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Ruthenium(II) ethylenediamine complexes with dipyridophenazine ligands: synthesis, characterization, DNA interactions, and antiproliferative activities
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Journal of Coordination Chemistry
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Ruthenium(II) ethylenediamine
complexes with dipyridophenazine
ligands: synthesis, characterization,
DNA interactions, and antiproliferative
activities
a
a
a
Mynam Shilpa , C. Shobha Devi , Penumaka Nagababu , J.
b
c
Naveena Lavanya Latha , Ramjee Pallela , Venkateshwara Rao
c
a
Janapala , K. Aravind & S. Satyanarayana
a
a
Department of Chemistry , Osmania University , Hyderabad ,
India
b
Department of Biotechnology , Krishna University ,
Machilipatnam , India
c
Toxicology Unit, Biology Division , Indian Institute of Chemical
Technology , Hyderabad , India
Accepted author version posted online: 22 Mar 2013.Published
online: 29 Apr 2013.
To cite this article: Mynam Shilpa , C. Shobha Devi , Penumaka Nagababu , J. Naveena
Lavanya Latha , Ramjee Pallela , Venkateshwara Rao Janapala , K. Aravind & S. Satyanarayana
(2013) Ruthenium(II) ethylenediamine complexes with dipyridophenazine ligands: synthesis,
characterization, DNA interactions, and antiproliferative activities, Journal of Coordination
Chemistry, 66:10, 1661-1675, DOI: 10.1080/00958972.2013.788154
To link to this article: http://dx.doi.org/10.1080/00958972.2013.788154
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Journal of Coordination Chemistry, 2013
Vol. 66, No. 10, 1661–1675, http://dx.doi.org/10.1080/00958972.2013.788154
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Ruthenium(II) ethylenediamine complexes with
dipyridophenazine ligands: synthesis, characterization, DNA
interactions, and antiproliferative activities
MYNAM SHILPA†, C. SHOBHA DEVI†, PENUMAKA NAGABABU†, J. NAVEENA
LAVANYA LATHA‡, RAMJEE PALLELA§, VENKATESHWARA RAO JANAPALA§,
K. ARAVIND† and S. SATYANARAYANAy*
yDepartment of Chemistry, Osmania University, Hyderabad, India
zDepartment of Biotechnology, Krishna University, Machilipatnam, India
xToxicology Unit, Biology Division, Indian Institute of Chemical Technology, Hyderabad,
India
(Received 9 October 2012; in final form 30 January 2013)
Ruthenium(II) complexes, [Ru(en)2dppz]2+ (1), [Ru(en)2qdppz]2+ (2), [Ru(en)2acdppz]2+ (3), and
[Ru(en)2actatp]2+ (4), have been synthesized and characterized by IR, 1H, 13C-NMR and LC–MS.
The interactions of these complexes with calf thymus (CT) DNA have been investigated by absorption, emission, viscosity, thermal denaturation, and circular dichroism. These techniques reveal that
the complexes bind strongly to DNA. The apparent binding constants for the complexes decrease
from 1 to 4 and are in the order of 8.5 ± 0.2 105 M1 (1), 7.3 ± 0.8 105 M1 (2), 4.3 ± 0.3 105
M1 (3), and 7.5 ± 0.5 104 M1 (4). The plot of log K versus log [Na+] yield slopes of 1.47,
1.44, 1.36, and 1.24 for 1, 2, 3, and 4, respectively. These complexes promote the photocleavage of pBR322 DNA. Cytotoxicities of these complexes suggest their possible anticancer activity.
Keywords: Ruthenium(II) complexes; DNA-Binding; Photocleavage; Anticancer activity
1. Introduction
Ruthenium(II) polypyridyl complexes have received attention as DNA-binding substrates
due to rich photochemical and photophysical properties [1]. In two decades of research on
the excited-state properties of ruthenium complexes [2], a variety of substituted 2,2′-bipyridine and 1,10-phenanthroline systems as well as other α,α′-diimine systems have been
investigated as ligands for ruthenium(II) complexes. There has been considerable interest
in DNA-binding properties of transition metal complexes [3–5].
Ruthenium polypyridyl complexes are promising DNA probes due to their intense
MLCT luminescence, excited-state redox properties, and DNA-binding properties [6].
When such binding is through intercalation, complexes can be quite useful. Many studies
have focused on the interaction of ruthenium complexes containing planar ligands which
*Corresponding author. Email: ssnsirasani@gmail.com
Ó 2013 Taylor & Francis
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have good biological activities [7–10]. Most of the well-known platinum anticancer complexes have amines as ligands [11]. Inventions [12] of platinum-diamine complexes with
excellent antitumor activity and lower renal toxicity than cisplatin have been reported.
