New Ru(II) complexes containing tris(2-pyridylmethyl)amine. Synthesis, structural, CT-DNA/albumin interaction, anti-oxidant and cytotoxicity studies

Article abstract of DOI:10.1016/j.ica.2017.12.010

Two new Ru(II) complexes containing tris(2-pyridylmethyl)amine was synthesised by reacting [RuCl₂ (EPh₃)₃] E = P, As with TPA.3HClO₄ in 1:1 ratio. The new complexes were characterized by various spectroscopic (I

Full text of DOI:10.1016/j.ica.2017.12.010

Accepted Manuscript
Research paper
New Ru(II) complexes containing Tris(2-pyridylmethyl)amine. Synthesis,
structural, CT-DNA/ albumin interaction, anti-oxidant and Cytotoxicity studies.
P. Naveen, F. Dallemer, R.J. Butcher, R. Prabhakaran
PII:
S0020-1693(17)31470-6
https://doi.org/10.1016/j.ica.2017.12.010
ICA 18044
DOI:
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Inorganica Chimica Acta
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21 September 2017
9 November 2017
3 December 2017
Please cite this article as: P. Naveen, F. Dallemer, R.J. Butcher, R. Prabhakaran, New Ru(II) complexes containing
Tris(2-pyridylmethyl)amine. Synthesis, structural, CT-DNA/ albumin interaction, anti-oxidant and Cytotoxicity
studies., Inorganica Chimica Acta (2017), doi: https://doi.org/10.1016/j.ica.2017.12.010
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New Ru(II) complexes containing Tris(2-pyridylmethyl)amine. Synthesis,
structural, CT-DNA/ albumin interaction, anti-oxidant and Cytotoxicity
studies.
P. Naveen,a F. Dallemer,b R. J. Butcher c and R. Prabhakarana*
a
Department of Chemistry, Bharathiar University, Coimbatore 641 046, India, E-Mail:
rpnchemist@gmail.com Tel.: +91-422-2428319; Fax: +91-422-2422387
bLaboratoire Chimie Provence-CNRS, UMR7246, Université of Aix-Marseille, Campus
Scientifique de Saint-Jérôme, Avenue Escadrille Normandie-Niemen, F-13397 Marseille
Cedex 20,France.
cInorganic and Structural Chemistry Howard University Washington DC 20059 Office: (202)
806-9892 Cell: (703) 585-6631 Fax: (202) 806-5442
Abstract
Two new Ru(II) complexes containing Tris(2-pyridylmethyl)amine was synthesised by
reacting [RuCl2 (EPh3)3] E=P, As with TPA.3HClO4 in 1:1 ratio. The new complexes were
characterized by various spectroscopic (IR, UV-Vis and NMR) techniques and the structure of
the complexes was confirmed by X-ray crystallography. Crystallographic studies of the
complexes 1 and 2revealed the octahedral geometry around ruthenium ion. The binding ability
of the complexes with CT-DNA was carried out by photophysical (absorption / emission
titration) methods in which an intercalative mode of binding was observed. Further, their
ability to interact with BSA protein has been explored. Investigations of antioxidant properties
showed that all the compounds have significant radical scavenging properties. The anticancer
activity of the Ru(II) complexes was probed in vitro cytotoxicity against human breast
(MCF7) and lung (A549) cancer cell lines by MTT assay and cytological changes observed in
(AO/EB and DAPI) staining method. Among the biological studies the compounds showed
significant activity.
Keywords: Ruthenium(II) complexes; Spectroscopy; X-ray crystallography; CT-DNA/BSA
protein binding; Anti-oxidant activity and cytotoxicity.
Corresponding author. Tel.: +91-422-2428319; Fax: +91-422-2422387.
E-mail address: rpnchemist@gmail.com (R. Prabhakaran)
1

1. Introduction
The field of metallopharmaceuticals has developed as important field of medicinal chemistry
due to the therapeutic uses of metal-based drugs [1]. Platinum complexes mostly cisplatin,
carboplatin and oxaliplatin are current most active chemotherapeutic agents, even though their
high toxicity and undesirable neuro-, hepato- and nephrotoxicity side effects [2,3]. However,
high systemic toxicity and resistance problems associated with platinum based drugs [4] have
opened up a new era of attention in the design and pharmacological development of
alternative metal-based anti-tumor agents [5]. In particular, ruthenium complexes represent a
new discussion of promising metal-based drugs with low toxicity and high activity in
cytotoxicity which does not respond well to platinum drugs [6].
The chemistry of ruthenium with unsaturated nitrogen ligands has been studied extensively in
recent times including the study of photophysical and photochemical properties [7]. The major
work has grown around N,N-chelating bipyridine bases and related species [8]. The number of
heteroatoms, the ring size and the substituents in the heterocyclic ring significantly modify the
π-acidity and regulate the physical and chemical properties of the compounds [9]. Recently,
the design of molecular architectures with imidazole ligands has contributed to the
understanding of biomolecular interactions with metal ions in biology and provides models for
the active sites of metalloproteins [10,11].
The tetradentate tripodal ligand, TPA [tris(2-pyridylmethyl)amine], and its derivatives
have been widely used in coordination chemistry during the last decades. Its σ-donating
central amine and its three π-accepting pyridyl methyl arms and the mobility of these arms
allow TPA to accommodate a variety of metal centres with different coordination geometries
and electronic structures [12]. These properties make it as a perfect ligand for the synthesis of
redox or catalytically active species, due to its adaptation to electronic and steric changes at
metal centre. The wide variety of TPA complexes that have been prepared and were found
applications as biological enzyme models [13], catalysts oxidation [14], oxygenation [15],
hydroxylation [16], epoxidation [17], aziridination spin-crossover species [18], phosphodiester
cleavage agents [19], dioxygen activation by iron non-hemeoxygenases. It can also act as
abridging ligand in a number of coordination polymers [20,21].
Herein, we describe the synthesis, spectral characterization, X-Ray crystallographic studies
and biological evaluation of two new Ru(II) Complexes containing TPA [tris(2-
pyridylmethyl)amine] ligand.
2

