Ruthenium(II) complexes of pyrrol-azo ligands: Cytotoxicity, interaction with calf thymus DNA and bovine serum albumin

Article abstract of DOI:10.1080/00958972.2013.814048

Two ruthenium(II) complexes of newly designed pyrrol-azo ligands(L) and bipyridine(bpy) formulated as [Ru(L)(bpy)₂]ClO₄, where HL¹= (4-chloro-phenyl)-(1H-pyrrol-2-yl)-diazene (1) complex 1 and HL²= (4-nitro-phenyl)-(1H-pyrrol-2-yl)-diazene for 2, were isolated in pure form. The complexes were characterized by physicochemical and spectroscopic methods. The electrochemical behavior of the complexes showed the Ru(III)/Ru(II) couple at different potentials with quasi-reversible voltammograms. The study of cytotoxicity effects of 1 and 2 on human breast cancer cells (,were isolated in pure form. The complexes were characterized by physicochemical and spectroscopic methods. The electrochemical behavior of the complexes showed the Ru(III)/Ru(II) couple at different potentials with quasi-reversible voltammograms. The study of cytotoxicity effects of 1 and 2 on human breast cancer cells (MCF 7, MDA-MB 231) and cervical cancer cell (HeLa) taking Cisplatin as a positive reference showed that 1 exhibited higher cytotoxicity against cancer cell lines than 2, but less activity than Cisplatin. The interaction of 1 with calf thymus DNA (CT-DNA) using absorption, emission spectral studies, viscosity-measurement, and electrochemical techniques has been used to determine the binding constant K_band the linear Stern-Volmer quenching constant K_SV. The results indicate that 1 strongly interacts with CT-DNA in groove binding mode. The interaction of bovine serum albumin (BSA) with 1 was also investigated with the help of spectroscopic tools. Absorption spectroscopy proved the formation of a BSA-[Ru(L¹)(bpy) ₂]ClO₄ complex.

Full text of DOI:10.1080/00958972.2013.814048

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Ruthenium(II) complexes of pyrrol-azo ligands:
Cytotoxicity, interaction with calf thymus DNA and
bovine serum albumin
HENA PAUL a , TITAS MUKHERJEE a , MANJIRA MUKHERJEE a , TAPAN K. MONDAL b
, ANURADHA MOIRANGTHEM c , ANUPAM BASU c , ENNIO ZANGRANDO d & PABITRA
CHATTOPADHYAY a
a Department of Chemistry , Burdwan University , Golapbag, Burdwan, 713104 , India
b Department of Chemistry , Jadavpur University , Jadavpur-32, India
c Department of Zoology , Burdwan University , Golapbag, Burdwan, 713104 , India
d Department of Chemical and Pharmaceutical Sciences , Via Licio Giorgieri 1, 34127 ,
Trieste , Italy
Accepted author version posted online: 14 Jun 2013.
To cite this article: HENA PAUL , TITAS MUKHERJEE , MANJIRA MUKHERJEE , TAPAN K. MONDAL , ANURADHA MOIRANGTHEM ,
ANUPAM BASU , ENNIO ZANGRANDO & PABITRA CHATTOPADHYAY (2013): Ruthenium(II) complexes of pyrrol-azo
ligands: Cytotoxicity, interaction with calf thymus DNA and bovine serum albumin, Journal of Coordination Chemistry,
DOI:10.1080/00958972.2013.814048
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Ruthenium(II) complexes of pyrrol-azo ligands: Cytotoxicity, interaction with
calf thymus DNA and bovine serum albumin
HENA PAUL1, TITAS MUKHERJEE1, MANJIRA MUKHERJEE1, TAPAN K. MONDAL2,
ANURADHA MOIRANGTHEM3 ANUPAM BASU3, ENNIO ZANGRANDO4 and
1
*
PABITRA CHATTOPADHYAY
1Department of Chemistry, Burdwan University, Golapbag, Burdwan-713104, India
2Department of Chemistry, Jadavpur University, Jadavpur-32, India
3Department of Zoology, Burdwan University, Golapbag, Burdwan-713104, India
4Department of Chemical and Pharmaceutical Sciences, Via Licio Giorgieri 1, 34127 Trieste, Italy
Two ruthenium(II) complexes of newly designed pyrrol-azo ligands (L) and bipyridine (bpy)
formulated as [Ru(L)(bpy)2]ClO4, where HL1 = (4-chloro-phenyl)-(1H-pyrrol-2-yl)-diazene (1)
complex 1 and HL2 = (4-nitro-phenyl)-(1H-pyrrol-2-yl)-diazene for 2, were isolated in pure
form. The complexes were characterized by physicochemical and spectroscopic methods. The
electrochemical behavior of the complexes showed the Ru(III)/Ru(II) couple at different
potentials with quasi-reversible voltammograms. The study of cytotoxicity effects of 1 and 2 on
human breast cancer cells (MCF 7, MDA-MB 231) and cervical cancer cell (HeLa) taking
Cisplatin as a positive reference showed that 1 exhibited higher cytotoxicity against cancer cell
lines than 2, but less activity than Cisplatin. The interaction of 1 with calf thymus DNA
(CT-DNA) using absorption, emission spectral studies, viscosity-measurement and
electrochemical techniques have been used to determine the binding constant Kb and the linear
Stern–Volmer quenching constant KSV. The results indicate that 1 strongly interacts with
CT-DNA in groove binding mode. The interaction of bovine serum albumin (BSA) with 1 was
also investigated with the help of spectroscopic tools. Absorption spectroscopy proved the
formation of a BSA-[Ru(L1)(bpy)2]ClO4 complex.
Keywords: Ruthenium(II) complex; Phenylazopyrrole; Cytotoxicity; DNA Interaction
1. Introduction
Aryl- and heteroaryl-azopyrroles have been used extensively in the textile, fiber, leather, paint
and printing industries due to their ability to absorb visible light, and for their easy synthesis [1].
These ligands are interesting mainly because of presence of the azo group (–N=N–) which can
*
Corresponding author. Email: pabitracc@yahoo.com
1

