Bipyrazole-based palladium(II) complexes as DNA intercalator and artificial metallonuclease

Article abstract of DOI:10.1007/s00706-018-2316-6

Abstract: Substituted 3′-methyl-1′,2-diphenyl-3,4-dihydro-1′H,2H-3,4′-bipyrazole and their square planar palladium(II) complexes of type [Pd(4n)Cl₂], where 4n = bipyrazole-based ligands, have been synthesized. The compounds have been characterized by various techniques like elemental analysis, mass, absorption, IR, ¹H NMR and ¹³C NMR spectroscopy. The complexes have been further analyzed by thermogravimetric analysis (TGA), conductance measurement and energy-dispersive X-ray spectroscopy (EDX). Interaction between compounds and herring sperm DNA has been studied by absorption titration, viscosity measurement, ethidium bromide displacement titration, and molecular docking study. The binding strength between compounds and DNA has been checked in terms of K_b, K_sv, and K_f values; while spontaneity of interaction has been checked by evaluating thermodynamic parameters like Gibb’s free energy, enthalpy change (?H°) and entropy change (?S°). The plasmid cleavage efficacy of complexes has been evaluated by gel electrophoresis technique. The minimum inhibitory concentration of compounds has been evaluated against pathogens such as Staphylococcus aureus, Escherichia coli, B. subtilis, S. marcescens and Pseudomonas aeruginosa. The in vivo and in vitro cytotoxic activity has been performed using cell viability assay and brine shrimp lethality bioassay. Graphical abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-018-2316-6

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-018-2316-6
ORIGINAL PAPER
Bipyrazole‑based palladium(II) complexes as DNA intercalator
and artifcial metallonuclease
Khyati P. Thakor1 · Miral V. Lunagariya1 · Bhupesh S. Bhatt1 · Mohan N. Patel1
Received: 15 June 2018 / Accepted: 10 October 2018
© Springer-Verlag GmbH Austria, part of Springer Nature 2019
Abstract
Substituted 3′-methyl-1′,2-diphenyl-3,4-dihydro-1′H,2H-3,4′-bipyrazole and their square planar palladium(II) complexes
of type [Pd(4n)Cl2], where 4n=bipyrazole-based ligands, have been synthesized. The compounds have been characterized
by various techniques like elemental analysis, mass, absorption, IR, 1H NMR and 13C NMR spectroscopy. The complexes
have been further analyzed by thermogravimetric analysis (TGA), conductance measurement and energy-dispersive X-ray
spectroscopy (EDX). Interaction between compounds and herring sperm DNA has been studied by absorption titration, vis-
cosity measurement, ethidium bromide displacement titration, and molecular docking study. The binding strength between
compounds and DNA has been checked in terms of Kb, Ksv, and Kf values; while spontaneity of interaction has been checked
by evaluating thermodynamic parameters like Gibb’s free energy, enthalpy change (∆H°) and entropy change (∆S°). The
plasmid cleavage efcacy of complexes has been evaluated by gel electrophoresis technique. The minimum inhibitory con-
centration of compounds has been evaluated against pathogens such as Staphylococcus aureus, Escherichia coli, B. subtilis, S.
marcescens and Pseudomonas aeruginosa. The in vivo and in vitro cytotoxic activity has been performed using cell viability
assay and brine shrimp lethality bioassay.
Graphical abstract
Keywords Absorption spectra · Pd(II) metal complexes · DNA interaction · Fluorescence · Thermodynamics · Bipyrazole
ligands · Antimicrobial activity
Introduction
DNA is the primary target to study the mode of action of any
Electronic supplementary material The online version of this
drugs since it carries the genetic information like growth,
article (https://doi.org/10.1007/s00706-018-2316-6) contains
development, functioning, and reproduction of the known liv-
ing organisms. The historical successful example of metal-
based drugs, cisplatin and carboplatin is currently used as
chemotherapeutic agents despite several side effects like
nephrotoxicity, neurotoxicity, ototoxicity, etc. Some tumor
cells get resistance to cisplatin during treatment [1]. These
facts indicate need of some alternatives of cisplatin and car-
boplatin having superior chemotherapeutic activity with
supplementary material, which is available to authorized users.
* Bhupesh S. Bhatt
bhupeshbhatt31@gmail.com
* Mohan N. Patel
jeenen@gmail.com
1
Department of Chemistry, Sardar Patel University,
Vallabh Vidyanagar, Gujarat 388 120, India
Vol.:(0123456789)
1 3

K. P. Thakor et al.
lesser side efects, and DNA as their primary target. The
group 10 transition metals are well-known DNA binders,
either via covalent binding or via non-covalent binding [2–4].
Palladium(II) complexes are isostructural and isoelectronic
with platinum(II) complexes. Therefore, the recent research
focuses on designing and synthesizing the new palladium(II)
complexes [5] of platinum(II) analog as antineoplastic agent
[6].
mass spectroscopy, and elemental analysis. The elemental
percentage of complexes and the presence of lattice water
molecules, magnetic and electrolytic nature of complexes
have been studied by EDX, TGA, magnetic moment meas-
urement by Gouy’s method, and conductance measurement,
respectively. The mode of interaction of DNA with metal
complexes has been evaluated by molecular docking study,
viscosity measurement, absorption and fuorescence titra-
tion. The binding strength has been assessed by absorption
and fuorescence spectroscopy, and diferent thermodynamic
parameters have been evaluated for DNA–metal complex
interaction. The nucleolytic, antimicrobial, in vivo and
in vitro cytotoxic studies of metal complexes have been car-
ried out by gel electrophoresis, MIC method, cell viability
assay, and brine shrimp lethality bioassay, respectively. In
our study, complex 5b having bipyrazole ligand with Cl as a
substituent atom is the most potent biological agent among
all the synthesized compounds, which can be attributed to
its smaller size and electronegative nature.
Most of the current pharmaceutical agents are organic mole-
cules. Among them, the pyrazoline derivatives are used as anti-
bacterial, antifungal, antiparasitic, antitubercular, and antiviral
and also possess anti-infammatory, analgesic, anti-diabetic,
and anesthetic properties. The attachement of an additional
lipophilic moiety to the pyrazoline ring enhances the antimi-
crobial as well as anti-infammatory activities of pyrazoline
moiety [7]. Coordination of small organic moiety to metal ion
creates the synergic efect and increases the pharmacologi-
cal potency of organic moiety [8, 9] as well as removes some
limitations such as faster hydrolysis of metal salt during DNA-
binding activity. The complexes interact with DNA and inter-
fere with DNA repair and DNA replication [10, 11]. Therefore,
during the last few decades, the interaction of small molecules
to biopolymer has been the prominent research area [12, 13].
Interaction of the small molecules to DNA is either covalent or
non-covalent. In covalent binding, complex binds to nitrogen
base of DNA, while in non-covalent binding, partial insertion of
aromatic moiety between DNA base pair takes place [14]. Also,
these complexes are capable of cleaving the DNA sequence,
i.e., phosphodiester linkage among the backbone of DNA; they
mimic the nuclease enzymes and are, therefore, called artifcial
metallonucleases. Thermodynamic parameters obtained from
the DNA interaction study are useful to understand the types
of binding between DNA and complexes.
