Ruthenium(II)/(III) complexes of 4-hydroxy-pyridine-2,6-dicarboxylic acid with PPh3/AsPh3 as co-ligand: Impact of oxidation state and co-ligands on anticancer activity in vitro

Article abstract of DOI:10.1039/c1dt11273b

With the aim to develop more efficient, less toxic, target specific metal drugs and evaluate their anticancer properties in terms of oxidation state and co-ligand sphere, a sequence of Ru^II, Ru^III complexes bearing 4-hydroxy-pyridine-2,6-dicarboxylic acid and PPh₃/AsPh ₃ were synthesized and structurally characterized. Biological studies such as DNA binding, antioxidant assays and cytotoxic activity were carried out and their anticancer activities were evaluated. Interactions of the complexes with calf thymus DNA revealed that the triphenylphosphine complexes could bind more strongly than the triphenylarsine complexes. The free radical scavenging ability, assessed by a series of in vitro antioxidant assays involving DPPH radical, hydroxyl radical, nitric oxide radical, superoxide anion radical, hydrogen peroxide and metal chelating assay, showed that the Ru^III complexes possess excellent radical scavenging properties compared to those of Ru^II. Cytotoxicity studies using three cancer lines viz HeLa, HepG2, HEp-2 and a normal cell line NIH 3T3 showed that Ru^II complexes exhibited substantial cytotoxic specificity towards cancer cells. Furthermore, the Ru^II complexes were found to be superior to Ru^III complexes in inhibiting the growth of cancer cells.

Full text of DOI:10.1039/c1dt11273b

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PAPER
Ruthenium(II)/(III) complexes of 4-hydroxy-pyridine-2,6-dicarboxylic acid
with PPh3/AsPh3 as co-ligand: Impact of oxidation state and co-ligands on
anticancer activity in vitro†
Thangavel Sathiya Kamatchi,a Nataraj Chitrapriya,b Hyosun Lee,*b Chris F. Fronczek,c Frank R. Fronczekc and
Karuppannan Natarajan*a
Received 5th July 2011, Accepted 31st October 2011
DOI: 10.1039/c1dt11273b
With the aim to develop more efficient, less toxic, target specific metal drugs and evaluate their
anticancer properties in terms of oxidation state and co-ligand sphere, a sequence of RuII, RuIII
complexes bearing 4-hydroxy-pyridine-2,6-dicarboxylic acid and PPh3/AsPh3 were synthesized and
structurally characterized. Biological studies such as DNA binding, antioxidant assays and cytotoxic
activity were carried out and their anticancer activities were evaluated. Interactions of the complexes
with calf thymus DNA revealed that the triphenylphosphine complexes could bind more strongly than
the triphenylarsine complexes. The free radical scavenging ability, assessed by a series of in vitro
antioxidant assays involving DPPH radical, hydroxyl radical, nitric oxide radical, superoxide anion
radical, hydrogen peroxide and metal chelating assay, showed that the RuIII complexes possess excellent
radical scavenging properties compared to those of RuII. Cytotoxicity studies using three cancer lines
viz HeLa, HepG2, HEp-2 and a normal cell line NIH 3T3 showed that RuII complexes exhibited
substantial cytotoxic specificity towards cancer cells. Furthermore, the RuII complexes were found to be
superior to RuIII complexes in inhibiting the growth of cancer cells.
ligands and isomers of the complex can all exert a critical influence
Introduction
on the biological activity of a metal complex.2–4 An understanding
Metal complexes offer infinite opportunities for the design of
of how these factors affect biological activity should enable the
compounds with bioactivity due to the large variety of available
design of metal complexes with specific medicinal properties. The
metals and the ability to tune the reactivity and structure of the
wide spectrum of contrasting biological activity amongst platinum
metal complexes by their ligand spheres.1 In general, the nature of
the metal ion, its oxidation state, the types and number of bound
complexes5–7 and the clinical success of PtII diam(m)ine complexes,
e.g. cisplatin, as anticancer drugs provide a good illustration of this
point.
aDepartment of Chemistry, Bharathiar University, Coimbatore, 641046,
India. E-mail: k_natraj6@yahoo.com; Fax: +91-422-2422387; Tel: +91-
422-2428319
Although 70% of all cancer patients receive cisplatin during
cancer treatment, chemotherapy with cisplatin and its analogues
still has several drawbacks; toxic side-effects and lack of activity
(drug resistance) against several types of cancer are problems
which need to be overcome.8 This provides the impetus for the
search for anticancer activity amongst complexes of other metals.
