Design, synthesis, characterization and antiproliferative activities of Ru(II) complexes of substituted benzimidazoles

Article abstract of DOI:10.14233/ajchem.2019.22162

Synthesis of [Ru(PPh3)2(BZM)2Cl2] (BZM= LS1, LS2, LS3, LS4 and LS5) where LS1 = (1H-benzo[d]imidazole-2-yl)methanethiol, LS2 = 2-(4-bromobutyl)-1H-benzo[d]imidazole, LS3 = 2-(4-nitrophenyl)-1H-benzo[d]imidazole, LS4 = 2-(4-chlorophenyl)-1H-benzo[d]imidazole and LS5= 4-(1H-benzo[d]imidazol-2-yl)aniline (BZM = benzimidazoles, PPh3 = triphenylphosphine) and metal complexes as MR, [Ru (PPh3)4Cl2], MLS1, MLS2, MLS3, MLS4 and MLS5 for use as potential anticancer compounds have been investigated. The complexes have been characterized by elemental analysis, IR, multinuclear NMR, UV-visible and ESI-MS spectroscopic techniques. The geometries of all complexes have been optimized by using density functional theory (DFT). The cytotoxicity effects of MR, MLS2 and LS1 were also investigated on Human cervical carcinoma cells (HeLa) by MTT assay, ROS generation and nuclear apoptosis assay. The percent cell viability assessed by MTT assay suggested that the synthesized MR, MLS2 and LS1 significantly reduce the viability of HeLa cells, in a dose-dependent manner. The inhibitory concentration (IC50) of MR, MLS2 and LS1 against HeLa cells was found 90.8, 81.8 and 115 μM, respectively. These compounds also induced the over production of intracellular reactive oxygen species (ROS) as well as the condensed and fragmented nucleus, which supports the molecular mechanism of cell death by apoptosis. The investigations suggested that the compounds MR, MLS2 and LS1 induce the cell death in HeLa cells through apoptotic pathway.

Full text of DOI:10.14233/ajchem.2019.22162

Asian Journal of Chemistry; Vol. 31, No. 10 (2019), 2311-2318
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2019.22162
Design, Synthesis, Characterization and Antiproliferative
Activities of Ru(II) Complexes of Substituted Benzimidazoles
1,*,
1,
2
3
3
ASHOK K. SINGH
, SNEHLATA KATHERIA , AMRENDRA KUMAR , ASIFF ZAFRI and MOHD. ARSHAD
1Department of Chemistry, University of Lucknow, Lucknow-226007, India
2Department of Physics, University of Lucknow, Lucknow-226007, India
3Department of Zoology, University of Lucknow, Lucknow-226007, India
*Corresponding author: E-mail: singhaks3@rediffmail.com
Received: 29 April 2019;
Accepted: 31 May 2019;
Published online: 30 August 2019;
AJC-19542
Synthesis of [Ru(PPh3)2(BZM)2Cl2] (BZM= LS1, LS2, LS3, LS4 and LS5) where LS1 = (1H-benzo[d]imidazole-2-yl)methanethiol, LS2
= 2-(4-bromobutyl)-1H-benzo[d]imidazole, LS3 = 2-(4-nitrophenyl)-1H-benzo[d]imidazole, LS4 = 2-(4-chlorophenyl)-1H-
benzo[d]imidazole and LS5= 4-(1H-benzo[d]imidazol-2-yl)aniline (BZM = benzimidazoles, PPh3 = triphenylphosphine) and metal
complexes as MR, [Ru (PPh3)4Cl2], MLS1, MLS2, MLS3, MLS4 and MLS5 for use as potential anticancer compounds have been
investigated. The complexes have been characterized by elemental analysis, IR, multinuclear NMR, UV-visible and ESI-MS spectroscopic
techniques. The geometries of all complexes have been optimized by using density functional theory (DFT). The cytotoxicity effects of
MR, MLS2 and LS1 were also investigated on Human cervical carcinoma cells (HeLa) by MTT assay, ROS generation and nuclear
apoptosis assay. The percent cell viability assessed by MTT assay suggested that the synthesized MR, MLS2 and LS1 significantly reduce
the viability of HeLa cells, in a dose-dependent manner. The inhibitory concentration (IC50) of MR, MLS2 and LS1 against HeLa cells
was found 90.8, 81.8 and 115 µM, respectively. These compounds also induced the over production of intracellular reactive oxygen
species (ROS) as well as the condensed and fragmented nucleus, which supports the molecular mechanism of cell death by apoptosis. The
investigations suggested that the compounds MR, MLS2 and LS1 induce the cell death in HeLa cells through apoptotic pathway.
Keywords: Ruthenium(II) complex, Benzimidazoles, Apoptosis, Cytotoxicity effect.
nition against ovarian carcinoma cells [5]. In particular, some
ruthenium derivative have been screened for their antineoplastic
activity and their derivative [H2Im][trans-Ru(III)Cl4(DMSO-
S)(HIm)] (NAMI-A) and imidazolium trans-[transchlorobis(1H-
indazole)-ruthenate(III)] (KP1019 or FFC14A) were the first
ruthenium based anticancer complexes that have been entered
into clinical trials [6,7]. Further, the ruthenium complexes compri-
sing of chalcones and flavones will be possible drugs against
breast cancer [8,9]. Since the toxicity of many drugs used in
chemotherapy limits their clinical sources, subsequently analyzed
the cytotoxic activity of the compounds using MTT assay [10].
