Novel cytotoxic oxovanadium(IV) complexes: Influence of pyrazole-incorporated heterocyclic scaffolds on their biological response

Article abstract of DOI:10.1002/aoc.3767

Homoleptic VO^IV complexes with bidentate heterocycles, namely bipyrazole/pyrimidin-2-amine/triazolopyrimidine derivatives, are novel square pyramidal oxovanadium(IV) complexes. Their identities have been established using physicochemical techni

Full text of DOI:10.1002/aoc.3767

Received: 28 November 2016
DOI: 10.1002/aoc.3767
Revised: 12 January 2017
Accepted: 21 January 2017
F U L L PA P E R
Novel cytotoxic oxovanadium(IV) complexes: Influence of
pyrazole‐incorporated heterocyclic scaffolds on their biological
response
Sanjay B. Gajera1 | Jugal V. Mehta1 | Darshana N. Kanthecha1 | Ravi R. Patel2 |
Mohan N. Patel1
1 Department of Chemistry, Sardar Patel
University, Vallabh Vidyanagar 388
Homoleptic VOIV complexes with bidentate heterocycles, namely bipyrazole/
120Gujarat, India
2 B. R. Doshi School of Bioscience, Sardar
Patel University, Vallabh Vidyanagar 388
120Gujarat, India
pyrimidin‐2‐amine/triazolopyrimidine derivatives, are novel square pyramidal
oxovanadium(IV) complexes. Their identities have been established using physico-
chemical techniques, namely elemental and spectral (ESI‐MS, IR, UV–visible) anal-
yses. The in vitro antibacterial activities against Gram‐negative and Gram‐positive
microorganisms have been studied for all compounds, exhibiting good inhibition
Correspondence
Mohan N. Patel, Department of Chemistry,
Sardar Patel University, Vallabh Vidyanagar
– 388 120, Gujarat, India.
as compared to the ligands. In addition, all compounds exhibit significant cytotox-
icity towards brine shrimp with LD50 values obtained in the range 8–24 μg ml−1
.
Email: jeenen@gmail.com
Cellular level cytotoxicity has been investigated using bioassay of
Schizosaccharomyces pombe, the complexes showing inevitable viability as concen-
tration increases. Notably, UV absorption spectral titrations of the synthesized com-
plexes with DNA reveal that the complexes bind to calf thymus DNA (CT‐DNA)
through intercalation mode (Kb = 104–105 M−1). Molecular docking reveals that
the compounds are stacked between the base pairs of DNA. The results suggest that
the complexes show promising binding affinity compared to the ligands towards
CT‐DNA.
KEYWORDS
antibacterial, CT‐DNA, molecular docking, oxovanadium(IV) complexes, S. pombe
1 | INTRODUCTION
if the treatment is predicted for long periods.[3] Great efforts
have therefore been made to synthesize oxovanadium(IV)
In the last few decades, much attention has been paid to the
chemistry of vanadium due to the discovery of its vital role
in biological systems. Vanadium complexes have numerous
applications in analytical, clinical, biological and pharmaco-
logical fields.[1] It has been observed that simple inorganic
vanadium compounds are more toxic than vanadium com-
pounds with organic ligands, and the efficacy of metal‐based
therapeutic agents changes markedly on making changes to
the organic ligands that are attached to the metal centre.[2]
For any complex considered for therapeutic use, it is impor-
tant to consider the intrinsic toxicity of the ligand, especially
complexes of high biological activity and low toxicity, which
are readily absorbed.
Pyrazole is a key motif in heterocyclic chemistry and
occupies a major position in medicinal and pesticide chemis-
try due to its wide range of bioactivities such as antibacte-
rial,[4] anticancer,[5] analgesic[6] and anticonvulsant[7]
activities. Moreover particularly N‐fused structures have
found augmented interest in recent years due to their plethora
of applications in various domains such as medicinal chemis-
try, organometallics and material chemistry. Among them,
pyrazolo[1,5‐a]pyrimidine motifs have received substantial
Appl Organometal Chem. 2017;e3767.
wileyonlinelibrary.com/journal/aoc
Copyright © 2017 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.3767

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GAJERA ET AL.
attention because of their ubiquity in several commercial
drugs such as zaleplon, indiplon (hypnotic) and ocinaplon.
Analogues of pyrazolo[1,5‐a]pyrimidines were found to have
a range of biological activities such as antifungal,[8]
antitumour,[9] analgesic[10] and anti‐inflammatory[11] activi-
ties and many more.
Earlier studies illustrated that some drugs exhibit
increased activity when administered in the form of metal
complexes. DNA is the primary target molecule for most
antiviral and anticancer therapies. Investigations of the inter-
action of DNA with other molecules are important for the
preparation of new pharmaceutical compounds.[12–14] Recent
advances in the study of catalytic and medicinal properties of
vanadium complexes have stimulated their design and syn-
thesis.[15] It is also suggested that vanadium could be consid-
ered as a representative of a new class of non‐platinum metal
antitumour agents.
2.2 | Instrumentation
1H NMR and 13C NMR spectra were obtained in 5 mm NMR
tubes at 298 K (unless stated otherwise) with Bruker Advance
1
(400 MHz) spectrometers. H NMR chemical shifts were
internally referenced to (CHD2)(CD3)SO (2.50 ppm) for
DMSO‐d6 and CDCl3 (7.26 ppm) for chloroform‐d1. 13C
NMR chemical shifts were internally referenced to CDCl3
(77.16 ppm) for chloroform‐d1 and (CD3)2SO (39.52 ppm)
for DMSO‐d6. Elemental analyses (C, H and N) of the synthe-
sized complexes were performed with a Thermo Finnigan ele-
mental analyser. IR spectra were recorded with an FT‐IR ABB
Bomen MB‐3000 spectrophotometer (Canada) as KBr pellets
in the range 400–4000 cm−1. Reflectance spectra of the metal
complexes were recorded with a UV‐160A UV–visible spec-
trophotometer (Shimadzu, Kyoto, Japan). Room temperature
magnetic measurements for the complexes were made using
a Gouy magnetic balance. The Gouy tube was calibrated
using mercury(II) tetrathiocyanatocobaltate(II) as the
calibrant (χg = 16.44 × 10−6 cgs units at 20 °C). A
Gallenkamp melting point apparatus (temperature range of
0–350 °C) was used to determine the melting points. Thermo-
grams of the complexes were recorded with a Mettler Toledo
TGA/DSC 1 thermogravimetric analyser. This analysis was
carried out at a heating rate of 10 °C min−1 in the range 20–
800 °C under nitrogen atmosphere. Antibacterial activity
was investigated using an incubator and laminar air flow cab-
inet (Toshiba, Delhi, India). Photo‐quantization of the DNA
nuclease activity was conducted using AlphaDigiDoc™ RT
version V.4.0.0 PC‐Image software (USA).
This report describes the synthesis and spectroscopic char-
acterization of nine new pyrazole‐incorporated substituted
pyrazoline/pyrimidin‐2‐amine/triazolopyrimidine
ligands.
These heterocyclic frameworks undergo coordination with
VOIV resulting in new square pyramidal oxovanadium(IV)
complexes. All the synthesized compounds have been
screened in terms of antimicrobial activity, DNA binding,
molecular docking, DNA cleavage, cell viability assay and
cytotoxicity. The results suggest that these new VOIV com-
plexes are well suited for development as anticancer agents.
2 | EXPERIMENTAL
2.3 | Syntheses
2.1 | Materials and reagents
Experiments were carried out under nitrogen atmosphere,
using a vacuum line. Dichloromethane and acetonitrile were
distilled over calcium hydride, methanol and ethanol over
magnesium under an argon atmosphere immediately before
use. Chromatographic separations were carried out on silica
gel 60–120 mesh (125–250 μM).
All reagents and solvents were purchased commercially and
used without further purification unless otherwise noted.
The purity of compounds was assessed using various analyt-
ical techniques, like electrospray ionization (ESI)‐MS, CHN
analysis, and 1H NMR, 13C NMR and infrared (IR) spectros-
copies. The measured molecular weights of ligands (interme-
diates) as well as complexes were consistent with expected
values which permitted unambiguous identification and
assessment of purity. Metal salt, guanidine hydrochloride,
2.3.1 | Synthesis of 5‐(1H‐imidazol‐1‐yl)‐3‐
methyl‐1‐phenyl‐1H‐pyrazole‐4‐carbaldehyde
(1b)
3‐methyl‐1‐phenyl‐2‐pyrazoline‐5‐one,
3‐amino‐1,2,4‐tri-
azole and potassium tert‐butoxide were purchased from
Sigma‐Aldrich. Agarose, Luria broth, ethidium bromide and
tris(hydroxymethyl)methylamine (Tris–HCl) were purchased
from Hi‐media Laboratories Pvt Ltd, India. Cultures of
pUC19 (MTCC 47), Staphylococcus aureus (S. aureus)
(MTCC‐3160), Bacillus subtilis (B. subtilis) (MTCC‐7193),
Serratia marcescens (S. marcescens) (MTCC‐7103), Pseudo-
monas aeruginosa (P. aeruginosa) (MTCC–1688) and
Escherichia coli (E. coli) (MTCC‐433) were purchased from
the Institute of Microbial Technology (Chandigarh, India).
5‐Chloro‐3‐methyl‐1‐phenyl‐1H‐pyrazole‐4‐carbaldehyde
(1a; 1 mmol), imidazole (1 mmol) and anhydrous potassium
carbonate (2 mmol) in dimethylformamide (DMF; 10 ml)
were charged in a 100 ml round‐bottom flask equipped with
a mechanical stirrer and a condenser. The reaction mixture
was heated at 90 °C for 2 h. The progress of the reaction
was monitored by TLC. After the completion of reaction as
confirmed by TLC, the reaction mixture was poured into
100 ml of ice–water, filtered, washed thoroughly with water,