Structural characterization of ligands 4a–4d
and corresponding complexes 5a–5b
1H NMR of ligands 4a–4d and complexes 5a–5d is embed-
1
ded in ESM 1a and 1b, respectively. In H NMR spectra
of ligands 4a–4d, three doublet of doublet in the range of
2.19–5.39 ppm are assigned to the 1,3,5-substituted-4,5-di-
hydro-1H-pyrazole moiety. Peak around 7.50 ppm shifted
to downfeld in all complexes 5a–5d indicates coordination
of ligands with palladium(II) metal. 13C NMR of ligands
4a–4d and complexes 5a–5d is embedded in ESM 2a and
2b, respectively. The EDX spectrum for complexes confrms
the presence of carbon, nitrogen, sulfur, chlorine, and pal-
ladium elements and the weight % of C, N, S, Cl and Pd is
provided in ESM 3. ESM 4 and 5 represent mass spectrum
and probable fragments of complex [Pd(4a)Cl2] (5a). Mass
spectrum of complex 5a exhibits molecular ion peak [M+]
at m/z = 595.59, [M+2] at 597.60, [M+4] at 601.52, and
[M+6] at 603.58, which suggest the presence of three chlo-
rine atoms in the complex and confrm the coordination of
ligand to the metal ion. The peak at m/z=526.58 is obtained
due to the loss of two chlorine atoms from the complex and
peak at m/z=418.72 is obtained due to the pyrazoline ligand
attached with palladium(II) metal.
Thus, with the aim to explore biological signifcance of
Pd(II) metal ion upon coordination with heterocyclic mole-
cules, we have synthesized bipyrazole-based ligands and their
Pd(II) complexes. The compounds have been characterized
by various spectroscopic and analytical techniques. All com-
pounds have been screened for various biological activities
like DNA binding, in vitro and in vivo cytotoxicity, nucleo-
lytic and antibacterial activity. Furthermore, thermodynamic
parameters have been evaluated by absorption spectroscopy
and free energy data have been evaluated by molecular dock-
ing study for DNA-Pd(II) complex interaction.
Thermogravimetric analysis is concerned with the change
in weight of a material as its temperature changes. It indi-
cates the temperature at which the material loses weight and
weight loss indicates sample decomposition. TGA has been
carried out at a 10 °C per min heating rate in the range of
20–800 °C under a nitrogen atmosphere. The thermogram of
the complex 5a shows no weight loss up to ∼200 °C, which
infers the absence of lattice and coordinated water molecules
(ESM 6). The characteristic thermogram of the complex 5a
Results and discussion
In the present article, we have synthesized four differ-
ent substituted 3′-methyl-1′,2-diphenyl-3,4-dihydro-
1′H,2H-3,4′-bipyrazole ligands and their square planar
palladium(II) complexes (Scheme 1). The compounds have
1
been characterized by IR, H NMR, 13C NMR, UV–Vis,
1 3

Bipyrazole-based palladium(II) complexes as DNA intercalator and artifcial metallonuclease
Scheme 1
shows two distinct mass losses. The mass loss (about 12%)
during the frst step between 200 and 350 °C corresponds to
the loss of two chlorine atoms from the complex 5a. The sec-
ond step (about 70%) corresponds to the loss of two ligands,
leaving behind the metal oxide as a residue.
sphere of the complexes. The above studies suggest neu-
tral and diamagnetic nature of all square planar Pd(II)
complexes.
DNA‑binding studies of palladium(II) complexes
5a–5d
Magnetic moments measurement has been carried out
at room temperature for all the Pd(II) complexes. Diamag-
netic behavior with zero B.M. µef value of Pd(II) complexes
suggests square planar geometry for all complexes. Elec-
tronic spectra of Pd(II) complexes have been recorded in
DMSO. The absorption peaks at around 300–390 nm and
200–280 nm are assigned to d–d transition and metal-to-
ligand charge-transfer transition (MLCT), respectively,
for square planar complexes [15]. The electrolytic nature
of palladium(II) complexes 5a–5d has been measured by
evaluating molar conductivities of complexes in DMSO.
The molar conductance (ɅM) values for the synthe-
sized palladium(II) complexes are found in the range of
9–12 cm2 Ω−1 mol−1, indicating non-electrolytic nature
and the absence of any counter ion outside the coordination
Absorption spectroscopy
Among all the spectroscopic techniques, absorption titra-
tion study is very useful, prominent, and reliable technique
to study the binding mode of small molecule to biopolymer
like DNA. Therefore, it is universally employed to evalu-
ate binding mode and binding constant. Absorption spectra
are very sensitive to change in the structure of compounds
and the structural change refects in the absorption maxima.
To determine the binding constant of compounds to her-
ring sperm DNA (HS DNA) interaction, we have monitored
the absorption maxima with gradual addition of DNA. For
non-covalent interaction, hypochromism with or without
1 3

K. P. Thakor et al.
redshift takes place [16, 17], while hyperchromism takes
place in electrostatic binding [18]. The observation of this
study refects the intercalative binding mode of compounds
to HS DNA. The strong stacking interaction is taking place
between the DNA base pair and chromophoric group of the
compound. This interaction slightly breaks the secondary
structure of DNA, which causes the hypochromism [19, 20].
The binding constant value (Kb) of ligand to DNA interaction
has been found in the range of 4.0×104 to 1.40×105 M−1
while Kb value of complex to DNA interaction has been
found in the range of 1.46×105 to 2.58×105 M−1. The per-
centage hypochromism (%H) observed in the above study
for DNA–metal complex binding is 9.35, 17.96, 17.14, and
11.01 for complex 5a–5d, respectively. Binding constants
of complexes are higher than antitumor drug cisplatin,
Binding constants are the measure of the stability, while
the free energy indicates the spontaneity or non-spontane-
ity of the binding. The free energy value (ΔG=−26.28 to
−30.87 kJ mol−1) of the compounds is negative. It indicates
the spontaneity of compound to DNA interaction. Binding
constant values and Gibb’s free energy of complexes are
higher than ligands. It indicates that upon complexation
binding ability of ligands is enhanced to a greater extent.
Binding constants (Kb) and free energy value (ΔG) of all
compounds are shown in Table 1.
From the above studies, complex 5b emerges as the most
efective DNA intercalator. So further studies, like evalua-
tion of temperature-dependent thermodynamic parameters,
i.e., change in enthalpy (∆H°), change in entropy (∆S°), and
Gibb’s free energy(∆G), have been calculated using van’t
Hof Eq. (2), to calculate the interaction forces between com-
plex 5b and DNA.
i.e., 5.51 × 104 M−1 and transplatin, i.e., 1.75 × 104 M−1
,
and lower than classical intercalator EB, i.e., 7 × 107 M−1
[21, 22]. The binding strength of the metal complexes
with DNA is comparable to reported N,S-donor thiosem-
icarbazone-derived Pd complexes such as [Pd(H-Msal-
mtsc)Cl(PPh3)] (Kb =1.44×105 M−1), [Pd(H-Msal-metsc)
Cl(PPh3)] (Kb = 1.69 × 105 M−1) [23] and [(Pd(H-Msal-
mtsc))2(µ-ppm)] (Kb =1.89×105 M−1) [24], while Kb values
of complexes are higher than reported complexes such as
[PdCl(dapdoH)] (Kb = 4.6 × 104 M−1) [25] and [Pd(Msal-
tsc)(PPh3)] (Kb =5.0×104 M−1) [23]. The intrinsic binding
constant Kb can be obtained from the ratio of the slope to the
intercept [26]. Figure 1 shows absorption titration spectra
of complex 5b upon the addition of increasing amount of
HS DNA. The absorption spectra of complex 5a, 5c, and
5d upon the addition of increasing amount of HS DNA are
shown in ESM 7.