The potential of different metal based anticancer agent
have been widely explored and especially ruthenium complexes
have been extensively studied in drug discovery [11]. The drug
resistance developed by cisplatin against cancer cells, have
led to new line of approach for development of new ruthenium
INTRODUCTION
Platinum complexes attracted researcher′s interests due
to its promising anticancer properties, because they are still
the most successful medical therapeutic metallodrugs [1,2].
Due to the serious side effects of platinum drugs, we encou-
raged the development of anticancer agents on the basis of
other metals with low side effects. In this direction, ruthenium
compounds also represent a promising metal-based anticancer
complex that can offer less toxicity than platinum based drugs
[3]. It demonstrated some exciting antitumor properties and
this may be the alternative option to platinum based antitumor
medicines. Additionally, ruthenium has the ability to mimic
iron cation binding to certain molecules, in view of rate of ligand
exchange and accessible oxidation states [4]. Further, imidazole
ligand shows imperative role with ruthenium to target site recog-
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2312 Singh et al.
Asian J. Chem.
complexes for metal based drug that can be used in the fields
of chemotherapy. In general, the drug discovery approach targets
the DNA or cellular mechanism [12]. Therefore, the interaction
of ruthenium complexes to adenine, guanine and cytosine,
through weak interaction and hydrogen bonding, signifying
the possibility to design compounds to interact through nucleotides
[12]. The binding modes and affinity of ruthenium complexes
also depends on the structure of DNA [13]. In this respect, we
have synthesized ruthenium complexes containing a triphenyl-
phosphine and substituted benzimidazole and assessed their
interaction with human cervical carcinoma cells. The cytotoxicity
of the complexes was assessed using cancer and normal cell
lines in vitro conditions.
Cellular morphology studies in HeLa cells:The morphol-
ogical changes in HeLa cells, treated with MR, MLS2 and LS1
were examined under the inverted contrast-phase microscope.
Briefly, HeLa cells was plated in 48 well culture plates and
treated with 10, 25, 50, 100 and 200 µM concentrations of MR,
MLS2 and LS1 for 24 h of incubation. Thereafter, the cell morp-
hology was examined by an inverted contrast-phase micro-
scope and photographed [14].
Detection of ROS in HeLa cells: The intracellular ROS
level was detected by DCFH-DA (2,7-dichlorodihydrofluo-
resce in diacetate) staining in accordance with the previous
report [15]. The HeLa cells were first seeded at the density 1
× 104 per well in a 96-well culture plate and incubated at 37
ºC for 24 h in 5 % CO2. Then, cells were exposed to 25, 50
and 100 µM concentrations of MR, MLS2 and LS1 for 12 h.
Subsequently, for the fluorescence microscopy imaging, the
cells were washed thrice with PBS (phosphate buffer saline)
and then incubated with fluorescence agent DCFH-DA (10
mM) for 25 min at 37 ºC. Later, cells were washed with MEM
(E) three times and were observed with a fluorescence inverted
microscope (Nikon ECLIPSE Ti-S, Japan). For the quantitative
analysis of ROS, HeLa cells (1 × 104 per well) were seeded in
96-well black bottom titer plate and treated with 25, 50 and
100 µM concentrations of the MR, MLS2 and LS1.Afterwards,
cells were trypsinized and washed thrice with PBS, then incub-
ated with DCFH-DA (10 mM) for 25 min. The cells were analyzed
for the fluorescence intensity by a multiwall microplate reader
(Synergy, BioTek) with an excitation and emission wavelength
at 485 and 528 nm, respectively. The values of fluorescence
intensity were expressed as the percentage of the treated group
with respect to the control group.
EXPERIMENTAL
The reagent and chemicals used for synthesis were procured
from Sigma Aldrich, Hi-media and Merck and used without
further purification. Column chromatography of all the synthe-
sized compounds was performed by silica gel 60 (40-60 Merck,
India) as stationary phase and acetonitrile as an eluent. FT-IR
spectra were recorded in KBr pellets on Perkin Elmer AC-1C
1
spectrometer from 4000 to 450 cm-1. H NMR spectra were
recorded in DMSO-d6 on Bruker Avance III HD spectrometer
at 300 MHz using TMS as an internal reference. ESI-MS mass
spectra were recorded on JEOL SX 102/DA-6000 spectro-
meter. Elemental analysis was performed on Exeter analytical
INC Model CE-440 C H NANALYSER. Electronic absorption
spectra were recorded on Labtronic 2900 UV-Vis spectrophoto-
meter and conductivity is carried out on microprocessor based
conductivity/TDS meter ESICO Model 1601.
Analysis of nuclear apoptosis in HeLa cells: For the
nuclear and chromatin structural change study, the HeLa cells
were seeded in 96 well culture plate and then treated with
different doses of MR, MLS2 and LS1 at 25, 50 and 100 µM
concentrations for 24 h [16]. Consequently, cells were washed
thrice with PBS and fixed in 4 % paraformaldehyde (used as a
fixative) for 10 min and then permeabilized with permeabili-
zing buffer (4 % paraformaldehyde and 0.5 % Triton X-100)
and stained with 10 mM of DAPI stain. After staining, cells
were examined with the help of an inverted fluorescence micro-
scope at the excitation wavelength of 360 nm and an emission
wavelength of 454 nm (Nikon ECLIPSE Ti-S, Japan) [16]. For
the quantitative analysis of apoptotic cells, the tests were perf-
ormed thrice for every treatment group and the 100 cells per
well were calculated in at least 10 random fields per well, to
count the percent apoptotic cells using an inverted fluorescent
microscope (Nikon ECLIPSE Ti-S, Japan).
