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Structurally different domains embedded half-sandwich arene Ru(II) complex: DNA/HSA binding and cytotoxic studies
Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: https://www.tandfonline.com/loi/gcoo20
Structurally different domains embedded halfsandwich arene Ru(II) complex: DNA/HSA binding
and cytotoxic studies
V. O. Yadhukrishnan, Mathiyan Muralisankar, Ramachandran Dheepika,
Ramaiah Konakanchi, Nattamai S. P. Bhuvanesh & Samuthira Nagarajan
To cite this article: V. O. Yadhukrishnan, Mathiyan Muralisankar, Ramachandran Dheepika,
Ramaiah Konakanchi, Nattamai S. P. Bhuvanesh & Samuthira Nagarajan (2020): Structurally
different domains embedded half-sandwich arene Ru(II) complex: DNA/HSA binding and cytotoxic
studies, Journal of Coordination Chemistry, DOI: 10.1080/00958972.2020.1782895
To link to this article: https://doi.org/10.1080/00958972.2020.1782895
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JOURNAL OF COORDINATION CHEMISTRY
https://doi.org/10.1080/00958972.2020.1782895
Structurally different domains embedded half-sandwich
arene Ru(II) complex: DNA/HSA binding and
cytotoxic studies
V. O. Yadhukrishnana, Mathiyan Muralisankara, Ramachandran Dheepikaa,
Ramaiah Konakanchib, Nattamai S. P. Bhuvaneshc and Samuthira Nagarajana
a
Department of Chemistry, Central University of Tamil Nadu, Thiruvarur, Tamilnadu, India;
Department of Chemistry, National Institute of Technology, Warangal, Telangana, India;
c
Department of Chemistry, Texas A & M University, College Station, TX, USA
b
ABSTRACT
ARTICLE HISTORY
In this investigation, we have designed and synthesized a new
Ru(II)-arene complex with triarylamine thiosemicarbazone hybrid
ligand to explore its cytotoxicity. The ligand and the complex
were well characterized by spectroscopic techniques. From the
single-crystal X-ray crystallography, the molecular structure of the
ligand was confirmed to be monoclinic lattice with C12/C1 space
group symmetry. X-ray photoelectron spectroscopy(XPS) studies
ensured that ruthenium is in þ2 oxidation state. The synthesized
Ru(II)-arene complex was thoroughly investigated for the in vitro
activity against human breast carcinoma (MCF-7), human colon
carcinoma (COLO 205), human neuroblastoma (IMR-32), murine
microphage (Raw 264.7) and embryonic kidney (HEK 293) using
MTT assay. The interaction of the Ru(II)-arene complex with DNA/
protein was also explored by absorption and emission spectral
methods. This investigation highlights the role of hybrid ligand
and its Ru(II) complex toward high cytotoxicity in vitro. The complex has high cytotoxic effect with IC50 value of 5.18 ± 1.128 mM
toward human breast carcinoma cell line.
Received 9 April 2020
Accepted 9 June 2020
CONTACT Samuthira Nagarajan
snagarajan@cutn.ac.in
Nadu, Thiruvarur 610005, India
Supplemental data for this article can be accessed here.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group
KEYWORDS
Triarylamine; thiosemicarbazone; arene Ru(II) complex:
DNA binding; cytotoxicity
Department of Chemistry, Central University of Tamil
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V. O. YADHUKRISHNAN ET AL.
1. Introduction
Metallo-anticancer drug development is an important and dynamic area of research
which drives the bioinorganic pharmaceutical findings toward life-changing inventions
[1–5]. Platinum-based drugs such as cisplatin, carboplatin, oxaliplatin, etc. have been
the benchmark agents to treat many types of cancers in the human body, but their
toxicity levels are relatively higher [6–9]. Developing new chemotherapeutics for treating platinum-resistant tumors with less toxicity and higher activity has gained global
attraction. The journey to find alternative anticancer metal-based drugs other than
platinum compounds is a very interesting research around the Globe. The ligand
exchange kinetics of ruthenium complexes is comparable with platinum(II) complexes;
the ligand exchange processes are quite slow and may take hours (rates 103 to 102
s1) [10–15].
