Investigation of DNA interaction and antiproliferative activity of mixed ligand dioxidomolybdenum(VI) complexes incorporating ONO donor aroylhydrazone ligands

Article abstract of DOI:10.1016/j.poly.2020.114533

Four new mixed ligand dioxidomolybdenum(VI) [Mo^VIO₂L^1-3(Q)] (1–3), [Mo^VIO₂L⁴(Q)]₂ (H₂O) (4) [where Q = MeOH for 1 and imidazole for 2–4] complexes have been synthesize

Full text of DOI:10.1016/j.poly.2020.114533

Polyhedron 183 (2020) 114533
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Polyhedron
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Investigation of DNA interaction and antiproliferative activity of mixed
ligand dioxidomolybdenum(VI) complexes incorporating ONO donor
aroylhydrazone ligands
a
a
a
a
Rupam Dinda a, , Arpita Panda , Atanu Banerjee , Monalisa Mohanty , Sagarika Pasayat ,
⇑
Edward R.T. Tiekink b
a Department of Chemistry, National Institute of Technology, Rourkela, 769008 Odisha, India
b Research Centre for Crystalline Materials, School of Science and Technology, 5 Jalan Universiti, Sunway University, Bandar Sunway, Selangor Darul Ehsan 47500, Malaysia
a r t i c l e i n f o
a b s t r a c t
Four new mixed ligand dioxidomolybdenum(VI) [MoVIO2L1-3(Q)] (1–3), [MoVIO2L4(Q)]2 (H2O) (4) [where
Q = MeOH for 1 and imidazole for 2–4] complexes have been synthesized using four different ONO donor
aroylhydrazone ligands (H2L1–4). All the derived ligands and complexes have been characterised by dif-
ferent physicochemical techniques, that is, elemental analysis, spectroscopic methods (UV–Vis, NMR
and IR), and cyclic voltammetry. The molecular geometries of 1–4 were established by X-ray crystallog-
raphy which reveals - - the Schiff base ligands coordinate - the distorted octahedral metal centres in a di-
Article history:
Received 3 December 2019
Accepted 26 March 2020
Available online 30 March 2020
Keywords:
Aroylhydrazone
Mixed ligand dioxidomolybdenum(VI)
complexes
DNA binding
negative tridentate fashion. The complexes have shown moderate binding affinity (103 to 104 Mꢀ1
)
towards CT-DNA. Further, in vitro cytotoxicity activity of all the complexes were determined against
HT-29 (colon cancer) and HeLa (cervical cancer) cell lines. Complex 4, due to the presence of a hetero-
cyclic 2-hydroxy-1-naphthyl moiety in the ligand backbone, was found to be more biologically active
in comparison to the others in the series.
Antiproliferative activity
Ó 2020 Elsevier Ltd. All rights reserved.
1. Introduction
through aldol condensation [10,11]. Schiff base ligands also have
the potential to stabilize various metal ions such as manganese,
Molybdenum is one of the major trace elements found in daily
diet and is also found in three different enzymes present in humans
namely, sulfite oxidase, xanthine oxidase and aldehyde oxidase that
have functions in detoxification [1–4]. It has been reported that the
lack of the required amount of molybdenum can increase the risk of
esophageal cancer [5]. Studies showed cell-growth inhibition prop-
erties of various polyoxidomolybdates on some specific carcinoma
cells, among which Mo(II) complexes produce good cytotoxicity
results [6,7]. Studies of molybdenum complexes as anticancer
agents reveal that with increased chelation in the structure there
is a decrease in anticancer properties [8]. Molybdenum can also
be considered a better replacement for cisplatin compared to other
transition metals, due its labile nature and comparatively low cyto-
toxicity against non-cancerous cells [9].
cobalt, copper, molybdenum, ruthenium, vanadium, etc. in their
different oxidation states [12–16]. Moreover, the presence of a
rigid, aromatic backbone provides specific spectroscopic properties
that make transition metal complexes potential probes for nucleic
acids. These metal complexes form intermediates in many enzy-
matic reactions that include the reaction of enzymes with amino
or carbonyl group of the complex, such as amine oxidases (tyra-
mine, histidine) containing copper used to oxidise primary amines,
molybdenum based xanthine oxidoreductase oxidises certain puri-
nes, pterins, and aldehydes to produce reactive oxygen which is
further reduced by cytochrome C having copper as an active com-
ponent, cobalt in cobalamin is found in fatty acid and amino acid
metabolism [17–31].
Aryl hydrazones form a class of Schiff base organic scaffolds
which show a wide range of biological properties including anti-
convulsant and antituberculosis properties along with others like
antibacterial, antifungal, antiinflammatory, antinociceptive and
antiplatelet activities [32–34]. Recently, Iproniazide, a hydrazone
derived drug, has been used in the treatment of tuberculosis
[35]. Also, an oral nitrofuran antibiotic named Nifuroxazide is used
for the treatment of anti-dehydration and for the treatment of
Molybdenum complexes of Schiff base ligands have gained sig-
nificant interest owing to their low-cost and ease of preparation.
Further, ligand preparation is basically facile: reacting sterically
and electronically varied amines and aldehydes, respectively,
⇑
Corresponding author.
E-mail address: rupamdinda@nitrkl.ac.in (R. Dinda).
https://doi.org/10.1016/j.poly.2020.114533
0277-5387/Ó 2020 Elsevier Ltd. All rights reserved.

2
R. Dinda et al. / Polyhedron 183 (2020) 114533
colitis [36]. Furazolidone is a synthetic nitrofuran derivative that is
well known for its antileishmanial activity and also clinically used
as an antiprotozoal and antibacterial agent [37–41]. The advanta-
geous electronic properties of arylhydrazones also give rise to com-
plexes with more effective DNA-binding and DNA cleavage
activities [18,23,25,26,28]. Molybdenum complexes of Schiff bases
of aryl hydrazones are attractive due to their various applications
including catalysis [42], luminescent probes [43], molecular sen-
sors [44] and also display impressive biological activities [45–49]
owing to the presence of the metal centre. On the other hand, as
substituted imidazoles are found to have antiprotozoal, antifungal
and antihypertensive properties [50–52], it has been employed as a
co-ligand in the present study, in order to increase the overall bio-
logical potential of the synthesized complexes.
In the past few years, our research group has investigated the
synthesis of several transition metal complexes incorporating –
ONO and –ONS donor ligand systems in order to explore their bio-
logical activity [15,53–68]. In continuation of this work, herein we
report four novel dioxidomolybdenum(VI) complexes with –ONO
donor, aroylazine ligand systems (H2L1–4). All the complexes are
characterized by various spectroscopic methods and their struc-
tural features have been elucidated by X-ray crystallography. Fur-
thermore, the complexes have been evaluated for their DNA
interaction activity which shows a moderate binding affinity.