These studies suggest that at least one ligand must be a N-donor and should possess one
hydrogen. Ruthenium(II) ethylenediamine complexes have similar DNA-binding properties
to complexes containing phen or bpy as co-ligands [13], hence we concentrate on ethylenediamine co-ligands. In our earlier study, we have reported that several transition metal
complexes containing ethylenediamine as co-ligands have good DNA-binding properties
and cytotoxicities [14].
We have synthesized more [Ru(en)2L]2+ complexes (where en = ethylenediamine, L = dppz
(1), qdppz (2), acdppz (3), and actatp (4) (figure 1)). Their synthesis, characterization, and
DNA-binding properties are analyzed by absorption, emission, viscosity, thermal denaturation, circular dichroism, photosensitizing properties, and the anticancer studies were performed by MTT assay.
2. Experimental
2.1. Materials and chemicals
1,10-Phenanthroline-5,6-dione [15], daa = [9-(3,4-diaminophenyl)acridine] [16], dppz,
qdppz, acdppz, and actatp [17, 18] were synthesized according to literature procedures.
Doubly distilled water was used to prepare buffers. All chemicals were reagent grade. The
Figure 1.
Molecular structures of 1–4.
Ruthenium(II) complexes
1663
DNA concentration per nucleotide was determined by using a molar absorption coefficient
(6600 M1 cm1) at 260 nm [19]. A ratio of 1.8–1.9 UV absorbance at 260 and 280 nm
indicates that the DNA was sufficiently free of protein [20].
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2.2. Physical measurements
IR spectra were recorded in KBr on a Perkin-Elmer FT-IR-1605. 1H and 13C-NMR spectra
were measured on a Varian XL-400 MHz spectrometer with DMSO-d6 as solvent at room
temperature and tetramethylsilane (TMS) as the internal standard. LC–MS were recorded
on a 2010A LQC system (Finnigan MAT) with MeCN as mobile phase. UV–Visible spectra were recorded on an Elico Bio-spectrophotometer model BL198. Emission spectra were
carried out by using an Elico Bio-spectrofluorimeter model SL174 at room temperature.
Circular dichroism spectra were recorded on a JASCO J-810 spectropolarimeter. Viscosity
experiments were carried out in an Ostwald viscometer maintained at 30.0 ± 0.1 °C in a
thermostatic water bath.
2.3. DNA-binding studies
2.3.1. Emission studies. In emission studies, fixed metal complex concentrations
(10 μM) were used and varying concentration (0–150 μM) of DNA was added. The excitation wavelength was fixed, and the emission range was adjusted before measurements. The
fraction of ligand bound was calculated from the relation Cb = Ct[(F F0)/Fmax F0)],
where Ct is the total complex concentration, F is the observed fluorescence emission intensity at a given DNA concentration, F0 is the intensity in the absence of DNA, and Fmax is
when the complex is fully bound to DNA. Binding constant of fluorescence was obtained
from a modified Scatchard equation using a plot of r/Cf versus r, where r is Cb/[DNA]
and Cf is the concentration of the free complex. Fluorescence quenching was carried out
by addition of 0.05 M of [Fe(CN)6]4 and 1.0 M of KI as quencher to the complex in the
presence and absence of DNA. According to the classical Stern–Volmer equation [21]
I0 =I ¼ 1 þ Ksv ½Q
where I0 and I are the luminescence intensities in the absence and presence of quencher
[Fe(CN)6]4 or KI, respectively. K is a linear Stern–Volmer quenching constant depending
on the ratio of the bound concentration of the complex to the concentration of DNA. [Q]
is the concentration of the quencher [Fe(CN)6]4 or KI.
2.3.2. Electronic absorption titration. Absorption titrations were carried out at room
temperature to determine the binding affinity between DNA and the complex. A complex
solution of 3.0 mL (20 μM) in a cuvette was placed in the sample compartment and then
spectrum was recorded from 200 to 700 nm. During the titration, small aliquots (10 μL) of
DNA solution were added to each cuvette (reference and sample) to eliminate the absorbance of DNA itself, and the solutions were mixed for 5 min, then the absorption spectra
were recorded. The titration process was repeated until there was no change in the spectra
(binding saturation achieved). The changes in the metal complex concentration due to
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dilution at the end of each titration were negligible. The intrinsic binding constant Kb was
calculated from [22]
½DNA=ðea ef Þ ¼ ½DNA=ðeb ef Þ þ 1=ðKb ðeb ef ÞÞ
where [DNA] is the concentration of DNA, ɛa, ɛf, and ɛb correspond to the extinction
coefficient for the free metal complex, complex in the presence of DNA, and complex in
fully bound form, respectively. In plots of [DNA]/(ɛa ɛf) vs [DNA], Kb is given by the
ratio of slope to intercept.
