New ruthenium(II) arene complexes of anthracenyl-appended diazacycloalkanes: Effect of ligand intercalation and hydrophobicity on DNA and protein binding and cleavage and cytotoxicity

Article abstract of DOI:10.1039/c3dt51641e

A series of half-sandwich Ru(II) arene complexes of the type [Ru(η⁶-arene)(L)Cl](PF₆) 1-4, where arene is benzene (1, 2) or p-cymene (3, 4) and L is N-methylhomopiperazine (L1) or 1-(anthracen-10-ylmethyl)-4-methylhomopiperazine (L2), has been isolated and characterized by using spectral methods. The X-ray crystal structures of 2, 3 and 4 reveal that the compounds possess a pseudo-octahedral piano- stool structure equipped with the arene ligand as the seat and the bidentate ligand and the chloride ion as the legs of the stool. The DNA binding affinity determined using absorption spectral titrations with CT DNA and competitive DNA binding studies varies as 4 > 2 > 3 > 1, depending upon both the arene and diazacycloalkane ligands. Complexes 2 and 4 with higher DNA binding affinities show strong hypochromism (56%) and a large red-shift (2, 10; 4, 11 nm), which reveals that the anthracenyl moiety of the ligand is stacked into the DNA base pairs and that the arene ligand hydrophobicity also dictates the DNA binding affinity. In contrast, the monocationic complexes 1 and 3 are involved in electrostatic binding in the minor groove of DNA. The enhancement in viscosities of CT DNA upon binding to 2 and 4 are higher than those for 1 and 3 supporting the DNA binding modes of interaction inferred. All the complexes cleave DNA effectively even in the absence of an external agent and the cleavage ability is enhanced in the presence of an activator like H₂O ₂. Tryptophan quenching measurements suggest that the protein binding affinity of the complexes varies as 4 > 2 > 3 > 1, which is the same as that for DNA binding and that the fluorescence quenching of BSA occurs through a static mechanism. The positive ΔH⁰ and ΔS ⁰ values for BSA binding of complexes indicate that the interaction between the complexes and BSA is mainly hydrophobic in nature and the energy transfer efficiency has been analysed according to the Foerster non-radiative energy transfer theory. The variation in the ability of complexes to cleave BSA in the presence of H₂O₂, namely, 4 > 2 > 3 > 1, as revealed from SDS-PAGE is consistent with their strong hydrophobic interaction with the protein. The IC₅₀ values of 1-4 (IC₅₀: 1, 28.1; 2, 23.1; 3, 26.2; 4, 16.8 μM at 24 h; IC ₅₀: 1, 19.0; 2, 15.9; 3, 18.1; 4, 9.7 μM at 48 h) obtained for MCF 7 breast cancer cells indicate that they have the potency to kill cancer cells in a time dependent manner, which is similar to cisplatin. The anticancer activity of complexes has been studied by employing various biochemical methods involving different staining agents, AO/EB and Hoechst 33258, which reveal that complexes 1-4 establish a specific mode of cell death in MCF 7 breast cancer cells. The comet assay has been employed to determine the extent of DNA fragmentation in cancer cells. The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c3dt51641e

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New ruthenium(II) arene complexes of anthracenyl-
appended diazacycloalkanes: effect of ligand
intercalation and hydrophobicity on DNA and
protein binding and cleavage and cytotoxicity†
Cite this: Dalton Trans., 2014, 43,
1203
Mani Ganeshpandian,a Rangasamy Loganathan,a Eringathodi Suresh,b
Anvarbatcha Riyasdeen,c Mohammad Abdulkadher Akbarshad,e and
Mallayan Palaniandavar*a
A series of half-sandwich Ru(II) arene complexes of the type [Ru(η6-arene)(L)Cl](PF6) 1–4, where arene is benzene
(1, 2) or p-cymene (3, 4) and L is N-methylhomopiperazine (L1) or 1-(anthracen-10-ylmethyl)-4-methylhomopipera-
zine (L2), has been isolated and characterized by using spectral methods. The X-ray crystal structures of 2, 3 and 4
reveal that the compounds possess a pseudo-octahedral “piano-stool” structure equipped with the arene ligand as
the seat and the bidentate ligand and the chloride ion as the legs of the stool. The DNA binding affinity determined
using absorption spectral titrations with CT DNA and competitive DNA binding studies varies as 4 > 2 > 3 > 1,
depending upon both the arene and diazacycloalkane ligands. Complexes 2 and 4 with higher DNA binding affinities
show strong hypochromism (56%) and a large red-shift (2, 10; 4, 11 nm), which reveals that the anthracenyl moiety of
the ligand is stacked into the DNA base pairs and that the arene ligand hydrophobicity also dictates the DNA binding
affinity. In contrast, the monocationic complexes 1 and 3 are involved in electrostatic binding in the minor groove of
DNA. The enhancement in viscosities of CT DNA upon binding to 2 and 4 are higher than those for 1 and 3 support-
ing the DNA binding modes of interaction inferred. All the complexes cleave DNA effectively even in the absence of
an external agent and the cleavage ability is enhanced in the presence of an activator like H2O2. Tryptophan quench-
ing measurements suggest that the protein binding affinity of the complexes varies as 4 > 2 > 3 > 1, which is the
same as that for DNA binding and that the fluorescence quenching of BSA occurs through a static mechanism. The
positive ΔH0 and ΔS0 values for BSA binding of complexes indicate that the interaction between the complexes and
BSA is mainly hydrophobic in nature and the energy transfer efficiency has been analysed according to the Förster
non-radiative energy transfer theory. The variation in the ability of complexes to cleave BSA in the presence of H2O2,
namely, 4 > 2 > 3 > 1, as revealed from SDS-PAGE is consistent with their strong hydrophobic interaction with the
protein. The IC50 values of 1–4 (IC50: 1, 28.1; 2, 23.1; 3, 26.2; 4, 16.8 µM at 24 h; IC50: 1, 19.0; 2, 15.9; 3, 18.1; 4,
9.7 µM at 48 h) obtained for MCF 7 breast cancer cells indicate that they have the potency to kill cancer cells in a
time dependent manner, which is similar to cisplatin. The anticancer activity of complexes has been studied by
employing various biochemical methods involving different staining agents, AO/EB and Hoechst 33258, which reveal
that complexes 1–4 establish a specific mode of cell death in MCF 7 breast cancer cells. The comet assay has been
employed to determine the extent of DNA fragmentation in cancer cells.
Received 20th June 2013,
Accepted 8th October 2013
DOI: 10.1039/c3dt51641e
www.rsc.org/dalton
Introduction
aDepartment of Chemistry, Central University of Tamil Nadu, Thiruvarur 610 004, India
bAnalytical Department and Centralized Instrument Facility, Central Salt and Marine
Chemical Research Institute, Council of Scientific and Industrial Research
(CSIR-CSMCRI), Bhavnagar 364 002, India
cDepartment of Animal Science, Bharathidasan University, Tiruchirappalli – 620 024,
Tamilnadu, India
dMahatma Gandhi-Doerenkamp Center for Alternatives to Use of Animals in Life
Science Education, Bharathidasan University, Tiruchirappalli, 620024 Tamilnadu, India
eDepartment of Food and Nutrition, King Saud University, Riyadh, Kingdom of
The most successful platinum-based anticancer drugs like cis-
platin and other multi-nuclear platinum compounds are
useful in the treatment of testicular and ovarian cancers and
are also widely employed for treating bladder, cervical, head
and neck, oesophageal and small cell lung cancers.1 However,
they show several side effects including nephrotoxicity,
emetogenesis and neurotoxicity during treatment of cancer2
and the activity is limited by both toxicity and acquired
resistance.3 The two Ru(III) complexes KP 1019 (indazolium
trans-[tetrachlorobis(1H-indazole)ruthenate(III)]) and NAMI-A
Saudi Arabia. E-mail: palaniandavar@cutn.ac.in, palanim51@yahoo.com
†Electronic supplementary information (ESI) available. CCDC 927197–927199
and 961677. For ESI and crystallographic data in CIF or other electronic format
see DOI: 10.1039/c3dt51641e
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[trans-RuCl4(DMSO)(Im)](ImH) are currently under clinical trials
for cancer chemotherapy and are anticipated to be approved as
drugs in the coming years.4 The complex KP 1019 is active
against primary tumors whereas NAMI-A is active against the
metastasis form of tumour.5 In this context, very recently,
attention has been focused on organometallic Ru(II) arene
complexes and their anticancer activities have been studied by
varying the arene, substituent ligand and leaving group.6
These “piano-stool” frameworks provide a handle for optimiz-
ing the design of drugs in terms of pharmacological properties
and reducing the side effects. Thus, Sadler et al. found that
the Ru(II)-arene–en (en, ethylenediamine) complex exhibits
efficient cytotoxic activity and also shows activity against cis-
platin-resistant cell lines.6 The Ru(II)-arene-PTA complex
(RAPTA-C) acts as an antimetastatic agent and shows a bio-
chemical mode of action against EAC cells, which is different
from that of platinum anticancer drugs. It inhibits specific
proteases, which plays a vital role in cancer drug develop-
ment.7 Very recently, Sheldrick and coworkers have prepared
half-sandwich Ru(II) and Rh(III) complexes with methyl-substi-
tuted polypyridyl ligands, which strongly bind to DNA and also
regulate apoptosis.8 Pandey et al. reported water soluble Ru(II)
and Rh(III) arene complexes, which show DNA binding and
topoisomerase II inhibitory activity.9 The antitumor activity of
many Ru(II)-arene complexes has been related to their
enhanced DNA binding affinity, which involves covalent and/
or noncovalent mode of DNA interaction.10–16
Scheme 1 Structures of the complexes and ligands used.
DNA and protein binding and cleavage ability on their cyto-
toxic activity (Scheme 1).