At this juncture, ruthenium, a rare transition metal of the platinum
group, has emerged as an attractive alternative due to several
favorable properties suited to rational anticancer drug design and
biological applications. Biologically compatible ligand-exchange
kinetics of RuII and RuIII similar to those of platinum complexes,
a higher coordination number that could potentially be used to
fine-tune the properties of the complexes, and lower toxicity (than
their platinum counterparts) towards healthy tissues by mimicking
iron in binding to many biological molecules are the advantages
of using ruthenium complexes.9,10
bDepartment of Chemistry and Green-Nano Materials Research Center,
Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Deagu, 702-
701, Republic of Korea. E-mail: hyosunlee@knu.ac.kr; Fax: +82-53-950-
6330; Tel: +82-53-950-5337
cDepartment of Chemistry, Louisiana State University, Baton Rouge, LA,
70803, USA
1
† Electronic supplementary information (ESI) available: H NMR spec-
trum of the complex [RuII-hpa-P] (Fig. S1). Cyclic voltammetric response
of [RuII-hpa-P] (Fig. S2). X-Ray crystal structure and atom numbering
scheme for [RuIII-hpa-As] (Fig. S3). Hydrogen-bonding distances and
angles (Table S1). Plot of (ea-ef)/(eb-ef) vs. [DNA] for the titration of DNA
with complexes. Emission spectra of EB bound to DNA in the absence and
presence of [RuIII-hpa-P]. Stern–Volmer quenching plot of EB bound to
CT-DNA by ruthenium complexes (Fig. S4–S6). Antioxidant activity of
all the complexes against various radicals (Fig. S7). Concentration effect
curves of the complexes against HeLa, HepG2, HEp-2 and NIH 3T3
cell lines (Fig. S8–S11). CCDC reference numbers 797111–797113. For
ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c1dt11273b
The entrance of two RuIII based drugs, NAMI-A11 and KP101912
(Fig. 1) into clinical trials for the treatment of metastatic tumors
2066 | Dalton Trans., 2012, 41, 2066–2077
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purchased from Sigma and dissolved in 5 mM Tris HCl buffer
(pH 7.0) containing 100 mM NaCl and 1 mM EDTA. It
was dialyzed several times against 5 mM Tris HCl buffer. All
experiments involving interactions of complexes with CT-DNA
were carried out in Tris HCl buffer (pH 7.0). The concentrations of
DNA, complexes [RuII(hpa)(PPh3)2CO], [RuII(hpa)(AsPh3)2CO],
[RuIII(hpa)(PPh3)2Cl] and [RuIII(hpa)(AsPh3)2Cl] were determined
spectrophotometrically using their extinction coefficients e258nm
=
6700 M-1 cm-1, e330nm = 9750 M-1 cm-1, e330nm = 26 330 M-1 cm-1,
e357nm = 8345 M-1 cm-1 and e330nm = 27 620 M-1 cm-1 respectively.
Fig. 1 Chemical structures of promising ruthenium drug candidates
under investigation: NAMI-A and KP1019.
Syntheses
increased the interest in this metal. Both complexes behave quite
differently from cisplatin in vivo. In addition, a number of RuII
compounds were recently shown to possess very encouraging
cytotoxic and antitumor properties in preclinical models13,14 and
are now under active investigation.
Synthesis of complexes [Ru(hpa)(EPh3)2D] (E = P or As; D = CO
or Cl). The new RuII and RuIII complexes were synthesized by the
following general procedure. An ethanolic (20 mL) solution of the
H2hpa (0.020 g, 0.099 mmol) was added to a refluxing solution of
[RuHCl(CO)(EPh3)3], (0.095 g, 0.099 mmol; 0.108 g, 0.099 mmol)
or [RuCl3(EPh3)3] (0.098 g, 0.099 mmol; 0.112 g, 0.099 mmol) in
ethanol (20 mL) (where E = P, As). The mixture was heated under
reflux for 3 h. The solution was filtered while hot, reduced to half
of its volume and left for slow evaporation. The semi crystalline
product that separated out was filtered off, washed with ethanol
and dried under vacuum.
