Biological applications of pyrazoline-based half-sandwich ruthenium(III) coordination compounds

Full text of DOI:10.1080/07391102.2016.1189360

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Biological applications of pyrazoline-based half-
sandwich ruthenium(III) coordination compounds
Jugal V. Mehta, Sanjay B. Gajera & Mohan N. Patel
To cite this article: Jugal V. Mehta, Sanjay B. Gajera & Mohan N. Patel (2016): Biological
applications of pyrazoline-based half-sandwich ruthenium(III) coordination compounds,
Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2016.1189360
To link to this article: http://dx.doi.org/10.1080/07391102.2016.1189360
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Date: 17 July 2016, At: 13:08

Journal of Biomolecular Structure and Dynamics, 2016
http://dx.doi.org/10.1080/07391102.2016.1189360
LETTER TO THE EDITOR
Biological applications of pyrazoline-based half-sandwich ruthenium(III) coordination
compounds
Jugal V. Mehta, Sanjay B. Gajera and Mohan N. Patel*
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar 388 120, Gujarat, India
Communicated by Ramaswamy H. Sarma
(Received 21 March 2016; accepted 10 May 2016)
1. Introduction
maximize their effectiveness as therapeutic agents. The
half-sandwich ruthenium(III) complexes with pyrazoline
ligand show comparison and contrasts to many of the
systems such as the actinomycins, porphyrins, Hoechst,
and benzo[a]pyrene derivatives and others in forming
complexes with the DNA or nucleic acids (Agarwal,
Chadha, & Mehrotra, 2015; Basu & Suresh Kumar,
2016; Bathaie, Ajloo, Daraie, & Ghadamgahi, 2015;
Jeon, Jin, Kim, & Lee, 2015; Monaselidze et al., 2015;
Nagaraj, Ambika, & Arunachalam, 2015).
Heterocyclic compounds have so far been synthe-
sized mainly due to the wide range of biological activi-
ties. Much attention has been paid to the synthesis of
heterocyclic compounds bearing nitrogen containing ring
system, like pyrazole mainly due to their higher pharma-
cological activity. Pyrazole and its derivatives are the
important structural motifs in heterocyclic chemistry and
occupy significant location in medicinal chemistry. They
exhibited a broad spectrum of pharmacological activities
(Mehta, Gajera, & Patel, 2016).
In the present study, a series of pyrazoline-based
half-sandwich organometallic ruthenium complexes have
been synthesized and well characterized. The synthesis
and structural characterization of compounds were car-
ried out with an aim to study their biological activity. A
satisfactory property of this type of ruthenium complexes
is the convenient piano-stool shape conferred by the
metal center. Such a characteristic provides considerable
opportunities for making new compounds with stimulat-
ing biological properties through functionalization and
rational ligand design. All synthesized complexes were
evaluated for their biological applications like DNA
binding, DNA cleavage, antimalarial, antimicrobial, and
cellular level cytotoxicity. These studies mainly focuses
on exploring the trend in DNA-binding affinities of
organometallic ruthenium complexes and the important
differences in some related properties.
The inclusion of biologically active ligands into metal
complexes deals much scope for the design of novel
drugs with improved and targeted activity. Studies on
such complexes indicate that new mechanisms of action
are favorable when combining the bioactivity of the
ligand with the properties inherent to the metal, leading
to the possibility of overcoming current drug resistance
pathways. It is well known that deoxyribonucleic acid
(DNA) is an important target in the organism for some
metal-based drugs or reagents. These reagents can inter-
act with DNA thereby changing the replication of DNA
and inhibiting the growth of the tumor cells. It has been
reported that the metal complexes can interact with DNA
non-covalently in the mode of intercalation, groove bind-
ing, and electrostatic effect.