Among the ruthenium complexes investigated, Ru(II)-arene half-sandwich complexes with a piano stool geometry have gained increased attention due to their
diverse activity with respect to the chelating ligands [16–19]. Sadler’s and coworkers
[20–22] complexes (N,N-chelating ligands) bind specifically to N7 of guanine and
exhibited high cytotoxicity against human ovarian cancer cell line (A2780). Likewise,
Ru(II)-arene complexes with various ligands such as quinoxalinone [23], phenanthroline
[24] and carboline [25] have been successfully investigated for their antitumor activity.
Recently, the study of thiosemicarbazone (TSC) is gaining attention due to its success
in clinical trials. Traynor et al. [26] and Stacy et al. [27] have reported on 3-aminopyridine-2-carboxaldehyde TSCs and their clinical trials (II Phase) for antineoplastic activity.
Prophylaxis against both vaccinia and smallpox viruses [28] by methisazone have been
reported by Hall et al. [29]. Among the various complexes studied imidazolium Ru(III)
complexes are in phase II trials. To be specific, in 2017, KP1019 has a new formulation
JOURNAL OF COORDINATION CHEMISTRY
3
to resolve the solubility issue and named as NKP-1339, approved by the FDA. In addition, it is very important to note that the former formulations are abandoned.
The combination of TSC ligands with many other biologically significant compounds
as co-ligands has been used in various applications [30–32]. Triarylamines (TAA) are
being studied for multiple applications, including electronic, bioinorganic, etc.; they are
easily soluble and highly fluorescent. Owing to these properties they have been successfully administrated as probes and markers for DNA [33, 34]. They possess a unique style
of interaction with DNA and can exclusively bind to quadruplex DNA [35, 36]. Chennoufi
et al. [37] have investigated on water-soluble pyridine substituted triphenylamine target
cytosolic organelles of living cells by apoptosis induced by two photon absorption. They
stated that photoactivation is related to mitochondrial apoptotic pathway by reactive
oxygen species (ROS) production.
In this work, in order to improve the activity of ruthenium(II)-arene complexes, we
have designed ligand with biologically active TSC and TAA. Hybrid architecture of ligand is acknowledged as sterically flexible owing to its intramolecular hydrogen bonding and co-ligand effect over the azomethine carbon atom. In addition, p-p stacking
interactions also help to improve the binding [38, 39]. The interaction of complex with
DNA and human serum albumin (HSA) was studied by spectroscopic methods. The
in vitro activity of these complexes is examined by MTT assay, antiproliferative assay
using human breast carcinoma cell line (MCF-7), human colon carcinoma cell line
(COLO 205), human neuroblastoma cell line (IMR-32) and murine microphage cell lines
(Raw 264.7). This investigation will give valuable idea about the role of hybrid ligand
and its cytotoxic activity to the organometallic research community.
2. Experimental
2.1. Materials and methods
All reagents were purchased from Sigma Aldrich (Bangalore, India) and used as
received. The melting point of the synthesized complex was measured by Lab India
instrument and produced as uncorrected. Electronic absorption spectra were taken
in the range 200–800 nm using a Jasco spectrophotometer. FT˗ IR spectra in the
range of 4000–500 cm˗ 1 were obtained using a Perkin-Elmer Frontier FT-IR spectrophotometer. Jasco V-630 was used for obtaining the emission spectra in dimethyl
formamide (DMF). Both 1H and 13C NMR spectra were recorded in CDCl3 solvent with
Tetramethylsilane (TMS) as an internal standard using a Bruker spectrometer (400
and 100 MHz, respectively). Electron spray ionization-mass spectrometry (ESI˗MS) analysis of ligand and complex were recorded on a Thermo ExactivePlus mass
spectrometer.
