Synthesis, characterization, DNA-binding, photocleavage, cytotoxicity and docking studies of Co(III) mixed ligand complexes

Article abstract of DOI:10.1007/s11243-013-9753-1

A new ligand, (2-ethoxy-6-(1H imadazo[4,5-f][1,10]phenanthroline-2-yl) phenol) (HEPIP) and its three Co(III) complexes [Co(phen)₂(HEPIP)] (ClO₄)₃ (1), [Co(bpy)₂(HEPIP)](ClO ₄)₃ (2) and [Co(dmb)₂(HEPIP)](ClO ₄)₃ (3) have been synthesized and characterized. All three Co(III) complexes exhibited antitumor activity against four human tumor cell lines. The interaction of these complexes with calf thymus DNA was studied by absorption and emission spectroscopy, viscosity measurements and DNA cleavage assays. The DNA-binding constants of complexes 1, 2 and 3 were determined as 6.13 × 10⁵, 4.46 × 10⁵ and 3.72 × 10⁵ M^-1, respectively. The complexes appear to interact with DNA through intercalation. Studies on the mechanism of photocleavage indicated that both superoxide anion radical and singlet oxygen may play an important role.

Full text of DOI:10.1007/s11243-013-9753-1

Transition Met Chem (2013) 38:811–819
DOI 10.1007/s11243-013-9753-1
Synthesis, characterization, DNA-binding, photocleavage,
cytotoxicity and docking studies of Co(III) mixed ligand complexes
•
Yata Praveen Kumar C. Shobha Devi
•
•
N. Deepika Nazar MD Gabra Nishant Jain
•
•
•
A. Srishailam Kotha Laxma Reddy S. Satyanarayana
•
Received: 28 May 2013 / Accepted: 1 August 2013 / Published online: 21 August 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract A new ligand, (2-ethoxy-6-(1H imadazo[4,5-
f][1,10]phenanthroline-2-yl)phenol) (HEPIP) and its three
Co(III) complexes [Co(phen)2(HEPIP)](ClO4)3 (1), [Co(bpy)2-
(HEPIP)](ClO4)3 (2) and [Co(dmb)2(HEPIP)](ClO4)3 (3)
have been synthesized and characterized. All three Co(III)
complexes exhibited antitumor activity against four human
tumor cell lines. The interaction of these complexes with
calf thymus DNA was studied by absorption and emission
spectroscopy, viscosity measurements and DNA cleavage
assays. The DNA-binding constants of complexes 1, 2 and
3 were determined as 6.13 9 105, 4.46 9 105 and
3.72 9 105 M-1, respectively. The complexes appear to
interact with DNA through intercalation. Studies on the
mechanism of photocleavage indicated that both superox-
ide anion radical and singlet oxygen may play an important
role.
diagnostic agents for medical applications, cleavage agents
for probing nucleic acid structure [1, 2] and identifiers of
transcription start sites [3]. Small molecules can bind to
DNA by different mechanisms, and binding studies are
important for the design of new and more efficient drugs
targeted to DNA [4]. Small molecules typically bind to
DNA by non-covalent interactions such as electrostatic
binding, groove binding and intercalative binding. Inter-
calating and groove binding molecules are important tools
in molecular biology, and many are clinically useful in the
treatment of cancer [5, 6]. Intercalation behavior is often
related to the antitumor activity of the compound [7, 8].
Cisplatin and its analogs are widely used as antitumor
drugs [9]. The search for new metallo-anticancer drugs,
which drives much current research [10], currently includes
a focus on ruthenium complexes. Octahedral complexes
with a dipyridophenazine ligand have been much studied,
because of the ‘‘Light switch effect’’ [11]. The crystal
structure of [Ru(phen)2DPPZ]2? (DPPZ = dipyrido[3,2-
a:20,30-c] phenazine) with an oligonucleotide was reported
by Niyazi et al. [12].
Introduction
Metal complexes that can bind to DNA are gaining con-
siderable attention owing to their diverse applications as
There have been intensive efforts to investigate factors
that determine affinity and selectivity in the binding of
small molecules to DNA [13], since information about
these factors would be valuable for the design of sequence-
specific DNA-binding molecules for applications in che-
motherapy and in the development of tools for biotech-
nology [14]. In our group, much effort has been devoted to
studying the DNA interactions and cytotoxicities of novel
polypyridyl complexes containing different intercalative
ligands [15–19].
Electronic supplementary material The online version of this
article (doi:10.1007/s11243-013-9753-1) contains supplementary
material, which is available to authorized users.