Statistical analysis: The data on cell viability, nuclear
condensation and ROS generation were represented as mean
standard error mean (SEM) from at least three independent
studies. Determination of difference in between the experimental
and the control groups was compared using one-wayANOVA
followed by Dunnett′s multiple range test of Graph Pad prism
software (Version 5.01). The p value of less than 0.05 was desi-
gnated to be statistically significant.
in vitro Cytotoxicity activity studies
Cervical carcinoma cell line (HeLa) culture: The cell line
of human cervix carcinoma (HeLa) was procured from the
National Centre for Cell Sciences (NCCS), Pune, India. The
cell was cultured at 37 ºC humidified condition and 5 % CO2
in MEM (E) culture media. The medium was supplemented
with 10 % fetal bovine serum (FBS), 1 % antibiotic solution
(10,000 U/mL penicillin and 10 mg/mL streptomycin), 0.1 mM
non-essential amino acids, 2.0 mM L-glutamine, 1.0 mM sodium
pyruvate and 1.5 g/L NaHCO3.
Cell viability assay on HeLa cells: HeLa cells (1 × 104
cells per mL) were seeded in 100 µL MEM (E) media per well
in 96 well titer plate for 24 h at 37 ºC and 5 % CO2 as per
previous protocol [13]. The treatment was conducted for 24 h
incubation with various concentrations (10, 25, 50, 100 and
200 µM) of the MR, MLS2 and LS1. Afterwards, 20 µL of MTT
reagent was added to each well and kept for 4 h at 37 ºC until
the development of formazan crystals. Subsequently, the obtained
formazan crystals were solubilized in 100 µL of DMSO and the
absorbance was recorded at 540 nm using a microplate reader
(BIORAD-680). The % cell viability has been presented as a
ratio of absorbance of treated groups to the absorbance of control
cells (without treatment). The % cell viability was calculated
following the given equation:
A540 (treated cells)
General procedure for synthesis of ligands (LS1-LS5):
All substituted benzimidazole ligands were prepared by the
Cell viability (%) =
×100
A540 (control cells)

Vol. 31, No. 10 (2019)
Design, Synthesis and Antiproliferative Activities of Ru(II) Complexes of Substituted Benzimidazoles 2313
previously reported procedure [17]. o-Phenylenediamine (0.25
mol) and appropriate carboxylic acid (0.34 mol) was heated
on an oil bath at 100 ºC for 8 h. The completion of the reaction
was monitored by TLC. After completion of the reaction, the
reaction mixture was cooled and its pH was adjusted between
7-8 using 10% NaOH solution. The crude benzimidazole was
filtered and washed with ice-cold water. The crude product
was dissolved in 40 mL of boiling water and 2 g of carbon
was added and digested for 15 min. The solution was filtered
while hot, cooled the filtrate to about 10 ºC. The pure product
obtained was filtered, washed with 25 mL of cold water and
dried (Scheme-I).
4-(1H-Benzo[d]imidazol-2-yl)aniline (LS5): Brown colour
solid; yield: 42.6 %; m.p.: 226 º, IR (KBr, νmax, cm-1) 3600 (N-
H str.), 1450 (C=N str.), 1650 (Ar-C=C), 2350 (Ar N-H(NH2)),
2900 (Ar C-H), 850 (Ar C-N). 1H NMR (300 MHz, DMSO-
d6, δ ppm): δ 6.40-7.89 (m, 8H, Ar-H), 6.40 (s, 1H, N-H),
2.50 (s, 2H, NH2). 13C NMR (300 MHz, DMSO-d6, δ ppm): δ
145, 142, 139, 128, 127, 102. UV-Vis (DMSO, λmax, nm (εmax
× 103 M-1 cm-1): 320 (0.5), 420 (4.0).
General procedure for synthesis of Ru(II) complexes:
The ruthenium precursor was prepared in accordance with the
previously reported method [18]. The precursor was synthe-
sized with the reaction of ruthenium trichloride trihydrate with
a methanolic solution of tryphinylphosphine in excess and
refluxed for 8 h under nitrogen,which gives the black precipi-
tate of [RuCl2(PPh3)4] (MR), washed with diethylether and
dried in vaccuo. Triphenylphosphine ligands are easily replaced
by other ligands in the precursor complexes [19]. The complexes
of general formula [Ru(PPh3)2(BZM)2Cl2] were synthesized
by the reaction of ruthenium dichlorotetratriphenylphosphine
with different substituted benzimidazole in 1:2 molar ratio in
methanol. Further, RuCl2(pph3)4 (0.265 g, 1 mmol) was added
to a methanolic solution of the ligands, 2 mmol (LS1, 0.328g;
LS2, 0.554 g; LS3, 0.478 g; LS4, 0.146 g; LS5, 0.456 g). The
solutions were refluxed for 10 h in nitrogen and reduced the
volume, thus a microcrystalline precipitate was obtained
(Scheme-I). The analytical data for these complexes are in good
agreement with their respective molecular formula. The new
complexes were formed by the substitution of two triphenyl
phosphine complexes by benzimidazole which indicates the labile
nature of phosphine bond and addition of Nitrogen based ligands,
this is due to the better sigma (σ) donating ability of the nitrogen
bases to that of triphenylphosphine [20]. The complexes were
sparingly soluble in organic solvent as acetone, alcohol and
methanol but soluble in DMSO/DMF. The molar conductivities
values were 0.055-0.163 ohm-1 cm2 mol-1. The observed values
indicates the non-electrolytic nature of complexes.