Experimental
Reagents and materials
The reagents and chemicals were obtained from commercial
sources (Sigma-Aldrich, USA; Himedia, India; Merck, India;
Genei, Bangaluru, India). RuCl3·3H2O (Arora Matthey), α-phel-
landrene, 1,3-cyclohexadiene, N-methylhomopiperazine (L1),
9-anthracene-10-carboxaldehyde, sodium cyanotrihydroborate,
ethidium bromide (EthBr), calf thymus (CT) DNA (highly poly-
merized, stored at −20 °C) (Aldrich), pUC19 supercoiled DNA,
agarose and bovine serum albumin (BSA) (Genei) were used as
received. Ultrapure MilliQ water (18.2 μΩ) was used in all
experiments. Tris(hydroxymethyl)aminomethane·HCl (Tris-
HCl) (pH 7.1) was prepared by the reported procedure.29 Com-
mercial solvents were distilled and then used for preparation
of complexes.
In recent years, there have been increasing investigations17
on the interaction of metallo-drugs with molecular targets
other than DNA for understanding their cytotoxic activities.
As serum proteins perform the transport, distribution,
accumulation and excretion properties of drugs in tumor
tissue, special consideration has been given to the study of
binding of metallo-drugs to the proteins.18 The solubility of
hydrophobic drugs in plasma effectively increases and the
drug binds to serum albumins and modulates their delivery
to cells in specific sites. In this connection significant reactiv-
ity of RAPTA-C compounds against two emerging protein
targets, thioredoxin reductase and cathepsin B, has been
explored very recently for their anticancer activity.19 Also, if the
Synthesis of 1-(anthracen-10-ylmethyl)-4-methyl-
homopiperazine (L2)
complex acts as artificial metallo-protease for disease-related To a solution of 9-anthracene-10-carboxaldehyde (2.06 g,
proteins, it will be a recognizing element20 or therapeutic 10.0 mmol) in methanol (50 mL), N-methylhomopiperazine
agent21 for the unknown active site of proteins by activating its (1.14 g, 10.0 mmol) and a small amount of acetic acid
cleavage.
(0.57 mL, 10 mM) were added. Sodium cyanotrihydroborate
In our earlier studies, we have correlated the cytotoxicity of (0.63 g, 10.0 mmol) in methanol (5 mL) was added dropwise to
certain mixed ligand Ru(II) complexes with the non-covalent the resulting solution with stirring. After the solution was
mode of DNA binding involving diimine co-ligands22–28 stirred for 3 days at 25 °C, it was acidified by adding conc. HCl
and different primary ligands like 1,8-bis(pyrid-2-yl)-3,6-di- and then evaporated almost to dryness under reduced
thiooctane
(pdto),22
1,8-bis(benzimidazol-2-yl)-3,6-dithio- pressure. The residue was dissolved in saturated aqueous solu-
octane (bbdo)22 and 2,2-dipyridylamine (Hdpa).23 Now we have tion (50 mL) of Na2CO3 and extracted with CHCl3 (3 × 50 mL).
embarked on studying Ru-arene complexes as anticancer The combined extracts were dried over anhydrous Na2SO4 and
agents and describe here a series of half-sandwich ruthe- filtered. On slow evaporation of the filtrate an orange brown
nium arene complexes of the type [Ru(η6-arene)(L)Cl](PF6), viscous solid was obtained. It was purified by recrystallization
where arene is benzene (1, 2) or p-cymene (3, 4) and L is from CH2Cl2. Yield: 1.88 g (62.0%); mp 194 °C; 1H NMR
N-methylhomopiperazine (L1) or 1-(anthracen-10-ylmethyl)- (400 MHz, CDCl3): δ 4.11 (s, N–CH2), 2.32 (s, N–CH3),
+
4-methyl-homopiperazine (L2), to explore the effect of their 7.37–7.79 (m, 9H) ppm. EI-MS m/z = 304 C21H24N2 .
1204 | Dalton Trans., 2014, 43, 1203–1219
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Isolation of ruthenium(II) complexes
Experimental methods
The starting materials [Ru(η6-arene)Cl2]2, where arene is
benzene or p-cymene, were prepared by using the procedures
already reported.30,31
Microanalyses (C, H, and N) were carried out using a Vario EL
elemental analyzer. UV-Vis spectra were recorded on
a
Shimadzu 2450 UV-Vis spectrophotometer using cuvettes of
1
1 cm path length. H NMR spectra were recorded on a Bruker
Isolation of [Ru(η6-benzene)(L1)Cl](PF6) (1)
400 MHz NMR spectrometer. Mass spectrometry was per-
formed using a ZQ ESI-MS spectrometer. Emission intensity
measurements were carried out using a JASCO F 6500 spectro-
fluorometer. Viscosity measurements were carried out using a
Schott Gerate AVS 310 automated viscometer. Cyclic voltamme-
try and differential pulse voltammetry on a platinum sphere
electrode were performed at 25.0 0.2 °C. The temperature of
the electrochemical cell was maintained by a cryocirculator
(HAAKE D8-G). Voltammograms were generated with the use
of an EG&G PAR Model 273 potentiostat. A Pentium IV compu-
ter along with EG&G M270 software was employed to control
the experiments and to acquire the data. A three-electrode
system consisting of a platinum sphere (0.29 cm2), a platinum
auxiliary electrode and a reference electrode were used. The
reference electrode for non-aqueous solution was Ag(s)/Ag+,
which consists of an Ag wire immersed in a solution of AgNO3
(0.01 M) and NBu4ClO4 (0.1 M) in acetonitrile placed in a tube
fitted with a Vycor plug using a sleeve.32 The cyclic voltammo-
grams (CV) and differential pulse voltammograms (DPV) of
1–4 were obtained in acetonitrile solutions with NBu4ClO4 as
the supporting electrolyte at ambient temperatures under N2.
Redox potentials were measured relative to the Ag/Ag+ refer-
ence electrode. All the complexes are electroactive with respect
to the metal as well as the ligand centres in the potential range
1.0 V. The redox potential E1/2 was calculated from the peak
potential (Epa) of the DPV response as Ep + ΔE/2 (ΔE is the
pulse height).
This complex was prepared by adding a methanolic solution of
L1 (0.23 g, 2 mmol) to a solution of the ruthenium dimer pre-
cursor [Ru(η6-benzene)Cl2]2 (0.50 g, 1 mmol) in methanol
(20 mL). This was stirred at ambient temperature for 5 h, fil-
tered and NH4PF6 (0.5 g, 3.07 mmol) was added. The volume
of the solvent was reduced, and the product formed as an
orange-yellow microcrystalline solid when left to stand at 4 °C.
The product separated out was collected by suction filtration,
washed with small amounts of cold methanol, water and
excess of ether and then dried in vacuum. Yield: 81%; anal.
calcd for [Ru(η6-benzene)(L1)Cl](PF6): C, 30.42; H, 4.25;
N, 5.91. Found: C, 30.49; H, 4.29; N, 5.98%. ESI-MS: [Ru-
(η6-benzene)(L1)Cl]+ displays a peak at m/z 328.75 (calcd
328.82). 1H NMR (400 MHz, DMSO-d6): δ 5.84 (s, η6-C6H6), 7.35
(s, –NH), 3.22 (s, –NCH3) ppm.
Isolation of [Ru(η6-benzene)(L2)Cl](PF6) (2)
This complex was prepared by adopting the procedure used for
the preparation of 1 but using L2 (0.61 g, 2 mmol) instead of
L1. Yield: 84%; anal. calcd for [Ru(η6-benzene)(L2)Cl](PF6):
C, 48.84; H, 4.55; N, 4.22. Found: C, 48.86; H, 4.59; N, 4.24%.
ESI-MS: [Ru(η6-benzene)(L2)Cl]+ displays
a peak at m/z
519.00 (calcd 519.06). 1H NMR (400 MHz, DMSO-d6): δ 6.25
(s, η6-C6H6), 3.20 (s, N–CH3), 7.35–8.93 (m, 9H) ppm.
Solutions of calf thymus (CT) DNA in the buffer 5 mM Tris-
HCl/50 mM NaCl in water gave a ratio of UV absorbance at 260
and 280 nm, A260/A280, of 1.9,33 indicating that the DNA was
sufficiently free of protein. Concentrated stock solutions of
DNA (14.8 mM) were prepared in buffer and sonicated for 25
cycles, where each cycle consisted of 30 s with 1 min intervals.
The concentration of DNA in nucleotide phosphate (NP) was
determined by UV absorbance at 260 nm after 1 : 100 dilutions
Isolation of [Ru(η6-p-cymene)(L1)Cl](PF6) (3)
This complex was prepared by adopting the procedure used for
the preparation of 1 but using the p-cymene dimer precursor
[Ru(η6-p-cymene)Cl2]2 (0.61 g, 1 mmol) instead of the benzene
dimer. Yield: 77%; anal. calcd for [Ru(η6-p-cymene)(L1)Cl]-
(PF6): C, 36.27; H, 5.33; N, 5.29. Found: C, 36.21; H, 5.39;
N, 5.22%. ESI-MS: [Ru(η6-p-cymene)(L1)Cl]+ displays a peak at
m/z 384.84 (calcd 384.93). 1H NMR (400 MHz, DMSO-d6):
δ 1.39 (d, CH(CH3)2), 2.35 (s, C–CH3), 3.19 (s, N–CH3),
5.42–6.18 (dd, C6H4), 7.22 (s, NH) ppm.
by taking the extinction coefficient, ε260 nm, as 6600 M−1 cm−1
.
Stock solutions of DNA were stored at 4 °C and used after not
more than 4 days. Supercoiled plasmid pUC19 DNA was stored
at −20 °C, and the concentration of DNA in base pairs was
determined by UV absorbance at 260 nm after appropriate
dilutions taking ε260 nm as 13 100 M−1 cm−1
.