Ruthenium complexes are also assumed to have a
biological mode of action that is significantly dissimilar
from those of platinum-based drugs. Additionally, the
rich synthetic chemistry, redox accessible oxidation
states, favorable ligand substitution reactions, and diverse
coordination geometries of ruthenium complexes have
been valuably considered for the design of new
pharmaceutical agents. Ruthenium-based organometallic
compounds are progressively gaining importance as
promising aspirants for the design of novel and
more effective metal-based drugs (Fricker, 2007). The
antitumor activity of many organometallic ruthenium
complexes is generally related to their enhanced DNA-
binding affinity, which involves covalent coordination or
simultaneous intercalation of extended aromatic groups
and specific hydrogen bonding depending on the particu-
lar type of ligands used. In this regard, diverse ligand
types are increasingly being developed and combined
with the ruthenium(III)–pentamethylcyclopentadienyl
ring moiety to enhance their DNA-binding properties, so
as to achieve different biological functions and to
*Corresponding author. Email: jeenen@gmail.com
© 2016 Informa UK Limited, trading as Taylor & Francis Group

2
J.V. Mehta et al.
2. Materials and methods
software, California (USA). The liquid chromatography-
mass spectroscopy (LC-MS) spectra were recorded using
make waters model micromass quattro micro API mass
spectrometer. The electronic spectra were recorded on a
UV-160A UV–vis spectrophotometer, Shimadzu, Kyoto
2.1. Materials and reagents
All the chemicals and solvents were of reagent grade and
used as purchased; double distilled water was used
throughout the studies. Pentamethylcyclopentadienyl
ruthenium(III) chloride [{(Cp*)Ru(μ-Cl)Cl}2], 2-acetyl
pyridine, 4-fluorobenzaldehyde, 4-chlorobenzaldehyde, 4-
bromobenzaldehyde, 4-methylbenzaldehyde, 3-fluoroben-
zaldehyde, 3-chlorobenzaldehyde, 3-bromobenzaldehyde,
phenyl hydrazine, potassium tert-butoxide, HS DNA, and
ethylenediaminetetraacetic acid disodium salt (edta) were
purchased from Sigma Aldrich Chemical Co. (India).
Agarose, Luria Broth (LB), ethidium bromide (EtBr), tris-
acetyl-edta (TAE), bromophenol blue, and xylene cyanol
FF were purchased from Himedia (India). Schizosaccha-
romyces Pombe Var. Paul Linder 3360 was obtained from
IMTECH, Chandigarh.
1
(Japan). The H NMR and 13C NMR were recorded with a
Bruker Avance (400 MHz). IR spectra were recorded on a
FT-IR Shimadzu spectrophotometer with sample prepared as
KBr pellets in the range of 4000–400 cm−1. C, H, and N ele-
mental analyses were performed using thermofinnigan at
CHN Lab, Panjab University, Chandigarh. Melting points
(°C, uncorrected) were determined in open capillaries on
ThermoCal10 melting point apparatus (Analab Scientific Pvt.
Ltd, India). Precoated silica gel plates (silica gel 0.25 mm,
60 G F 254; Merck, Germany) were used for thin-layer chro-
matography. 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),
Citizen balance. Antibacterial study was carried out by
means of laminar air flow cabinet Toshiba, Delhi (India).
The thermogram of complexes was recorded with a Mettler
Toledo TGA/DSC 1 thermo gravimetric analyzer.
2.2. Physical measurements
Photo quantization of the gel after electrophoresis was done
using AlphaDigiDoc™ RT. Version V.4.0.0 PC-Image
Scheme 1. Synthesis of tri-substituted pyrazoline ligands (5a‒5g) and its complexation with ruthenium metal ion.

Half-sandwich ruthenium(III) coordination compounds
3
2.3. Synthesis of the pyrazoline ligands and Ru(III)
complexes
Flow time for buffer alone was measured and was
termed as t0. The detailed experimental process is
reported in published literatures (Mehta et al., 2015).
Detailed process for the synthesis and characterization of
pyrazoline ligands as well as their Ru(III) complexes are
shown in the Supplementary material 1. The proposed
reaction for the synthesis of ligands (5a–5g) and Ru(III)
2.4.2.3. By molecular docking with HS DNA. The rigid
molecular docking study has been performed using HEX
8.0 software to determine the orientation of the
organometallic Ru(III) complexes binding to DNA.