H
N
N
NH2
NH2
Refluxed
R
R COOH
in ethanol
PPh3
Ru
NH
H
N
Cl
N
Refluxed
in ethanol
R
2
R + [Ru(PPh3)4Cl2]
R
N
Cl
N
HN
PPh3
R = SH (LS1), Br (LS2), NO2 (LS3), Cl (LS4), NH2 (LS5)
Scheme-I: Synthetic route for synthesis of ligand and its ruthenium(II) complexes
(1H-Benzo[d]imidazole-2-yl) methanethiol (LS1): Pale
yellow solid; yield: 42.90 %, m.p.: 158 ºC, IR (KBr, νmax, cm-1):
3445 (N-H str.), 2963 (Ar C-H), 2600 (S-H str.), 1700 (Ar-C),
1600 (C=N), 2900 (Ar C-H). 1H NMR (300 MHz, DMSO-d6, δ
ppm): δ 7.01-7.51 (m, 4H Ar-H), 4.41 (s, 1H, N-H), 2.01 (s,
2H, CH2), 2.2(s 1H, SH). 13C NMR (300 MHz, DMSO-d6, δppm):
133, 132, 122, UV-Vis (DMSO, λmax, nm (εmax × 103 M-1 cm-1):
250 (2.2), 300 (2.4), 348 (0.1).
2-(4-Bromobutyl)-1H-benzo[d]imidazole (LS2): Reddish
brown solid; yield: 40.4 %, m.p.: 166 ºC, IR (KBr, νmax, cm-1):
3300 (N-H str.), 1527(C=N str.), 2900( ArH, C-H), 1700 (Ar
1
[Ru(PPh3)2(LS1)2Cl2]·3H2O (MLS1): Dark brown colour,
yield: 46 %; m.p.: 327 ºC, IR (KBr, νmax, cm-1): 1143 (P-Ph),
1328 ν(-NH), 2963 (NH), 1H NMR (300 MHz, DMSO-d6, δ
ppm): δ 6.80 (m, PPh3), 7.01-7.66 (m, 4H, Ar-H), 4.01 (s, 1H,
NH), 2.40 (2H, CH2); 2.49 (s, 1H, sh); 13C NMR (300 MHz,
DMSO-d6, δppm): δ 135, 122; MS (ESI) m/z: 1077 (1076).Anal.
calcd. (found) (%) RuP2Cl2C52H52N4S2O3: C, 57.09 (57.09); H,
4.83 (4.83); N, 5.20 (4.80). 31P NMR (500 MHz, DMSO-d6, δ
C=C). H NMR (300 MHz, DMSO-d6, δ ppm): δ 6.57-7.90
(m, 8H,Ar-H), 6.96 (s, 1H, N-H), 2.50-2.54 (m, 8H, CH2); 13C
NMR (300 MHz, DMSO-d6, δ ppm): δ 146, 142, 141, 136, 133,
128, 126, 123, 121, 118, 115, 101. UV-Vis (DMSO, λmax, nm
(εmax × 103 M-1 cm-1): 240 (0.9), 270 (0.95), 275 (0.8).
2-(4-Nitrophenyl)-1H-benzo[d]imidazole (LS3): Brown
solid ; yield: 40.50 %, m.p.: 220 ºC, IR (KBr, νmax, cm-1): 3100
(N-H str.), 3000 (Ar C-H), 1350 (N-O sym str.), 1400 (C-N str.),
(C-Br), 1700 (C=C ring str.), 1602 (N-O asym str.), 1539 (C=N
str.). 1H NMR (300 MHz, DMSO-d6, δ ppm): δ 7.57-8.22 (m,
8H, Ar-H), 6.87 (s, 1H , N-H). 13C NMR (300 MHz, DMSO-d6,
δ ppm): δ 167, 150, 146, 141, 138, 137, 131, 128, 126, 124, 101.
UV-Vis (DMSO, λmax, nm (εmax × 103 M-1 cm-1): 250 (1.7), 340
(0.1), 450 (0.5).
ppm): δ 40.34 ppm (s, PPh3). UV-Vis (DMSO, λmax, nm (εmax
×
103 M-1 cm-1): 240 (3.5), 260 (3.2), 450 (1.0).
[Ru(PPh3)2Cl2(LS2)2] (MLS2): Dark red colour: yield:
62 %; m.p.: 296 ºC, IR (KBr, νmax, cm-1): 1156 (P-Ph), 1371 ( NH2),
3056 (NH2 str.), 1H NMR (300 MHz, DMSO-d6, δ ppm): δ 6.67
(s, PPh3), 7.26-7.66 (m, 8H, Ar-H), 6.60 (2H, NH), 1.25-3.79
(m,16H,CH2). 13C NMR (300 MHz, DMSO-d6, δ ppm): δ 134,
133, 132, 131, 129, 128. ESI-MS m/z: 1177 (1178).Anal. calcd.