2.2. Synthesis
2.2.1. Synthesis procedure of ligand
Triarylamine (TAA) aldehyde was added to a methanolic solution of 4-cyclohexyl thiosemicarbazide with a few drops of glacial acetic acid, and the resulting mixture was
heated at 60–70 C for 6 h. The solution was cooled to room temperature to afford a
4
V. O. YADHUKRISHNAN ET AL.
yellow solid, and was filtered, washed with ethanol, and dried in vacuum. Methanol
and acetonitrile (1:1) mixture was allowed to evaporate slowly to get suitable crystals
of compound for XRD analysis. Yield: 86%. Yellow solid. M.p. 236 C. ESI˗ MS (m/z)
Calcd. for C34H41N7S2 [M þ H]þ 612.2943, found 612.2855. Anal. Calc. for C34H41N7S2: C,
66.74; H, 6.75; N, 16.02; S, 10.48. Found: C, 66.91; H, 6.73; N, 16.01; S, 10.43. UV–vis
(DMF), kmax (nm): 213, 386. FT-IR (ATR, cm1): m(N-H); 3328(m), m(C-H); 2930(s),
m(C ¼ N); 1589(s), m(C ¼ S); 1267(s). 1H NMR (400 MHz, CDCl3), d(ppm): 10.23 (s, 2H, ¼N˗
NH), 7.90 (s, 2H, HC ¼ N), 7.52 (d, J ¼ 8.0 Hz, 4H), 7.32 (d, J ¼ 6.8 Hz, 3H), 7.14 (d,
J ¼ 8.0 Hz, 2H), 7.08 (d, J ¼ 8.4 Hz, 4H), 4.31–4.24 (m, 2H), 2.09 (s, 2H, cyclohexyl-NH))
1.76–1.63 (m, 8H), 1.46–1.39 (m, 4H), 1.34–1.21 (m, 8H). 13C NMR (100 MHz, CDCl3),
d(ppm): 175.44 (C ¼ S), 146.32 (C ¼ N), 129, 128, 127, 125, 124, 123 (aromatic carbons),
52.83, 32.77, 25.50, 24.75 (aliphatic carbons).
2.2.2. Synthesis of [(g6˗p˗cymene)-RuII(ligand)Cl]Cl
[(g6˗p˗cymene)-RuII(Cl)2]2 was synthesized as per the reported method [40]. The prepared Ru(II)-(g6˗ p˗ cymene) dimer (0.100 g, 0.2 mmol) and ligand were combined in
20 mL of CH2Cl2 and the resultant mixture was stirred for 12–14 h at room temperature. The color of the reaction mixture changed to dark red. The dark red solution was
concentrated under reduced pressure, and the addition of hexane (20 mL) gave an
orange solid. The product was collected by filtration, washed with hexane, and dried
in vacuum. Yield: 78%. Orange solid. M.p: 216 C. ESI˗ MS (m/z) Calcd. for
C44H56Cl2N7RuS2 [M˗2Cl-H]þ 846.2926, found 846.2816. UV–vis (DMF), kmax (nm): 214,
398, 540. Anal. Calc. for C44H55N7RuS2: C, 59.88; H, 6.28; N, 11.11; S, 7.21. Found: C,
60.02; H, 6.27; N, 11.08; S, 7.29. FT-IR (ATR, cm1): m(N-H); 3347(m), m(C-H); 2968(s),
m(C ¼ N); 1580(s), m(C ¼ S); 1241(s). 1H NMR (400 MHz, CDCl3), d(ppm): 14.42 (s, 1H,
¼N NH), 8.73 (s, 1H, HC ¼ N), 8.08 (d, J ¼ 8.4 Hz, 1H), 7.37 (t, J ¼ 8.0 Hz, 4H), 7.20 (d,
J ¼ 7.2 Hz, 6H), 7.14 (d, J ¼ 8.4 Hz, 1H), 7.08 (d, J ¼ 8.4 Hz, 2H), 5.49 (d, J ¼ 6.0 Hz, 1H,
p-cym-H), 5.10 (d, J ¼ 6.0 Hz, 1H, p-cym-H), 5.01 (d, J ¼ 5.2 Hz, 1H) 4.97 (d, J ¼ 1.6 Hz,
1H, p-cym-H), 2.72–2.66 (m, 2H, p-cym CH(CH3)2), 2.12 (s, 3H, p-cym (CH3)), 2.02 (s,
cyclohexane attached N 3H) 1.20 (d, J ¼ 6.8 Hz, 3H, p-cym C(CH3)2), 1.14 (d, J ¼ 6.8 Hz,
3H, p-cym C(CH3)2), 1.77–1.61 (m, 4H), 1.61–1.57 (m, 8H), 1.50–1.46 (m, 2H), 1.44–1.35
(m, 8H). 13C NMR (100 MHz, CDCl3), d(ppm): 175.59 (C ¼ S), 148.82(C ¼ N), 132, 129,
128, 125, 124, 123 (aromatic carbons), 103.50, 88.55, 87.94, 82.66 (carbons of p-cymene), 52.87, 32.76, 30.71, 25.51, 24.76, 21.40, 18.56 (aliphatic carbons).
2.3. DNA-binding studies
