Synthesis, Characterization, and Biological Activities of Pendant Arm-Pyridyltetrazole Copper(II) Complexes: DNA Binding/Cleavage Activity and Cytotoxic Studies

Article abstract of DOI:10.1002/cbdv.201400369

2-(1H-Tetrazol-5-yl)pyridine (L) has been reacted separately with Me₂NCH₂CH₂Cl·HCl and ClCH₂CH₂OH to yield two regioisomers in each case, N,N-dimethyl-2-[5-(pyridin-2-yl)-1H-tetrazol-1-yl]ethanamine (L1)/N,N-dimethyl-2-[5-(pyridin-2-yl)-2H-tetrazol-2-yl]ethanamine (L2) and 2-[5-(pyridin-2-yl)-1H-tetrazol-1-yl]ethanol (L3)/2-[5-(pyridin-2-yl)-2H-tetrazol-2-yl]ethanol (L4), respectively. These ligands, L1-L4, have been coordinated with CuCl₂·H₂O in 1:1 composition to furnish the corresponding complexes 1-4. EPR Spectra of Cu complexes 1 and 3 were characteristic of square planar geometry, with nuclear hyperfine spin 3/2. Single X-ray crystallographic studies of 3 revealed that the Cu center has a square planar structure. DNA binding studies were carried out by UV/VIS absorption; viscosity and thermal denaturation studies revealed that each of these complexes are avid binders of calf thymus DNA. Investigation of nucleolytic cleavage activities of the complexes was carried out on double-stranded pBR322 circular plasmid DNA by using a gel electrophoresis experiment under various conditions, where cleavage of DNA takes place by oxidative free-radical mechanism (OH^·). In vitro anticancer activities of the complexes against MCF-7 (human breast adenocarcinoma) cells revealed that the complexes inhibit the growth of cancer cells. The IC₅₀ values of the complexes showed that Cu complexes exhibit comparable cytotoxic activities compared to the standard drug cisplatin.

Full text of DOI:10.1002/cbdv.201400369

1516
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Synthesis, Characterization, and Biological Activities of Pendant Arm-
Pyridyltetrazole Copper(II) Complexes: DNA Binding/Cleavage Activity and
Cytotoxic Studies
by Shaik Mustafaa), Bommuluri Umamaheswara Raob), Manubolu Surya Surendrababu*a),
Kalidindi Krishnam Rajuc), and Gollapalli Nageswara Raob)
a) Department of Chemistry, GITAM University Hyderabad campus, Hyderabad 502329, India
(e-mail: manabolu@gmail.com)
b) Department of Inorganic and Analytical Chemistry, Andhra University, Visakhapatnam, India
c) Department of Biotechnology, Gokaraju Rangaraju Institute of Technology, Hyderabad, India
2-(1H-Tetrazol-5-yl)pyridine (L) has been reacted separately with Me2NCH2CH2Cl·HCl and
ClCH2CH2OH to yield two regioisomers in each case, N,N-dimethyl-2-[5-(pyridin-2-yl)-1H-tetrazol-1-
yl]ethanamine (L1)/N,N-dimethyl-2-[5-(pyridin-2-yl)-2H-tetrazol-2-yl]ethanamine (L2) and 2-[5-(pyr-
idin-2-yl)-1H-tetrazol-1-yl]ethanol (L3)/2-[5-(pyridin-2-yl)-2H-tetrazol-2-yl]ethanol (L4), respectively.
These ligands, L1–L4, have been coordinated with CuCl2 ·H2O in 1:1 composition to furnish the
corresponding complexes 1–4. EPR Spectra of Cu complexes 1 and 3 were characteristic of square planar
geometry, with nuclear hyperfine spin 3/2. Single X-ray crystallographic studies of 3 revealed that the Cu
center has a square planar structure. DNA binding studies were carried out by UV/VIS absorption;
viscosity and thermal denaturation studies revealed that each of these complexes are avid binders of calf
thymus DNA. Investigation of nucleolytic cleavage activities of the complexes was carried out on double-
stranded pBR322 circular plasmid DNA by using a gel electrophoresis experiment under various
conditions, where cleavage of DNA takes place by oxidative free-radical mechanism (OH·). In vitro
anticancer activities of the complexes against MCF-7 (human breast adenocarcinoma) cells revealed that
the complexes inhibit the growth of cancer cells. The IC50 values of the complexes showed that Cu
complexes exhibit comparable cytotoxic activities compared to the standard drug cisplatin.
Introduction. – Polyazole rings are versatile ligands [1–3] for coordinating
transition metals, therefore, synthesis of transition metal complexes containing
polyazole rings, particularly tetrazoles and their derivatives, has been ascribed
enormous significance due to their practical applications [4–7]. There is an increasing
interest in tetrazole derivatives for the development of ꢀclickꢁ chemistry which was
reported by Sharpless and co-workers [8][9]. Conversely, tetrazole-based compounds
have interesting structures with special functionalities [10][11]. Tetrazole derivatives
have found applications in therapeutics as antihypertensive agents [12], antibiotics
[13][14], and drugs for AIDS treatment [15][16].
Even though many tetrazole-containing derivatives are reported in the literature,
there is always an increasing demand for the development of novel and effective
tetrazole-containing therapeutic agents.
In continuation of our ongoing research on DNA binding and cleavage activities of
transition metal complexes [17][18], we herein report the synthesis, crystal structures,
DNA binding, DNA cleavage, and cytotoxic activities of CuII complexes which are
ꢂ 2015 Verlag Helvetica Chimica Acta AG, Zürich

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1517
obtained by the reaction of pyridyltetrazole derivatives containing pendant arms like
HOCH2CH2 or Me2NCH2CH2 groups.
Results and Discussion. – Synthesis of 2-(1H-tetrazol-5-yl)pyridine (L) and its
derivatives L1–L4 were carried out with the procedure described in the literature
[19][20] with a small modification, as shown in Scheme 1.
Scheme 1. Synthetic Route for Ligands L1–L4
a) NaN3, LiCl, NH4Cl, DMF, reflux, 10 h. b) K2CO3, MeCN, reflux, 24 h.
A signal at d(C) 154.9 in the 13C-NMR spectrum of L indicated the formation of a
1,5-disubstituted tetrazole and the presence of a signal at 2220 cmÀ1 in the IR spectrum
1
indicated an azide bond (NÀN or N¼N band) [21][22]. The H-NMR spectrum of L
showed the expected signals for the pyridine ring, while the NH H-atom of the tetrazole
ring appeared as broad signal at d(H) 7.1 (Figs. S1 and S2 in the Supporting
Information1)). Treatment of ligand L with Me2NCH2CH2Cl·HCl and ClCH2CH2OH
in basic medium afforded regioiosmers L1–L4 by alkylation at either the N1 or N2
positions (see Scheme 1). The structures of the isomers were readily assigned by their
1H- and 13C-NMR spectra. The signals of the Cq-atom of the tetrazole ring appeared in
the range of d(C) ca. 150.1–154.7 in the N1 and N2 isomers, respectively (Fig. S3). The
N1 isomer gave the signal at d(C) 150.45 for L1 and 152.15 for L3, respectively, while
the N2 isomer gave the signal at 151.35 for L2 and 154.77 for L4, respectively. The
1H-NMR spectra of L1–L4 showed four separate signals corresponding to the pyridyl
H-atoms and two triplets for the CH2 groups. A singlet was observed in the spectra of L1
and L2 at d(H) 2.29 and 2.27, respectively, which indicated the presence of six H-atoms
1
)
Supporting material is available upon request from the authors.