GAJERA ET AL.
3 of 13
dried and recrystallized from ethanol to afford a white solid
(Scheme 1).
flask. The reaction mixture was refluxed for 5 h. After the
completion of the reaction as monitored by TLC (ethyl ace-
tate–hexane, 1:5), the reaction mixture was cooled to room
temperature washed with water and dried over Na2SO4. The
ensuing product upon purification by chromatography (silica
gel 60–120 mesh, eluent 20% EtOAc–hexanes) afforded the
desired compound as a white solid (Scheme 1). Yield 87%;
m.p. 213 °C; mol. wt 413.14 g mol−1. LC–MS: 414 (M+)
and 416 (M + 2)+. Elemental analysis (%): calcd for
C29H22ClN5: C, 69.64; H, 4.87; N, 16.92; found: C, 69.70;
H, 4.82; N, 16.96. IR (KBr, νmax, cm−1): 2924 (aromatic ring
─CHstretching), 1500 (─CH2 scissoring), 1597 (─C═N), 1388
(─CH3 rocking), 870 (C─Cl), 1126 (C─N), 771 (aromatic
2.3.2 | Synthesis of 3‐methyl‐5‐phenoxy‐1‐
phenyl‐1H‐pyrazole‐4‐carbaldehyde (1c)
Compound 1a (1 mmol), phenol (1 mmol) and anhydrous
potassium carbonate (2 mmol) in DMF (10 ml) were charged
in a 100 ml round‐bottom flask equipped with a mechanical
stirrer and a condenser. The reaction mixture was heated at
90 °C for 2 h. The progress of the reaction was monitored
by TLC. After the completion of reaction as confirmed by
TLC, the reaction mixture was poured into 100 ml of ice–
water, filtered, washed thoroughly with water, dried and
recrystallized from ethanol to afford a white solid.
1
ring ─CHbending). H NMR (400 MHz, DMSO‐d6, δ, ppm):
2.096 (s, 3H, CH3), 3.305 (dd, 1H, J = 7.2 Hz, 18.4 Hz, C4‐
Ha of pyrazoline), 3.970 (dd, 1H, J = 12.8 Hz, 18 Hz, C4‐Hb
of pyrazoline), 5.543 (dd, 1H, J = 6.8 Hz, 13.2 Hz, C5‐H of
pyrazoline), 6.794–8.605 (m, 14H, Ar─H). 13C{1H} NMR
(100 MHz, DMSO‐d6, δ, ppm): 13.31 (─CH3 of pyrazole),
42.21 (─CH2 of pyrazoline), 54.81 (─CH of pyrazoline);
117.84, 124.33, 138.00, 144.00, 147.55, 148.96, 151.65 (C
of aromatic); 113.50, 119.94, 120.62, 123.68, 125.29,
128.77, 129.56, 129.70, 136.97, 149.76 (CH of aromatic).
2.3.3 | Synthesis of (E)‐3‐(5‐chloro‐3‐methyl‐
1‐phenyl‐1H‐pyrazol‐4‐yl)‐1‐(pyridin‐2‐yl)prop‐
2‐en‐1‐one (3a)
To 1a (5.0 mmol), 2‐acetylpyridine (5.0 mmol) and 20%
ethanolic NaOH (5 ml) were added. The reaction mixture
was stirred at room temperature until formation of precipitate.
The product obtained was isolated by filtration, washed with
cold ethanol and recrystallized from CHCl3.
2.3.7 | Synthesis of 5′‐(1H–imidazol‐1‐yl)‐3′‐
methyl‐1′,2‐diphenyl‐5‐(pyridin‐2‐yl)‐3,4‐
dihydro‐1′H,2H‐3,4′‐bipyrazole (bpy‐N; 4b)
The synthesis was performed as for 4a using 3b (1.0 mmol)
and phenylhydrazine (1.0 mmol). Yield 79%; m.p. 259 °C;
mol. wt. 445.53 g mol−1. LC–MS (m/z): 446 (M+). Elemental
analysis (%): calcd for C27H23N7: C, 72.79; H, 5.20; N,
2.3.4 | Synthesis of (E)‐3‐(5‐(1H‐imidazol‐1‐
yl)‐3‐methyl‐1‐phenyl‐1H‐pyrazol‐4‐yl)‐1‐
(pyridin‐2‐yl)prop‐2‐en‐1‐one (3b)
To 1b (5.0 mmol), 2‐acetylpyridine (5.0 mmol) and 20%
ethanolic NaOH (5 ml) were added. The reaction mixture
was stirred at room temperature until formation of precipitate.
The product obtained was isolated by filtration, washed with
cold ethanol and recrystallized from CHCl3.
22.01; found: C, 72.85; H, 5.15; N, 22.05. IR (KBr, νmax
,
cm−1): 2924 (aromatic ring ─CH), 1498 (─CH2 scissoring),
1597 (─C═N), 1388 (─CH3 rocking); 1118 (C─N), 756
(C─H ar. bending). 1H NMR (400 MHz, CHCl3‐d1, δ,
ppm): 2.356 (s, 3H, CH3), 3.434 (dd, 1H, J = 7.2 Hz,
18 Hz, C4‐Ha of pyrazoline), 3.918 (dd, 1H, J = 13.2 Hz,
18.0 Hz, C4‐Hb of pyrazoline), 5.240 (dd, 1H, J = 7.2 Hz,
12.8 Hz, C5‐H of pyrazoline), 6.788–8.557 (m, 17H,
Ar─H). 13C{1H} NMR (100 MHz, CHCl3‐d1, δ, ppm):
13.13 (─CH3 of pyrazole), 41.25 (─CH of pyrazoline),
54.67 (─CH2 of pyrazoline), 117.03, 131.98, 137.92,
143.58, 147.15, 147.87, 151.71 (C of aromatic); 113.55,
120.18, 120.59, 120.65, 122.39, 122.86, 127.85, 129.11,
129.34, 130.53, 136.03, 137.41, 149.05 (CH of aromatic).
2.3.5 | Synthesis of (E)‐3‐(3‐methyl‐5‐
phenoxy‐1‐phenyl‐1H‐pyrazol‐4‐yl)‐1‐(pyridin‐
2‐yl)prop‐2‐en‐1‐one (3c)
To 1c (5.0 mmol), 2‐acetylpyridine (5.0 mmol) and 20%
ethanolic NaOH (5 ml) were added. The reaction mixture
was stirred at room temperature until formation of precipitate.
The product obtained was isolated by filtration, washed with
cold ethanol and recrystallized from CHCl3.
2.3.6 | Synthesis of 5′‐chloro‐3′‐methyl‐1′,2‐
diphenyl‐5‐(pyridin‐2‐yl)‐3,4‐dihydro‐1′H,2H‐
3,4′‐bipyrazole (bpy‐Cl; 4a)
Compound 3a (1.0 mmol) and phenylhydrazine (1.0 mmol)
were thoroughly mixed in ethanol (5 ml) with a catalytic
amount of potassium tert‐butoxide in a 50 ml round‐bottom
2.3.8 | Synthesis of 3′‐methyl‐5′‐phenoxy‐1′,2‐
diphenyl‐5‐(pyridin‐2‐yl)‐3,4‐dihydro‐1′H,2H‐
3,4′‐bipyrazole (bpy‐O; 4c)
The synthesis was performed as for 4a using 3c (1.0 mmol)
and phenylhydrazine (1.0 mmol). Yield 75%; m.p. 249 °C;