DNA is recognized as the primary target of many metal-based anticancer drugs. Most
of the metal complexes exert their effects through binding with DNA, which is the critical step to evaluate the effectiveness of metal-based drugs. As a result, the elucidation of non-covalent interactions between DNA and complex was investigated with
the help of different techniques. The UV–vis absorption spectroscopic studies for DNAbinding analysis were performed at room temperature. CT-DNA sample was dissolved
in 50 mM NaCl/5 mM TrisHCl (pH 7.2) solution. The CT-DNA solution (without complex)
showed an absorbance at 260 nm with 6600 M1 cm1 extinction coefficient, indicating
JOURNAL OF COORDINATION CHEMISTRY
5
that the CT-DNA was in protein-free form [41]. Stock solutions were stored at 4 C and
used within 4 days. About 0–50 lM of CT-DNA was added to the fixed concentration
of complex. The spectra were recorded after equilibration for three min, allowing the
compounds to bind with CT-DNA. Complex of required concentration was prepared by
dissolving the calculated amount of complex in 5% DMF/TrisHCl/NaCl.
The competitive binding of each complex with EB was investigated by fluorescence
spectroscopic techniques, thus examining whether the complex can displace EB from
its CT-DNA-EB complex. EB solution was prepared using TrisHCl/NaCl buffer (pH 7.2).
The test solution was added in aliquots of 2.5 lM concentration to DNA-EB and the
change in fluorescence intensities at 596 nm (excitation at 450 nm) was recorded.
2.4. HSA-binding studies
The fluorescence emission spectroscopy was utilized to study the binding of Ru(II)arene complex with HSA. The experiments were carried out at a fixed excitation wavelength (corresponding to HSA) at 280 nm and monitoring the emission at 335 nm.
Stock solution of HSA was prepared in Tris-buffer (50 mM NaCl/5 mM Tris-HCl, pH 7.2)
and stored in the dark at 4 C. HSA (2.5 mL) solution was titrated by consecutive additions of 106 M of complex. The synchronous fluorescence spectra measurements
were obtained at the same concentration of HSA and the complex. The spectra were
measured at two different Dk (difference between the excitation and emission wavelengths of HSA) values of 15 and 60 nm.
2.5. Antiproliferative activity and MTT assay
The human breast carcinoma cell line (MCF-7), human colon carcinoma cell line (COLO
205), human neuroblastoma cell line (IMR-32) and murine microphage cell line (Raw
264.7) were obtained from National Centre for Cell Science (NCCS), Pune and grown in
Dulbecco’s Modified Eagles Minimum (DMEM) containing 10% fetal bovine serum
(FBS), amphotericin (3 lg mL1), gentamycin (400 lg mL1), streptomycin (250 lg
mL1) and penicillin (250 units mL1) in a carbon dioxide incubator at 5% CO2. About
700 cells/well were seeded in a 96-well plate using culture medium, the viability was
tested using trypan blue dye with the help of a haemocytometer and 95% of viability
was confirmed. After 24 h, the new medium with compounds in the concentration of
100, 10 and 1 lg mL1 were added at respective wells and kept at incubation for 48 h.