stabilize ruthenium in lower oxidation states due to its strong π-acidic character. Such complexes
are useful in metal-assisted organic transformations, and as metallochromic indicators and also
show photochromic behavior [2-6]. Azophenyl complexes have been extensively used as
catalysts for transfer hydrogenation of ketones [7, 8]. Among the different metals catalyzing
reaction, ruthenium-based systems are effective [9-11].
Variable oxidation states of ruthenium complexes containing nitrogen heterocycles are
useful as building blocks for supramolecular assemblies, photophysical properties, directional
electron and energy transfer, DNA intercalation, and potential antitumor activities [12-15]. A
wide range of ruthenium compounds have been used as potential anticancer drugs [16-20]. The
study of their interaction with proteins, DNA and lipids have received considerable attention
because of the spectroscopic and redox properties of ruthenium(II) complexes with heterocyclic
N-donors, the higher coordination number of ruthenium compared with platinum providing
additional coordination sites, and due to the possible application in biochemistry and clinical
diagnosis [21-23]. Bovine serum albumin has a wide range of physiological functions involving
binding, transport and delivery of fatty acids, porphyrins, bilirubin, steroids, etc. It has specific
binding sites for metals and pharmaceutical dyes [24].
Considering the above facts and our ongoing interest towards the study of interaction of
metal chelates with CT-DNA [25-28] and BSA [29], herein we report synthesis and
characterization, cytotoxicity of 1 and 2 towards human breast cancer cells (MCF 7,
MDA-MB 231) and cervical cancer cell (HeLa), DNA binding and protein binding study of 1.
The redox behavior of these complexes in acetonitrile by cyclic voltammetry has also been
studied. Binding of 1 and 2 with bovine serum albumin (BSA) was studied by absorption and
fluorescence spectroscopy.
2. Experimental
2.1. Materials and physical measurements
All chemicals and reagents were obtained from commercial sources and used as received.
Solvents were distilled from an appropriate drying agent. RuCl3·3H2O (Aldrich) was used
without purification. [RuIICl2(bpy)2]·2H2O was synthesized according to the literature method
[30]. Tetra-n-butylammonium perchlorate (TBAP) was prepared by addition of sodium
2

perchlorate (taking the usual precaution of handling perchlorate salts !) to a hot solution of tetra-
n-butylammoniumbromide (Aldrich).
The C, H and N elemental analyses were performed on a Perkin Elmer model 2400
elemental analyzer and ruthenium analyses were carried out by a Varian atomic absorption
spectrophotometer (AAS) model-AA55B, GTA using a graphite furnace. Electronic absorption
spectra were recorded on a JASCO UV-Vis/NIR spectrophotometer model V-570 from 1100–
200 nm. IR spectra (KBr discs, 4000–300 cm-1) were recorded using a Perkin-Elmer FTIR model
RX1 spectrometer. 1H NMR spectra were recorded on a Bruker AC300 spectrometer using TMS
as internal standard in CDCl3. Room temperature magnetic susceptibility measurements were
performed with a vibrating sample magnetometer PAR 155 model. Molar conductances (ΛM)
were measured in a Systronics conductivity meter (model 304) in acetonitrile with ~10-3 mol·L-1.
The measurement of pH was done with a Systronics digital pH meter (Model 335).
Electrochemical measurements were recorded on a CH-Instrument electrochemical system
(Model 620D) using Pt-wire and Ag/AgCl as working and reference electrodes, respectively,
taking TBAP as supporting electrolyte. All measurements were made at 298 K with 10-3-10-4 M
complex in acetonitrile purged with dry nitrogen for 3-4 min in order to remove dissolved
oxygen.
2.2. Synthesis of compounds
2.2.1. Preparation of HL1 and HL2. Ligands were synthesized following the common
procedure of coupling pyrrole with the corresponding diazotized para-substituted aniline
(scheme 1). The procedure for preparation of (4-chloro-phenyl)-(1H-pyrrol-2-yl)-diazene (HL1)
is described in detail; the other ligand was obtained as solid product in the same way.
(4-Chloro-phenyl)-(1H-pyrrol-2-yl)-diazene (HL1): Initially a diazotized solution of
4-chloro-aniline (2.55 g, 20.0 mmol) was prepared using ice-cold 2.0 mL conc. HCl and aqueous
NaNO2 (0.56 g). Then the diazotized solution was added dropwise to the solution of pyrrole
(1.35 g, 20.9 mmol) in aqueous Na2CO3 solution at 0-5 °C with continuous stirring. The pH of
the reaction mixture was maintained at 7-9 by adding Na2CO3 solution dropwise if necessary.
The red compound that precipitated was filtered (scheme 1). Finally the compound was purified
through recrystallization from a mixture of dichloromethane and n-hexane (1:1). The reddish-
3