◦
◦
ΔH
ΔS
ln Kb =
+
,
(2)
RT
R
◦
◦
◦
ΔG = ΔH − TΔS ,
(3)
where Kb is the intrinsic binding constant measured at the
diferent absolute temperature (301.5, 303, 308, 313 K) and
R is the gas constant in J mol−1 K−1. Enthalpy change and
entropy change have been evaluated from the linear van’t
Hof plot (ln Kb versus 1/T), where slope gives ∆H° and
intercept gives ∆S°. The Gibb’s free energy has been calcu-
lated from the Eq. (1) and verifed by Eq. (3). All thermo-
dynamic parameters for complex to DNA binding are shown
in Table 2. The DNA–complex interaction contribution to
thermodynamic parameters can be the result of hydrogen
bonding, hydrophobic interactions, conformational change
of DNA or diferent coupled processes (ion release, proton
transfer, changes in the hydration water, etc.). The positive
entropy and enthalpy changes arise from ionic and hydro-
phobic interactions, while the negative enthalpy and entropy
changes arise from van der Waals interactions and hydrogen
bond formation.
From the values of the binding constant (Kb), free energy
(ΔG) of complex to DNA binding has been calculated using
Eq. (1):
ΔG = − RT ln Kb.
(1)
Table 1 Binding constant Kb of complex to DNA interaction and
Gibb’s free energy (∆G°) of compounds
Compounds
Kb × 105/M−1
∆G°/kJ mol−1
4a
4b
4c
4d
5a
5b
5c
5d
0.40
0.49
1.40
0.52
1.71
2.58
1.46
2.38
−26.28 0.14
−26.77 0.12
−29.35 0.13
−26.90 0.11
−29.85 0.09
−30.87 0.08
−29.46 0.11
−30.67 0.11
Fig. 1 Representative absorption titration spectra of complex 5b upon
addition of increasing amount of HS DNA at ambient temperature.
Inset: plot of [DNA]/(εa–εf) vs. [DNA]
1 3

Bipyrazole-based palladium(II) complexes as DNA intercalator and artifcial metallonuclease
Table 2 Thermodynamic
parameters of complex 5b to HS
DNA binding at the diferent
temperature
T/K
Kb × 105/M−1
∆G°/kJ mol−1
∆H°/kJ mol−1
∆S°/J K−1 mol−1
301.5
303
308
2.60
2.38
1.85
1.35
−31.25 0.11
−31.18 0.14
−31.06 0.11
−30.74 0.12
−44.12 0.14
−42.64 0.13
313
The negative value of ΔG° and ΔH° for complex–DNA
interaction indicates that interaction is thermodynamic
favored and spontaneous. The negative values of ΔH° and
ΔS° represent the enthalpy favored exothermic interaction,
which suggests the involvement of hydrogen bond and van
der Waals forces as a driving force for DNA binding, which
is the considerable evidence for intercalative binding. Also,
the negative value of entropy change confers that intercala-
tion of complex into stacks of DNA base pairs results in
stable conformational changes in DNA and the reaction is
in a more ordered state, possibly due to the fxing of the
motional freedom of both the complex and DNA [27]. The
stabilization of DNA conformation after intercalation of
complex may be explained by the existence of dipole inter-
action induced due to polarization caused by Cl atoms, and
H-bonding, as many H-bonding sites are available with
ligand molecules, which contribute to large negative entropy
change.
viscosity for ligands is 4c>4d>4b>4a and for complexes
is 5b>5d>5a>5c. This order is similar to the binding con-
stant order of compounds.
Ethidium bromide (EB) displacement titration
The successive addition of complexes 5a–5d to EB–DNA
system at diverse “r” value [Complex]/[DNA]) resulted
in the remarkable decrease in fluorescence maxima of
EB–DNA system (Fig. 3, ESM 8). The displacement of EB
by complexes, at 610 nm suggests the intercalative bind-
ing mode of complex to DNA. The extent of fuorescence
intensity quenching is the measure of the strength of bind-
ing between DNA base pair and complexes [33–35]. In our
study, inner flter efect is corrected using Lakowics equation
[36].
The observed fuorescence quenching data give a linear
plot analogous to Stern–Volmer equation. The calculated
quenching constant (KSV) shows the ability of complexes to
displace the EB [37]. The binding constant (Kf), quenching
constants (KSV), number of binding sites (n) are listed in
Table 3. The Kf values calculated from the Scatchard plots
indicate that complex 5b exhibits the highest binding pro-
pensity and the order of binding strength of the complexes to
herring sperm DNA (HS DNA) is 5b>5d>5a>5c, which
is the same as observed in the UV titration study and vis-
cosity measurement study. The values of “n” are observed
around 1.0 indicating the 1:1 molar ratio between the com-
plexes and HS DNA.
Hydrodynamic volume measurement
To support the absorption titration study, we have carried
out relative viscosity measurement by successive addition
of complexes to a fxed concentration of DNA. The relative
viscosity of the DNA has been increased upon the addition
of free ligands as well as their respective complexes (Fig. 2)
due to the increase in the chain length of DNA with base
pair separation [28–30]. The observation supports absorp-
tion titration data and suggests intercalation of compounds
into stacks of DNA base pairs [31, 32]. Order of increase in
Fig. 2 A plot of relative viscos-
ity titration upon incremental
addition of complexes at 27 °C
1 3

K. P. Thakor et al.
Fig. 3 Emission spectra of
EB-bound DNA solutions in
the absence and presence of
increasing concentrations of
complex 5b (3.33–33.3 µM)
in Tris–HCl. [EB]=33.3 µM,
[DNA]=10.0 µM. The arrow
shows the change in intensity
upon increasing amounts of the
complex
Table 3 The quenching constant (KSV), binding constant (Kf), and the
number of binding sites (n) of complex to DNA binding
Complexes
Ksv/M−1
Binding sites/n
Kf/M−1
5a
5b
5c
5d
1.62×104
1.14×104
1.15×104
0.90×104
0.8157
1.0147
0.7869
0.9638
2.29×103
1.32×104
1.24×103
6.07×103
Molecular docking study
Molecular docking can provide some insight of preferred
binding mode between macromolecules and ligands. To
determine the relative binding energy of complex to DNA,
compounds have been docked to the B-DNA (PDB ID:
1BNA). In the docking method, out of 2000 docking solu-
tions, only the lowest relative binding energy of docked
structure is obtained. From the ensuing docked structures,
it is clear that all compounds ft well into the G–C rich
minor groove of the targeted DNA, stabilized by either
van der Waals interaction, H-bonding or through hydro-
phobic contacts. The negative entropy change value for
DNA–complex intercalation as suggested by tempera-
ture-dependent UV–Vis studies rules out the existence of
hydrophobic interaction.