After incubation MTT assay was performed [42].
After 48 h of drug treatment, the medium was changed again for all groups and
10 lL of MTT (5 mg mL1 stock solution) was added and the plates were incubated for
an additional 4 h. The medium was discarded and the formazan blue, which was
formed in the cells, was dissolved with 50 lL of DMSO. The optical density was measured on a micro plate spectrophotometer at a wavelength of 570 nm. The percentage
of cell inhibition was calculated using the following formula [43]:
% Growth inhibition ¼ 100 – ðAi =Ao Þ 100
where Ai is the absorbance of the sample and Ao is the absorbance of the control.
6
V. O. YADHUKRISHNAN ET AL.
Scheme 1. Synthesis of ligand.
Scheme 2. Synthesis of complex.
Non-linear regression graph was plotted between % cell inhibition and Log10 concentration and IC50 was determined using Graph Pad Prism software.
3. Results and discussion
The hybrid ligand was synthesized through condensation reaction between 4-cyclohexyl thiosemicarbazide and triarylamine dialdehyde as shown in Scheme 1.
Ruthenium(II)(g6-p-cymene) complex of the type [RuCl(g6-p-cymene)ligand]Cl was
synthesized by the reaction between [RuCl(m-Cl)(g6-p-cymene)]2 and triarylamine-based
TSC ligand (Scheme 2). Synthesized ligand and complex were characterized using
UV–vis, FT-IR, NMR spectroscopic and mass spectrometric studies. The structure of the
ligand was determined by single-crystal X-ray diffraction study.
3.1. Spectroscopy and crystal structure
The absorption spectrum of the ligand in DMF exhibited two characteristic bands (supporting information Figure S1). The bands at 213 and 318 are assigned to p-p and
n-p transitions, respectively [44]. The complex also exhibited two prominent bands at
214 and 286 nm which correspond to p-p and n-p transitions, respectively (supporting information Figure S2). The additional bands at 392 and 540 nm were assigned to
MLCT and d-d transitions [45]. Absorption spectra of the complex were taken in DMF
JOURNAL OF COORDINATION CHEMISTRY
7
Figure 1. Thermal ellipsoid (50%) plot of ligand with atomic labeling.
for fresh solution and after five days. There were no changes observed in the spectra,
which shows that ruthenium(II) complex is stable in the test solution [46]. FT-IR data
of the TSC ligands show a characteristic peak at 3453 cm1 due to m(N H). Other
characteristic peaks are obtained at 1582 cm1 and 1321 cm1 which correspond to
m(C ¼ N) and m(C ¼ S), respectively. After the complex formation, the stretching frequency of thiocarbonyl m(C ¼ S) decreases, indicating that the sulfur is coordinated
with ruthenium ion [47].
1
H NMR spectrum of ligand exhibits (¼N-NH) and azomethine (HC ¼ N) protons
were at 10.26 and 7.90 ppm (supporting information Figure S8). The same protons in
the complex were deshielded and appeared at 14.56 ppm. The aromatic protons of
the complex resonate in the range of 7.37–7.08 ppm. The new signals observed in the
region 5.50–4.97 ppm are due to occurrence of p-cymene complex [48]. The signal due
to cyclohexyl protons in the ligand and complex were displayed in the range of
4.30–3.89 ppm and 2.04–1.23 ppm (supporting information Figure S10). In the 13C
spectrum of ligand, thiocarbonyl (C ¼ S) and imine (C ¼ N) carbon signals appeared at
175.99 ppm and 146.32 ppm (supporting information Figure S9). All aromatic carbons
in the ligand and complex appeared in the range of 149.61–119.74 ppm and
150.69–119.23 ppm. 13C NMR spectrum of the complex, new signals at
103.95–82.12 ppm confirmed the presence of p-cymene group in complex (supporting
information Figure S11).