Docking was performed and the most stable configura-
tion was chosen as input for investigation. The coordi-
nates of metal complexes were taken from their
optimized structure as a .mol file and were changed to
.pdb format. HS DNA used in the experimental work
was too large for current computational resources to
dock, therefore, the structure of the DNA of sequence d
1
complexes (6a–6g) is shown in scheme 1. H NMR and
13C NMR spectra of ligands are shown in Supplementary
materials 2 and 3, respectively.
2.4. Biological study of synthesized compounds
2.4.1. In vitro antibacterial study
The in vitro antibacterial activities of pyrazoline ligands
and organometallic ruthenium complexes were tested
against two Gram positive: Staphylococcus aureus,
Bacillus subtilis and three Gram negative: Serratia mar-
cescens, Escherichia coli, Pseudomonas aeruginosa
micro-organisms. The MIC value was determined by
broth dilution technique. The detailed experimental pro-
cess is reported in newly published literatures (Mehta,
Gajera, & Patel, 2015).
(ACCGACGTCGGT)2 (PDB id: 423D,
a familiar
sequence used in oligodeoxynucleotide study) obtained
from the Protein Data Bank (www.rcsb.org/pdb). All
calculations were carried out on an Intel CORE i5-, 2.5-
GHz-based machine running MS Windows 8, 64bit as
the operating system. By default, parameters were used
for the docking proposal with correlation type shape
only, FFT mode at 3D level, grid dimension of 6 with
receptor range 180 and ligand range 180 with twist range
360 and distance range 40.
2.4.2. DNA-binding study
2.4.2.1. By absorption titration. DNA-mediated hypo-
chromic and bathochromic shifts under the influence of
the organometallic ruthenium complexes were measured
with the help of UV–vis absorbance spectra. UV–vis
spectral titration of organometallic ruthenium complexes
(in DMSO) with HS DNA in phosphate buffer was
carried out to inspect the binding mode for the com-
plexes. The concentration of HS DNA was determined
by measuring absorbance at 260 nm and using
12,858 L mol−1cm−1 as the molar extinction coefficient
value. In this experiment, fixed amount of DNA solution
(100 μL) in phosphate buffer was added to sample cell
holding in definite concentration of complex solution
(20 μM) and reference cell to nullify the effect of HS
DNA, and allowed to incubate for 10 mins prior to the
spectra being recorded. DMSO was also added into the
reference cell as a control to nullify the effect of DMSO.
The intrinsic binding constant, Kb, was determined by
equation reported in published literatures (Mehta et al.,
2015).
2.4.3. DNA cleavage study by agarose gel
electrophoresis
Gel electrophoresis of pUC19 DNA was carried out in
TAE buffer (0.04 M tris-acetate, pH 8, .001 M edta). The
15 μL reaction mixture containing 300 mg/L plasmid
DNA in TE buffer (10 mM tris, 1 mM edta, pH 8.0) and
200 μM complex solution. Reactions were allowed to pro-
ceed for 3 h at 37°C in dark and reactions were satiated by
the addition of 5 μL loading buffer (0.25% bromophenol
blue, 40% sucrose, 0.25% xylene cyanol, and 200 mM
edta). The aliquots were loaded directly onto 1% agarose
gel and electrophoresed at 50 V in 1× TAE buffer. Gel
was stained with 0.5 mg/L ethidium bromide and was
photographed on a UV illuminator. The percentage of each
form of DNA was determined. After electrophoresis, the
proportion of DNA in each fraction was estimated
quantitatively from the intensity of the bands using
AlphaDigiDoc™ RT. Version V.4.0.0 PC-Image software.
The degree of DNA cleavage activity was expressed in
terms of the percentage of conversion of the SC-DNA to
OC-DNA and L-DNA according to the equation reported
in published literatures (Mehta et al., 2015).
2.4.2.2. By viscosity measurements. An Ubbelohde
viscometer maintained at a constant temperature of
27 0.1°C in a thermostatic jacket, was used to measure
the flow time of DNA in phosphate buffer (Na2HPO4/
NaH2PO4, pH 7.2) with accuracy of 0.01 s and precision
of 0.1 s. DNA samples, approximately 200 base pairs in
average length, were prepared by sonicating in order to
minimize complexities arising from DNA flexibility.