Y. P. Kumar ꢀ C. S. Devi ꢀ N. Deepika ꢀ
N. M. Gabra ꢀ A. Srishailam ꢀ K. L. Reddy ꢀ
S. Satyanarayana (&)
Department of Chemistry, Osmania University,
Hyderabad 500007, Andhra Pradesh, India
e-mail: ssnsirasani@gmail.com
In this article, we report three Co(III) mixed ligand
polypyridyl complexes, [Co(phen)2HEPIP](ClO4)3 (1),
[Co(bpy)2HEPIP](ClO4)3 (2) and [Co(dmb)2HEPIP](ClO4)3
(3), their DNA-binding behavior, and their abilities to
N. Jain
Chemical Biology Division, Indian Institute of Chemical
Technology, Hyderabad 50060, Andhra Pradesh, India
123

812
Transition Met Chem (2013) 38:811–819
absorbance at 260 and 280 nm of about 1.8–1.9:1, indi-
cating that the DNA was sufficiently free of protein [23].
The concentration of CT-DNA was determined spectro-
photometrically using the molar absorption coefficient
6,600 M-1 cm-1 (260 nm) [24]. Human tumor cell lines
were obtained from NCCS, Pune, and maintained in RPMI
1640 medium (Sigma Aldrich) supplemented with 10 %
fetal bovine serum, 1 % penicillin, and streptomycin in a
humidified 5 % CO2 atmosphere.
induce cleavage of pBR322 DNA. Cell viability experiments
indicated that the Co(III) complexes showed significant
dose-dependent cytotoxicities against four human tumor cell
lines, namely, human cervical cancer (HeLa), human alve-
olar adenocarcinoma (A549), prostate cancer (DU145) and
hepatocellular carcinoma (HEPG). The complexes were also
tested for antimicrobial activity and docked into DNA base
pairs using a docking program [20, 21].
Experimental
Synthesis and characterization
Materials and methods
1,10-Phenanthroline-5,6-dione [25], cis-[Co(phen)2Br2]-
Brꢀ2H2O, cis-[Co(bpy)2Br2]Brꢀ2H2O and cis-[Co(bpy)2Br2]-
Brꢀ2H2O [26, 27] were prepared according to the methods
used in our previous study and the literature, respectively
[28, 29]. The syntheses of the free ligands and their Co(III)
complexes are shown in Scheme 1.
CoCl2ꢀ6H2O, 1,10-phenanthroline monohydrate, 2,20-
bipyridine and 4,40-dimethyl-2,20-bipyridine were pur-
chased from Merck. CT-DNA and supercoiled (CsCl
purified) pBR322 DNA (Bangalore Genei, India) were used
as received. All other common chemicals and solvents
were procured from locally available sources; solvents
were purified before use by standard procedures [22].
Deionized, double-distilled water was used for preparing
various buffers. Solutions of DNA in 5 mM Tris-HCl
buffer (pH = 7.2) and 50 mM NaCl gave a ratio of UV
Synthesis of HEPIP
A
solution of 1,10-phenanthroline-5,6-dione (0.25 g,
1.2 mmol), 3-ethoxy salicylaldehyde(0.31 g, 1.9 mmol)
N
Co(phen)2Br2
HO
O
N
N
N
N
Co
N
N
H
N
HO
O
HO
O
N
OHC
N
N
O
HO
N
Co
H
N
Co(bpy)2Br2
N
N
N
H
N
N
N
O
O
N
N
N
AcOH
NH4OAC
4 hr
N
HO
O
N
N
N
Co(dmb)2Br2
Co
N
N
N
H
N
Scheme 1 Synthetic routes of ligand and Co(III) complexes
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Transition Met Chem (2013) 38:811–819
813
Synthesis of complex 3
and ammonium acetate (1.9 g, 25 mmol) in glacial acetic
acid (10 mL) was refluxed for 4 h. The light yellow solu-
tion so obtained was cooled, diluted with water (25 cm3)
and neutralized with ammonia. The precipitate was filtered
off, washed with H2O and Me2CO and then dried. Yield:
0.75 g (72 %).
This complex was obtained by a procedure similar to that
described above, but using cis-[Co(dmb)2Br2]Brꢀ
2H2O(0.587 g, 1.0 mmol) in place of cis-[Co(phen)2Br2]-
Brꢀ2H2O. Yield: (65 %). C45H40Cl3CoN8O14; Calcd (%):
C, 49.4; H, 3.8; N, 10.5. Found (%): C, 48.9; H, 3.2; N,
10.8. ESI–MS (in DMSO), m/z; 1,082 (Calcd 1,080). IR
(KBr cm-1): 1,438 (C=N), 1,307 (C=C), 629 (Co–N(HE-
PIP)), 485 (Co–N(dmb)); UV–Vis (CH3OH kmax, nm (log
e): 261 (3.60), 271 (3.58), 429 (3.28);1H-NMR (DMSO-d6,
25 °C, d ppm, J = Hz): 9.1 (2H, s); 9.1 (2H,d, J = 6.8);
8.5 (2H, d, J = 7.2); 7.5–8.8 (1H, m); 7.1 (1H, d,
J = 7.2);6.8–7.4 (1H, m) 6.9 (1H, d, J = 6.6); 5.80 (1H,
NH, s); 3.2–3.8 (2H, q), 2.2–2.9 (3H, t), 2.1 (4 methyl);
13C[1H]-NMR (DMSO-d6, d ppm): 153, 150, 146, 143,
138, 134, 131, 127, 128, 117, 114, 112, 109, 67 and 20.
