DJ Journal of Engineering Chemistry and Fuel (DJ JECF)
Ruthenium(III) Thiosemicarbazone Complexes: Synthesis,
Characterization, DNA Binding, Antibacterial, Invitro Anticancer and
Antioxidant Studies
*
1
K Sampath1, C Jayabalakrishnan2
Department of Chemistry, Kumaraguru College of Technology, Coimbatore, 641049, Tamil Nadu,
India.
2
Post Graduate and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya
College of Arts and Science, Coimbatore, 641020, Tamil Nadu, India.
*
[email protected], +919965671803.
ABSTRACT
Ruthenium(III) complexes of the type [RuBr2(PPh3)2L] (where L = monobasic bidentate
thiosemicarbazone ligand) have been synthesized. Structural features of the complexes were
determined by various physico-chemical and spectral techniques. DNA binding of the complexes was
investigated by absorption spectroscopy which indicated that the complexes bind to DNA via
intercalation. The complexes have shown significant antibacterial activity than the ligands against a
panel of bacteria. The efficiency of the complexes to arrest the growth of MCF-7 cancer cell line has
been studied along with cell viability test under in vitro conditions. Finally, antioxidative property of
the complexes against DPPH radical was investigated.
Keywords: Ruthenium(III) complexes, Thiosemicarbazones, DNA binding, Cytotoxicity, DPPH.
[7, 8] Moreover, some thiosemicarbazones
have shown to increase their biological activity
by their ability to form chelates with specific
metal ions. [9] In the case of pharmaceuticals,
the binding capacity of thiosemicarbazones is
further increased by condensation of the
thiosemicarbazide with an aldehyde containing
heteroatom.[10,11] With this background in
mind, we herein report on the synthesis and
characterization of ruthenium(III) complexes
of 2-chloro/nitro benzaldehyde 4-methyl-3thiosemicarbazones. The investigation of the
biological properties of the complexes focused
on the binding properties with CT-DNA was
performed by UV spectroscopy. The in vitro
cytotoxicity studies against a panel of bacteria,
and MCF-7 cancer cell line, and antioxidative
property against DPPH radical were
investigated.
1. INTRODUCTION
Metal complexes remain an important
resource for the generation of chemical
diversity in the search for novel therapeutic
and diagnostic agents, especially in the arena
of anticancer drug development.[1,2] It has
been demonstrated that free radicals can
damage proteins, lipids, and DNA of biotissues, leading to increased rates of cancer.[3]
Fortunately, antioxidants can prevent this
damage, due to their free radical scavenging
activity.[4] Hence, it is very important to
develop compounds with both strong
antioxidant and DNA-binding properties for
effective
cancer
therapy.
Ruthenium
complexes offer the potential of reduced
toxicity, a novel mechanism of action, noncross resistance, significant radical scavenging
activity and a different spectrum of activity
compared
to
platinum
containing
compounds.[5] In addition, non-nuclear targets
have also been implicated in the antineoplastic
activity of ruthenium complexes, particularly
in the case of the clinically investigated
ruthenium(III) antimetastatic drug, NAMIA.[6] Thiosemicarbazones form an important
class of compounds because of their promising
pharmacological
properties,
such
as
trypanocidal activity, antitubercular activity,
antioxidative activity and antitumor activities.
doi:10.18831/djchem.org/2015011004
2. EXPERIMENTAL
2.1. Materials and methods
All the chemicals used were
chemically pure and AR grade. Solvents were
purified and dried according to the standard
procedure. [12] Calf-thymus (CT-DNA) was
purchased from Bangalore Genei, Bangalore,
India. The metal precursor, [RuBr3(PPh3)3] and
thiosemicarbazone ligands, HL1 and HL2, were
39
DJ Journal of Engineering Chemistry and Fuel (DJ JECF)
(KBr, cm-1): 1521 ν(C=N); 741 ν(C–S); 523
ν(Ru–N); 1434 ν(Ru–triphenylphosphine).
UV-vis (DMSO), λmax (nm): 321, 368, 407
(ILCT), 434 (LMCT). EPR (300 K, ‘g’ value):
2.17. µeff (300 K): 1.70 µB.
prepared by literature methods.[13,14] Micro
analyses (C, H, N & S) were performed on a
Vario EL III CHNS analyser at STIC, Cochin
University of Science and Technology, Kerala,
India. IR spectra were recorded as KBr pellets
in the 400-4000 cm-1 region using a Perkin
Elmer FT-IR 8000 spectrophotometer.