brown single crystals of HL1 suitable for X-ray crystallography were obtained from a hexane-
toluene mixture of the compound on slow evaporation at room temperature.
C10H8ClN3: Anal. Found: C, 58.39; H, 3.89; N, 20.44; Calc.: C, 58.41; H, 3.92; N, 20.43.
m.p. 72 1 °C. IR (KBr, cm-1): νN-H, 3453, νN=N, 1425. 1H NMR (δ, ppm; J, cps; in CDCl3): 9.75
(s, 1H); 7.351 (d, 1H, J = 5.1); 6.753 (d, 2H, J = 7.3); 6.624 (d, 2H, J = 8.53); 6.326 (m, 1H);
6.173 (s, 1H). ESI-MS (m/z): [M+H]+, 206.34. Yield: 85%.
(4-nitro-phenyl)-(1H-pyrrol-2-yl)-diazene (HL2): L2H was prepared taking 4-nitro-aniline
(2.76 g, 20.0 mmol) instead of para-chloro-aniline following similar method.
C10H8N4O2: Anal. Found: C, 55.47; H, 3.70; N, 25.74; Calc.: C, 55.55; H, 3.73; N, 25.91.
1
m.p. 111 1 °C. IR (KBr, cm-1): νN-H, 3450, νN=N, 1420. H NMR (δ, ppm; J, cps; in CDCl3):
9.77 (s, 1H); 7.472 (d, 1H, J = 5.4); 7.324 (d, 2H, J = 5.5); 7.063 (d, 2H, J = 7.45); 6.532
(m, 1H); 6.410 (s, 1H). ESI-MS: [M+H]+, m/z, 217.15; Yield: 80%.
2.2.2. Preparation of ruthenium(II) complexes (1 and 2). To an ethanolic solution of the
respective organic moiety (0.205 g, 1.0 mmol of HL1 or 0.216 g, 1.0 mmol of HL2), a solution of
[RuCl2(bpy)2] (0.484 g, 1.0 mmol) in dry EtOH (20 mL) previously purged with N2 was added
dropwise and the resulting solution was stirred for ∼ 6 h under N2. The solvent volume of the
resulting solution was reduced to one third of the total and a water solution of NaClO4 (0.140 g,
1 mmol) was added dropwise with stirring. The resulting mixture was stirred for ∼ 2 h and then a
red solid was obtained by filtration. The product was dissolved in minimum dichloromethane and
pure ruthenium complex was obtained by chromatographic separation over a silica gel column
prepared in dichloromethane (scheme 1).
[Ru(L1)(bpy)2]ClO4 (1): C30H23Cl2N7O4Ru: Anal. Found: C, 50.11; H, 3.16; N, 13.75;
Ru, 14.17; Calc.: C, 50.22; H, 3.23; N, 13.66; Ru, 14.09. IR (KBr, cm-1): νN=N, 1385; ν(ClO4-)
,
1
1088 and 624. Conductance Λo (Ohm-1 cm2 mol-1) in methanol: 91. H NMR (δ, ppm; J, cps; in
CDCl3): 8.372 (t, 2H); 8.255 (d, 1H, J = 5); 8.177 (d, 1H, J = 8.1); 8.115 (d, 1H, J = 8); 7.905-
7.832 (m, 3H); 7.719 (m, 2H); 7.584 (d, 1H, J = 5.3); 7.410 (d, 1H, J = 5.25); 7.293-7.236 (m,
5H); 6.863 (d, 2H, J = 8.75); 6.759 (d, 2H, J = 8.75); 6.439 (q, 1H); 6.281 (s, 1H). ESI-MS (m/z):
[Ru(L1)(bpy)2] + H+, 618.2 (100% abundance); [Ru(L1)(bpy)2]ClO4 + K+, 756 (10% abundance).
Yield: 70-72%.
4

[Ru(L2)(bpy)2]ClO4 (2): C30H23ClN8O6Ru: Anal. Found: C, 49.45; H, 3.15; N, 15.38; Ru,
13.82; Calc.: C, 49.49; H, 3.18; N, 15.39; Ru, 13.88. IR (KBr cm-1): νN=N, 1384; ν(ClO4-), 1091
1
and 626. Conductance Λo (Ohm-1 cm2 mol-1) in methanol: 102. H NMR (δ, ppm; J, cps; in
CDCl3): 8.453 (m, 2H); 8.287 (d, 1H, J = 5); 8.201 (m, 2H); 8.016-7.959 (m, 3H); 7.927 (m,
1H); 7.866-7.830 (m, 4H); 7.677 (d, 1H, J = 4.5); 7.501 (d, 1H, J = 5.5); 7.461 (d, 1H, J = 4.5);
7.389-7.354 (m, 3H); 7.102 (d, 2H, J = 9); 6.640 (q, 1H); 6.507 (s, 1H). ESI-MS (m/z):
[Ru(L2)(bpy)2] + H+, 632.2 (60% abundance). Yield: 70-72%.
2.3. X-ray Crystal structure analysis
Single crystals suitable for X-ray diffraction study of L1H were grown by slow evaporation of a
methanolic solution at ambient temperature. Diffraction data collection of L1H was carried out at
150(2) on an Oxford Diffraction X-Calibur CCD System using Mo-Kα radiation (λ =
0.71073 Å). Cell refinement, indexing and scaling of the data set were performed using CrysAlis
[31], Denzo and Scalepack [32]. The structure was solved by direct methods and refined by full-
matrix least-squares based on F2 with all observed reflections [33]. Hydrogens were fixed at
geometrical positions. All calculations were performed using the WinGX System, Ver 1.80.05
[34]. Details of the crystal data and structure refinement are given in table 1.
2.4. DFT Calculation
Full geometry optimization was carried out using density functional theory at the B3LYP level
for 1 and 2 [35]. All elements except ruthenium were assigned the 6-31G(d) basis set. The SDD
basis set with effective core potential was employed for ruthenium [36]. The vibrational
frequency calculations were performed to ensure that the optimized geometries represent local
minima and there are only positive eigenvalues. All calculations were performed with the
Gaussian03 program package. Vertical electronic excitations based on B3LYP optimized
geometry were computed using the time-dependent density functional theory (TD-DFT)
formalism [37] in acetonitrile using conductor-like polarizable continuum model (CPCM).
GaussSum [38] was used to calculate the fractional contributions of various groups to each
molecular orbital.
5