ESI–MS (in DMSO), m/z; 358 (Calcd 357);Calcd for
C21H16N4O2 %: C, 70.5; H, 4.7; N, 15.6; Found (%); C,
70.1; H, 4.4; N, 15.2. IR (KBr cm-1): 1,655 (C=N), 1,510
1
(C=C). HNMR (DMSO-d6, 25 °C, d ppm, J = Hz); 9.1
(1Hs); 9.0 (2H, d, J = 7.2); 8.9 (2H, d 7.6); 6.5–7.8
(1H, m); 7.1 (1H,d J = 6.9); 6.8–7.2 (1H, m) 6.9 (1H, d,
J = 7.5); 5.8 (1H, N H, s); 3.2 (2H,q),1.4 (3H,t); 13C[1H]-
NMR (DMSO-d6, d ppm): 150, 148, 145, 139, 135, 132,
130, 125, 124, 116, 115, 113, 110, 65 and 16.
Synthesis of complex 1
Physical measurements
A
mixture of cis-[Co(phen)2Br2]Brꢀ2H2O (0.57 g,
1.0 mmol) and HEPIP (0.48 g, 1.5 mmol) in EtOH
(50 cm3) was refluxed for 4 h to give a yellow solution.
After filtration, the complex was precipitated by addition of
a saturated ethanolic solution of NaClO4. The complex was
filtered off and dried under vacuum before recrystallization
(Me2CO–Et2O). Yield: (79 %). C45H32Cl3CoN8O14; Calcd
(%); C, 48.9; H, 3.1; N, 10.4. Found (%): C, 49.1; H, 3.4;
N, 10.2. IR (KBr cm-1): ESI–MS (in DMSO), m/z;
1,073 (Calcd 1,072). 1,424 (C=N), 1,337 (C=C), 625
UV–Visible spectra were recorded with an Elico Bio-
spectrophotometer, model BL198. IR spectra were recor-
ded in KBr discs on
a Perkin-Elmer FT-IR-1605
spectrometer. 1H and 13C [1H]NMR spectra were measured
on a Bruker Z-Gradient single axis fitted with a high-res-
olution probe and 400 MHz standard spectrometer using
DMSO-d6 as the solvent and TMS as an internal standard.
Microanalysis was performed on a Perkin-Elmer 240 ele-
mental analyzer. Fluorescence spectra were recorded with
an Elico spectrofluorimeter model SL 174.
1
(Co–N(HEPIP)), 456 (Co–N(phen)). H-NMR (DMSO-d6,
25 °C, d ppm, J = Hz): 9.1 (2H s); 9.0 (2H,d, J = 7.8); 8.9
(2H, d, J = 6.8); 6.7–7.9 (1H, m); 7.1 (1H,d,
J = 6.8);6.9–7.6 (1H, m) 6.9 (1H, d, J = 7.7); 5.7 (1H,
NH,); 3.5–4.2 (2H,q),1.6–2.1 (3H,t);13C[1H]-NMR
(DMSO-d6, d ppm): 151, 149.5, 146, 143, 138, 135, 131,
128, 129, 117, 116, 112, 110, 66.5 and 20.
The DNA-binding experiments were performed in Tris–
HCl buffer at 25 °C. The absorption titrations were per-
formed at a fixed complex concentration, to which the
DNA stock solution was gradually added up to the point of
saturation. The mixture was allowed to equilibrate for
5 min before the spectra were recorded. The emission
intensities were recorded in the range of 520–720 nm. In
these emission studies, fixed complex concentrations
(10 lM) were taken and to this, varying concentrations
(0–100 lM) of DNA were added. The excitation wave-
length was fixed, and the emission range was adjusted
before measurements. The fraction of the ligand bound was
calculated from the relation,
Synthesis of complex 2
This complex was obtained by a procedure similar to that
described above, except that [Co(bpy)2Br2]Brꢀ2H2O
(0.53 g, 1.0 mmol) was used in place of cis-[Co(phen)2Br2]-
Brꢀ2H2O. (Yield: 72 %). C41H32Cl3CoN8O14; Calcd (%):
C, 46.5; H, 3.2; N, 10.8. Found (%): C, 46.2; H, 3.1; N,
11.1. ESI–MS (in DMSO), m/z; 1,025 (Calcd 1,024). IR
(KBr cm-1): 1,466 (C=N), 1,374 (C=C), 627 (Co–N
(HEPIP)), 476 (Co–N(bpy)).1H-NMR (DMSO-d6, 25 °C, d
ppm, J = Hz): 9.4 (2H s); 9.1 (2H,d, J = 7.6); 8.6 (2H, d,
J = 6.9); 7.5–7.9 (1H, m); 7.0 (1H, d); 6.8–7.2 (1H, m) 6.6
(1H, d, J = 7.3); 5.68 (1H, NH, s); 3.4–3.8 (2H, q),1.6–1.9
(3H, t); 13C[1H]-NMR (DMSO-d6, d ppm): 152, 155, 146,
143, 138, 134, 131, 127, 128, 117, 115, 112, 110, 66
and 18.
