Evaluation of the cell cytotoxicity and DNA/BSA binding and cleavage activity of some dioxidovanadium(V) complexes containing aroylhydrazones

Article abstract of DOI:10.1016/j.jinorgbio.2014.12.018

Three dioxidovanadium(V) complexes [VO₂L^1-3] (1-3) [HL¹ = 1-napthoyl hydrazone of 2-acetyl pyridine, HL² = 2-furoyl hydrazone of 2-acetyl pyridine and H₂L³ = isonicotinoyl hydrazone of 2-hy

Full text of DOI:10.1016/j.jinorgbio.2014.12.018

Evaluation of the cell cytotoxicity and DNA/BSA binding and cleavage
activity of some dioxidovanadium(V) complexes containing aroylhydrazones
Subhashree P. Dash, Alok K. Panda, Sagarika Pasayat, Sudarshana Ma-
jumder, Ashis Biswas, Werner Kaminsky, Subhadip Mukhopadhyay, Sujit K.
Bhutia, Rupam Dinda
PII:
DOI:
Reference:
S0162-0134(14)00311-0
doi: 10.1016/j.jinorgbio.2014.12.018
JIB 9641
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
25 September 2014
17 December 2014
17 December 2014
Please cite this article as: Subhashree P. Dash, Alok K. Panda, Sagarika Pasayat, Sudar-
shana Majumder, Ashis Biswas, Werner Kaminsky, Subhadip Mukhopadhyay, Sujit K.
Bhutia, Rupam Dinda, Evaluation of the cell cytotoxicity and DNA/BSA binding and
cleavage activity of some dioxidovanadium(V) complexes containing aroylhydrazones,
Journal of Inorganic Biochemistry (2014), doi: 10.1016/j.jinorgbio.2014.12.018
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ACCEPTED MANUSCRIPT
Evaluation of the cell cytotoxicity and DNA/BSA binding and
cleavage activity of some dioxidovanadium(V) complexes containing
aroylhydrazones
a
b
a
a
b,
Subhashree P. Dash , Alok K. Panda , Sagarika Pasayat , Sudarshana Majumder , Ashis Biswas *,
c
d
d
a,
Werner Kaminsky , Subhadip Mukhopadhyay , Sujit K. Bhutia , Rupam Dinda *
a
Department of Chemistry, National Institute of Technology, Rourkela 769008, Odisha, India.
b
School of Basic Sciences, Indian Institute of Technology Bhubaneswar, Bhubaneswar 751 013, Odisha, India.
c
Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195, USA.
d
Department of Life Science, National Institute of Technology, Rourkela 769008, Odisha, India.
*Corresponding authors: (R. Dinda): Department of Chemistry, National Institute of Technology,
Rourkela, 769008, Odisha, India. Tel.: + (91) 661 246 2657; Fax: + (91) 661 246 2022; E-mail:
rupamdinda@nitrkl.ac.in; (A. Biswas): School of Basic Sciences, Indian Institute of Technology
Bhubaneswar, Bhubaneswar 751013, Odisha, India. Tel.: + (91) 674 257 6051; Fax: + (91) 674 230 6202;
E-mail: abiswas@iitbbs.ac.in
1

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ABSTRACT
___________________________________________________________________________
_
1
−3
1
Three dioxidovanadium(V) complexes [VO L ] (1−3) [HL = 1-napthoyl hydrazone of 2-acetyl
2
2
3
pyridine, HL = 2-furoyl hydrazone of 2-acetyl pyridine and H L = isonicotinoyl hydrazone of
2
2
-hydroxy benzaldehyde] have been reported. All the complexes were characterized by various
spectroscopy (IR, UV-visible and NMR) and the molecular structures of 1 and 2 were
characterized by single crystal X-ray diffraction technique. Structural report established five-
coordinate geometries, distorted toward square pyramidal for each of 1 and 2, based on a
tridentate -O,N,N coordinating anion and two oxido-O atoms. The experimental results show that
the complexes interact with calf-thymus DNA (CT-DNA) possibly by a groove binding mode,
5
−1
with binding constants of ~ 10 M . All complexes show good photo-induced cleavage of
pUC19 supercoiled plasmid DNA with complex 1 showing the highest photo-induced DNA
3
4
cleavage activity of ~ 68%. 1−3 also exhibit moderate binding affinity in the range of 10 −10
−
1
M towards bovine serum albumin (BSA), while all the complexes show good photo-induced
BSA cleavage activity. Moreover the antiproliferative activity of all these complexes was
studied, which reveal all compounds are significantly cytotoxic towards the HeLa cell line.
_
____________________________________________________________________________
Keywords: Dioxidovanadium(V) aroylhydrazone complexes / X-ray crystal structure / DNA
binding and photo-induced cleavage / BSA activity / Cytotoxicity.
2

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1
. Introduction
Metal complexes such as cisplatin and carboplatin are metal based drugs, which are widely used
in cancer chemotherapy. The great success in the clinical treatment of human malignancies has
stimulated research in the area of inorganic antitumor agents [1]. However, their use is extremely
hampered by severe toxicity and development of resistance during the therapy [2–5]. In order to
overcome these disadvantages, current strategies in the development of novel metallo drugs
focused more and more on the use of transition metal complexes containing improved organic
ligands [6–8]. The molecular mechanism of action of such DNA-targeting drugs involves
covalent binding to nucleobase moieties and a low degree of selectivity [9]. In order to overcome
the challenges put forth by platinum drugs, researchers in the area of medicinal inorganic
chemistry are developing non-platinum based chemotherapeutic agents that exhibit enhanced
selectivity and non-covalent DNA binding interaction modes (intercalation, groove binding and
external electrostatic binding), are of considerable interest [10,11]. Recently, there is a great
interest on the binding of transition metal complexes with DNA because of their utility as DNA
structural probes, DNA foot printing, sequence-specific cleavage agent and potential anticancer
drug [10].
Among the nonplatinum complexes for metal based chemotherapy, focus turned to vanadium
complexes due to the fact that vanadium is a bioessential element responsible for numerous
bioactivities in living organism [12−23]. Interaction of vanadium complexes with DNA has
recently attracted much attention [24−28], owing to their possible applications as new cancer
therapeutic agents and their photochemical properties, which make them potential probes of
DNA structure and conformation. Vanadium complexes with diverse biological activity viz.
antibacterial [29−31], anti-inflammatory [32], and antitumor [33−35] activity have been
3

