is subjected
to gel electrophoresis, relatively fast migration will be observed for
the intact supercoil form (Form I). If scission occurs on one strand
(nicking), the supercoil will relax to generate a slower moving open
circular form (Form II). If both strands are cleaved, a linear form
(Form III) that migrates between forms I and II will be generated.
The cleavage of plasmid pBR322 DNA induced by Cu(II) and Zn(II)
complexes is performed in the absence of external cofactor agents
(either oxidant or reductant). The Cu(II) complexes are found to
promote the cleavage of plasmid pBR322 DNA from the supercoiled
form (I) to the nicked form (II) and linear form (III) (Fig. 6). No DNA
cleavage has been observed for the control in which the metal
Fig. 5. Differential pulse voltammograms of 1 (a) and 7 (b) in buffer (pH ¼ 7.2) at 25 ꢂ
C
in the presence of increasing amount of DNA. Arrow indicates the changes in vol-
tammetric currents upon increasing the DNA concentration.

682
N. Pravin, N. Raman / European Journal of Medicinal Chemistry 85 (2014) 675e687
Table 5
Moreover, the DNA cleavage activity of complexes 1 and 5 has
been evaluated in the presence and absence of activators viz.; H2O2,
AH2, MPA and GSH as shown in Fig. 7. The results reveal that the
cleavage activity of complexes are drastically enhanced in the
presence of these activators and follows the order:
Minimum inhibitory concentration of the synthesized compounds against the
growth of fungi ( M).
m
Compound Minimum inhibitory concentration (MIC) (ꢁ104
m
M) SEM ¼
2
Aspergillus Fusarium Curvularia Rhizoctonia
niger
Candida
albicans
solani
lunata
bataticola
MPA
>
H2O2
¼
GSH
>
AH2 for complex
1
and
L1
L2
L3
L4
1
2
3
4
5
16.2
18.9
20.3
19.2
9.4
11.3
12.1
12.6
12.9
15.4
18.1
13.1
17.4
18.5
21.6
19.0
10.3
11.7
15.2
15.0
12.1
14.3
14.0
15.3
1.7
15.5
17.6
19.5
18.1
10.9
13.0
11.3
13.6
14.4
18.2
16.3
15.5
1.2
14.9
15.8
17.5
16.9
12.2
14.1
15.2
14.3
15.5
17.1
17.6
16.9
1.5
16.4
17.6
18.4
17.9
14.6
15.5
16.1
17.1
16.4
18.1
16.5
18.9
1.8
MPA > GSH > AH2 z H2O2 for complex 5. The complex 1 in the
presence of activators exhibits momentous DNA cleavage activity,
followed by complete degradation of DNA.
If the nuclease activity is quenched by the presence of any
radical scavengers, it may be due to presence of reactive oxygen
species (ROS). In order to investigate the role of reactive oxygen
species which is responsible for the DNA cleavage mediated by
complexes 1 and 5, reactions are carried out in the presence of
hydroxyl radical scavengers (DMSO and EtOH), singlet oxygen
quencher (NaN3) and superoxide oxygen scavenger (SOD) under
identical conditions. As shown in Fig. 8, the addition of singlet
oxygen scavenger (NaN3) and superoxide scavenger (SOD) does not
show any inhibitory effect (Fig. 8(a) lanes 8 and 9; Fig. 8(b) lanes 2
and 3) to the complexes 1 and 5, while the addition of hydroxyl
radical scavengers (EtOH and DMSO) shows significant inhibitory
effects (Fig. 8(a) lanes 6 and 7; Fig. 8(b) lanes 4 and 5) and hence
hydroxyl radical (OHꢄ) is one of the ROS. Nevertheless, hydroxyl
radical (OHꢄ) is likely to be the reactive species responsible for the
nuclease activity of plasmid DNA. From these observations, both
the complexes 1 and 5 display efficient DNA cleavage activity
through a hydrolytic cleavage induced by reactive oxygen species
(ROS).
6
7
8
aFluconazole 1.4
a
Fluconazole is used as the standard. Average of 3 determinations, 3 replicates.
complex is absent (lanes 2 and 3) and in presence of free CuCl2 and
ZnCl2 (100 mM). When the concentrations of the Cu(II) and Zn(II)
complexes are increased the amount of form I of pBR322 DNA di-
minishes gradually, whereas the amounts of form II and III increase.