Fig. 4 Representative image of molecular docking of complex 5b
with B-DNA
(5a), − 297.50 (5b), − 293.74 (5c), and − 296.61 (5d)
kJ mol−1.
Gel electrophoresis
This experiment is carried out to measure the infuence of
complexes on the electrophoretic mobility and cleavage of
the supercoiled form of pUC19 DNA [38, 39]. When circu-
lar plasmid DNA is subjected to electrophoresis, relatively
fast migration will be observed for the intact supercoil form
(form I, SC). If one strand of DNA is cleaved, the super-
coiled form is relaxed to slower moving open circular or
nicked form (form II, OC) and if both strands are cleaved
linear form (form III, LC) is generated which migrates in
between [40]. Complexes have been found to promote the
The DNA minor groove is the primary site of inter-
action between DNA and compounds, followed by inter-
calation of compounds as suggested by absorption titra-
tion, hydrodynamic measurement and EB displacement
studies. The representative docked structure is shown in
Fig. 4 and other docked structures are embedded in ESM
9. The relative binding energy of the docked structures
of ligands is − 277.62 (4a), − 281.35 (4b), − 285.66 (4c),
and −282.60 (4d) kJ mol−1 and of complexes are −293.95
1 3

Bipyrazole-based palladium(II) complexes as DNA intercalator and artifcial metallonuclease
Fig. 5 Gel electrophoresis image of cleavage of pUC19 DNA by syn-
Fig. 7 MIC values of compounds at fve diferent concentrations
thesized complexes
cleavage of pUC19 DNA from supercoiled form I to the
nicked form II (Fig. 5). As shown in Fig. 5, the intensity
of the circular supercoiled DNA (form I) band is found to
decrease, while that of the nicked (form II) band apparently
increases in lanes 3–8. The gel electrophoretic separation
shows the cleavage of pUC19 DNA induced by the com-
plexes. The complex 5b shows the cleavage of the pUC19
DNA similar to cisplatin and it possesses the highest cleav-
age percentage. Percentage cleavage of all compounds is
embedded in ESM 10. Percentage comparison of DNA
cleavage from SC form to OC form is shown in Fig. 6.
syndrome (TSS) and staphylococcal scalded skin syndrome
(SSSS) are associated with Staphylococcus aureus. Most of
the pathogens acquired resistance to the antibiotic; therefore,
the urgent need of new antibiotics has arisen. The antibacte-
rial activity of the compounds has been tested against two
Gram-negative bacterial strains: E. coli and P. aeruginosa,
and against three Gram-positive bacterial strains: B. subti-
lis, S. aureus, and S. marcescens using the broth dilution
method. The MIC (minimum inhibitory concentration) val-
ues are presented in ESM 11. The MIC values of all synthe-
sized ligands are in the range of 305–475 µM and complexes
are in the range of 50–105 µM (Fig. 7). Out of the eight
compounds, 5b displayed broad-spectrum antimicrobial
activity against all tested bacterial strains with MIC values
50–85 µM. Moreover, compound 5c showed the most potent
activity with MIC values of 55 µM for S. aureus and 5d
showed the most potent activity against E. coli with MIC
value 75 µM. As a result, we can also conclude that the
chelation of heterocycle to metal also reinforces the antimi-
crobial activity of heterocyclic molecules [41, 42].
Antimicrobial activity of ligands 4a–4d
and corresponding complexes 5a–5b
Escherichia coli cause gastrointestinal symptoms, ranging
from mild to severe and bloody diarrhea, mostly without
fever. Pseudomonas aeruginosa can cause infection in the
lung, bloodstream. Some Bacillus species can cause food
poisoning. S. marcescens causes central nervous system dis-
eases such as meningitis, urinary tract infection. Toxic shock
Cellular level cytotoxicity using S. pombe
Schizosaccharomyces pombe, the sixth model eukaryotic
organism, has been used as an important organism in study-
ing the cellular response to DNA damage and process of
replication because many genes of these organisms are
homologous to human diseases genes. Therefore, we have
used this organism to carry out primary in vivo cytotoxicity
test. The basic principle of staining is that staining dye could
not penetrate through the live cell wall but it could pen-
etrate through the dead cell wall. Because of this, the dead
S. pombe cells found in blue and live cells are transparent
under the microscope using trypan blue dye. Cellular level
cytotoxicity on S. pombe cells has been carried out using
compounds, transplatin and anticancer drug cisplatin used as
Fig. 6 Percentage cleavage of pUC19 DNA from SC to OC by com-
plexes in the form
1 3

K. P. Thakor et al.
Fig. 8 Percentage viability of
S. pombe cell upon treated with
compound at diferent concen-
tration
Fig. 9 S. pombe cell upon treated with compound observed under the
microscope
Fig. 10 LC50 value of compounds in µg cm−3
the standard. This result refects that toxicity is found to be
proportional to the concentration of the compounds (Fig. 8).
Compounds 5b and 5c are found to have the maximum
toxicity. After 17 h of the treatment, many of the S. pombe
cells died due to the toxic nature of the compounds (Fig. 9).
From the experiment, it is observed that the toxicity of
ligands is increased upon chelation with palladium(II).
from the plot. The LC50 of the compounds are in the range
of 8.31–16.80 µg cm−3 (ESM 12). The mortality of larvae
is found to increase with increasing the concentration of
compounds. From the data recorded, complex 5b is the most
potent among all the compounds. From Fig. 10, it is con-
cluded that the synthesized complexes are a good cytotoxic
agent than that of respective ligands. The order of potency
of compounds is 5b>5c>5d>5a.
In vitro cytotoxicity using brine shrimp
Brine Shrimp Lethality Bioassay technique is a useful tool
to carry out primary analysis for cytotoxicity of synthesized
compounds. The main advantages of this method are: this
method is economically benefcial, less time consuming than
other cytotoxicity test and more reliable. Procedure for this
method has been carried out according to the protocol of
Meyer et al. [43]. A plot of the log of sample concentration
versus percentage of mortality has been shown the linear
relationship and LC50 of compounds have been calculated
Conclusions
Substituted pyrazoline ligands coordinated to palladium(II)
have been synthesized, characterized and tested for their bio-
logical efect. Absorption titration, viscosity measurement,
EB displacement, and molecular docking studies results
suggest that all complexes are very efcient DNA inter-
calator. Hydrodynamic study data, Gibb’s free energy and
1 3

Bipyrazole-based palladium(II) complexes as DNA intercalator and artifcial metallonuclease
binding constant values obtained from absorption titration
study, and binding energy obtained from molecular dock-
ing study are consistent with the EB displacement data. All
the studies suggest compounds as efcient DNA intercala-
tor with the highest propensity observed for complex 5b.
The temperature-dependent thermodynamic parameters of
complex 5b support the above studies. The large negative
value of entropy change suggests that H-bonding between
DNA base pairs and ligand molecules and dipole interac-
tion induced due to polarization caused by Cl atoms may
stabilize the DNA conformational changes due to the inter-
calation of complex into the stacks of DNA base pairs. The
result of gel electrophoresis study reveals that all complexes
are very efective metallonuclease and the percentage cleav-
age of complexes 5b and 5d is compatible with cisplatin.