2. Experimental
2.1 Materials and measurements
The ligand TPA.3HClO4 [tris(2-pyridylmethyl)amine] [22] and the ruthenium
precursor [RuCl2(PPh3)3] [23] were synthesized according to the standard literature
procedures. All the reagents used in this study were analar grade and the solvents were
purified and dried according to the standard procedure [24]. Infrared spectra were measured as
KBr pellets with a Nicolet Avatar Model FT-IR spectrophotometer in the 400-4000 cm-1
range. The electronic spectra of the complexes were recorded in dichloromethane by using
Jasco V-630 spectrophotometer in the 200-800 nm range. 1H NMR spectra were taken in
DMSO at room temperature with a Bruker 400 MHz instrument with the chemical shift
relative to tetramethylsilane(TMS). Melting points were recorded by using Lab India melting
point apparatus. The collection and correction of single-crystal data for the new Ru(II)
complexes were performed at 293 K with a CCD Kappa diffractometer using graphite-
monochromated Mo Kα (λ = 0.71073 Å) radiation [25]. The structures were solved using
SHELXS-97 [26] and refined by full-matrix least-squares on F2 using SHELXL-2014 and 2016
[27].
2.2 Preparation of Ligand [TPA.3HClO4]
Tris(2-pyridyl methyl)amine was synthesized by literature method [28]. A solution of
18.0 g of picolyl chloride hydrochloride in 100 ml of deionized water was cooled in 0 ºC in
an ice bath. To this solution 45 cm3 of a 5.3 N aqueous solution of NaOH was added with
stirring. The resulting free amine appeared as a bright red emulsion following the
neutralization. To this mixture was then added a solution of 6.4 g of 2-(amino methyl)
pyridine in 100 cm3 of dichloromethane. The mixture was then allowed to warm to room
temperature and over a 48 hours period, an additional 45 cm3 of 5.3 N aqueous NaOH was
added. During the addition of the NaOH solution, the pH of the aqueous portion of the
reaction mixture was not allowed to exceed 9.5. The crude mixture was then washed with 100
cm3 of 15 % NaOH to this solution n number of times adding perchloric acid gave pure white
product which was settled down, was filtered and the compound was characterized by IR, Uv-
Vis and NMR spectroscopic techniques.
Yield: 97%. M.p. 225 °C. FT-IR (cm−1) in KBr: 1638 (νC=N), 1086 (νClO4); UV-Vis (DMSO),
1
λmax (ε): 271 (11,624) nm (dm3mol−1cm−1); 340 (12,565) nm (dm3mol−1cm−1); H NMR
3

(DMSO), (d, δ8.82-8.84 ppm, C2-H),(t, δ 7.82- 7.87 ppm, C3-H), (t, δ 8.36- 8.41 ppm C4-H),
(d, δ 7.97- 7.99 ppm (C5-H), (s, δ 2.99 ppm (-CH2-) protons).
2.3 Synthetic procedure for new ruthenium(II) complexes
To the solution of [RuCl2 (EPh3)3] E=P, As [0.16 g (PPh3), (0.18 g (AsPh3), 0.1681 mmol] in
dichloromethane (15 cm3), TPA.3HClO4 (0.1 g; 0.1681 mmol) in methanol (15 cm3) was
added under reflux condition for 6 h. The resulting solution was filtered and concentrated.
Evaporation of solvent gave orange colour crystals suitable for X-ray crystallographic studies.
Complex: 1
Yield: 82%. M.p. 185°C. FT-IR (cm−1) in KBr: 1619 (νC=N), 625, 1443 (Ru-PPh3),1086
(νClO4);UV-Vis (DMSO), λmax (ε): 265 (21,021) nm (dm3mol−1cm−1) (intra-ligand transition);
1
327 (16,624) nm (dm3mol−1cm−1) (LMCT).; H NMR (DMSO), (m, δ 7.1-8.5 ppm aromatic
protons overlap of pyridine and PPh3), (s, δ 2.50 ppm (–CH2–) protons).
Complex: 2
Yield: 78%. M.p. 178°C. FT-IR (cm−1) in KBr: 1621 (νC=N),622, 1455 (Ru-AsPh3), 1086
(νClO4); UV-Vis (DMSO), λmax (ε): 279 (33,167) nm (dm3mol−1cm−1) (intra-ligand transition);
331 (28,237) nm (dm3mol−1cm−1) (LMCT).1H NMR (DMSO), (m, δ 7.0-8.3 ppm aromatic
protons overlap of pyridine and AsPh3), (s, δ 2.51 ppm (–CH2–) protons).
2.4 DNA binding study
The CT DNA binding studies were carried out in deionised water with the buffer
tris(hydroxymethyl)- aminomethane (Tris, 5 mM) and sodium chloride (50 mM) and pH
adjusted to 7.2 with hydrochloric acid at room temperature. The concentration of CT-DNA
was determined by UV absorbance at 260 nm. Solutions of CT-DNA in Tris-HCl buffer gave
a ratio of UV absorbance at 260 and 280 nm, A260/ A280, of approximately 1.9, indicating that
the DNA was sufficiently free of protein. The molar absorption coefficient, λ260, was taken as
6600 M−1 cm−1. Various concentrations of CT-DNA were added to the ligand and complexes
(25 µM dissolved in DMSO/Tris HCl buffer, 1 % DMSO in the final solution). 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. Control experiments
with DMSO were performed and no changes in the spectra of CT-DNA were observed.
4