The single-crystal XRD of the ligand shows the monoclinic lattice with C12/C1 space
group symmetry. The experimental details, crystallographic refinement parameters and
selected bond lengths and angles are presented in supporting information Tables S1
and S2. Thermal ellipsoid plot of ligand with the atomic labeling scheme is shown in
Figure 1. The ligand is typically composed of three different domains: TAA group connected to cyclohexyl by TSC moiety. The structure reveals that the central nitrogen
atom of triarylamine is in sp2 hybridization and the three phenyl rings are slightly
tilted from the plane of the ring, existing in a propeller-like fashion [14]. The molecule
has E-conformation with respect to the N2 N3, the bond length is 1.370, 1.385, 1.380
and 1.386 Å and dihedral angle is 177.0 , 164.7 , 173.3 and 173.2 for ligand.
ESI-MS spectra of ligand and complex are shown in supporting information Figures
S12 and S13, respectively, in positive scan mode. The spectrum of ligand shows
8
V. O. YADHUKRISHNAN ET AL.
Figure 2. The narrow scan XPS spectra of complex.
[M þ H]þ peak as the base peak at 612.2865 m/z. Molecular ion peak for the complex
was not observed due to possible fragmentation. The complex exhibits base peak at
846.2816 m/z due to [M 2Cl-H]þ, suggesting that the chloro (2Cl) group is
labile [49].
The narrow scan XPS spectra of the elements Ru 3d, Ru 3p, C 1 s, S 2p and S 2 s are
given in Figure 2. The presence of Ru 3d5/2 (280.64 eV) and Ru 3p3/2 (462 eV) peaks
confirms that the ruthenium is present in the complex as Ru(II). S 2p (100 eV) and S
2 s (160 eV) suggest the formation of ruthenium-sulfur bond [50] and 1 s peak was
obtained at 284.15 eV.
3.2. DNA interaction studies
3.2.1. Uv–visible titration
The electronic spectra of complex with and without CT-DNA are given in Figure 3. The
changes observed in the electronic spectra accounts for the complex–CT–DNA interaction. From the spectra, it is found that the complex upon addition of CT-DNA exhibits hypochromism with 2-3 nm red-shift which can be indicative of intercalation mode
of binding [51, 52]. The equilibrium constant (Kb) was found from the linear plot
obtained using the equation:
JOURNAL OF COORDINATION CHEMISTRY
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Figure 3. Absorption spectra of complex with CT-DNA in Tris-HCl buffer upon addition of CT-DNA.
[Complex] ¼ 1.5 105 M, [DNA] ¼ 0 50 lM.
½DNA
¼ ½DNA=ðeb ef Þ þ 1=Kb ðeb ef Þ
ðea ef Þ
where [DNA] is the concentration on DNA in base pairs, ea is the apparent extinction
coefficient calculated as A(observed)/[complex], ef is the extinction coefficient for the free
complex and eb is the extinction coefficient for the complex in the completely bound
form. Kb was calculated from the plot of [DNA]/(ea-ef) versus [DNA] by taking the ratio
of slope and intercept given in supporting information Figure S3. The binding constant was found to be 3.2 104 M1. The Kb found is matching with already reported
similar complexes [53].