2.5. Cytotoxicity assay
For this study, human breast cancer epithelial cell (MCF 7) and cervical cancer cell (HeLa) were
cultured in DMEM medium supplemented with 10 % fetal bovine serum and 1 % penicillin-
streptomycin at 37 °C in a humidified atmosphere containing 5 % CO2. The mesenchymal type
of breast cancer cell (MDA-MB 231) was cultured in L-15 medium supplemented with 10 %
fetal bovine serum and 1 % penicillin-streptomycin at 37 °C in a humidified atmosphere without
CO2.
The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide (MTT) was used to
assess the cytotoxic effect of 1 and 2. Human cancer cells (MCF7, MDA MB 231 and HeLa)
were seeded in 24 well culture plate with a seeding density of 10 × 104 cells per well in 500.0 µl
of complete medium. Cells were treated with different concentrations of the ruthenium
complexes (0.2 µg/ml to 60.0 µg/ml) for 24 h along with controlled experiments with standard
Cisplatin for comparison. At the end of the treatment period, 50.0 µl of MTT (5.0 mg/ml in PBS)
was added in each well and incubated for 3 h at 37 °C. Media containing MTT was removed and
the formazon was dissolved with DMSO. Absorbance was measured spectrophotometrically at
590 nm.
2.6. DNA Binding experiments
The tris–HCl buffer solution (pH 7.4), used in all the experiments involving CT-DNA, was
prepared using deionized and sonicated HPLC grade water (Merck). The CT-DNA was
sufficiently free from protein, with the ratio of UV absorbance of DNA in tris–HCl solution at
260 and 280 nm (A260/A280) of ca. 1.9 [39]. The concentration of DNA was determined with
its extinction coefficient ε of 6600 L mol-1 cm-1 at 260 nm [40]. The stock solution of DNA was
always stored at 4 °C and used within 4 days. Concentrated stock solution of complex was
prepared by dissolving 1 in DMSO and suitably diluting with tris–HCl buffer to the
concentration required for all the experiments. Absorption spectral titration was performed by
keeping constant the concentration of 1 while varying the CT-DNA concentration. To eliminate
the absorbance of DNA itself, an equal solution of CT-DNA was added to the ruthenium(II)
complex solution and to the reference. In the fluorescence displacement experiment with
ethidium bromide (EB), 5.0 μL of EB solution (1.0 mmol L-1) in tris–HCl was added to 1.0 mL
of DNA solution at saturated binding levels [41], and stored in the dark for 2.0 h. Solution of 1
6

was titrated into the DNA/EB mixture and then diluted in tris–HCl buffer to 5.0 mL, making
solutions with varied mole ratio of the metal complex to CT-DNA. Prior to measurements, the
mixture was shaken and incubated at room temperature for 30 min. The fluorescence spectra of
EB bound to DNA were obtained at an emission wavelength of 584 nm.
To adjudge the binding mode (groove/intercalative) of 1 with DNA, the well-known
method using Ostwald’s viscometer was used. Titrations were performed by introducing 1 (0.5–
3.5 µM) into a CT-DNA solution (5.0 µM) present in the viscometer. The viscosity values of the
solutions were calculated from the observed flow time of CT-DNA-containing solution corrected
from the flow time of buffer alone (t0), η = t-t0. The calculated data were used to plot the
(η / η0)1/3 versus the ratio of the concentration of 1 and CT-DNA, where η is the viscosity of
CT-DNA in the presence of the compound and η0 is the viscosity of CT-DNA alone.
2.7. Protein (bovine serum albumin) binding experiments
Samples for spectroscopic measurements were prepared by dissolving bovine serum albumin in
water and administering the appropriate concentration of 1. The samples were carefully degassed
using pure nitrogen for 15 min. Quartz cells with high vacuum Teflon stopcocks were used for
degassing.
2.8. Cyclic voltammetry (CV) experiments
Voltammetric measurements were carried out in a one-compartment cell using a Pt disk (area,
0.023 cm2) working electrode, a Pt flag counter electrode, and a Ag/AgCl reference electrode.
Typical CV curves for 0.1 mM 1 and 2 in tris-buffer in absence and presence of CT-DNA were
carried out.
3. Results and discussion
3.1. Synthesis and characterization
HL1 and HL2 were prepared by coupling pyrrole with the diazonium ion obtained from the
corresponding para-substituted aniline. These organic moieties are bidentate NN donors,
interesting due to the presence of hard donor azo-N and borderline base pyrrolic-N centres. The
ligand has been characterized by spectroscopic tools and X-ray diffraction studies. Complexes 1
and 2 were obtained in good yield from reaction of [RuCl2(bpy)2] with equimolar amount of HL1
7