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GAJERA ET AL.
mol. wt 471.56 g mol−1. LC–MS (m/z): 472 (M+). Elemental
analysis (%): calcd for C30H25N5O: C, 76.41; H, 5.34; N,
Elemental analysis (%): calcd for C22H18N8: C, 66.99; H,
4.60; N, 28.41; found: C, 66.85; H, 4.15; N, 28.45. IR
(KBr, νmax, cm−1): 2924 (─CH aromatic ring), 1581
(─C═N), 1373 (─CH3 rocking); 1149 (C─N), 763 (C─H
ar. bending). 1H NMR (400 MHz, CHCl3‐d1, δ, ppm):
2.379 (s, 3H, CH3), 5.230 (s, 2H, NH2), 6.940–7.670 (m,
13H, Ar─H). 13C{1H} NMR (100 MHz, CHCl3‐d1, δ,
ppm): 12.86 (─CH3 of pyrazole); 120.19, 127.67, 128.35,
132.15, 136.57, 136.91, 137.65, 148.32 (C of aromatic);
119.07, 121.25, 122.69, 123.06, 125.02, 129.29, 129.91,
136.09, 138.55, 148.38, 149.53 (CH of aromatic).
14.85; found: C, 76.55; H, 5.35; N, 14.90. IR (KBr, νmax
,
cm−1): 2924 (aromatic ring ─CH), 1504 (─CH2 scissoring),
1597 (─C═N), 1389 (─CH3 rocking); 1126 (C─N), 995
(C─O─C) 771 (C─H ar. bending). 1H NMR (400 MHz,
DMSO‐d6, δ, ppm): 2.196 (s, 3H, CH3), 3.405 (dd, 1H,
J = 7.2 Hz, 18.4 Hz, C4‐Ha of pyrazoline), 4.070 (dd, 1H,
J = 13.2 Hz, 18.0 Hz, C4‐Hb of pyrazoline), 5.643 (dd, 1H,
J = 6.8 Hz, 13.2 Hz, C5‐H of pyrazoline), 6.894–8.705 (m,
19H, Ar─H). 13C{1H} NMR (100 MHz, DMSO‐d6, δ,
ppm): 12.33 (─CH3 of pyrazole), 42.25 (─CH2 of
pyrazoline), 54.57 (─CH of pyrazoline); 117.03, 124.67,
132.88, 137.11, 143.98, 147.15, 147.17, 151.81 (C of aro-
matic); 114.50, 121.38, 121.52, 121.63, 123.37, 123.76,
128.85, 130.11, 130.32, 131.55, 136.03, 137.42, 149.55
(CH of aromatic).
2.3.11 | Synthesis of 4‐(3‐methyl‐5‐phenoxy‐1‐
phenyl‐1H‐pyrazol‐4‐yl)‐6‐(pyridin‐2‐yl)
pyrimidin‐2‐amine (pml‐O; 5c)
The synthesis was performed as for 5a using 3c (1.0 mmol)
and guanidine hydrochloride (1.0 mmol). Yield 79%; m.p.
249 °C; mol. wt 420.48 g mol−1. LC–MS (m/z): 421 (M+).
Elemental analysis (%): calcd for C25H20N6O: C, 71.41; H,
4.79; N, 19.99; found: C, 71.45; H, 4.85; N, 19.95. IR
(KBr, νmax, cm−1): 2924 (─CH aromatic ring), 1574
(─C═N), 1381 (─CH3 rocking), 1195 (C─N), 1026
2.3.9 | Synthesis of 4‐(5‐chloro‐3‐methyl‐1‐
phenyl‐1H‐pyrazol‐4‐yl)‐6‐(pyridin‐2‐yl)
pyrimidin‐2‐amine (pml‐Cl; 5a)
Compound 3a (1.0 mmol) and guanidine hydrochloride
(1.0 mmol) were thoroughly mixed in ethanol (5 ml) with a
catalytic amount of potassium tert‐butoxide in a 50 ml
round‐bottom flask. The reaction mixture was refluxed for
6 h. After the completion of the reaction as monitored by
TLC (ethyl acetate–hexane, 1:5), the reaction mixture was
cooled to room temperature washed with water and dried
over Na2SO4. The ensuing product upon purification by chro-
matography (silica gel 60–120 mesh, eluent 20% EtOAc–
hexanes) afforded the desired compound as a white solid
(Scheme 1). Yield 80%; m.p. 181 °C; mol. wt 362.10 g mol
−1. LC–MS: 363 (M+), 365 (M + 2)+. Elemental analysis
(%): calcd for C24H17ClN6: C, 62.90; H, 4.17; N, 23.16;
found: C, 62.85; H, 4.15; N, 23.14. IR (KBr, νmax, cm−1):
3309 (1° NH2) 2929 (aromatic ring ─CHstretching), 763 (aro-
matic ring ─CHbending), 1510 (─C═N), 840 (C─Cl), 1226
1
(C─O─C), 756 (C─H ar. bending). H NMR (400 MHz,
CHCl3‐d1, δ, ppm): 2.360 (s, 3H, CH3), 5.239 (s, 2H,
NH2), 6.857–7.641 (m, 15H, Ar─H). 13C{1H} NMR
(100 MHz, CHCl3‐d1, δ, ppm): 13.58 (─CH3 of pyrazole);
111.04, 115.26, 115.62, 119.10, 136.41, 138.04, 145.34,
148.41, 157.22 (C of aromatic); 122.04, 122.70, 122.83,
123.19, 123.40, 126.64, 129.00, 129.12, 129.67, 129.77,
148.69 (CH of aromatic).
2.3.12 | Synthesis of 7‐(5‐chloro‐3‐methyl‐1‐
phenyl‐1H‐pyrazol‐4‐yl)‐5‐(pyridin‐2‐yl)‐[1,2,4]
triazolo[1,5‐a]pyrimidine (tpml‐Cl; 6a)
An oven‐dried 25 ml round‐bottom flask was charged with 3a
(1.0 mmol), 3‐aminotriazole (1.2 mmol), KOH (0.1 mmol)
and DMF (5 ml). The resulting solution was stirred at 110 °
C for 20 min. On completion, the reaction mass was allowed
to cool to ambient temperature, diluted with water (20 ml)
and extracted into ethyl acetate (2 × 20 ml). The combined
organic layers were dried over anhydrous Na2SO4, and
organic solvent was evaporated on a rotatory evaporator.
The crude residue was purified by chromatography (silica
gel 60–120 mesh, eluent 20% EtOAc–hexanes) affording
compound 6a (Scheme 1). Yield 85%; m.p. 189 °C; mol. wt
387.83 g mol−1. LC–MS: 388 (M+), 390 (M + 2)+. Elemental
analysis (%): calcd for C25H16ClN7: C, 61.94; H, 3.72; N,
1
(C─N), 1543 (C═C). H NMR (400 MHz, DMSO‐d6, δ,
ppm): 2.344 (s, 3H, CH3), 5.196 (s, 2H, NH2), 7.416–8.207
(m, 10H, Ar─H). 13C{1H} NMR (100 MHz, DMSO‐d6, δ,
ppm): 14.97 (─CH3 of pyrazole); 125.97, 126.57, 137.99,
149.66, 154.52, 160.39, 163.91, 164.20 (C of aromatic);
118.65, 121.32, 125.92, 129.26, 129.76, 129.98, 137.82,
149.97 (CH of aromatic).
2.3.10 | Synthesis of 4‐(5‐(1H‐imidazol‐1‐yl)‐3‐
methyl‐1‐phenyl‐1H‐pyrazol‐4‐yl)‐6‐(pyridin‐2‐
yl)pyrimidin‐2‐amine (pml‐N; 5b)
The synthesis was performed as for 5a using 3b (1.0 mmol)
and guanidine hydrochloride (1.0 mmol). Yield 79%; m.p.
250 °C; mol. wt 394.44 g mol−1. LC–MS (m/z): 395 (M+).
25.28; found: C, 61.85; H, 3.75; N, 25.27. IR (KBr, νmax
,
cm−1): 2931 (aromatic ring ─CHstretching), 763 (aromatic ring
1
─CHbending), 1550 (─C═N), 800 (C─Cl), 1203 (C─N). H
NMR (400 MHz, DMSO‐d6, δ, ppm): 2.344 (s, 3H, CH3),