Electronic spectra were recorded in DMSO
solution with a Systronics double beam UV-vis
spectrophotometer 2202 in the range 200-800
nm. Magnetic susceptibility measurements of
the complexes were recorded using Guoy
balance. EPR spectra were recorded on a
varian E-112 ESR spectrophotometer at Xband microwave frequencies for powdered
samples at room temperature. EI mass spectra
of the complexes were recorded on a JEOL
GCMATE II mass spectrometer. Melting
points were recorded with Veego VMP-DS
heating table and are uncorrected.
2.3. DNA binding - Titration experiments
All the experiments involving the
binding of complexes with CT-DNA were
carried out in a doubly distilled water buffer
with tris(hydroxymethyl)-aminomethane (Tris,
5 mM) and sodium chloride (50 mM) and
adjusted to pH 7.2 with hydrochloric acid. A
solution of CT-DNA in the buffer gave a ratio
of UV absorbance of about 1.8-1.9 at 260 and
280 nm, indicating that the DNA was
sufficiently free of protein. The CT-DNA
concentration per nucleotide was determined
spectrophotometrically by employing an
extinction coefficient of 6600 M-1 cm-1 at 260
nm. The complexes were dissolved in a mixed
solvent of 5 % DMSO and 95 % Tris-HCl
buffer. Stock solutions were stored at 4 °C and
used within 4 days. Absorption titration
experiments were performed with fixed
concentrations of the complexes (25 µM) with
varying concentration of DNA (0-50 µM).
While measuring the absorption spectra, an
equal amount of DNA was added to both the
test solution and the reference solution to
eliminate the absorbance of DNA itself.
2.2. Synthesis of Ruthenium (III)
Thiosemicarbazone complexes
A methanolic solution (20 mL)
containing thiosemicarbazone ligands (0.1100.138 g,0.5 mmol) was added to
[RuBr3(PPh3)3] (0.520g, 0.5 mmol) in benzene
(20 mL). The resulting solution was refluxed
for 8 h. The reaction mixture was then cooled
to room temperature, which results in the
formation of precipitate. It was filtered off and
the purity of the complexes was checked by
TLC. This solid was recrystallized from
CH2Cl2/Hexane mixture. Our sincere effort to
obtain single crystal of the complexes went
unsuccessful.
[RuBr2(PPh3)2L1] (1): Yield: 57 %.
M.P:
268
°C.
Anal.
calcd.
for
C45H39Br2ClN3P2RuS (%): C, 53.45; H, 4.00;
N, 4.22; S, 3.25. Found (%): C, 53.40; H, 3.88;
N, 4.15; S, 3.17. EI-MS: Found m/z = 1012.48
(M+) (calculated m/z = 1012.15 for M+). IR
(KBr, cm-1): 1578 ν(C=N); 745 ν(C–S); 540
ν(Ru–N); 1433 ν(Ru–triphenylphosphine).
UV-vis (DMSO), λmax (nm): 316, 369
(ILCT), 413 (LMCT). EPR (300 K, ‘g’ value):
2.04. µeff (300 K): 1.71 µB.
[RuBr2(PPh3)2L2] (2): Yield: 59 %.
M.P:
295
°C.
Anal.
calcd.
for
C45H39Br2N4O2P2RuS (%): C, 60.30; H, 4.15;
N, 5.63; S, 3.22. Found (%): C, 52.94; H, 3.75;
N, 5.55; S, 3.12. EI-MS: Found m/z = 1022.65
(M+) (calculated m/z = 1022.71 for M+). IR
3. CYTOTOXICITY STUDIES
3.1. Antibacterial activity
The in vitro antibacterial screenings of
the free ligands and its complexes were tested
for their effect on certain human pathogenic
bacteria by disc diffusion method. The ligands
and complexes were stored dry at room
temperature and dissolved in dimethyl
sulfoxide. The bacteria (Escherichia coli,
Staphylococcus
aureus
and
Klebsiella
pneumonia) were grown in nutrient agar
medium and incubated at 37 ºC for 24 h
followed by frequent subculture to fresh
medium, and were used as test bacteria. Then
the petriplates were inoculated with a loop full
of bacterial culture and spread throughout the
petriplates uniformly with a sterile glass
spreader. To each disc the test samples and
reference antiobiotic (Cotrimazole) were added
with a sterile micropipette. The plates were
40
DJ Journal of Engineering Chemistry and Fuel (DJ JECF)
then incubated at 35 ºC for 24 h and at
27
ºC for bacteria and fungus, respectively. Plates
with disc containing respective solvents served
as control. Inhibition was recorded by
measuring the diameter of the inhibitory zone
after the period of incubation.