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reported. Thus, the DNA-binding, DNA photocleavage and anti-proliferative activity of
oxidometal complexes based on vanadium and multifunctional ligands has been of recent interest
[
36−39].
On the other hand, hydrazones, –NH–N=CRR' (R and R = H, alkyl, aryl), are versatile ligands
and have applications in the fields of analytical [40] and medicinal chemistry [41−43].
Hydrazone moieties are important pharmacophoric cores of several anti-cancer, anti-
inflammatory, anti-nociceptive and anti-platelet drugs [44−51]. Moreover, the electronic effects
of hydrazone complexes are also important in the design of complexes with better DNA-binding
and cleaving characteristics [52−59].
Keeping in view biological and medicinal properties of aroylhydrazones and vanadium, we
found it vital to join both moieties in search of designing new oxidovanadium complexes that
could aggressively work against tumor cells. As a part of our studies on design and synthesis of
novel vanadium complexes with potential pharmaceutical activities [60,61], herein we have
reported three novel dioxidovanadium(V) complexes with aroylhydrazone ligand, viz. 1-
1
2
napthoyl hydrazone of 2-acetyl pyridine (HL ), 2-furoyl hydrazone of 2-acetyl pyridine (HL )
3
and isonicotinoyl hydrazone of 2-hydroxy benzaldehyde (H L ). The crystal structures of the two
2
1
2
complexes [VO L ] (1), and [VO L ] (2) have been determined by X-ray crystallography. The
2
2
interaction of these complexes with calf-thymus DNA (CT-DNA) was investigated using UV-
Vis absorption titration and thermal denaturation studies. Their photo-cleavage reactions with
pUC19 supercoiled plasmid DNA were investigated by gel electrophoresis, and the protein
binding and cleavage activity were also tested. Furthermore, the cytotoxicity of all the complexes
has been evaluated by the MTT assay.
4

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2
. Experimental
.1. Materials and methods
2
All chemicals were purchased from commercial sources and used without further purification.
VO(acac) ] (acac = 2,4-pentanedionate ion) was prepared as described in the literature [62].
[
2
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) and DAPI (4′,6-diamidino-2-
phenylindole dihydrochloride) were purchased from Sigma Aldrich (USA). Minimal essential
medium (MEM) was purchased from Gibco, India. Reagent grade solvents were dried and
distilled prior to use. The supercoiled (SC) pUC19 DNA was purified from E. coli cells with the
aid of GeneJET Plasmid Isolation Kit (Thermo Scientific, USA). CT-DNA was purchased from
SRL (India) (biochemistry grade). Agarose (molecular biology grade) and bovine serum albumin
(
BSA) was purchased from Sigma Aldrich (USA).
.2. Instrumentation
Elemental analyses were performed on a Vario ELcube CHNS Elemental analyzer and IR
2
1
13
spectra were recorded on a Perkin-Elmer Spectrum RXI spectrophotometer. H and C NMR
spectra were recorded with a Bruker Ultrashield 400 MHz spectrometer using SiMe as the
4
internal standard. Electronic spectra were recorded on a Perkin-Elmer Lamda25
spectrophotometer. Cyclic voltammograms were recorded in DMF solutions, containing 0.1 (M)
TEAP (tetraethyl ammonium perchlorate) as supporting electrolyte, using a CH1120A
potentiostat, with Pt working electrode, Pt wire as counter electrode and Ag, AgCl/saturated KCl
as reference electrode.
5

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2
2
.3. Preparation of ligands and compounds
1
−2
.3.1. Synthesis of HL
1
Schiff base ligands, 1-napthoyl hydrazone of 2-acetyl pyridine (HL ) and 2-furoyl hydrazone of
2
2
-acetyl pyridine (HL ) were prepared by the condensation of 2-acetyl pyridine and its
corresponding acid hydrazide in equimolar ratio in ethanol by a standard procedure [61]. The
resulting white compound was filtered, washed with ethanol and dried over fused CaCl2.
1
13
Elemental analysis results, NMR ( H and C) and IR data for all of these verified their
preparation.
1
HL . Yield: 77%. Anal. calcd. for C H N O: C 74.72, H 5.23, N 14.52: Found: C 74.74, H
1
8
15
3
–
1
1
5
.21, N 14.55%. IR (KBr pellet, cm ): 3163 ν(N–H); 1651 ν(C=O); 1580 ν(C=N). H NMR (400
MHz, DMSO-d ppm): δ 11.19 (s, 1H, NH), 8.63–7.44 (m, 11H, Aromatic), 2.43 (s, 3H, CH ).
6
,
3
1
3
C NMR (100 MHz, DMSO-d , ppm): δ 166.15, 155.42, 154.48, 149.64, 148.98, 137.35,
6
1
33.40, 130.87, 130.56, 128.79, 128.16, 127.51, 126.84, 126.73, 125.59, 124.77, 121.63, 13.26.
2
HL . Yield: 74%. Anal. calcd. for C H N O : C 62.87, H 4.84, N 18.33: Found: C 62.88, H
1
2
11
3
2
–
1
1
4
.87, N 18.31%. IR (KBr pellet, cm ): 3121 ν(N–H); 1683 ν(C=O); 1576 ν(C=N). H NMR (400
MHz, DMSO-d , ppm): δ 11.04 (s, 1H, NH), 8.71–6.70 (m, 7H, Aromatic), 2.51 (s, 3H, CH ).
6
3
1
3
C NMR (100 MHz, DMSO-d , ppm): δ 149.31, 146.87, 146.59, 141.13, 138.43, 128.26,
6
1
25.73, 122.73, 121.88, 112.60, 112.56, 13,72.
3
2
.3.2. Synthesis of H2L
3
Schiff base ligand, isonicotinoyl hydrazone of 2-hydroxy benzaldehyde (H L ), was prepared by
2
the condensation of 2-hydroxy benzaldehyde and isonicotinoyl hydrazide in equimolar ratio in
1
–2
ethanol by the procedure adopted similar to HL . The resulting white compound was filtered,
6