GAJERA ET AL.
5 of 13
7.566–8.620 (m, 11H, Ar─H). 13C{1H} NMR (100 MHz,
DMSO‐d6, δ, ppm): 13.39 (─CH3 of pyrazole); 107.31,
122.44, 132.15, 133.69, 137.58, 144.16, 149.91, 153.28 (C
of aromatic); 105.13, 115.64, 119.89, 120.63, 123.06,
123.69, 134.52, 143.13, 150.32 (CH of aromatic).
2.3.13 | Synthesis of 7‐(5‐(1H‐imidazol‐1‐yl)‐3‐
methyl‐1‐phenyl‐1H‐pyrazol‐4‐yl)‐5‐(pyridin‐2‐
yl)‐[1,2,4]triazolo[1,5‐a]pyrimidine (tpml‐N; 6b)
FIGURE 1 Bipyrazole‐incorporated oxovanadium(IV) complexes
The synthesis was performed as for 6a using 3b (1.0 mmol),
3‐aminotriazole (1.2 mmol), KOH (0.1 mmol) and DMF
dried in vacuo. The synthesis of the complex is shown in
Scheme 1. Yield 67%; m.p. > 300 °C; mol. wt 990.82 g
mol−1. MS (ESI, m/z): 894.76 [M − SO4]+. ΩM: 89
Ω−1 cm2 mol−1; μeff: 1.89 BM. Elemental analysis (%): calcd
for C48H40Cl2N10O5SV: C, 58.19; H, 4.07; N, 14.14; V, 5.14;
found: C, 58.24; H, 4.10; N, 14.24; V, 5.16. UV–visible
(DMSO, λmax, nm (cm−1)): 543 (18 416), 758 (13 192). IR
(KBr, νmax, cm−1): 2916 (aromatic ring ─CH), 1381 (─CH3
rocking), 771 (aromatic bending ─CH), 1597 (─C═N)
1126 (C─N), 871 (C─Cl), 1026 (V═O).
(5 ml). Yield 75%; m.p. 241 °C; mol. wt 419.45 g mol−1
.
LC–MS (m/z): 419 (M+). Elemental analysis (%): calcd for
C23H17N9: C, 65.86; H, 4.09; N, 30.05; found: C, 65.85; H,
4.15; N, 30.08. IR (KBr, νmax, cm−1): 2931 (aromatic ring
─CH), 1558 (─C═N), 1381 (─CH3 rocking), 1203 (C─N).
1H NMR (400 MHz, DMSO‐d6, δ, ppm): 2.348 (s, 3H,
CH3), 7.243–9.310 (m, 14H, Ar─H). 13C{1H} NMR
(100 MHz, DMSO‐d6, δ, ppm): 14.65 (─CH3 of pyrazole);
110.29, 123.53, 133.69, 137.58, 144.29, 149.91, 153.21,
159.73 (C of aromatic); 109.13, 118.34, 120.89, 121.63,
125.60, 126.02, 127.01, 134.52, 143.13, 150.32, 155.85,
156.84 (CH of aromatic).
2.3.16 | Synthesis of [VOIV(bpy‐N)2]SO4 (7b)
The synthesis was performed as for 7a using 4b. Yield 69%;
m.p. > 300 °C; mol. wt 1054.06 g mol−1. MS (ESI, m/z):
958.00 [M − SO4]+. ΩM: 92 Ω−1 cm2 mol−1; μeff: 1.75
BM. Elemental analysis (%): calcd for C54H46N14O5SV: C,
61.53; H, 4.40; N, 18.60; V, 4.83; found: C, 61.58; H, 4.34;
N, 18.69; V, 4.89. UV–visible (DMSO, λmax, nm (cm−1)):
558 (17 921), 746 (13 404). IR (KBr, νmax, cm−1): 2916 (aro-
matic ring ─CH), 1388 (─CH3 rocking), 763 (aromatic bend-
ing ─CH), 1597 (─C═N), 1126 (C─N), 1033 (V═O).
2.3.14 | Synthesis of 7‐(3‐methyl‐5‐phenoxy‐1‐
phenyl‐1H‐pyrazol‐4‐yl)‐5‐(pyridin‐2‐yl)‐[1,2,4]
triazolo[1,5‐a]pyrimidine (tpml‐O; 6c)
The synthesis was performed as for 6a using 3c (1.0 mmol),
3‐aminotriazole (1.2 mmol), KOH (0.1 mmol) and DMF
(5 ml). Yield 75%; m.p. 236 °C; mol. wt 445.49 g mol−1
.
LC–MS (m/z): 446 (M+). Elemental analysis (%): calcd for
C26H19N7O: C, 70.10; H, 4.30; N, 22.01; found: C, 70.05;
H, 4.15; N, 22.08. IR (KBr, νmax, cm−1): 2931 (aromatic ring
─CH), 1551 (─C═N), 1381 (─CH3 rocking), 1203 (C─N),
2.3.17 | Synthesis of [VOIV(bpy‐O)2]SO4 (7c)
1
1048 (C─O─C). H NMR (400 MHz, DMSO‐d6, δ, ppm):
The synthesis was performed as for 7a using 4c. Yield 71%;
m.p. > 300 °C; mol. wt 1106.13 g mol−1. MS (ESI, m/z):
1010.07 [M − SO4]+. ΩM: 91 Ω−1 cm2 mol−1; μeff: 1.79
BM. Elemental analysis (%): calcd for C60H50N10O7SV: C,
65.15; H, 4.56; N, 12.66; V, 4.61; found: C, 65.18; H, 4.60;
N, 12.70; V, 5.56. UV–visible (DMSO, λmax, nm (cm−1)):
546 (18 315), 751 (13 315). IR (KBr, νmax, cm−1): 2916 (aro-
matic ring ─CH), 1381 (─CH3 rocking), 763 (aromatic bend-
ing ─CH), 1597 (─C═N), 1126 (C─N), 1087 (C─O─C),
1033 (V═O).
2.421 (s, 3H, CH3), 7.194–9.211 (m, 16H, Ar─H). 13C{1H}
NMR (100 MHz, DMSO‐d6, δ, ppm): 12.15 (─CH3 of
pyrazole); 110.39, 123.57, 132.15, 133.69, 137.58, 144.16,
149.91, 153.28, 159.71 (C of aromatic); 108.18, 117.34,
120.39, 121.63, 124.64, 125.06, 126.01, 134.52, 143.13,
150.32, 155.85, 156.64 (CH of aromatic).
2.3.15 | Synthesis of [VOIV(bpy‐Cl)2]SO4 (7a)
(Figure 1)
A previously prepared methanolic solution of 4a (5 mmol)
was added to a hot solution of VOSO4 (2.5 mmol) in MeOH
(25.0 ml), with constant stirring. The resulting solution was
refluxed for 10 h and concentrated under vacuum. Upon the
addition of Et2O, a brown solid precipitate was obtained,
which was collected by filtration, washed with Et2O and
2.3.18 | Synthesis of [VOIV(pml‐Cl)2]SO4 (8a)
(Figure 2)
The synthesis was performed as for 7a using 5a. Yield 65%;
m.p. > 300 °C; mol. wt 888.64 g mol−1. MS (ESI, m/z):
792.08 [M − SO4]+. ΩM: 95 Ω−1 cm2 mol−1; μeff: 1.76