or HL2 in refluxing dry ethanol under dinitrogen, followed by addition of aqueous solution of
sodium perchlorate to the resulting reaction mixture after cooling (viz. scheme 1).
Complexes 1 and 2 are soluble in DCM, acetonitrile, methanol and DMF.
Microanalytical data confirm the formulation of 1 and 2. Both 1 and 2 are 1:1 electrolytes as
determined by the observed conductivity (Λo) of 91-102 mho.cm2.mol-1 in methanol, and the
magnetic moment studies demonstrate that the complexes are diamagnetic in nature.
3.2. Structure of HL1
Single crystals of HL1 were obtained from a hexane-toluene solvent mixture of the compound on
slow evaporation at room temperature. The crystal structure of the ligand is shown in figure S1
together with the atom labelling scheme for all non-H atoms, and a selection of bond lengths and
angles appear in table 2.
The crystallographic independent unit comprises molecules A and B, which are
comparable with respect to bond distances and angles, as indicated in table 2 where values of A
and B agree within their esd’s. The molecules are arranged about a pseudo inversion center, but
oriented so that their mean planes form a dihedral angle of 53.1°. This disposition allows the
double H-bond interactions N-H…N of 3.012 and 3.074 Å (see figure S1). Both molecules
present coplanar atoms, indicating an electron delocalization inside each molecule. The crystal
packing shows A parallel aligned in a head-tail arrangement (being referred by a crystallographic
center of symmetry) to form weak π-π interactions between the phenyl and pyrrole ring of the
adjacent symmetry related molecule (and similarly do the molecules B, viz. figure S2).
3.3. DFT Computations: Explanation of spectral and redox properties
DFT calculation has been performed for 1 and 2. The optimized structures of these molecules are
developed using GAUSSIAN 03 (figure 1). The calculated bond angles and lengths for 1 and 2
are given in table S1. The orbital energies along with contributions from the ligands and metal
are given in table S2 and figure S3, which depicts selected occupied and unoccupied frontier
orbitals for 1). The HOMO of 1 is constituted by 80% contribution from L, whereas to HOMO-2
bpy contributes 19% and the metal ion 75%. The LUMO-2 is composed of L by 74%. Thus,
HOMO-2→LUMO is considered as major transition corresponding to Ru(dπ)→bpy(π*). The
other transitions are HOMO→LUMO [Ru(dπ)/L(π)→bpy(π*), HOMO-1→LUMO
8

[Ru(dπ)/L(π)→bpy(π*) and HOMO-2→LUMO+1 [Ru(dπ)→bpy(π*)], that are intraligand and
ligand-to-ligand charge transfer transitions. All these transitions are observed for 1, and the
calculated transitions are reported in table S3. Solvent polarity stabilizes occupied MOs more
efficiently than unoccupied MOs. Thus, the energy separation (DE) between HOMO and LUMO
increase on going from gas phase to MeCN solution.
Figure S4 depicts selected occupied and unoccupied frontier orbitals of 2. The metal
significantly contributes to the occupied MOs (76%, comparable to 75% of 1). Bipyridine
contributes 18% to HOMO-2 and 95% to LUMO in 2. The orbital energies of 2 along with
contributions from the ligands and metal are given in table S4 and the calculated transitions are
in table 3. The intensity of these transitions has been assessed from oscillator strength (f). In
MeCN the longest wavelength band is calculated at >550 nm (f, 0.0067) for 1 followed by
transitions at 502.3, 467.2, 448.9 and 395.4 nm along with a large number of transitions in the
UV region (< 400 nm). In 2 the calculated transitions are 558.0 (f, 0.0435), 512.0 (f, 0.0894),
458.1 (f, 0.0872) and 444.5 (f, 0.0773) nm. The observed transitions are in agreement with the
calculated ones for both complexes.
3.4. Spectral properties
IR spectra of the complexes are compared with the free ligands to confirm ligand coordination.
The IR spectrum of HL1 showed a band at ∼ 3455 cm-1 for νN-H, which is absent in spectra of 1,
and that for L2H at ∼ 3210 cm-1 is also absent in spectra of 2 [42]. The νN=N in HL1 is 1425 cm-1
and shifts to lower wavenumber in 1, suggesting coordination. Similar result was obtained for 2.
Bands at 1380–1385 cm-1 are assigned to ν(N=N) of coordinated azo (L1 / L2) in the complexes
[43]. Going from –N=N–(σ2π2) to (σ2π2 π*1), the bond order decreases from 2 to 1.5; therefore,
the frequency of ν(N=N) decreases significantly from free ligand (1420-1425 cm-1). Here back-
bonding from Ru(dπ) to azo(π*) orbitals is expected to become unimportant upon azo anion
radical formation. The complexes [Ru(L)(bpy)2]ClO4 show sharp absorptions at 1090 cm-1 and
626 cm-1 assignable to ν(ClO4-), which are absent for the organic moieties.
1
The H NMR spectra of ligands and [Ru(L)(bpy)2]ClO4 were recorded in CDCl3. The
signals for L1 and L2 appeared in the spectra as usual in support of the proposed structural
1
formulas. In the H NMR spectra of the complexes the protons appeared at higher δ values with
respect to the free organic moieties. The NH-pyrrole signal is at δ = ~9.75 ppm, which is absent
9