(found) (%) RuP2Cl2C56H56N4Br2: C, 57.09 (56.59); H, 4.75
(4.25); N, 4.75 (4.55). 31P NMR (500 MHz, DMSO-d6, δppm):
δ 40.38. UV-Vis (DMSO, λmax, nm (εmax × 103 M-1 cm-1): 240
(2.53), 260 (0.6), 450 (1.0).
2-(4-Chlorophenyl)-1H-benzo[d]imidazole (LS4): Black
colour solid; yield: 39.6 %, m.p. 234 ºC, IR (KBr, νmax, cm-1):
3150(NH str.), 3000 (ArH), (C-Cl),1525 (C=N),1600 (C=C
ring system), 1429 (C-N str.). 1H NMR (300 MHz, DMSO-d6,
δ ppm): δ 7.52-8.12 (m, 8H,Ar-H), 6.80 (s,1H N-H). 13C NMR
(300 MHz, DMSO-d6, δ ppm): δ 165, 149, 145, 141, 138, 130,
128, 126, 123, 101. UV-Vis (DMSO, λmax, nm (εmax × 103 M-1
cm-1): 270 (2.4), 360 (0.1), 420 (4.0).
[Ru(PPh3)2Cl2(LS3)2]H2O (MLS3): Brownish, yield: 42
%; m.p.: 320 ºC, IR (KBr,νmax, cm-1): 1171 (PPh), 1585 (C=N),

2314 Singh et al.
Asian J. Chem.
1926 (NO), 1H NMR (300MHz, DMSO-d6, δppm): 6.90 (s, PPh3),
7.56-8.26 (16H, Ar-H), 6.60 (2H, NH). 13C NMR (300 MHz,
DMSO-d6, δ ppm): 166, 150, 145, 141, 138, 136, 131, 127,
127, 124, 101. ESI-MS m/z: 1191 (1192). Anal. calcd. (found)
(%) RuP2Cl2C62H50N6O5: C, 62.46 (62.92); H, 4.19 (4.16); N,
7.16 (6.66). 31P NMR (500 MHz, DMSO-d6, δ ppm): δ 40.53.
UV-Vis (DMSO, λmax, nm (εmax × 103 M-1 cm-1): 220 (2.6), 260
(2.65), 275 (2.4), 350 (1.8), 450 (0.5).
complexes at 3145 cm-1 was observed in all the cases which
could be assigned to ν(N-H) of benzimidazole. This feature sugg-
ested that the group NH is not coordinated nor deprotonated
during complex formation. Though, there was shift in ν(C=N)
in the spectra of the complexes containing benzimidazole, suggests
coordination of ruthenium metal ion through nitrogen. Further,
in all complexes, band position in the range 1199-1143 of M-PPh3
suggests the coordination of metal-phosphorus [24].
1
[Ru(PPh3)2(LS4)2Cl2]·H2O (MLS4): Black, yield: 42 %,
m.p.: 308 ºC, IR (KBr, νmax, cm-1): 1199 (P-Ph), 1320 (NH2),
3700 (NH2 str.), 1H NMR (300 MHz, DMSO-d6, δ ppm): 7.13
(m, PPh3), 7.28-8.04 (m,ArH), 6.80 (s, NH), 13C NMR (300MHz,
DMSO-d6, δppm): 168, 137, 135, 133, 132, 131, 129, 128, 121,
ESI-MS m/z: 1099 (1100).Anal. calcd. (found) (%) RuP2Cl2C62
H50N4O: C, 67.69 (67.09); H, 4.54 (4.04); N, 0.36 (0.26). 31P
NMR (500 MHz, DMSO-d6, δ ppm): 40.54, UV-Vis (DMSO,
NMR studies: The H NMR spectra, display aromatic
protons in the range, δ 8.0-6.0 ppm in all complexes and there
integration indicates the coordination of two benzimidazole
moiety. In MLS1, two multiplet in the range δ 7.23-7.26 and
proton signals at δ 6.52 (4H) and singlet at δ 4.0 (2H) indicated
the presence of amino phenyl group. In MLS2, 1H NMR spectrum
showed a singlet at δ 1.5(2H) and δ 3.82 (3H) protons confirmed
the presence of methane thiol group. In MLS3, multiplet at δ
3.12 for one proton and doublet at δ1.29 for six protons indicate
the presence of propane group. In case of MLS4, two multiplets
at δ 8.25 and δ 7.74 for two protons indicated the substitution
of nitro phenyl at C2 of benzimidazole nucleus. One weak singlet
at δ12.40 may be assigned to the N-H proton of benzimidazole.
31P spectra of all the ruthenium(II) complexes were recorded
in DMSO-d6 in order to confirm the presence of triphenyl phos-
phine groups and to determine the geometry of the complexes.
The observation of sharp singlet peak in the range δ 7.50-
6.40, suggest the only one possible geometric isomer of the
complexes is formed [25]. However, IR spectrum showed
single absorption band around 1199-1143 cm-1, suggests that
triphenyphosphine is in trans-position [26].
λ
max, nm (εmax × 103 M-1 cm-1): 240 (2.5), 255 (0.6), 450 (0.5),
550 (0.8).