3.2.2. EB displacement studies
The prepared complex does not possess fluorescence in solution with CT-DNA at
room temperature, thus the binding of complex with CT-DNA cannot be studied directly. The quenching of fluorescence of ethidium bromide (EB) bound to CT-DNA with
the addition of complex is investigated. A decrease in the fluorescence is observed
when the complex replaces EB from the CT-DNA and it gives an indirect evidence for
the DNA binding. Figure 4 shows the interaction of complex with CT-DNA treated
with EB. The fluorescence decreased upon each addition of the complex. The quenching of fluorescence confirms the displacement of EB from the CT-DNA by the ruthenium complex. The interactions obtained by fluorescence studies were quantitatively
measured using the Stern-Volmer equation, F0/F ¼ 1þKq[Q] [53], where F0 and F are
the fluorescence intensities in the presence and in the absence of the complex, Kq is
the linear Stern-Volmer quenching constant and [Q] is the concentration of complex.
10
V. O. YADHUKRISHNAN ET AL.
Figure 4. Fluorescence quenching curves of EB bound to DNA in the presence of complex.
[DNA] ¼ 5 lM, [EB] ¼ 5 lM, and [complex] ¼ 0 50 lM. Arrow shows decrease in emission upon
increasing DNA concentration.
Kq is found from the slope of F0/F versus [Q] and given in supporting information
Figure S4.
The equation KEB[EB]¼Kapp[complex] is used to calculate the apparent binding constant Kapp [54], where [complex] is concentration of complex when the fluorescence
intensity of EB is reduced to 50%. [EB] ¼ 5 mM and KEB ¼ 1.0 107. The results suggest
that the complex binds with CT-DNA through intercalation mode interaction. Kq and
Kapp values were found to be 2.08 104 and 1.02 106 M1, respectively.
3.3. Protein-binding studies
3.3.1. Fluorescence spectroscopic studies
Emission spectra were obtained in a broad range of 290–450 nm on exciting at
280 nm. The fluorescence quenching of HSA upon adding the ruthenium complex was
observed at 310 nm with 2–3 nm hypochromic shift. Changes in the spectra of complex are illustrated in Figure 5. The decrease in fluorescence intensity confirmed the
interaction of complex with HSA. The shift in wavelength has proved that the interaction of complex and protein takes place at hydrophobic environment. Using the
Stern-Volmer equation the quenching constant (Kq) was found to measure the interaction quantitatively from the plot of F0/F versus [Q] (supporting information Figure
S5). The Scatchard equation was used to find the equilibrium binding constant.
Scatchard equation is given by log[(F0-F)/F] ¼ logKb þ nlog[Q], where F0 and F are the
fluorescence in the absence and presence of the complex, Kb is the equilibrium binding constant and n is the number of binding sites [52]. The Kb is found from a plot of
log[(F0-F)/F] versus log [Q] (supporting information Figure S6). The ligand and complex
showed binding constant values toward HSA of 1.18 105 and 9.78 105 M1,
JOURNAL OF COORDINATION CHEMISTRY
11
Figure 5. Fluorescence quenching curves of HSA in the absence and presence of complex.
[HSA] ¼ 1 mM and [complex] ¼ 0–20 mM.
respectively. The values obtained from fluorescence data show that the complex has
good binding affinity toward the HSA.
Beckford et al. [56] studied the biological behavior of a ruthenium-arene complex
containing piperonal-based TSC ligands. The obtained binding constants for CT-DNA
were in the range of 103 M1, where complex in this study shows better binding in
the range of 104 M1. The combination of ligands with Ru metal has exhibited DNA
and protein-binding constants which is comparable with our complex [54, 55]. Subasi
et al. [57] reported a similar kind of study on ruthenium-arene complex with pyrrole
ring containing TSC ligand gives binding constant in the range 104 M1 toward
CT-DNA. Similarly, in another report Muralisankar et al. [58] reported DNA- and protein-binding studies of TSC containing complexes are in the range of 104 M1 and
protein-binding constant in the range of 105 M1.