It shows, both the complexes are able to induce significant cleavage
of the plasmid DNA even at the low concentration (5
concentration of 30 M, Cu(II) and Zn(II) complexes almost pro-
mote the complete conversion of DNA from form I to forms II and III
mM). At the
m
(Fig. 6; lanes 4e11).
Fig. 6. Agarose gel electrophoresis pattern for the cleavage of pBR322 plasmid DNA (10
m
M) by complexes 1e8 at 37 ꢂC after incubation for 45 min at 30
M) ZnCl2 þ DNA; lane 4: complex 1 þ DNA; lane 5: complex 2 þ DNA; lane 6:
complex 3 þ DNA. lane 7: complex 4 þ DNA; lane 8: complex 5 þ DNA; lane 9: complex 6 þ DNA; lane 10: complex 7 þ DNA; lane 11: complex 8 þ DNA.
mM concentration with
histogram of % cleavage forms. lane 1: DNA control; lane 2: (100
mM) CuCl2þ DNA; lane 3: (100
m

N. Pravin, N. Raman / European Journal of Medicinal Chemistry 85 (2014) 675e687
683
Fig. 7. Agarose gel electrophoresis pattern for the cleavage pattern of pBR322 DNA (10
m
M) by complexes 1 and 5 at 37 ꢂC after incubation for 45 min 30
mM concentration with
histogram of % cleavage forms. In absence and presence of complex 1 with different activators: lane 1, DNA control; lane 2, DNA þ Asc (100
m
M); lane 3, DNA þ H2O2 (100
M); lane 9, DNA þ 1 þ H2O2
M).
mM); lane
4, DNA þ GSH (100
(100
m
M); lane 5, DNA þ MPA (100
m
M); lane 6, DNA þ 1 þ Asc (100
m
M); lane 7, DNA þ 1 þ MPA (100
m
M); lane 8, DNA þ 1 þ GSH (100
m
m
M); lane 10, DNA þ 5 þ GSH (100
mM); lane 11, DNA þ 5 þ H2O2 (100
mM); lane 12, DNA þ 5 þ MPA (100
mM); lane 13, DNA þ 5 þ AH2 (100
m
Fig. 8. Agarose gel electrophoresis pattern for the cleavage pattern of pBR322 DNA (10
m
M) by complex 1 and 5 at 37C after incubation for 45 min 30
m
M concentration with
M); lane 3,
M); lane 8, DNA þ 1 þ NaN3
M); lane 9, DNA þ 1 þ SOD (1U); (b) In the presence of complex 5 with different radical scavengers; lane 1, DNA control; lane 2, DNA þ 5 þ SOD (1U); lane 3, DNA þ 5 þ NaN3
M); lane 4, DNA þ 5 þ DMSO (100 M); lane 5, DNA þ 5 þ EtOH (100 M).
histogram of % cleavage forms. (a) In the absence and presence of complex 1 with different radical scavengers; lane 1, DNA control; lane 2, DNA þ DMSO (100
m
DNA þ EtOH (100
m
M); lane 4, DNA þ SOD (1U); lane 5, DNA þ NaN3 (100
m
M); lane 6, DNA þ 1 þ DMSO (100
mM); lane 7, DNA þ 1 þ EtOH (100
m
(100
(100
m
m
m
m

684
N. Pravin, N. Raman / European Journal of Medicinal Chemistry 85 (2014) 675e687
Table 6
complexes may be attributed to the extended planar structure
induced by the * conjugation resulting from the chelation of
the Cu(II) ion with the ligands. These findings confirm that the
binding of the complex to DNA, which consequently leads to cell
death.
IC50
(mM) values of complexes, cisplatin and carboplatin against various cancer cell
pep
lines.
Compound
IC50 values (
mM)
HeLa
HEp-2
Hep-G2
MCF-7
NIH 3T3
176 12
CuCl2
524 11
637 17
465 11
521 13
1
2
3
4
12.1 1.6 11.8 2.2
14.2 1.2 13.6 1.8
13.3 1.5 12.7 2.1
12.8 1.3 12.2 2.6
3.4 0.21 13.8 2.3 378 4.2
4.8 0.51 15.6 1.9 346 3.8
4.2 0.40 14.8 2.2 357 4.5
3.8 0.32 14.3 2.7 366 5.3
4.11. In vivo effect on tumor screening
In vivo, there is a significant (P < 0.001) increase in mean sur-
Cisplatin
12.6 0.4 13.6 0.9 12.1 0.5
10.8 1.6 198 10
9.7 1.3 243 11
vival time and increase in life span of the synthesized complexes
Carboplatin 12.3 0.8 12.5 1.2
6.9 0.9
1e4 on EAC (41.34e31.21 and 135.75e112.87 at 100 mg kgꢀ1
,
Average of 3 determinations, 3 replicates.