Isolation of [Ru(η6-p-cymene)(L2)Cl](PF6) (4)
X-Ray crystallography
This complex was prepared by adopting the procedure used for
the preparation of 3 but using L2 instead of the L1 ligand. Crystals 2–4 of suitable size selected from the mother liquor
Yield: 79%; Anal. calcd for [Ru(η6-p-cymene)(L2)Cl](PF6): were mounted on the tip of a glass fiber and cemented using
C, 51.70; H, 5.32; N, 3.89. Found: C, 51.77; H, 5.38; N, 3.85%. epoxy resin. Intensity data for the crystals were collected using
ESI-MS: [Ru(η6-p-cymene)(L2)Cl]+ displays a peak at m/z 574.98 MoKα (λ = 0.71073 Å) radiation on a Bruker SMART APEX dif-
(calcd 575.06). 1H NMR (400 MHz, DMSO-d6): δ 1.35 (d, CH- fractometer equipped with a CCD area detector at 296 K. The crys-
(CH3)2), 2. 50 (s, C–CH3), 3.14 (s, N–CH3), 5.64–6.61 (dd, C6H4), tallographic data are shown in Table 1. The SMART34 program
7.50–8.91 (m, 9H) ppm.
was used for collecting frames of data, indexing the reflections,
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Computational studies
Table 1 Selected interatomic distances [Å] and bond angles [°] for com-
plexes 2, 3 and 4
The DFT calculations were carried out using the Gaussian03
program package36 using the hybrid density functional theory
(B3LYP) method. The 6-31G* basis set was used to describe C,
N, and hydrogen atoms. The Ru atom was described using the
LANL2DZ basis set. The full geometry optimizations were
carried out for complexes 1–4.
2
3
4
Bond distance
Ru(1)–N(1)
Ru(1)–N(2)
Ru(1)–Cl(1)
Ru(1)–C(1)
Ru(1)–C(2)
Ru(1)–C(3)
Ru(1)–C(4)
Ru(1)–C(5)
Ru(1)–C(6)
Ru–cent
2.157(3)
2.171(3)
2.413(8)
2.219(3)
2.193(3)
2.191(3)
2.172(3)
2.212(3)
2.202(3)
1.689(3)
2.193(2)
2.164(2)
2.418(7)
2.186(3)
2.197(3)
2.193(3)
2.186(3)
2.240(3)
2.194(3)
1.688(7)
2.166(2)
2.178(19)
2.414(6)
2.175(5)
2.255(3)
2.183(3)
2.205(3)
2.263(3)
2.200(3)
1.712(4)
DNA binding and cleavage experiments
Concentrated stock solutions of metal complexes were pre-
pared using acetonitrile–water mixed solvent (1 : 50 v/v) and
then diluting them suitably with 5 mM Tris-HCl/50 mM NaCl
buffer at pH 7.1 to the required concentrations for all the
experiments. For absorption and emission spectral experi-
ments the DNA solutions were pretreated with the solutions
of metal complexes to ensure no change in the concentration
of the metal complexes. Absorption spectral titration experi-
Bond angle
N(1)–Ru(1)–N(2)
N(1)–Ru(1)–Cl(1)
N(2)–Ru(1)–Cl(1)
73.61(10)
87.59(7)
88.64(7)
71.49(9)
88.79(7)
89.22(7)
73.53(8)
86.42(6)
87.22(5)
and determining the lattice parameters. The SAINT34 program
was used for integration of the intensity of reflections and
scaling, and the SHELXTL35 program for space group and
structure determination, and least-squares refinements on F2.
The structure was solved by a heavy atom method. All the non-
hydrogen atoms were located in successive difference Fourier
syntheses. The final refinement was performed by full-matrix
least-squares analysis. Hydrogen atoms attached to the ligand
moiety were placed at fixed positions using the HFIX instruc-
tion. The crystallographic data and details of data collection
for 2–4 are given in Table 2. In the case of the structure of 4,
four F atoms in one of the PF6 anions was highly disordered
and the coordinates/occupany factor for the disordered atom
positions were determined using ‘FVAR’/‘PART’ of SHELXTL
and the disordered atoms were refined anisotropically till con-
vergence is reached. The contribution of the disordered lattice
solvent molecules in 4 was removed from the final data using
‘SQUEEZ’ of the PLATON package.
ments were performed by maintaining
a constant con-
centration of the complex and varying the nucleic acid
concentration. This was achieved by dissolving an appropriate
amount of the metal complex and DNA stock solutions while
maintaining the total volume constant (1 mL). This results in
a series of solutions with varying concentrations of DNA but
with a constant concentration of the complex. The absorbance (A)
of the most red-shifted band of each investigated complex was
recorded after successive additions of CT DNA.
The Tris buffer was used as a blank to make preliminary
adjustments. The excitation wavelength was fixed and the emis-
sion range was adjusted before measurements. DNA was pre-
treated with ethidium bromide in the ratio [NP/EthBr] = 10 for
30 min at 27 °C. The metal complexes were then added to this
mixture and their effect on the emission intensity was measured.
For viscosity measurements, a Schott Gerate AVS 310 auto-
mated viscometer was thermostated at 25 °C in a constant
temperature bath. The concentration of DNA was 500 mM in
Table 2 Crystal data and structure refinement for complexes 2, 3 and 4
2
3
4
Formula
Crystal system
Space group
a (Å)
b (Å)
c (Å)
C27H30ClN2RuF6P
Monoclinic
C2/c
38.416(3)
7.5479(5)
20.9677(13)
90
C16H28ClF6N2PRu
Orthorhombic
Pna21
20.6176(2)
11.0391(1)
8.7929(1)
90
C124H152Cl4F24N8P4Ru4
Monoclinic
P21/c
15.8707(2)
19.8808(2)
20.3162(2)
90
α (°)
β (°)
γ (°)
120.4000(10)
90
90
90
101.317(1)
90
V [Å]
5243.9(6)
8
0.825
2001.26(3)
4
1.055
6285.58(12)
2
0.694
1.522
0.71073
2.287–23.407
R1 = 0.0418
wR2 = 0.0812
0.0904
Z
μ (mm−1
)
D(calc) [g cm−3
λ (Å) (MoKα)
]
1.682
1.755
0.71073
1.9–28.0
R1 = 0.0376
wR2 = 0.0864
0.0403
0.71073
2.0–33.1
R1 = 0.0222
wR2 = 0.0564
0.0238
θ [Min–max °]
Final R indices
a
R1
b
wR2
0.0864
0.0573
0.1007
a R1 = ∑||Fo| − |Fc||/∑|Fo|. b wR2 = {∑w[(Fo2 − Fc2)2]/∑w[(Fo2)2]}1/2.
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NP and the flow times were noted from the digital timer were then loaded on a 3% polyacrylamide (stacking) gel. Gel
attached to the viscometer (1/R = [Ru]/[DNA] = 0.5). electrophoresis was done initially at 60 V until the dye passed
Circular dichroic spectra were obtained using a 1 or 0.2 cm into the separating gel (12% polyacrylamide) from the stacking
path length quartz cell. Each CD spectrum was collected after (3%) gel, followed by setting the voltage to 110 V for 1.5 h.
averaging over at least four accumulations using a scan speed Staining was done with a Coomassie Brilliant Blue R-250
of 100 nm min−1 and a 1 s response time. Machine plus solution (acetic acid–methanol–water = 1 : 2 : 7 v/v) and destain-
cuvette baselines were subtracted and the resulting spectrum ing was done with a water–methanol–acetic acid mixture (5 : 4 : 1
zeroed outside the absorption bands.
v/v) for 4 h. The gels, after destaining, were scanned with a HP
The cleavage of supercoiled pUC19 plasmid DNA (form I, Precision Scan LTX scanner and the images were further pro-
40 μM) in 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.1 in the cessed using the Adobe Photoshop 7.0 software package.
absence and presence of activating agents like ascorbic acid
Cell culture
(10 μM) or H2O2 (20–200 μM) was monitored using agarose gel
electrophoresis. The samples were incubated for 1 h at 37 °C.
A loading buffer containing 25% bromophenol blue, 0.25%
xylene cyanol and 30% glycerol (3 μL) was added and electro-
phoresis was performed at 60 V for 5 h in Tris-acetate-EDTA
(TAE) buffer (40 mM Tris-base, 20 mM, acetic acid, 1 mM
EDTA) using 1% agarose gel containing 1.0 μg mL−1 ethidium
bromide. The gels were viewed in an Alpha Innotech Corpor-
ation Gel doc system and photographed using a CCD camera.
Densitometric calculations were done using the AlphaEaseFC
StandAlone software. The intensities of supercoiled DNA were
corrected by a factor of 1.47 as a result of its lower staining
capacity by ethidium bromide.37 The cleavage efficiency was
measured by determining the ability of the complex to convert
the supercoiled DNA (SC) to nicked circular form (NC) and
linear form (LC). Inhibition reactions were carried out by prior
incubation of the SC pUC19 DNA (40 μM) with DMSO (6 µM),
sodium azide (100 µM), distamycin (100 µM) SOD (4 units)
and catalase (6 units).
The MCF 7 human breast cancer cell lines were obtained from
the National Center for Cell Science (NCCS), Pune, India. The
cells were cultured in RPMI 1640 medium (Biochrom AG, Berlin,
Germany) supplemented with 10% fetal bovine serum (Sigma,
USA), cisplatin (Getwell Pharmaceuticals, India), mitomycin C
(Sigma, USA) and 100 U mL−1 penicillin and 100 μg mL−1 strep-
tomycin as antibiotics (Himedia, Mumbai, India) in 96 well
culture plates at 37 °C in a humidified atmosphere of 5% CO2 in
a CO2 incubator (Heraeus, Hanau, Germany). All experiments
were performed using cells from passage 15 or less.