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2.4. Viscosity experiments
Viscosity experiments were carried out in an Ostwald viscometer maintained at 30.0
± 0.1 °C in a thermostatic water bath. CT-DNA samples with approximately 200 base pairs
were prepared by sonicating to minimize complexities arising from DNA flexibility [23].
Flow time was measured with a digital stop watch, each sample was measured three
times, and an average flow time was calculated. Data were presented as (η/η0)1/3 versus
[complex]/[DNA], where η is the viscosity of DNA in the presence of complexes and η0
is the viscosity of DNA alone. Viscosity values were calculated from the observed flow
time of DNA and DNA containing complex [24]
g0 ¼ t t0 =t0 and g ¼ t 1 t0 =t0
2.5. Thermal denaturation studies
Thermal denaturation studies were carried out with an Elico Bio-spectrophotometer model
BL198 by monitoring the absorbance at 260 nm with complex (10 μM) and CT-DNA
(100 μM) [25]. Salt dependence studies were performed in tris buffer by titrating preformed complex-DNA adduct with various NaCl concentrations as indicated.
2.6. DNA photocleavage experiment
Photocleavage studies were carried out in a total volume of 10 μL containing pBR322
DNA (0.1 μg), and different concentrations of ruthenium(II) complexes were incubated for
30 min in the dark and then irradiated at room temperature with a UV lamp 365 nm for
60 min. Samples were analyzed by electrophoresis for 2.5 h at 40 V on a 0.8% agarose gel
in buffer (pH 8.2). The gel was stained with 1 μg/mL ethidium bromide and then photographed under UV light.
2.7. Circular dichroism
Circular dichroism spectra of DNA were obtained by using a JASCO J-810 spectropolarimeter operating at 25 °C with 3 cm3 of CT-DNA (90 μM dm3) sealed in a dialysis bag
and 6 cm3 of the complex (30 μM dm3) outside the bag and the system agitated on a
shaker bath for 24 h. The region between 210 and 350 nm was scanned for each sample.
Molecular ellipticity values were calculated according to the formula
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½hk ¼ ½hk =Cl 100
where [h]λ is the molecular ellipticity value at a particular wavelength expressed in degrees
cm2M1, C is the concentration of nucleotide phosphates per liter, l is the length of the
cell in cm, and hλ is observed rotation in degrees.
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2.8. In-vitro cytotoxicity studies
The cell line HL-60 (human myeloid leukemia) was obtained from the National Center for
Cellular Sciences (NCCS), Pune, India. HL-60 cells were cultured in RPMI 1640 media supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin
and 100 μg/mL streptomycin. HL-60 cell lines were maintained in culture at 37 °C in an
atmosphere of 5% CO2. Cells were seeded in each well containing 100 μL medium at a final
density of 2 104 cells/well, in 96-well micro titer plates at identical conditions. After overnight incubation, the cells were treated with different concentrations of testing complexes
(20-100 μg/mL in RPMI 1640 medium and filtered) in a final volume of 200 μL with five
replicates each. After 24 h, 10 μL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasolium bromide] (5 mg/mL) was added to each well and the plate was incubated at 37 °C in the
dark for 4 h. The formazan crystals were solubilized in DMSO (100 μL/well), and the reduction in MTT was quantified by absorbance at 570 nm in a spectrophotometer (Spectra MAX
Plus; Molecular Devices; supported by SOFT max PRO 3.0). Effects of test complexes on
cell viability were calculated using untreated cells as the control. The data were subjected to
linear regression analysis, and the regression lines were plotted for the best straight-line fit.
The IC50 (50% inhibition of cell viability) concentrations were calculated using the respective regression equation.
2.9. Synthesis of complexes
2.9.1. [Ru(en)2dppz]2+·H2O (1). This complex was prepared according to the method
given in the literature [26]. A mixture of cis-[Ru(en)2Cl2]Cl (0.074 g, 0.25 mM) and dppz
(0.070 g, 0.25 mM) were placed in a 100 mL round-bottom flask containing 20 mL of
methanol and refluxed for 2 h. The resulting brownish-red solution was allowed to cool at
room temperature, and then, NaClO4 in methanol was added. The thick crystalline precipitate of [Ru(en)2dppz]2+ that formed was collected and recrystallized from acetone/water.