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1
13
washed with ethanol and dried over fused CaCl . Elemental analysis results, NMR ( H and C)
2
and IR data for this verified its preparation.
3
H L . Yield: 62%. Anal. calcd for C H N O : C 64.72, H 4.59, N 17.41: Found C 64.68, H
2
13 11
3
2
–
1
4
1
.58, N 17.45%. (KBr pellet, cm ): 3167 ν(O–H), 3002 ν(N–H), 1683 ν(C=O), 1612 ν(C=N),
1
567 ν(C=C/aromatic). H NMR (400 MHz, DMSO-d , ppm): δ 12.33 (s, 1H, OH), 11.14 (s, 1H,
6
13
NH), 8.69 (s, 1H, CH), 8.80–6.89 (m, 8H, C H ). C NMR (100 MHz, DMSO-d , ppm): δ
6
4
6
1
1
2
61.83, 157.96, 150.81, 150.11, 149.56, 140.40, 132.19, 129.76, 123.17, 121.98, 119.88, 119.09,
16.90.
1
−3
.3.3. Synthesis of [VO L ](1−3)
2
1
2
3
To the hot solution of ligand [HL (0.371g), HL (0.311g) and H L (0.323g)] (1 mmol) in 30 mL
2
methanol, [VO(acac) ] (0.265g, 1 mmol) in 5 mL N,N-dimethylformamide (DMF) was added.
2
The color changed instantly to brown. After 6 h of reflux, it was cooled, filtered off and kept for
crystallization. Slow evaporation of the filtrate over 4 days produced yellow X-ray quality
crystals. The crystals were filtered and washed with methanol. Elemental analysis results, NMR
1
13
51
(
H, C, V), UV-visible (UV-Vis) and IR data for all of these verified their preparation.
may also be prepared by a different synthetic method described in a recent report of
3
preliminary crystallographic communication [63].
1
[
VO L ](1). Yield: 54%. Anal. calcd. for C H N O V: C 58.23, H 3.80, N 11.32: Found: C
2
18 14
3
3
–
1
5
9
8.25, H 3.82, N, 11.29%. IR (KBr pellet, cm ): 1595 ν(C=N); 1253 ν(C–O)enolic; 1034 ν(N–N);
–
1
–1
56, 941 ν(V=O). UV-Vis (DMF) [λ max, nm (ε, M cm )]: 397 (20640); 286 (14065); 263
1
(
14986). H NMR (400 MHz, DMSO-d , ppm): δ 8.93–7.60 (m, 11H, Aromatic), 2.71 (s, 3H,
6
1
3
CH ). C NMR (100 MHz, DMSO-d , ppm): δ 178.27, 164.60, 155.04, 153.50, 143.73, 133.98,
3
6
7

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1
33.06, 130.88, 130.29, 129.21, 128.53, 127.90, 127.68, 126.76, 126.70, 126.33, 125.60, 14.35.
5
1
V NMR (DMSO-d , ppm): δ –508.
6
2
[
VO L ](2). Yield: 51%. Anal. calcd. for C H N O V: C 46.32, H 3.24, N 13.50: Found: C
2
12 10
3
4
–
1
4
6.31, H 3.25, N, 13.51%. IR (KBr pellet, cm ): 1600 ν(C=N); 1254 ν(C–O)enolic; 1044 ν(N–N);
–
1
–1
9
35 ν (V=O). UV-Vis (DMF) [λ max, nm (ε, M cm )]: 396 (13248); 297 (8019); 260 (7836).
1
13
H NMR (400 MHz, DMSO-d , ppm): δ 8.79–6.72 (m, 7H, Aromatic), 2.71 (s, 3H, CH ). C
6
3
NMR (100 MHz, DMSO-d , ppm): δ 173.34, 168.32, 159.60, 155.20, 147.22, 145.19, 145.47,
6
51
1
33.18, 127.81, 125.41, 123.21, 13.41. V NMR (DMSO-d , ppm): δ –509.
6
3
[
VO L ](3). Yield: 62%. Anal. calcd. for C H N O V: C 48.31, H 3.12, N 13.00: Found: C
2
13 10
3
4
–
1
4
8.34, H 3.11, N 13.02%. IR (KBr pellet, cm ): 1604 ν(C=N); 1257 ν(C–O)enolic; 1047 ν(N–N);
–
1
–1
9
70 ν (V=O). UV-Vis (DMF) [λ max, nm (ε, M cm )]: 410 (9875); 328 (13751); 264 (15294).
1
13
H NMR (400 MHz, DMSO-d , ppm): δ 9.01(s, 1H, CH), 8.74–6.89 (m, 8H, Aromatic). C
6
NMR (100 MHz, DMSO-d , ppm): δ 168.69, 164.35, 150.54, 150.00, 149.43, 135.25, 133.76,
6
51
1
22.68, 122.14, 120.44, 119.92, 116.90, 116.32. V NMR (DMSO-d , ppm): δ –542.
6
2
.4. X-ray diffraction studies
Suitable single crystal of 1 and 2 was chosen for X-ray diffraction studies. Crystallographic data
and details of refinement are given in Table 1. The unit cell parameters and the intensity data for
the complexes (1 and 2) were collected at 100 K, on a Bruker APEX II single crystal X-ray
diffractometer using graphite monochromated Mo K radiation (λ = 0.71073 Å). The intensity
data were corrected for Lorentz, polarization and absorption effects. The structures were solved
using the SHELXS97 [64] and refined using the SHELXL97 [65] computer programs. The non-
hydrogen atoms were refined anisotropically.
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2
2
.5. DNA binding experiments
.5.1. Absorption spectral studies
The UV-Vis titration experiments were performed with Perkin-Elmer Lamda35
spectrophotometer using a fixed concentration of metal complex (25 M) but variable CT-DNA
concentrations ranging from 0 to 70 M in 10 mM Tris–HCl buffer (pH 8.0) containing 1%
DMF. Binding of ligands to CT-DNA was also studied. For this, a fixed concentration of ligand
[
25 M in 10 mM Tris–HCl buffer (pH 8.0) containing 1% DMF] was titrated with variable
DNA concentration ranging from 0 to 350 M.
.5.2. Thermal denaturation studies
Thermal denaturation studies of CT-DNA (160 M) in the absence and presence of complexes
2
(150 M) were carried out by monitoring the absorbance at 260 nm in the temperature range of
o
o
3
0−90 C with a ramp rate of 1 C/min in 10 mM Tris-HCl buffer (pH 8.0) containing 1% DMF.
The experiments were carried out using a Chirascan CD spectropolarimeter (Applied
Photophysics, UK) in absorbance mode equipped with peltier temperature controller. The
melting temperature (T ) was determined from the derivative plot (dA /dT vs T) of the melting
m
260
profile [66].
.6. DNA cleavage experiments
2
For DNA cleavage experiments, 300 ng (SC) pUC19 DNA was used and all experiments were
carried out in 50 mM Tris-HCl buffer (pH 8.0) containing 1% DMF and 10 mM phosphate
buffer (pH 7.8) containing 1% DMF.
9