Cb ¼ Ct½ðF ꢁ F0Þ=ðFmax ꢁ F0Þꢂ
where Ct is the total complex concentration, F is the
observed fluorescence emission intensity at a given DNA
concentration, F0 is the intensity in the absence of DNA
and Fmax is when the complex is fully bound to DNA. The
binding constant (Kb) was obtained from a modified Scat-
chard equation [30], from a Scatchard plot of r/Cf versus r,
where r is Cb/[DNA] and Cf is the concentration of free
complex.
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Transition Met Chem (2013) 38:811–819
Viscosity experiments were carried out with an Ostwald
complexes 1–3, cells were placed in 96-well micro-assay
culture plates (8 9 103 cells per well) and grown overnight
at 37 °C in a 5 % CO2 incubator. The test complexes were
dissolved in DMSO and diluted with RPMI 1640
(RPMI = Roswell Park Memorial Institute medium) and
then added to the wells to achieve final concentrations
ranging from 10 to 100 lM. After treatment of tumor cells
with the complexes for 48 h, the plates were washed twice
with culture medium, then MTT was added and the plates
were incubated for another 4 h. Cells without added Co(III)
complexes were used as negative control. Cisplatin was
used as the positive control. The IC50 values were deter-
mined by plotting the percentage viability versus concen-
tration on a logarithmic graph and reading of the
concentration at which 50 % of cells remained viable rel-
ative to the control. Each experiment was repeated at least
three times to obtain mean values.
viscometer. DNA samples approximately 200 base pairs in
average length were prepared by sonication in order to
minimize complexities arising from DNA flexibility [31].
Data were analyzed as (g/g°)1/3 versus [Co]/[DNA], where
g is viscosity of DNA in the presence of complex and g° is
the viscosity of DNA alone. Viscosity values were calcu-
lated from the observed flow time of DNA-containing
solutions (t [ 100 s) corrected for the flow time of buffer
alone (t°), g = t-t0 [32].
For gel electrophoresis experiments, supercoiled
pBR322 DNA (100 lM) was treated with the appropriate
complex in 50 mM Tris–HCl and 18 mM NaCl buffer
pH 7.8, and the solutions were then irradiated at room
temperature with a UV lamp (10 W). The samples were
analyzed by electrophoresis for 2.5 h at 40 V on a 1 %
agarose gel in Tris–acetic acid–EDTA buffer, pH 7.2. The
gels were stained with 1 mg mL-1ethidium bromide and
photographed under UV light.
The molecular docking calculations on all three com-
plexes were done using the 3.01 GOLD (Genetic Optimi-
zation for Ligand Docking) program 55, which is based on
Genetic Algorithms. This method allows partial flexibility of
the hydroxyl groups of the respective DNA molecule and full
flexibility of the ligand. The DNA sequences used for the
docking simulations were obtained from the Protein Data
Bank and are double helices associated with ligands.
Using Discovery Studio 3.0, we built the DNA sequence
(CGATTAATCG) obtained from the Protein Data Bank
(PDB: 1D49) double helix DNA decamer. The DNA struc-
tures were chosen so as to allow an evaluation of the binding
preference for CG sequences. In order to evaluate the GOLD
scoring function, all water molecules were removed from the
DNA molecules. The function fitted was Gold Score.
Hypothetical structures resulting from the initial docking
were energy-minimized. The docking procedure depended
on two principal features: (1) an energy (or scoring) function
for evaluating trial configurations of the two interacting
molecules and (2) an algorithm for seeking the best achiev-
able minimum of this function. The two interacting mole-
cules were considered as rigid bodies, and the sum of the van
der Waals, hydrogen bonding and electrostatic energy terms
were used as the scoring function.
Antimicrobial tests were performed by the standard disc
diffusion method [33]. The complexes were screened for
antifungal activity against Aspergillus niger and Fusarium
oxysporium, which were isolated from the infected parts of
host plants grown on M test agar medium. The cultures of
the fungi were purified by single-spore isolation technique.