(0.3 mM, 1 mL) and the final volume was
made up to 4 mL with double distilled water.
DPPH solution with methanol was used as a
positive control and methanol alone acted as a
blank. The solution was incubated at 37 °C for
30 min in dark. The decrease in absorbance of
DPPH was measured at 517 nm. The tests were
run in triplicate, and various concentrations
(20-100 µg/ mL) of the complexes used to fix a
concentration at which the complexes showed
50 % of activity. In addition, the percentage of
activity was calculated using the formula, % of
suppression ratio = [(A0 - AC)/A0] X 100. A0
and AC are the absorbance in the absence and
presence of the tested complexes respectively.
The 50 % activity (IC50) can be calculated
using the percentage of activity.
3.2. In Vitro anticancer activity
Cytotoxicity of the complexes was
carried out on human breast cancer cell line
(MCF-7). Cell viability was carried out using
the MTT assay method. [15] The MCF-7 cells
were grown in eagles minimum essential
medium (EMEM) containing 10 % fetal bovine
serum (FBS). For the screening experiment,
the cells were seeded onto 96-well plates at
plating density of 10,000 cells/ well and
incubated to allow for cell attachment at 37 °C,
5 % CO2, 95 % air and 100 % relative
humidity for 24 h prior to the addition of the
complexes. The complexes were dissolved in
DMSO and diluted in the respective medium
containing 1 % FBS. After 24 h the medium
was replaced with the respective medium with
1 % FBS containing the complexes at various
concentrations and incubated at 37 °C under
conditions of 5 % CO2, 95 % air and 100 %
relative humidity for 48 h. Triplication was
maintained and the medium not containing the
complexes served as control. After 48 h, 15 µL
of MTT (5 mg mL-1) in phosphate buffered
saline (PBS) was added to each well and
incubated at 37 °C for 4 h. The medium with
MTT was then removed and the formed
formazan crystals were dissolved in 100 µL of
DMSO. The absorbance was then measured at
570 nm using micro plate reader. The % cell
inhibition was determined using the following
formula and a graph was plotted with the
percentage of
cell inhibition versus
concentration. From this, the IC50 was
calculated: % cell Inhibition = [mean OD of
untreated cells (control)/mean OD of treated
cells (control)] x100.
4. RESULTS AND DISCUSSION
Analytical and spectroscopic data for
the complexes indicate a 1:1 metal-ligand
stoichiometry for all the complexes. The
synthetic route of the complexes and the
proposed structure of the complexes are shown
in figure B1. The complexes are soluble in
most common organic solvents like CH2Cl2,
CHCl3, DMF, DMSO, etc.
4.1. Infrared spectra
The IR bands for the metal complexes
derived
from
the
4-methyl-3thiosemicarbazone ligands are most useful in
attempting to determine the mode of
coordination. A medium sharp band observed
at 1585 and 1530 cm-1 due to the azomethine
C=N stretching frequency of the free ligands
HL1 and HL2 respectively was shifted to lower
frequency in the spectra of the complexes at
1521-1578
cm-1
indicating
that
the
coordination through N atom.[17] A band
observed at 951 cm-1 for HL1 and 850 cm-1 for
HL2 due to vibration of the C=S double bond
which disappeared in the spectra of the
complexes and a new band, C–S appeared at
741-745 cm-1 indicating that the other
coordination is through thiolate sulphur after
enolization followed by deprotonation on
sulphur. [18] In all the complexes, the medium
intensity band in the region 523-540 cm-1 is
attributed to Ru–N. [19] In addition, the
characteristic absorption band due to
triphenylphosphine was also observed for all
the complexes at 1433-1434 cm-1. Overall, the
3.3. Antioxidant activity
The
2,2-diphenyl-2-picryl-hydrazyl
(DPPH) radical scavenging activity of the
complexes was measured according to the
method of Elizabeth. [16] The DPPH radical is
a stable free radical having a λmax at 517 nm. A
fixed concentration of the complex (100 µL)
was added to a solution of DPPH in methanol
41
DJ Journal of Engineering Chemistry and Fuel (DJ JECF)
complexes contain monobasic NS coordinated
thiosemicarbazones.
present in the complexes. All the complexes
exhibit a single isotropic resonance with g
values in the range 2.04-2.17. Although the
complexes have some distortion in their
octahedral geometries, the observation of
isotropic lines in the EPR spectra may be due
to the occupancy of the unpaired electron in a
degenerate orbital. [22] Hence, trans positions
are assigned for the triphenylphosphine group.