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2
.6.1. Chemical-induced DNA cleavage
For chemical nuclease studies, the reactions were performed in the dark using hydrogen peroxide
(0.5 mM) as the oxidising agent in absence and presence of complexes (1 – 500 µM). The
o
solutions were incubated at 37 C for 3 h and analysed for DNA cleaved products by agarose gel
electrophoresis.
2
.6.2. Photo-induced DNA cleavage
The photolytic DNA cleavage experiments were carried out on (SC) pUC19 DNA (300 ng)
with complexes (1 – 500 µM) in 50 mM Tris HCl buffer (pH 8.0) containing 1% DMF and in 10
mM phosphate buffer (pH 7.8) containing 1% DMF. The extent of DNA cleavage was measured
from the intensities of the bands using UVP Gel Documentation System. The observed error in
measuring the band intensities ranged between 3% − 6%. The mechanistic studies were done
using four different additives among them two are singlet oxygen quenchers (sodium azide and
L-histidine) and other two are hydroxyl radical quenchers (KI and D-mannitol) prior to the
addition of the complex. The concentration of each additive was 0.5 mM.
2
2
.7. Protein binding and cleavage experiments
.7.1. Bovine serum albumin (BSA) interaction studies
The BSA binding study was performed by tryptophan fluorescence quenching experiments using
Fluoromax 4P spectrofluorimeter (Horiba Jobin Mayer, USA). Briefly, 2 M of BSA was taken
in 10 mM Tris-HCl buffer (pH 8.0) containing 1% DMF and the quenching of the emission
intensity of tryptophan residues of BSA at 344 nm (excitation at 295 nm) was monitored with
increasing complex concentration [1 – 8 M in 10 mM Tris-HCl buffer (pH 8.0) containing 1%
DMF].
1
0

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2
.7.2. BSA photo-induced cleavage studies
Freshly prepared solution of BSA in 10 mM Tris-HCl buffer (pH 8.0) containing 1% DMF was
used for photochemical protein cleavage studies. BSA (5 M) solution in absence and presence
of complexes [1 – 500 M in 10 mM Tris-HCl buffer (pH 8.0) containing 1% DMF] was photo-
irradiated using UVA light 350 nm (Luzchem Photoreactor Model LZC-1, Ontario, Canada)
fitted with 14 UVA tubes (84 W) for a duration of 90 min. The extent of BSA cleavage was
measured from the intensities of the bands using UVP Gel Documentation System. Molecular
weight markers were used in each gel to calibrate the molecular weight of BSA. The BSA
cleavage activity of the ligands (300 M) under the same experimental conditions and the
complexes (300 M) in dark were also studied. The mechanistic studies were done using four
different additives among them two are singlet oxygen quenchers (sodium azide and L-histidine)
and other two are hydroxyl radical quenchers (KI and D-mannitol) prior to the addition of the
complex. The concentration of each additive was 0.3 mM.
2
.8. In vitro cytotoxic activity
2
.8.1. MTT assay
Human cervical cells HeLa were obtained from National Centre of Cell Science (NCCS), Pune,
India, and were maintained in minimal essential medium supplemented with 10% fetal bovine
serum, penicillin-streptomycin solution and incubated at 37 °C in 5% CO and 95% humidified
2
incubator. HeLa cells were harvested from maintenance cultures in logarithmic phase, after
counting in a hemocytometer using trypan blue solution. The cell concentration was adjusted to
4
5
7
x10 cells/ml and the cells were plated in a 96 well flat bottom culture plate and incubated for
2 h with various concentrations of the test complexes which were dissolved in a 10% (v/v)
DMF solution. The effect of the drugs on the cancer cell viability was studied using MTT dye
1
1

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reduction assay by measuring the optical density at 595 nm using micro-plate reader
spectrophotometer (Perkin-Elmer 2030) [67]. A 10% (v/v) DMF solution which was used to
dissolve the drugs was used in the control group treatment.
2
.8.2. Nuclear Staining
Nuclear staining using DAPI stain was performed according to the method previously described
68]. Briefly, HeLa cells either treated or untreated with test compounds were smeared on a clean
[
glass slide, cells were fixed with 3.7% formaldehyde for 15 min, permeabilised with 0.1% Triton
X-100 and stained with 1 μg/ml DAPI for 5 min at 37 °C. 10% DMF solution which was used to
dissolve the drugs was also used in control group treatment. The cells were then washed with
PBS and examined by fluorescence microscopy (Olympus IX 71) to ascertain any condensation
or fragmentation of the nuclei indicating cells undergoing apoptosis.
1
2

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3
. Results and discussion
.1. Synthesis
3
1
−3
Three dioxidovanadium(V) complexes [VO L ](1–3) have been prepared in the present study
2
by taking different aroylhydrazones as ligands (Scheme 1) in order to observe their influence, if
any, on the pharmacological activities of the complexes. Reactions of the selected
aroylhydrazones with VO(acac) proceed in refluxing DMF+MeOH afforded yellow or orange
2
crystalline products in good yield. Complexes (1–3) were completely soluble in DMF, DMSO
and partially soluble in CH CN and H O. All the complexes were stable in both solid and
3
2
solution phases. The solution phase stability over the period of the biological assays, i.e. 72 h, of
the complexes was confirmed by electronic absorption and NMR spectral studies. The
representative spectra are given in Figs S1 and S2.
3
3
.2. Spectral Characteristics
.2.1. IR spectroscopy
1
–2
3
The IR data of all the ligands, (HL and H L ) and their corresponding metal complexes, 1–3
2
are given in the experimental section. All the ligands posses two bands in the region of 3163–
–
1
–1
3
002 cm and 1683–1651 cm due to –NH and –C=O groups respectively are found to be
absent in the corresponding metal complexes. The disappearance of characteristic –NH and
C=O bands in the ligand spectra, and the appearance of new bands in the range 1257–1253
–
–
1
cm in the complexes indicates the enolisation of these two groups forming a –N=C–O bond
–
1
sequence. The strong and sharp peak displayed by the complexes in the range 1604–1595 cm is
likely to be associated with the –C=N–N=C– moiety [69]. The presence of two sharp bands in
−
1
−1
the range 956−941 cm for complex 1 and broad bands at 935 and 970 cm for 2 and 3
1
3