Finally, the in vitro antiproliferative activities of 1–4 were assayed
against the HT-29 (colon cancer) and HeLa (cervical cancer) cell
lines and the mechanism of cell death for 4 ascertained by DAPI
staining, i.e. apoptosis.
with the corresponding hydrazides (1.0 mmol) [1-naphthoic
hydrazide (H2L1), 2-furoic hydrazide (H2L2) and 2-thiophenecar-
boxylic acid hydrazide (H2L3)], while, H2L4 was obtained by the
condensation of 2-hydroxy-1-naphthaldehyde with isonicotinic
hydrazide in equimolar ratio (1.0 mmol, each) in EtOH medium
by following standard procedures [15,56,65]. The resulting com-
pounds were then filtered, washed thoroughly with ethanol and
dried over fused CaCl2 in a desiccator. Elemental analysis results
NMR (1H and 13C), UV–Vis and IR data verified their preparation.
H2L1: Yield: 62% (0.169 g). Anal. calcd. for C14H14N2O4: C, 61.31;
H, 5.14; N, 10.21. Found: C, 61.40; H, 5.36; N, 10.01. IR (KBr pellet,
cmꢀ1): 3336
m(OAH); 2998 m(NAH); 1657 m(C@O); 1556 m(C@N).
1H NMR (400 MHz, DMSO d6): d 12.13 (s, 1H,–OH), 10.81 (s, 1H,
NH), 8.65 (s, 1H, HC = N–), 7.97–6.72 (m, 6H, aromatic), 4.06 (m,
2H, –OCH2), 1.35 (t, 3H, –CH3). 13C NMR (100 MHz, DMSO d6): d
166.18 (CO–N), 159.18 (N = CH–), 152.36–113.59 (10C, aromatic),
69.33 (–OCH2), 19.95 (–CH3).
H2L2: Yield: 71% (0.237 g). Anal. calcd. for C20H18N2O3: C, 71.84;
H, 5.43; N, 8.38. Found: C, 71.78; H, 5.42; N, 8.41. IR (KBr pellet,
cmꢀ1): 3392
m(OAH); 3042 m(NAH); 1657 m(C@O); 1571 m(C@N).
1H NMR (400 MHz, DMSO d6): d 12.02 (s, 1H,–OH), 10.62 (s, 1H,
NH), 9.13 (s, 1H, HC = N–), 8.02–6.97 (m, 10H, aromatic), 4.07
(m, 2H, –OCH2), 1.35 (t, 3H, –CH3). 13C NMR (100 MHz, DMSO d6):
d 169.16 (CO–N), 158.67 (N = CH–), 154.16–118.21 (16C, aromatic),
68.31 (–OCH2), 14.56 (–CH3).
H2L3: Yield: 74% (0.214 g). Anal. calcd. for C14H14N2O3S: C,
57.92; H, 4.86; N, 9.65; S, 11.04. Found: C, 57.83; H, 4.91; N,
9.64; S, 10.99. IR (KBr pellet, cmꢀ1): 3378
m
(OAH); 2998
m(NAH);
1663
m(C@O); 1561 m
(C@N). 1H NMR (400 MHz, DMSO d6): d
12.11 (s, 1H,–OH), 10.86 (s, 1H, NH), 8.86 (s, 1H, HC = N–), 7.92–
6.98 (m, 6H, aromatic), 4.06 (m, 2H, –OCH2), 1.35 (t, 3H, –CH3).
13C NMR (100 MHz, DMSO d6): d 167.02 (CO–N), 156.23 (N = CH–),
151.52–115.23 (10C, aromatic), 66.34 (–OCH2), 16.52 (–CH3).
H2L4: Yield: 74% (0.215 g). Anal. calcd. for C17H15N3O2: C, 69.61;
H, 5.15; N, 14.33. Found: C, 69.24; H, 5.44; N, 14.41. IR (KBr pellet,
2. Experimental section
2.1. Materials and methods
The chemicals used herein were purchased from commercial
sources, while the solvents were distilled under a dry nitrogen
atmosphere according to the standard literature procedures
[69,70] prior to use. [MoO2(acac)2] was used as the metal precursor
and synthesized as per the reported procedure [70]. Elemental
analysis (CHNS) measurements were carried out in a Vario EL cube
elemental analyser instrument. FT–IR spectra were recorded by
employing a Perkin Elmer Spectrum RX I spectrophotometer. Elec-
tronic spectra were recorded on a PerkinElmer Lambda 25 spec-
trophotometer. 1H and 13C NMR spectra where measured on a
400 MHz Bruker Ultrashield spectrometer with SiMe4 as the stan-
dard. CH-Instruments (model no. CHI6003E) electrochemical anal-
yser was used to analyse the redox behaviour with Pt as the
working and auxiliary electrodes, saturated calomel electrode
(SCE) as the reference electrode and TBAP (tetrabutylammonium
perchlorate) (0.1 M) as the supporting electrolyte under a dry
nitrogen atmosphere at 298 K. Calf thymus (CT) DNA (biochemistry
grade) was procured from SRL (India), methyl green, ethidium bro-
mide, Dulbecco’s phosphate buffered saline (DPBS), Dulbecco’s
modified Eagle medium (DMEM), fetal bovine serum (FBS), trypsin
EDTA solution and antibiotic-antimitotic solution were procured
from the Himedia (India). 3-[4,5-Dimethylthiazol-2-yl]- 2,5-
diphenyltetrazolium (MTT) and 40,6-diamidino-2-phenylindole
dihydrochloride (DAPI) were purchased from Sigma-Aldrich
(India). For the DNA binding study, ultrapure water used for the
biological assay was obtained through the purification system Mil-
lipore MilliQ Academic.
cmꢀ1): 3389
m(OAH); 2988 m(NAH); 1653 m(C@O); 1569 m(C@N).
1H NMR (400 MHz, DMSO d6): d 12.53 (s, 1H,–OH), 12.41 (s, 1H,
NH), 9.49 (s, 1H, HC = N–), 8.85–7.24 (m, 10H, aromatic). 13C
NMR (100 MHz, DMSO d6): d 166.89 (CO–N), 160.41 (N = CH–),
151.52–114.91 (13C, aromatic).
2.3. Synthesis of (1)
[MoO2(acac)2] (1.0 mmol) was added to a refluxing solution of
H2L1 (1.0 mmol) taking methanol as the solvent. The reaction mix-
ture was refluxed for 4 h after which the solution turned dark-
orange. The resultant reaction mixture was filtered while hot and
kept undisturbed. After 2–3 days of slow evaporation, a fine orange
crystalline complex, [MoO2L1(Q)] (where, Q = MeOH) (1), suitable
for X-ray crystallographic measurements was obtained.
[MoO2L1(Q)] (1): Yield: 66% (0.286 g). Anal. Calcd for C15H16
MoN2O7: C, 41.68; H, 3.73; N, 6.48; Found: C, 41.66; H, 3.79; N,
6.51. IR (KBr pellet, cmꢀ1): 1584
(C@N), 945, 934 (Mo = O).
, dm3 molꢀ1 cmꢀ1): 442 (1011),
-
m
m
UV ꢀ Vis (DMSO): kmax, nm (
e
310 (9758). 1H NMR (400 MHz, DMSO d6): d (ppm): 8.89 (s, 1H,
HC = N–), 7.96–6.71 (m, 6H, aromatic), 4.14 (s, 1H, –HO (enolic)),
4.096 (m, 2H, –OCH2), 3.18 (d, 3H, –OCH3), 1.35 (t, 3H, –CH3). 13C
NMR (100 MHz, DMSO d6): d (ppm): 161.98 (CO = N), 156.32
(N = CH–), 149.78–112.98 (10C, aromatic), 64.82 (–OCH2), 49.07
(–OCH3), 15.19 (–CH3).