Chelating efect minimizes high lability and hydrolysis rate
of palladium complexes in the biological atmosphere and
these improve their clinical efect. All compounds exhibit
noticeable MIC value against fve diferent pathogens.
model number E-660A. Ethidium bromide displacement
experiments were performed using Fluoromax-4 spectro-
fuorometer, Horiba. Photo quantization of the gel after
electrophoresis was done using AlphaDigiDoc™ RT version
V.4.0.0 PC-Image software, California, USA.
5‑Chloro‑3‑methyl‑1‑phenyl‑1H‑pyrazole‑4‑carbaldehyde
(2) An air-dried fask charged with DMF (10 mmol) was
kept under the ice bath until the temperature reaches 0 °C
and then carefully dropwise addition of POCl3 (70 mmol)
was carried out. To generate Vilsmeier reagent, we stirred
the reaction mixture for half an hour with maintaining tem-
perature 0–5 °C. Then, 5-methyl-2-phenyl-2,4-dihydro-
3H-pyrazole-3-one (1, 30 mmol) was added to this mixture
and the reaction mass was refuxed at 60 °C. Formylation
was completed within 3–4 h. The completion of the reaction
was checked on the TLC using a mixture of ethyl acetate
and hexane (30:70) as a mobile phase. The reaction mix-
ture was poured into ice-cold water. Slowly lime yellow
colored product 5-chloro-3-methyl-1-phenyl-1H-pyrazole-
4-carbaldehyde (2) was generated. M.p.: 140–142 °C [44].
Experimental
Synthesis of chalcones 3a–3d
All the chemicals and solvents were of analytical grade and
used as purchased. Na2PdCl4, 2-acetylthiophene, 5-chloro-
2-acetylthiophene, 5-bromo-2-acetylthiophene, 3-bromo-
2-acetylthiophene, 5-methyl-2-phenyl-2,4-dihydro-3H-pyra-
zole-3-one, potassium tert-butoxide, HS DNA, and EDTA
were purchased from Sigma Aldrich Chemical Co. (India).
Phosphorus oxychloride (POCl3) and dimethylformamide
(DMF) were purchased from SDFCL, India. Agarose, Luria
Broth (LB), ethidium bromide (EB), bromophenol blue, and
xylene cyanol FF were purchased from Himedia (India). S.
pombe Var. Paul Linder 3360 was obtained from IMTECH,
Chandigarh.
To a solution of appropriate substituted 2-acetylthio-
phene (10 mmol) in 20 cm3 of methanol, freshly prepared
methanolic KOH solution (20%, w/v) was added, stirred
for 15 min and 5-chloro-3-methyl-1-phenyl-1H-pyrazole-
4-carbaldehyde (10 mmol) was added. The reaction mix-
ture was stirred at room temperature for 2 h. The reaction
mixture was poured into ice-cold water and neutralized with
dilute hydrochloric acid. The of-white precipitate was sepa-
rated by fltration and washed with distilled water to give
the crude product. The obtained product was recrystallized
from methanol. The purity of the products was checked on
TLC using a mixture of ethyl acetate and hexane (20:80) as
the mobile phase [45].
The 1H NMR (400 MHz) and 13C NMR (100 MHz) were
recorded with a Bruker Avance. IR spectra were recorded
on an FT-IR Shimadzu spectrophotometer in the range
4000–400 cm−1 with the sample prepared as KBr pellets.
C,H,N-elemental analyses were performed with a model
EURO EA3000 Elemental Analyser. Precoated silica gel
plates (silica gel 0.25 mm, 60 G F 254; Merck, Germany)
were used for thin-layer chromatography. The LC–MS spec-
tra were recorded using Thermo scientifc mass spectropho-
tometer (USA). The electronic spectra were recorded on a
UV-160A, UV–Vis spectrophotometer, Shimadzu, Kyoto
(Japan). The magnetic moments were measured by Gouy’s
method using mercury tetrathiocyanatocobaltate(II) as the
calibrant (χg =16.44×10−6 cgs units at 20 °C). The antibac-
terial study was carried out using laminar air fow cabinet
Toshiba, Delhi (India). The thermogram of complexes was
recorded with an SDT Q600 V20.9 Build 20. Conductance
measurement was carried out using conductivity meter
3‑(5‑Chloro‑3‑methyl‑1‑phenyl‑1H‑pyrazol‑4‑yl)‑
1‑(thiophen‑2‑yl)prop‑2‑en‑1‑one (3a, C17H13ClN2OS)
Prepared by the above method from 2-acetylthiophene and
5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde.
Yield: 81%; white solid; m.p.:>250 °C; 1H NMR (400 MHz,
CDCl3): δ=2.41 (s, 3H, –CH3), 6.82 (d, 1H, J=15.60 Hz,
3-H), 7.18 (d, 1H, J=15.20 Hz, 2-H), 7.25–7.73 (m, 8H, 2′,
3′, 4′, 2″′–6″′-H) ppm; 13C NMR (100 MHz, CDCl3): δ=
13.7 (–CH3), 111.4, 115.6, 121.5, 126.2, 127.1, 128.5,
132.7, 139.2 (CH), 118.1, 135.8, 141.5, 145.5, 154.7, 172.9
(C) ppm.
3‑(5‑Chloro‑3‑methyl‑1‑phenyl‑1H‑pyrazol‑4‑yl)‑1‑(5‑chlo‑
rothiophen‑2‑yl)prop‑2‑en‑1‑one (3b, C17H12Cl2N2OS) Pre-
pared by the above method from 2-acetyl-5-chlorothiophene
1 3

K. P. Thakor et al.
and 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-car-
(1H, dd, J=8.0 Hz, 8.0 Hz, 10.0 Hz, 4-Ha), 3.85 (1H, dd,
J=12.8 Hz, 12.0 Hz, 5.2 Hz, 4-Hb), 5.38 (1H, dd, J=8.0 Hz,
8.0 Hz, 4.0 Hz, 5-H), 6.86-7.72 (13H, m, 2′, 3′, 4′, 2″′-6″′,
2″′-6″′-H) ppm; 13C NMR (100 MHz, CDCl3): δ=13.3 (–
CH3), 41.8 (–CH2 of pyrazoline), 55.4 (–CH of pyrazoline),
112.5, 113.4, 119.6, 124.8, 125.2, 125.8, 126.6, 127.4,
128.1, 129.0, 129.3, 129.3 (aromatic CH), 113.9, 117.2,
120.0, 138.0, 143.6, 147.6, 156.9 (aromatic C) ppm; IR
(KBr): V =3055, υ(C–H)ar, 1542, υ(C=N), 1319, υ(C=C),
1118, υ(C–N), 1064, υ(C–Cl), 748, δ(C–H)ar, 871, υ(C–S–
C)thiophene cm−1; MS: m/z (%)=418.02 (100) [M+].
1
baldehyde. Yield: 77%; cream solid; m.p.: > 250 °C; H
NMR (400 MHz, CDCl3): δ = 2.40 (s, 3H, –CH3), 6.90
(d, 1H, J = 15.20 Hz, 3-H), 7.12 (d, 1H, J = 14.80 Hz,
2-H), 7.57–7.82 (m, 7H, 3′, 4′, 2″′–6″′-H) ppm; 13C NMR
(100 MHz, CDCl3): δ=13.6 (–CH3), 112.1, 115.8, 121.9,
127.9, 128.8, 134.5, 139.5 (CH), 118.7, 135.1, 136.7, 142.9,
146.8, 155.2, 174.2 (C) ppm.