Yield: 70%. Analytical data: Elemental analysis (Expt. and Calcd for C22H28N8O9Cl2Ru:
Calcd (%): C, 52.47; H, 5.20; N, 22.25. Found (%): C, 52.15; H, 5.13; N, 22.14. IR (KBr,
cm1): 1560 (C=N), 1450 (C=C), 616 (M L), 473 (M N (en)). LC–MS: 720 (found
721). 1H-NMR (400 MHz, DMSO-d6, TMS, δ): 9.2 (d, 2H, H1, 1′), 9.10 (d, 2H, H4, 4′),
8.4 (d, 2H, H3, 3′), 8.2 (d, 2H, H5, 5′), 6.6 (d, 2H, H2, 2′), 3.22 (s, 4H, A, A′) and 2.87
(m, 4H, B, B′). 13C[1H]-NMR (100 MHz, DMSO-d6, δ): 166.26 (2C, a, a′), 141.18 (2C, f,
f′), 136.02 (2C, g, g′), 134.9 (2C, c, c′), 130.2 (4C, d, d′, h, h′), 129.4 (2C, e, e′), 127.4
(2C, b, b′), 52.64 (2C, C), 27.5 (2C, C′).
2.9.2. [Ru(en)2qdppz]2+·H2O (2). cis-[Ru(en)2qdppz]2+ was prepared as above by
replacing dppz with qdppz. The mixture was refluxed for 5 h with recrystallization from
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acetone/water. Yield: 50%. Analytical data: Elemental analysis (Expt. and Calcd for
C30H30N8O11Cl2Ru) Calcd (%): C, 56.86; H, 4.45; N, 17.68. Found (%): C, 56.35; H,
4.29; N, 17.42. IR (KBr, cm1): 1640 (C=O), 1540 (C=N), 1496 (C=C), 636 (M L), 490
(M N (en)). LC–MS: 852 (found 851). 1H-NMR (DMSO-d6, TMS, δ): 9.01 (d, 2H, H1,
1′), 8.2 (d, 2H, H4, 4′), 7.95 (d, 1H, H5), 7.81 (d, 2H, H3, 3′), 7.5 (d, 2H, H6, 6′), 7.4 (s,
2H, H7, 7′), 6.5 (d, 2H, H2, 2′), 3.64 (s, 4H, A, A′), and 2.85 (m, 4H, B, B′). 13C[1H]NMR (100 MHz, DMSO-d6, δ): 175 (2C, l, l′), 159 (4C, a, a′, f, f′), 140 (6C, m, m′, g, g′,
i, k), 132 (5C, c, c′, h, o, o′), 126.9 (6C, n, n′, d, d′, e, e′), 125.3 (3C, b, b′, j), 58.32 (2C,
C), 27.4 (2C, C′′).
2.9.3. [Ru(en)2acdppz]2+·H2O (3). This complex was prepared as above by replacing
dppz with acdppz. The mixture was refluxed for 8 h and recrystallized from 20 mL of water.
Yield: 55%. Analytical data: Elemental analysis (Expt. and Calcd for C35H35N9O9Cl2Ru:
Calcd (%): C, 61.75; H, 4.89; N, 18.52. Found (%): C, 61.53; H, 4.56; N, 18.69. IR (KBr,
cm1): 1546 (C=N), 1460 (C=C), 679 (M L), 491 (M N (en)). LC–MS: 899 (found
890). 1H-NMR (DMSO-d6, TMS, δ): 8.23 (d, 2H, H1, 1′), 8.22 (s, 1H, H4), 7.95 (d, 2H,
H3, 3′), 7.70 (d, 4H, H9, 9′), 7.62 (s, 1H, H6), 7.56 (s, 1H, H7), 7.54 (t, 4H, H8, 8′), 7.35 (s,
1H, H5), 7.20 (s, 1H, H10), 7.15 (t, 2H, H2, 2′), 2.6 (s, 4H, A, A′), and 2.2 (m, 4H, B, B′).
13 1
C[ H]-NMR (100 MHz, DMSO-d6, δ): 149.9 (6C, a, a′, f, f′ g, g′), 147.8 (6C, n, n′, o, i),
132.3 (3C, c, c′, j), 124.5 (5C, k, k′, h, e, e′), 123.4 (4C, l, l′, d, d′, e, e′), 123.16 (8C, b, b′,
k, k′, m, m′), 65.48 (2C, C), 29.5 (2C, C′).