6 of 13
GAJERA ET AL.
FIGURE
2
Pyrimidine‐2‐amine‐incorporated oxovanadium(IV)
FIGURE
3
Triazolopyrimidine‐incorporated oxovanadium(IV)
complexes
complexes
BM. Elemental analysis (%): calcd for C38H30Cl2N12O5SV:
C, 51.36; H, 3.40; N, 18.91; V, 5.73; found: C, 51.32; H,
3.42; N, 18.96; V, 5.78. UV–visible (DMSO, λmax, nm (cm
−1)): 518 (19 305), 719 (13 908). IR (KBr, νmax, cm−1):
3664 (─NH2), 3055 (aromatic ring ─CH), 1350 (─CH3
rocking), 756 (aromatic bending ─CH), 1504 (─C═N), 833
(C─Cl), 964 (V═O).
3.10; N, 20.96; V, 5.46. UV–visible (DMSO, λmax, nm (cm
−1)): 538 (18 587), 742 (13 477). IR (KBr, νmax, cm−1):
3055 (aromatic ring ─CH), 1381 (─CH3 rocking), 763 (aro-
matic bending ─CH), 1558 (─C═N), 794 (C─Cl), 995
(V═O).
2.3.22 | Synthesis of [VOIV(tpml‐N)2]SO4 (9b)
2.3.19 | Synthesis of [VOIV(pml‐N)2]SO4 (8b)
The synthesis was performed as for 7a using 6b. Yield 73%;
m.p. > 300 °C; mol. wt 1001.90 g mol−1. MS (ESI, m/z):
905.84 [M − SO4]+. ΩM: 86 Ω−1 cm2 mol−1; μeff: 1.86
BM. Elemental analysis (%): calcd for C46H34N18O5SV: C,
55.15; H, 3.42; N, 25.16; V, 5.08; found: C, 55.23; H, 3.48;
N, 25.26; V, 5.16. UV–visible (DMSO, λmax, nm (cm−1)):
548 (18 248), 742 (13 477). IR (KBr, νmax, cm−1): 3055 (aro-
matic ring ─CH), 1381 (─CH3 rocking), 763 (aromatic bend-
ing ─CH), 1550 (─C═N), 995 (V═O).
The synthesis was performed as for 7a using 5b. Yield 64%;
m.p. > 300 °C; mol. wt 951.88 g mol−1. MS (ESI, m/z):
855.82 [M − SO4]+. ΩM: 94 Ω−1 cm2 mol−1; μeff: 1.81
BM. Elemental analysis (%): calcd for C44H36N16O5SV: C,
55.52; H, 3.81; N, 23.54; V, 5.35; found: C, 55.56; H, 3.86;
N, 23.56; V, 5.30. UV–visible (DMSO, λmax, nm (cm−1)):
516 (19 379), 725 (13 793). IR (KBr, νmax, cm−1): 3649
(─NH2), 3078 (aromatic ring ─CH), 1381 (─CH3 rocking),
771 (aromatic bending ─CH), 1504 (─C═N), 964 (V═O).
2.3.23 | Synthesis of [VOIV(tpml‐O)2]SO4 (9c)
2.3.20 | Synthesis of [VOIV(pml‐O)2]SO4 (8c)
The synthesis was performed as for 7a using 6c. Yield 67%;
m.p. > 300 °C; mol. wt 1053.97 g mol−1. MS (ESI, m/z):
957.91 [M − SO4]+. ΩM: 89 Ω−1 cm2 mol−1; μeff: 1.87
BM. Elemental analysis (%): calcd for C52H38N14O7SV: C,
59.26; H, 3.63; N, 18.61; V, 4.83; found: C, 59.28; H, 3.56;
N, 18.66; V, 4.88. UV–visible (DMSO, λmax, nm (cm−1)):
544 (18 382), 759 (13 175). IR (KBr, νmax, cm−1): 3055 (aro-
matic ring ─CH), 1334 (─CH3 rocking), 763 (aromatic bend-
ing ─CH), 1504 (─C═N), 1078 (C─O─C), 979 (V═O).
The synthesis was performed as for 7a using 5c. Yield 69%;
m.p. > 300 °C; mol. wt 1003.95 g mol−1. MS (ESI, m/z):
907.89 [M − SO4]+. ΩM: 92 Ω−1 cm2 mol−1; μeff: 1.82
BM. Elemental analysis (%): calcd for C50H40N12O7SV: C,
59.82; H, 4.02; N, 16.74; V, 5.07; found: C, 59.86; H, 4.10;
N, 16.69; V, 5.16. UV–visible (DMSO, λmax, nm (cm−1)):
522 (19 157), 731 (13 679). IR (KBr, νmax, cm−1): 3657
(─NH2), 3055 (aromatic ring ─CH), 1350 (─CH3 rocking),
756 (aromatic bending ─CH), 1504 (─C═N), 1072
(C─O─C), 964 (V═O).
2.4 | Biological evaluation
2.4.1 | Absorption spectroscopy in presence of
2.3.21 | Synthesis of [VOIV(tpml‐Cl)2]SO4 (9a)
calf thymus DNA
(Figure 3).
The absorption spectra were recorded with a Shimadzu UV‐
2450 UV–visible spectrophotometer using cuvettes of 1 cm
path length. Absorption spectral titration experiments were
performed by maintaining a constant concentration of the
complex and varying the nucleic acid concentration. It was
achieved by dissolving an appropriate amount of the metal
The synthesis was performed as for 7a using 6a. Yield 71%;
m.p. > 300 °C; mol. wt 938.66 g mol−1. MS (ESI, m/z):
842.60 [M − SO4]+. ΩM: 96 Ω−1 cm2 mol−1; μeff: 1.85
BM. Elemental analysis (%): calcd for C40H28Cl2N14O5SV:
C, 51.18; H, 3.01; N, 20.89; V, 5.43; found: C, 51.21; H,