[23,24] Moreover, the trans structure is more
favoured structure than a cis structure, due to
the bulky nature of two PPh3 groups.[17] The
nature of spectra obtained is in good agreement
with that of previously reported ruthenium(III)
complexes.[18,24]
4.2. Electronic spectra
The electronic spectra of the
complexes were recorded in DMSO. The
electronic spectra of the complexes show three
to four bands in the region 316-434 nm. These
peaks have been shifted when compared to free
ligands (305-396 nm) which reveal that the
ligands are involved in the coordination with
ruthenium metal ion. The ground state of
ruthenium(III) (t52g configuration) is 2T2g,
while the first excited doublet levels in the
order of increasing energy are 2A2g and 2T1g,
which arise from t2g4 eg1 configuration.[20] The
lower wavelength bands at 312-315 and 368408 nm are characterized as ligand centered
transitions, π-π* and n-π* transitions,
occurring within the ligand orbitals.[21] Since
in a d5 system, and especially in ruthenium(III)
that has relatively high oxidising properties,
the charge transfer bands of the type Lπy→t2g
are prominent in the low energy region
obscuring the weaker bands due to d-d
transition. Therefore, it becomes difficult to
assign conclusively the bands of ruthenium(III)
complexes appearing in the visible region.
Hence, all the bands that appear in this region
(413-434 nm) have been assigned to charge
transfer transitions, which are in conformity
with the assignments made for similar
ruthenium(III) complexes.[17,18]
4.4. Mass spectral analysis
The mass spectrum of the ruthenium
(III) complexes is in good agreement with the
proposed molecular structure and the EI-mass
spectrum of the complexes is shown in figure
B3 and B4. The molecular ion peak, [M +]
appears at m/z = 1012.48 and 1022.65 confirms
the stoichiometry of the complexes, 1 and 2,
respectively.
5. DNA BINDING – TITRATION
EXPERIMENTS
Since DNA is generally accepted as
the primary target, interactions between small
molecules and DNA rank among the primary
action mechanisms of antitumor activity. [25]
Electronic absorption spectroscopy is an
effective method to elucidate the nature of the
interaction of the metal complexes with DNA.
In general, hypochromism and a red shift are
associated with the binding of metal complexes
to DNA, due to the intercalative mode
involving a strong stacking interaction between
the aromatic chromophore of the complexes
and the base pairs of DNA. [26] The results of
absorption spectra of the complexes in the
absence and presence of CT-DNA are given in
figure B5. As shown in figure B5, when
titrated with CT-DNA the absorption bands of
the complexes 1 and 2 exhibited
hypochromism of 18.65 % and 15.42 % with
red shifts of 2 nm at 360 and 359 nm,
respectively. These results suggested an
intimate association of the test complexes with
CT-DNA, and it is also likely that these
complexes bind to the DNA helix via
intercalation. After the complexes intercalate
4.3. Magnetic moment and EPR spectra
The room temperature magnetic
susceptibility
measurements
of
the
ruthenium(III) complexes shows that they are
paramagnetic. The magnetic moment value, μB
= 1.70-1.71 corresponds to single unpaired
electron in a low-spin 4d5 configuration and
confirms that ruthenium is in +3 oxidation
state in all the complexes.[19]
All the complexes are uniformly
paramagnetic
with magnetic
moments
corresponding to one unpaired electron at room
temperature (low-spin Ru(III), t2g5 ). The Xband EPR spectra of powdered samples of the
complexes were recorded at room temperature
and the EPR spectrum of the complex 1 is
shown in figure B2.The nature of the spectra
revealed the absence of any hyperfine splitting
due to interactions with any other nuclei
42
DJ Journal of Engineering Chemistry and Fuel (DJ JECF)
to the base pairs of DNA, the π* orbital of the
intercalated complexes could couple with π
orbitals of the base pairs, thus decreasing the π
 π* transition energies, hence resulting in
hypochromism. [27] In order to compare
quantitatively the binding strength of the
complexes, the intrinsic binding constants (Kb)
of them with CT-DNA were determined from
the following equation.