respectively) in a dose dependent manner (Table 7). The results are
almost comparable to that of cisplatin and carboplatin (40.66/44.28
and 151.60/162.37, respectively). In addition, the complexes 1e4
significantly (P < 0.01) reduce the tumor volume in dose dependent
manner when compared to tumor control groups. The EAC tumor
cells from ascitic fluid of different treatment groups are stained
with Lieshman stain. They show marked cytological changes and
cytolytic activity when compared to the respective tumor control
cells (Fig. 10). The complexes 1e4 delay the cell division, thereby
suggesting the reduction in EAC volume and increase survival time
in mice. At the dose level 100 mg kgꢀ1, they are significantly
improved the MST in tumor bearing mice. The prolongation of life
span is a reliable criterion for judging efficacy of anticancer drugs
[44,45] and the complexes 1e4 are able to meet this criterion.
Myelosuppression and anemia (reduced hemoglobin) have been
frequently observed in ascites carcinoma [46,47]. Anemia encoun-
ters in ascites carcinoma mainly due to iron deficiency, either by
hemolytic or myelopathic conditions which finally lead to reduced
RBC number [48]. In this study, elevated WBC count, reduces he-
moglobin and RBC count are observed (Table 8) in EAC control mice
and the oral administration of complex 1 restores hemoglobin
content and maintains normal values of RBC and WBC, thus sup-
porting its hematopoietic protecting activity without inducing
myelotoxicity, the most common side effects of cancer chemo-
therapy. Therefore, further investigation in order to explore the
potential of the complex 1 in tumor treatment may prove to be
worthwhile.
IC50, Drug concentration inhibiting 50% cellular growth following 72 h of drug
exposure.
4.10. In vitro cytotoxic profile
Cytotoxicity is a common limitation in stipulations of the
introduction of new compounds into the pharmaceutical industry.
The positive results obtained from the previous biological studies,
namely, antimicrobial, DNA fastening and ripping studies for
complexes 1e4, encourage us to test their in vitro cytotoxic activity
against a panel of human cancer cell lines along with normal mouse
embryonic fibroblasts (NIH 3T3) cells by the MTT assay method.
Cisplatin and carboplatin are used as positive controls to assess the
cytotoxicity of the test compounds. The IC50 values show that the
complexes 1e4 exhibit significant activity against HeLa, HEp-2,
Hep-G2 and MCF-7 cell lines, which are almost equal to the activ-
ity of the well-known anticancer drugs, cisplatin and carboplatin
(Table 6). Interestingly, on comparison of the IC50 value of the
complexes 1e4 with cisplatin against Hep-G2, the inhibitory ac-
tivity is about three times higher than that of cisplatin and two
times than that of carboplatin (Fig. 9). These results also indicate
that the IC50 value of the complexes 1e4 against NIH 3T3 (normal
cells) is found to be lying in the range 346e378
that the complexes are very specific for cancer cells and even less
toxic compared to standard drugs (IC50 ¼ 198 M for cisplatin and
243 M for carboplatin). It is clearly noted that the free CuCl2 does
not show any significant activity on all the cancer cells
M), which confirms that the chelation of the
mM, which confirms
m
m
(IC50 ¼ 465e642
m
ligand (nature of p-substituents) with the Cu(II) ion is the respon-
sible factor for the observed varying cytotoxic properties of the
complexes 1e4. Also the better cytotoxic properties of the
5. Experimental protocols
The materials and methods for the newly synthesized mixed-
ligand complexes are depicted as supplementary materials S1
(Supplementary Files). We acquired the probable composition of
complexes as [ML(CBDCA)] (where
M
¼
Cu(II) and Zn(II);
CBDCA ¼ cyclobutane-1,1-dicarboxylic acid and L ¼ Knoevenagel
condensed Schiff bases) which were confirmed through elemental
analyses, magnetic susceptibility, molar conductivity, NMR, UV, IR,
EPR and mass spectra since no single crystals suitable for X-ray
determination could be isolated.