GAJERA ET AL.
7 of 13
complex, DNA stock solutions and maintaining the total vol-
ume constant. The absorbance (A) of the most red‐shifted
band for each complex was recorded after successive addi-
tions of calf thymus DNA (CT‐DNA). The stock solution
was prepared by dissolving CT‐DNA in a Tris–HCl buffer
(pH = 7.2) which was kept overnight at 4 °C for complete
dissolution. The DNA concentration per nucleotide was
determined by absorption spectroscopy using the molar
absorption coefficient (6600 M−1 cm−1) at 260 nm.[12,16]
i.e. MIC. The MIC value was determined using the two‐fold
serial dilution technique in triplicate. The lowest compound
concentration inhibiting visible bacterial growth is reported
as MIC.[18,19]
2.4.5 | In vitro cytotoxicity bioassay
The cytotoxicity assay was performed on brine shrimp
nauplii using the Meyer method. The lethal concentrations
of compounds resulting in 50% mortality (LC50) of the brine
shrimp from the 24 h and the dose–response data were trans-
formed into a straight line by means of a trend line fit linear
regression analysis; LC50 was determined from the best‐fit
line obtained by plotting % mortality versus concentration.
All data were collected from at least three independent exper-
iments and LC50 determined using OriginPro 8 software as
previously described.[13,20,21]
2.4.2 | Molecular docking of complexes with
DNA duplex with sequence d(CGCGAATTC
GCG)2
A rigid molecular docking study was performed using HEX
8.0 software to determine the orientation of the VOIV com-
plexes binding to DNA.[17] Docking was performed and the
most stable configuration was chosen as input for investiga-
tion. The coordinates of metal complexes were taken from
their optimized structure as a molecule and were converted
to .pdb format using ChemBio3D Ultra 12.0 software. CT‐
DNA used in the experimental work was too large for the cur-
rent computational resources to dock; therefore, the structure
of the DNA of sequence d(ACCGACGTCGGT)2 (PDB id:
423D, a familiar sequence used in oligodeoxynucleotide
study) obtained from the Protein Data Bank (http://www.
rcsb.org/pdb) was used. All calculations were carried out
using an Intel CORE i5, 2.5 GHz based machine running
MS Windows 8 64 bit as the operating system.
2.4.6 | Cellular level cytotoxicity
Due to eukaryotic characteristics and fairly large size of
Schizosaccharomyces pombe (S. pombe), it has become an
important tool for studying cell biology. Cytotoxicity of the
synthesized compounds was tested using bioassay of S.
pombe cells at the cellular level as previously described.[14]
3 | RESULTS AND DISCUSSION
Molecular design
As shown in Figure 4, the typical active N–N functionality
embedded in pyridyl–pyrazole, pyridyl–pyrimidine and pyri-
dyl–triazolopyrimidine will serve as a basic core ligand with
a readily available metalation site. The designed molecule
was synthesized using a developed strategy reported ear-
lier.[22–24] This incorporated imidazole and phenol features
an installation of different biological application.
2.4.3 | Chemical nuclease activity
DNA cleavage study was performed using pUC19 DNA with
the synthesized compounds. The samples were incubated for
1 h at 37 °C. The samples were analysed using 1% agarose
gel electrophoresis (Tris–acetate–EDTA buffer, pH = 8.0)
for 3 h at 100 mV. The gel was stained with (0.5 mg ml−1
)
ethidium bromide. The gels were viewed in an Alpha
Innotech Corporation Gel Doc system and photographed
using a CCD camera. The cleavage efficiency was measured
by determining the ability of the complex to convert
supercoiled (SC) DNA to nicked circular (NC) and linear
(L) forms.
3.1 | Synthesis and general aspects
In the 1H NMR spectra of bipyrazoles, the pro‐chiral methy-
lene protons of pyrazoline appear as two distinct doublets of
a doublet, thereby indicating that both the protons are mag-
netically non‐equivalent and diastereotopic. The chiral C─H
1
protons of pyrazoline appear as doublets of a doublet. In H
2.4.4 | In vitro antibacterial activity
NMR spectra of pyrimidin‐2‐amines, ─NH2 protons resonate
as singlet at around 5.5–5.7 ppm (supporting information 1).
The ESI‐MS of complexes were acquired to understand the
relative composition and stability of the complexes. Notably
this displayed the most abundant peaks for complex 7b at
m/z 958.09 ([M − SO4]+) suggesting cleavage of the
M─SO4 bond and loss of sulfate group from outer sphere
of respective complexes (supporting information 2). The
The minimal inhibitory concentration (MIC) was determined
against three Gram‐negative microorganisms, namely E. coli
(MTCC 433), P. aeruginosa (MTCC P‐09) and S.
marcescens (MTCC 7103), and two Gram‐positive bacteria,
namely B. subtilis (MTCC 7193) and S. aureus (MTCC
3160). The antibacterial activity for the test compounds was
investigated to determine the bacteriostatic concentration,

8 of 13
GAJERA ET AL.
DNA from 0 to 500 μl and with constant volume of the com-
pound (125 μl). In order to eliminate the absorbance of DNA
itself; a reference cell containing DNA alone was prepared at
the same concentration to which increments of the DNA
stock solution were added. The binding of the compounds
to DNA led to a decrease in the absorption of the UV–visible
spectrum, indicating that the π* orbital of the compounds can
couple with the π orbital of the DNA base pairs, resulting in a
decrease in the π–π* transition energy and causing a
bathochromic shift. The binding constants (Kb) of the com-
pounds to CT‐DNA were determined by monitoring the
changes in absorbance with increasing concentration of CT‐
DNA (Figure 5). The absorption data were analysed to eval-
uate Kb determined from the spectroscopic titration data
using the following equation:
FIGURE 4 Modifications of the ‘square pyramidal’ complexes
characteristic thermogram (mass loss with temperature) of
complex 7a (supporting information 3) shows two distinct
mass losses. For any complex, the water molecules are elim-
inated, namely crystallization water up to 120 °C and coordi-
nated water at 120–180 °C, but here no mass losses occur up
to 200 °C. So we concluded the absence of crystallization and
coordinated water molecules in the VOIV complexes. The
mass loss (9.62%) during the first step up to 265 °C corre-
sponds to decomposition of SO42− ion complex. The second
step (83.42%) corresponds to decomposition of bidentate
ligands, leaving behind the metal oxide as residue.
½DNAꢀ ½DNAꢀ
1
¼
þ
εa−εf
εb−εf
Kbðεb−εfÞ
3.2 | DNA binding from absorption spectral
studies
There are a number of ways in which small molecules can
interact with DNA; for example, groove binding, which is
primarily an electrostatic interaction between a cationic mol-
ecule and the negative phosphate backbone of DNA, covalent
binding, in which a molecule coordinates to one of the base
pairs of DNA (cisplatin covalently binds to DNA), and inter-
calation, resulting from insertion of a molecule or part of a
molecule between the base pairs of DNA. Intercalation typi-
cally requires an extended flat aromatic group to accomplish
this type of DNA interaction.[25] Because of the
photophysical properties of metal complexes, a great deal of
research has been conducted, beginning with the initial stud-
ies by Barton and co‐workers in the mid‐1980s,[26,27] associ-
ated with the development of ruthenium(II) complexes as
DNA probes and phototherapeutics.
To this end, we evaluated the interaction of VOIV com-
plexes with CT‐DNA by spectroscopic means.[28–30] The
DNA binding experiment was performed in Tris–HCl buffer
using DMSO solution of all compounds. The DNA concen-
tration per nucleotide was determined by absorption spectros-
copy using the molar absorption coefficient of 6600 M−1 cm
−1
at 260 and 280 nm, with A260/A280 of 1.9, indicating that
the DNA was sufficiently free of protein.[31] The stock solu-
tions were stored at 4 °C and used within four days. Absorp-
tion titrations were carried out by varying the volume of CT‐
FIGURE 5 (A) Absorption spectra of [VOIV(bpy‐Cl)2SO4] (7a) in
5 mM Tris–HCl buffer at pH = 7.1, in the absence and presence of
increasing amounts of DNA. (B) Plot of [DNA]/(εa − εf) versus [DNA]