2.9.4. [Ru(en)2actatp]2+H2O (4). [Ru(en)2actatp]2+ was prepared as above by replacing
dppz with actatp. The mixture was refluxed for 8 h and recrystallized from 20 mL of water.
Yield: 40%. Analytical data: Elemental analysis (Expt. and Calcd for C32H32N8O9Cl2Ru:
Calcd (%): C, 61.23; H, 4.82; N, 17.85. Found (%): C, 61.12; H, 4.56; N, 17.56. IR (KBr,
cm1): 1565 (C=N), 1430 (C=C), 605 (M L), 497 (M N (en)). LC–MS: 847 (found
845). 1H-NMR (DMSO-d6, TMS, δ): 9.13 (d, 2H, H1, 1′), 9.08 (s, 1H, H7), 8.37 (d, 2H,
H3, 3′), 7.9 (d, 2H, H6, 6′), 7.5 (m, 4H, H4, 4′, H5, 5′), 7.24 (t, 2H, H2, 2′), 2.9 (s, 4H, A,
A′), and 2.4 (m, 4H, B, B′). 13C[1H]-NMR (100 MHz, DMSO-d6, δ): 138.3 (2C, a, a′),
137.5 (4C, f, f′, g, g′), 135.6 (2C, c, c′), 133.4 (4C, k, k′), 132.1 (5C, j, j′, d, d′), 131.4
(4C, h, h′, e, e′), 129.8 (4C, i, i′), 126.3 (2C, b, b′), 68.22 (2C, C), 30.2 (2C, C′).
3. Results and discussion
3.1. Emission studies
In the absence of DNA, the complexes upon excitation at 457, 468, 420, and 468 nm have
emission at 610, 536, 540, 520 nm for 1, 2, 3, and 4. Upon addition of CT-DNA, the
emission intensities of the complexes increase by 2.35, 2.12, 1.68, and 1.53 for 1, 2, 3,
and 4 (figure 2). This implies that the complexes strongly interact with DNA and are protected by DNA, since the hydrophobic environment inside the DNA helix reduces the
accessibility of water and the complexes mobility is restricted at the binding site, leading
to decrease in the vibrational modes of relaxation and hence fluorescence intensity
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Ruthenium(II) complexes
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Figure 2. Fluorescence emission spectra of [Ru(en)2dppz]2+ in tris buffer in the presence of CT DNA, [Ru]
= 10 μL (3 106), [DNA] = 0–150 μM. λexc = 457 nm, λemi = 610 nm. The insert shows the best fit of data to r/Cf
vs. r.
increases. Fluorescence binding constant calculated for all complexes are 8.9 ± 0.5 105
M1 (1), 7.6 ± 0.8 105 M1 (2), 5.2 ± 0.2 105 M1 (3), and 8.2 ± 0.4 104 M1 (4).
This observation is further supported by fluorescence quenching experiments using
[Fe(CN)6]4 (0.1 M) and KI (1 M) as quenchers. In the plot of I0/I versus [Q], the slope is
the Ksv. Ksv values are smaller in the presence of DNA as complexes bound to DNA can
be protected from the quencher, because highly negatively charged [Fe(CN)6]4 would be
repelled by the negative DNA phosphate backbone, hindering quenching of the emission
of the bound complex [27], hence quenching is small. At higher concentration of DNA,
the slope is almost zero, indicating that the bound species is inaccessible to quencher.
Comparing with ferrocyanide, KI shows less quenching affinity, because KI is mononegative, whereas ferrocyanide is tetra negative. Ferrocyanide quenching and KI fluorescence
quenching curves of complexes when bound to DNA are given in figure 3 and Ksv values
are given in table 1. From absorption and fluorescence spectroscopy, the binding of
complexes with DNA is in the order 1 > 2 > 3 > 4.
3.2. Absorption studies
DNA-binding studies can be conveniently monitored by absorption spectroscopy [28]. The
electronic absorption spectra of complexes in the presence of increasing amount of DNA
in tris buffer are shown in figure 4. In the UV region, intense absorptions at 346 nm for
[Ru(en)2dppz]2+, 318 nm for [Ru(en)2qdppz]2+, 305 nm for [Ru(en)2acdppz]2+, and 312 nm
for [Ru(en)2actatp]2+ were attributed to intra ligand π–π⁄ transition of the complexes. The
broad MLCT absorptions at 466, 463, 445, and 463 nm for 1, 2, 3, and 4, respectively,
attributed to Ru(dπ)→L–L(π⁄) transitions of the complex were significantly perturbed, indicating interaction of complex with DNA. This band is bathochromically shifted relative to
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Figure 3. Emission quenching curves of 1, 3 with KI and 2, 4 with [Fe(CN)6]4 in absence of DNA (a),
presence of DNA 1:50 (b) and 1:200 (c).