[Ru(PPh3)2(LS5)2Cl2]·2H2O (MLS5): Black crystalline
colour: yield: 38 %, m.p.: 348 ºC, IR (KBr,νmax, cm-1): 1186 (P-
Ph), 1314 (NH2), 3300 (NH2 str.); 1H NMR (300 MHz, DMSO-
d6, δ ppm): 6.91 (m, PPh3), 7.54-7.90 (m, ArH), 2.50 (s, 2H,
NH2), 6.42 (s, H, NH), 13C NMR (300 MHz, DMSO-d6, δppm):
144, 142, 139, 127, 101. ESI-MS m/z: 1149 (1151). Anal.
calcd. (found) (%): C, 64.75 (64.25); H, 4.87 (4.07); N, 7.31
(7.01). 31P NMR (500 MHz, DMSO-d6, δ ppm): 40.58. UV-Vis
(DMSO, λmax, nm (εmax × 103 M-1 cm-1): 250 (3.3), 405 (2.3),
460 (1.5).
Computational details:The Gaussion-09 program package
was used to perform density functional theory (DFT) calculations
[21]. The gas phase geometry of complexes were fully optimized
using B3LYP hybrid functional using 6-31G** basis set for
all the atoms expect Ru. For Ru, LANL2DZ basis set was used
[22,23].
Electronic absorption spectroscopy:The UV-visible absor-
ption spectral assignments of complexes are presented in Table-
1. The intense high energy band below 300 nm is assigned to
ligand centered transitions. The low energy absorptions at 600
nm arise due to Ru based MLCT transitions. The nature of the
observed electronic spectra and the positions of absorption
band are consistent with those of other similar octahedral Ru(II)
complexes [27]. Further, all ruthenium complexes are found
to be diamagnetic which suggest that their geometry ought to
be octahedral as revealed by geometry optimization. The absor-
ption band in the range 220-450 nm, based on the values of
RESULTS AND DISCUSSION
Infrared studies: Infrared spectra of the ligands showed
the characteristic band at 1600-1450 and 3600-3100 cm-1
which corresponds to ν(C=N) and ν(N-H) for LS1-LS5. The
vibrational spectra of the metal complexes were compared with
the corresponding ligands to study the binding behaviour of
ruthenium metal to the ligand. The band position of metal
*
extinction coefficient (2.2-0.50) both band assigned to π-π
transitions arising from an excitation of an intra ligand orbitals.
TABLE-1
UV-VISIBLE SPECTRAL DATA OF THE LIGANDS AND Ru(II) COMPLEXES IN METHANOL
max (nm) (εmax × 103 M–1 cm–1)
Compounds
LS1
Λ
244 (0.17), 230 (0.34), 2390.61), 250 (1.05), 258 (1.14), 260 (1.15), 265 (1.12), 270 (1.10), 277 (1.10), 280 (1.09), 283 (0.01)
213 (1.45), 244 (1.42), 251 (1.66), 267 (1.69), 279 (1.42), 293 (0.14), 3469 (0.01), 441 (0.52)
LS2
LS3
LS4
236 (1.92), 241 (2.02), 249 (2.28), 258 (2.39), 265 (2.29), 281 (2.31), 291 (2.43), 295 (2.36), 326 (0.26), 341 (0.19), 347 (0.17)
212 (0.72), 230 (0.73), 240 (1.51), 253 (1.42), 267 (1.37), 273 (1.48), 275 (1.11), 279 (1.18)
LS5
227 (1.79), 231 (1.92), 243 (2.23), 247 (2.38), 252 (2.48), 260 (2.42) 265 (2.43), 269 (2.38), 280 (2.41), 299 (2.53), 310 (2.20),
332 (2.20), 429 (0.63)
MLS1
213 (1.15), 223 (1.60), 230 (2.04), 234 (2.15), 250 (2.50), 257 (2.90), 272 (2.96), 279 (2.90), 301, 339 (1.50), 350 (2.15), 359
(2.00), 363 (0.75), 399 (0.59)
MLS2
MLS3
209 (0.39), 221 (0.29), 229 (0.30), 259 (0.53), 277 (0.33), 300 (0.22), 340 (0.12), 400 (0.17), 420 (0.20), 460 (0.20), 500 (0.06)
205 (0.60), 213 (1.53), 217 (1.51), 223 (2.03), 226 (2.13), 238 (1.30), 255 (0.75), 268 (0.76), 287 (0.45), 449 (0.35), 540 (0.51),
601 (0.35), 652 (0.25), 703 (0.16)
MLS4
MLS5
212 (0.73), 230 (0.78), 240 (1.49), 250 (1.49), 267 (1.37), 273 (1.50), 324, 357, 372
215 (2.20), 223 (2.36), 230 (2.64), 248 (2.67), 256 (2.90), 263 (2.07), 273 (2.96), 281 (3.01), 294 (3.01), 309 (1.96), 338 (2.11),
350 (2.15), 354 (2.27), 359 (2.59), 363 (2.00), 369 (1.93), 399 (0.75), 448 (0.58)

Vol. 31, No. 10 (2019)
Design, Synthesis and Antiproliferative Activities of Ru(II) Complexes of Substituted Benzimidazoles 2315
The electronic absorption spectra of [Ru(PPh3)2(BZM)2Cl2]
in methanol generally showed two absorption bands in 550 to
450 nm range with ε of the order 103. The band around 550
nm in MLS4 is weaker than that around 450 nm. The band around
550 nm may have some MLCT character and also certain
amount of d-d character.