2.4. Synthesis of mixed-ligand complexes (2–4)
The precursor [MoO2(acac)2] was added to an refluxing
methanolic solution of H2L2-4 in a equimolar ratio (1.0 mmol, each)
and a dark-orange solution was obtained in each case after 1 h of
reflux. Then a stoichiometric amount of the co-ligand imidazole
(Q) (1.0 mmol) was added. Each solution was filtered while hot,
2.2. Synthesis of ligands (H2L1–4
)
Substituted aryl hydrazone ligands H2L1–3 were prepared by the
condensation of 3-ethoxy-2-hydroxybenzaldehyde (1.0 mmol)

R. Dinda et al. / Polyhedron 183 (2020) 114533
3
after a reflux period of ca 3 h. After slow evaporation of the filtrate
over 4 days, orange-red crystals of [MoO2L2-3(Q)] (2 and 3) and
[MoO2L4(Q)]2(H2O) (4), were obtained and washed thoroughly
with methanol and dried.
tions, based on a gaussian (1–3 [71]) or multi-scan (4 [72]) tech-
niques, were applied. The structures were solved by direct
methods [73] and refined on F2 with anisotropic displacement
parameters and C-bound H atoms in the riding model approxima-
tion [74]. For 1, the oxygen-bound H atoms were refined with a
distance restraint OAH = 0.84 0.01 Å while for 2–4, the nitro-
[MoO2L2(Q)] (2): Yield: 68% (0.389 g). Anal. Calcd for C23H22
-
MoN4O5: C, 52.28; H, 3.82; N, 10.60. Found: C, 52.17; H, 3.88; N,
10.71. IR (KBr pellet, cmꢀ1): 1567
(C@N); 922, 906 (Mo = O).
, dm3 molꢀ1 cmꢀ1): 441 (1300),
m
m
gen-bound
H
atoms were refined with
a distance restraint
UV ꢀ Vis (DMSO): kmax, nm (
e
NAH = 0.88
0.01 Å. A weighting scheme of the form w = 1/
307 (10124). 1H NMR (400 MHz, DMSO d6): d (ppm): 9.06 (s, 1H,
HC = N–), 8.83–7.02 (m, 13H, aromatic), 4.12 (m, 2H, –OCH2),
1.38 (t, 3H, –CH3). 13C NMR (100 MHz, DMSO d6): d (ppm):
170.78 (CO = N), 157.32 (N = CH–), 150.10–119.24 (19C, aromatic),
64.83 (–OCH2), 15.22 (–CH3).
[r
2(F2o) + (aP)2 + bP] where P = (Fo2 + 2F2c)/3) was introduced in each
refinement. For 2, the maximum and minimum residual electron
density peaks of 2.69 and 1.03 eÅꢀ3, respectively, were located
0.84 and 0.72 Å from the Mo atom, respectively. This is an artefact
of the data and not indicative of unresolved chemistry. For 3, owing
to poor agreement, the (1 1 3) reflection was omitted from the last
cycles of refinement. The thienyl ring was disordered over two co-
planar, anti-parallel orientations with the site occupancy factor of
the major component refining to 0.875(4). The C9, C11 and C12
positions were common to both components and were refined
anisotropically. The S1 and C10 atoms of the major component
were refined anisotropically but those for the minor component
were refined isotropically. The C9–C10 and C10–C11 bond lengths
of the minor component were constrained to be equivalent to
those for the major component. Finally, the absolute structure
was determined based on differences in Friedel pairs included in
the data set. In 4, there is half a water molecule disordered over
two positions about a centre of inversion; the OAH distances were
fixed at 0.84 Å. There is some evidence for disorder in the imida-
zole ring as seen in the shapes of the displacement ellipsoids. How-
ever, an alternate position was not discerned for this molecule. The
molecular structure diagrams were generated by ORTEP for Win-
dows [75] and the packing diagrams with DIAMOND [76]. Addi-
tional data analysis was made with PLATON [77].
[MoO2L3(Q)] (3): Yield: 64% (0.309 g). Anal. Calcd for C17H16
-
MoN4O5S: C, 42.16; H, 3.33; N, 11.57; S, 6.62. Found: C, 42.11; H,
3.39; N, 11.58; S, 6.71. IR (KBr pellet, cmꢀ1): 1557
(C@N); 928,
906
, dm3 molꢀ1
m
m
(Mo = O). UV ꢀ Vis (DMSO): kmax, nm (
e
cmꢀ1): 442 (1425), 322 (8986). 1H NMR (400 MHz, DMSO d6): d
(ppm): 8.87 (s, 1H, HC = N–), 7.86–6.99 (m, 6H, aromatic), 4.07
(m, 2H, –OCH2), 1.34 (t, 3H, –CH3). 13C NMR (100 MHz, DMSO d6):
d (ppm): 165.90 (CO = N), 155.69 (N = CH–), 150.27–119.71 (10C,
aromatic), 65.22 (–OCH2), 15.19 (–CH3).
[MoO2L4(Q)]2 (H2O) (4): Yield: 61% (0.603 g). Anal. Calcd for
C
40H32Mo2N10O9: C, 48.60; H, 3.26; N, 14.17. Found: C, 48.66; H,
3.21; N, 14.18. IR (KBr pellet, cmꢀ1): 1557
(C@N); 928, 906.
, dm3 molꢀ1 cmꢀ1): 453
m
m
(Mo = O). UV ꢀ Vis (DMSO): kmax, nm (
e
(2983), 328 (9456), 260 (10531). 1H NMR (400 MHz, DMSO d6): d
(ppm): 9.82 (s, 1H, HC = N–), 8.79–7.03 (m, 13H, aromatic). 13C
NMR (100 MHz, DMSO d6): d (ppm): 166.87 (CO = N), 161.33
(N = CH–), 154.31–112.00 (16C, aromatic).
2.5. Single crystal X-ray structure determination
Crystal data and refinement details for 1–4 are collated in
Table 1. Intensity data (100 K) for 1–3 were measured on a
Rigaku/Oxford Diffraction XtaLAB Synergy diffractometer (Dual-
2.6. In vitro cytotoxic activity
flex, AtlasS2) fitted with CuK
those for 4 were measured on an Agilent Technologies SuperNova
Dual CCD with an Atlas detector fitted with Mo K radiation
a
radiation (k = 1.54178 Å) while
2.6.1. Cell culture
HT-29 (colon cancer) and HeLa (cervical cancer) cells were
obtained from National Centre of Cell Science (NCCS), Pune, India.
The cells were maintained in DMEM supplemented with 10% FBS
a
(k = 0.71073 Å). Data reduction and empirical absorption correc-
Table 1
Crystal data and refinement details for complexes 1–4.