in 1H NMR spectra of the complexes. Mass spectra of the complexes (figures S4 and 5) are also
in support of these molecular structures.
Electronic absorption spectra of the complexes were recorded at room temperature using
acetonitrile and data are reported in table 4. The spectra of the complexes exhibit the
characteristic transitions at 295-297 nm and 375-378 nm corresponding to intramolecular π→π∗
and n→π∗ transitions, respectively. Bands at 489 and 493 nm in 1 and 2, respectively, were
observed due to the d(Ru) → π* (ligand) MLCT transitions.
3.5. Electrochemical behavior
The redox properties of 1 and 2 were examined by cyclic voltammetry using a Pt-disk working
electrode, a Pt-wire auxiliary electrode and Ag/AgCl electrode as reference in dry MeCN using
[n-Bu4N]ClO4 (0.1 M) as the supporting electrolyte. Voltammetric data are given in table 4. Each
complex exhibits a quasi-reversible RuII/RuIII redox couple around a redox wave at ca. E1/2 of
0.845 V (versus Ag/AgCl) for 1 and at ca. E1/2 of 1.11 V (versus Ag/AgCl) for 2 at 25 °C (viz.
figure 2). No cathodic response for RuII→RuI reduction was obtained for the complexes up to
-1.50 V. The voltammetric parameters were studied in the scan rate interval 50-400 mV s-1. The
ratio between the cathodic peak current and the square root of the scan rate (Ipc/ν1/2) is
approximately constant. The peak potential shows a small dependence on the scan rate. The ratio
Ipc to Ipa is close to unity. From these data, it can be concluded that the redox couple is related to
a quasi-reversible one-electron transfer controlled by diffusion.
3.6. Cytotoxicity
The cytotoxicities of 1 and 2 were tested on human cervical cancer HeLa cell line taking the
Cisplatin as a positive reference. The activity is expressed as the concentration of the complexes
required to inhibit the cellular survival fraction to 50% (LC50). The results presented in table 5
indicate that 1 exhibits higher cytotoxicity against HeLa cell line and 2 is much less active than
Cisplatin. The experiment with another cell lines shows that 1 is also the dose dependent
inhibition of the proliferation of human breast cancer cells MCF 7 and MDA-MB 231. The LC50
value of 1 for HeLa, MCF 7 and MDA-MB 231 cells were 22 µg/mL, 22 µg/mL and 20 µg/mL,
respectively, whereas that of Cisplatin for MCF 7 and MDA-MB 231 were 50 µg/mL and
56 µg/mL, respectively. This study clearly indicates that 1 is better than Cisplatin in terms of
10

cytotoxicity. The activity of 1 over 2 may be caused by 1 being more lipophilic than 2,
facilitating the passive uptake of drug molecules across the lipidic cell membrane and hence
enhancing the cytotoxicity [44]. However, both these ruthenium(II) complexes (1 and 2) possess
better antitumor activity than earlier reports of cytotoxicity of octahedral ruthenium(II)
complexes having two 2,2’-bipyridine moieties as coligands [45, 46].
3.7. DNA Binding experiments
3.7.1. Electronic absorption titration. From the cytotoxic assay 1 has very significant cytotoxic
effect compared to 2 and even better than Cisplatin. As a result, the interaction of CT-DNA with
1 was examined.
The binding mode of 1 with calf thymus DNA was examined by electronic absorption
titration with CT-DNA. As shown in figure 3, the spectra indicate a significant hyperchromism
centered around 375 nm, suggesting a strong interaction between 1 and DNA. The spectral
change might be interpreted as due to groove binding [47] since 1 contains azo-pyrrole, which
could facilitate formation of van der Waals contacts or hydrogen bonds during interaction with
DNA grooves. From these titration data the intrinsic binding constant (Kb) of 1 with CT-DNA
has been determined using the following equation (1) [48],
[DNA]/(εa-εf) = [DNA]/(εb-εf) + 1/[kb(εb-εf)]
(1)
where [DNA] represents the DNA concentration, εf and εb are the extinction coefficients for the
free and fully bound 1, respectively, and εa the complex extinction coefficient during each
addition of DNA. The [DNA]/(εa-εf) plot against [DNA] gave a linear relationship (figure 4). The
intrinsic binding constant (Kb) for 1 is calculated from the slope to intercept ratio (Kb =
1.0×105 M-1, R = 0. 0.95469 for five points). The value is in agreement with those of established
groove binding rather than classical intercalation [49].
3.7.2. Ethidium bromide fluorescence displacement experiments. Ethidium bromide (EB)
fluorescence displacement experiments were also performed to investigate the interaction mode
of the complexes with CT-DNA. EB fluorescence intensity (at λex = 522) will be enhanced in the
presence of DNA because of its intercalation into the helix, and quenched by the addition of
11

another molecule that displaces EB from DNA. Here, the fluorescence intensity of EB bound to
DNA at 620 nm decreases by increasing the concentration of 1, as shown in figure 5. EB
fluorescence quenching due to release of some EB from the EB-DNA system is supportive to
interaction of 1 with CT-DNA through groove binding. The quenching trend of EB bound DNA
by 1 is in agreement with the linear Stern–Volmer equation (2) [50]:
I0/I = 1 + KSV [complex]
(2)
where I0 and I represent the fluorescence intensities in the absence and presence of quencher,
respectively. KSV is the linear Stern–Volmer quenching constant and [complex] the molar
concentration of the quencher. From the slope of the regression line in the derived plot of I0/I vs
[complex] (figure 6), the KSV value for the complex was determined as 1.125 × 104 for 1 (R =
0.99023 for five points), indicating strong affinity of 1 to CT-DNA.
3.7.3. Viscosity. From spectroscopy it was observed that 1 has a strong interaction with DNA.
To further clarify the interactions between 1 and DNA, viscosity measurements were carried out.
From this experiment there is almost no effect on the relative viscosity of the DNA solution,
suggesting that the binding mode is groove binding, in support of the results obtained in the
spectroscopic study. If 1 intercalated, increase of the relative viscosity of the DNA solution
would be observed because intercalation leads to lengthening the DNA helix. Non-classical
intercalation could bend (or kink) the DNA helix and reduce its effective length and,
concomitantly, its viscosity [51, 52].
3.7.4. Cyclic voltammetric studies. Cyclic voltammetry has also been introduced to study
interaction between metal complexes and DNA, providing a useful complement to spectral
studies [53]. The binding of 1 with DNA is shown in figure 7. Cyclic voltammograms of 1 in the
absence and presence of CT-DNA exhibited significant shifts in the anodic peak potentials
followed by decrease in peak currents, indicating interaction between 1 and CT-DNA. The shift
in the value of the formal potential (ΔE0) is used to calculate the ratio of equilibrium binding
constants K2+/K3+ according to the following equation described by Bard and Carter [54],
12