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respectively are assigned to V=O stretching, which indicates the dioxido nature of the complexes
[
60].
.2.2. Electronic spectra
The electronic spectra of the complexes 1–3 were recorded in DMF at 1.53 x 10
3
−
4
M
concentration, and are quite similar for all three complexes. Strong absorptions are observed in
the wavelength range 410–396 nm, assignable to the ligand-to-metal charge transfer transitions
whereas the other bands in the higher energy region (328–260 nm) are likely to be due to ligand
centred transitions [60]. A representative spectrum of 1 is shown in Fig. 1.
3
.2.3. NMR spectra
1
13
1–2
3
The H and C NMR data of the free ligands (HL
and H L ) and corresponding
2
dioxidovanadium(V) complexes (1–3) were recorded using DMSO-d and the data are given in
6
the Experimental Section. The spectra of all the ligands exhibit resonances in the range δ =
1
1.19–11.04 ppm due to –NH group, which disappear in the spectra of the complexes confirming
enolisation of the ligands. The absence of peaks at δ = 12.33 for complexes 3 indicates the
deprotonation of the –OH group that used to be present in its ligand fragment. 3 displays a
singlet at δ = 9.01 ppm due to –CH and 1 and 2 display singlets at δ = 2.71 ppm due to –CH3
groups. All the aromatic protons of the complexes are clearly observed in the expected region, δ
=
8.93–6.72 ppm [69].
5
1
The V NMR spectra of 1–3 displays a singlet in the range δ = –542 to –508 ppm. These
chemical shifts are normal for complexes containing dioxidovanadium(V) complexes [60].
3
.3. Electrochemical properties
The electrochemical properties of 1–3 have been examined in DMF solution (0.1 M TEAP) by
cyclic voltammetry using a Pt working electrode, Pt auxiliary electrode, and an Ag/AgCl
1
4

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reference electrode. The voltammogram pattern is similar for 1–3, which includes two reduction
processes corresponding to one electron transfer each. The potential data are listed in Table 2 and
Fig. 2 depicts a representative voltammogram of 2. In the cathodic region, complexes show two
quasi reversible single electron wave at E1/2 values within the potential window −1.16 to −1.07 V
and −1.52 to −1.49 V, which are assigned to the V(V)/V(IV) and V(IV)/V(III) reduction of the
metal respectively [70].
3
3
.4. X-ray crystallographic studies
.4.1. Molecular structures of [VO L ] (1) and [VO L ] (2)
1
2
2
2
1
Though the preliminary characterization data (microanalysis, H NMR and IR) indicated the
presence of the ligand and two oxido group in complexes 1 & 2, they could not point to any
definite stereochemistry of the complexes, as well as the coordination mode of HL in those. For
an unambiguous characterization of these complexes, structures of both have been determined by
X-ray crystallography, revealing that the complexes are isostructural cis-dioxidovanadium(V)
species; except that the naphthoyl groups in 1 has been substituted by a furoyl group in 2. Hence,
details of 2 are not discussed here. Only complex 1 is discussed here as a representative case.
Selected bond angles and bond lengths for both are given in Table 3 for comparison purposes.
The molecular structure and the atom numbering scheme of 1 and 2 are shown in Fig. 3 and
Fig. 4 respectively. In 1 (Fig. 3), the penta-coordinated vanadium atom is in an N O donor
2
3
environment, arranged in a square pyramidal geometry and is bound by a tridentate uninegative
anion, coordinating via enolate-O(1), azo-N(2) and pyridyl-N(1) atoms, and two oxido-O(2) and
1
O(3) atoms. The monobasic ligand (HL ) occupies three positions of the basal plane, forming
two five membered chelate ring O(1)−V(1)−N(2) and N(1)−V(1)−N(2) around the V(V) center
with bite angles of 73.30(4) and 72.99(4)º, respectively. The terminal V–O distances of
1
5

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1
.6269(11) and 1.6098(11) Å towards oxygen atoms O(2) and O(3) are slightly longer than the
values found in the monooxidoalkoxido vanadium(V) complexes, which contain only one
terminal oxygen atom [71]. The angle between the two terminal oxygen atoms, O(2)−V(1)−O(3)
is 109.87(6)º. Along with O(3), the basal plane is formed by the enolate oxygen O(1) and the azo
nitrogen N(2), the fourth corner of the square plane being occupied by the pyridyl nitrogen, N(1),
and the vanadium atom is displaced by 0.0322 Å from the plane towards the axial oxygen O(2).
The metal-ligand distances are in the normal range [60,72] and follow the order V=O
−
{
O(2),O(3)} < V−O {O(1)} < V−N {N(1),N(2)}.
The structure of complex 3 has recently been reported in a crystallography journal [63] which
described that the V atom in (3) is coordinated by the O,N,O- tridentate Schiff base ligand and
two oxido-O atoms. The resulting NO donor set is based on a square pyramid with one of the
4
oxido- atom in the axial position and the Schiff base ligand is present as a pyridinium cation.
3
3
.5. DNA binding studies
.5.1. Absorption spectroscopic studies
The binding propensity of the complexes 1–3 to CT-DNA was studied using different
spectroscopic techniques. The complexes 1–3 shows absorption bands in the region 440−400 nm
and 340-300 nm which are attributed to L−V(d) ligand to metal charge transfer (LMCT) and
intraligand transitions respectively [69]. UV-Vis titration experiments were carried out to
determine the equilibrium binding constant (K ) of the complexes to CT-DNA (Table 4 and Fig.
b
5
). The UV-Vis absorption bands in the region 340-300 nm which attributes mainly to
intraligand transitions, upon the addition of CT-DNA, showed a hypochromic shift for complex
and 2, whereas complex 3 exhibited hyperchromism (Fig. 5c). The extent of hypochromism
observed in complex 1 and 2 ranged between 5−7%, whereas complex 3 showed a higher
1
1
6