2.4.4. In vitro cytotoxic study using brine shrimp
lethality bioassay (BSLB)
Brine shrimp (Artemia cysts) eggs were hatched in a
shallow rectangular plastic dish (22×32 cm), filled with

4
J.V. Mehta et al.
artificial seawater, which was prepared with commercial
salt mixture and double-distilled water. An unequal parti-
tion was made in the plastic dish with the help of a per-
forated device. Approximately, 50 mg of eggs were
sprinkled into the large compartment and was opened to
ordinary light. After two days, nauplii were collected by
a pipette from the lighted side. The detailed experimental
process is reported in newly published literatures (Mehta
et al., 2015).
3.2. Biological study of synthesized compounds
3.2.1. In vitro antibacterial screening
The antimicrobial activity of free pyrazoline ligands
(5a–5g) and synthesized complexes (6a–6g) has been
tested against five different bacteria in terms of the mini-
mum inhibitory concentration (MIC). Results shows that
all the ruthenium complexes are active against all the
micro-organisms than those of respective pyrazoline
ligands (Supplementary material 5). The MIC values of
all synthesized complexes ranges from 67 to 102 μM.
The mechanism of toxicity of the complexes may be
ascribed to the increase in the lipophilic nature of the
complexes arising from chelation. Another mechanism of
toxicity of these complexes to micro-organisms may be
due to the inhibition of energy production or ATP pro-
duction, by inhibiting respiration or by the uncoupling of
oxidative phosphorylation.
The increase in antimicrobial activity of metal com-
plexes can be the result of: (I) chelate effects, (II) nature
of the ligands, (III) total charge of the complex, (IV) nat-
ure of the ion neutralizing the complex, and (V) nuclear-
ity of the metal center in the compounds. The inhibition
activity seems to be governed in certain degree by the
facility of coordination at the metal center as well as
electronic nature of the ligands. The lower MIC value of
complex (6a) is probably due the greater lipophilic nat-
ure of the complex (6a) as well as might be due to pres-
ence of fluorine atom as a substituent. Presence of more
electronegative environment in complex (6a) enhances
its antimicrobial property. Such increased activity of the
metal chelates and possible mode of increased toxicity of
the Ru(III) complexes compared to that of the free pyra-
zoline and diverse biological activity due to coordination
of the pyrazoline to the metal can be explained on the
basis of Tweedy’s chelation theory and Overtone’s con-
cept (Mehta et al., 2016).
2.4.5. Cellular level bioassay using S. pombe cells
Cellular level bioassay was done using S. pombe cells,
which were grown in liquid yeast extract media in
150 mL Erlenmeyer flask containing 50 mL of yeast
extract media. Flask was incubated at 30°C on shaker
at 150 rpm till the exponential growth of S. pombe
obtained (24–30 h). Then the cell culture was treated
with the different concentrations (2, 4, 6, 8, 10 mg/L)
of synthesized complexes, free ligands, and also with
dimethylsulphoxide (DMSO) as a control and further
allowed to grow for 16–18 h. Next day, by centrifuga-
tion at 10,000 rpm for 10 mins; treated cells were col-
lected and dissolved in 500 μL of PBS. The 80 μL of
yeast culture dissolved in PBS and 20 μL of 0.4% try-
pan blue prepared in PBS were mixed and cells were
observed in a compound microscope (40X). The dye
could enter the dead cell only so they appeared blue,
whereas live cells resisted the entry of dye. The
number of dead cells and number of live cells were
counted in one field. Cell counting was repeated in two
more microscopic fields and average percentage of
cells which died due to synthesized compounds were
calculated.
2.4.6. In vitro antimalarial study using strain of
Plasmodium falciparum
All the synthesized organometallic ruthenium complexes
and ligands were screened for their antimalarial study
against the P. falciparum strain and was acquired from
Shree R. B Shah Mahavir superspeciality hospital, Surat,
Gujarat, India was used in in vitro tests. The detailed
experimental process is reported in published literatures
(Mehta et al., 2015).
3.2.2. DNA-binding study
Since DNA is the crucial target for many metal-based
drugs, and DNA-binding is the critical step for the study
of effective metal-based drugs, which is of prominence
in illuminating the mechanisms involved in the site-
specific recognition of DNA, and designing new types of
pharmaceutical molecules, the binding behaviors of the
synthesized compounds toward DNA are discovered both
theoretically and experimentally with the aid of different
procedures and techniques.