Table 7
Effect of Cu(II) complexes treatment on the survival of tumor-bearing mice.
Treatment
MST
Increase in life span
Tumor control
cis-platin
carboplatin
1
2
3
4
16.26 0.52
40.66 0.81*
44.28 0.81*
41.34 0.27*
31.21 0.57*
34.87 0.71*
38.82 0.34*
e
151.60
162.37
135.75
118.14
112.87
127.57
N ¼ 6; d of drug treatment ¼ 9, *p < 0.01versus tumor control.
Fig. 9. The in vitro cytotoxicity of Cu(II) complexes against a panel of human cell lines.
Data were analyzed by one-way ANOVA followed by Dunnett's test.

N. Pravin, N. Raman / European Journal of Medicinal Chemistry 85 (2014) 675e687
685
Fig. 10. (a) Smear showing matured EAC control cells (for 24 h) with definite cell wall and structure without degeneration, (b) EAC tumor cells treated with cisplatin for 24 h, (c) EAC
tumor cells treated with carboplatin for 24 h and (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.
The Schiff base ligands (L1eL4) were synthesized by the method
reported in the literature by us [22]. To a stirred ethanolic solution
of the above Schiff base(s) (5 mmol), a solution of copper(II)/zinc(II)
chloride (0.67 g/0.68 g; 5 mmol) in ethanol was added dropwise.
After the reaction for 1 h at 60 ꢂC, a solution of cyclobutane-1,1-
dicarboxylic acid (0.72 g; 5 mmol) in ethanol was added. The re-
action 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.
n
(eCeO); 513
n
(MꢀO); 430
n
(MꢀN). MS m/z: 545 (56.4%) [Mþ1]þ.
LM10ꢀ3
(U
ꢀ1 cm2 molꢀ1) ¼ 9.6. lmax (cmꢀ1) in DMF, 40,362, 29,642,
21,786 (ε ¼ 1102 L Mꢀ1 cmꢀ1). meff (BM): 1.89.
[CuL3(CBDCA)] (3) Yield: (1.6 g, 50%). m.pt: 180e187 ꢂC. Anal.
Calc. for C30H28N2O6Cu: Cu, 11.7; C, 62.6; H, 4.8; N, 4.9; Found: Cu,
11.6; C, 62.4; H, 4.7; N, 4.8 (%). IR (KBr pellet, cmꢀ1): 3434
n
(eOH);
1689 n(non-coordinated C]O in CBDCA); 1609 n(eC]N); 1529
n
(eHC]C); 1325
n
(eCeO); 529
n
(MꢀO); 433 (MꢀN). MS m/z: 577
n
(51.8%) [Mþ1]þ. LM 10ꢀ3
(
Uꢀ1 cm2 molꢀ1) ¼ 11.3. lmax (cmꢀ1) in
[CuL1(CBDCA)] (1) Yield: (1.9 g, 56%). m.pt: 182e187 ꢂC. Anal.
Calc. for C30H26N4O8Cu: Cu, 10.0; C, 57.0; H, 4.1; N, 8.8; Found: Cu,
DMF, 41,424, 29,302, 21,186 (ε ¼ 1084 L Mꢀ1 cmꢀ1). meff (BM): 1.86.
[CuL4(CBDCA)] (4).Yield: (1.7 g, 51%). m.pt: 184e190 ꢂC. Anal.
Calc. for C32H32N2O6Cu: Cu, 10.5; C, 63.6; H, 5.3; N, 4.6; Found: Cu,
9.9; C, 56.8; H, 3.9; N, 8.7 (%). IR (KBr pellet, cmꢀ1): 1686
n
(non-
coordinated C]O in CBDCA); 1606
n
(eC]N); 1507
n
(eHC]C);
10.4; C, 63.5; H, 5.2; N, 4.5 (%). IR (KBr pellet, cmꢀ1): 1680
n
(non-
1473, 1312, 833 (eCeN str; eNO2); 1329
n
n
(eCeO); 520
n
(MꢀO);
coordinated C]O in CBDCA); 1607n(eC]N); 1522 n(eHC]C);
439
n
(MꢀN).
MS
m/z:
635
(54.5%)
[Mþ1]þ.