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hyperchromism of ~30%, which indicated all these three dioxidovanadium(V) complexes
interacted with CT-DNA. The binding affinity of the interaction between CT-DNA and each of
1
–3, the binding constant (K ) was calculated using the following equation [58].
b
ꢀ
ꢁꢂꢃ
ꢀꢁꢂꢃ
ꢌ
ꢉꢇꢄꢊꢆꢇꢄꢈ ꢋꢇ
;
ꢍ ꢎꢄ ꢆꢇꢄ ꢏ
ꢊ ꢊ ꢈ
Eq. 1
ꢄꢅꢆꢇꢄꢈ
where [DNA] is the concentration of DNA base pairs, ε ε and ε correspond to the apparent
a,
f
b
absorption coefficient coefficient for the complex for the complex i.e. Abs/[complex] in presence
of DNA, in absence of DNA and to fully bound DNA respectively. The plot of [DNA]/(ε − ε ) vs
a
f
[
DNA] gave a slope and the intercept equal to 1/(ε − ε ) and 1/K (ε − ε ), respectively. The
b f b b f
binding constant K is calculated from the ratio of the slope to the intercept. The data reported in
b
Table 5 reveal that the DNA binding constant (K ) of these three dioxidovanadium(V)
b
5
5
−1
complexes varies within 2.30 × 10 to 1.31 × 10 M with the order of DNA binding strength as
3
> 2 > 1. The DNA binding activity of similar type oxidovanadium complexes has been
reported by Banerjee et al. [73] and Sasmal et al. [74], and the present results are in accordance
with their reported results. The binding affinity of ligands to CT-DNA was also estimated. All
2
the ligands showed ~ 10 folds less DNA binding affinity than their corresponding complexes
(Fig. S3 and Table S1).
3
.5.2. Thermal denaturation studies
To investigate whether the stability of DNA alters upon interaction with the complexes, DNA
melting experiments were performed in absence and presence of complexes 1–3 [66]. The mid-
point transition temperature or melting temperature (T ) of CT-DNA in absence of any complex
m
is ~65.7º C. The T increased very slightly upon interaction with complexes 2 and 3 i.e. 1.65 and
m
1
.74º C respectively, whereas complex 1 showed a slight decrease (1.50º C) in the thermal
melting of CT-DNA (Fig. 6 and Table 4). The low ΔT values for 1−3 suggest primarily groove
m
1
7

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binding of the complexes to CT DNA rather than an intercalative mode of binding to DNA
which usually gives large positive ΔT values [73,75]. On the other hand complex 1 shows a
m
slight negative ∆T value of −1.50 (Table 4 and Fig. 6), which may be due to destabilisation of
m
the DNA double helix by this complex [76].
3
3
.6. DNA cleavage studies
.6.1. Chemical-induced DNA cleavage
The DNA cleavage activity of the complexes 1–3 (1−500 μM) was studied in the dark in
presence of hydrogen peroxide (500 μM) as an oxidizing agent using (SC) pUC19 DNA (300
ng) in 50 mM Tris-HCl buffer (pH 8.0) containing 1% DMF. The complexes do not show any
chemical-induced DNA cleavage activity.
3
.6.2. Photo-induced DNA cleavage
To investigate whether the DNA binding properties of the complexes were accompanied with
photo nuclease activity, a photo-induced DNA cleavage activity assay was performed. Photo-
induced DNA cleavage activity of 1–3 was studied using (SC) pUC19 DNA in 50 mM Tris-HCl
buffer (pH 8.0) containing 1% DMF, upon irradiation of UVA light of 350 nm in the presence
and absence of the complexes (Fig. 7). DNA cleavage was indicated by the decrease in the (SC)
pUC19 DNA (Form I) and subsequent formation of nicked circular DNA (Form II) and
linearized DNA (Form III). The percentage of net DNA cleavage by the complexes was
estimated using the following equation:
ꢛꢜꢝꢞꢇꢟꢟꢠꢡꢢꢇꢣꢛꢜꢝꢞꢇꢟꢟꢟꢠ
ꢛꢜꢝꢞꢇꢟꢟꢥꢡꢢꢇꢣꢛꢜꢝꢞꢇꢟꢟꢟꢥ
ꢐꢑꢒꢇꢓꢐꢔꢇꢕꢖꢑꢗꢘꢗꢙꢑꢇꢚ ꢉ
ꢤꢇ
Eq.2
ꢛꢜꢝꢞꢇꢟꢠꢡꢇꢛꢜꢝꢞꢇꢟꢟꢠꢡꢢꢇꢣꢛꢜꢝꢞꢇꢟꢟꢟꢠ
ꢛꢜꢝꢞꢇꢟꢥꢡꢇꢛꢜꢝꢞꢇꢟꢟꢥꢡꢢꢇꢣꢛꢜꢝꢞꢇꢟꢟꢟꢥ
The subscripts “s” and “c” refers to the sample and control respectively [77]. Then, the net DNA
cleavage by complexes 1–3 was plotted in a concentration dependent manner (Fig. 8).
Approximately 10% DNA cleavage was observed in presence of 10 M complexes 1–3 (Fig. 8,
1
8