MTT assay
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay was carried out as described previously.39 The
complexes in the concentration range of 0.05–50 μg mL−1, dis-
solved in DMSO (Sigma-Aldrich, St. Louis, MO, USA), were
added to the wells 24 h after seeding 5 × 103 cells per well in
200 μL of fresh culture medium. DMSO was used as the vehicle
control. After 24 and 48 h, 20 μL of an MTT solution
[5 mg mL−1 in phosphate-buffered saline (PBS)] was added to
BSA binding and cleavage experiments
The protein binding study was performed by tryptophan fluo- each well and the plates were wrapped with aluminum foil and
rescence quenching experiments using bovine serum albumin incubated for 4 h at 37 °C. The purple formazan product
(BSA, 5 µM) as the substrate in phosphate buffer (pH 6.8). formed was dissolved by addition of 100 μL of 100% DMSO to
Quenching of the emission intensity of tryptophan residues of each well. The absorbance was monitored at 570 nm (measure-
BSA at 344 nm (excitation wavelength at 295 nm) was moni- ment) and 630 nm (reference) using a 96 well plate reader (Bio-
tored using complexes 1–4 as quenchers with increasing Rad, Hercules, CA, USA). Stock solutions of the metal com-
complex concentration. The F0/F versus [complex] plot was con- plexes were prepared in DMSO and in all the experiments the
structed using the corrected fluorescence data taking into percentage of DMSO was maintained in the range of 0.1–1%.
account the effect of dilution. The excitation and emission slit DMSO by itself was found to be non-toxic to the cells till 1%
widths were 3 and 1.5 nm, respectively. Fluorescence measure- concentration. Data were collected for four replicates each and
ments were performed using a 1 cm quartz cell on a JASCO F were used to calculate the mean. The percentage inhibition
6500 spectrofluorimeter.
was calculated from these data using the formula:
Mean OD of untreated cells ðcontrolÞ ꢀ Mean OD of treated cells
ꢁ 100
Mean OD of untreated cells ðcontrolÞ
Protein cleavage experiments were carried out by incubating
The IC50 values were calculated using Table Curve 2D,
BSA (15 µM) with 1–4 in Tris-HCl buffer for 4 h at 37 °C accord- version 5.01.
ing to the literature.38 The samples were dissolved in the
Hoechst 33258 staining
loading buffer (24 µL) containing SDS (7% w/v), glycerol (4%
v/v), Tris-HCl buffer (50 mM, pH 6.8), mercaptoethanol (2%
v/v) and bromophenol blue (0.01% w/v). The protein solutions
were then denatured on heating to boil for 3 min. The samples
The cell pathology was detected by staining the nuclear chro-
matin of trypsinized cells (4.0 × 104/mL) with 1 μL of Hoechst
33258 (1 mg mL−1) for 10 min at 37 °C. Staining of suspension
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cells with Hoechst 33258 was used to detect apoptosis. A drop DNA content of individual nuclei and to evaluate the degree of
of cell suspension was placed on a glass slide, and a coverslip DNA damage representing a fraction of the total DNA in the tail.
was overlaid to reduce light diffraction. At random, 300 cells
were observed in a fluorescent microscope (Carl Zeiss, Jena,
Germany) fitted with a 377–355 nm filter and observed at
×400 magnification.
Results and discussion
Synthesis and characterization of ruthenium(II) complexes
A new series of half-sandwich organometallic complexes of the
AO/EB staining
type [Ru(η6-arene)(L)Cl](PF6) (1–4), where arene = benzene
For both suspension and adherent cells, 96-well plates were
centrifuged at 1000 rpm (129 g) for 5 min using a Beckman
model TJ-6 centrifuge with inserts for 96-well plates. An EB/AO
dye mix (8 μL) was added to each well, and cells were observed
in a fluorescent microscope (Carl Zeiss, Jena, Germany) fitted
with a 377–355 nm filter, at ×400 magnification, and the per-
centage of cells reflecting pathological changes were calcu-
lated. Tests were done in triplicate, counting a minimum of
100 cells in total for each.
(1, 2) or p-cymene (3, 4), L = N-methylhomopiperazine (L1) or
1-(anthracen-10-ylmethyl)-4-methylhomopiperazine (L2), has
been prepared by the reaction of the appropriate dimeric
complex [Ru(η6-arene)Cl2]2 with a slight excess of equimolar
amount of either L1 or L2 in methanol at room temperature.
The products were isolated as hexafluorophosphate salts in
excellent yields. The formulae of the complexes [Ru(η6-arene)-
(L)Cl](PF6), as determined by elemental analysis and ESI-MS,
are supported by the X-ray crystal structures of 2, 3 and 4
(cf. below). All the complexes are soluble in Tris-HCl–NaCl
(5 : 50 mM) buffer at pH 7.1 and in acetonitrile–water mixed
solvent (1 : 50 v/v). They exhibit low energy ligand field (LF)
bands, which confirm the low-spin d6 configuration of Ru(II)
complexes. The three intense absorption bands (354, 372, and
393 nm) observed for 2 and 4 are characteristic of intra-ligand
(π → π*) transitions located on the anthracene moiety.42 Com-
plexes 1 and 3 show an absorbance tail (360–430 nm) due to
mixed 1MC (metal-centered) and 1MLCT metal-to-ligand
charge transfer transitions and a low energy band (312 nm)
due to a weak MLCT transition43 (Table S1†). The ESI-MS data
reveal that the complexes retain their identity even in solution
(Fig. S1†), which is supported by values of molar conductivity
in acetonitrile (ΛM/Ω−1 cm2 mol−1: 81–87), falling in the
range44 for 1 : 1 electrolytes. The 1H NMR spectra of 1–4 in
DMSO-d6 show downfield shifts in the positions of the arene
protons due to the incorporation of the L1/L2 ligand, which
modifies the electron density around metal centre as
compared to the corresponding precursor complexes.45 The
N-methyl groups are observed as singlets at 3.14–3.22 ppm and
the aromatic protons of the anthracene moiety show up
between 7.50 and 8.93 ppm. The cyclic (CV) and differential
pulse voltammetric (DPV) responses obtained in acetonitrile
solution reveal that the Ru(II)–Ru(III) redox couples of 1–4 are
far from reversible (Table S1,† E1/2, 0.326 to 0.344 V). The
Ru(II)–Ru(III) redox potential of 4 is more negative than those
for 1, 2 and 3. This is expected of the electron repelling alkyl
groups present in arene and L2 ligands, which increase the
electron density on ruthenium metal and destabilize the lower
oxidation state.
Comet assay
DNA damage was quantified by means of the comet assay as
described in the literature.40,41 Assays were performed under
red light at 4 °C. Cells used for the comet assay were sampled
from a monolayer during the growing phase, 24 h after seeding.
Cells were treated with Ru(II) complexes at IC50 dose, and cells
were harvested by a trypsinization process at 12 and 24 h.
A total of 200 μL of 1% normal agarose in PBS at 65 °C was
dropped gently onto a fully frosted microslide, covered immedi-
ately with a coverslip, and then placed over a frozen ice pack for
about 5 min. The coverslip was removed after the gel had set.
The cell suspension from one fraction was mixed with 1% low-
melting agarose at 37 °C in a 1 : 3 ratio. A total of 100 μL of this
mixture was applied quickly on top of the gel, coated over the
microslide, and allowed to set as before. A third coating of
100 μL of 1% low-melting agarose was placed on the gel con-
taining the cell suspension and allowed to set. Similarly, slides
were prepared (in duplicate) for each cell fraction. After solidifi-
cation of the agarose, the coverslips were removed, and the
slides were immersed in an ice-cold lysis solution (2.5 mM
NaCl, 100 mM Na2EDTA, 10 mM Tris, NaOH, pH 10, 0.1%
Triton X-100) and placed in a refrigerator at 4 °C for 16 h. All of
the above operations were performed under low lighting con-
ditions in order to avoid additional DNA damage. The slides,
after removal from the lysis solution, were placed horizontally
in an electrophoresis tank. The reservoirs were filled with an
electrophoresis buffer (300 mM NaOH and 1 mM Na2EDTA,
pH > 13) until the slides were just immersed in it. The slides
were allowed to stand in the buffer for about 20 min (to allow
DNA unwinding), after which electrophoresis was carried out at
0.8 V cm−1 for 15 min. After electrophoresis, the slides were
removed, washed thrice in a neutralization buffer (0.4 M Tris,
pH 7.5), and gently dabbed to dry. Nuclear DNA was stained
Description of the crystal structures of [Ru(η6-benzene)(L2)Cl]-
(PF6) 2, [Ru(η6-p-cymene)Ru(L1)Cl](PF6) 3 and
[Ru(η6-p-cymene)(L2)Cl](PF6) 4
with 20 μL of EthBr (50 μg mL−1). Photographs were taken using The ORTEP representations of the structures of the complex
an epifluorescence microscope (Carl Zeiss). A total of 200 cells monocations of 2, 3 and 4 including the atom numbering
from each treatment were digitalized and analyzed by image scheme are shown in Fig. 1A, 1B and 1C respectively. The crys-
analysis (CASP Software). The images were used to estimate the tallographic data and selected bond lengths and bond angles
1208 | Dalton Trans., 2014, 43, 1203–1219
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ligands and a chloride ion occupying the remaining coordi-
nation sites. The complex cations adopt the familiar “three-
leg piano-stool” structure46,47 with the η6-arene ring forming
the seat and the two N1 and N2 atoms of L1/L2 and Cl− con-
stituting three legs of the stool, as evident from the nearly 90°
bond angles N(1)–Ru(1)–Cl(1) (86.42–88.79°) and N(2)–Ru(1)–
Cl(1) (87.22–89.22°). The six Ru–C bonds have almost similar
bond distances (2: 2.172(3)–2.219(3) Å; 3: 2.186(3)–2.240(3) Å;
4: (2.175(5)–2.263(3) Å) with an average bond length of 2.204 Å
and the ruthenium–arene centroid ring distance (Ru–Ct,
1.689(7) Å) is almost the same for all the complexes and is
similar to those in the related Ru(II) arene complex [Ru(η6-p-
cymene)(en)Cl](PF6) containing chelated en (1.669(14) Å).46
The Ru–Namine bonds formed by the bidentate ligands L1 and
L2 and the Ru–Cl1 bond in 2 and 4 are comparable to those
for other Ru(II) arene complexes.47 The Ru–Namine bonds
formed by L2 in 4 (Ru–N1, 2.166(2); Ru–N2, 2.178(19) Å) are
longer than those formed by L2 in 2 (Ru–N1, 2.157(3); Ru–N2,
2.171(3) Å) and the Ru–Cl1 bond distance in 2 (2.413(8) Å) is
the same as that in 4. The N1 atom of L2 is coordinated to the
metal centre slightly more strongly than the N2 atom in 2
(Ru–N1, 2.157(3); Ru–N2, 2.171(3) Å) and 4 (Ru–N1, 2.166(2);
Ru–N2, 2.178(19) Å). The bite angle of L2 in 2 (N(1)–Ru(1)–
N(2): 73.61(10)°) is lower than the bond angles involving chlor-
ide ions (N1–Ru–Cl1, 87.59(7)°, N2–Ru–Cl1 (88.64(7)°), as
expected.46 In 3 the N1 atom of L1 (Ru–N1, 2.193(2) Å) is co-
ordinated to Ru(II) weaker than the N2 atom (Ru–N2, 2.164(2)
Å) due to improper orientation of the lone-pair orbital of the
sterically hindered N1 atom towards Ru(II) orbitals. Also, the
Ru–N bonds (Ru–N1, 2.193(2), Ru–N2, 2.164(2) Å) formed by
1,4-diazepane in 3 are longer than those (Ru–N1, 2.130, Ru–
N2, 2.136 Å) formed by ethylenediamine in [Ru-(η6-p-cymene)-
(en)Cl][PF6]46 on account of improper orientation of the lone-
pair orbital of the N1 atom of L1 with unfavourable steric inter-
action between the alkane chains of the macrocyclic ligand
backbone towards Ru(II) orbitals. This is consistent with the
bite angle (71.49(9)°) of 1,4-diazepane in 3, which is much less
than that (78.9(10)°) of en in [Ru(η6-p-cymene)(en)Cl][PF6].