3. Results and discussion
3.1. Characterization of Ru(III) complexes
Characterization of Ru(III) complexes by magnetic
moments, electronic spectra, conductance measurements,
LC–MS spectrum analysis, and thermal analysis tech-
niques are shown in Supplementary material 4.
3.2.2.1. By absorption titration. Electronic absorption
spectroscopy is one of the most useful techniques for the
study of binding mode and binding strength of the metal
complexes with DNA. The binding ability of the

Half-sandwich ruthenium(III) coordination compounds
5
complexes with HS DNA can be characterized by
measuring their effects on absorption spectra. An absorp-
tion spectrum of synthesized complexes with HS DNA
was recorded for a constant concentration of complexes
i.e. 20 μM with varying concentration of DNA i.e.
100 μM to obtain different DNA/complex mixing ratio is
shown in Figure 1.
The magnitude of the hypochromism and red shift is
commonly found to depend on the strength of the inter-
calative interaction. The extent of spectral change is
related to the strength of binding. The absorption spectral
alterations are observed in the intraligand charge transfer
(ILCT) band around 260–290 nm for the examination of
DNA-binding mode and strength. As the DNA concentra-
tion is increased, the ILCT transition bands of synthesized
complexes exhibit hypochromicity [hypochromicity,
H% = [(Afree − Abound)/Afree] × 100%] of about 18.30
0.74%−22.63 0.86%, and bathochromicity of 2–4 nm.
The complex (6a) has highest percentage of hypochromic-
ity (22.63 0.86%). These spectral characteristics may
suggest a mode of binding that involves a stacking
interaction between the aromatic chromophore and the
DNA base pairs.
Kb values of complexes are comparable with classical
intercalators ethidium bromide
(7.16 × 107 M−1),
but higher than [RuCl(AsPh3)L3] (3.2 × 104 M−1)
(Prakash, Manikandan, Viswanathamurthi, Velmurugan, &
Nandhakumar, 2014) and from the Kb value and observed
red shift, it is clear that the complexes bind to the DNA by
intercalation mode and complex (6a) has the highest bind-
ing ability (Supplementary material 6) than the other com-
plexes due to an existence of fluorine atom. Presence of
fluorine atoms act as not only chemical isosteres for the
oxygen atoms in the heterocyclic base of thymidine, but
shows strong affinity of binding due to strong van der
Waals force of interaction between fluorine and hydrogen
atom compared to other substituents. As a result, it shows
superior antimicrobial and DNA interaction activity than
other compounds. The binding mode is further confirmed
by viscosity measurement study.
3.2.2.2. By viscosity measurements. Viscosity measure-
ment is regarded as the most critical means for studying
the binding mode of metal complexes with DNA in solu-
tion and provides stronger fights for intercalative binding
mode.
In order to elucidate the binding strength of the com-
plexes, the DNA-binding constants Kb were determined by
monitoring the changes of absorbance in the ILCT band
with increasing concentration of HS DNA. The Kb values
of ruthenium complexes are found in the range 1.04
( 0.08) × 105‒7.28 ( 0.25) × 105 M−1 and are higher than
that of free pyrazoline ligand (Supplementary material 6).
The effects of ruthenium complexes on the viscosity
of HS DNA are shown in Figure 2. As illustrated in this
figure, on increasing the amount of the complexes the rel-
ative viscosity of HS DNA increases progressively, which
confirms that the complexes are bound to HS DNA by
intercalation. This phenomenon may be explained by the
insertion of the compounds between the DNA base pairs,
leading to an increase in the separation of base pairs at
intercalation sites and thus an increase in overall DNA
length. It is clear from the figure that all these complexes
show an increase in the relative viscosity of HS DNA.
The viscosity results may reveal the tendency of each
Figure 1. Absorption spectral changes on addition of HS
DNA to the solution of complex 6a after incubating it for
10 mins at room temperature in phosphate buffer (Na2HPO4/
À
Á
NaH2PO4, pH 7.2). Inset: plot of ½DNAꢀ=ð ea ꢁ ef ) vs.