ACCEPTED MANUSCRIPT
inset). The cleavage activity was saturated for each of the complexes at a concentration of 100
M. At this concentration, the photo nuclease activity of complexes 1, 2, and 3 was 68%, 40%
1
–2
and 49%, respectively. Moreover, we found that HL (100 M) showed very slight (~ 5−10 %)
3
photo-induced DNA cleavage activity, while H L (100 M) was not able to cleave DNA under
2
identical experimental conditions (Fig. S4). These data indicate that the extent of DNA cleavage
under UVA light increases (~30−55%) upon complexation where complex 1 has the highest
photo nuclease activity among three dioxidovanadium(V) complexes. The photo-induced DNA
cleavage experiments were also carried out in the presence of phosphate buffer. The buffer had
no effect on the photo nuclease activity of the complexes and exhibited similar activity as with
tris buffer which is in coherence with our previous studies with dioxidovanadium complexes
[
60]. Control experiments suggest that DMF (1%) alone failed to cleave DNA under similar
experimental conditions (Fig. S4).
In order to understand the mechanistic pathways involved in the photo cleavage reactions, we
systematically investigated the photo-induced DNA cleavage activity of complexes 1–3 in
presence of various additives. The DNA cleavage reaction involving molecular oxygen can
proceed in two mechanistic pathways, (1) a type-II process involving singlet oxygen species
1
(
O ) or (2) by a photo-redox pathway involving reactive hydroxyl radicals (*OH) [78]. The
2
addition of NaN (singlet oxygen quencher) inhibited the DNA cleavage activity of 1, 2 and 3 by
3
~5, 3 and 10 % respectively (Fig. 9). In contrast, the other singlet oxygen quencher (L-histidine)
slightly inhibited the photo-induced DNA cleavage activity of complexes 1–3, in the range of 2–
5
%. KI, a hydroxyl radical scavenger inhibited the photo-nuclease activity of complexes 1, 2 and
3
by 6, 5 and 14 %, respectively (Fig. 9). D-mannitol did not show any appreciable inhibition in
the photo-induced DNA cleavage activity (Fig. S5 and Fig. 9). Therefore, it is suggested that
1
9

ACCEPTED MANUSCRIPT
complex 3 exhibit photo-induced DNA cleavage activity probably via both singlet oxygen and
hydroxyl radical pathways, but for other two complexes (1 & 2), the mechanistic pathway
involved in photo-induced DNA cleavage could not be stated with certainty. The photo-induced
DNA cleavage activity of similar type oxidovanadium complexes has been reported by Sasmal et
al. [74,78], and the present results are in accordance with the reported results.
3
3
.7. Protein binding and cleavage experiments
.7.1. BSA binding studies
The interaction of the complexes with BSA was studied by examining the quenching of
tryptophan fluorescence emission spectra of BSA in presence of 1, 2 and 3. The fluorescence of
the protein is specifically caused by three amino acid residues namely tryptophan, tyrosine and
phenylalanine. BSA has two tryptophan residues in its primary sequence and exhibits tryptophan
fluorescence at an excitation of 295 nm with an emission maximum at 344 nm. The interaction
with BSA was monitored by the quenching of tryptophan fluorescence upon the addition of
complexes (Fig. 10). The intrinsic tryptophan fluorescence intensity of BSA quenched gradually
on increasing the complex concentration (Fig. 10). The Stern-Volmer quenching constant (KSV)
and Scatchard analysis were done using corrected fluorescence data taking into account the
effect of dilution. Linear fit of the data were done using the Stern-Volmer equation,
ꢛꢦ
ꢉ ꢧ ꢋꢇꢨꢩꢪꢇ ꢫꢃ
Eq.3
ꢛ
and the Scatchard equation,
ꢖꢬꢙꢇꢭ
ꢛꢦꢆꢇꢛ
ꢛ
ꢮ ꢉ ꢖꢬꢙꢇꢨꢯꢩꢂ ꢋ ꢇꢰꢇꢖꢬꢙ ꢫꢃ
Eq.4
where F and F are the emission intensity of BSA in absence and in presence of the quencher
0
(complexes) concentration [Q] respectively, gave the Stern-Volmer quenching constant (KSV),
the association binding constant (KBSA) and the number of binding sites (n) [60].
2
0

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

The quenching constants for 1, 2 and 3 are 4.04  , 4.29  and 9.21



  respectively (Table 5), which revealed that the quenching ability of the complexes are
in the order 3 > 2 > 1. Similar results have also been reported by Khan et al. for the interaction of
some oxidovanadium(V) complexes with BSA [26]. The extent of quenching of the fluorescence
intensity shows a measure of association of the complex with BSA. Small molecules bind
independently to a set of sites in a protein molecule and the equilibrium between the free and the
bound molecules is given by the Scatchard equation (Eq. 4). The association binding constant
(KBSA) as obtained from the Scatchard equation showed similar trends as that of quenching


constant i.e 3 > 2 > 1, with 3 exhibiting the highest binding constant of 9.25   and 1,


8
.03   althoughthe binding stochiometry/site (n) for the complexes on the BSA
molecule is lower than 1 (Fig S6 a–c and Table 5)
.7.2. BSA cleavage activity
3
In some recent reports it is established that the vanadium complexes can exhibit photo-induced
BSA cleavage activity [60,79,74]. In present study the photo-induced BSA cleavage activity of
dioxidovanadium(V) complexes in UVA light of 350 nm was carried out using 5 M of BSA in
1
0 mM Tris-HCl buffer (pH 8.0) containing 1% DMF and was monitored by SDS-PAGE gel
electrophoresis in the presence and absence of 1, 2 and 3 (Fig. 11a–c). All three
dioxidovanadium(V) complexes cleaved BSA more than 15% even at a concentration of 1 M in
presence of UVA light (84 W), and this photo-induced BSA cleavage activity saturated when the
complex concentration was 300 M in the reaction mixture (Fig 11d). At this concentration (300
M), the maximum BSA cleavage activity was shown by complex 3 (~72%) followed by
complex 2 and 1 which were ~57% and ~51% respectively. All three failed to cleave BSA in the
2
1