1518
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
of the Me2N group. The CH2 groups of L1–L4 linked to the tetrazole ring appeared at
d(H) 5.12, 5.06, 5.08, and 4.87, respectively. The presence of a OH group in L3 and L4
was confirmed by a broad peak at 3350–3200 cmÀ1 in the IR spectra.
Ligands L1–L4 were treated with CuCl2 ·2 H2O salt in MeOH under reflux under
N2 atmosphere for 2–3 h. All reactions were carried out using a 1:1 metal/ligand
stoichiometry ratio to give corresponding complexes 1–4 (see Scheme 2). Physical
properties of the Cu complexes are shown in Table 1. Elemental analyses of the
obtained complexes showed that they have 1:1 composition. All Cu complexes showed
magnetic moments in the range of 1.82–1.96 BM, slightly higher than the spin only
values (meff 1.73) expected for a d9 system with one unpaired electron [23] in CuII
complexes.
ESI-MS of Cu complexes were used to compare the stoichiometric composition.
The molecular-ion peaks (Mþ ) were observed at m/z 324.5 and 325.5 for the Cu
complexes 1 and 3, respectively (Fig. S4), suggesting a 1:1 stoichiometry of Cu/ligand
(CuLCl2). Elemental analysis values were in close agreement with the values calculated
from the molecular formula assigned to these complexes, which was further supported
by ESI-MS studies of representative complexes.
Scheme 2. Synthesis of N1- and N2-Substituted Cu Tetrazole Complexes 1–4
Table 1. Physical Properties of Pyridyltetrazole Cu Complexes 1–4
No.
Complex
Colour
M.p. [8]
meff (BM)
1
2
3
4
Cu(L1)Cl2
Cu(L2)Cl2
Cu(L3)Cl2
Cu(L4)Cl2
Green
Light green
Green
180–182
170–174
186–188
174–175
1.82
1.88
1.94
1.96
Light green

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1519
The electronic absorption spectral data with the molar extinction coefficient emax of
the Cu complexes recorded in DMF are shown in Table 2. Electronic spectra of Cu
complexes 1–4 showed bands in the region 405–418 nm with high emax values in the
range of 8500–7500 mÀ1 cmÀ1 which are assigned to metal-to-ligand charge transfer
(MLCT; n!p*), while a single broad band observed in the region 654–648 nm is
assigned to d!d transition. Weak d-d bands in low intensity with emax values in the
2
2
range of 150–70 mÀ1 cmÀ1 are assigned to the Eg ! T2g electronic transition, these
assignments suggest a square planar geometry for the CuII complexes [24–26].
Table 2. Electronic Spectral Data lmax [nm] (emax [mÀ1 cmÀ1]) of the CuII Complexes
Complex
MLCT
d!d [cmÀ1
]
1
2
3
4
418 (8450)
415 (7100)
405 (8350)
415 (7500)
654 (110)
652 (70)
648 (140)
650 (90)
IR Data of the metal complexes are given in Table 3. IR Spectra of ligands L3 and
L4 showed a broad band in the range of 3350–3100 cmÀ1 corresponding to OH, and
bands at 1650–1500 cmÀ1, as well as two similar bands at ca. 1150–900 cmÀ1
,
correspond to the tetrazole group [27]. The formation of coordination bonds between
the tetrazole ring and CuII was confirmed by observing the IR frequencies of the
tetrazole ring. Ligands L1–L4 showed characteristic absorption bands at 1633–
1606 cmÀ1 which were shifted to lower frequencies in all Cu complexes. Additional
bands at ca. 1340–1200 and 800–600 cmÀ1 appeared due to the coordination of the
pyridine ring with the CuII-atom.
Table 3. Selective IR Bands [cmÀ1] of CuII Complexes with Tentative Assignments
Vibration
L1
1
L2
2
L3
3
L4
4
ÀOH
–
–
–
–
3355
1633
1523
1454
3134
1595
1400
1334
3325
1626
1508
1443
3126
1592
1398
1324
C¼N (tetrazole)
C¼C (tetrazole)
Py/MÀPy
1614
1498
1433
1575
1416
1298
1606
1486
1434
1568
1408
1294
Solid-state electroparamagnetic resonance spectra of CuII complexes 1 and 3 were
recorded in the X-band region at room temperature (258), and the data are
summarized in Table 4. Complexes 1 and 3 exhibit gk values of 2.18 and 2.16 and
g? values of 2.07 and 2.05, respectively. From the observed values, it is clear that
Table 4. EPR Spectral Assignments for Complexes 1 and 3 at Room Temperature
Complex
gk
g?
gav
G
1
3
2.18
2.16
2.07
2.05
2.10
2.08
2.62
3.30