1‑(5‑Bromothiophen‑2‑yl)‑3‑(5‑chloro‑3‑methyl‑1‑phe‑
nyl‑1H‑pyrazol‑4‑yl)prop‑2‑en‑1‑one (3c, C17H12BrClN2OS)
Prepared by the above method from 2-acetyl-5-bromothio-
phene and 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-car-
5′‑Chloro‑5‑(5‑chlorothiophen‑2‑yl)‑3′‑methyl‑1′,2‑diphen‑
yl‑3,4‑dihydro‑1′H,2H‑3,4′‑bipyrazole (4b, C23H18Cl2N4S) Pre-
pared by the above method from (E)-3-(5-chloro-3-methyl-
1-phenyl-1H-pyrazol-4-yl)-1-(5-chlorothiophen-2-yl)
prop-2-en-1-one (5 mmol) and phenyl hydrazine (5 mmol)
after 3 h reflux. Yield: 69%; yellow crystalline solid;
m.p.:>250 °C; 1H NMR (400 MHz, CDCl3): δ=2.19 (3H,
s, –CH3), 3.19 (1H, dd, J = 7.6 Hz, 7.6 Hz, 9.2 Hz, 4-Ha),
3.78 (1H, dd, J=12.8 Hz, 12.8 Hz, 4.4 Hz, 4-Hb), 5.38 (1H,
dd, J=7.6 Hz, 8.0 Hz, 5.2 Hz, 5-H), 6.84–7.56 (12H, m, 3′,
4′, 2″′-6″′, 2″′-6″′-H) ppm; 13C NMR (100 MHz, CDCl3):
δ=13.2 (–CH3), 41.1 (–CH2 of pyrazoline), 55.4 (–CH of
pyrazoline), 113.2, 113.4, 119.8, 124.7, 124.8, 126.5, 128.2,
129.0, 129.1, 129.2, 129.2, 130.2 (aromatic CH), 114.0,
117.3, 120.8, 131.8, 138.4, 143.9, 147.9, 157.6 (aromatic C)
ppm; IR (KBr): V =3055, υ(C–H)ar, 1596, υ(C=N), 1373,
υ(C=C), 1180, υ(C–N), 1049, υ(C–Cl), 756, δ(C–H)ar, 864,
υ(C–S–C)thiophene cm−1; MS: m/z (%)=452.97 (100) [M+].
1
baldehyde. Yield: 74%; yellow solid; m.p.: >250 °C; H
NMR (400 MHz, CDCl3): δ=2.41 (s, 3H, –CH3), 6.90 (d,
1H, J=15.60 Hz, 3-H), 7.30–7.81 (m, 8H, 2, 3′, 4′, 2″′-6″′-H)
ppm; 13C NMR (100 MHz, CDCl3): δ=13.9 (–CH3), 112.5,
117.1, 121.9, 126.1, 128.4, 132.8, 139.1 (CH), 118.5, 134.8,
136.8, 142.2, 145.2, 153.9, 172.4 (C) ppm.
1‑(3‑Bromothiophen‑2‑yl)‑3‑(5‑chloro‑3‑methyl‑1‑phe‑
nyl‑1H‑pyrazol‑4‑yl)prop‑2‑en‑1‑one (3d, C17H12BrClN2OS)
Prepared by the above method from 2-acetyl-3-bromothio-
phene and 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-car-
1
baldehyde. Yield: 72%; yellow solid; m.p.: > 250 °C; H
NMR (400 MHz, CDCl3): δ=2.42 (s, 3H, –CH3), 6.96 (d,
1H, J=16.00 Hz, 3-H), 7.25-7.81 (m, 8H, 2, 2′, 3′, 2″′–6″′-
H) ppm; 13C NMR (100 MHz, CDCl3): δ = 13.5 (–CH3),
111.2, 115.8, 121.4, 126.7, 128.6, 132.6, 138.8 (CH), 118.2,
134.7, 135.7, 142.2, 146.3, 155.4, 173.6 (C) ppm.
Synthesis of ligands 4a–4d
5‑(5‑Bromothiophen‑2‑yl)‑5′‑chloro‑3′‑methyl‑1′,2‑diphen‑
yl‑3,4‑dihydro‑1′H,2H‑3,4′‑bipyrazole (4c, C23H18BrClN4S)
Prepared by the above method from (E)-1-(5-bromothio-
phen-2-yl)-3-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)
prop-2-en-1-one (5 mmol) and phenyl hydrazine (5 mmol)
after 3 h refux. Yield: 75%; yellow crystalline solid; m.p.:
>250 °C; 1H NMR (400 MHz, CDCl3): δ=2.19 (3H, s, –
CH3), 3.19 (1H, dd, J=7.6 Hz, 7.2 Hz, 9.6 Hz, 4-Ha), 3.78
(1H, dd, J=12.8 Hz, 13.2 Hz, 4.0 Hz, 4-Hb), 5.38 (1H, dd,
J = 8.0 Hz, 7.2 Hz, 5.2 Hz, 5-H), 6.82-7.56 (12H, m, 3′,
4′, 2″′-6″′, 2″′-6″′-H) ppm; 13C NMR (100 MHz, CDCl3):
δ=13.3 (–CH3), 41.1 (–CH2 of pyrazoline), 55.4 (–CH of
pyrazoline), 113.4, 113.4, 119.8, 124.8, 125.6, 128.0, 128.2,
129.0, 129.1, 129.2, 130.2 (aromatic CH), 113.8117.3,
120.8, 129.6, 138.0, 143.3, 147.8, 157.2 (aromatic C)
ppm; IR (KBr): V =3047, υ(C–H)ar, 1596, υ(C=N), 1365,
υ(C=C), 1180, υ(C–N), 1049, υ(C–Cl), 756, δ(C–H)ar, 864,
υ(C–S–C)thiophene cm−1; MS: m/z (%)=497.01 (100) [M+].
To a solution of the appropriate enone 3a–3d (5 mmol) in
10 cm3 of methanol, phenyl hydrazine (5 mmol) and freshly
prepared methanolic potassium tert-butoxide (5 mmol)
solution were added. The reaction mixture was refuxed for
3–4 h. The reaction was monitored for every 60-min interval
with precoated silica TLC plates using a mixture of ethyl
acetate and hexane (20:80) as the mobile phase. The excess
of solvent was removed under reduced pressure and the reac-
tion mixture was poured into ice-cold water. The lime yellow
products precipitated out at low temperature were washed
with an excess of distilled water, recrystallized in a mini-
mum amount of methanol and dried under reduced pressure.
5′‑Chloro‑3′‑methyl‑1′,2‑diphenyl‑5‑(thiophen‑2‑yl)‑3,4‑di‑
hydro‑1′H,2H‑3,4′‑bipyrazole (4a, C23H19ClN4S) Prepared
by the above method from (E)-3-(5-chloro-3-methyl-1-
phenyl-1H-pyrazol-4-yl)-1-(thiophen-2-yl)prop-2-en-1-one
(5 mmol) and phenyl hydrazine (5 mmol) after 3 h refux.