[DNA]/(εa – εf) = [DNA]/(εb – εf) + 1/Kb (εb –
εf)
where [DNA] is the concentration of DNA in
base pairs and the apparent absorption
coefficient εa, εf and εb correspond to Aobs/[
complex], the extinction coefficient of the free
complex and the extinction coefficient of the
complex when fully bound to DNA,
respectively. The plot of [DNA]/(εa – εf) versus
[DNA] gave a slope and the intercept
which
are equal to 1/(εb – εf) and 1/Kb (εb – εf),
respectively; Kb is the ratio of the slope to the
intercept. The magnitudes of intrinsic binding
constants (Kb) were calculated to be 1.5 X 10 5
M-1 and 5.6 X 104 M-1 for complexes, 1 and 2,
respectively. The observed values of Kb
revealed that the ruthenium (III) complexes
bind strongly than the respective ligands HL1
(1.7 X 104 M-1) and HL2 (4.6 X 103 M-1) to
DNA via intercalative mode. From the results
obtained, it has been found that complex 1
strongly bound with CT-DNA relative to that
with complex 2. Interestingly, the Kb values
obtained for the above ruthenium (III)
complexes are comparable to those of the other
known ruthenium complexes, Kb at 2.3-4.7 X
104 M-1. [28]
facilitates the ability of a complex to cross a
cell membrane which leads alteration in the
cell permeability and subsequent injury to the
cell membrane. [29, 30] Moreover, the mode
of action of the compounds may involve the
hydrogen bond through >C=N group with
active centers of all cell constituents resulting
in interference with normal cell process. [9]
Among the bacteria studied the complexes
shows higher activity against Staphylococcus
aureus. Even though the complexes possess
higher activity than the free ligands, they could
not reach the effectiveness of standard drug
Cotrimazole.
6.2. In Vitro anticancer activity
The optimistic results obtained from
antibacterial activity of the complexes
encouraged us to test their anticancer
properties against MCF-7 by means of a
colorimetric assay (MTT assay). The MTT
assay is based on the mitochondrial reduction
of the tetrazolium salt by actively growing
cells to produce blue insoluble formazan
crystals i.e. only live cells reduce yellow MTT
to blue formazan products. The test complexes
were dissolved in DMSO and blank samples
containing the same amount of DMSO are
taken as controls. The effects of the complexes
on the viability of these cells were evaluated
after an exposure period of 48 h. The results
were analyzed by means of cell viability
curves and expressed with IC50 values in the
studied concentration range from 0.1-100 µM.
The activity that corresponds to the inhibition
of cancer cell growth at a maximum level is
shown in figure B6. Upon increasing the
concentration of complexes, the results of
MTT assays revealed that the complex 1 (IC50
= 75.6 µM) showed a higher cytotoxic effect
than complex 2 (IC50 = 98.8 µM) against the
selected cancer cell line. The ligands does not
show any significant activity (IC50 > 100 µM),
it confirms that the chelation of the ligands
with the ruthenium (III) ion is the only
responsible factor for the observed cytotoxic
property of the complexes. There are reports in
the literature on the cytotoxic effects of the
complexes with longer incubation time periods
(72 h). [31] The longer incubation period may
result in the development of cellular resistance
for that particular compound and also have
harmful effects such as affecting non-target
sites in the body when they are used for
6. CYTOTOXIC ACTIVITY
6.1. Antibacterial activity
From the DNA binding experiments it
can be understood that the complexes did
interact with DNA. Hence, it is considered
worthwhile to study the cytotoxic activity of
the complexes. The antibacterial activity of the
ligands and complexes were tested against the
human
pathogens
(Escherichia
coli,
Staphylococcus
aureus
and
Klebsiella
pneumonia). The screening data are reported in
table A1. The complexes possess significant
antibacterial activity than the ligands. The
inhibition activity of ruthenium complexes
against the bacteria suggesting that chelation
43
DJ Journal of Engineering Chemistry and Fuel (DJ JECF)
clinical purposes, the complexes that show
activity in shorter time are preferred. Hence,
the data obtained for our complexes with lower
incubation period (48 h) are highly significant.