1520
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
gk >g? >2.00, which suggests that the unpaired electron lies predominantly in the
dx2Ày2 orbital, which is characteristic of a square planar geometry in CuII complexes.
The geometric parameter G, which is a measure of the exchange interaction
between the Cu centers in the polycrystalline compound, is calculated using the
equation:
G¼(gk À2.0023)/(g? À2.0023)
According to Hathaway and Tomlinson [28], if G>4.0, considerable exchange
interaction is negligible because the local tetragonal axes are aligned parallel or are
slightly misaligned. If G <4.0, exchange is considerable, and the local tetragonal axes
are misaligned. The observed G values of complexes 1 and 3 (2.62 and 3.30, resp.)
suggest that there is exchange interaction in the CuII complexes.
Crystal Structure of Complex 32). Dark green crystals of complex 3 suitable for X-
ray diffraction were obtained from MeOH solution. Crystal data and structure
refinements are shown in Table 5. The complex crystallizes in a monoclinic system, the
space group is P21/c. The coordination sphere of the CuII-atom comprises the pyridine
N-atom, one tetrazole N-atom, and two Cl-atoms (Fig. 1). Selected bond distances and
bond angles are listed in Table 6. Ligand L3 binds to the CuII-atom through the
Fig. 1. ORTEP Diagram of complex 3
2
)
CCDC-1014885 contains the crystallographic data for complex 3 reported herein. These data can be
obtained free of charge via the Cambridge Crystallographic Data Centre, 12, Union road,
Cambridge CB2IEZ, UK or via www.ccdc.cam.ac.uk.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1521
Table 5. Crystal Data and Structure Refinement for Complex 3
Empirical formula
Mr
C8H9Cl2CuN5O
325.64
Crystal size [mm]
Temperature [K]
Wavelength [Š]
Crystal system
Space group
Z
0.16Â0.14Â0.12
293(2)
1.54184
Monoclinic
P21/c
8
Unit cell parameters:
a [Š]
b [Š]
c [Š]
a [8]
b [8]
g [8]
16.2372(6)
12.0116(3)
12.1616(2)
90
95.375(2)
90
V [Š3]
2361.51(11)
1.832
6.723
Dx (calc.) [gcmÀ3
]
Absorption coefficient [mmÀ1
F(000)
]
1304
q Range for data collection
Limiting indices
Reflections collected
Independent reflections
4.586–70.7998
À17ꢀhꢀ19, À10ꢀkꢀ14, À14ꢀlꢀ14
9237
4422 [Rint ¼0.0294]
Completeness to q¼67.6848 [%]
99.3
Absorption correction
Multi-scan
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I>2s(I)]
R Indices (all data)
1.00000; 0.71939
Full-matrix least-squares on F2
4422/2/311
1.140
R1 ¼0.0400, wR2 ¼0.0985
R1 ¼0.0515, wR2 ¼0.1047
Table 6. Bond Lengths [Š] and Angles [8] for Complex 3
Cu(1)ÀN(2)
2.017(3)
2.061(3)
2.438(3)
2.438(3)
0.844(10)
1.416(5)
95.63(9)
170.21(9)
83.83(10)
97.22(7)
93.42(5)
N(2)ÀCu(1)ÀN(1)
Cu(1)ÀCl(1)
79.06(12)
2.2363(11)
2.2261(11)
1.388(6)
0.9300
1.371(6)
Cu(1)ÀN(1)
Cu(1)ÀO(1)a)
Cu(1)ÀCl(2)
O(1)ÀCu(1)b)
C(1)ÀC(2)
O(1)ÀH(7)
C(1)ÀH(27)
O(1)ÀC(8)
C(2)ÀC(3)
N(1)ÀCu(1)ÀCl(2)
N(1)ÀCu(1)ÀCl(1)
N(1)ÀCu(1)ÀO(1)a)
Cl(1)ÀCu(1)ÀO(1)a)
Cl(2)ÀCu(1)ÀCl(1)
N(2)ÀCu(1)ÀCl(1)
N(2)ÀCu(1)ÀCl(2)
N(2)ÀCu(1)ÀO(1)a)
Cl(2)ÀCu(1)ÀO(1)a)
C(1)ÀN(1)ÀCu(1)
91.51(9)
171.42(10)
80.01(12)
106.29(9)
126.2(3)
Symmetry transformations used to generate equivalent atoms: a) x, Àyþ3/2, zÀ1/2. b) x, Àyþ3/2,
zþ1/2.

1522
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Fig. 2. a) Close packing diagram of complex 3 showing H-bonding between pendant-OH and Cu-atom of
other complex. b) Close packing diagram of complex 3 showing H-bonding between pendant-OH and
Cl-atom of other complex.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1523
tetrazole N-atom at the N1 position of the tetrazole ring and through the pyridyl N-
atom to generate a five-membered chelating ring. The tetrazole ring is coplanar with
the pyridyl ring. This coplanarity is also observed in other metal complexes containing
pyridyltetrazole ligands [29–31]. Two Cl-atoms are coordinating the CuII-atom, they
are located in plane with the pyridyltetrazole rings. The bond distances are 2.2363(11)
for Cu(1)ÀCl(1) and 2.2261(11) for Cu(1)ÀCl(2).
There are two crystallographic independent centrosymmetric Cu centres, each of
which forms a centrosymmetric dimer held together by H·Cl H-bonding and other
alcoholic O-atoms (present on the pendant arm) act as weak donor to Cu of another
dimer. The bond distance of CÀO is 1.416 Š and O· · ·Cu is 2.438 Š (Fig. 2), suggesting
that the pendant arm OH group shows weak interaction with the Cu atom of the other
dimer, resulting in a chain linkage arrangement structure [32]. These interactions are
further established by Hirshfeld interactions studies.
Hirshfeld surface and 2D fingerprint plots generated from the crystal data of
complex 3 using CrystalExplorer 3.1 are shown in Fig. 3. The color mapping and the
shape of the 3D Hirshfeld surfaces of 3 show the different intermolecular interactions
Fig. 3. Hirshfeld surface and 2D fingerprint plot associated with a) close contact interaction with all atoms
and b) close contact interactions of O· · ·Cu only in complex 3

1524
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
present in the crystal packing. The red spots on the surfaces indicate the close-contact
interactions, which are mainly responsible for the molecular packing in the crystals.
Apart from H-bonding, the crystal packing of 3 is controlled by dominant interactions
involving O· · ·H, H· · ·Cl, N· · ·C, and Cl· · ·Cl contacts, which are observed as red
spots on the Hirshfeld surfaces shown in Fig. 3 [33–35]. The close contact data with O
vs. other atoms is given in Table 7. From the data it is clear that the pendant arm OH
group is associated with O· · ·Cu week interaction (Fig. 2,a and b).
Table 7. Percentage Interactions of O-Atom vs. Other Atoms in Complex 3
S. No.
Interaction
%
1
2
3
4
5
OÀCu
OÀN
OÀC
OÀH
OÀO
3.3
0.8
0.5
0.0
0.0
DNA Binding Studies. The binding interactions of complexes 1–4 with calf thymus
DNA (CT-DNA) were monitored by UV/VIS spectroscopy. The absorption spectra of
the Cu complexes are compared with and without CT-DNA at 400–450 nm. Electronic
absorption data upon addition of CT-DNA and binding constants of these complexes
are given in Table 8. The typical absorption spectrum of complex 3 is shown in Fig. 4. In
the presence of increasing amounts of CT-DNA, the UV/VIS absorption spectra of
complexes 1–4 showed increase in absorbance exhibiting bathochromic shift (1.9–
3.4 nm) with hyperchromism (10.6–12.5%) with respect to control (0 ml DNA). The
change in absorbance values with increasing amount of CT-DNA was used to evaluate
the intrinsic binding constant Kb (Table 8).
It is evident from Table 8 that all the complexes bind to DNA with high affinities,
and that the estimated binding constants are in the range of 4.2–6.8·105 mÀ1. The
binding order of the complexes is 1>2>3>4. The Kb values of complexes 1–4 are
comparable with the reported values for redox-active CuII pyridine-based tetrazolo[1,5-
a]pyrimidine complexes [36]. The Kb value for cisplatin, which shows a hyperchromic
shift, after subsequent addition of CT-DNA, is reported as 3.20·104 mÀ1 [37].
The binding nature of the synthesized complexes is significant due to p-stacking
interaction of planar pyridyltetrazole rings. Binding of complexes to CT-DNA by exter-
Table 8. Effect of CT-DNA on the Absorption and Binding Constant of Cu Complexes
Complex
lmax [nm]
Dl [nm]a)
H [%]b)
Kb [mÀ1
]
Free
Bound
1
2
3
4
438.2
438.0
438.8
438.3
440.9
439.9
442.2
440.3
2.7
1.9
3.4
2.0
12.4
11.3
12.5
10.6
6.8·10À5
6.3·10À5
5.6·10À5
4.2·10À5
a) Dl, Bathochromic shift. b) H, Hyperchromism.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1525
Fig. 4. Absorption spectra of complex 3 in the absence and in the presence of increasing concentration of
CT-DNA; bottom spectrum is recorded in the absence of DNA and above spectra after addition of 10 ml
DNA each time
nal contact (electrostatic or groove binding) brings about bathochromic shift with hy-
perchromism absorption intensity [38]. A strong binding constant for 1 may be attrib-
uted to greater symmetric nature and electrostatical interaction with calf thymus [39].
Thermal Denaturation Studies. The transition temperature from helix to coil can be
determined by monitoring the absorbance of the DNA bases at 260 nm. The thermal
melting studies were performed at [DNA]/[complex]¼25. The Tm values were
determined by monitoring the absorbance of DNA at 260 nm as a function of
temperature. The melting point of free CT-DNA was 60.18 under the employed
experimental conditions. Under the same conditions, addition of CuII complexes 1–4
increased the Tm values (Æ0.5–1.08) by 4, 3, 5, and 38, respectively. This increase of the
Tm values of CT-DNA in the presence of the complexes indicated that pyridyltetrazole
CuII complexes stabilize the double helix of DNA [40][41].