Complex
1
2
3
4
Formula
C
15H16MoN2O7
C23H20MoN4O5
528.37
C17H16MoN4O5S
484.34
light-brown
0.13 ꢁ 0.16 ꢁ 0.17
Orthorhombic
P212121
C40H32Mo2N10O9
988.64
red-brown
0.10 ꢁ 0.25 ꢁ 0.30
Monoclinic
P21/n
Formula weight
Crystal colour
Crystal size/mm3
Crystal system
Space group
432.24
light-brown
0.08 ꢁ 0.10 ꢁ 0.11
light-brown
0.03 ꢁ 0.04 ꢁ 0.08
Triclinic
Triclinic
ꢀ
ꢀ
P1
P1
a/Å
b/Å
c/Å
10.2487(2)
12.2450(2)
13.2350(4)
90.236(2)
97.070(2)
90.6870(10)
1648.16(7)
4
7.98370(10)
9.8896(2)
13.9897(2)
91.2290(10)
90.0870(10)
104.721(2)
1068.03(3)
2
8.09320(10)
12.0195(2)
19.6535(2)
90
90
90
1911.82(4)
4
1.683
976
6.972
9.4568(2)
13.0259(2)
15.9707(3)
90
106.320(2)
90
1888.05(6)
2
1.739
a/°
b/°
c
/°
V/Å3
Z
Dc/g cmꢀ3
F(0 0 0)
1.742
872
6.901
1.643
536
5.410
996
0.738
m (MoK
a
)/mmꢀ1
Measured data
h range/°
Unique data
39,123
3.4–67.1
5893
25,342
3.2–67.1
3817
13,028
4.3–67.1
3399
9628
2.3–29.3
4403
Observed data (I ꢂ 2.0
No. parameters
R, obs. data; all data
a; b in weighting scheme
Rw, obs. data; all data
r
(I)) 5652
3707
461
0.022; 0.059
0.039; 1.022
0.023; 0.600
3386
302
0.037; 0.096
0.052; 2.843
0.038; 0.097
3665
266
0.019; 0.047
0.030; 0.698
0.019; 0.047
283
0.031; 0.074
0.037; 2.383
0.043; 0.083
Range of residual electron
density peaks/eÅꢀ3
ꢀ0.74–0.42
ꢀ1.03–2.69
ꢀ0.65–0.26
ꢀ0.57–0.63

4
R. Dinda et al. / Polyhedron 183 (2020) 114533
and penicillin–streptomycin solution at 37 °C in a 95% humidified
and 5% CO2 incubator. Working concentrations of 5, 10, 50 and
597 nm (excitation 520 nm) with an increasing amount of the
complex concentration (0–100 M) in a Fluoromax 4P spectro-
l
100
l
g/ml were prepared in culture medium from a stock concen-
fuorimeter (Horiba Jobin Mayer, USA).
tration of 3 mg/ml of each complex. Also, the concentration of
DMSO was maintained at less than 0.1% (v/v) in all experiments
to avoid any toxicity due to DMSO.
3. Results and discussion
3.1. Synthesis
2.6.2. MTT assay
For the MTT assay, both HT-29 and HeLa cells were harvested
from their maintenance cultures in logarithmic phase. Following
cell counting in a hemocytometer, cells were seeded into a 96 well
plate, at a concentration of 1 ꢁ 104 cells per well. These cells were
then incubated with various concentrations of the test compounds
for 48 h. The result of the treatment of the complexes on viability
of the cancer cells was studied using MTT dye reduction assay by
measuring the optical density at 595 nm using micro-plate reader
spectrophotometer (Perkin-Elmer 2030) [69].
Here, four new mixed ligand dioxidomolybdenum [MoVIO2L1-3(Q)]
(1–3), [MoVIO2L4(Q)]2 (H2O) (4) [where Q = MeOH for 1 and
imidazole for 2–4] complexes were synthesized from four differ-
ently substituted ONO donor aroylhydrazone ligands, H2L1–4, as
indicated in Scheme 1. The present study was undertaken as it
would be interesting to observe the biological activity of molybde-
num complexes with different substituents. The complexes were
successfully characterized by UV ꢀ vis, IR, NMR, cyclic voltamme-
try and the molecular and crystal structures were determined by
X-ray crystallography. The aqueous phase stability of the com-
plexes has been investigated by time-dependent UV–vis spec-
2.6.3. DAPI staining
Nuclear morphology of the cancer cells in response to treatment
with the complexes was studied through DAPI staining by follow-
ing a reported procedure [59]. Treated and untreated cells were
fixed with 4% formaldehyde for 15 min followed by staining with
troscopy for 72
depicted in Fig. SI 1.
h and a representative spectrum, for 1, is
3.2. Spectral characteristics
1 lg/ml DAPI for 5 min at 37 °C. The cells were then washed with
PBS and finally examined by fluorescence microscopy (Olympus IX
71) to determine if any nuclear condensation or fragmentation had
occurred, indicating the cells underwent apoptosis.
Detailed spectral (IR, UV–Vis and NMR) data of H2L1ꢀ4 and their
respective corresponding complexes (1–4) are given in the Experi-
mental section. Infrared spectra of the ligands display three broad
band in the ranges 3392–3336, 3042–2988 and 1663–1653 cmꢀ1
2.7. DNA binding experiments
which are attributed to
m
(OAH) stretching of hydroxy group of 2-
(C@O) stretching,
hydroxy aldehyde, (NAH) stretching and
m
m
2.7.1. Absorption spectral studies
Interaction of the dioxidomolybdenum(VI) complexes with CT-
DNA was studied through UV–Vis absorption titration experi-
respectively. Disappearance of characteristic bands of OAH, NAH
and C@O in the complex spectra indicates the coordination of the
ligands to the metal centre in their phenolate form after enoliza-
tion [68,80]. Also, the presence of two new stretching bands in
the range 928–906 cmꢀ1 in the complex spectra indicate the diox-
ido nature (Mo = O) of the complexes.
Electronic spectra of 1–4 were recorded in DMSO. The spectra of
all the complexes were similar in nature and as a representative
UV–Vis spectrum of 4 is depicted in Fig. 1. Strong absorptions
observed in the range 453–442 nm are assignable to ligand-to-
metal charge transfer (LMCT) transitions, the presence of this extra
peak as compared to that of the ligand suggest coordination of the
ligand upon complexation. Whereas bands in the higher energy
region 328–260 nm are likely to be due to ligand centred transi-
tions [66,68].
ments. A fixed concentration of the metal complex (25
titrated against variable concentration of CT-DNA (0 to
110 M) in 50 mM Tris-HCl buffer (pH 8.0). Readings were
recorded after each addition of CT-DNA (10 M) to the metal. Data
lM) was
a
l
l
were then calculated using the following equation (Eqn 1) to
obtain the binding constant Kb [28,57,64].