X-ray diffraction studies: Structural studies were carried
out by powder X-ray diffraction using a 1.4 kW Cu-rotating
anode based Rigaku (Tokyo, Japan) powder diffractometer
operating in Bragg-Brentano geometry and fitted with a curved
crystal graphite monochromator in the diffraction beam and a
high temperature attachment. The XRD data in the 2θ range
of 20-80º at a step of 0.02º were collected at room temperature.
Fig. 1 depicts the XRD patterns of samples MLS1, MLS2,
MLS3, MLS4 and MLS5. It is evident that samples MLS1,
MLS2 and MLS5 are stabilized in the crystalline form at room
temperature. However samples MLS3, MLS4 are most like
amorphous in nature as no sharp Bragg′s peaks were observed.
In order to investigate the crystal structure of the samples
MLS1, MLS2 and MLS3, Lebail profile fittings were carried
out using FULLPROF package [28]. In present profile fittings,
pseudo-Voigt function and a sixth order polynomial wereused
30
40
50
60
(°)
Fig. 1. X-Ray diffraction pattern of metal complex
70
80
90
2θ
to define the profile shape and background, respectively. During
the profile fittings, parameters, such as the scale factor, zero
correction, background, half-width parameters, the mixing
parameters, lattice were varied. Table-2 depicts the observed,
calculated and difference profiles obtained after the Lebail
analysis of the full XRD pattern for MLS2 using monoclinic
space group P21/c. The fit between the observed and calculated
profiles is quite good suggest that the sample MLS5 most likely
MLS1
MLS2
MLS3
MLS4
MLS5
Fig. 2. Perspective view of optimized geometry of metal complexes

2316 Singh et al.
Asian J. Chem.
TABLE-2
UNIT CELL PARAMETER OBTAINED AFTER PROFILE FITTING OF X-RAY DIFFRACTION
DATA COLLECTED AT ROOM TEMPERATURE USING MONOCLINIC SPACE GROUP p21/c
Sample
a (Å)
b (Å)
c (Å)
α (°)
β (°)
γ (°)
MLS1
MLS2
MLS3
17.521(2)
17.996(3)
18.010(2)
21.562(4)
22.086(5)
22.076(3)
10.719(1)
11.0178(2)
10.978(3)
90.00
90.00
90.00
91.05(1)
90.94(2)
91.01(1)
90.00
90.00
90.00
stabilized in the monoclinic phase at room temperature with
space group P21/c symmetry. The unit cell parameters obtained
after the Lebail profile fittings for the samples MLS1, MLS2
and MLS3 are given in Table-2.
Optimized geometry of complexes: In order to gain struc-
tural insight regarding the synthesized Ru(II) complexes, density
functional theory (DFT) calculations were performed using
suitable functional (vide infra). Perspective views of optimized
geometry of these complexes have been presented in Fig. 2.
The relevant optimized geometrical parameters of these complexes
are comparable with the analogues Ru(II) complexes [19,29-32].
The geometry optimization results indicate that in all of the
complexes, Ru(II) centre acquires the octahedral geometry. The
peculiar feature that is being observed in all the optimized
geometry is that two PPh3 moieties adopts trans-position with
respect to each other. This can be attributed to the inherent bulk-
iness associated with the PPh3 ligand and also because of the
relatively strong trans directing influence by chloro ligands
[19]. The pertinent optimized geometrical parameters for the
Ru(II) complexes is shown in Table-3.
(A) MR
TABLE-3
OPTIMIZED GEOMETRICAL PARAMETERS FOR Ru(II)
COMPLEXES [BOND LENGTH (Å) AND BOND ANGLE (°)]
(b) MLS2
Parameter
MLS1
MLS2
MLS3
MLS4
MLS5
Ru-N1
Ru-N2
2.230
2.307
2.319
2.257
2.330
2.261
2.268
2.322
2.314
2.258
Ru-P1
2.529
2.515
2.511
2.552
2.551
Ru-P2
2.513
2.562
2.534
2.506
2.502
Ru-Cl1
2.513
2.510
2.534
2.472
2.502
Ru-cl2
2.541
2.543
2.473
2.539
2.427
Ru-N1-C
Ru-N2-C
Ru-P1-C
Ru-P2-C
P1-Ru-P2
Cl1-Ru-Cl2
126.94
126.72
117.92
122.52
174.45
170.63
125.70
125.85
115.44
124.21
177.53
176.78
125.21
123.81
112.30
123.07
176.40
175.47
123.85
125.19
114.37
117.53
176.19
175.21
125.60
122.25
110.88
123.61
177.42
177.57
(C) LS1
Fig. 3. Photomicrograph showing morphological changes in HeLa cells
after treatment with 10, 25, 50, 100 and 200 µM concentrations of
(A)MR (B)MLS2 and (C) LS1, at 24 h by inverted phase contrast
microscopy
Morphological cytotoxicity study: The morphological
changes in HeLa cells were observed under inverted phase
contrast microscope after treatment of MR, MLS2 and LS1
at 10, 25, 50, 100 and 200 µM concentrations for 24 h. As
depicted in Fig. 3, severe changes were observed in the morph-
ology of HeLa cells treated with MR, MLS2 and LS1. The
treated cells showed a typical feature of apoptosis viz. cyto-
plasmic vacuolization, cellular shrinkage, became round shape
and nuclear condensation as compared to the untreated cells
which were also reported by an earlier study [33]. While the
untreated control cells exhibit even cell surface, flat and remained
smooth, signifying the morphology of healthy cells.