[DNA]. Error bars represent standard deviation of three
replicates. (Arrow shows the change in absorption with increase
in concentration of DNA).
Figure 2. Effect on relative viscosity of HS DNA under the
influence of increasing amounts of complexes at 27( 0.1)°C in
phosphate buffer at pH 7.2. Error bars represent standard
deviation of three replicates.

6
J.V. Mehta et al.
ligand to intercalate into DNA base pairs. The increase in
DNA viscosity observed in the complexes suggests a
classical intercalative mode. The increase in the degree of
viscosity of all compounds depends on their affinity to
DNA, with an order as follows: 6a > 6b > 6c > 6d =
6f > 6g > 6e > ligands (Supplementary material 7). This
behavior is similar to that of ruthenium complexes
reported by Haq et al. (1995).
nuclease. The general accepted mechanism of the DNA
hydrolysis reaction is a nucleophilic attack at the DNA
phosphate backbone, to form a five-coordinate intermedi-
ate, which can be stabilized by the catalyst. Subsequent
cleavage of either the 3′-PO or the 5′-PO results in a
strand scission. After this nucleophillic attack, one group
leaves as an alcohol (Sangeetha Gowda, Mathew,
Sudhamani, & Naik, 2014).
The principle of this method is that molecules
migrate in the gel as a function of their mass, charge,
and shape, with supercoiled DNA migrating faster than
open circular molecules of the same mass and charge.
The native DNA remains in the supercoiled (SC) form,
also known as covalently coiled coil DNA, here desig-
nated as Form I. Single strand cleavage results in so
called nicked or open circular (OC) form of DNA
(designated as Form II), whereas the double-strand cleav-
age results in linear (L) form of DNA (designated as
Form III).
3.2.2.3. By molecular docking with HS DNA. In order
to test the intercalation binding mode and DNA
intercalating potential of the studied compounds, we
investigated their interactions with the double-stranded
helical DNA. Molecular docking studies of the ruthe-
nium complexes with the DNA duplex of sequence d
(ACCGACGTCGGT)2 were performed to predict the
chosen binding site along with the preferred orientation
of complex inside the DNA helix (Supplementary
material 8).
The study shows that the complexes under investiga-
tion interact with DNA via an intercalation mode involv-
ing outside edge stacking interaction with oxygen atom
of the phosphate backbone. From the ensuing docked
structures, it is clear that the complexes fit well into the
intercalative mode of the targeted DNA and A−T rich
region stabilized by van der Waal’s interaction and
hydrophobic contacts. The resulting binding energies of
docked complexes (6a−6g) are found to be −250.95,
−249.33, −242.78, −259.73, −262.87, −261.64, and
−254.93 kJ mol−1, respectively. Also, resulting binding
energies of docked pyrazoline ligands 5a–5g are found
to be −237.34, −220.06, −238.09, −224.23, −238.05,
−228.28, and −230.15 kJ mol−1, respectively.
Figure 3 illustrates the cleavage of pUC19 DNA
induced by the compounds under aerobic conditions.
This clearly shows that the relative binding efficacy of
complexes to DNA is much higher than the binding effi-
cacy of ruthenium salt and free pyrazoline ligands
(Supplementary material 9). The difference in DNA
cleavage efficiency of complexes was due to the differ-
ence in binding affinity of complexes to DNA. The simi-
lar behavior of ruthenium complexes with plasmid DNA
was shown by reported compounds of type cis,fac-[RuCl
(dmso-S)3(L)] (Gaur et al., 2011).
3.2.4. In vitro cytotoxic study using BSLB
The BSLB is considered as a useful method for prelimi-
nary calculation of toxicity of compounds and develop-
ment in the assay procedure of bioactive compound,
which indicates cytotoxicity as well as a wide range of
pharmacological activities (such as antiviral, insecticidal,
anticancer, pesticidal, etc.) of the compounds. All the
synthesized compounds were studied for their cytotoxic-
ity using the protocol of Meyer et al. (1982). The
method is inexpensive, rapid, reliable, and economical.