Bidentate carboxylate ligands have been used to endow cis-
platin derivatives with high aquatic solubility and resistance to
hydrolysis.15 Two such examples, carboplatin and oxaliplatin, are
used routinely in clinical practice. So, as a chelating partner to
ruthenium, 4-hydroxy-pyridine-2,6-dicarboxylic acid (H2hpa) is
chosen due to its effective contribution in anti HIV investigation16
and its valuable participation in anticancer activities.
Since the metal ion, its oxidation state and chelating ligand
sphere play vital roles in determining chemical properties, and
hence biological activities, a series of RuII and RuIII complexes
enclosing 4-hydroxy-pyridine-2,6-dicarboxylic acid were synthe-
sized and structurally characterized. To obtain insight into the
influence of the co-ligands, triphenylphosphines are replaced by
triphenylarsines. All the complexes were subjected to biological
studies such as DNA binding, antioxidant assays and cytotoxic
activity testing.
Synthesis of [Ru(hpa)(PPh3)2(CO)] ([RuII-hpa-P]). The com-
plex was synthesized from [RuHCl(CO)(PPh3)3] and H2hpa.
Orange crystals of the product were obtained by recrystallization
from a chloroform/ethanol mixture. Yield = 0.076 g, 92%; m.p.
>300 ◦C; elemental analysis calcd.(%) for C44H33NO6P2Ru: C,
63.31; H, 3.98; N, 1.68%. Found: C, 63.38; H, 3.99; N, 1.67%;
UV-visible: lmax (5%DMSO/H2O)/nm 263 (e/dm3 mol-1 cm-1
34 253) and 330 (9750); IR (KBr disks, nmax/cm-1) 3446 (OH); 1635
(COO, asymm); 1429 (COO, symm); 1917 (C O); 1H NMR; dH
(500MHZ; [D6] DMSO; 25 ◦C, TMS): 11.72 (1H, s, OH), 6.60
(2H, s, CH), 7.16–7.49 (30H, m, Ph), 1.06 (6H, t, J = 6.5 Hz,
CH3CH2OH), 3.42–3.47 (4 H, m, CH3CH2OH), 4.35 (2H, t, J = 5
Hz, CH3CH2OH).
Experimental section
Equipment and techniques
Melting points were determined with a Lab India instrument.
Elemental analyses (C, H, N, S) were performed on a Vario EL
III Elementar elemental analyzer. A Nicolet Avatar Model FT-IR
spectrophotometer was used to record the IR spectra (4000–400
cm-1) of the free ligand and the complexes. Fluorescence spectra
Synthesis of [Ru(hpa)(AsPh3)2(CO)] ([RuII-hpa-As]). The com-
plex was synthesized from [RuHCl(CO)(AsPh3)3] and H2hpa.
Yield = 0.087g, 95%; m.p. >300 ◦C; elemental analysis calcd.
(%) for C44H33NO6As2Ru: C, 57.28; H, 3.61; N, 1.52%. Found:
C, 57.17; H, 3.60; N, 1.51%; lmax (5%DMSO/H2O)/nm 257
(e/dm3 mol-1 cm-1 126 070) and 330 (26 330); IR (KBr disks,
1
were measured on a Perkin Elmer spectrofluorimeter. H NMR
spectra were recorded on Bruker AMX 500 at 500 MHz using
tetramethylsilane as an internal standard. The electrochemical
analyzer (CHI 1120A) equipped with a three electrode compart-
ment consisting of platinum disc working electrode, platinum wire
counter electrode and Ag/AgCl reference electrode was used to
record the cyclic voltammograms of the complexes.
n
max/cm-1) 3410 (OH); 1620 (COO, asymm); 1443 (COO, symm);
1960 (C O); 1H NMR dH (500MHZ; [D6] DMSO; 25 ◦C, TMS):
11.69 (1H, s, OH), 6.58 (2H, s, CH), 7.13–7.49 (30H, m, Ph).