Figure 4. Absorption spectra of [Ru(en)2dppz]2+ (a) and [Ru(en)2qdppz]2+ (b) in tris buffer upon addition of CT
DNA in absence (top) and presence of CT DNA (lower), [Ru] = 10 μM; [DNA] = 0–126 μM. Insert: plots of
[DNA]/(ɛa–ɛf) vs. [DNA] for the titration of DNA with complex. Solid line is linear fitting of the data. Arrow
shows change in absorption with increasing DNA concentration.
those of [Ru(phen)3]2+ (448 nm) [29], in accord with extension of the framework. Upon
increasing the CT-DNA concentration, hypochromism was 13% with a red shift of 6 nm
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Ruthenium(II) complexes
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for [Ru(en)2dppz]2+, 10% with a red shift of 5 nm for [Ru(en)2qdppz]2+, 7.6% with a red
shift of 3 nm for [Ru(en)2acdppz]2+, and 7% with a red shift of 3 nm for [Ru(en)2actatp]2+.
Since the ligand intercalates into the base pairs of DNA, the π⁄ orbital of the intercalating
ligand can couple with the π orbital of the base pairs; the coupling π⁄ orbital is partially
filled by electrons, thus decreasing the transition probabilities, resulting in hypochromism.
These data imply that these complexes bind to DNA in an intercalative mode [30].
In order to further investigate the binding strength of the complexes, the intrinsic binding
constants Kb of the complexes with CT-DNA were calculated as 8.5 ± 0.2 105 M1 (1),
7.33 ± 0.8 105 M1 (2), 4.3 ± 0.3 105 M1 (3), and 7.5 ± 0.5 104 M1 (4); these values
were slightly higher than those obtained by the emission titration method. The difference
between Kb values obtained by absorption and fluorescence techniques is in agreement with
earlier reports [31]. The binding constants of these complexes are smaller when compared
to the similar complexes with different ancillary ligands, in the range of 105–106 M1 [32];
comparing with ethylenediamine, bpy and phen are aromatic and more planar. Among these
four intercalating ligands, dppz is more planar than the other ligands. The binding constant
data confirm that 1 > 2 > 3 > 4 as the order and all bind strongly to CT-DNA.
Generally, the intercalative ligand should contain an aromatic heterocyclic functionality
such as dppz (extended basic ligands) and imidazole (extended planar ligands) that can
insert and stack between the base pairs of double helical DNA. Extension of intercalative
ligand increases the strength of interaction and binding constant of the complex with DNA
[9, 33].
Since an octahedral complex binds to DNA in three dimensions, its ancillary ligand can
also be modified or functionalized to tune the DNA binding; if the ancillary ligand is bulky
with nonaromatic groups such as –CH3, the DNA binding of the complex will be weakened. The ancillary ligand phen expands the π-delocalization and thus decreases the sigma
donor capacity of the metal ion, leading to a decrease in the electron density on the metal
ion and in turn stabilization of metal dπ orbital but destabilization of the ligand π⁄ orbital
[34]. Additionally, the increased hydrophobicity of the complexes leads to self-stacking in
solution, and this effect may reduce the net binding affinity. The binding constants of the
complexes follow the trend of [Ru(ip)2dppz]2+ (2.1 107 M1) > [Ru(phen)2dppz]2+
(7.5 106 M1) > [Ru(bpy)2dppz]2+ (4.9 105 M1) > [Ru(en)2dppz]2+ (8.5 105 M1).
This may be explained by the planar area of the ancillary ligand ip is larger than that of the
phen, bpy, and en. In contrast, [Ru(phen)3]2+ and [Ru(dip)3]2+ show strong affinity for
DNA, following the order en < bpy < phen 6 dip < ip [13, 14, 35].
3.3. Viscosity measurements
To further clarify the interactions between complexes and DNA, viscosity measurements
were carried out. Hydrodynamic measurements which are sensitive to length increases
(i.e. viscosity, sedimentation) are regarded as the least ambiguous and the most critical
tests of binding model in solution in the absence of crystallographic structural data [36].
Intercalation results in lengthening the DNA helix, as base pairs are separated to accommodate the binding ligand, leading to increase in DNA viscosity. Figure 5 shows the
effects of 1, 2, 3, and 4 and ethidium bromide on the viscosity of rod-like DNA. Ethidium
bromide is a known DNA classical intercalator and increases the relative specific viscosity
by lengthening of the DNA double helix through intercalation. Upon increasing the amount
of complex, the relative viscosity of DNA increases steadily, similar to the behavior of
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M. Shilpa et al.