Absorption spectra were recorded after equilibrium at 20 °C for 10 min. The intrinsic binding
constant Kb was determined by using following equation (1), [29,30]
[DNA]/[εa-εf]) = [DNA]/[εb-εf]+1/Kb[εb-εf]
(1)
The absorption coefficients εa, εf and εb correspond to Aobsd/[DNA], the extinction coefficient
for the free compound and the extinction coefficient for the compound in the fully bound form
respectively. The intrinsic binding constant Kb can be obtained from the ratio of the slope to
the intercept from the plot of [DNA]/[εa −εf] versus [DNA]. In order to find out the mode of
attachment of CT DNA to the compounds, fluorescence quenching experiments with EB-DNA
were carried out by adding the complexes to Tris-HCl buffer solution of EB-DNA. The
change in the fluorescence intensity was recorded. Before measurements, the system was
shaken well and incubated at room temperature for 5 min.
2.5 Serum albumin binding study
Bovine Serum Albumin (BSA)was purchased from HiMedia, The protein binding study was
performed by tryptophan fluorescence quenching experiments using bovine serum albumin
(BSA, 10 µM) as the substrate in phosphate buffer (pH = 7.2). Quenching of the emission
intensity of tryptophan residues of BSA at 346 nm (excitation wavelength at 280 nm) was
monitored using ligand and complexes as quenchers with increasing concentration (10-
100µM). Synchronous fluorescence spectra of BSA with various concentrations of the
complexes were obtained from 300 to 400 nm when ∆λ = 60 nm and from 290 to 500 nm
when ∆λ = 15 nm. For synchronous fluorescence spectra, the same concentrations of BSA and
the compounds were used and the spectra were measured at two different ∆λ values such as 15
and 60 nm [31].
2.6 Antioxidant property:
2.6.1 DPPH radical scavenging activity [32]
The potential antioxidant activity of ligand and ruthenium(II) complexes (1mg/1ml)
was evaluated by DPPH radical-scavenging assay according to the procedure described by
Braca et al., DPPH free radicals are used for rapid analysis of antioxidants. While scavenging
the free radicals, the antioxidants donate hydrogen and form a stable DPPH–H molecule.
Compounds at various concentrations (100, 200, 300, 400, 500 µL) were taken and about 3
mL of 0.1 mM methanolic solution of DPPH was added to the aliquots of samples and
standards (BHT) and the mixture was shaken vigorously. Negative control was prepared by
5

adding 100 µL of methanol in 3 mL of 0.1 mM methanol solution of DPPH. The tubes were
allowed to stand for 30 min at 27oC. The absorbance of the sample was measured at 517 nm
and expressed as percentage of inhibition.
2.6.2 ABTS•+ scavenging activity [33]
The total antioxidant activity of the samples was measured by ABTS●+ decolourization
assay according to the method of Re et al., ABTS●+ was produced by reacting 7 mM ABTS
aqueous solution with 2.4 mM potassiumpersulphate in the dark for 12-16 hrs at room
temperature. Prior to assay, this solution was diluted in ethanol (about 1:89 v/v) and
equilibrated at 25oC to give an absorbance of 0.700 0.02 at 734 nm. The stock solution of the
sample extract was diluted and after introduction of 20 µL of samples into the assay, they
produced between 20 % and 80% inhibition of the blank absorbance. After the addition of 1
mL of diluted ABTS solution to 30-50 µL of samples (final concentration 0.1 - 5 mM)
o
incubated at 30 C exactly for 30 min and the absorbance was measured after the mixing and
determined in triplicates. The results were expressed in % of inhibition.
2.6.3 Total antioxidant activity [34]
The antioxidant activity of compounds (1mg/1ml) was evaluated by the green
phosphomolybdenum complex formation according to the method of Prieto et al., Different
concentrations of samples and standard (ascorbic acid in 1 mM dimethyl sulphoxide) were
added with 3 mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4
mM ammonium molybdate) in a test tube. The test tubes were incubated in a water bath at
95oC for 90 min. After the samples had cooled to room temperature, the absorbance of the
mixture was measured at 695 nm against the reagent blank. A typical blank solution contained
3 mL of reagent solution and water in place of sample, and it was incubated under same
conditions as the rest of the samples. The results are reported in mean values expressed as mg
of ascorbic acid equivalents per gram compounds.
2.7 Cytotoxicity
2.7.1 Cell culture
The Human breast cancer (MCF-7) and human Lung cancer cells (A-549) were
procured from the National Centre for Cell Sciences (NCCS), Pune, India. The selected cancer
cells were maintained in Dulbecco’s modified eagles medium (DMEM) supplemented with
2mM l-glutamine and balanced salt solution (BSS) adjusted to contain 1.5 g/L Na2CO3, 0.1
mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM l-glutamine, 1.5 g/L glucose, 10
6