Synthesis of [Ru(hpa)(PPh3)2(Cl)] ([RuIII-hpa-P]). This was
synthesized from [RuCl3(PPh3)3] and H2hpa. Red crystals
of X-ray quality were grown by recrystallization from a
dichloromethane/acetonitrile mixture. Yield = 0.070, 84%; m.p.
>300 ◦C; elemental analysis calcd. (%) for C43H33NO5ClP2Ru: C,
61.32; H, 3.95; N, 1.66%. Found: C, 61.45; H, 3.93; N, 1.67%;
lmax (5%DMSO/H2O)/nm 259 (e/dm3 mol-1 cm-1 18 380), 327
(10 244) and 357 (8345); NIR (KBr disks, nmax/cm-1) 3433 (OH);
1650 (COO, asymm); 1442 (COO, symm).
Materials
Commercially available RuCl3·3H2O (Himedia) was used
without further purification. The starting complexes [RuHCl-
(CO)(PPh3)3],50
[RuHCl(CO)(AsPh3)3],51
[RuCl3(PPh3)3],52
[RuCl3(AsPh3)3],53 were prepared as reported earlier. 4-Hydroxy-
pyridine-2,6-dicarboxylic acid was purchased from Acros organics
and used as supplied. Solvents were purified and dried according
to standard procedures.54 Calf thymus DNA (CT-DNA) was
Synthesis of [Ru(hpa)(AsPh3)2(Cl)] ([RuIII-hpa-As]). This was
prepared from [RuCl3(AsPh3)3] and H2hpa. Red crystals of
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X-ray quality were obtained by recrystallization from
a
antioxidants, including the natural antioxidant vitamin C and the
synthetic antioxidant BHT.
dichloromethane/acetonitrile mixture. Yield = 0.081g, 88%; m.p.
>300 ◦C; elemental analysis calcd. (%) for C43H33NO5ClAs2Ru:
C, 55.53; H, 3.58; N, 1.51%. Found: C, 55.42; H, 3.59; N, 1.52;
lmax (5%DMSO/H2O)/nm 256 (e/dm3 mol-1 cm-1 77 760) and
330 (27 620); IR (KBr disks, nmax/cm-1) 3419 (OH); 1638 (COO,
asymm); 1432(COO, symm).
DPPH∑ scavenging assay
The DPPH radical scavenging activity of the compounds was
measured according to the method of Blios.59 The DPPH radical
is a stable free radical. Because of the odd electron, DPPH shows
a strong absorption band at 517 nm in the visible spectrum. As
this electron becomes paired off in the presence of a free radical
scavenger, the absorption vanishes and the resulting decolorization
is stoichiometric with respect to the number of electrons taken up.
Various concentrations of the experimental complexes were taken
and the volume was adjusted to 100 mL with methanol. About
5 mL of a 0.1 mM methanolic solution of DPPH was added to
the aliquots of samples and standards (BHT and vitamin C) and
shaken vigorously. A negative control was prepared by adding
100 mL of methanol in 5 mL of 0.1 mM methanolic solution
DPPH. The tubes were allowed to stand for 20 min at 27 ◦C. The
absorbance of the sample was measured at 517 nm against the
blank (methanol).
Crystallography: Data collection and structure determination
X-ray diffraction measurements were performed on a Nonius
KappaCCD diffractometer equipped with an Oxford Cryostream
chiller and graphite monochromated Mo-Ka radiation. The
structures of all the three complexes were solved by direct methods
and refinements were carried out by full matrix least-squares
techniques. The hydrogen atoms were placed in idealized positions,
except for those on OH, for which positions were refined. All three
structures were solvates, [RuII-hpa-P] with ordered EtOH and dis-
ordered CHCl3, [RuIII-hpa-P] with disordered MeCN/DCM, and
[RuIII-hpa-As] with ordered MeCN. Contribution to the structure
factors by disordered solvent in [RuII-hpa-P] was removed for
refinement using the SQUEEZE55 procedure, amounting to 0.64
molecules of disordered chloroform per Ru complex. Solvent in
[RuIII-hpa-P] was modeled as MeCN and DCM sharing a site in a
9 : 1 ratio. The following computer programs were used: structure
solution SIR-97,56 refinement SHELXL-97,57 molecular diagrams,
ORTEP-358 for Windows.