Upon the incorporation of the anthracenyl-methyl group in the
N2 position of the diazepane ligand in 3 to obtain 4, the Ru–
N2 bond distance (Ru–N2: 3, 2.164(2); 4, 2.178(19) Å) in 4
becomes longer than that in 3 suggesting that the sterically
demanding anthracenyl-methyl group renders the coordi-
nation of N2 to the metal weaker due to improper orientation
of nitrogen orbitals. However, N1 binds to ruthenium a little
stronger in 4 than in 3 (Ru–N1: 2.193(2) 3; 2.166(2) Å 4). In 3
the hydrogen atom on the secondary amine nitrogen (N1) in
L1 is involved in hydrogen bonding with a fluorine atom of the
neighbouring PF6 molecule (N–H⋯F: 2.294 Å).
Fig. 1 ORTEP representation of the crystal structures of [Ru(η6-benzene)-
(L2)Cl]+ (1A), [Ru(η6-p-cymene)(L1)Cl]+ (1B) and [Ru(η6-p-cymene)(L2)-
Cl]+ (1C) showing the atom numbering scheme and displacement
ellipsoid (50% probability level). (Hydrogen atoms and PF6 anions are
omitted for clarity.)
are listed in Tables 1 and 2 respectively. The asymmetric units
of 2, 3 and 4 contain a cationic complex molecule and a
counter ion. In the complex cations Ru(II) is coordinated to all
the six carbon atoms (C1–C6) of the arene ring, both the nitro-
gen atoms (N1 and N2) of bidentate L1 and L2 ligands and
one chloride ion (Cl1). The coordination geometry around
Ru(II) in the complexes is best described as pseudo-octahedral
with the arene ring occupying the three coordination sites in
a η6-fashion and the two nitrogen atoms of the bidentate
Structures of complexes: density functional theory calculations
Geometry optimization for 1–4 has been carried out at the DFT
level. The Ru atom was described using the LANL2DZ basis set
while all the non-metal atoms were described using the 6-31G*
basis set (Fig. S2†). The initial geometries were taken from the
single-crystal X-ray data of 2–4 and subjected to optimization.
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Fig. 2 Frontier molecular orbital diagram for the HOMO and LUMO of 1–4.
The geometrical parameters viz. bond lengths, bond angles DNA binding studies: absorption spectral studies
and bond energies were calculated (Tables S2, S3†) using the
Gaussian 03 package. Contour plots of molecular orbitals of
the complexes were generated using Gauss view 3.0 and the
frontier molecular orbitals in 1–4 were calculated (Fig. 2). The
The intense absorption spectral band around 372 nm
observed for 2 and 4 and the less intense band around
312 nm for 1 and 3 were used to characterize the interaction
of the complexes with CT DNA in 5 mM Tris-HCl/50 mM
DFT calculations reproduce the familiar “three-leg piano-stool”
NaCl buffer at pH 7.1. On adding a solution of CT DNA to 2
structures of 2–4, confirming the optimized structure of 1.
and 4, a strong decrease in absorption intensity (hypochro-
It is seen that the highest occupied molecular orbital
mism, Δε, ∼56%, Fig. 3, Table 3) is observed with red shifts
(2, 10; 4, 11 nm) in the band position, suggesting strong
intercalation of the anthracenyl ligand moiety appended to
the diazepane ligand in between DNA base pairs. In contrast,
a lower hypochromism (Δε, ∼37%) of the 312 nm band with
no shift in band position is observed for 1 and 3 suggesting
an electrostatic mode of DNA binding of the complexes. The
observed order of hypochromism, viz., 4 > 2 > 3 > 1, reflects a
(HOMO) is localized largely on the Ru atom and Cl− ion in 1
and 3 while the same is localized more on the anthracenyl
group in 2 and 4. However, the lowest unoccupied molecular
orbital (LUMO) is localized largely on the arene and N-methyl
group of the diazacycloalkane ligand. The calculated HOMO
energies of the complexes vary as 1 (−9.3371) < 2 (−7.7236) > 3
(−9.0277) < 4 (−7.6100 eV) (Table S3†) and those of LUMO
exhibit a similar trend: 1 (−5.2706) < 2 (−4.9569) > 3 (−5.0031) <
decrease in DNA binding affinity along this series, as the
4 (−4.8254 eV). Upon introducing the anthracenylmethyl group
in 1 to obtain 2, both HOMO and LUMO energies increase,
and interestingly the increase in HOMO energy is more pro-
nounced than that in LUMO energy. Similar trends are
observed for 3 and 4. It is noteworthy that upon incorporating
methyl and isopropyl groups into the benzene ring in 1 and 2
to obtain 3 and 4 respectively, the changes in both HOMO and
LUMO energies are not significant revealing that the coordi-
nation of the arene ligand does not affect the HOMO and
LUMO energy levels. This clearly supports that the HOMO
orbitals in 2 and 4 are localized on the anthracenyl moiety.
The HOMO–LUMO energy gaps in 1 (4.0665 eV) and 3 (4.0246 eV)
are almost the same, which is consistent with almost the same
Ru(II)–Ru(III) redox potential of the complexes (Tables S1, S3
and Fig. S3†). The higher HOMO energies of the anthacenyl-
methyl group in complexes 2 and 4 result in intercalative inter-
action between the DNA base pairs leading to DNA binding
Fig. 3 Absorption spectra of [Ru(η6-benzene)(L2)Cl]+ (12 × 10−5 M) in
5 mM Tris-HCl buffer at pH 7.1, in the absence (R = 0) and presence (R =
affinities higher than 1 and 3, which contain only a simple di-
azacycloalkane (cf. below).
1–20) of increasing amounts of CT DNA. Inset: the plot of [DNA]/(εa − εf)
vs. [DNA] for [Ru(η6-benzene)(L2)Cl]+.
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Table 3 Ligand-based absorption spectral propertiesa and fluorescence spectral propertiesb of ruthenium(II) complexes bound to CT DNA
Ligand-based
Complex
λmax (nm)
R
Change in absorbance
Δε (%)
Red-shift (nm)
Kb (×104 M−1
)
Kapp b (×104 M−1
)
[Ru(η6-benzene)(L1)Cl]+ 1
[Ru(η6-benzene)(L2)Cl]+ 2
[Ru(η6-p-cymene)(L1)Cl]+ 3
[Ru(η6-p-cymene)(L2)Cl]+ 4
312
372
312
372
20
20
20
20
Hypochromism
Hypochromism
Hypochromism
Hypochromism
36.2
55.8
37.6
56.1
—
10
—
11
0.6 0.02
6.2 1.0
0.8 0.05
7.8 0.4
2.8
24.2
3.1
26.8
a Measurements were made at R = 20, where R = [DNA]/[complex]; the concentration of solutions of Ru(II) complexes = 12 × 10−5 M (2 and 4) and
1 × 10−3 M (1 and 3). b Apparent DNA binding constant from the ethidium bromide displacement assay using increasing concentrations (0–60 µM)
of 1–4.
percentage of hypochromism is commonly associated with on the coordinated p-cymene enhances the DNA binding
the strength of DNA binding interaction.22,23,48,49 In order affinity slightly higher than those for complexes containing a
to quantitatively compare the DNA binding affinities the simple coordinated benzene ligand.
intrinsic binding constant Kb was calculated using the
equation:50
Competitive DNA binding studies
The competitive DNA binding of 1–4 has been studied by
½DNAꢂ=ðεa ꢀ εf Þ ¼ ½DNAꢂ=ðεb ꢀ εf Þ þ 1=Kbðεb ꢀ εf Þ
monitoring the emission intensity of DNA-bound EthBr (λex,
450; λem, 595 nm) upon adding the complexes. Note that the
anthracene moiety does not fluoresce in this region, and the
where [DNA] is the concentration of DNA in base-pairs, εa is
the apparent extinction coefficient calculated as Aobs
/
excitation wavelength is chosen to selectively excite EthBr only.