GAJERA ET AL.
9 of 13
TABLE 1 Absorption spectral properties of ligands and VOIV com-
plexes bound to CT‐DNA with an error of ꢁ3% for three replicates
The ‘apparent’ extinction coefficient (εa) was obtained by
calculating the absorbance of the compounds. The terms εf
and εb correspond to the extinction coefficients of free
(unbound) and the fully bound compounds. Kb was calcu-
lated using a plot (Figure 5).of [DNA]/(εa − εf) versus
[DNA] from the ratio of slope to intercept.[31] In the present
study, the heteroatom present in the aromatic moiety can eas-
ily bind to DNA base pairs.[32] The interaction of complex 7a
with an increase in CT‐DNA is shown in Figure 5. In the
absorption spectrum, the ligands and complexes exhibit more
intense absorption bands, which are attributed to strong
stacking interaction between the planar extended π‐system,
facilitating a non‐covalent interaction of the compounds with
the base pairs of the DNA molecule.[33,34] On increasing the
concentration of CT‐DNA, the absorption band of the com-
pounds is affected, resulting in hypochromism and
bathochromic red shift. Complexes 7b, 8b and 9b exhibited
strong hydrophobic CT‐DNA interaction due to the presence
of an extended π‐system of the aromatic imidazole ring and
planarity of the molecule. Kb of 7b, 8b and 9b with CT‐
DNA was found to be 1.31 × 105, 3.43 × 105 and
2.14 × 105, respectively, which are greater than those of the
rest of the complexes due to the presence of imidazole ring.
Addition of CT‐DNA to the compounds leads to a decrease
in absorbance of the π–π* band, which indicates that the
binding of ligands and complexes with DNA is due to a
strong interaction. Table 1 gives the static binding constants
of ligands and complexes with CT‐DNA.
Binding
Wavelength Shift Hypochromism
constant
Compound
(nm)
(nm)
(%)
(Kb × 105)
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
8a
8b
8c
9a
9b
9c
379
375
379
265
265
268
313
314
316
390
392
332
269
280
279
370
340
338
3
2
2
3
2
3
2
2
3
3
4
3
2
5
3
2
4
3
28 ꢁ 0.84
12 ꢁ 0.36
11 ꢁ 0.33
19 ꢁ 0.57
13 ꢁ 0.39
12 ꢁ 0.36
19 ꢁ 0.57
15 ꢁ 0.45
14 ꢁ 0.42
30 ꢁ 0.90
23 ꢁ 0.69
37 ꢁ 1.11
32 ꢁ 0.96
20 ꢁ 0.60
25 ꢁ 0.75
44 ꢁ 1.32
19 ꢁ 0.57
22 ꢁ 0.66
0.618 ꢁ 0.019
0.356 ꢁ 0.010
0.144 ꢁ 0.004
0.382 ꢁ 0.012
0.447 ꢁ 0.013
0.269 ꢁ 0.008
0.657 ꢁ 0.019
0.574 ꢁ 0.017
0.486 ꢁ 0.014
0.571 ꢁ 0.017
1.31 ꢁ 0.039
1.22 ꢁ 0.037
0.413 ꢁ 0.012
3.43 ꢁ 0.102
0.274 ꢁ 0.008
0.327 ꢁ 0.010
2.14 ꢁ 0.064
1.87 ꢁ 0.056
3.3 | Molecular docking of complexes with
DNA duplex with sequence d(CGCGAATTC
GCG)2
In addition to the wet laboratory activity, we also performed
computational work (molecular docking) for all synthesized
compounds. Molecular docking of the complexes with the
DNA duplex with sequence d(ACCGACGTCGGT)2 was
investigated. The molecular docking technique is important
for understanding drug–DNA interactions for rational drug
design and discovery, as well as in mechanistic studies by
placing a small molecule in the binding site of the target spe-
cific region of DNA mainly in a non‐covalent fashion.[35]
Docking was performed to preliminarily predict the binding
affinity and the chosen binding site along with the sterically
acceptable conformations. It is generally accepted that the
lower the binding free energy, the more potent the binding
affinity between the receptor (DNA) and the ‘ligand’
molecules.
FIGURE 6 Most probable 2D/3D model of docking pose to active
site of compounds with CT‐DNA
docking energy presented in Table 2, the complexes reason-
ably bind to DNA as compared to ligands. The study shows
that the compounds under investigation interact with DNA
via an intercalation mode involving outside edge stacking
interaction with oxygen atoms of the phosphate backbone.
Thus, we can say that there is a mutual complement between
Amongst all possible conformations, the one having the
most optimal energy was chosen for the final binding orienta-
tion analysis. The docked pose with minimum energy
(Figure 6).clearly shows that minor groove mode plays a pre-
dominant role in the interaction. As can be seen from the

10 of 13
GAJERA ET AL.
TABLE 2 Molecular docking results in terms of binding energy
with that of the ligands and salt under the same conditions.
DNA cleavage can occur by hydrolytic and oxidative path-
ways,[36] in which hydrolytic DNA cleavage involves cleav-
age of phosphodiester bonds to generate fragments and
converting SC form of DNA to the open circular (OC) form
and then L form,[37] being used for identifying the percentage
of cleavage as a function of concentration of nuclease. Oxida-
tive DNA cleavage involves either oxidation of the deoxyri-
bose moiety by abstraction of sugar hydrogen or oxidation
of nucleobases.[38] The results of these experiments are
depicted in Figure 7.
The percentage cleavage data show that all compounds
show better cleavage activity as compared to vanadium salt
andligands (Figure 8). The principle of this method is that mol-
ecules migrate in the gel as a function of their mass, charge and
shape, withSCDNAmigrating faster thanOC molecules ofthe
samemassandcharge.ThenativeDNAremainsintheSCform.
Single‐strand cleavage results in so‐called NC or OC form of
DNA whereas double‐strand cleavage results in L form of
DNA. The migration rate during agarose gel electrophoresis
depends on both size (base pairs) and conformation, with
smaller or condensed DNA migrating faster than larger or
unfolded DNA. SC DNA has a tightly packed conformation
and therefore migrates faster through agarose gels than L
DNA (intermediate migration) or OC DNA (slowest migra-
tion).[39] Resultsshowthat all complexes arebetter DNAcleav-
ing agents as compared to ligands and salt. The pertinent
cleavage data are presented in supporting information 4.
Binding energy
Binding energy
Ligand
(kcal mol−1
)
Complex
(kcal mol−1
)
4a
4b
4c
5a
5b
5c
6a
6b
6c
−289.61
−285.39
−288.90
−277.21
−280.23
−291.92
−280.25
−287.18
−293.05
7a
7b
7c
8a
8b
8c
9a
9b
9c
−364.90
−365.62
−330.02
−377.23
−367.75
−392.37
−384.68
−382.66
−367.10
spectroscopic techniques and molecular docking modelling,
which substantiates our spectroscopic results and provides
further evidence of intercalative binding.
3.4 | Chemical nuclease activity
The activity of the complexes as chemical nucleases was
studied using supercoiled pUC19 DNA in 1% agarose gel at
100 V for 2 h. The efficiency of the complexes was compared
3.5 | Antibacterial activity
FIGURE 7 Cleavage of SC pUC19 DNA with series of ligands and
complexes (50 μM) using 1% agarose gel containing 0.5 μg ml−1
ethidium bromide. All reactions were incubated in tris–acetate–EDTA
buffer (pH = 8) in a final volume of 20 μl, for 24 h at 37 °C. Lane 1,
DNA control; lane 2, DNA + salt; lanes 3–5, DNA + (4a–4c); lanes 6–
8, DNA + (5a–5c); lanes 9–11, DNA + (6a–6c); lanes 12–14, DNA +
(7a–7c); lanes 15–17, DNA + (8a–8c); lanes 18–20, DNA + (9a–9c).
NC, nicked circular; SC, supercoiled; L, linear
The antibacterial activities of all newly synthesized
bipyrazole, pyrimidin‐2‐amine and triazolopyrimidine deriv-
atives and complexes were tested against arbitrarily selected
two Gram‐positive and three Gram‐negative bacterial stains.
When tested for antibacterial activity, vanadium salt has been
found to be inactive (MIC >1500 μM). The aim was to com-
bine pyrazole derivatives with vanadium ions to investigate
FIGURE 8 Cleavage of SC, NC and L
forms of DNA with an error of ꢁ3% for three
replicates represented by black error bars