LM
1313
n
(eCeO); 1272, 1081,
n
(eCeOeCe); 532
n
(MꢀO); 440 (MꢀN).
n
10ꢀ3
(
Uꢀ1 cm2 molꢀ1) ¼ 9.8. lmax in DMF, 43,759, 28,326, 21,413
MS m/z: 605 (57.3%) [Mþ1]þ. LM 10ꢀ3
(
U
ꢀ1 cm2 molꢀ1) ¼ 10.1. lmax
(ε ¼ 1058 L Mꢀ1 cmꢀ1). meff (BM): 1.87.
(cmꢀ1) in DMF, 41,549, 27,821, 21,598 (ε ¼ 1113 L Mꢀ1 cmꢀ1). meff
(BM): 1.85.
[CuL2(CBDCA)] (2) Yield: (1.7 g, 58%.). m.pt: 180e185 ꢂC. Anal.
Calc. for C30H28N2O4Cu: Cu, 11.9; C, 66.2; H, 5.2; N, 5.1; Found: Cu,
[ZnL1(CBDCA)] (5) Yield: (2.0 g, 59%). m.pt: 180e186 ꢂC. Anal.
Calc. for C30H26N4O8Zn: Zn, 10.3; C, 57.; H, 4.1; N, 8.8; Found: Zn,
11.8; C, 66.1; H, 5.1; N, 5.0 (%). IR (KBr pellet, cmꢀ1): 1681
n
(non-
coordinated C]O in CBDCA); 1603
n
(C]N); 1503
n
(eHC]C); 1328
10.2; C, 56.9; H, 4.0; N, 8.7 (%). IR (KBr pellet, cmꢀ1): 1677
n(non-
Table 8
Effect of Cu(II) complexes on hematological parameters of EAC tumor bearing mice.
Design of treatment Hb (g %)
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 0.15
5.78 0.42a
12.18 0.21d
11.34 0.12d
4.9 0.12
2.46 0.14a
4.18 0.19d
3.42 0.15a,e
6.78 0.14
5.73 0.26
12.23 0.34a
7.19 0.17b,d
7.63 0.27a,d
8.56 0.11a,d
9.96 0.41a,d
16.86 0.55
25.98 0.56a
17.64 0.24d
16.98 0.51d
17.18 0.45d
66.46 1.36
26.23 1.16a
32.6 1.61 1.25 0.45
72.8 1.07a 1.20 0.45
18.74 0.47a
15.58 0.27a,d
14.95 0.54a,d
12.16 0.63a,d
15.67 0.42a,d
66.08 0.86d 36.12 1.24d 1.17 0.32
64.63 1.09d 31.60 1.23d 0.94 0.24
63.42 1.28d 33.27 1.06d 1.03 0.15
11.09 0.16a,d 3.98 0.17b,d
13.12 0.14d
4.14 0.15d
19.98 0.58b,d 65.84 1.24d 38.71 1.13d 1.06 0.23
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 comp.

686
N. Pravin, N. Raman / European Journal of Medicinal Chemistry 85 (2014) 675e687
coordinated C]O in CBDCA); 1618
n
(eC]N); 1509
n
(eHC]C);
1 shows that the activity is higher than that of other complexes
(2e4) and standard drugs. Particularly, admirable cytotoxic speci-
ficity against Hep G2 (human liver carcinoma cells) and the IC50
value of the complex 1 shows that the activity is about three times
higher than that of cisplatin and two times higher than that of
carboplatin. Further studies are needed to appraise its pharmaco-
logical properties in vivo and to elucidate the actual mechanism of
its biological activity. These findings clearly indicate that the Cu(II)
complexes may have potential with the biological target DNA,
thereby affecting cellular responses akin to DNA strap interaction,
damage and cytotoxic profile. Thus, the Cu(II) complexes are
dominating over the others and proving their worth as robust and
better choice for potent and safe chemotherapeutic drug design.
1461, 1304, 839
n
(eCeN str; eNO2); 1312
n
(eCeO); 511
n
(MꢀO);
436
n
(MꢀN). 1H NMR (ppm): (aromatic) 6.9e7.6 (m); (CH, 1H) 6.9
(q), CBDCA [(CH2, J ¼ 7.6, 2H) 3.2 (q), (CH2, J ¼ 14.8, 2H) 2.3
(q)],(eCH3, 6H), 2.1 (s). 13C NMR (ppm):176.5 (C13), 170.6 (C7), 153.1
(C9), 146.8 (C12), 136.8 (C5), 134.3 (C4), 127.6e129.2 (C1 to C3), 124.4
(C11), 123.5 (C10), 114.3 (C6), 61.3 (C14), 25.7 (C15), 20.7 (C8), (19.1)
(C16).