½DNAꢃ
½DNAꢃ
1
¼
þ
ð1Þ
ea
ꢀ
ef eb
ꢀ
ef Kbðeb
ꢀ
ef
Þ
Where [DNA] is represents the concentration of CT–DNA base pairs
whereas the parameters ea ef and eb correspond to the apparent
,
extinction co-efficient for the complexes, i.e. Abs/[complex] in the
presence and absence of CT–DNA and to fully bound DNA, respec-
The 1H and 13C NMR data of H2L1–4 and their corresponding
complexes (1–4) were recorded in DMSO d6. The ligand spectra
exhibit two singlets in the ranges d = 12.53–12.02 and 12.41–
10.62 ppm due to the presence of phenolic –OH and NH protons,
respectively. The disappearance of these two resonances in the
spectra of the complexes confirms their deprotonation and coordi-
nation to the central metal atom. The presence of a singlet reso-
nance for the ligands and complexes in the region d = 9.82–
8.65 ppm is attributed to the azomethine –CH proton. One quartet
in the range d = 4.12–4.06 ppm and a triplet in the range d = 1.38–
1.34 ppm is observed for H2L1-3 and their complexes 1–3 due to the
presence of the ethoxy group in the ligand moiety. On the other
hand, due to presence of a coordinated MeOH molecule, complex
1 exhibits an OH (enolic) resonance at d = 4.14 ppm and a –OCH3
resonance at d = 4.09 ppm. Moreover, the increased number of res-
onances in the aromatic region for 2–4 as compared to H2L2-4 indi-
cates the binding of co-ligand imidazole to the metal atom. 1H
NMR spectra of all the complexes (1–4) are given in Fig. SI 2–5.
In the 13C NMR spectra of 1–4, signals for the aromatic
carbons are found in the downfield region in the range
tively. A plot of [DNA]/(ea
and the intercept equal to (1/Kb)(eb
to the intercept gives the value of binding constant Kb. Binding
ꢀ
e
f) vs [DNA] gave a slope of 1/(ea
ꢀ
ef)
ꢀ
e
f). Thus, the ratio of the slope
affinity of the ligands towards CT-DNA was studied in a similar
fashion by taking a fixed concentration of ligand [25
50 mM Tris-HCl buffer (pH 8.0)] against variable CT-DNA concen-
trations ranging from 0 to 100 M.
lM in
l
2.8. Competitive DNA binding by fluorescence measurements
To understand the exact mode of binding of the complexes with
CT-DNA, competitive DNA binding experiments with three fluores-
cent dyes namely 40,6-diamidino-2-phenylindole (DAPI), methyl
green (MG) and ethidium bromide (EB) was carried out. Among
these fluorescent dyes EB tends to bind DNA through intercalation
whereas DAPI and MG bind to the minor and major grooves,
respectively [57,78,79]. This was studied by measuring the fluores-
cence emission intensities of DAPI, MG and EB bound CT-DNA at
455 nm (excitation 358 nm), 663 nm (excitation 600 nm) and

R. Dinda et al. / Polyhedron 183 (2020) 114533
5
Scheme 1. Schematic pathway for the formation of [MoO2L1-3(Q)] (1–3), [MoO2L4(Q)]2 (H2O) (4).
irreversible single electron reductive response is observed in the
cathodic region at a potential window ꢀ0.74 to ꢀ0.69 V that can
be attributed to the Mo(VI) ? Mo(V) reduction [59,65,67,81]. Also,
a single electron irreversible cathodic response in the region ꢀ1.04
to ꢀ0.95 V and anodic response at a potential window 0.44 to
0.12 V are observed that may be attributed to the ligand centered
reduction and oxidation, respectively. The cyclic voltammetry
experiments of the ligands have also been performed and a repre-
sentative voltammogram of H2L1 is shown in Fig. SI 6. The redox
potential data of all the complexes are listed in Table 2 and the sin-
gle electron processes were also confirmed by comparing the cur-
rent height to the standard ferrocene–ferrocenium couple under
identical experimental conditions.
3.4. Molecular structures
The molecular structures of 1–4 have been established by X-
crystallography and selected geometric parameters are collated
in Table 3. The crystallographic asymmetric unit of 1 comprises
two independent molecules, the first of which is shown in Fig. 3
(a) and the second in Fig. SI 7(a); the molecules have very similar
conformations as seen in the overlay diagram of Fig. SI 7(b). The
molecules of 1 comprise a Mo(=O)2 core complexed by a di-nega-
tive, tridentate Schiff base ligand, via the phenoxide-O1, amide-
O4 and imine-N1 atoms, with the sixth site occupied by an O-
bound methanol molecule. The oxo groups are cis, the phenox-
ide-O1 and amide-O4 atoms trans, the oxido-O2 atom, occupying
a position in the approximate plane of the tridentate ligand, is
trans to the imine-N1 atom, and the oxido-O3 atom is trans to
the methanol-O6 atom. The resulting NO5 donor set is based on
an octahedron. The mode of coordination of the tridentate ligand
gives rise to five- and six-membered chelate rings and it is the
Fig. 1. UV–vis spectra of [MoO2L4(Q)]2ꢄH2O (4) in DMSO employing the complex
concentration 1.3 ꢁ 10ꢀ4 M.
d = 170.78–112.98 ppm. Signals for the aliphatic carbons of –OCH2
and –CH3 of 1–3 appear in the range d = 65.22–15.19 ppm.
Whereas complex 1 exhibits an additional aliphatic carbon signal
at d = 49.07 due to the presence of methanolic –OCH3 carbon.
3.3. Electrochemical properties
Electrochemical properties of the complexes have been studied
by cyclic voltammetry in DMSO solution. Voltammograms of all
the complexes show a similar type of redox property and represen-
tative voltammograms for 1 and 2 are depicted in Fig. 2. An

6
R. Dinda et al. / Polyhedron 183 (2020) 114533
Fig. 3. Molecular structure of the first independent molecule comprising the
asymmetric unit of 1, showing the atom labelling scheme and anisotropic
displacement parameters at the 70% probability level.
The Mo-O1(phenoxide) bond length of 1.9170(14) Å is consider-
ably shorter than the Mo-O4(amide) bond length of 2.0299(14) Å,
an observation correlated with delocalisation of
over the amide chromophore.
p-electron density
As mentioned above, the five-membered, i.e. Mo,O4,N1,N2,C8,
and six-membered, i.e. Mo,O1,C1,C6,C7,N1, chelate rings are
formed upon complexation of the di-anion. The five-membered
ring is practically planar, exhibiting a r.m.s. deviation for the five
atoms of 0.0087 Å. By contrast, the best description for the six-
membered ring is that of an envelope whereupon the Mo1 atom
lies 0.504(2) Å out of the plane of the five remaining atoms, which
exhibit a r.m.s. deviation of 0.0435 Å. The dihedral angle between
the least-squares planes through the chelate rings is 7.82(9)°, indi-
cating the backbone of the tridentate ligand is approximately pla-
nar. The appended furanyl ring makes a dihedral angle of 6.11(13)°
with the five-membered chelate ring, again indicating a co-planar
arrangement; the dihedral angle between the outer rings is 5.26
(14)°.
The molecular structures of 2 and 3 are closely related to that of
1 in that the methanol molecule has been replaced by an imidazole
in each case, and with the furanyl substituent in 1 replaced by
naphthyl (2) and thienyl (3), leading to cis-N2O4 distorted octahe-
dral geometries. In 4, imidazole is still present but both peripheral
aromatic systems differ, being based on naphthyl and 4-pyridyl; 4
was isolated as a mono-hydrate. The molecular structures of 2–4
are shown in Fig. 4(a)-(c) and selected geometric parameters are
Fig. 2. Cyclic voltammogram of [MoO2L1(Q)] (1) (a) and [MoO2L2(Q)] (2) (b) in
DMSO.