1526
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Viscosity Measurement. Binding interactions of CuII complexes with CT-DNA were
also investigated by viscosity measurements [42]. A classical intercalation model
usually resulted in lengthening the DNA helix, as base pairs were separated to
accommodate the binding ligand leading to the increase of DNA viscosity. As seen in
Fig. 5, the viscosity of DNA increased with increase in the ratio of complexes to DNA.
This result further suggested an intercalative binding mode of the complex with DNA
and is in agreement with spectroscopic results, such as hyperchromism and bath-
ochromism, of complexes in the presence of DNA.
DNA Cleavage Activity. The nuclease activities of complexes 1–4 were investigated
on pBR322 super coiled plasmid DNA by agarose gel electrophoresis in the absence
and presence of oxidant (H2O2) at 120 min incubation period. The gel electrophoresis
diagrams are shown in Fig. 6; all even lanes were run in the presence and all odd lanes
were run in the absence of oxidant (H2O2), the data are shown in Table 9. The gel
mainly contained 1 in lanes 3 and 4, 2 in lanes 5 and 6, 3 in lanes 7 and 8, and complex 4
in lanes 9 and 10, respectively. All complexes in micromolar concentration with 120 min
incubation period, in the absence and presence of oxidant (H2O2), showed significant
cleavage activities at physiological temperature and pH (7.2).
From Fig. 6 it is clear that all complexes showed higher cleavage activities in the
presence of oxidant (H2O2; even lanes) converting form I to forms II and III. This may
be attributed to the formation of free OH· radicals. The production of OH· due to the
reaction between metal complex and oxidant may be explained as shown below [43].
.
(L)Cu2þ þH2O2 !(L)Cu3þ þOHÀ þOH
Fig. 5. Effect of CuII complexes on the viscosity of DNA; relative specific viscosity (R) vs. [complex]/
[DNA]

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1527
Fig. 6. Agarose gel (1%) showing the results of electrophoresis of 1 ml of pBR322 plasmid DNA
(0.10 mgmlÀ1) and 2 ml of 0.1m Tris ·HCl buffer (pH 8.0): 70.5 mm complex in DMF; 10 mm H2O2 were
added, respectively; incubation at 378 (120 min). Lane 1, DNA (control); lane 2, DNA þH2O2 (control);
lane 3, DNA þ1; lane 4, DNA þ1þH2O2; lane 5, DNA þ2; lane 6, DNA þ2þH2O2; lane 7, DNA þ3;
lane 8, DNA þ3þH2O2; lane 9, DNA þ4; lane 10, DNAþ4þH2O2.
Table 9. Selected SC pBR322 DNA Cleavage Data of Cu Complexes (c¼70.5 mm)
Lane no.
Reagents
Percentage ofa)
Form I
Form II
Form III
1
2
3
4
5
6
7
8
9
DNA
DNAþH2O2
96.3
93.4
88.6
80.3
8.2
4.2
12.5
–
3.7
6.6
ND
ND
ND
ND
10.2
15.6
ND
–
DNAþComplex 1
DNAþComplex 1þH2O2
DNAþComplex 2
DNAþComplex 2þH2O2
DNAþComplex 3
DNAþComplex 3þH2O2
DNAþComplex 4
DNAþComplex 4þH2O2
11.4
19.7
81.6
80.2
88.5
100
76.3
ND
23.7
57.3
ND
42.7
10
a) Ca. Æ7–10% due to neglecting of discernible cleavage of pBR322. ND, Not determined.
Cleavage Mechanism Studies. The nuclease cleavage mechanism of complex 3 was
investigated in the presence of EDTA (chelating agent; 1·10À6 m), DTT (reducing
.
agent; 1·10À6 m), DMSO (OH radical scavenger), and NaN3 (singlet oxygen
quencher; 1·10À6 m); the data are shown in Table 10. In Fig. 7, lanes 1 and 2 are
control and lanes 3 and 4 are runs with complex 3 in absence and presence of H2O2,
respectively. Complex 3 cleaves DNA more efficiently in the presence of H2O2, which
may be due to reaction of OH· with DNA [44]. In lane 5, the presence of the chelating
agent EDTA diminishes the nuclease activity of Cu complex 3, whereas in the presence
of reducing agent (DTT) in lane 6 the cleavage activity of 3 is enhanced. This may be
due to the formation of CuI complex by catalytic reduction. In the presence of EDTA
the cleavage activities of the complexes were considerably reduced. Lane 7 in the
presence of DMSO and lane 8 in the presence of NaN3 showed not much effect in
cleavage activities confirming the absence of singlet oxygen involvement.