Figure 5. Effect of increasing amounts of ethidium bromide (EB), 1, 2, 3 and 4 on the relative viscosity of CTDNA at 25 (±0.1) °C. [DNA] = 0.5 mM.
ethidium bromide. The increased degree of viscosity may depend on the intercalative affinity for DNA and follows the order EB > 1 > 2 > 3 > 4.
3.4. Thermal denaturation studies
The melting of DNA is an important parameter to study the interaction of transition metal
complexes with nucleic acids. Thermal denaturation of DNA in the presence of complexes
can give insight into their conformational changes when temperature is raised and offer
information about the interaction strength of complexes with DNA. The melting temperature Tm, at which 50% of the DNA has become single strand, can be determined from the
thermal denaturation curves of DNA by monitoring absorption changes at 260 nm. According to the literature [37], the intercalation of natural or synthesized organics and metallointercalators generally results in considerable increase in melting temperature (Tm). DNA
melting revealed that Tm of CT-DNA was 60 °C and Tm in the presence of 1, 2, 3, and 4
(20 μV) are 71 ± 0.1, 69 ± 0.1, 66 ± 0.1, and 65 ± 0.1 °C, respectively, under our
experimental conditions. The observed melting temperature in the presence of complexes
reveals strong classical intercalation [38] and shows that interaction of [Ru(en)2dppz]2+
with DNA is the strongest.
3.5. Salt dependence studies
The salt dependence binding of 1, 2, 3, and 4 to DNA is shown in figure 6. As the concentration of NaCl increases, the binding constant decreases. The dependence of binding
constant for these complexes upon Na+ concentration is a consequence of the linkage of
complex and Na+ binding to DNA and may be analyzed by polyelectrolyte theory [39].
The slope of the lines in figure 6 provides an estimate of Zψ, where ψ is the fraction of
counter ions associated with each DNA phosphate (ψ = 0.88 for DNA) and Z is the charge
on the complex (Z = +2). The slopes of the lines in figure 6 are 1.47, 1.44, 1.36, and
1.24 for 1, 2, 3, and 4, respectively. These values are less than the theoretically expected
values of Zψ (2 0.88 = 1.76). Such lower values could arise from coupled anion release
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Ruthenium(II) complexes
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Figure 6. Salt dependence of the equilibrium binding constants for DNA binding of 1, 2, 3 and 4. The lines
indicate the slope of the linear square fit to the data as 1.47 (1), 1.44 (2), 1.36 (3) and 1.24 (4).
or from change in complex or DNA hydration upon binding. The knowledge of Zψ allows
for a quantitative estimation of the nonelectrostatic contribution to the DNA binding
constant for these complexes.
3.6. Photoactivated cleavage of Ru(II) complexes
There is substantial and continuing interest in DNA endonucleolytic cleavage activated by
metal ions [40, 41]. Control runs in the agarose gel electrophoresis experiments suggest
that untreated plasmid pBR322 DNA does not show cleavage. Complexes exhibit concentration-dependent, single-strand cleavage of supercoiled Form I to the nicked Form II
DNA (figure 7). Upon increasing the concentration of complexes, the amount of Form II
increases gradually, while Form I diminished gradually. This is the result of single-stranded
cleavage of pBR322 DNA. That neither irradiation of DNA alone at 365 nm (without Ru
(II)) nor incubation with Ru(II) without light yields significant strand scission indicates Ru
(II) complexes play an important role in the DNA cleavage. To identify the nature of the
Figure 7. Photocleavage of pBR322 DNA in the absence and presence of 1, 2, 3 and 4 after 60 min irradiation
at 365 nm. Lane 0 control plasmid DNA (untreated pBR322), lanes a to c addition of complexes, in amounts of
20, 30, 40 μM of concentration, and lane d complexes in presence of histidine (2 mM).
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M. Shilpa et al.
reactive species responsible for photoactivated cleavage of plasmid DNA, we have
investigated with singlet oxygen inhibiting agent histidine. Figure 7 shows the photocleavage of pBR322 DNA in the presence of complex alone and complex + histidine. Indeed,
plasmid DNA cleavage by 1–4 was inhibited in the presence of histidine (lane d).