Table 2
Cyclic voltammetric resultsa for 1–4 at 298 K in DMSO.
Complex
EcP (V)
Ea (V)
P
1
2
3
4
ꢀ0.74, ꢀ0.99
ꢀ0.69, ꢀ1.04
ꢀ0.71, ꢀ1.04
ꢀ0.70, ꢀ0.95
0.12
0.39
0.21
0.44
a
In DMSO at a scan rate 100 mV sꢀ1. Where EPc and EaP are cathodic and anodic
peak potentials vs. SCE, respectively.
acute bite angles subtended by these (Table 3) that are responsible
for the major distortions from the ideal octahedral geometry.
Table 3
Selected geometric parameters (Å, °) for complexes 1–4.
Complex
1
1a
2
3
4
(Y = O6)
(Y = O6)
(Y = N3)
(Y = N3)
(Y = N3)
Parameter
Mo-O1
Mo-O2
Mo-O3
Mo-O4
Mo-N1
Mo-Y
O1-C1
O4-C
N1-N2
N1-C
N2-C
O1-Mo-O4
O1-Mo-N1
O2-Mo-O3
O2-Mo-N1
O3-Mo-Y
O4-Mo-N1
1.9170(14)
1.7083(13)
1.6956(15)
2.0299(14)
2.2457(16)
2.3207(14)
1.351(2)
1.312(2)
1.395(2)
1.295(3)
1.309(3)
150.17(6)
81.27(6)
106.63(7)
157.75(6)
169.24(6)
72.09(6)
1.9242(14)
1.7068(13)
1.6966(15)
2.0351(14)
2.2423(16)
2.3221(14)
1.347(2)
1.314(2)
1.391(2)
1.296(3)
1.308(3)
149.39(6)
80.85(6)
105.78(7)
157.86(6)
171.72(6)
71.49(5)
1.947(2)
1.714(3)
1.701(3)
2.010(2)
2.231(3)
2.347(3)
1.357(4)
1.327(4)
1.403(4)
1.275(5)
1.293(5)
149.59(10)
81.66(10)
105.94(13)
161.21(12)
169.43(12)
71.77(10)
1.947(2)
1.709(2)
1.698(2)
2.016(2)
2.248(2)
2.330(2)
1.353(4)
1.322(4)
1.393(3)
1.296(4)
1.307(4)
149.33(8)
81.61(9)
104.69(10)
163.61(9)
170.54(10)
71.93(9)
1.9367(17)
1.7064(18)
1.7040(19)
2.0169(19)
2.228(2)
2.336(2)
1.343(3)
1.320(3)
1.403(3)
1.291(3)
1.297(3)
148.77(8)
80.41(7)
106.42(10)
161.96(9)
168.97(9)
72.25(7)
a
The parameters for the second independent molecule in 1 follow the numbering scheme shown in Fig. 3(a).

R. Dinda et al. / Polyhedron 183 (2020) 114533
7
3.5. Molecular packing
The geometric parameters associated with the specified inter-
molecular interactions discussed in the following are collated in
the respective captions of Fig. 5 and SI 8–11. In the molecular pack-
ing of 1, each independent molecule self-associates into a dimeric
aggregate via hydroxy-O–H. . .N(imine) hydrogen bonds as shown
in Fig. 5(a) and Fig. SI 8((a) for the first and second independent
molecules, respectively. The supramolecular dimers are assembled
into a layer in the ab-plane via furanyl- and imine-C–H. . .O(oxido)
interactions occurring between Mo1- and Mo2-containing mole-
cules, Fig. SI 8(b). The layers inter-digitate along the c-axis with
the closest atom-to-atom contact being a weak methyl-C–H. . .
(oxido) contact, Fig. SI 8(c).
O
Centrosymmetric supramolecular dimers are also found in the
crystal of 2. Here, the imidazole-N–H atom is bifurcated forming
hydrogen bonds with the phenoxide-O1 and ethoxy-O5 atoms to
form a five-membered {. . .H. . .OC2O} synthon, Fig. 5(b). Each com-
ponent of the dimer is connected into a layer in the ab-plane by
naphthyl- and imidazole-C–H. . .O(oxido) and methylene-C–H. . .
O
(amide) interactions resulting in a double-layer, Fig. SI 9(a).
Alternatively, the packing can be described as comprising
supramolecular layers sustained by the aforementioned CAH. . .
O
interactions, see Fig. SI 9(b), which are clipped into a double-
layer by the NAH. . .O hydrogen bonds. The double-layers stack
along the c-axis direction without directional interactions
between them.
In the molecular packing of 3, analogous imidazole-N–H. . .
O
-
(phenoxide, ethoxy) hydrogen bonding and five-membered {. . .H. . .
OC2O} synthons are formed as seen for 2 but, in this case, these
extend to form a one-dimensional supramolecular chain as shown
in Fig. 5(c). The chain is orientated along the a-axis and has a heli-
cal topology, being propagated by screw (21) symmetry; additional
stability to the chain is provided by imidazole-C–H. . .O(oxido)
interactions. Chains are connected laterally to form a supramolec-
ular layer in the ab-plane via phenyl-C–H. . .O(oxido) contacts, see
Fig. SI 10(a). The most prominent points of contact between layers
along the c-axis direction are S. . .O secondary bonding interactions
(S1. . .O2i = 3.068(2) Å for (i): 1-x, ½+y, ½-z). Such interactions are
well-known but, are gaining increasing recognition for their impor-
tance in stabilising molecular packing [82–84]; a view of the unit
cell contents is shown in Fig. SI 10(b).
The inclusion of a water molecule, albeit statistically disordered
about a centre of inversion, provides the cohesion to the molecular
packing in the crystal of 4. The imidazole-N–H forms hydrogen
bonds to each of the disordered water-O atoms, with the water-
O–H donor atoms forming hydrogen bonds to oxido-O and pyri-
dyl-N atoms. In this way, six molecules of 4 are assembled about
the water molecule. As there are peripheral oxido, imidazole and
pyridyl residues in the seven-molecule aggregate, a three-dimen-
sional architecture is generated as illustrated in Fig. SI 11.
Fig. 4. Molecular structures of (a) 2, (b) 3 (minor component of the disordered
thienyl ring is omitted) and (c) 4 (water molecule omitted) showing atom labelling
schemes and displacement parameters at the 50, 70 and 50% probability levels,
respectively.
3.6. In vitro cytotoxic activity
given in Table 3. The molecular geometry and trends in geometric
parameters for 2–4 match closely those established for 1. The nota-
ble exception is the equivalence of the Mo-O(oxido) bond lengths
in 4. The conformations of the chelate rings also match those seen
in 1; geometric data are given in Table SI 1. Across the series, the
five-membered rings are most planar in the structures of 2 and
3, and in terms of the deviations of the Mo atoms in the envelope
conformations for the six-membered rings, these are intermediate
to those seen in the independent molecules of 1. In the same way,
the dihedral angles between the chelate rings in each of 2–4 are
intermediate to those seen in 1. The dihedral angles between the
outer aromatic rings is greater than those in 1 in the case of 2,
i.e. 14.77(11)°, or smaller, in 3 (2.13(17)°) and 4 (4.77(19)°).