ACCEPTED MANUSCRIPT
dark. Control experiment performed with 1% DMF and the ligands suggest that the DMF and
ligands alone were cleavage inactive under similar experimental conditions (Fig S6d).
In order to explore the mechanistic pathways involved in the photo cleavage of BSA, we
systematically investigated the photo-induced BSA cleavage activity of complexes 1–3 (300 M)
in presence of various additives (Fig 12a) [74,78,79]. Complex 1 apparently did not show any
inhibition in presence of any additives while 3 showed slight inhibition of around 5 – 9 % in the
presence of various additives (Fig 12b). On the other hand, the addition of NaN and L-histidine
3
(
singlet oxygen quencher) inhibited the BSA cleavage activity of 2 by ~11 and 5 % respectively
Fig 12b). Similarly, the photo-induced BSA cleavage activity of complexes 2, in the presence of
(
KI and D-mannitol (hydroxyl radical scavengers), were inhibited by ~ 19 and 10 %, respectively
Fig 12b). Therefore, it is concluded that complex 2 exhibited photo-induced BSA cleavage
activity possibly via both singlet oxygen and hydroxyl radical pathways while for complex 1 and
(
3
3
3
the mechanistic pathways could not be determined with a certain degree of confidence.
.8. In vitro antiproliferative activity
.8.1. MTT assay
The in vitro cell cytotoxicities of complexes 1–3 have been studied on human cervical cancer
1
–2
3
cells HeLa using MTT assay. The ligands, (HL , H L ) and VO(acac) are less cytocytotoxic
2
2
giving IC values of > 100 μM, whereas 1–3, show good cytotoxicity in the range 10 – 22 μM
5
0
(Table 5). The results of DNA binding/cleavage ability of the ligands (Figs S3 and S4) have also
considered and are consistent with the observation that metal complexes can exhibit greater
pharmacological activities than the free ligands [80]. The significant increase in the cytotoxicity
of complex to that of free ligand clearly indicates that incorporation of vanadium in the ligand
moiety has a marked effect on the antiproliferative activity. A possible explanation is that by
2
2

ACCEPTED MANUSCRIPT
coordination, the polarity of the ligand and the central metal ion are reduced through the charge
equilibration, which favours permeation of the complexes through the lipid layer of the cell
membrane [81,82]. Under the same experimental conditions commonly used chemotherapeutic
drugs like cisplatin, gefitinib, gemcitabine, 5-fluorouracil and vinorelbine, are comparably
effective in HeLa cells with an IC value of 13, 20, 35, 40 and 48 μM, respectively [83].
5
0
Comparing the percentage of cell viability, all the complexes show good cytotoxicity as reflected
from their IC values, with their dose dependency illustrated in Fig. 13, which is in accordance
5
0
with the DNA binding results. Apart from targeting DNA, it is well known that vanadium
compounds exhibit anti-cancer activity via various other pathways such as targeting
mitochondria and ion-pumps etc. Prasad et al. showed that oxidovanadium(IV) complexes target
mitochondria and lead to apoptosis [84]. Ohlin and his co-workers demonstrated that
decavanadate compounds exhibit anti-cancer activity by inhibiting ion pumps [85]. Additionally,
vanadium compounds induce apoptosis via reactive oxygen species [86]. In order to find out the
exact pathway involved in the present study, further work needs to be explored. Moreover, the
variation in results of cytotoxicity of the complexes may be attributed to the effect of various
functional groups attached to the aroylhydrazone derivative. In our recent report, the
antiproliferative activity of some vanadium compounds are evaluated [60,61] using HeLa cells,
and the present results are in accordance with the reported values.
3
.8.2. Nuclear Staining Assay
To gain insight in to the pathway of cell death, DAPI staining was performed. Chromatin
condensation during the process of apoptosis (type I programmed cell death) is a characterising
marker of nuclear alteration. HeLa cells were treated with 15, 5, 5 μM of complexes 1–3,
respectively. The cells were incubated for 24 h before DAPI nuclear staining. Cells were
2
3

ACCEPTED MANUSCRIPT
examined under a fluorescent microscope fitted with a DAPI filter. Control cells (treated with
1
0% (v/v) DMF) hardly showed any sort of condensation in comparison to the treated cells, as
shown in Fig. 14. All images, taken in grayscale demonstrate the brightly condensed chromatin
bodies and the nuclear blebbings as marked by arrows in Fig. 14. Besides showing nuclear
change, the treated cells exhibited a shrinking morphology, which is another important hallmark
of apoptosis.
2
4

ACCEPTED MANUSCRIPT
4
. Conclusions
In conclusion, we report here three novel dioxidovanadium(V) complexes with aroylhydrazone
ligands, where the penta-coordinated vanadium atom is in an N O (1, 2) and NO (3) donor
2
3
4
environment, arranged in a square pyramidal geometry. The dioxidovanadium(V) complexes,
–3 showed good DNA binding propensity. Their DNA binding activities were investigated
1
using UV-Vis absorption titration and thermal denaturation studies. The experimental results
show that the complexes interact with CT-DNA possibly by a groove binding mode, with
5
-1
binding constants of ~ 10 M . All complexes show good photo-induced cleavage of (SC)
pUC19 DNA with complex 1 showing the highest photo induced DNA cleavage activity of ~
6
8%. The results from the mechanistic study, suggested that among the three complexes, the
photolytic DNA cleavage of complexes 3 proceeded possibly via both singlet oxygen and
hydroxyl radical pathways. Complexes 1–3 also exhibit moderate binding affinity in the range of
3
4
-1
1
0 −10 M towards BSA, while all the complexes show good photo-induced BSA cleavage
activity. Moreover the antiproliferative activity of all these complexes was studied, which reveal
all compounds are significantly cytotoxic towards the HeLa cell line. The results reported herein
will inspire further work on dioxidovanadium(V) complexes for the development of metal-based
agents for anti-cancer applications.
2
5

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Acknowledgments
The authors thank the reviewers for their comments and suggestions, which were helpful in
preparing the revised version of the manuscript. Funding for this research was provided by
Department of Science and Technology, India [Grant SR/WOS-A/CS-145/2011 (S.P.D), grant
SR/FT/CS-016/2008 (R.D)] and Council of Scientific and Industrial Research, India [Grant
3
7(1535)/12/EMR-II) (AB)]. A.K.P acknowledges the receipt of ICMR, India grant 45/25/2012-
Bio/BMS for providing fellowship. R. D. thanks Prof. S. K. Chattopadhyay for electrochemical
studies and fruitful discussion.
Appendix A. Supplementary material
The crystallographic data for the structural analysis of 1 and 2 have been deposited with the
Cambridge Crystallographic Data Centre, CCDC No. for 1 is 1015982, for 2 is 1015983. A copy
of this information may be obtained free of charge from the CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK (Tel.: +44 (0) 1223 762911; E-mail: deposit@ccdc.cam.ac.uk). Other
supplementary data related to this article can be found at http://dx.doi.org/10
2
6

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