GAJERA ET AL.
11 of 13
the antibiotic activity. The ligands (4a, 4b, 4c, 5a, 5b, 5c, 6a,
6b and 6c) exhibited MIC values in the range 248–365 μM
(Figure 9). These results suggest that neither vanadium nor
pyrazole‐based ligands are active individually, but the coordi-
nation of both is indispensable for antibacterial activity. A sat-
isfactory reason for this increase in antibacterial activity may
be considered in the light of Overtone's concept[12] and chela-
tion theory. According to Overtone's concept of cell perme-
ability, the lipid membrane that surrounds a cell favours the
passage of only lipid‐soluble materials, so that liposolubility
is an important factor which controls bacteriostatic activity.
Onchelation, thepolarityoftheVOIV ionwillbeloweredto
a greater extent due to the overlap of the ligand orbital and par-
tial sharing of the positive charge of the VOIV ion with donor
groups. Further, it increases the delocalization of π‐electrons
over the whole chelate ring and enhances lipophilicity of the
complexes. This increased lipophilicity enhances the penetra-
tion of the complexes into lipid membranes which can then
block the metal binding sites in bacterial enzymes. The com-
plexes also disturb the respiratory processes of the cell and thus
blockthesynthesisofproteins,whichrestrictsfurthergrowthof
the organism. The antibacterial activity was evaluated by MIC
values. The pertinent MIC data are presented in supporting
information 5. It is worth mentioning that all the compounds
showed more promising activity against Gram‐negative bacte-
ria compared to Gram‐positive bacteria. This might be due to
thepresence ofa thickouter membrane inGram‐positivebacte-
ria, which hinders access of many compounds to the cytoplas-
mic membrane where complexes exert their activity.
3.6 | Cytotoxicity
All the synthesized compounds were screened for their cyto-
toxicity (brine shrimp bioassay) using the protocol of Meyer
et al.[21] Brine shrimp lethality bioassay is a development in
the assay procedure of bioactive compounds, which indicates
cytotoxicity as well as a wide range of pharmacological activ-
ities of compounds. Results for the lethality were noted in
terms of deaths of larvae. The mortality rate of brine shrimp
nauplii was found to increase with increasing concentration
of complexes. A plot of the log of sample concentration ver-
sus percentage mortality showed a linear correlation. From
the graph, the LC50 values of the compounds were calculated.
It is observed from the results (Figure 10) that 6a, 6b and 6c
are good cytotoxic agents (12.07, 12.02 and 12.09 μg ml−1
,
respectively) as compared to all other ligands.
FIGURE 9 Antimicrobial activity with an
error of ꢁ3% for three replicates represented
by black error bars
FIGURE 10 Influence of compounds on
brine shrimp lethality bioassay with an error
of ꢁ3% for three replicates represented by
black error bars

12 of 13
GAJERA ET AL.
The results indicate that the triazolopyrimidines are more
by toxicity of the synthesized compounds could be easily
monitored by vital staining. The toxicity was found to vary
with the type of substituent, principal moieties and concen-
trations of the synthesized compounds. A general observation
is that as the concentration of compounds increased the cyto-
toxicity also increased.[14] After treatment, many of the S.
pombe cells were killed due to the toxic nature of the
compounds.
cytotoxic than pyrazolines and pyrimidin‐2‐amines. Another
observation is that the compounds containing imidazole
nucleus are more potent than those containing phenoxy
nucleus and chloro‐substituted at the 5‐position on 1‐phe-
nyl‐3‐methylpyrazole. The order of potency of pyrazole‐
incorporated compounds is 5a < 5c < 5b < 4a < 4c
<4b<6c<6a<6b<8a<8c<8b<7b<7c<7a<9c<9a=9b.
The tested VOIV complexes have strong cytotoxic activity,
but this examination is a preliminary one and further tests
are required to investigate the actual mechanism of cytotoxic-
ity and probable effects on higher animal model and on can-
cer cell lines. The pertinent cytotoxicity data are presented in
supporting information 6.
From Table 3, we observed that the cytotoxicity of VOIV
complexes is better than that of the respective free ligands.
The compounds bearing imidazole ring show better results
than the others. Compound 6b is more potent amongst the
ligands and complex 9b is most toxic amongst all the synthe-
sized compounds.
3.7 | Cell viability assay
4 | CONCLUSIONS
In the present study, S. pombe cells were used to study the
cytotoxic effect of the compounds at a cellular level. S.
pombe cells have become an important tool for studying cell
biology due to the eukaryotic characteristics and fairly large
size. A comparative study of cellular level cytotoxicity of
the free ligands and their complexes indicates that the metal
complexes show better activity against S. pombe cells com-
pared to the pyrazole‐incorporated ligands. Cell death caused
The goal of the present study was to explore the rational
design and biological evaluation of 3d1 VOIV complexes.
Moreover, pyrazole‐based heterocyclic frameworks can have
a major effect on the chemical and biological behaviour of
[VOIV(XY)2]2+ complexes. This appears to be the first
time that bipyrazole/pyrimidin‐2‐amine/triazolopyrimidine
ligands have been used in vanadium complexes. In general,
the introduction of pyrazole‐based ligands to metal enhances
the cytotoxic activity against brine shrimp and S. pombe, with
complexes 9a, 9b and 9c being most cytotoxic. This is
because of the triazolopyrimidine derivatives present in these
compounds. The chelating ligand appears to determine the
selectivity of nucleobase binding and complexes 7b, 8b and
9b show promising binding affinity because of the imidazole
substituent present on the 5‐position of pyrazole. Molecular
docking indicates intercalation binding and proves that the
complexes have better binding affinity than the ligands. In
addition, VOIV complexes show altered inhibition against
bacterial species as compared to ligands. In addition com-
plexes 7b, 8b, 9a, 9b and 9c have excellent inhibition which
is ascribed to triazolopyrimidine moiety of the compounds as
well as imidazole substituent. The complexes of pyrimidin‐2‐
amine derivative, 8a, 8b and 8c, show DNA cleaving effi-
ciency. The work reported here demonstrates that rational
chemical design can be applied to VOIV complexes to
achieve potent biological activity. It is notable that pyrazole
substituents can also play a major role in controlling the
chemical and biological properties of metal complexes. In
general, vanadium complexes offer much promise for the
design of novel therapeutic agents.
TABLE 3 Viability of S. pombe cells with an error of ꢁ3% for three
replicates
Viability (%) for each concentration
Compound
2 μM
4 μM
6 μM
8 μM
10 μM
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
8a
8b
8c
9a
9b
9c
83
81
83
85
82
85
81
79
81
77
75
81
80
77
79
76
73
75
78
76
78
80
77
80
76
74
76
73
70
73
75
72
74
71
68
70
72
70
71
74
72
74
70
68
70
67
64
66
69
66
68
65
62
64
67
65
67
69
66
69
65
63
65
62
59
61
64
61
63
60
57
59
63
61
62
65
63
65
61
59
61
58
55
57
60
57
59
56
53
55
ACKNOWLEDGEMENTS
This work was supported by UGC, New Delhi providing
BSR‐one time grant fellowship and UGC‐BSR JRF/SRF

GAJERA ET AL.
13 of 13
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and CISST, Vallabh Vidyanagar for LC‐MS.
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