3.6.1. MTT assay
In order to understand the in vitro cytotoxic potential of the
synthesized complexes, an MTT assay was performed against two
cancer cell lines HT-29 and HeLa after 48 h of incubation. As the
test complexes were dissolved in DMSO, DMSO was taken as a con-
trol experiment so as to identify the role of solvent in cytotoxicity
of the complexes. The results obtained were analyzed through a %
cell viability profile and expressed in terms of their IC50 values, as
depicted in Fig. 6 and Table 4. The aroylhydrazone ligands gave
high IC50 values of > 300
to be the most active against both the cell lines. It was capable of
killing almost 50% of the cell population even at a low concentra-
lM. Among all the complexes, 4 proved

8
R. Dinda et al. / Polyhedron 183 (2020) 114533
Fig. 5. Supramolecular aggregates in the crystals of 1–4 sustained by hydrogen bonding (shown as orange dashed lines): (a) dimer in 1 stabilised by hydroxy-O–H. . .N(imine)
hydrogen bonds [O6–H6o. . .N2i: H6o. . .N2i = 1.907(19) Å, O6. . .N2i = 2.732(2) Å with angle at H6o = 168(2)° for symmetry operation (i) 2-x, 2-y, 1-z], (b) dimer in 2 mediated by
imidazole-N–H. . .O(phenoxide, ethoxy) hydrogen bonds [N4–H4n. . .O1ii: H4n. . .O1ii = 2.28(3) Å, N4. . .O1ii = 2.953(4) Å with angle at H4n = 133(4)°; N4–H4n. . .O5ii:
H4n. . .O5ii = 2.26(3) Å, N4. . .O5ii = 2.983(4) Å with angle at H4n = 139(4)° for (ii) 1-x, 1-y, 1-z], (c) helical chain in 3 sustained by imidazole-N–H. . .O(phenoxide, ethoxy)
hydrogen bonds [N4–H4n. . .O1iii: H4n. . .O1iii = 2.19(3) Å, N4. . .O1iii = 2.947(3) Å with angle at H4n = 144(3)°; N4–H4n. . .O5iii: H4n. . .O5iii = 2.23(3) Å, N4. . .O5iii = 2.927(3) Å with
angle at H4n = 136(3)° for (iii) -½+x, ½-y, 1-z] and (d) detailed view and seven-molecule aggregate in 4 sustained by imidazole-N–H. . .O(water), water-O–H. . .O(oxido) and
water-O–H. . .N(pyridyl) hydrogen bonds [O1w–H1w. . .O3iv: H1w. . .O3iv = 2.30 Å, O1w. . .O3iv = 3.028(5) Å with angle at H1w = 145°;O1w–H2w. . .N5v: H2w. . .N5v = 1.91 Å,
O1w. . .N5v = 2.708(5) Å with angle at H2w = 158°; N4–H4n. . .O1w: H4n. . .O1w = 1.93(2) Å, N4. . .O1w = 2.795(7) Å with angle at H2w = 170(4)°; N4–H4n. . .O1wvi
:
H4n. . .O1wvi = 2.16(3) Å, N4. . .O1wvi = 3.016(7) Å with angle at H4n = 166(5)° for (iv) ½-x, -½+y, ½-z, (v) ½+x, ½-y, -½+z and (vi) -x, -y, -z]; the latter are shown as blue dashed
lines. For reasons of clarity, non-participating hydrogen atoms are removed. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
Fig. 6. Cell viability profile of 1-against HT-29 and HeLa after 48 h of incubation. Data are reported as the mean SD for n = 4 and *p < 0.05 statistical differences between
treatment of complexes 1–4.
tion of 5
potent.
lg/ml. While the rest of the complexes proved to be less
Table 4
IC50 values of 1–4.
Lately, remarkable cytotoxic activities have been reported for
molybdenum complexes against various human cancer cell lines
Complexes
HT-29
HeLa
such as HT-29 (IC50 values 2.62–221.81
l
M), HeLa (IC50 values
1
2
3
4
177.92 5.41
150.89 2.65
96.26 0.80
20.63 0.71
84.63 3.26
162.55 1.54
60.64 3.81
4.41 0.98
12ꢀ>300
l
M) and MCF-7 (IC50 values 8.9–294.6 l
M) [85-89].
Cytotoxicity of 4 was found to be comparable and even better than
those of some clinical drugs and various previous reports of molyb-

R. Dinda et al. / Polyhedron 183 (2020) 114533
9
Fig. 7. Morphology of HeLa and HT-29 control and cells treated with complex 4 for 24 h. The cells were stained with DAPI and visualized under fluorescent microscope. Scale
bar corresponds to 20 m.
l
denum complexes as cytotoxic agents [90-93]. After the cytotoxic-
ity result it was observed that among the four complexes, mixed
ligand complexes 2–4 showed better biological activity than that
of complex 1, with a coordinated methanol molecule. While com-
plex 4 showed the highest cytotoxicity among the three mixed
ligand complexes, probably due to the presence of heterocyclic
2-hydroxy-1-naphthyl functional group attached to the ligand
backbone in comparison to 2 and 3 [17,23,25].
3.6.2. DAPI staining
As complex 4 proved to be the most potent among the series, it
was logical to study its effect on the nuclear morphology of both
cancer cell lines. This was studied through DAPI staining. Cells
were treated at a concentration of 50
lg/ml of 4 for a period of
24 h, after which they were stained with DAPI and examined under
a fluorescence microscope. From the images (Fig. 7) it could be
deduced that there were condensed chromatin bodies, nuclear
blebbings and shrunken morphology in the cells treated with 4,
whereas there were hardly any condensation observed in the con-
trol image. These phenomena are indicative of apoptosis taking
place as the mechanism of cell death in response to treatment with
4.
Fig. 8. Electronic absorption spectra of [MoO2L4(Q)]2ꢄH2O (4) (25
lM) upon the
lM) in 50 mM Tris-HCl buffer (pH 8.0). The arrow
titration of CT-DNA (0–110
indicates the changes in absorbance intensity with respect to an increase in the
concentration of CT-DNA. The inset shows the linear fit of [DNA]/(ea f) vs. [DNA].
ꢀ
e
mic shifts may be due to the interaction between the electronic
states of ligand chromophores and the DNA bases [96-98]. The
hypochromism or hyperchromism observed in these cases were
used to estimate the binding strength of the complexes to the
CT-DNA. For all complexes, the 260–290 nm range showed a signif-
icant hypochromism which may be due to the higher binding affin-
ity towards CT-DNA in the lower wavelength region. The
equilibrium binding constant (Kb) between CT-DNA and each of
the complexes 1–4 was calculated using Eqn 1 (given above).
The Kb values are shown in Table 5 and revealed that 4 has the
highest binding affinity of 3.57 ꢁ 104 Mꢀ1 and 1 has the lowest
binding affinity of 9.91 ꢁ 103 Mꢀ1. All complexes display moderate
binding affinity, and the DNA binding strength of the complexes
are in the order of 4 > 3 > 2 > 1 (Table 5). There were no significant
interactions observed for the ligands towards CT-DNA.