mM (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid) (HEPES) and 10% fetal bovine
serum (GIBCO, USA). Penicillin and streptomycin (100 IU/100µg) were adjusted to 1mL/L.
The cells were maintained at 37oC with 5% CO2 in a humidified CO2 atmosphere.
2.7.2 Evaluation of cytotoxicity (MTT assay)
The inhibitory concentration (IC50) value was evaluated using an MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay [35]. Both the cancer cells were
grown (1×104 cells/well) in a 96-well plate for 48 h in to 80% confluence. The medium was
replaced with fresh medium containing serially diluted compounds of AGE and AGM, and the
cells were further incubated for 48h. The culture medium was removed, and 100µL of the
MTT [3-(4,5-dimethylthiozol-2-yl)-3,5-diphenyl tetrazolium bromide] (Hi-Media) solution
was added to each well and incubated at 37oC for 4 h. After removal of the supernatant, 50µL
of DMSO was added to each of the wells and incubated for 10 min to solubilize the formazan
crystals. The optical density was measured at 620 nm in an ELISA multi well plate reader
(Thermo Multiskan EX, USA). The OD value was used to calculate the percentage of viability
using the following formula.
% of viability = OD value of experimental sample/OD value of experimental control ×100
2.7.3 Fluorescence microscopic analysis of apoptotic cell death
Approximately 1µL of a dye mixture (100 mg/mL acridine orange (AO) and 100
mg/mL ethidium bromide (EB) in distilled water) was mixed with 0.9ml of cell suspension
(1×105 cells/mL) on clean microscope cover slips. The pretreated cancer cells were collected,
washed with phosphate buffered saline (PBS) (pH 7.2) and stained with 10 µL of AO/EB.
After incubation for 2 min, the cells were washed twice with PBS (5 min each) and visualized
under a fluorescence microscope (Nikon Eclipse, Inc, Japan) at 400× magnification with an
excitation filter at 580nm. Similarly the cells were placed on glass coverslip in a 6-well plate
and treated with complex for 24h. The fixed cells were permeabilised with 0.2% triton X-100
(50µl) for 10min at room temperature and incubated for 3min with 10µl of DAPI by placing a
coverslip over the cells to enable uniform spreading of the stain. The cells were observed
under (Nikon Eclipse, Inc, Japan) fluorescent microscope.
3. Result and Discussion
3.1. Synthesis of new Ruthenium (II) complexes
The reactions of tris(2-pyridylmethyl)amine[TPA.3HClO4] with RuCl2(EPh3)3 E=P, As
in 1:1 methanol-dichloromethane resulted in the formation of new Ru(II) complexes (Scheme
7

1). The analytical data of which confirmed their stoichiometry (complexes 1 and 2). The
complexes are soluble in common organic solvents such as dichloromethane, chloroform,
ethanol, methanol, dimethylformamide and dimethylsulfoxide.
-
. ClO4
N
N
N
.3HClO4
Cl
CH2Cl2 / Methanol
, 6 h
N
N
Ru
RuCl2(EPh3)3
+
N
EPh3
N
N
E=P, As
Scheme 1: Preparation of new Ruthenium(II) complexes
3.2. Spectroscopic Studies
The IR spectra of the ligand and the corresponding Ru(II) complexes provided significant
information about the metal ligand bonding. A sharp band at 1638 cm-1 in the ligand
corresponding to νC=N group of tris pyridyl shifted to lower frequency at 1619 and 1621 cm-1 in
the complex indicating the coordination of nitrogen to metal atom [36]. There is a new broad
band at 1086 cm-1 in the ligand and complexes was due to perchlorate ion. Furthermore, new
absorption bands were observed in the range 1443–1455 cm-1 which are assigned to (Ru-EPh3
E= P, As) [37]. Electronic spectra of the ligand and new ruthenium(II) complexes were
recorded in DMSO displayed two bands in the region 265-340 nm. The bands at 265 nm has
been assigned to the intra ligand transition which take place between the orbitals of the ligand
→
[38]. However, the bands observed at 327-340 nm was notified to LMCT (s d) [39,40]. The
1H-NMR spectra of the ligand and new Ru(II) complexes have been taken in DMSO and
shown in (Fig.S1-S3). The spectrum of the ligand showed overlap of signals as a multiplet at δ
7.82-8.41 ppm range corresponding to aromatic protons [41,42]. A doublet appeared at δ 8.82-
8.84 ppm was due to aromatic C(2) proton and a triplet appeared at δ 7.82-7.87 ppm was due
to aromatic C(3) proton and another triplet appeared at δ 8.36-8.41 ppm was due to aromatic
C(4) proton, however a doublet appeared at δ 7.97-7.99 ppm was due to aromatic C(5) proton.
In addition, a singlet appeared at δ 2.99 ppm was assigned to methylene protons [43]. Whereas,
8

in both complex 1 and 2 overlap of signals as a multiplet at range of δ 7.20-8.41 ppm are
corresponding to aromatic protons of pyridine group and PPh3/AsPh3 protons. Further, a singlet
appears at the range of δ 2.50- 2.51ppm due to –CH2 protons.
3.3. X-Ray Crystallography
The ORTEP diagram with the atom labelling for the complexes are shown in (Fig. 1 and 2) A
summary of the structure refinement and selected geometrical parameters (inter atomic
distances and angles) are given in (Table 1 and 2) respectively. Both the complexes1 and 2
crystallized in a monoclinic space group ‘P21/c’. In the complexes the ligand was coordinated
to ruthenium ion by utilizing all the nitrogen atoms with bond distances at the range of
[Ru(1)–N(1), Ru(1)–N(2), Ru(1)–N(3) and Ru(1)–N(4) of 2.090(3) Å - 2.123(3) Å]. The
remaining sites were occupied by chlorine atom Ru(1)–Cl(1) with bond distances of
2.4489(7)/2.4410(10) and phosphorous atom of triphenylphosphine or arsine with Ru(1)–P(1)/
Ru(1)–As(1) bond distances of 2.3768(8)/ 2.4741(4).The observed bond distances are
comparable with those found in other reported ruthenium complexes containing
PPh3/AsPh3[44]. The bite angle N(3)-Ru(1)-N(1), N(2)-Ru(1)-N(1), N(3)-Ru(1)-N(4) and
N(2)-Ru(1)-N(4) of 81.42°- 91.08° of both complexes acted as neutral tetradentate manner.
The presence of a perchlorate ion outside the coordination sphere is compensated the charge
of ruthenium as Ru(II) resulting in the formation of octahedral environment. The N(4)-Ru(1)-
P(1) bond angle [173.58 (7)º], N(1)-Ru(1)-Cl(1) bond angle [169.93(7)º], N(1)-Ru(1)-As(1)
bond angle [176.49(10)º] and N(3)-Ru(1)-Cl(1) bond angle [170.23(9) º] are deviate
considerably from the ideal angle of 180°. The variation in bond angles and bond lengths in
both the complexes indicates considerable distortion in the octahedral geometry.
9