5‑(3‑Bromothiophen‑2‑yl)‑5′‑chloro‑3′‑methyl‑1′,2‑diphen‑
yl‑3,4‑dihydro‑1′H,2H‑3,4′‑bipyrazole (4d, C23H18BrClN4S)
P r e p a r e d by t h e a b ove m e t h o d f r o m
1
Yield: 70%; yellow crystalline solid; m.p.: > 250 °C; H
NMR (400 MHz, CDCl3): δ = 2.21 (3H, s, –CH3), 3.26
1 3

Bipyrazole-based palladium(II) complexes as DNA intercalator and artifcial metallonuclease
(E)-1-(3-bromothiophen-2-yl)-3-(5-chloro-3-methyl-1-phenyl-
1H-pyrazol-4-yl)prop-2-en-1-one (5 mmol) and phenyl hydra-
zine (5 mmol) after 3 h refux. Yield: 67%; yellow crystalline
solid; m.p.: >250 °C; 1H NMR (400 MHz, CDCl3): δ=2.22
(3H, s, –CH3), 3.55 (1H, dd, J=6.8 Hz, 8.0 Hz, 10.4 Hz, 4-Ha),
4.11 (1H, dd, J=12.8 Hz, 12.8 Hz, 4.8 Hz, 4-Hb), 5.41 (1H,
dd, J=7.2 Hz, 8.0 Hz, 5.6 Hz, 5-H), 6.82-7.58 (12H, m, 2′, 3′,
2″′-6″′, 2″′-6″′-H) ppm; 13C NMR (100 MHz, CDCl3): δ=13.2
(–CH3), 42.8 (–CH2 of pyrazoline), 55.7 (–CH of pyrazoline),
113.4, 113.5, 119.9, 124.8, 125.7, 126.2, 128.2, 129.0, 129.2,
129.3, 132.0 (aromatic CH), 113.9, 117.7, 121.8, 130.2, 138.2,
145.4, 149.0, 159.1 (aromatic C) ppm; IR (KBr): V =3020,
υ(C–H)ar, 1542, υ(C=N), 1396, υ(C=C), 1134, υ(C–N), 1041,
υ(C–Cl), 717, δ(C–H)ar, 833, υ(C–S–C)thiophene cm−1; MS: m/z
(%)=497.10 (100) [M+].
4-Hb), 5.38 (1H, dd, J=8.0 Hz, 8.0 Hz, 5.2 Hz, 5-H), 6.84-
7.57 (12H, m, 3′, 4′, 2″′-6″′, 2″′-6″′-H) ppm; 13C NMR
(100 MHz, DMSO-d6): δ = 13.3 (–CH3), 41.1 (–CH2 of
pyrazoline), 56.0 (–CH of pyrazoline), 113.6, 113.7, 120.0,
124.8, 125.0, 126.3, 128.5, 129.4, 129.5, 129.6, 130.4 (aro-
matic CH), 114.2, 118.2, 121.1, 135.2, 139.1, 146.0, 149.0,
160.6 (aromatic C) ppm; IR (KBr): V = 3055, υ(C–H)ar,
1598, υ(C=N), 1380, υ(C=C), 1203, υ(C–N), 1118, υ(C–
Cl), 748, δ(C–H)ar, 871, υ(C–S–C)thiophene cm−1; MS: m/z
(%)=630.01 (100) [M+]; conductance: 12 Ω−1 cm2 mol−1;
UV–Vis: λmax (ε) = 280 (34,320), 415 (18,641) nm (M−1
cm−1).
Dichloro[5‑(5‑bromothiophen‑2‑yl)‑5′‑chloro‑3′‑methyl‑1′,2‑
diphenyl‑3,4‑dihydro‑1′H,2H‑3,4′‑bipyrazole]palladium(II)
(5c, C23H18BrCl3N4PdS) Prepared by the above method from
4c (5 mmol) and Na2PdCl4 (5 mmol). Yield: 64%; brownish
yellow solid; m.p.:>300 °C; 1H NMR (400 MHz, DMSO-
d6): δ=2.19 (3H, s, –CH3), 3.20 (1H, dd, J=8.4 Hz, 8.0 Hz,
10.0 Hz, 4-Ha), 3.79 (1H, dd, J=12.4 Hz, 12.0 Hz, 5.2 Hz,
4-Hb), 5.39 (1H, dd, J=8.0 Hz, 8.0 Hz, 5.2 Hz, 5-H), 6.82-
7.57 (12H, m, 3′, 4′, 2″′-6″′, 2″′-6″′-H) ppm; 13C NMR
(100 MHz, DMSO-d6): δ = 13.8 –CH3), 41.5 (–CH2 of
pyrazoline), 55.9 (–CH of pyrazoline), 113.5, 113.6, 120.0,
125.3, 126.0, 128.8, 129.0, 129.5, 129.6, 130.6 (aromatic
CH), 114.1, 118.3, 124.1, 129.8, 138.2, 146.8, 149.6, 160.0
(aromatic C) ppm; IR (KBr): V = 3078, υ(C–H)ar, 1600,
υ(C=N), 1380, υ(C=C), 1180, υ(C–N), 1064, υ(C–Cl), 748,
δ(C–H)ar, 871, υ(C–S–C)thiophene cm−1; MS: m/z (%)=474.52
(100) [M+]; conductance: 11 Ω−1 cm2 mol−1; UV–Vis: λmax
(ε)=280 (36,471), 415 (18,113) nm (M−1 cm−1).
General synthesis of complexes 5a–5d
The square planar metal complexes 5a–5d of type [Pd(4n)
Cl2] were synthesized by the reactions of Na2PdCl4 with the
respective pyrazoline ligands 4a–4d in a 1: 1 molar ratio in
1: 1 methanol chloroform system. The reaction mixture was
refuxed for 30 min followed by stirring for 24 h at ambient
temperature.
Dichloro[5′‑chloro‑3′‑methyl‑1′,2‑diphenyl‑5‑(thiophen‑
2‑yl)‑3,4‑dihydro‑1′H,2H‑3,4′‑bipyrazole]palladium(II)
(5a, C23H19Cl3N4PdS) Prepared by the above method from
4a (5 mmol) and Na2PdCl4 (5 mmol). Yield: 65%; brownish
yellow solid; m.p.: >300 °C; 1H NMR (400 MHz, DMSO-
d6): δ=2.22 (3H, s, –CH3), 3.26 (1H, dd, J=8.4 Hz, 7.6 Hz,
10.0 Hz, 4-Ha), 3.85 (1H, dd, J=12.0 Hz, 12.0 Hz, 6.0 Hz,
4-Hb), 5.38 (1H, dd, J=8.0 Hz, 8.0 Hz, 4.0 Hz, 5-H), 6.86-
7.73 (13H, m, 2′, 3′, 4′, 2″′-6″′, 2″′-6″′-H) ppm; 13C NMR
(100 MHz, DMSO-d6): δ = 13.9 (–CH3), 42.0 (–CH2 of
pyrazoline), 55.8 (–CH of pyrazoline), 112.8, 113.8, 120.0,
125.0, 126.0, 126.2, 127.1, 128.4, 129.4, 129.5, 129.5,
129.6 (aromatic CH), 114.0, 118.0, 120.2, 130.4, 138.2,
145.9, 149.0, 159.4 (aromatic C) ppm; IR (KBr): V =3062,
υ(C–H)ar, 1596, υ(C=N), 1380, υ(C=C), 1149, υ(C–N),
1072, υ(C–Cl), 748, δ(C–H)ar, 910, υ(C–S–C)thiophene cm−1;
MS: m/z (%)=595.98 (100) [M+]; conductance: 9 Ω−1 cm2
mol−1; UV–Vis: λmax (ε)=280 (32,641), 415 (14,924) nm
(M−1 cm−1).