3.4. In vitro antiproliferative evaluation
The antiproliferative activity of the newly synthesized compounds was evaluated
against different cancer cell lines such as IMR-32 (neuroblastoma), MCF-7 (breast),
COLO 205 (colon), Raw 264.7 (murine) and HEK293 (embryonic kidney) by using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method [53]. The MTT
assay results were shown in IC50, expressed in micromolar units and summarized in
Table 1. The percentage of cell viability versus concentration graphs is shown in
supporting information Figure S7. Cisplatin was used as a positive control. The results
clearly indicate that the complex exhibited potent antiproliferative activity against
MCF-7 and Raw 264.7 with IC50 values of 5.18 ± 1.128 mM [38] and 13.29 ± 1.0.21 mM
[39], respectively, compared to standard drug cisplatin (IC50 ¼ 3.78 ± 1.088 and
7.24 ± 0.156 mM). Similarly the complex showed good activity against IMR-32
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V. O. YADHUKRISHNAN ET AL.
Table 1. IC50 values of synthesized compounds against IMR-32, COLO 205, MCF-7, RAW 264.7 and
HEK 293 cell lines.
IC50 (mM)
Comp.
IMR-32
COLO 205
MCF-7
Raw 264.7
HEK 293
Ligand
Complex
STD
59.24 ± 1.068
30.88 ± 1.047
4.29 ± 1.125
57.42 ± 0.514
28.07 ± 1.342
5.68 ± 1.057
64.57 ± 1.893
5.18 ± 1.128
3.78 ± 1.088
63.23 ± 1.023
13.29 ± 1.021
7.24 ± 0.156
ND
38.72 ± 1.121
ND
(30.88 ± 1.047 mM) [54] and COLO 205 (28.07 ± 1.342 mM) [41], respectively. In addition,
we have also tested the toxicity of the complex against HEK293 normal cell line, the
IC50 values of 38.72 ± 1.121 mM. Interestingly, the complex has superior cytotoxic effect
with IC50 value of 5.18 ± 1.128 mM toward human breast carcinoma cell line (MCF-7)
among the cell lines studied, which is comparable with the standard drug cisplatin
and few similar molecules. Demoro et al. [59] have investigated binuclear TSC ruthenium complexes with nitrofuryl groups as potential antitumor agents, where the IC50
value was in the range of 8.7–30 mM against MCF-7. In another study, Anitha et al. [60]
have shown that complexes with TSC/semicarbazone bearing 9,10-phenanthrenequinone groups exhibited IC50 from 13 to 39 mM. The complex has better cytotoxicity
than the 9-anthraldehyde TSC substituted Ru complexes reported by Beckford et al.
[56]. The impressive cytotoxicity of complex against MCF-7 cancer lines has proven
them to be new promising candidate in anticancer research.
4. Conclusion
Triarylamine-TSC hybrid ligand and its Ru(II) complex as an anticancer agent has been
synthesized and characterized by various techniques. The single-crystal X-ray diffraction study confirmed the structure of the ligand; the monoclinic lattice with C12/C1
space group symmetry. XPS spectra evidently showed that ruthenium is present in the
complex in þ2 oxidation state. The interaction of complex with CT-DNA was calculated to be 3.2 104 M1. The complex showed 9.78 105 M1 binding constant values, toward HSA. In vitro studies of the ligand and complex against five different cell
lines, IMR-32, COLO 205, MCF-7, RAW 264.7 and HEK 293, are analyzed. The complex
showed moderate activity against COLO 205, IMR-32 and HEK293. Interestingly, the
complex has superior cytotoxic effect with IC50 value of 5.18 ± 1.128 mM toward human
breast carcinoma cell line (MCF-7) among the cell lines studied, which is comparable
with the standard drug cisplatin. On summation of the studies, the cytotoxicity role of
hybrid combination of ligands and its Ru(II)-arene complex have been explained and
they can be potentially used as ideal anticancer agents.
Disclosure statement
No potential conflict of interest was reported by the authors.
Acknowledgements
M.S. thanks the Department of Science and Technology and Ministry of Science and
Technology, Government of India for Post-Doctoral Fellowship under SERB-NPDF program.
JOURNAL OF COORDINATION CHEMISTRY
13
Funding
S.N. gratefully acknowledges DST-SERB for the financial support.
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