the typical morphological and biological changes in cells like
shrinkage of the cell, condensation of nuclear chromatin and
fragmented apoptotic bodies [34]. In this study, we observed
concentration dependent nuclear apoptosis in HeLa cells in
MR, MLS2 and LS1. As evident from photomicrographs of
HeLa cells (Fig. 4), synthesized compounds exhibit apoptosis
in HeLa cells evident by the occurrence of chromatin conden-
sation and nuclear bounded apoptotic bodies.As evident from
the pictorial graph representing quantitative % apoptotic cells
at 25, 50 and 100 µM of MR showed approximately 9.39, 16.347
and 23.34 % of apoptotic cells in HeLa cells. Furthermore, at
Nuclear condensation and apoptosis: Induction of apop-
tosis in cancerous cells is considered as a valuable method to
cure the various cancers. Apoptosis is usually characterized by

Vol. 31, No. 10 (2019)
Design, Synthesis and Antiproliferative Activities of Ru(II) Complexes of Substituted Benzimidazoles 2317
Fig. 4. Photomicrograph showing morphological changes, nuclear condensation and cellular apoptosis on HeLa cells, treated with 25, 50 and
100 µM of MR, MLS2 and LS1 against HeLa cells
100
25, 50 and 100 µMdoses of MLS2, showed around 14.34, 21.85
MR
MLS2
LS1
and 30.67 % of apoptotic cells and LS1 showed approximately
6.67, 12.34 and 20.85 % apoptotic cells with respect to control
(Fig. 5). The photomicrograph of HeLa cells having condensed
and fragmented nuclei suggest that MR, MLS2 and LS1induced
the cell death by the process of apoptotic. The anti-proliferative
effects of MR, MLS2 and LS1 on HeLa cells were inspected
using the MTT assay after 24 h exposure. As revealed from
Fig. 6, data of cell viability on HeLa cells displayed that MR
at 10, 25, 50, 100 and 200 µM concentrations significantly
reduced the viability 92.01, 79.18, 63.99, 52.31 and 39.18 %
(p < 0.001) respectively, as compared to untreated cells (taken
as 100 % viable). Likewise at 10, 25, 50, 100 and 200 µM doses
80
60
40
20
0
0
10
25
50
100
200
Concentration (µM)
Fig. 6. Column graph demonstrating as the % cell viability at different
concentrations of MR, MLS2 and LS1on HeLa cells, calculated by
MTT assay after 24 h incubation. Data are represented as mean
SEM of three independent experiments and value of p*** ≤ 0.001
35
MR
MLS2
LS1
30
25
20
15
10
5
of MLS2 treatment reduced the cell viability in 89.43, 77.01,
60.00, 47.87 and 34.37 %, while LS1 showed approximately
96.21, 86.38, 74.40, 58.87 and 42.79 % (p < 0.001) reduction
of viable cells, with respect to the control. The results clearly
recommended that MR, MLS2 and LS1 significantly reduced
the viability of cervical carcinoma cell line depending upon
dose. The IC50 value of MR, MLS2 and LS1 against HeLa
cells was estimated to be 90.8, 81.8 and 115 µg/mL, respec-
tively and suggested that MLS2 was found to be more toxic
as compared to MR and LS1. This study also supported the
previous report that metal complexes were more toxic than
that of ligand [35].
0
0
25
50
100
Concentration (µM)
Fig. 5. Column graph representing % apoptotic cells in HeLa cells at 25,
50 and 100 µM concentrations of MR, MLS2 and LS1. Data were
represented as mean SEM of three independent experiments and
value of p*** ≤ 0.001

2318 Singh et al.
Asian J. Chem.
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terized by drastic alterations in expression and action of some
potent apoptotic markers [36]. ROS production has also been
involved in an initial incident of the cellular apoptosis and
various cancer chemotherapeutic drugs induce apoptosis by
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mediated cellular apoptosis in HeLa cells with the increasing
concentrations of MR, MLS2 and LS1 as compared to untreated
cells (Fig. 4). Similarly, the results obtained from the quantitative
fluorescence intensities showed that 25 µM concentration of
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The cellular apoptosis by MTT assay proposed that MR,
MLS2 and LS1 significantly reduced the viability of HeLa
cells in a dose-dependent manner. The inhibitory concentration
(IC50) value of MR, MLS2 and LS1 was estimated to be 90.8,
81.8 and 115 µg/mL for HeLa cells, respectively. The excessive
ROS generation supports the molecular mechanism of apoptosis
as well as the data of nuclear condensation assay, also suggested
that these synthesised compound induced cell death in HeLa
cells by an apoptotic manner.
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ACKNOWLEDGEMENTS
The authors gratefully acknowledge Rajiv Gandhi National
Fellowship for financial Support and DST-PURSE, Department
of Chemistry, Lucknow University, Lucknow, India.
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CONFLICT OF INTEREST
The authors declare that there is no conflict of interests
regarding the publication of this article.
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