Results for the lethality are noted in terms of deaths
of larvae. The mortality rate of brine shrimp nauplii is
found to increase with increasing concentration of com-
plexes. A plot of the log of sample’s concentration ver-
sus percentage of mortality showed a linear correlation.
From the graph, the LC50 values of the compounds are
calculated, and they were found in the range of
5.714−83.368 mg/L (Supplementary material 6). Com-
plex (6a) is the most potent amongst all the compounds.
From the data recorded, it is concluded that the synthe-
sized complexes are good cytotoxic agent than that of
respective ligands. The order of potency of compounds
is 6a > 6b > 6d > 6c > 6g > 6e > 6f > pyrazole ligands.
3.2.3. DNA cleavage study by agarose gel
electrophoresis
The cleavage activity on supercoil form of pUC19 DNA
by synthesized compounds has been monitored by agar-
ose gel electrophoresis. To investigate the DNA-binding
property of the synthesized pyrazoline and ruthenium
complexes that are associated with further pharmacologi-
cal activities, chemical nuclease activity assay has been
performed. The DNA cleavage can occur by two major
pathways, i.e. oxidative cleavage and hydrolytic cleav-
age: (Mehta et al., 2015) (I) oxidative DNA cleavage
involves either oxidation of the deoxyribose moiety by
abstraction of sugar hydrogen or oxidation of nucle-
obases. (II) Hydrolytic DNA cleavage involves cleavage
of phosphodiester bond to generate fragments which can
be subsequently relegated. Hydrolytic cleavage which
started in a modest way of converting supercoil (SC)
form of DNA to the open circular (OC) form and last in
linear (L) form, is now being used for identifying the
percentage of cleavage as a function of concentration of

Half-sandwich ruthenium(III) coordination compounds
7
Figure 3. Photogenic view of cleavage of pUC19 DNA (300 μg/cm3) with series of compounds using 1% agarose gel containing
0.5 μg/cm3 EtBr. All reactions were incubated in TE buffer (pH 8) at a final volume of 15 mm3 for 24 h at 37°C.
Table 1. Effect of compounds on viability of S. Pombe cells at different concentrations with error uncertainty in the value 5%.
Concentration (mg/L)
2
4
6
8
10
Compounds
% Viability
5a
5b
5c
5d
5e
5f
5g
6a
6b
6c
6d
6e
6f
6g
72
73
74
76
79
82
85
62
64
67
70
72
73
75
2
2
2
2
2
3
3
1
1
2
2
2
2
2
68
69
67
69
75
77
78
58
60
61
63
66
66
70
2
2
2
2
2
2
3
1
1
1
1
2
2
2
62
65
64
66
72
74
72
55
57
56
56
60
61
64
96
97
1
1
1
1
2
2
2
1
1
1
1
1
1
1
4
4
59
61
61
62
68
72
70
50
54
52
51
55
56
56
1
1
1
1
1
2
2
1
1
1
1
1
1
1
56
1
1
1
1
1
2
2
1
1
1
1
1
1
1
58
59
60
63
70
67
46
47
48
48
49
50
50
DMSO
Untreated cells
The degree of lethality is found to be directly propor-
tional to the concentration of the compounds. The mor-
tality rate of brine shrimp nauplii is found to increase
with increasing concentration of complexes.
chemically synthesized compounds could be easily
observed by vital staining (Supplementary material 10).
The toxicity is found to vary with the type of substituent
present and concentrations of the synthesized com-
pounds. General observation is that as concentration of
compounds increased, the cytotoxicity is also increased.
After treatment, many of the S. pombe cells are killed
due to toxic nature of the compound. Complex (6a) is
the most active amongst all the compounds.
3.2.5. Cellular level bioassay using S. pombe cells
The cellular level cytotoxicity bioassay of free pyrazoline
(5a–5g) and synthesized ruthenium complexes (6a–6g)
has been tested against S. pombe in terms of the % via-
bility. Table 1 shows that all the ruthenium complexes
are active against S. pombe cells than those of respective
pyrazoline ligands.
3.2.6. In vitro antimalarial study using strain of
P. falciparum
S. pombe cells have become an important tool to
study cell biology due to its eukaryotic and fairly big
size characteristics. Cell death caused by toxicity of the
Malaria is one of the parasitic infections that cause vast
medical, financial, and emotional problem in the world.