Upon adding the complexes (0–60 µM) to CT DNA (125 µM)
pretreated with ethidium bromide (EthBr, 12.5 µM) ([DNA]/
[EthBr] = 10) in 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.1,
the emission intensity of DNA-bound EthBr decreases. From a
plot of the observed intensities against complex concentration
the value of the apparent DNA binding constant (Kapp) was cal-
culated53 using the equation:
[complex], εf corresponds to the extinction coefficient of the
complex in its free form and εb refers to the extinction coeffi-
cient of the complex in the bound form. Each set of data,
when fitted into the above equation, gives a straight line with a
slope of 1/(εb − εf) and a y-intercept of 1/Kb(εb − εf) and Kb is
determined from the ratio of the slope to the intercept. The
values of Kb vary in the order 4 > 2 > 3 > 1, which is the same
as that observed for the hypochromism. The Kb values of 2 and
4 (2, 6.2; 4, 7.8 × 104 M−1) are respectively 10-fold higher than
1 and 3 (1, 6.0; 3, 8.0 × 103 M−1), strongly supporting the inter-
calative mode of interaction of the appended anthracenyl ring
(cf. above). It has been found that the simple anthracene itself
is involved in intercalative interaction with the DNA base
KEthBr½EthBrꢂ ¼ Kapp½Complexꢂ
where KEthBr is 4.94 × 105 M−1 54
[Complex] is the concentration
,
of the complex used to obtain a 50% reduction in fluorescence
intensity of EthBr. Addition of a second DNA binding molecule
either replaces the DNA-bound EthBr (if it binds to DNA more
strongly than EthBr) and/or destabilizes the excited state of
DNA-bound EthBr by means of molecular collision,55 which
would quench the DNA-bound EthBr emission. The observed
Kapp values of the complexes decrease in the order 4 (26.8) > 2
(24.2) > 3 (3.1) > 1 (2.8 × 104 M−1), which is consistent with the
order of the above Kb values obtained by UV-Vis absorption
spectral titration (Fig. 4, Table 3) and a plot of Kb vs. Kapp is
linear (Fig. S4†). This result indicates that both 2 and 4 at
higher concentrations would compete with DNA-bound EthBr
more efficiently than 1 and 3 through intercalative interaction
of the anthracenyl ring as well as hydrophobic interaction of
methyl and isopropyl moieties of arene ring with the hydro-
phobic DNA surface.
stack,42,51 and the order of Kb values (3.8–4.8 × 105 M−1
)
observed for other complexes with appended anthracenyl
ligands is of higher order than those for 2 and 4.51 The
observed Kb values of 2 and 4 are lower than those of Ru-poly-
pyridyl complexes like [Ru(pdto)(dppz)]2+ (Kb, 3.0 × 106 M−1),22
[Ru(phen)2(dppz)]2+ (5.1 × 106 M−1),52 and [Ru(bpy)2(dppz)]2+
(Kb, 1.3 × 106 M−1),52b which is expected of the deeper inter-
calation of dppz with a larger aromatic surface area into DNA
base pairs. But the values are higher than that for rac-
[Ru(phen)3]2+ (Kb, 6.2 × 103 M−1 24
due to partial insertion of the
)
phen ring into the DNA base pairs. Certain Ru(II)-arene com-
plexes like [Ru(η6-arene)(CPI)Cl2],9 where CPI is 1-(4-cyano-
phenyl)imidazole, also exhibit higher order of Kb values. Also,
the DNA binding affinity of 4 is higher than that of 2, due to
hydrophobic interaction of the methyl and isopropyl groups of
coordinated p-cymene with DNA. This is supported by the
Viscosity measurements
similar trend in Kb values of 1 and 3, both involved in electro- Measurement of the viscosity of CT DNA after treatment with
static binding to phosphate groups on the DNA surface. Thus varying concentrations of complexes provides reliable evidence
the complexes containing the anthracenyl–methyl ligand for the DNA binding mode. Thus intercalation of complexes
moiety show 10-fold higher DNA binding affinities and the with DNA and DNA groove binding increases the length of
hydrophobic interaction of the methyl and isopropyl groups DNA22,23 and hence enhances the DNA viscosity whereas the
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consistent with their weak electrostatic interaction with DNA.
On the other hand, upon addition of 2 and 4, an increase in
intensities of both the positive and negative bands of DNA is
observed, which is characteristic23,57c of intercalative mode of
DNA interaction. This supports the above suggestion that the
anthracene ring of the complexes is intercalated in between
DNA base pairs (Fig. S6, Table S4†).
DNA cleavage studies
The DNA cleavage activity of 1–4 was studied (Fig. S7†) by incu-
bating the complexes (150 μM) with pUC19 supercoiled (SC)
DNA (40 μM) in the absence of an external agent in 5 mM Tris-
HCl/50 mM NaCl buffer at pH 7.1 for 1 h at 37 °C. All the com-
plexes convert SC DNA (form I) into nicked circular (NC) DNA
(form II) to almost the same extent (∼54%) (Table S5†).
Control experiments performed using DNA or RuCl3·3H2O
alone in separate lanes did not reveal any apparent cleavage of
DNA. Previous studies have shown that Ru(II)-arene complexes
of the type [Ru(arene)(NN)Cl]+ undergo rapid aquation to form
[Ru(arene)(NN)(H2O)]2+ in an aqueous environment,58 which
can bind to nitrogen of a DNA heterocyclic base by replacing
the aqua ligand or generate RuIVvO active species for DNA
cleavage through the C8-guanine/C1-sugar oxidation.59 In the
presence of H2O2 (30 μM), the complex 4 shows oxidative DNA
cleavage ability higher than 1–3 (Fig. 5, Table 4). When the
concentration of 4 is increased from 20 to 100 μM, the amount
of form I decreases and that of form II increases, and the
maximum degradation occurs at 80 μM concentration (Fig. S8,
Table S6†). Also, interestingly, when the cleavage ability of 4
(100 μM) was studied as a function of time (10–60 minutes) in
the presence of H2O2 under similar conditions (Fig. 6), 50%
DNA cleavage is reached within 30 minutes, and the amount
of form II reaches the maximum and then remains constant. It
is well known that in the presence of H2O2 as an oxidizing
agent, Ru(II) species is oxidised to Ru(III) species leading to the
formation of more accessible and reactive oxygen species
(OH•) by the Fenton type mechanism,60 causing DNA cleavage.
When the standard radical scavengers are added to the reac-
tion mixture a dramatic (DMSO, a hydroxide radical quencher)
to significant (NaN3, a singlet oxygen quencher) inhibition in
DNA cleavage is observed (Fig. S9†) supporting the involve-
ment of •OH radicals rather than 1O2 or O2− or H2O2 species in
the oxidative DNA cleavage reaction.
Fig. 4 Effect of addition of 2 (A) and 4 (B) on the emission intensity of
the CT DNA-bound ethidium bromide (12.5 μM) at different concen-
trations (0–60 µM) in a Tris buffer (pH 7.1).
complexes that bind to the DNA surface do not alter the length
of the DNA helix due to kinking or bending.51 The values of
the relative specific viscosity (η/η0), where η and η0 are the
specific viscosities of DNA in the presence and absence of the
complexes, are plotted against 1/R (=[Ru complex]/[DNA] =
0.0–0.5) (Fig. S5†). The order of the ability of the complexes to
increase the viscosity of DNA depends on the intercalating
ligand and hydrophobicity of the arene moiety and varies as:
4 ≥ 2 > 3 ≥ 1. The insertion of the anthracenyl ring of 2 and 4
in between the DNA base pairs leads to an increase in separ-
ation of the base pairs at intercalation sites and, hence, an
increase in overall DNA contour length resulting in an increase
in viscosity. In contrast, complexes 1 and 3 with no inter-
calating group show a negligible increase in viscosity upon
their binding to DNA and the Ru-arene–en analogues of 1 and
3 have been reported to show only DNA surface binding.56
Protein binding studies
Circular dichroic spectral studies
Tryptophan quenching measurements. The majority of
The CD spectrum of CT DNA shows a positive band at 275 nm intrinsic fluorescence of BSA when excited at 295 nm is
(UV: λmax, 258 nm) arising due to base stacking and a negative provided by tryptophan residues alone and other amino
band at 245 nm due to helicity of DNA, which are very sensitive acid residues like tyrosine and phenylalanine contribute to
to the mode of DNA interaction of small molecules.57 Upon fluorescence only weakly.61,62 In order to determine the
addition of 1–4 to DNA at 1/R = [Ru complex]/[DNA] = 1, protein binding affinity of complexes, tryptophan emission-
changes in both the positive and negative signals of DNA are quenching experiments were carried out by adding the com-
observed. Complexes 1 and 3 show no change in intensities of plexes in increasing concentration (10–50 μM) to BSA (5 μM) at
the negative band but a decrease in intensity of the positive 298 K and following the decrease in fluorescence intensities.