Dichloro[5‑(3‑bromothiophen‑2‑yl)‑5′‑chloro‑3′‑methyl‑1′,2‑
diphenyl‑3,4‑dihydro‑1′H,2H‑3,4′‑bipyrazole]palladium(II)
(5d, C23H18BrCl3N4PdS) Prepared by the above method from
4d (5 mmol) and Na2PdCl4 (5 mmol). Yield: 62%; brownish
yellow solid; m.p.: >300 °C; 1H NMR (400 MHz, DMSO-
d6): δ=2.23 (3H, s, –CH3), 3.55 (1H, dd, J=7.6 Hz, 7.6 Hz,
10.0 Hz, 4-Ha), 4.11 (1H, dd, J=12.8 Hz, 12.8 Hz, 4.8 Hz,
4-Hb), 5.42 (1H, dd, J=8.0 Hz, 8.0 Hz, 4.8 Hz, 5-H), 6.82–
7.58 (12H, m, 2′, 3′, 2″′-6″′, 2″′-6″′-H) ppm; 13C NMR
(100 MHz, DMSO-d6): δ = 13.6 (–CH3), 42.8 (–CH2 of
pyrazoline), 55.9 (–CH of pyrazoline), 113.8, 113.9, 120.1,
125.1, 126.1, 126.9, 128.7, 129.5, 129.6, 129.7, 132.2 (aro-
matic CH), 114.0, 117.9, 120.2, 130.4, 138.2, 145.9, 149.0,
159.4 (aromatic C) ppm; IR (KBr): V = 3078, υ(C–H)ar,
1600, υ(C=N), 1380, υ(C=C), 1180, υ(C–N), 1072, υ(C–
Cl), 748, δ(C–H)ar, 871, υ(C–S–C)thiophene cm−1; MS: m/z
(%)=674.79 (100) [M+]; Conductance: 8 Ω−1 cm2 mol−1;
UV–Vis: λmax (ε) = 280 (30,471), 415 (10,679) nm
(M−1 cm−1).
Dichloro[5′‑chloro‑5‑(5‑chlorothiophen‑2‑yl)‑3′‑methyl‑1′,2‑
diphenyl‑3,4‑dihydro‑1′H,2H‑3,4′‑bipyrazole]palladium(II)
(5b, C23H18Cl4N4PdS) Prepared by the above method from
4b (5 mmol) and Na2PdCl4 (5 mmol). Yield: 71%; brownish
yellow solid; m.p.:>300 °C; 1H NMR (400 MHz, DMSO-
d6): δ=2.20 (3H, s, –CH3), 3.20 (1H, dd, J=7.6 Hz, 8.0 Hz,
10.0 Hz, 4-Ha), 3.78 (1H, dd, J=12.4 Hz, 12.0 Hz, 5.2 Hz,
1 3

K. P. Thakor et al.
of EB–DNA in the presence of quencher and [Q] is the
quencher concentration.
Complex‑DNA interaction by absorption
spectroscopy
To determine the strength of the interaction of complexes
with DNA, the value of the binding constant (Kf) was calcu-
lated using the Scatchard Eq. (5):
UV–Vis absorption titration was carried out using known
concentration of herring sperm DNA (HS DNA) in phos-
phate buffer and complexes (solution in DMSO). HS
DNA was dissolved in the phosphate bufer and the con-
centration of DNA is measured at 260 nm wavelength
(ε = 12,858 cm−1). The experiment procedure was carried
out according to our previously published literature [46].
(I0 − I)
log
= log Kf + n log[Q],
(5)
I
where I0 and I are the fluorescence intensities of the
EB–DNA in the absence and presence of diferent concen-
trations of complexes, respectively, and n is the number of
binding sites.
Hydrodynamic volume measurement
The hydrodynamic volume measurement study was car-
ried out using an ubbelohde viscometer kept under the
thermostatic bath at constant temperature 27 0.1 °C. The
detailed experiment was carried out according to the lit-
erature [47, 48].
Molecular docking of compounds with B‑DNA
To determine the theoretical binding energy of compounds
to DNA, docking study was performed using HEX 6.0 soft-
ware. The .pdb fles of complex coordinates were obtained
by converting their .mol fle using CHIMERA 1.5.1 soft-
ware. The structure of B-DNA (1 BNA: 5′-D(*CP*GP*CP
*GP*AP*TP*TP*CP*GP*CP*G)-3′) was obtained from the
Protein Data Bank (http://www.rcsb.org/pdb). All solvents
were removed before docking. Grid dimension 0.6 with FFT
mode 3D and correlation type shape only were used. The
other parameters were kept at their default values.
EB–DNA replacement titration
Complexes showed no fuorescence at room temperature
in solution or in the presence of HS DNA, and their bind-
ing to DNA could not be directly predicted through the
emission spectra. Intense fuorescent light is emitted from
EB in the presence of HS DNA due to the strong inter-
calation between adjacent DNA base pairs in the double
helix; therefore, EB is considered to be a typical indicator
of intercalation [49]. Hence, a competitive binding study
of each complex with EB was done to understand whether
the complex can displace EB from its HS DNA–EB com-
plex and the mode of DNA interaction with the com-
plexes. The HS DNA–EB complex was prepared by add-
ing 33.3 µM EB and 10 µM HS DNA in bufer (phosphate
bufer at pH 7.0). The possible intercalating efect of the
complexes was studied by adding a certain amount of a
solution of the complex step by step into a solution of the
DNA–EB complex. The infuence of the addition of each
complex to the DNA–EB complex solution was obtained
by recording the variation of fuorescence emission spec-
tra with emission wavelength at 610 nm (excitation wave-
length at 540 nm). The reaction time has been studied and
the results showed that 4 min was enough for stabiliza-
tion. So the change in fuorescence emission intensity was
measured within 4 min after each addition. The values of
the Stern–Volmer constant (KSV, in M−1) were calculated
according to the linear Stern–Volmer Eq. (4) and the plots
I0/I vs. [Q].
DNA cleavage and antibacterial studies
Gel electrophoresis was performed to monitor cleavage of
pUC19 DNA by synthesized complexes. The experiment
was performed according to the previously published litera-
ture [50]. The in vitro antibacterial activity of free ligands
and synthesized palladium(II) complexes was performed
according to the literature [51].
In vitro and in vivo cytotoxicity
In vitro and in vivo cytotoxicity was performed according to
our previously published literature [46].
Acknowledgements The authors are thankful to the Head, Department
of Chemistry, Sardar Patel University, Vallabh Vidyanagar, Gujarat,
India, for providing the laboratory facilities, U.G.C., New Delhi for
providing fnancial assistance of UGC BSR Grant no. C/2013/BSR/
Chemistry/59 and “BSR UGC One Time Grant”, vide UGC letter no.
F.19-119/2014 (BSR).
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