1528
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Table 10. Selected SC pBR322 DNA Cleavage Data of Cu Complex 3 (c¼70.5 mm)
Lane no.
Reagents
Percentage of
Form I
Form II
Form III
1
2
3
4
5
6
7
8
DNA
96.3
93.4
34.6
10.2
79.6
6.5
3.7
6.6
ND
ND
ND
ND
ND
ND
ND
ND
DNAþH2O2 (10 mm)
DNAþComplex 3
65.4
89.7
20.4
93.5
63.5
61.8
DNAþComplex 3þH2O2 (10 mm)
DNAþComplex 3þEDTA (10 mm)
DNAþComplex 3þDTT (10 mm)
DNAþComplex 3þDMSO (0.5 mm)
DNAþComplex 3þNaN3 (10 mm)
36.5
38.2
Fig. 7. Agarose gel (1%) electrophoresis of 1 ml of pBR322 plasmid DNA (0.10 mg mlÀ1) and 2 ml of 0.1m
Tris·HCl buffer (pH 80). Lane 1, DNA (control); lane 2, DNA þH2O2 (250 mm); lane 3, DNA þ3; lane 4,
DNAþ3þH2O2; lane 5, DNAþ3þEDTA; lane 6, DNA þ3þDTT; lane 7, DNA þ3þDMSO; lane 8,
DNAþ3þNaN3.
Cytotoxicity Studies. The results of the investigation of the anticancer activities of
the Cu complexes on MCF-7 cell lines are shown in Fig. 8. The cell proliferation was
measured by using the standard 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-
lium-5-carboxanilide (XTT) assay [45][46] with four different concentrations of the
compounds (5, 10, 50, and 100 mm) and cisplatin (as positive control) were tested in
triplicate for 48 h. The results obtained by the XTT assay are expressed as the average-
standard deviation of two experiments. The IC50 values of the complexes exhibited
differential and dose-dependent inhibitory activities on the growth of MCF-7 cancer
cells. All Cu complexes effectively inhibited MCF-7 cell growth, but the ligands showed
no activity. The complexes showed excellent activities with IC50 values of 32 (Æ0.8), 36
(Æ0.6), 28 (Æ1.4), 30 (Æ0.8), and 38 (Æ0.8) for complexes 1–4 and cisplatin,
respectively. When comparing the inhibitory effects with respect to the IC50 values,
complex 3 inhibited cell growth to 28Æ1.4 mgmlÀ1 at a lower concentration range after
72 h of treatment of MCF-7 cells (Fig. 8). It is clear from the IC50 values that other
complexes show less inhibition on the growth of MCF-7 cells; the inhibition order
is 3>4>1>2.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1529
Fig. 8. Cytotoxic activities of a)–c) cisplatin (control), d)–f) complex 3, and g)–i) complex 4 after 12, 24,
and 48 h, resp.
Inhibitory Effects of Complexes on Survival of MCF-7 at Different Concentrations.
Survival studies of MCF-7 at different concentrations were carried out by constantly
incubating cells with Cu complexes 1–4 and then, the resulting mixture was washed to
remove the metal complex. Cu complexes decreased MCF-7 cell proliferation
significantly in a concentration-dependent manner. Cell survival was determined at
complex concentrations of 5, 10, 50, and 100 mm. At these concentrations, complex 3
was able to kill 55, 65, 75, and 79% of the cells, respectively (Fig. 9). All Cu complexes
showed better toxicities at low concentration (5 mgmlÀ1) with 35, 32, 55, and 45% for
complexes 1–4, respectively.
Conclusions. – Mononuclear CuII complexes of novel bidentate ligands (pyridylte-
trazole pendant arm containing OH and Me2N groups) were synthesized and

1530
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
Fig. 9. Cytotoxic activities of complexes 1–4 at different concentrations after 24 h on MCF-7 cells
characterized. Physicochemical and spectral studies revealed that the mononuclear
complexes have distorted square planar geometry. Single crystal X-ray diffraction
studies of 3 confirmed the suggested structure. All Cu complexes are avid binders of
CT-DNA. Complexes 1 and 3 showed higher Kb values which may be due to the more
symmetric nature. In the presence of H2O2, Cu complexes cleaved DNA more
effectively, which may be due to the reaction of OH· with DNA. All complexes cleaved
DNA via an oxidative path. All Cu complexes were tested for anticancer studies on
MCF-7 human cell line. The highlights of the present investigations are: i) mononuclear
complexes were synthesized with pyridyltetrazole pendant arm ligands, ii) different
regioisomeric and symmetric nature of synthesized copper complexes are attractive
features to develop chemotherapeutic drugs, iii) DNA binding and cleavage activities
of the complexes were investigated, iv) anticancer activities of the synthesized
complexes are good and encourage to design better and efficient metal-based drugs.
M. S. S. B. and S. M. thank UGC, New Delhi, for sanctioning the project F.No.42-306/2013(SR).
Experimental Part
General. Chemicals were purchased from SigmaÀAldrich, and metals used in the preparation of the
complexes were of reagent grade. Solvents used in the synthesis of the ligands and metal complexes were
distilled before use. All other chemicals were of AR grade and were used without further purification.
Agarose, used in gel electrophoresis, was purchased from SigmaÀAldrich, CT-DNA and plasmid pBR322
were purchased from Genie Biolabs, Bangalore, India. M.p.: Sisco melting apparatus Model 935;
uncorrected. Column chromatography (CC): silica gel (SiO2, 60–120 mesh). FT-IR Spectra: Bruker IFS
1
66V; in KBr and PE medium; n˜ in cmÀ1. H- and 13C-NMR spectra: Bruker 300 MHz; d in ppm rel. to
Me4Si as internal standard, J in Hz. ESI-MS: Pexciex API 2000, Q1MSQ1; in m/z. Elemental analyses:

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1531
PerkinElmer 2400 CHNS elemental analyzer; in %. Magnetic moments were determined in the
polycrystalline state on a PAR model-155 vibrating sample magnetometer operating at field strength of
2–8 kG. High purity Ni metal (saturation moment 55 emugÀ1) was used as standard. Electro-
paramagnetic resonance spectra (EPR): Shimadzu UV-1800 spectrophotometer; in DMF.
Preparation of Compounds. The preparation of L was carried out as described in the literature
[19][20]. M.p. 221–2238. 1H-NMR ((D6)DMSO): 8.56 (d, J¼7.9, 1 H of Py); 8.00 (d, J¼7.8, 1 H of Py);
7.79 (t, J¼7.8, 1 H of Py); 7.26 (t, J¼7.9, 1 H of Py); 7.10 (s, NH).
Synthesis of L1 and L2. To L (1.0 g, 6.8 mmol) dissolved in MeCN (30 ml) was added K2CO3 (4.6 g,
34 mmol). The resulting soln. was heated under reflux for 30 min, and to the hot soln. was added
Me2NCH2CH2Cl (3.1 g, 22 mmol). The mixture was then stirred under reflux for further 24 h. After
cooling, the solvent was removed under reduced pressure to afford a white precipitate, which was purified
by CC (SiO2; AcOEt/hexane 20 :80) to give isomers L1 and L2.
N,N-Dimethyl-2-[5-(pyridin-2-yl)-1H-tetrazol-1-yl]ethanamine (L1). Yield: 0.55 g (26%). White-
brown solid. M.p. 72–768. 1H-NMR: 8.72 (d, J¼4.5, 1 H); 8.47 (d, J¼8.1, 1 H); 7.91 (dt, J¼7.8, 1.8, 1 H);
7.45 (dd, J¼7.5, 5.1, 1 H); 5.12 (t, J¼6.9, 2 H); 2.87 (t, J¼6.9, 2 H); 2.29 (s, 6 H). 13C-NMR: 150.45
(C(16)); 148.53 (C(9)); 144.66 (C(34)); 137.16 (C(17)); 123.15 (C(19)); 122.24 (C(20)); 48.24 (C(4));
43.56 (C(6)); 35.34 (Me2N).
N,N-Dimethyl-2-[5-(pyridin-2-yl)-2H-tetrazol-2-yl]ethanamine (L2). Yield: 0.55 g (26%). White-
brown solid. M.p. 66–688. 1H-NMR: 8.64 (d, J¼4.5, 1 H); 8.36 (d, J¼8.1, 1 H); 7.88 (dt, J¼7.8, 1.8, 1 H);
7.35 (t, J¼7.5, 5.1, 1 H); 5.06 (t, J¼6.9, 2 H); 2.82 (t, J¼6.9; 2 H); 2.27 (s, 6 H). 13C-NMR: 151.35 (C(16));
149.44 (C(9)); 145.36 (C(34)); 138.08 (C(17)); 124.64 (C(19)); 122.12 (C(20)); 49.54 (C(4)); 44.34 (C(6));
35.68 (Me2N).
Synthesis of L3 and L4. The above mentioned preparation of L was followed by addition of
ClCH2CH2OH (2.0 g, 24 mmol) to give L3 and L4.
2-[5-(Pyridin-2-yl)-1H-tetrazol-1-yl]ethanol (L3). Yield: 0.35 g (17%). White solid. M.p. 56–588.
1H-NMR: 8.71 (d, J¼4.8, 1 H); 8.36 (d, J¼7.8, 1 H); 7.97 (dt, J¼7.8, 1.5, 1 H); 7.53 (dd, J¼6.9, 5.1, 1 H);
5.08 (m, 2 H); 4.20 (m, 3 H). 13C-NMR: 152.15 (C(16)); 149.73 (C(9)); 144.39 (C(34)); 138.14 (C(17));
125.79 (C(19)); 124.35 (C(20)); 59.42 (C(6)); 51.39 (C(4)).
2-[5-(Pyridin-2-yl)-2H-tetrazol-2-yl]ethanol (L4). Yield: 0.35 g (17%). White solid. M.p. 54–568.
1H-NMR: 8.77 (d, J¼4.2, 1 H); 8.24 (d, J¼7.8, 1 H); 7.86 (t, J¼6.6, 1 H); 7.42 (dd, J¼7.5, 4.8, 1 H); 4.87
(t, J¼4.8, 2 H); 4.26 (t, J¼5.1, 2 H). 13C-NMR : 154.77 (C(16)); 150.24 (C(9)); 145.72 (C(34)); 138.81
(C(17)); 124.81 (C(19)); 123.33 (C(20)); 60.52 (C(6)); 51.43 (C(4)).
General Procedure for the Synthesis of Cu Complexes. The appropriate ligand (L1–L4; 1.36 mmol)
was dissolved in MeOH (30 ml) and added to a CuCl2 ·H2O (1.36 mmol) soln. in MeOH (10 ml). The
resulting pale green- to green-colored solns. were then heated under reflux for 2 to 3 h, the soln. was left
over night at r.t. and filtered to collect the respective precipitate.
Dichloro{N,N-dimethyl-2-[5-(pyridin-2-yl-kN)-1H-tetrazol-1-yl-kN4]ethanamine}copper (1). Yield:
0.28 g (58%). Pale green precipitate. IR (KBr): 3245, 2945, 1648, 1594, 1575, 1496, 1416, 1298, 1196, 1147,
1023, 835, 785. Anal. calc. for C10H14Cl2CuN6 (352.71): C 34.05, H 4.00, N 23.83; found: C 33.84, H 3.92, N
23.62.
Dichloro{N,N-dimethyl-2-[5-(pyridin-2-yl-kN)-2H-tetrazol-2-yl-kN4]ethanamine}copper (2). Yield:
0.28 g (58%). Pale green precipitate. IR (KBr): 3254, 2938, 2852, 1638, 1580, 1568, 1482, 1408, 1294, 1226,
1167, 1139, 1019, 855, 735. Anal. calc. for C10H14Cl2CuN6 (352.71): C 34.05, H 4.00, N 23.83; found: C
33.90, H 3.91, N 23.73.
Dichloro{2-[5-(pyridin-2-yl-kN)-1H-tetrazol-1-yl-kN4]ethanol}copper (3). Yield: 0.24 g (51%).
Green precipitate. IR (KBr): 3134, 2923, 2856, 1610, 1595, 1483, 1400, 1334, 1165, 1019, 803, 721. Anal.
calc. for C8H9Cl2CuN5O (325.64): C 29.51, H 2.79, N 21.51; found: C 29.37, H 2.75, N 21.13.
Dichloro{2-[5-(pyridin-2-yl-kN)-2H-tetrazol-2-yl-kN4]ethanol}copper (4). Yield: 0.19 g (43%).
Green precipitate. IR (KBr): 3126, 2923, 2856, 1648, 1592, 1398, 1324, 1278, 1119, 824, 754. Anal. calc.
for C8H9Cl2CuN5O (325.64): C 29.51, H 2.79, N 21.51; found: C 29.42, H 2.35, N 21.26.
Single-Crystal X-Ray Structure Determination. Single crystals of 3 were obtained from MeOH soln.
at r.t. A crystal of suitable dimensions was mounted on a CryoLoop (Hampton Research Corp.) at IIT
Hyderabad with a layer of light mineral oil and placed in a N2 stream at 150(2) K. All measurements were