CD signal is observed with a small blue-shift (5 nm), which is Generally, fluorescence quenching can be illustrated by the
1212 | Dalton Trans., 2014, 43, 1203–1219
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Fig. 5 Oxidative cleavage of supercoiled pUC19 DNA (40 μM) in the presence of H2O2 (30 μM) by ruthenium(II) complexes 1–4 (100 μM) in a buffer
containing 5 mM Tris-HCl/50 mM NaCl at 37 °C; lane 1, DNA; lane 2, DNA + H2O2 (30 μM); lane 3, DNA + H2O2 + 1; lane 4, DNA + H2O2 + 2; lane 5,
DNA + H2O2 + 3; lane 6, DNA + H2O2 + 4. SC and NC are supercoiled and nicked circular forms of DNA, respectively.
of the linear plot of F0/F vs. [Q] follows the order 4 (11.6 × 104) >
Table 4 Oxidative cleavage data of SC pUC19 DNA (40 μM) by com-
plexes 1–4 (100 μM) in the presence of H2O2 (30 μM) for an incubation
time of 1 h
2 (9.9 × 104) > 3 (6.2 × 104) > 1 (4.8 × 104 L mol−1) (Fig. 7,
Table S7†), which is in agreement with the trend in DNA
binding affinities. The anthracenyl-appended complexes (2, 4)
are capable of quenching the tryptophan fluorescence 1.8
times more strongly than those containing simple diazepane
ligands (1, 3) by binding to the hydrophobic region of protein.
Similarly, p-cymene complexes (3, 4) show 1.2 times higher
fluorescence quenching than benzene complexes (1, 2) due to
enhanced hydrophobic interaction of methyl and isopropyl
groups of p-cymene with tryptophan site. These observations
are supported by the blue shifts for 2 (4 nm) and 4 (7 nm) and
no such shift for 1 and 3, which is due to the increase in hydro-
phobicity of the microenvironment around tryptophan site in
the protein.64 Thus the ligand hydrophobicity contributes to the
enhanced BSA protein binding affinities of the complexes. The
value of bimolecular quenching constant (kq) calculated from
Form (%)
SC
Lane number
Reaction conditions
NC
2.4
9.1
72.2
88.8
1
2
3
4
DNA
97.6
90.9
27.8
11.2
DNA + H2O2
DNA + H2O2 + 1
DNA + H2O2 + 2
KSV and τo (6.2 ns)65 falls in the range 0.7–1.8 × 1013 L mol−1 s−1
,
which is higher than the maximum possible value for dynamic
quenching (2.0 × 1010 L mol−1 s−1),66 suggesting the involvement
of static quenching mechanism by the present Ru(II) arene com-
plexes. Also, the linear fit obtained for Stern–Volmer quenching
analysis represents a single quenching mechanism for all the
complexes. In order to investigate further whether the quenching
mechanism is static or dynamic the quenching of tryptophan
emission of BSA was studied at two different temperatures, viz.,
298 and 308 K (Fig. 8, Table S7†). It is found that the KSV and kq
values for the present complexes decrease with increase in temp-
erature. In dynamic quenching, the number of collisions
between the quencher and the excited state of fluorophore
increases with increase in temperature leading to an increase in
fluorescence quenching. In contrast, if static quenching
occurs, the quencher molecule forms a non-fluorescent adduct
with BSA in the ground state and with an increase in tempera-
Fig. 6 Time course of pUC19 supercoiled plasmid DNA (40 μM) clea-
vage by complex 4 (100 μM) in the presence of H2O2 (30 μM) containing
5 mM Tris-HCl/50 mM NaCl at pH = 7.1 and 37 °C with incubation times
of 0, 10, 20, 30, 40, 50 and 60 min for lanes 1–7. SC and NC are super-
coiled, nicked circular forms of DNA, respectively.
well-known Stern–Volmer equation:63
F0=F ¼ 1 þ KSV½Qꢂ ¼ 1 þ kqτo½Qꢂ
where F0 and F are the fluorescence intensities of BSA in the ture the concentration of the BSA–complex adduct decreases
absence and presence of the complexes respectively, KSV is the leading to a decrease in fluorescence quenching.67 It is obvious
Stern–Volmer quenching constant, kq is the quenching rate that fluorescence quenching for the present complexes occurs
constant of the biomolecule, τo is the average fluorescence life- through a static rather than a dynamic mechanism. Also, inter-
time of BSA without the quencher and [Q] is the concentration estingly, the complexes containing anthracenyl ligand moiety
of the quenching complex. The value of Ksv obtained as a slope show higher decrease of KSV values than those of simple
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Fig. 7 (a) Fluorescence quenching spectra of BSA at different concentrations of complex 4 at room temperature; excitation wavelength, 295 nm. (b)
Plots of relative integrated emission intensity (Fo/F) vs. [complex] for 1–4.
⊗
●
Fig. 8 The Stern–Volmer curves of BSA with complexes 1 and 2 at 298 K ( ) and 308 K ( ).
Table 5 Binding and relative thermodynamic parameters of the interaction of Ru(II) complexes with BSA at two different temperatures
Temp
(K)
Binding constant
b
Number of
binding sites (n)
ΔG0
ΔS0
ΔH0
Complexes
(K′ × 104)
(kJ mol−1
)
(J mol−1
)
(kJ mol−1
)
[Ru(η6-benzene)(L1)Cl]+ 1
[Ru(η6-benzene)(L2)Cl]+ 2
[Ru(η6-p-cymene)(L1)Cl]+ 3
[Ru(η6-p-cymene)(L2)Cl]+ 4
298
308
298
308
298
308
298
308
1.70
1.46
3.72
2.77
2.14
1.78
4.48
3.79
1.20
1.11
1.09
1.06
1.02
1.01
1.18
1.16
−24.13
−24.55
−26.07
−26.19
−24.70
−25.06
−26.53
−26.99
333.12
323.66
336.71
326.16
334.32
324.64
341.30
331.17
75.14
74.27
74.93
75.18
diazepane ligands when the temperature is increased from 298 By fitting the linear plot of log((F0 − F)/F) vs. log[Q] (Fig. S10,†
to 308 K.
Table 5) at two different temperatures (298, 308 K) the K′ values
b
for 1–4 were calculated, which confirm that Ru(II) complexes
having a large hydrophobic area can interact efficiently with BSA
through a static pathway (cf. above) and a plot of Kb (DNA) vs. Kb′
(BSA) is linear (Fig. S11†). Also, the value of n (1.02–1.20), which
is approximately equal to 1, indicates that the binding site in
BSA is unique and that the hydrophobic environment of trypto-
phan residue is easily accessible by the complexes.69
Determination of binding parameters and binding force
When static quenching interaction occurs, if it is assumed that
the complex binds independently to a set of equivalent
binding sites in BSA, the binding parameters can be deter-
mined according to the Scatchard equation:68
ðF0 ꢀ FÞ
In order to identify the type of interaction force including
hydrophobic force of interaction, electrostatic interactions, van
log
¼ log K′b þ n log½Qꢂ
F
where K′ is the binding constant for the binding of the complex der Waals interactions, hydrogen bonds, etc.,70 that acts
b
with BSA and n is the number of binding sites per BSA molecule. between complexes and BSA, various thermodynamic
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parameters like free energy changes (ΔG0), enthalpy changes
(ΔH0), and entropy changes (ΔS0) of the interactions were
calculated from the following equations:
lnðK2=K1Þ ¼ ½ð1=T1Þ ꢀ ð1=T2ÞꢂΔH 0=R
ΔG0 ¼ ꢀRT ln K ¼ ΔH 0 ꢀ TΔS0
K1 and K2 are the binding constants at temperatures T1 and T2,
respectively, and R is the gas constant. The various types of
forces of interaction that act between the complex and the
protein are indicated by the sign and magnitude of the ther-
modynamic parameters: (i) the positive values for both ΔH0
and ΔS0 correspond to the involvement of hydrophobic forces
in protein binding, (ii) the negative values for both ΔH0 and
ΔS0 correspond to van der Waals and hydrogen bonding inter-
actions and (iii) the negative value of ΔH0 and the positive
value of ΔS0 indicate electrostatic interaction.70 From the
results presented in Table 5, it is evident that the binding
process is spontaneous, since the values of ΔG0 are negative.
Also, the positive values obtained for both ΔH0 and ΔS0
support the presence of strong hydrophobic forces of inter-
action between the ligands of Ru-arene complexes and BSA.
When the hydrophobic ligands bind to the tryptophan
environment, ordered water molecules contained in the hydro-
phobic pocket of BSA are expelled out leading to positive
values of ΔS0. However, the role of hydrogen bonding and elec-
trostatic interaction cannot be excluded completely.
Fig. 9 SDS-PAGE diagram of the cleavage of bovine serum albumin
(BSA, 15 μM) using various concentrations of [Ru(η6-p-cymene)(L2)Cl]+
in the presence of H2O2 (100 μM) in phosphate buffer. Lane 1: BSA; lane
2: BSA + H2O2 (100 μM); lane 3: BSA + 4 (100 μM) + H2O2; lane 4: BSA +
4 (200 μM) + H2O2; lane 5: BSA + 4 (300 μM) + H2O2; lane 6: BSA + 4
(400 μM) + H2O2; lane 7: BSA + 4 (500 μM) + H2O2, lane 8: BSA mole-
cular weight marker.
non-specifically to the protein and cleave BSA into very small
fragments. When the concentration of 4, which shows more
efficient oxidative DNA cleavage than other complexes, is
enhanced, complete degradation of BSA is observed even at
300 μM concentration in the presence of H2O2 (100 μM)
(Fig. 9). The hydrophobic substituents of the coordinated
ligand in complexes recognize the more exposed hydrophobic
regions of BSA for binding and facilitate the protein cleavage
in an oxidative rather than a hydrolytic pathway.
Energy transfer between BSA and 4
As complex 4 exhibits higher protein binding affinity than 1–3,
fluorescence resonance energy transfer (FRET) from protein to
complex 4 has been verified by overlapping the UV-Vis absorp-
tion spectrum of 4 with the fluorescence emission spectrum of
BSA (Fig. S12†) according to the Förster theory of non-radio-
active energy transfer (ESI†).71,72 The distance between BSA
(donor) and the bound complex (acceptor) (r0, 1.93 nm), the
energy transfer efficiency (E, 0.56) and the critical distance
between the donor and the acceptor when the transfer
efficiency is 50% (R0, 1.68 nm) have been calculated. The
experimentally determined average distance r0 (1.93 nm)
between the donor fluorophore (BSA) and the acceptor (4) lies
within the scale of 2 to 8 nm as expected,73 and the fulfillment
Cytotoxicity studies
MTT assay. The cytotoxic activity of 1–4 against the
MCF-7 human breast cancer cell line has been investigated in
aqueous buffer solution in comparison with the widely used
drug cisplatin under identical conditions by using the MTT
assay. All the complexes are found to kill cancer cells more
efficiently at 48 h incubation than at 24 h incubation clearly
indicating that the cell killing activity is time-dependent. The
cytotoxic ability of 1, 2 and 3 are similar to, while that of 4 is
higher than, that of cisplatin for both 24 and 48 h incubation.