Fig. 1. ORTEP diagram for Complex 1
Fig. 2. ORTEP diagram for Complex 2
10

Table. 1. Crystallographic data of Ru(II) complexes
Complex 1
Complex 2
1574011
CCDC No
1574010
Empirical formula
Formula weight
C36H33Cl2N4O4PRu
788.6
C36H33Cl2N4O4AsRu
832.55
Temperature
293 K
295 K
Wavelength
Crystal system
Space group
0.71073Å
Monoclinic
P 21/c
0.71073Å
Monoclinic
P 21/c
a
b
c
α
10.6466 (3) Å
16.5811 (3) Å
19.7906 (3) Å
90°
8.8972 (4) Å
21.0580 (9) Å
18.4484(8) Å
90 °
99.75°
99.38°
β
90°
90°
γ
Volume
Z
Calculated density
Absorption coefficient
F(000)
3443.19(8) Å3
4
3410.2(3) Å3
4
1.521 Mg/m3
0.703 mm-1
1608
1.622 Mg/m3
1.626 mm-1
1608
Crystal size
Theta range for data
collection
0.3 x 0.24x 0.24 mm
2.383 to 29.026 °
0.33 x 0.24x 0.17 mm
1.934 to 36.492 °
-14<=h<=14, -21<=k<=21, -
26<=l<=26
-14<=h<=14, -35<=k<=34, -
30<=l<=23
Limiting indices
Reflections collected /
unique
78365/ 9029
74772/ 15932
Absorption correction
Max. and min.
transmission
0.8467 and 0.8025
1.00000 and 0.77952
Refinement method
Data / restraints /
parameters
Full-matrix least-squares on F2 Full-matrix least-squares on F2
9029/ 0 / 433
15932/ 0 / 433
Goodness-of-fit on F2
Final R indices
[I>2sigma(I)]
1.079
1.219
R1 = 0.0432, wR2 = 0.1065
R1 = 0.0888, wR2 = 0.1332
R1 = 0.1137, wR2 = 0.1410
0.947 and -1.458 e.A-3
R indices (all data)
Largest diff. peak and
hole
R1 = 0.06, wR2 = 0.115
1.361 and -0.718 e.A-3
11

Table. 2. Bond lengths and Bond angles of Ru(II) complexes
Bond lengths
Ru(1)-N(1)
Bond angles
N(1)-Ru(1)-N(2)
Complex 1
2.094(2)
79.40(10)
Ru(1)-N(2)
Ru(1)-N(3)
Ru(1)-N(4)
Ru(1)-Cl(1)
Ru(1)-P(1)
2.090(3)
2.088(2)
2.123(3)
2.4489(7)
2.3768(8)
N(1)-Ru(1)-N(3)
N(1)-Ru(1)-N(4)
N(2)-Ru(1)-N(3)
N(2)-Ru(1)-N(4)
N(3)-Ru(1)-N(4)
N(1)-Ru(1)-Cl(1)
81.42(9)
80.7(10)
160.2(10)
91.1(10)
81.0(10)
169.92(7)
N(2)-Ru(1)-Cl(1)
N(3)-Ru(1)-Cl(1)
N(4)-Ru(1)-Cl(1)
N(1)-Ru(1)-P(1)
N(2)-Ru(1)-P(1)
N(3)-Ru(1)-P(1)
N(4)-Ru(1)-P(1)
97.09(8)
100.98(7)
89.95(8)
102.91(7)
94.80(8)
94.29(7)
173.59(8)
Ru(1)-N(1)
Ru(1)-N(2)
Ru(1)-N(3)
Ru(1)-N(4)
Ru(1)-Cl(1)
Ru(1)-As(1)
2.093(3)
2.096(3)
2.081(3)
2.086(3)
2.4410(10)
2.4741(4)
N(3)-Ru(1)-N(4)
N(3)-Ru(1)-N(1)
N(3)-Ru(1)-N(2)
N(4)-Ru(1)-N(1)
N(4)-Ru(1)-N(2)
N(1)-Ru(1)-N(2)
N(1)-Ru(1)-Cl(1)
N(2)-Ru(1)-Cl(1)
N(3)-Ru(1)-Cl(1)
N(4)-Ru(1)-Cl(1)
N(1)-Ru(1)-As(1)
N(2)-Ru(1)-As(1)
N(3)-Ru(1)-As(1)
N(4)-Ru(1)-As(1)
86.51(12)
81.62(13)
91.08(12)
81.21(13)
161.67(12)
80.46(13)
89.09(10)
90.34(9)
170.23(9)
89.11(9)
176.49(10)
98.53(9)
Complex 2
101.79(8)
99.75(8)
3.4 DNA binding studies
For evaluating the biological property of any new compound, DNA binding is the major
property for pharmacology and hence, the kind of the interaction between DNA and a
compound to be examined is of paramount importance. Hence, the mode and tendency for the
binding of the Ru(II) complexes to CT-DNA were studied with different physicochemical
methods.
12