(
MS
m/z:
636
(62.4%)
[Mþ1]þ.
LM
10ꢀ3
Uꢀ1 cm2 molꢀ1) ¼ 14.5. lmax (cmꢀ1) in DMF, 42,257, 33,169. meff
(BM): diamagnetic.
[ZnL2(CBDCA)] (6) Yield: (1.8 g, 61%). m.pt: 176e183 ꢂC. Anal.
Calc. for C30H28N2O4Zn: Zn, 11.9; C, 66.0; H, 5.8; N, 5.1; Found: Zn,
11.8; C, 65.9; H, 5.7; N, 5.0 (%). IR (KBr pellet, cmꢀ1): 1681
n(non-
coordinated C]O in CBDCA). 1607
1322 (eCeO); 519
(MꢀO), 448
n(eC]N); 1523 n(eHC]C);
n
n
n
(MꢀN). 1H NMR (ppm): (aro-
Acknowledgments
matic) 6.9e7.4 (m); (CH, 1H) 6.9 (q), CBDCA [(CH2, J ¼ 7.1, 2H) 3.2
(q), (eCH3, 6H), 2.2 (s), (CH2, J ¼ 14.2, 2H) 2.1 (q)]. 13C NMR
(ppm):176.4 (C13), 170.1 (C7), 136.8 (C16), 136.7 (C5), 136.2 (C9), 134.4
(C4), 130.3 (C11), 127.8e129.2 (C1 to C3), 127.2 (C12), 119.4 (C10), 114.4
(C6), 61.3 (C14), 25.6 (C15), 20.7 (C8), (19.4) (C16). MS m/z: 546 (65.2%)
The authors express their heartfelt thanks to the Science &
Engineering Research Board (SERB), DST (File No. SR/S1/IC-27/
2012) New Delhi, India 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 Pharmaceutical Chemistry, Swami Vivekanandha
College of Pharmacy, Elayampalayam, Tiruchengodu, for in vitro and
in vivo anti-cancer studies.
[Mþ1]þ. LM 10ꢀ3
(
Uꢀ1 cm2 molꢀ1) ¼ 16.3. lmax (cmꢀ1) in DMF,
40,762, 26,395. meff (BM): diamagnetic.
[ZnL3(CBDCA)] (7).Yield: (1.9 g, 60%). m.pt: 186e192 ꢂC. Anal.
Calc. for C30H28N2O6Zn: Zn, 11.3; C, 62.3; H, 4.9; N, 4.8; Found: Zn,
11.2; C, 62.2; H, 4.8; N, 4.7(%). IR (KBr pellet, cmꢀ1): 3433
n(eOH);
1684
n
(non-coordinated C]O in CBDCA), 1611
(eCeO); 519
(MꢀO), 431
n(eC]N); 1526
n
(eHC]C); 1329
n
n
n
(MꢀN). 1H NMR
Appendix A. Supplementary data
(ppm):(CeOH, 1H) 10.4 (s), (CH, 1H) 6.9 (q), (aromatic) 6.8e7.4 (m);
CBDCA [(CH2, J ¼ 7.4, 2H) 3.1(q), (CH2, J ¼ 14.8, 2H) 2.2 (q)],(eCH3,
6H), 2.1 (s). 13C NMR (ppm):175.9 (C13), 170.2 (C7), 157.2 (C12), 145.2
(C9), 136.7 (C16), 136.5 (C5), 133.7 (C4), 127.6e129.4 (C1 to C3), 123.1
(C10), 118.3 (C11), 114.6 (C6), 61.2 (C14), 25.7 (C15), 20.7 (C8), (19.3)
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.08.036.
(C16). MS m/z: 578 (56.8%) [Mþ1]þ. LM 10ꢀ3
(
U
ꢀ1 cm2 molꢀ1) ¼ 16.1.
References
lmax (cmꢀ1) in DMF, 41,550, 32,659. meff (BM): diamagnetic.
[ZnL4(CBDCA)] (8) Yield: (1.9 g, 58%) m.pt: 191e197 ꢂC. Anal.
Calc. for C32H32N2O6Zn: Zn, 10.7; C, 63.4; H, 5.3; N, 4.6; Found: Zn,
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