1532
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
made on an Oxford Supernova X-calibur Eos CCD detector with graphite-monochromated CuKa
radiation (l¼1.54184 Š), CrysAlisPro version 1.171.35.19 (Agilent Technologies). The structures were
solved by direct methods (SIR92) and refined on F2 by full-matrix least-squares using SHELXL-97
[47][48]. ORTEP-3 was used to draw the molecule.
Hirshfeld Surface Analysis. Hirshfeld surfaces and 2D fingerprint plots were generated using
CrystalExplorer 3.1 [49] based on results of single crystal X-ray diffraction studies. The function dnorm is a
ratio encompassing the distances of any surface point to the nearest interior (di) and exterior (de) atom
and the Van der Waals radii of the atoms [50–52]. The negative value of dnorm indicates the sum of di and
de shorter than the sum of the relevant Van der Waals radii, which was considered as closest contact and
visualized as red color in the Hirshfeld surfaces. The white color denotes an intermolecular distance close
to Van der Waals contacts with dnorm equals to zero whereas contacts longer than the sum of Van der Waals
radii with positive dnorm values are colored with blue. A plot of di vs. de is a 2D fingerprint plot which
recognizes the existence of different types of intermolecular interactions.
DNA Binding and Cleavage Experiments. All measurements with CT-DNA were performed in 5 mm
Tris·HCl buffer (pH 7.2)/50 mm NaCl. The UV absorbance ratio 260/280 was 1.8–1.9, indicating the
DNAwas sufficiently free of protein [53]. The concentration of CT-DNA per nucleotide was determined
from the absorption intensity at 260 nm with the known value of 6600 mÀ1 cmÀ1. The absorption titrations
were performed by adding increasing amounts of CT-DNA to a soln. of the complex at a fixed
concentration contained in a quartz cell and recording the UV/VIS spectrum after each addition. The
absorption of CT-DNA was subtracted by adding the same amount of DNA to a blank. The data were
then fitted to Eqn. 1 to obtain the intrinsic binding constant Kb [54].
[DNA]/(ea Àef)¼[DNA]/(eb Àef)þ1/Kb(eb Àef)
(1)
[DNA] is the molar concentration of CT-DNA, ea, eb, and ef are apparent, free, and bound metal
complex extinction coefficients, resp. Thus, Kb is the ratio of the slope to the y-intercept. DNA melting
experiments were performed using a spectrophotometer connected to a thermostat. The absorbance of
DNA (60 mm) at 25–808 in both the absence and presence of 10.5 mm of the complex was recorded at
260 nm. Tm was calculated by plotting the temp. vs. relative absorption intensity (A/A0). Viscosity
experiments were carried out on an Ostwald viscometer, immersed in a thermostated water-bath
maintained at a constant temp. at 30.0Æ0.18. CT-DNA samples of ca. 0.5 mm were prepared by sonicating
in order to minimize complexities arising from CT-DNA flexibility [55]. Flow time was measured with a
digital stopwatch three times for each sample and an average flow time was calculated. Data were
presented as (h/h0)1/3 vs. the concentration of the Cu complexes, where h is the viscosity of CT-DNA soln.
in the presence of the complex, and h0 is the viscosity of CT-DNA soln. in the absence of the complex.
Viscosity values were calculated after correcting the flow time of buffer alone (t0) [56]:
h¼(tÀt0)/t0
A DMF soln. containing the metal complexes (250 mm) in a clean Eppendorf tube was treated with
pBR322 plasmid DNA (3.3 ml of 150 mgmlÀ1) in Tris·HCl buffer (0.10m, pH 8.0) containing NaCl
(50 mm) in presence and absence of additives. The contents were incubated for 1 h at 378 and loaded onto
a 1% agarose gel after mixing with 5 ml of loading buffer (0.25% bromophenol blueþ25% xylene
cyanolþ30% glycerol, sterilized). The electrophoresis was performed at a constant voltage (80 V) until
bromophenol blue had travelled through 75% of the gel. Subsequently, the gel was stained for 10 min by
immersion in ethidium bromide soln. The gel was then distained for 10 min by keeping it in sterile dist.
H2O. The plasmid bands were visualized by viewing the gel under a transilluminator and photographed.
A control experiment was done in the presence of OH· radical scavenger DMSO, EDTA, DTT, and
singlet oxygen quencher azide ion (NaN3).
Cell Culture. Human cancer cell lines (MCF-7 cell line) used in this study were procured from the
National Centre for Cell Science, Pune. All cells were grown in minimal essential medium (MEM;
GIBCO) supplemented with 4.5 glÀ1 glucose, antibiotics (benzylpencillin (50 unitsmlÀ1), streptomycin
(50 mgmlÀ1), and amphotericin B (50 mgmlÀ1)), 2 mm l-glutamine, and 5% fetal bovine serum (growth
medium) at 378 in 5% CO2 incubator.

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
1533
XTT Assay. The biochemical procedure is based on the activity of mitochondrial enzymes which are
inactivated shortly after cell death. This method was found to be very efficient in assessing the viability of
cells. A colorimetric method based on the tetrazolium salt (XTT assay) was first described by Scudiero
[57]. In brief, the trypsinized cells from T-25 flask were seeded in each well of a 96-well flat-bottomed
tissue culture plate at a density of 5·103 cells/well in growth medium and cultured at 378 in 5% CO2 to
adhere. After 24 h incubation, the supernatant was discarded and the cells were pretreated with growth
medium and were subsequently mixed with different concentrations of test compounds (5, 10, 50, and
100 mgmlÀ1) in triplicates to achieve a final volume of 100 ml and then cultured for 72 h. The compound
was prepared as 1.0 mgmlÀ1 concentration stock solns. in DMSO. Culture medium and solvent were used
as controls. Each well then received 50 ml of fresh XTT (0.9 mgmlÀ1 in RPMI along with XTT activator
reagent) followed by incubation for 2 h at 378. At the end of the incubation, the 96-micro well plate was
shaken for 15 s. OD490 Values of the culture plate (OD630 values as ref.) were measured on an ELISA
reader, Anthos 2020 spectrophotometer.
% cell survival¼100À[(At ÀAb)/(Ac ÀAb)]·100
At, Absorbance of test; Ab, absorbance of blank; Ac, absorbance of control; % cell inhibition,
100À% cell survival.
REFERENCES
[1] P. Lin, W. Clegg, R. W. Harrington, R. A. Henderson, Dalton Trans. 2005, 2388.
[2] T. Hu, L. Liu, X. Lü, X. Chen, H. He, F. Dai, G. Zhang, D. Sun, Polyhedron 2010, 29, 296.
[3] Y. Qiu, B. Liu, G. Peng, J. Cai, H. Deng, M. Zeller, Inorg. Chem. Commun. 2010, 13, 749.
[4] R. N. Butler, in ꢀComprehensive Heterocyclic Chemistry IIꢁ, Eds. A. R. Katritzky, C. W. Rees,
E. F. V. Scriven, Pergamon, Oxford, 1996, Vol. 4, p. 621.
[5] R. J. Herr, Bioorg. Med. Chem. 2002, 10, 3379.
[6] J. McGinley, A. Fleming, J. Inclusion Phenom. Macrocyclic Chem. 2008, 61, 1.
[7] G. Aromí, L. A. Barrios, O. Roubeau, P. Gamez, Coord. Chem. Rev. 2011, 255, 485.
[8] Z. P. Demko, K. B. Sharpless, J. Org. Chem. 2001, 66, 7945.
[9] F. Himo, Z. P. Demko, L. Noodleman, K. B. Sharpless, J. Am. Chem. Soc. 2003, 125, 9983.
[10] Y. Tao, J.-R. Li, Z. Chang, X.-H. Bu, Cryst. Growth Des. 2010, 10, 564.
[11] M.-F. Wu, M.-S. Wang, S.-P. Guo, F.-K. Zheng, H.-F. Chen, X.-M. Jiang, G.-N. Liu, G.-C. Guo, J.-S.
Huang, Cryst. Growth Des. 2011, 11, 372.
[12] T. Mavromoustakos, A. Kolocouris, M. Zervou, P. Roumelioti, J. Matsoukas, R. Weisemann, J. Med.
Chem. 1999, 42, 1714.
[13] R. A. Powers, B. K. Shoichet, J. Med. Chem. 2002, 45, 3222.
[14] S. Y. Kang, S.-H. Lee, H. J. Seo, M. E. Jung, K. Ahn, J. Kim, J. Lee, Bioorg. Med. Chem. Lett. 2008,
18, 2385.
[15] G. C. G. Pais, X. Zhang, C. Marchand, N. Neamati, K. Cowansage, E. S. Svarovskaia, V. K. Pathak,
Y. Tang, M. Nicklaus, Y. Pommier, T. R. Burke Jr., J. Med. Chem. 2002, 45, 3184.
[16] B. C. H. May, A. D. Abell, J. Chem. Soc., Perkin Trans. 1 2002, 172.
[17] M. S. Surendra Babu, K. H. Reddy, J. Chin. Chem. Soc. 2012, 59, 843.
[18] M. S. Surendra Babu, T. B. Patrudu, K. Hussain Reddy, E-J. Chem. 2011, 8, S309.
[19] A. D. Bond, A. Fleming, J. Gaire, F. Kelleher, J. McGinley, V. McKee, U. Sheridan, Polyhedron
2012, 33, 289.
[20] A. Fleming, F. Kelleher, M. F. Mahon, J. McGinley, V. Prajapati, Tetrahedron 2005, 61, 7002.
[21] H. Gallardo, R. Magnago, A. J. Bortoluzzi, Liq. Cryst. 2001, 28, 1343.
[22] P. A. Bethel, M. S. Hill, M. F. Mahon, K. C. Molloy, J. Chem. Soc., Perkin Trans. 1 1999, 3507.
[23] A. D. Kulkarni, S. A. Patil, V. H. Naik, P. S. Badami, Med. Chem. Res. 2011, 20, 346.
[24] R. N. Butler, A. F. M. Fleming, J. Heterocycl. Chem. 1997, 34, 691.
[25] W. J. Geary, Coord. Chem. Rev. 1971, 7, 81.