A concentration of 1.5 mg mL-1 of each cobalt complex in
DMSO was prepared for testing against spore germination
of each fungus. Filter paper discs of 5 mm were prepared
using Whatman filter paper no. 1 (sterilized in an auto-
clave) and saturated with 10 mL of the cobalt complex
dissolved in DMSO. The fungal culture plates were inoc-
ulated and incubated at 25 2 °C for 48 h. The plates
were then observed, and the diameters of the inhibition
zones (in millimeters) were measured and tabulated. The
results were also compared with the standard antifungal
drug fluconazole at the same concentration. The antibac-
terial activities of the complexes were studied against
Staphylococcus aureus (MTCC 96) and Escherichia coli
(MTCC 443). Each complex was dissolved in DMSO at
1 mg mL-1. Paper discs of Whatman filter paper no. 1
were cut and sterilized in an autoclave. The paper discs
were saturated with 10 mL of the cobalt complex dissolved
in DMSO or DMSO as negative control and placed asep-
tically in Petri dishes containing M test agar media inoc-
ulated with S. aureus or E. coli. The Petri dishes were
incubated at 37 °C, and the inhibition zones were recorded
after 24 h. The experiments were repeated, and the average
of the two runs was taken. The results were also compared
with the standard antibacterial drug streptomycin at the
same concentration.
Results and discussion
Characterization
The ESI–MS spectrum of HEPIP shows a molecular ion
peak at m/z 358, equivalent to its molecular weight (Calcd
1
357). The H-NMR spectrum of HEPIP gave a peak at 9.1
The MTT (MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay was used [34] to
determine the viability of tumor cells; upon treatment with
(singlet) corresponding to OH, a quartet at 3.2 and triplet at
1.4 ppm corresponding to CH2 and CH3 protons and a
broad peak at 5.8 (singlet) corresponding to –NH. The
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Transition Met Chem (2013) 38:811–819
815
remaining signals belong to the ring protons between 9.0
and 6.9 ppm and were observed with proper multiplicity.
The protons next to nitrogen appeared downfield as a
doublet at 9.0 ppm. The 13CNMR spectrum of HEPIP gave
a peak at 148 ppm corresponding to COH carbon, 145
corresponding to carbon next to nitrogen and the ethoxy
carbon at 65 ppm, and 16(methyl) other peaks were
observed in the aromatic region as expected.
bands [35]. The electronic spectra of these complexes in
the absence and presence of CT-DNA are illustrated in
Fig. 1, and the data are summarized in Table 1. Bands at
kmax 430 nm (1), 428 nm (2) and 425 nm (3) are assigned
to MLCT [36], while the bands at kmax 265 nm (1), 262 nm
(2) and 263 nm (3) are due to p–p* transitions of HEPIP.
Addition of increasing quantities of CT-DNA results in
decreasing peak intensities. As the DNA concentration is
increased, the MLCT bands of complexes 1, 2 and 3 exhibit
hypochromism of 20.6, 11.5 and 8.9 %, respectively, and
bathochromism of about 3–6 nm. Intrinsic DNA-binding
constants K were determined by monitoring the change of
absorbance of the MLCT bands of the complexes with
increasing concentration of DNA [37]. According to fol-
lowing equation [38],
The ESI–MS spectrum of [Co(phen)2(HEPIP)](ClO4)3ꢀ
2H2O showed the molecular ion peak at m/z of 1,073 (Calcd
1,072). The IR spectrum of this complex showed bands at
1,424 (C=N) and 1,337 (C=C), shifted to a lower frequency
when compared to free ligand consistent with complexation.
New bands at 625 and 456 cm-1 assigned to Co–N (HEPIP)
and Co–N(phen), respectively, support complex formation. In
the1HNMRspectrumpeaksdue tothe variousprotonsofphen
and HEPIP are shifted downfield upon complexation. The
13CNMR spectrum showed that the carbon next to nitrogen
shifted downfield to 146 ppm. The carbon attached to OH was
shifted downfield to 151, and ethoxy and methyl carbons
resonate at 66 and 20 ppm, respectively. Aromatic peaks are
also shifted downfield. The ESI–MS spectrum of
[Co(bpy)2(HEPIP)](ClO4)3ꢀ2H2O shows a molecular ion
peak at m/z of 1,032 which is equivalent to its molecular
weight (Calcd 1,031). The IR spectrum of this complex
includes bands at 1,466 (C=N) and 1,374 (C=C), which are
shifted to lower frequency when compared to the free ligand,
indicating complexation. New bands at 630 and 580 cm-1
assigned to Co–N(HEPIP) and Co–N(bpy), respectively,
support complex formation. The 1HNMR spectrum of
[Co(bpy)2 (HEPIP)] (ClO4)3ꢀ2H2O shows the various protons
of bpy and HEPIP are shifted downfield upon complexation.
In the 13CNMR spectrum of this complex, the signal at
152 ppm, whichisnexttonitrogen, isshifteddownfield, while
the carbonattachedtoOHisshifteddownfieldto150 ppm and
the ethoxy carbon resonates at 66 ppm. Peaks in the aromatic
region are also shifted downfield.