OH∑ scavenging assay
The hydroxyl radical scavenging activity of the complex has been
investigated by using the Nash method.60 In vitro hydroxyl radicals
were generated by an Fe3+/ascorbic acid system. The detection of
hydroxyl radicals was carried out by measuring the amount of
formaldehyde formed from the oxidation reaction with DMSO.
The formaldehyde produced was detected spectrophotometrically
at 412 nm. A mixture of 1.0 mL of iron-EDTA solution (0.13%
ferrous ammonium sulphate and 0.26% EDTA), 0.5 mL of EDTA
solution (0.018%), and 1.0 mL of DMSO (0.85% DMSO (v/v)
in 0.1 M phosphate buffer, pH 7.4) were sequentially added in
the test tubes. The reaction was initiated by adding 0.5 mL of
ascorbic acid (0.22%) and was incubated at 80–90 ◦C for 15 min in
a water bath. After incubation, the reaction was terminated by the
addition of 1.0 mL of ice-cold trichloroacetic acid (17.5% w/v).
Subsequently, 3.0 mL of Nash reagent was added to each tube and
left at room temperature for 15 min. The intensity of the colour
formed was measured spectrophotometrically at 412 nm against
reagent blank.
DNA binding experiments
The experiments were carried out in 5 mM Tris-HCl buffer (pH
7.0) at ambient temperature and the complexes were dissolved
in 5 mM Tris-HCl buffer containing 5% DMSO. Changes in the
fluorescence emission spectrum of the ethidium bromide (referred
to as EB)-DNA complex were recorded under various complex
concentrations. The fluorescence spectra were obtained at an
excitation wavelength of 522 nm and an emission wavelength
of 584 nm. Melting profiles were measured at 260 nm by a
Cary 300 spectrophotometer. Readings were recorded for every
1 ◦C raise in temperature per minute. The viscosity measurement
was carried out using an Ubbelodhe viscometer immersed in a
thermostatic water bath maintained at 25( 0.l) ◦C. DNA samples
approximately 200 base pairs in length were prepared by sonication
in order to minimize complexities arising from DNA flexibility.
Flow times were measured with a digital stopwatch; each sample
was measured three times, and an average flow time was calculated.
Relative viscosities for CT-DNA in the presence and absence of
the complex were calculated from the relation h = (t-t0)/t0, where t
is the observed flow time of DNA-containing solution and t0 is the
flow time of Tris-HCl buffer alone. Data are presented as (h/h0)1/3
versus binding ratio, where h is the viscosity of CT-DNA in the
presence of complex and h0 is the viscosity of CT-DNA alone.
NO∑ scavenging assay
The assay of nitric oxide (NO∑) scavenging activity is based on
a method61 in which sodium nitroprusside in aqueous solution
at physiological pH spontaneously generates nitric oxide, which
interacts with oxygen to produce nitrite ions. These can be
estimated using the Greiss reagent. Scavengers of nitric oxide
compete with oxygen leading to reduced production of nitrite ions.
For the experiment, sodium nitroprusside (10 mM) in phosphate
buffered saline was mixed with a fixed concentration of the
complex and standards and incubated at room temperature for
150 min. After the incubation period, 0.5 mL of Griess reagent
containing 1% sulfanilamide, 2% H3PO4 and 0.1% N-(1-naphthyl)
ethylenediamine dihydrochloride was added. The absorbance of
the chromophore formed was measured at 546 nm.