3.4.1 Electronic absorption titration
In general, electronic absorption spectroscopy is one of the most suitable techniques to
investigate the mode of interaction of metal complexes with DNA [45,46]. Normally, a
compound binds to DNA through intercalation usually results in the hypochromism with or
without a small red or blue shift, due to a strong stacking interaction between the planar
aromatic chromophore of the compound and the base pairs of DNA [47,48]. The results of
absorption spectra of the ligand and new Ru(II) complexes in the absence and presence of CT-
DNA are shown in (Fig. 3) In the present investigation, the interaction of ligand and Ru(II)
complexes with CT DNA was investigated. Absorption spectroscopic titration experiments of
the ligand and complexes in buffer were performed using a fixed metal concentration to which
increasing amounts of the DNA stock solution was gradually added. The ligand centred
transitions at 258 nm and 318 nm, 432nm for complexes 1 and 2 respectively, were considered
for the corresponding absorptivity changes upon the incremental addition of DNA. In ligand a
band around 258 nm shows hyperchromism by about 2.1% and a blue shift of 2 nm. For
complex 1 under the same conditions, upon the addition of DNA, the band at about 318 nm
exhibits hypochromism of 16.4% with a 4 nm red shift. For complex 2 under the same
conditions, upon the addition of DNA, the band at about 432 nm exhibits hypochromism of
16.6% with a 9 nm red shift. The changes in the spectrum indicate that the complex possesses
a strong affinity for CT-DNA. In order to compare quantitatively the binding strength of the
ligand and complexes, the intrinsic binding constant, Kb of the ligand and complexes with CT-
DNA was calculated (Table 3). It can be determined by monitoring the changes in the
absorption in the IL band at the corresponding λmax with increasing concentration of DNA and
is given by the ratio of the slope to the Y intercept in plots of [DNA] / (Ɛa - Ɛf) versus [DNA]
(Fig. 4). The magnitudes of the intrinsic binding constants (Kb) for ligand 3.33 0.30×105 M−1
4.75 0.27×105 M−1and 1.40 0.32×106 M−1for complexes 1 and 2 respectively. The observed
values of Kb revealed that the ligand and the Ru(II) complexes bind to DNA via an
intercalative mode. These results are similar to those reported earlier for the intercalative
mode of various metallo intercalators [49].
13

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.8
0.6
0.4
0.2
Complex 1
Ligand
300
300
400
Wavelength (nm)
Wavelength (nm)
0.8
0.6
0.4
0.2
0.0
Complex 2
200
300
400
500
Wavelength (nm)
Fig. 3. Changes in the electronic absorption spectra of ligand and complexes (1 and 2) (10
µM) with increasing concentrations (5-50 µM) of CT-DNA (tris HCl buffer, pH 7.2)
0.005
Ligand
Complex 1
Complex 2
0.004
0.003
0.002
0.001
0.000
0
10
20
30
40
50
[DNA]
Fig. 4. Plot of [DNA] vs {[DNA]/(εa-εf)} for ligand and complexes (1 and 2)
14

Table. 3. The Kb and Ksv values for the interactions of ligand and complexes with CT-DNA
Compound
Ligand
Kb/M-1
Ksv/M-1
3.33 0.30×105
4.75 0.27×105
1.40 0.32×106
1.42 0.01×103
1.57 0.01×103
1.98 0.08×103
Complex 1
Complex 2
3.4.2 Ethidium Bromide-Displacement Assay
The binding mode of ligand and new Ru(II) complexes with DNA have been
confirmed further by ethidium bromide displacement studies. Competitive binding studies by
using ethidium bromide (EB) bound to DNA were carried out for the ligand and complexes.
(Fig. 5) shows the variation of emission spectra of DNA pre-treated with EB ([DNA]/ [EB] =
1:1) on increasing concentration of the ligand and Ru(II) complexes. The above results
revealed that the quenching of the initial fluorescence intensity about 10.7% for ligand and
11.1% and 27.34 %with increasing concentration of the complexes 1 and 2 respectively.
Quenching data were calculated by using Stern-Volmer plot and Kq value is obtained as a
slope from the plot of Iₒ/Icorr versus [Q] (Fig. S4; Table 3). The quenching plots illustrate that
the quenching of EB bound to CT-DNA by free ligand and the complexes are in good
agreement with the linear Stern–Volmer equation. The Kq values for the ligand, complexes 1
and 2 were found to be 1.42 0.01×103 M−1, 1.57 0.01×103 M−1 and 1.98 0.08×103 M−1,
respectively. These results are comparable with those reported earlier for the intercalative
mode of various ruthenium intercalators [50].
15

100
80
Complex 1
100
80
60
40
20
0
Ligand
60
40
20
550
600
650
700
750
550
600
650
700
750
Wavelength (nm)
Wavelength (nm)
100
80
60
40
20
Complex 2
550
600
650
700
Wavelength (nm)
Fig. 5. Fluorescence quenching curves of ethidium bromide bound to DNA of ligand and
complexes (1 and 2). [DNA] = 10 µM, [EB] = 10 µM and [compound] = 0-50 µM.
3.4.3 UV absorption spectra of BSA
A common method to distinguish between static and dynamic quenching is by careful
examination of the absorption spectra of the BSA in the presence of ligand and complexes
[51]. The UV absorption spectra of BSA in the presence of three compounds (Fig. S5) showed
that the absorption intensity of BSA was decreased with the addition of these compounds from
248 to 250 nm. The changes in the absorbance spectra for BSA+ compounds indicate that test
compounds interact with the BSA [52]. It is to be noted that dynamic quenching only affects
the excited state of fluorophore and does not change the absorption spectrum. However, the
complexes in ground-state induced the change in absorption spectrum of fluorophore. Thus,
possible quenching mechanism of BSA by compounds may be static quenching [53].
16