3.7. DNA binding studies
3.7.1. Absorption spectroscopic studies
Applying spectral techniques, the DNA binding affinity of the
complexes 1–4 to CT-DNA was studied and the equilibrium bind-
ing constant (Kb) of the complexes to CT-DNA was established
(Table 5 and Fig. 8). Complexes 1–4 exhibit absorption bands in
the regions 450–400, 340–300 that are attributed to L–Mo(dp)
and LMCT transitions while bands at 260–290 nm are due to
intraligand transitions [67]. On adding CT-DNA, complex 1 showed
a hyperchromic shift in the L–Mo(dp) LMCT region whereas hypo-
chromic shifts were observed in both the 340–300 and 260–
290 nm ranges; Fig. SI 12(a). For 2–4, hypochromic shifts were
found in all three ranges; Fig. 6 and Fig. SI 12(b)–(c). Both the
hyperchromic and hypochromic shifts indicate the interaction of
the CT-DNA with the dioxidomolybdenum complexes. The hyper
chromic shift observed may be either due to electrostatic binding
of the complexes to the DNA i.e. the external contact or to the
major and minor grooves of DNA [94,95]. The observed hypochro-
3.7.2. Competitive binding studies
In an attempt to determine the exact mode of binding of the
complexes to CT-DNA, competitive binding experiments were per-
formed with three fluorescent dyes namely 40,6-diamidino-2-
phenylindole (DAPI), methyl green (MG) and ethidium bromide
(EB). Among the three, EB tends to bind DNA through an intercala-
tion mode whereas DAPI and MG bind the minor and major
grooves, respectively [62]. Titration of EB with increasing concen-
tration of complex show the quenching of emission intensity of
EB bound to CT-DNA at 596 nm with a bathochromic shift. Thus,
the indicated displacement of CT-DNA bound EB by 1–4 shows
the binding through intercalation mode; Fig. 9 and Fig. SI 13–14.
Table 5
CT-DNA Binding parameters for complexes 1–4.
Complex
Binding Constant (Kb) (Mꢀ1
)
1
2
3
4
9.91 ꢁ 103
1.49 ꢁ 104
2.61 ꢁ 104
3.57 ꢁ 104

10
R. Dinda et al. / Polyhedron 183 (2020) 114533
Fig. 9. (a) Fluorescence quenching of EB (10 lM) upon the titration of complex 4 (0–100 lM). The arrows indicate the decrease in fluorescence intensity with respect to an
increase in the complex concentration; the inset shows the Stern-Volmer plot of the respective complexes and (b) is the Scatchard plot of 4.
plexes. Also, the result of an antiproliferative study revealed 4 to
Table 6
Competitive Binding binding parameters of EB with complexes 1–4.
be the most cytotoxic towards both HT-29 and HeLa cancer cell
lines among the series. The presence of imidazole as a co-ligand
along with a heterocyclic 2-hydroxy-1-naphthyl moiety in the
ligand backbone of 4 may be responsible for a better interaction
with DNA as well as cytotoxicity in comparison to the other com-
plexes. The outcome of the present study will definitely boost fur-
ther research on the design of dioxidomolybdenum complexes as
metal-based agents for anticancer studies.
Complex
KSV (Mꢀ1
)
kq (Mꢀ1 sꢀ1
)
Ka (Mꢀ1
)
n
1
2
3
4
3.86 ꢁ 103
5.04 ꢁ 103
5.29 ꢁ 103
7.84 ꢁ 103
6.25 ꢁ 1011
8.16 ꢁ 1011
8.58 ꢁ 1011
1.27 ꢁ 1012
4.65 ꢁ 103
2.95 ꢁ 105
8.98 ꢁ 104
7.81 ꢁ 104
1.02
1.42
1.29
1.24
The bimolecular rate constant (Kq) of EB quenching was deter-
mined by following the Stern–Volmer equation and was found to
be in the range 6.25 ꢁ 1011–1.27 ꢁ 1012 Mꢀ1 sꢀ1 (Table 6). Further,
on titration of DAPI bound CT-DNA with increasing concentration
of the complexes, 1 and 3 showed the quenching of emission inten-
sity at 451 nm with a visible hypsochromic shift, whereas 2 shows
a bathochromic shift at the same range in the emission maxima
(Fig. SI 15). From the data it also shows that except for 4, 1–3 bind
to CT-DNA through minor groove binding mode. All complexes
exhibited the quenching of ~53% for 1 followed by ~56% and
~62% for 2 and 3, respectively at the fluorescence intensity of
451 nm. On the other hand, only for complex 4 was a proportionate
binding observed on titration with MG bound CT-DNA. A batho-
chromic shift is observed at 663 nm, confirming the major groove
binding mode of 4 to CT-DNA (Fig. SI 16). Hence, these data prove
that 1–3 along with an intercalative mode of binding also bind to
CT-DNA through minor groove binding, while 4 binds through
intercalation and in the major groove binding mode.
CRediT authorship contribution statement
Rupam Dinda: Conceptualization, Visualization, Supervision,
Funding acquisition. Arpita Panda: Investigation. Atanu Banerjee:
Investigation, Validation, Writing - original draft, Writing - review
& editing. Monalisa Mohanty: Writing - original draft, Writing -
review & editing. Sagarika Pasayat: Investigation. Edward R. T.
Tiekink: Investigation, Writing - original draft, Writing - review
& editing.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
The authors thank the reviewers for their comments and sug-
gestions, which were helpful in preparing the revised version of
the manuscript. R. D thanks CSIR, Govt. of India [Grant No. 01
(2963)/18/EMR-II] and DBT, Govt. of India [Grant No. 6242-P112/
RGCB/ PMD/DBT/RPDA/2015] for funding this research. The
authors also thank Sunway University Sdn Bhd (Grant no. STR-
RCTR-RCCM-001-2019) for support of crystallographic studies.
4. Conclusions
In summary, this article presents the synthesis and characteri-
zation of four new mixed ligand dioxidomolybdenum(VI) [MoVIO2-
L1-3(Q)] (1–3), [MoVIO2L4(Q)]2 (H2O) (4) [where Q = MeOH for 1 and
imidazole for 2–4] complexes from tridentate arolyazine ligand
systems (H2L1–4). Single crystal X-ray crystallography revealed a
distorted octahedral structure in each case. Hydrogen bonding fea-
tures in the molecular packing of 1–4, lead to supramolecular
dimeric aggregates in 1 and 2, a helical chain in 3 and a three-
dimensional architecture in 4.
The present series of complexes were tested for biological prop-
erties in terms of their DNA binding ability and in vitro cytotoxicity.
The study revealed that the complexes bind moderately with CT-
DNA via intercalative, minor and major groove modes, while 4
showed the highest binding affinity compared to the other com-
Appendix A. Supplementary data
Crystallographic data for 1–4 reported in this paper have been
deposited with the Cambridge Crystallographic Data Centre (CCDC)
as supplementary publication nos 1920543–1920546 for 1–4,
respectively. These data can be obtained free of charge via www.
ccdc.cam.ac.uk/getstructures. Geometric data characterising the
chelate rings are given in Table SI 1 and crystallographic diagrams

R. Dinda et al. / Polyhedron 183 (2020) 114533
11
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