½DNAꢂ=ðea ꢁ ef Þ ¼ ½DNAꢂ=ðeb ꢁ ef Þ þ 1=ðKðeb ꢁ ef ÞÞ
where [DNA] is the concentration of the base pairs, the
apparent absorption coefficients ea, ef and eb correspond to
A
obsd/[Co], the extinction coefficients for the free cobalt
complex, the complex in the presence of DNA and the
cobalt complex in the fully bound form, respectively. In
plots of [DNA]/(ea-ef) versus [DNA], K is given by the
ratio of slope to intercept. The values of K so obtained were
6.13 9 105, 4.46 9 105 and 3.72 9 105 M-1, for complex
1, 2 and 3, respectively. These spectroscopic characteristics
suggest a stacking interaction between the complexes and
the base pairs of DNA. The difference in binding strength
of complexes 1 and 2 could be attributed to the different
ancillary ligands; phenanthroline is more planar than
bipyridyl; hence, the binding constant of complex 1 is
higher than complex 2. Similarly, the two additional
The ESI–MS spectrum of [Co(dmb)2(HEPIP)](ClO4)3ꢀ
H2O shows a molecular ion peak at m/z of 1,082 which is
equivalent to its molecular weight (Calcd 1,080). In the
1HNMR spectrum peaks due to various protons of the dmb
and HEPIP ligands are shifted downfield upon complexa-
tion. In the 13CNMR spectrum, the C next to nitrogen is
shifted downfield to 153 ppm, while the carbon attached to
OH shifts downfield to 156 ppm, and the ethoxy and
methyl carbons resonate at 67 and 18 ppm respectively.
The aromatic peak are also shifted downfield.
Fig. 1 Absorption spectrum of [Co(bpy)22-HEPIP]3? (1) in
Tris–HCl buffer at 25 °C in the presence of increasing amount of
CT-DNA, [Co] = 10 mM, [DNA] = 0–120 mM. The arrows indi-
cate the change in absorbance upon increasing the DNA concentra-
tion. Insert: Plot of [DNA]/(ea - ef) versus [DNA] for titration of the
Co(III) complexes
Electronic absorption
For metallo-intercalators, DNA binding is associated with
hypochromism and a red shift in the MLCT and ligand
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Transition Met Chem (2013) 38:811–819
Table 1 Results of absorption titration experiments
400
380
360
340
320
300
280
260
240
220
200
180
160
Complexes
Hypochromicity Absorption kmax (nm)
(%)
Free
Bound
Dk
CT-DNA alone
[Co(phen)2 2-HEPIP]3? 20.6
–
–
–
10
8
276
258
261
286
266
266
[Co(bpy)2 2-HEPIP]3?
[Co(dmb)22-HEPIP]3?
11.5
8.9
5
540
560
580
600
620
640
660
680 700
720
methyl groups of dimethyl bipyridyl in complex 3 exert
steric hindrance; hence, complex 2 binds stronger than
complex 3. The HEPIP ligand contains a free hydroxy
group, which may form intramolecular hydrogen bonds
with the nitrogen of DNA.
Wavelength (nm)
Fig. 2 Emission spectra of, [Co(phen)22-HEPIP]3? in Tris–HCl
buffer at 25 °C upon addition of CT-DNA, [Co] = 20 lM,
[DNA] = 0–120 lM. The arrow shows the increase in intensity
upon increasing CT-DNA concentrations
[Fe(CN)6]4- would be repelled by the negative DNA
phosphate backbone. The method essentially consists of
titrating a given amount of the DNA–metal complex with
increasing concentrations of [Fe(CN)6]4-and measuring
the change in fluorescence intensity (Fig. 3); in the absence
of DNA, the complex is efficiently quenched by
[Fe(CN)6]4-, resulting in linear Stern–Volmer plots. The
Stern–Volmer quenching constant Ksv can be determined
by using the Stern–Volmer equation [39];
Fluorescence spectroscopic studies
To further understand the nature of the complex binding to
DNA, luminescence titration experiments were performed
at a fixed metal complex concentration (5 lM) in Tris
buffer (pH = 7.2) at ambient temperature. The change of
emission intensity is related to the extent to which the
complex enters into the hydrophobic environment within
the DNA. Figure 2 shows the fluorescence excitation and
emission spectra for the free and bound complexes in the
presence of different amounts of CT-DNA. Excitation
wavelengths of 433, 430 and 428 nm and emission wave-
lengths of 610, 618 and 627 nm for complexes 1, 2 and 3,
respectively, were used for fluorescence measurements.
Addition of DNA to a solution of each complex resulted in
an increase in fluorescence intensity, by a factor of 1.54,
1.48 and 1.25 times, respectively. The intrinsic binding
constant was obtained from the fluorescence data using a
modified form of the Scatchard equation [30] with a plot of
r/cf versus r, where r is the binding ratio Cb/[DNA] and Cf
is the free ligand concentration. These plots gave binding
constants (Kb) of (5.45 0.1) 9 105 M-1, (4.16 0.1) 9
105 M-1, and (3.18 0.1) 9 105 M-1 for complexes 1, 2
and 3, respectively. The order of Kb values agrees with the
results of the absorption studies.