3.5. Protein binding studies
Fluorescence spectroscopy is a valuable method to conclude the interactions between
metal complexes and serum albumins. The intrinsic fluorescence of BSA is generally due to
the presence tryptophan residue. The emission spectra of BSA solution exhibit an intense
fluorescence emission maximum at 346 nm and 345 nm, respectively, due to the tryptophan
residues [54-56].Fig. 6 shows the effect of increasing concentration of the complexes on the
fluorescence emission intensity ofBSA. On increasing the concentration of the ligand and
complexes to BSA solution, a steady decrease in the emission band at 349 nm was observed.
The quenching induced about 50.9 % for ligand 91% and 53.2 % for complex 1 and 2 of
quenching of the initial fluorescence intensity of BSA with 2-5 nm red shifts.The results
revealed that the complex 1 showed better interaction with the BSA than ligand and complex
2.
The quenching constant KSV value was obtained from the plot of I0/Icorrversus [Q] in
Stern–Volmer equation (4) (Fig. 7 and Table 4).
I0/Icorr = 1+ KSV [Q]
(4)
From KSV values, it is seen that the ligand and new complexes (1 and 2) exhibited strong
protein-binding ability with enhanced hydrophobicity.
3.5.1 Binding constants and the number of binding sites
For the static quenching interaction, it is assumed that there are similar and
independent binding sites in the biomolecule, the binding constant (Kb) and the number of
binding sites (n) can be determined according to the Scatchard equation (5) [57].
log [(I0−I)/I]= log Kb+ n log [Q]
(5)
From Fig.S6 and Table 4 shows that the ligand and complexes exhibited potent activity for
Bovine serum albumin(BSA). Among ligand and complexes, Complex 1 shows better binding
activity.
3.5.2 Synchronous fluorescence spectroscopic studies of BSA
To investigate the structural changes occurred to BSA upon the addition of our
compounds, synchronous fluorescence spectra of BSA were measured before and after the
addition of test compounds to get the valuable information on molecular microenvironment
[58]. Synchronous fluorescence spectra showed tyrosine residues of BSA only at the
17

wavelength interval ∆λof 15 nm whereas tryptophan residues of BSA at ∆λof 60 nm. While
increase the concentration of complexes (0–50 µM) added to BSA (10 µM), a decrease in the
fluorescence intensity with a blue shift in the tryptophan emission maximum is observed for
ligand and complexes1 and 2 (Fig. S7). The results show that the emission intensity of
tyrosine residue increases without any change in the wavelength of emission. These
observations indicate that the compounds did not affect the microenvironment of tyrosine
residues and also both ligand and complexes bind to active site in the protein which makes
them as potential molecules for biological applications.
300
300
Ligand
Complex 1
200
200
100
100
0
0
-100
-200
-100
300
320
340
360
380
400
420
300
320
340
360
380
400
420
440
Wavelength (nm)
Wavelength (nm)
300
Complex 2
200
100
0
-100
300
320
340
360
380
400
Wavelength (nm)
Fig. 6. Fluorescence quenching of BSA (1 × 106 M; λexi = 276; λemi = 349 nm) in the absence
and presence of various concentration of ligand and complexes (0-50 µM).
18

6
5
4
3
2
1
Ligand
Complex 1
Complex 2
0
20
40
60
80
100
Q
Fig. 7. Stern–Volmer plot of fluorescence titration of the ligand and complexes (1 and 2) (0 –
50 µM) with BSA (10 µM)
Table 4. Quenching constant (Ksv), binding constant (Kbin) and number of binding sites (n)
for the interactions of ligand and complexes (1 and 2) with BSA
Compounds
Ligand
KSV/M-1
1.21 0.08 x 104
Kbin/M-1
5.80 0.08×104
‘n’
1.18 0.07
Complex 1
Complex 2
5.67 0.60×104
1.42 0.08×104
6.48 0.12×105
6.33 0.08×104
1.28 0.12
1.16 0.07
3.6. Antioxidant property
Free radicals are the reactive species, their overproduction result in stimulation of
disease like cardiovascular disease and cancer (Deighton et al., 2000). Hence, antioxidants are
recommended to overcome this issue. Hence the antioxidant activity of ligand and new Ru(II)
complexes were studied.
3.6.1 DPPH radical scavenging activity
The DPPH radical scavenging activity of ligand and complexes (1 and 2) is
represented in Fig.8. Both complexes showed the radical scavenging activity and their
reduction capability of DPPH radical is determined by the decrease in absorbance at 517 nm
[59]. Synthetic antioxidant BHT used as standards. The complex 2 was higher in potential to
reduce the stable DPPH radical, but lower than the BHT antioxidant potential of BHT. The
DPPH radical scavenging of new Ru(II) having better some of the other reported ruthenium
complexes [60].
19

100
90
80
70
60
50
40
30
20
10
0
Complex 1
Ligand
Complex 2
BHT
100
200
300
400
500
Concentration
Fig. 8DPPH radical scavenging activity of ligand and Ru(II) complexes.
3.6.2 ABTS cation radical scavenging activity
An important attribute of antioxidants is the proton radical scavenging. A well-known
protonated radical like 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) has
characteristic absorbance maxima at 734ꢀnm which decreases with the scavenging of the
proton radicals [61]. The ABTS cation radical scavenging activity is shown in Fig. S8. Here,
both ligand and complexes showed various concentration dependent linear scavenging
activity. Remarkably, the Complex 2 showed higher percentage of inhibition of radical
generation in DPPH radical scavenging activity as compared to ligand and BHT standard.
3.6.3 Total antioxidant activity
The total antioxidant activity of compounds was determined by measuring the
formation of green phosphomolybdenum complex, by reducing Mo(VI) to (V) [62]. Fig. 9.
Among the ligand and complexes1 and 2, the complex 2 having high total antioxidant activity
than ligand and complex 1.The antioxidant activity is expressed in mg Ascorbic acid
Equivalents/g of compounds.
20