ΔE° = Eb° – Ef° = 0.059 log(K2+/K3+)
where Eb° and Ef° are the formal potentials of the bound and free complexes, respectively, and
K2+ and K3+ are the corresponding binding constants for the binding of reduction and oxidation
species to DNA, respectively. Ratio of equilibrium binding constants K2+/K3+ is calculated to be
3.35 that indicates strong binding of DNA with Ru(II).
3.8. Protein (bovine serum albumin) binding experiments
3.8.1. Absorption characteristics of BSA–Ru(II) complex. The absorption spectra of BSA in
the absence and presence of 1 at different concentrations were studied (figure 8). Upon
increasing the concentration of the complex, absorption of BSA increases regularly, due to
adsorption of BSA on the surface of the complex. From these data the apparent association
constant (Kapp) determined for complexes with BSA has been determined using the following
equation (3) [46]:
1/(Aobs – A0) = 1/(Ac – A0) + 1/ Kapp(Ac – A0)[comp]
(3)
where Aobs is the observed absorbance of the solution containing different concentrations of the
complex at 280 nm, A0 and Ac are the absorbances of BSA and the complex at 280 nm,
respectively, with a concentration of C0, and Kapp represents the apparent association constant.
The enhancement of absorbance at 280 nm was due to absorption of the surface complex, based
on the linear relationship between 1/(Aobs - A0) vs reciprocal concentration of the complex with a
slope equal to 1/Kapp(Ac - A0) and an intercept equal to 1/(Ac - A0) (figure 8, inset). The value of
the apparent association constant (Kapp) determined from this plot is 3.7 × 103 M-1.
3.8.2. Fluorescence quenching of BSA by 1. The effect of increasing the concentration of the
complex on the fluorescence emission spectrum of BSA was studied (figure 9). With the addition
of 1, BSA fluorescence emission is quenched. The fluorescence quenching is described by the
Stern–Volmer relation (eqn. (2)). Here, I0 and I represent the fluorescence intensities of BSA in
the absence and presence of quencher, respectively. KSV is the linear Stern–Volmer quenching
constant and [complex] the molar concentration of the quencher. A linear plot (figure 9, inset)
13

between I0/I against [complex] was obtained and from the slope we calculated the KSV as
2.17 × 105.
4. Conclusion
Synthesis and characterization of two new azopyrrole ligands, HL1 = (4-chloro-phenyl)-(1H-
pyrrol-2-yl)-diazene and HL2
=
(4-nitro-phenyl)-(1H-pyrrol-2-yl)-diazene and their
corresponding ruthenium(II) complexes along with the detailed structural analysis of HL1 by
single crystal X-ray crystallography is reported. The cytotoxicities of 1 and 2 on human cervical
cancer HeLa cell line and human breast cancer epithelial cell (MCF 7 and MDA-MB 231) lines
taking the Cisplatin as a positive reference clearly indicate that 1 has a higher cytotoxic acitivity
against cancer cells than Cisplatin but 2 is less active due to more electron withdrawing nitro
(NO2) group in L2 compared to chloro in L1 at para position of the azo. The spectroscopic study
of interaction of 1 with CT-DNA showed groove binding, in accord with unchanged values of
the viscosity of the DNA solution upon addition of 1. This study clearly indicates that 1 binds to
calf thymus DNA in a groove binding interaction [55, 56] but not in a intercalative mode like
reported octahedral ruthenium(II) complexes [57-60]. However, the Kb of 1 is comparable to that
of the reported ruthenium(II) complexes [55, 56]. Electrochemical studies indicate that the
reduced form of the ruthenium complex (Ru(II)) has a stronger association to DNA than the
oxidized Ru(III) form. Absorption and emission spectroscopy, as well as fluorescence, are used
to study interactions of BSA-[Ru(L1)(bpy)2]ClO4.
Supporting material
Crystallographic data of HL1 have been deposited with the Cambridge Crystallographic Data
Centre, CCDC No. 885460. Copies of this information are available on request at free of charge
from CCDC, 12 Union Road, Cambridge, CB21EZ, UK (Fax: +44-1223-336-033; E-mail:
deposit@ccdc.ac.uk or http://www.ccdc.cam.ac.uk).
Acknowledgements
Financial support from Council of Scientific and Industrial Research (CSIR), New Delhi, India is
gratefully acknowledged. A.B. and A.M. are acknowledging DBT, New Delhi for the work in
cancer cells.
14

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17

Table 1. Crystal data and details of refinement for HL1.
Empirical formula
C10H8N3Cl
205.64
Formula weight
Crystal system
Space group
Triclinic
P
1
a (Å)
b (Å)
c (Å)
7.6867(4)
9.9521(6)
12.7722(6)
82.262(4)
89.479(4)
78.194(5)
947.51(9)
1.442
α
β
γ
(
°
)
(°
)
(°)
Volume (Å3)
calc [g/cm3]
ρ
Z
F
θ
4
(000)
424
3.07 - 25.00
0.362
range (°)
(Mo Kα) (mm-1)
μ
Collected / unique reflections
20544 / 6672
3313
Reflections
Parameters
I
>2 (I)
σ
253
Goodness-of-fit
Final indices [
indices (all data)
Residuals (e.Å-3)
0.848
R
I>2
σ
(I
)]
R
R
1 = 0.0330, wR2 = 0.0592
1 = 0.0614, wR2 = 0.0630
R
0.432, -0.453
18