I0=I ¼ 1 þ Ksv½Qꢂ
where I0 and I are the fluorescence intensities in the
absence and presence of the quencher, respectively, Q is
the concentration of the quencher and Ksv is a linear Stern–
Volmer quenching constant. Figure 3 shows the Stern–
Volmer plots, which are linear for all three complexes [40,
41]. Ferrocyanide quenching curves for the three
Quenching studies
We next carried out emission quenching experiments using
[Fe(CN)6]4-, which permits distinguishing of bound
Co(III) species. Positively charged free complex ions
should be readily quenched by [Fe(CN)6]4-,whereas when
bound to DNA, the complex will be protected from the
quencher because the highly negative charge of
Fig. 3 Emission quenching of Co(III) complexes [Co(phen)22-HE-
PIP]3? with K4[Fe(CN)6]4- in the presence and absence of DNA.
[Co] = 10 mM, [DNA]/[Co] = 40:1
123

Transition Met Chem (2013) 38:811–819
817
complexes in the presence and absence of CT-DNA are
shown in Fig. 3. The absorption, fluorescence and
quenching studies all indicate that the binding constants are
in the order 1 [ 2 [ 3.
electrophoresis; on the other hand, simple electrostatic
interaction of small molecules with DNA does not signif-
icantly influence the supercoiled form; thus, the mobility of
the supercoiled DNA does not change.
Plasmid pBR322 DNA was subjected to gel electro-
phoresis after incubation with the cobalt(III) complexes
and irradiation at 365 nm. In control experiments where the
complex was absent (lane 1) or the DNA-complex mixtures
were incubated in the dark, no photocleavage was notice-
able (Fig. 5). In contrast, irradiation with increasing con-
centrations of all three complexes (lanes 2–5) resulted in a
decrease in the amount of supercoiled DNA, whereas the
nicked (form II) increased, which is slow moving [45].
These results indicate that scission occurs on one strand
(nicked). All three complexes are effective for photo-sen-
sitized cleavage of DNA.
Viscosity studies
A hydrodynamic measurement such as viscosity is sensi-
tive to DNA length change and is regarded as the least
ambiguous and most critical test of a binding model. In
classical intercalation, the DNA helix lengthens as base
pairs are separated to accommodate the bound ligand,
leading to an increase in the viscosity of the DNA solution
[42, 43]. On the other hand, partial and/or nonclassical
intercalation of the ligand may bind the DNA helix,
resulting in a decrease in its effective length and con-
comitantly its viscosity. The effects of the three complexes
on the viscosity of DNA are shown in Fig. 4. As the con-
centration of the complexes increases, the relative viscosity
of DNA also increases, similar to the behavior of the
proven DNA intercalator [Ru(phen)2dppz]2? [44].
Although the intercalating ligand is the same in all three
complexes, there are small differences in the viscosity, due
to the difference in ancillary ligands. These results again
suggest that these complexes show an intercalative binding
mode to CT-DNA.
Antimicrobial activities
The antifungal activity data (Table 2) indicate that the
complexes show appreciable activity against A. niger and
F. oxysporium at 1.5 mg mL-1 concentration. The results
for the compounds were compared with DMSO as control
and are expressed as inhibition zone diameter (in milli-
meters) versus control. Complex 1 showed the highest
activity against A. niger and moderate activity against F.
oxysporium. This complex exhibited greater antifungal
activity against A. niger compared to the standard drug
fluconazole. Complexes 2 and 3 showed less activity than
fluconazole. The antibacterial activity data (Table 3) indi-
cate that the complexes have high activity against both S.
aureus and E. coli at 1 mg mL-1 concentration. Complex 1
shows the highest activity (19 mm) against S. aureus and
18-mm inhibition against E. coli. This complex exhibits
greater antibacterial activity against S. aureus than the
Photo-activated cleavage of pBR322 DNA
Plasmid pBR 322 DNA is mainly in the closed-circle
supercoiled form (Form I). Intercalation of small molecules
into plasmid DNA can cleave the supercoiled form, which
decreases its mobility and can be visualized by gel
4.5
a
4
3.5
b
3
2.5
2
c
d
1.5
1
0.5
0
0
0.02
0.04
0.06
0.08
0.1
0.12
[complex]/[DNA]
Fig. 4 Effect of increasing amount of ethidium bromide (a) com-
plexes [Co(phen)2(2-HEPIP)](ClO4)3 (b) [Co(bpy)22-HEPIP](ClO4)3
(c) and [Co(dmb)22-HEPIP](ClO4)3 (d) on relative viscosity of CT-
DNA at 30 0.1 °C. The total concentration of DNA is 0.25 mM,
[Co] = 20 lM
Fig. 5 Photo-activated cleavage of pBR 322 DNA in the presence
of [Co(phen)22-EHPIP]3?](1), [Co(bpy)22-HEPIP]3?](2) and
[Co(dmb)22-HEPIP]3?](3)complexes, after irradiation at 365 nm.