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3.7. Circular dichroism
Circular dichroism is useful in diagnosing changes in DNA morphology during drug-DNA
interactions, as the band due to base stacking (+275 nm) and that due to right-handed
helicity (248 nm) are quite sensitive to the mode of DNA interactions with small molecules [42]. The changes in CD signals of DNA observed on interaction with drugs may be
assigned to the corresponding changes in DNA structure [42]. Intercalation shows more
perturbation on the base stacking and increasing of molecular ellipticity, whereas electrostatic interactions show less/no perturbation on the base stacking and decrease in molecular
ellipticity. The CD spectrum of the complexes is shown in figure 8 and molecular ellipticity values are given in table 1 as 1 > 2 > 3 > 4.
Figure 8.
dialysis.
Circular dichroism spectra of CT-DNA in the absence (0) and presence of 1, 2, 3 and 4 after 24 h
Table 1. Quenching constant (Ksv) data of complexes in the absence and presence of DNA (1 : 50, 1 : 200) with
[Fe(CN)6]4 and KI as quencher and molecular ellipticity values of complexes in the presence of DNA after 24 h
dialysis.
[K4(Fe(CN)6]4 (0.01 M)
Complex
[Ru(en)2dppz]2+
[Ru(en)2qdppz]2+
[Ru(en)2acdppz]2+
[Ru(en)2actatp]2+
KI (1 M)
Only complex
1 : 50
1 : 200
Only complex
1 : 50
1 : 200
Molecular ellipticity
3328
1023
875.3
763.3
249.3
371.5
334.2
375.6
46.9
58.2
85.3
103
620
580
452
420
15.2
20.6
35.2
42.3
0.43
0.57
1.27
7.25
56,452
52,753
48,256
40,238
Ruthenium(II) complexes
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3.8. Antiproliferative activity
Cell proliferation or viability was measured using the MTT assay [43]. The cytotoxic
effects of test complexes were investigated on human leukemia cell lines (HL-60).
After 24 h of treatment, the number of live cells was measured by MTT assay and
IC50 values for each testing complex were determined (table 2). It is evident from
table 2 that HL-60 cells were moderately sensitive to 1, with an IC50 value of
22.30 μg/mL, as compared with other complexes. Complex 2 did not show any activity against the HL-60 cells, and the decreasing order of activity among the test complexes was in the order 1 > 3 > 4. DMSO stock solution was used for all complexes
to perform a proper comparison among the complexes; untreated cells containing the
same amount of DMSO are taken as negative controls. Cisplatin was used as the
positive control. These complexes exhibited dose-dependent growth inhibitory effect
(figure 9) against the tested cell lines. The structure of the mixed ligand complexes
could be a factor for exhibiting differential anti-proliferative activities on the cancerous cell lines (figure 10). These results are in agreement with reported ruthenium
polypyridyl complexes [7, 8].
Table 2. Cytotoxic activity of mixed ligand ruthenium(II) complexes on HL-60 cells.
Complexes
(IC50 values in μg/mL)
(IC50 values in μM)
1
2
3
4
Cisplatin
21.30 ± 0.81
No activity
25.72 ± 1.13
56.59 ± 1.46
18.15 ± 1.16
37.85 ± 1.62
No activity
40.89 ± 1.55
81.74 ± 1.26
21.12 ± 1.26
Figure 9. In-vitro cytotoxity of 1, 3 and 4 on tumor cell line HL-60 after treatment of 24 h in the absence
(control) and presence of different concentrations.
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M. Shilpa et al.
Figure 10. The morphological effects exerted by complexes on HL-60 cells 24 h after treatment. Photographs
were taken using a Nikon inverted light microscope (20 objective). a shows the untreated cells and b, c and d
show cells treated with 0.5 mM of 1, 3 and 4 complexes, respectively.
4. Conclusion
Four ethylenediammine Ru(II) complexes with extended planar aromatic ligands have
been synthesized, characterized, and their interaction with CT-DNA examined. Absorption,
emission, thermal denaturation, and viscosity experiments were performed and the results
suggest an intercalative mode of DNA binding. The planarity of the modified dipyridophenazine plays an important role in dictating DNA-binding affinity. Thus, the dppz complex binds to DNA more strongly and follows the order qddpz > acdppz > actatp. The
present study demonstrates that the ancillary ligands with hydrogen bonding potential
support the intercalative interaction of ligands with extended aromatic rings and enhances
the DNA-binding affinity. However, for ethylenediamine complexes with ligands like
actatp, a decrease in binding affinity has been observed illustrating that minor changes in
the ligand architecture and electronic structure can remarkably change the DNA-binding
affinity. The results of the present study show that the complexes could be potential anticancer agents.
Acknowledgements
We are grateful to the DST (Department of Science and Technology) India for providing
financial support and also thankful to Dr. Bhanuprakash (Scientist, NIN) for providing CD
facilities.
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