1534
CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)
[26] B. J. Hathaway, in ꢀComprehensive Coordination Chemistryꢁ, Eds. G. Wilkinson, R. D. Gillard, J. A.
McCleverty, Pergamon, Oxford, 1987, Vol. 5, p. 583.
[27] P. N. Gaponik, S. V. Voitekhovich, A. S. Lyakhov, Chem. Heterocycl. Compd. (N.Y., NY, U.S.) 2000,
36, 326.
[28] B. J. Hathaway, A. A. G. Tomlinson, Coord. Chem. Rev. 1970, 5, 1.
[29] B. K. Tripuramallu, R. Kishore, S. K. Das, Inorg. Chim. Acta 2011, 368, 132.
[30] X. He, C.-Z. Lu, D.-Q. Yuan, Inorg. Chem. 2006, 45, 5760.
[31] H. Zhao, Z.-R. Qu, H.-Y. Ye, R.-G. Xiong, Chem. Soc. Rev. 2008, 37, 84.
[32] A. M. Thomas, A. Mukherjee, M. K. Saha, M. Nethaji, A. R. Chakravarthy, Indian J. Chem., Sect. A
2004, 43, 1626.
[33] R. Soman, S. Sujatha, C. Arunkumar, J. Fluorine Chem. 2014, 163, 16.
[34] F. Arjmand, M. Muddassir, J. Photochem. Photobiol., B 2010, 101, 37.
[35] J. Liu, H. Zhang, C. Chen, H. Deng, T. Lu, L. Ji, J. Chem. Soc., Dalton Trans. 2003, 114.
[36] A. Haleel, P. Arthi, N. D. Reddy, V. Veena, N. Sakthivel, Y. Arun, P. T. Perumal, A. K. Rahiman,
RSC Adv. 2014, 4, 60816.
[37] F. Arjmand, F. Sayeed, M. Muddassir, J. Photochem. Photobiol., B 2011, 103, 166.
[38] K. C. Skyrianou, F. Perdih, I. Turel, D. P. Kessissoglou, G. Psomas, J. Inorg. Biochem. 2010, 104, 161.
[39] L. A. Lipscomb, F. X. Zhou, S. R. Presnell, R. J. Woo, M. E. Peek, R. R. Plaskon, L. D. Williams,
Biochemistry 1996, 35, 2818.
[40] K. Jeyalakshmi, N. Selvakumaran, N. S. P. Bhuvanesh, A. Sreekanth, R. Karvembu, RSC Adv. 2014,
4, 17179.
[41] S. Satyanarayana, J. C. Dabrowiak, J. B. Chaires, Biochemistry 1993, 32, 2573.
[42] J. SantaLucia Jr., Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1460.
[43] K. Yamamoto, S. Kawanishi, J. Biol. Chem. 1989, 264, 15435.
[44] A. Hangan, A. Bodoki, L. Oprean, G. Alzuet, M. Liu-Gonzµlez, J. Borrµs, Polyhedron 2010, 29,
1305.
[45] W. N. Roehm, G. H. Rodgers, S. M. Hatfield, A. L. Glasebrook, J. Immunol. Methods 1991, 142, 257.
[46] H. H. Aung, S. R. Mehendale, C. Z. Wang, J.-T. Xie, E. McEntee, C.-S. Yuan, Cancer Chemother.
Pharmacol. 2007, 59, 369.
[47] G. M. Sheldrick, SHELXS-97, Program for Crystal Structure Solution, University of Gçttingen,
Gçttingen, Germany, 1997.
[48] G. M. Sheldrick, SHELXL-97, Program for Crystal Structure Refinement, University of Gçttingen,
Gçttingen, Germany, 1997.
[49] S. K. Wolff, D. J. Grimwood, J. J. McKinnon, M. J. Turner, D. Jayatilaka, M. A. Spackman,
CrystalExplorer 3.1, University of Western Australia, Crawley, Western Australia, 2013, ꢂ 2005–
2013, http://hirshfeldsurface.net/.
[50] F. L. Hirshfeld, Theor. Chim. Acta 1977, 44, 129.
[51] M. A. Spackman, D. Jayatilaka, CrystEngComm 2009, 11, 19.
[52] M. A. Spackman, J. J. McKinnon, CrystEngComm 2002, 4, 378.
[53] M. E. Reichmann, S. A. Rice, C. A. Thomas, P. Doty, J. Am. Chem. Soc. 1954, 76, 3047.
[54] A. Wolfe, G. H. Shimer Jr., T. Meehan, Biochemistry 1987, 26, 6392.
[55] F. Q. Liu, Q. X. Wang, K. Jiao, F. F. Jian, G. Y. Liu, R. X. Li, Inorg. Chim. Acta 2006, 359, 1524.
[56] J. B. Chaires, N. Dattagupta, D. M. Crothers, Biochemistry 1982, 21, 3933.
[57] D. A. Scudiero, R. H. Shoemaker, K. D. Paull, A. Monks, S. Tierney, T. H. Nofziger, M. J. Currens,
D. Seniff, M. R. Boyd, Cancer Res. 1988, 48, 4827.
Received October 6, 2014