Lane a control plasmid DNA (untreated pBR 322), lanes 1–4,
addition of complexes 20, 40, 60 80 lM
123

818
Transition Met Chem (2013) 38:811–819
standard drug streptomycin. Complexes 2 and 3 showed
less activity against these bacteria than streptomycin.
Earlier studies have given results which were also similar
to this study [10, 11].
Table 2 Antifungal activity of the Cobalt(III) complexes
Complex
Inhibition zone diameter (mm) of
bacterial species
A. niger
F. oxysporium
[Co(phen)2 2-HEPIP]3? (3)
[Co(bpy)2 2-HEPIP]3? (1)
[Co(dmb)2 2-HEPIP]3? (2)
Fluconazole (standard)
22.0 0.3
9.0 0.2
12.0 0.1
15–18
20.0 0.1
11.0 0.4
11.0 0.2
15–18
Cytotoxicity studies
The positive results obtained from the DNA-binding and
cleavage experiments encouraged us to test the cytotoxic-
ities of the complexes against a panel of human cancer cell
lines, namely, human cervical cancer cell line HeLa, A549,
DU145 and HEPG tumor by colorimetric (MTT) assay.
The complexes were dissolved in DMSO, and blank sam-
ples containing the same volume of DMSO were taken as
controls. The results were analyzed by means of cell via-
bility and expressed as IC50 values as shown in Table 4.
The results of the in vitro cytotoxicity studies further
confirm the binding of the complexes to DNA, which
consequently leads to cell death. The MTT assay was
employed to measure the metabolic activity of mitochon-
dria in the cells, based on the principle that living cells are
capable of reducing the lightly colored tetrazolium salt into
an intensely colored formazan derivative [46]. Figure 6
shows the viability of HeLa, A549, DU145 and HEPG cells
upon treatment with complexes 1–3 for 48 h; all three
complexes show only slight cytotoxicity toward HeLa,
A549, DU145 and HEPG tumor cells. Complex 1 exhibits
more cytotoxicity than 2 and 3, and the complexes are less
cytotoxic than the standard drug cisplatin. These results are
in accordance with those reported previously for analogous
cobalt polypyridyl complexes [47]. For example, the IC50
value of [Co(phen)2 pip]3? is 54.6 lM L-1 [47].
Table 3 Antibacterial activity of the Cobalt(III) complexes
Complex
Inhibition zone diameter (mm) of
bacterial species
S. aureus
E. coli
[Co(phen)2 2-HEPIP]3? (3)
[Co(bpy)2 2-HEPIP]3? (1)
[Co(dmb)2 2-HEPIP]3? (2)
Streptomycin (standard)
18.0 0.1
10.0 0.3
09.0 0.4
13–17
17.0 0.2
09.0 0.1
85.0 0.4
13–17
Values of zone of inhibition (mm, including the diameter of the disc)
Table 4 The Cytotoxic activity of the compounds
S. No
A549
DU145
HELA
HEPG2
Complex-1
Complex-2
Complex-3
Cisplatin
23 1.4
87 1.8
35 2.3
12 1.0
24 0.8
58 0.6
32 0.4
30 1.0
[ 100
27 1.0
[100
[ 100
19 1.2
[100
6
0.5
7
0.3
IC50 values are given in lM, and cisplatin is included for compari-
son. Data are presented as mean values standard deviations, and cell
viability assessed after 48 h of incubation
Fig. 6 In vitro cytotoxicity of
complexes 1–3 on four different
tumor cells. Cytotoxicity was
measured by MTT reduction
assay after 48 h. Untreated cells
are used as the control
123

Transition Met Chem (2013) 38:811–819
819
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These Co(III) complexes can interact with DNA by
hydrogen bonding, particularly involving N7 of adenine,
N3 of guanine, N1 of cytosine and thymine and phosphate
oxygen. Also, van der Waal’s attractions and p–p stacking
between the complex and DNA chain is possible. Our
modeling results showed that these complexes can bind to
DNA with three strong hydrogen bonds, with GOLD fitness
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order of GOLD fitness scores from molecular docking
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Conclusion
A new ligand HEPIP and three of its cobalt(III) complexes
were prepared and characterized by elemental analysis,
1
ESI–MS, IR and H NMR. These complexes have high
DNA-binding affinity, interacting with DNA by intercala-
tion. Complex 1 shows the highest cytotoxicity against the
four tumor cell lines. Upon irradiation at 365 nm, all three
complexes can cleave plasmid DNA.
Acknowledgments We are grateful to UGC New Delhi for financial
support and CFRD Osmania University for recording NMR and also
grateful to Dr. Shasi Vardhan Kalivendi Senior Scientist Indian
Institute of Chemical Technology (IICT), Hyderabad, for helping
biological studies.
31. Satyanarayana S, Dabrowiak JC, Chaires JB (1993) Biochemistry
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