Table 2. Selected bond lengths (Å) and angles (
of HL1.
°
) of the two independent molecules
Molecule B
Molecule A
C(1a)-N(7a)
N(7a)-N(8a)
N(8a)-C(9a)
C(9a)-N(10a)
C(9a)-C(13a)
N(10a)-C(11a)
1.429(2)
1.280(2)
1.374(2)
1.384(2)
1.387(3)
1.362(2)
C(1)-N(7)
N(7)-N(8)
N(8)-C(9)
C(9)-N(10)
C(9)-C(13)
N(10)-C(11)
1.435(2)
1.281(2)
1.382(2)
1.377(2)
1.388(3)
1.356(2)
C(2a)-C(1a)-N(7a)
C(6a)-C(1a)-N(7a)
N(8a)-N(7a)-C(1a)
N(7a)-N(8a)-C(9a)
N(10a)-C(9a)-N(8a)
N(10a)-C(11a)-C(12a)
C(11a)-C(12a)-C(13a)
N(10a)-C(9a)-C(13a)
N(8a)-C(9a)-C(13a)
C(11a)-N(10a)-C(9a)
124.08(19)
116.52(18)
113.81(16)
114.85(16)
126.01(18)
108.99(18)
107.42(18)
107.45(18)
126.53(19)
108.48(17)
C(2)-C(1)-N(7)
C(6)-C(1)-N(7)
N(8)-N(7)-C(1)
N(7)-N(8)-C(9)
N(10)-C(9)-N(8)
N(10)-C(11)-C(12)
C(11)-C(12)-C(13)
N(10)-C(9)-C(13)
N(8)-C(9)-C(13)
C(11)-N(10)-C(9)
125.51(17)
115.54(17)
113.61(16)
113.86(17)
126.60(18)
109.10(18)
107.27(17)
107.48(17)
125.92(18)
108.79(17)
19

Table 3. Some selected vertical electronic excitations of 2.
Eexcitation
(eV)
Osc.
strength (f)
λexcitation
(nm)
λexpt.
(nm)
Key transitions
Character
2.2219
2.4216
2.7060
2.7888
3.0804
3.5166
4.1865
558.0
512.0
458.1
444.5
402.4
357.6
296.2
0.0435
0.0894
0.0872
0.0773
0.2219
0.1302
0.0411
(86%)HOMO→LUMO
Ru(dπ)/L(π)→bpy(π*)
Ru(dπ)/L(π)→bpy(π*)
Ru(dπ)/bpy(π)→bpy(π*)
Ru(dπ)/bpy(π)→bpy(π*)
Ru(dπ)/L(π)→bpy(π*)
Ru(dπ)/L(π)→bpy(π*)
L(π)→L(π*)
495
(87%)HOMO-1→LUMO
(77%)HOMO-2→LUMO+1
(68%)HOMO-3→LUMO+1
(61%)HOMO-1→LUMO+3
(69%)HOMO-3→LUMO+4
379
299
(47%)HOMO-6→LUMO+2
(34%)HOMO-7→LUMO+2
4.3734
283.5
0.1145
(43%)HOMO-7→LUMO+2
(27%)HOMO-6→LUMO+2
L(π)→L(π*)
20

Table 4. UV-vis spectral and electrochemical dataa.
Electrochemical data
E1/2, E (V) for Ru(II)/Ru(III)
nm (ε) (ε
, dm3·mol-1·cm-1)
Compd.
λ
Δ
489(2277), 375(3886), 297(4485)
495(2536), 379(3963), 299(4563)
0.845 (0.101)
1.11 (0.084)
1
2
ain acetonitrile
21

Table 5. Cytotoxicity (LC50, µg/mL) of
1
and
2
(24 h).
Complex
HeLa
42.6
22
MCF 7
50
MDA MB 231
Cisplatin
56
20
22
1
2
75.4
Not significant
Not significant
22

1
2
Figure 1. Optimized structure of
1
and
2
at DFT-B3LYP/6-31G(d) level of theory.
23

1.5
0.0
-1.5
-3.0
-4.5
-6.0
1.2 1.1
1.0 0.9 0.8 0.7
0.6 0.5
Potential (V)
Figure 2. Cyclic voltammogram of
1 in MeCN (0.1 M n-Bu4NClO4) at 298 K, Ag/AgCl
reference electrode, and scan rate 100 mV/s.
24

0.30
0.27
0.24
0.21
0.18
0.15
0.12
f
a
300
350
400
450
500
Wavelength(nm)
Figure 3. Electronic spectra of
1 through titration with CT-DNA in tris-HCl. The increase of
DNA concentration is indicated by an arrow.
25

2.2
2.1
2.0
1.9
1.8
1.7
1.6
1.5
0.8
1.6
2.4
3.2
4.0
4.8
[DNA] x 106
Figure 4. Plot of [DNA]/(εa
-εf) vs [DNA] for titration of CT-DNA with 1 in tris-HCl buffer;
binding constant Kb = 1
×
105 M-1 (R = 0. 0.95469 for five points).
26

900
750
600
450
300
150
a
f
550
600
650
700
Wavelength(nm)
Figure 5. Emission spectra of CT-DNA-EB system in tris–HCl buffer based on titration of
complex. ex = 522 nm.
λ
27

2.4
2.1
1.8
1.5
1.2
2
4
6
8
10 12 14 16
[Complex] x 105 (M)
Figure 6. Plot of I0/I vs [complex] for titration of CT-DNA–EB system with
1
using
spectrofluorimeter; linear Stern–Volmer quenching constant (Ksv) = 1.125
for five points).
×
104 (R = 0.99023
28

Figure 7. Cyclic voltammograms of the ruthenium(II) complex,
(a) absence (black curve) and (b) presence of CT-DNA (red curve).
1 in tris–HCl buffer in
29