Though the above set of complexes are active
against the tumor cell lines under in vitro
cytotoxicity experiments, none of them could
reach the effectiveness shown by the standard
drug cisplatin (IC50 = 14 μM). [32]
Comparison of anticancer activity of the
ruthenium (III) complexes reveals that the
complexes
of
chlorobenzaldehyde
thiosemicarbazone moiety exhibited higher
cytotoxic effects than the complexes of
nitrobenzaldehyde thiosemicarbazone.
capacity to stabilize the unpaired electrons and
thereby scavenge the free radicals.[33]
7. CONCLUSION
The present contribution describes the
synthesis of new ruthenium(III) complexes
comprising versatile benzaldehyde 4-methyl-3thiosemicarbazones
ligands
and,
were
characterized by various spectroscopic
techniques. An octahedral geometry has been
tentatively assigned for all the complexes. The
DNA binding ability of the complexes assessed
by absorption spectroscopy suggested an
intercalative mode of binding with binding
constants 1.5 X 105 M-1 and 5.6 X 104 M-1 for
complexes 1 and 2 respectively. The
complexes possess significant antibacterial
activity against panel of bacteria. The in vitro
antitumor activity of the complexes against
MCF-7 tumor cells showed that the complex 1
has higher ability towards the inhibition of
tumor cell growth than the other complexes
and ligands. The antioxidant activity showed
that all the ruthenium(III) complexes can serve
as potential antioxidants than the ligands
against DPPH radical. All encouraging
chemical and biological findings indicate that
these complexes are very promising candidates
as live cell imaging reagents that could
contribute to the understanding of cellular
uptake of metal complexes. Further studies are
needed to assess their pharmacological
properties in vivo and to elucidate the actual
mechanism of their biological activity.
6.3. Antioxidant activity
Since the experiments conducted so far
reveals that the complexes exhibit significant
cytotoxic activity, it is considered worthwhile
to study the antioxidant activity of these
complexes. Hence, we carried out experiments
to explore the free radical scavenging activity
of the complexes, with the hope of developing
potential antioxidants and therapeutic reagents
for respiratory diseases, such as asthma,
emphysema and asbestosis.[33] 2,2-Diphenyl2-picryl-hydrazyl (DPPH) assay is widely used
for assessing the ability of radical scavenging
activity and it is measured in terms of IC50
values. Because of the presence of odd
electron, DPPH shows a strong absorption
band at 517 nm in the visible spectrum. As this
electron becomes paired off in the presence of
a free radical scavenger, this absorption
vanishes, and the resulting decolourization is
stoichiometric with respect to the number of
electrons taken up. The DPPH assay of the
tested ligands and complexes is shown in
figure B7, it is seen from the results that the
complex, 1 exhibited comparable activity
compared to the standard, ascorbic acid (Aca).
The IC50 values indicated that the complexes
showed antioxidant activity in the order of 1 >
2 > HL1 > HL2. Complex 1 showed a higher
antioxidant activity than the complex 2. From
the these results, the scavenging effect of the
free ligands is significantly less when
compared to that of their corresponding
ruthenium (III) complexes. This might be due
to the d5 low spin electronic configuration and
the availability of an odd electron in
ruthenium(III) complexes, which increases the
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APPENDIX A
Table A1.Antibacterial activity of the ligands and complexes
Diameter of inhibition zone (mm)a
Ligands and
Complexes
E. coli
S. aureus
K. pneumoniae
HL1
9
11
10
HL2
8
10
9
1
20
23
22
2
19
20
21
Ciprofloxacine
23
26
24
DMSO
a
No activity
Values are an average of triplicate runs.
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APPENDIX B
R
C
HN
HN
C
CH3
N
R
H
R
1
HL
HL1
ClCl
HL
HL22
NO22
CH PPh3
[RuBr3(PPh3)3]
N
S
Ligand
Methanol/Benzene, TEA
HN
C
CH3
N
Br
Ru
Br
S
PPh3
Figure B1.Synthetic route of the ruthenium(III) thiosemicarbazone complexes
Figure B2. EPR spectrum of the complex 1
49
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Figure B3.EI-mass spectrum of the complex 1
Figure B4.EI-mass spectrum of the complex 2
50
DJ Journal of Engineering Chemistry and Fuel (DJ JECF)
Figure B5.Electronic spectra of complexes 1 and 2 in Tris-HCl buffer upon addition of CT-DNA. [Complex] =
25 µM, [DNA] = 0-50 µM. Arrow shows the absorption intensities decrease upon increasing DNA
concentrations. (Inset: Plot between [DNA] and [DNA]/ [ε a-εf] X 10-8).
51
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Figure B6.% Growth inhibition of MCF-7 cell line as a function of concentration of the complexes 1 and 2
Figure B7.Antioxidant activity of the ligands and complexes, 1 and 2, and standard, ascorbic acid (Aca) against
DPPH radical
52