Also, the observed IC50 values (Table 6) follow the order 4 > 2 >
3 > 1, revealing that cytotoxicity depends upon the mode and
extent of interaction of the complexes with DNA and protein.
The p-cymene complexes 3 and 4 show cell killing activities
higher than the benzene complexes 1 and 2 respectively, which
may be traced to the hydrophobicity of p-cymene being higher
74
of the required condition 0.5R0 < r < 1.5R0 indicates that the
energy transfer from BSA to Ru(II) complexes occurs with high
probability,75,76 confirming the static quenching mechanism.
BSA cleavage studies
The protein cleavage activity of 1–4 was studied using BSA than benzene effective in penetrating the cell membrane.
(15 μM). In the absence of an activator like hydrogen peroxide, Similar observations46 have been made for [Ru(arene)(en)Cl]+
all the complexes fail to show any protein cleavage even at complexes against A2780 human ovarian cancer cells (IC50
:
500 μM concentration of complexes (Fig. S13†). When the clea- η6-p-cymene, 9.0; η6-benzene, 17.0 μM). We have earlier found
vage experiment was carried out under the same conditions that mixed ligand copper(II) complexes of hydrophobic 5,6-
(500 μM complex) in the presence of H2O2 (100 μM) at pH 7.2 dmp co-ligand show cell killing activity higher than those of
(Fig. S14†) all the complexes show significant smearing or phen irrespective of the cell lines used.77 A molecule with a
fading of the BSA band suggesting that the complexes bind higher hydrophobicity would possess a higher lipophilicity
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Table 6 In vitro cytotoxicity assay for complexes 1–4 against the
MCF-7 breast cancer cell line. IC50 values are in μM. (Data are mean%
SD% of each triplicate)a
IC50 (μM)
Complex
24 h
48 h
[Ru(η6-benzene)(L1)Cl]+ 1
[Ru(η6-benzene)(L2)Cl]+ 2
[Ru(η6-p-cymene)(L1)Cl]+ 3
[Ru(η6-p-cymene)(L2)Cl]+ 4
cisplatin
28.1 1.1
23.1 0.8
26.2 0.3
16.8 0.3
28.5 0.8
19.0 0.4
15.9 0.2
18.1 0.9
9.7 1.0
19.3 1.3
a IC50 = concentration of drug required to inhibit the growth of 50% of
the cancer cells (in μM).
and hence a higher ability to penetrate the cell membrane.
Also, 1 and 3 exhibit cell killing activity lower than the Ru-
arene–en analogue possibly due to the lack of hydrogen
bonding N–H functionality on the N-methylhomopiperazine
moiety. Interestingly, 2 and 4 show a cytotoxic activity higher
than 1 and 3 respectively, revealing that the incorporation of
the intercalating anthracenyl ligand moiety as in 2 and 4 leads
to ten times stronger DNA binding and 1.8–2.0 times stronger
protein binding (cf. above) and hence 1.2–1.7 times higher cell
Fig. 10 (A) AO/EB- and Hoechst 33258-stained MCF-7 breast cancer
killing activity at 24 h incubation. Similarly, complexes 3 and 4 cells at 24 h and 48 h incubation by the treatment of 4. (The arrow head
shows chromatin fragmentation, chromatin condensation and late
apoptosis indication of apoptotic bodies.) (B) AO/EB fluorescent study of
1–4 induced apoptosis of MCF-7 breast cancer cells. The graph shows
manual count of apoptotic cells in percentage. (Data are mean% SD%
with the p-cymene ligand show both DNA and protein binding
affinity 1.2–1.3 times higher than complexes 1 and 2, respect-
ively, with the benzene ligand, which is similar to 1.2–1.4
times higher cytotoxicity at 24 h incubation than the latter. We
have shown previously that the complex [Ru(Hdpa)2(dppz)]2+
(IC50, 2.5 μM) with the intercalating ligand dppz exhibits
higher cytotoxicity against cervical epidermoid carcinoma cell
lines than [Ru(Hdpa)2(phen)]2+ (IC50, 30 μM)23 with phen
showing weaker intercalation. Also, the lower oxidation state of
ruthenium metal and the ability of complexes to strongly inter-
act with both DNA and protein play vital roles in conferring
enhanced cytotoxicity on the complexes.10–16,78 Thus the lipo-
philicity of both the arene ligand and the appended anthra-
cene ring dictates the cytotoxicity of complexes by facilitating
their penetration into the cell membrane more than the DNA
and protein binding affinity of complexes.
Fluorescent staining studies. The design of chemotherapeu-
tic drugs in order to understand the complexities of apoptosis
evolved by cancer cells and the development of strategies to
selectively induce apoptosis in cancer cells have turned into a
unique target in cancer drug development.79 The characteristic
morphological changes induced by 1–4 have been evaluated by
adopting fluorescent microscopic analysis of Hoechst 33258-
and AO/EB-stained MCF-7 breast cancer cells. All the com-
plexes induce cell death through different pathways like apop-
tosis and necrosis upon treatment of the cells with the
corresponding IC50 concentrations at different incubation
times (24 and 48 h). Interestingly, 4 exhibits a higher percen-
tage of apoptotic cell death than other complexes (Fig. 10).
When Hoechst 33258 staining is adopted, chromatin fragmen-
tation, bi- and/or multinucleation, cytoplasmic vacuolation,
of each triplicate.)
nuclear swelling, cytoplasmic blebbing and late apoptosis indi-
cation of dot-like chromatin condensation are the morphologi-
cal changes observed.80 The cytological changes observed are
classified into four types according to the fluorescence emis-
sion and morphological features of chromatin condensation in
the AO/EB stained nuclei: (i) viable cells having uniformly
green fluorescing nuclei with highly organized structure;
(ii) early apoptotic cells (which still have intact membranes but
have started undergoing DNA fragmentation) having green
fluorescing nuclei, but peri-nuclear chromatin condensation is
visible as bright green patches or fragments; (iii) late apoptotic
cells having orange to red fluorescing nuclei with condensed
or fragmented chromatin; and (iv) necrotic cells, swollen to
large sizes, having uniformly orange to red fluorescing nuclei
with no indication of chromatin fragmentation. All these mor-
phological changes indicate that the cells are made to die in
such a way that the complexes induce both apoptosis and
necrosis and one particular mode of cell death (apoptosis) is
preferred during 48 h treatment than during 24 h treatment. It
has been reported that the anticancer activity of certain ruthe-
nium complexes depends upon their interference with specific
proteins81 and their modes of binding to duplex DNA14 even
though binding to plasma proteins might result in drastic
modifications, or even loss, of the biological properties of the
complex molecules. It is clear that both the hydrophobic
1216 | Dalton Trans., 2014, 43, 1203–1219
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currently used drug cisplatin. The facile transport of the
complex cation across the cell membrane on account of the
efficient hydrophobicity of both the arene and the L2 ligand
also contributes to the prominent anticancer activity of the
complex. The complex may also bind to the cellular proteins
involved in inducing cancer and cause cell death mainly
through the apoptotic mode. Thus, a suitable intercalating
ligand moiety such as anthracene along with the coordinated
p-cymene ligand may be incorporated into ruthenium-based
organometallic compounds as DNA as well as protein recog-
nition elements when designing efficient cytotoxic agents.
Fig. 11 Comet assay of EB-stained (a) control (untreated) and (b and c)
4 treated breast cancer cells at 12 h and 24 h incubation.
methyl and isopropyl groups of the arene ligand as well as the
appended anthracenyl moiety in 4 enhance the permeability of
the complex across the cell membrane and enhance the DNA
binding affinity of the complexes so as to interfere with the
cellular function of DNA and exhibit enhanced anticancer
activity.
Comet assay. Single cell gel electrophoresis (comet assay) in
an agarose gel matrix was used to study DNA fragmentation,
which is a hallmark of apoptosis.82 When cancer cells are
treated with an IC50 concentration of 4 showing the highest
cell killing activity, statistically significant and well-formed
comets are observed, while the control (untreated) cells fail to
show a comet like appearance (Fig. 11). Also, the extent of DNA
damage, which is estimated from the length of the comet tail,
increases in a time-dependent manner for 12 h and 24 h
incubation. This clearly indicates that 4 indeed induces DNA
fragmentation, which is further evidence for induction of
apoptosis by the complex.
Acknowledgements
Professor M. Palaniandavar is a recipient of a Ramanna
National Fellowship of the Department of Science and Tech-
nology, New Delhi. The DST Ramanna Fellowship (grant
no. SR/S1/RFIC/01/2007-2010 & SR/S1/RFIC/01/2010-2013) is
acknowledged for a Junior Research Fellowship to M. G and
R. L. The present work was supported by DST Nano Mission
[scheme no. SR/NM/NS-110/2010(G)] and CSIR projects (CSIR/
01(2462)/11/EMR-II). The X-ray diffraction facility was created
by funding from the Department of Science and Technology
(DST-FIST), New Delhi and NMR and fluorescence spectral
facilities were created by the University Grants Commission
(SAP), New Delhi in the Department of Chemistry, Bharathida-
san University, Tiruchirappalli. We thank Professor A. Ramu,
Madurai Kamaraj University, Madurai for providing the CD
facilities. We thank the Bharathidasan University Informatics
Centre for providing geometry computational facilities.
Conclusions
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