1000
900
800
700
600
500
400
300
200
100
0
Complex 1
Ligand
Complex 2
BHT
100
200
300
400
500
Concentration
Fig. 9 Total antioxidant activity of ligand and Ru(II) complexes.
3.7 Cytotoxicity assay
We examined the effect of synthesized compounds on human breast cancer (MCF-7)
and Human lung Cancer (A-549) cell line by using the MTT assay and found that the
compounds efficacy in inhibit the growth of the cancer cells significantly that the standard
(Fig.S9). From IC50 values (Table S1) of compounds against breast cancer cells were
calculated and it found to be 26 1.0µM/ml for ligand and 22 1.5µM/ml and 20 1.0µM/ml
for complex
1
and 2respectively. Whereas theIC50 values 27
1.0µM/ml for
ligand24 1.5µM/ml and 26 1.0µM/ml for complex 1 and 2 were obtained for A549 lung
cancer cells [63,64]. The observed IC50 values of synthesized complexes are significantly
higher than standard.
3.7.1 Fluorescence microscopy analysis of nuclear fragmentation
Acridine orange /Ethidium bromide (AO/EBr) staining Method
In order to elucidates the apoptotic activity of synthesized compounds apoptotic
staining fluorescence microscopic analysis was carried out. Fluorescence microscopy images
of A-549 and MCF-7 cancer cells in the absence and in the presence of compounds are shown
in (Fig. 10) which shows that the untreated A-549 and MCF-7 cells (control) did not show any
21

significant adverse effect compared to the compounds treated with cancer cells and it can be
observed that the addition of compounds with their respective IC50 concentration to the cancer
cells, the green colour of cells are turned into orange/yellow colour. The colour change may be
due to the induced apoptosis and also the nuclear condensation effect of the cells by the
treated compounds [65].
Fig.10. AO/EB stained A-549 and MCF-7 (control and cells treated with ligand and
complexes 1 and 2) cells at 24 h incubation. The yellow or red colour cells show early
apoptotic cells
3.7.2 DAPI staining method
In order to further confirm the nuclear condensation and fragmentation of compounds
on selected cancer cells, DAPI staining method was followed. Fluorescence microscopy
images of Lung and breast cancer cells after 24 h stained with DAPI in the absence and
presence of compounds are shown in (Fig. 11). The untreated cells didn’t show any significant
changes whereas compounds treated cancer cells shows bright fetches in which it indicates the
condensed chromatins and nuclear fragmentations in the cancer cells. Further the bright fetch
observed in compounds are higher when compared with MCF-7 and A 549 cells. Thus, the
results from MTT assay and Fluorescence microscopy analysis evident the potential
therapeutic activity of the compounds and they can be further subjected to explore them as
drug [66].
22

Fig.11. DAPI(MCF-7) and (A-549) (control and cells treated with ligand and complexes 1 and
2) after 24 h incubation. The blue fetches show nuclear fragmentations in the cancer cells.
4. Conclusion
The present article describes the synthesis of two new ruthenium(II) complexes
containing Tris(2-pyridylmethyl)amine [TPA.3HClO4]. The two new complexes have been
characterized by using various spectroscopic techniques (NMR, UV-Vis, IR) and X-ray
crystallography. The interaction of the ligand and complexes with calf thymus (CT-DNA) has
been examined by photophysical studies, which revealed that the complexes bound to DNA
through an intercalative mode. The protein binding studies revealed the alteration of the
secondary structure of the protein by the complexes through static quenching. The ligand and
complexes proved their effectiveness in scavenging the free radicals better than the standards
BHT and ascorbic acid. Further, in vitro cytotoxicity of ligand and complexes against human
breast (MCF7) and lung (A549) cancer cell lines by MTT assay and cytological changes
observed in (AO/EB and DAPI) staining method was significant activity as compared with
standard cisplatin.
ACKNOWLEDGEMENT
The authors greatly acknowledged Department of Science and Technology
(SR/FT/CS-32-/2011 dated 04.05.2012) DST-SERB, New Delhi, India and Council of
Science and Industrial Research (01(2553)/12/EMR-II dated 03.04.2012) New Delhi, India
for their financial support. The authors thanks J. P. Jasinski, Professor of Physical &
23

Structural Chemistry, Department of Chemistry, USA for NSF-MRI program (grant CHE-
1039027) for funds to purchase the X-ray diffractometer for collection of X-ray
crystallographic data.
Supplementary material:
Crystallographic data for complexes 1 and 2 have been deposited at the Cambridge
Crystallographic Data Centre as supplementary publication (CCDC No. 1574010 and
1574011).
The
data
can
be
obtained
free
of
charge
at
www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallographic Data
Centre, 12, Union Road, Cambridge CB2 1EZ, UK [fax: +44-1223/336-033; e-mail:
deposit@ccdc.cam.ac.uk].
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27

HIGHLIGHTS
ꢀ Two new Ru(II) complexes have been synthesized and characterized by spectral and
X-ray crstallography.
ꢀ The DNA and protein interactions of these complexes were studied by a variety of
techniques.
ꢀ The antioxidant activity against various radicals have also been examined
ꢀ The cytotoxic activity was evaluated against a human breast cancer cell line (MCF-7)
and Human lung Cancer (A-549). The complexesexhibited a significant anticancer
activity over the standard drug, Cisplatin
ꢀ Fluorescence microscopy analysis of nuclear fragmentation[(AO/EBr) staining
Method, DAPI staining method for ligand and complexes are also investigated.
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Graphical abstract
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