P. falciparum is the parasite responsible for most malaria

8
J.V. Mehta et al.
cases up to 80%, which often proves harmless. As part
of this search for novel drugs against malaria, we report
encouraging results for new compounds resulting from
the modification of pyrazoline moiety by coordination to
ruthenium metal centers; the new complexes are highly
active against a chloroquine resistant strain of P. falci-
parum. The results of the pharmacological screening are
expressed as the drug concentration resulting in 50%
inhibition (IC50) of parasite growth. The mean values of
IC50 in mg/L for all complexes and respective ligands
are given in Supplementary material 6.
Results of data show that all complexes exhibit
good antimalarial activity than respective ligands
(0.54‒2.15 mg/L). The highest activity is observed for
the complex (6a) which exhibits the lowest IC50 value
(0.54 mg/L). It is thus clear that the combination of pyra-
zoline ligand and ruthenium metal in a single molecule
does produce an enhancement of the activity against
resistant strains of the parasite, demonstrating the validity
of our concept in the search for novel antimalarial drugs
capable of overcoming resistance. The IC50 values of all
ruthenium complexes are comparable to [Ru(A)2(B)]Cl2
(10 mg/L) (Anchuri et al., 2013).
in UV–vis absorption curve and suggests classical
intercalation for all the ruthenium complexes, where
complex (6a) binds more strongly than rest of the com-
plexes. Molecular docking studies of the complexes with
the DNA duplex of sequence d(ACCGACGTCGGT)2
were performed to predict the chosen binding site, which
suggests intercalation between complex and DNA base
pairs. The DNA cleavage study of pUC19 shows that all
complexes have high cleavage ability than metal salt and
ligands. From the biological activity, complex (6a) is
found to be more potent than other due to the presence
of fluorine atom at fourth position of aromatic ring as a
substituent. Presence of more electronegative environ-
ment in complex (6a) improves its biological property.
The preliminary studies encourage for carrying out fur-
ther in vivo experiments. The results are of importance
toward further designing and developing ruthenium-
based complexes and systematic assessment of biological
activity for their potential applications as therapeutic
agents.
Supplementary material
The supplementary material for this paper is available online at
http://dx.doi.org/10.1080/07391102.2016.1189360
4. Conclusions
A series of substituted pyrazoline-based half-sandwich
organometallic ruthenium complexes have been synthe-
sized and well characterized. The synthesis and structural
characterization of compounds were carried out with an
aim to study their biological activity. Likewise, arene
ring, pentamethylcyclopentadienyl ring derivatives
occupy three coordination sites at the octahedral metal
center and provide to the metal a lipophilic protecting
face. On the other hand, the remaining three coordination
sites allow the introduction of ligands with hydrophilic
character. By analogy with their structure to a piano
stool, these are known as piano stool complexes, with
the metal center being coordinated by pentamethylcy-
clopentadienyl ring, a chlorido ligand and a chelating
pyrazole ligand. The molar conductivities values of all
organometallic Ru(III) complexes are indicates, one
counter ion present outside the coordination sphere
which accomplish all complexes are ionic in nature. The
antimicrobial activity of the complexes has been tested
on five different micro-organisms and the results show
an enhanced biological activity in relation to the free
ligands. All the ruthenium complexes show good in vitro
cytotoxic as well as in vitro antimalarial activity. A com-
parative study of cellular level cytotoxicity values of the
all compounds indicates that the metal complexes show
better activity against S. pombe cells compared to the
pyrazoline ligand. The viscosity data are in good agree-
ment with bathochromicity and hypochromicity observed
Acknowledgements
The authors are thankful to the Head, Department of Chemistry,
Sardar Patel University, Vallabh Vidyanagar, Gujarat, India, for
providing the laboratory facilities, Dhanji P. Rajani, microcare
laboratory, Surat for in vitro antimalarial activity, U.G.C., New
Delhi for providing financial assistance of UGC BSR grant
number C/2013/BSR/Chemistry/59 and “BSR UGC One Time
Grant,” vide UGC letter No.F.19-119/2014 (BSR).
Disclosure statement
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper.
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