Antioxidant assays
The ability of ruthenium complexes to act as hydrogen donors or
free radical scavengers was explored by conducting a series of in
vitro antioxidant assays involving DPPH radical, hydroxyl radical,
nitric oxide radical, hydrogen peroxide, superoxide anion radical,
metal chelating assay and comparing the results with standard
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H2O2 scavenging assay
screening experiments, the cells were seeded into 96-well plates
in 100 mL of the respective medium containing 10% FBS, at a
plating density of 10 000 cells/well and incubated at 37 ◦C, 5%
CO2, 95% air and 100% relative humidity for 24 h prior to the
addition of compounds. The compounds were dissolved in DMSO
and diluted in the respective medium containing 1% FBS. After
24 h, the medium was replaced with the respective medium with
1% FBS containing the compounds at various concentrations and
incubated at 37 ◦C, 5% CO2, 95% air and 100% relative humidity
for 48 h. Experiments were performed in triplicate and the medium
without the compounds served as control. After 48 h, 10 mL of
MTT (5 mg mL-1) in phosphate buffered saline (PBS) was added
to each well and incubated at 37 ◦C for 4 h. The medium with
MTT was then removed and the formed formazan crystals were
dissolved in 100 mL of DMSO and the absorbance measured at
570 nm using a micro plate reader. The % cell inhibition was
determined using the following formula, and a graph was plotted
between % of cell inhibition and concentration. From this plot,
the IC50 value was calculated.
The ability of the complexes to scavenge hydrogen peroxide
was determined using the method of Ruch et al.62 A solution
of hydrogen peroxide (2.0 mM) was prepared in phosphate
buffer (0.2 M, pH 7.4) and its concentration was determined
spectrophotometrically from absorption at 230 nm with molar
absorptivity 81 M-1 cm-1. The complexes (100 mg mL-1), BHT and
vitamin C (100 mg mL-1) were added to 3.4 mL of phosphate buffer
together with hydrogen peroxide solution (0.6 mL). An identical
reaction mixture without the sample was taken as negative control.
Absorbance of hydrogen peroxide at 230 nm was determined after
10 min against the blank (phosphate buffer).
∑
-
O2 scavenging assay
The superoxide anion radical (O2 ∑) scavenging assay was based
-
on the capacity of the complexes to inhibit formazan formation
by scavenging the superoxide radicals generated in the riboflavin-
light-NBT system.63 Each 3 mL reaction mixture contained 50 mM
sodium phosphate buffer (pH 7.6), 20 mg riboflavin, 12 mM
EDTA, 0.1 mg NBT and 1 mL complex solution (20–100 mg mL-1).
The reaction was started by illuminating the reaction mixture with
different concentrations of complex for 90 s. Immediately after
illumination, the absorbance was measured at 590 nm. The entire
reaction assembly was enclosed in a box lined with aluminum foil.
Identical tubes with the reaction mixture kept in the dark served
as blanks.
% inhibition = [mean OD of untreated cells (control) / mean OD
of treated cells (control)] ¥ 100.
Results and discussion
Reactions of H2hpa with [RuCl3(EPh3)3] and [RuHCl(CO)(EPh3)3]
gave new complexes of the type [Ru(hpa)(EPh3)2D] as shown in
Scheme 1. The analytical data confirmed the molecular formulae
for the new complexes. It has been observed that one molecule
of the dicarboxylic acid replaced a chloride ion, a hydride ion
and a PPh3/AsPh3 in the case of RuII complexes, two chlorides
and a PPh3/AsPh3 in the case of RuIII complexes. X-ray crystal
determinations revealed that the dicarboxylic acid coordinated to
the metal ion as dibasic tridentate ONO donor.
Metal chelating activity
The chelation with ferrous ions by the experimental complexes was
estimated by the method of Dinis et al.64 Initially, about 100 mL
the samples and the standards were added to 50 mL solution of
2 mM FeCl2. The reaction was initiated by the addition of 200 mL
of 5 mM ferrozine and the mixture was shaken vigorously and
left standing at room temperature for 10 min. Absorbance of the
solution was then measured spectrophotometrically at 562 nm
against the blank (deionized water).
Electrochemistry
Redox behaviors of the ligand and new RuII and RuIII complexes
have been evaluated by means of cyclic voltammetry using their
methylene chloride solutions containing 0.1 M tetrabutylam-
monium perchlorate (~1 ¥ 10-3 M) as a supporting electrolyte.
The solution was degassed with a continuous flow of nitrogen
before scanning. All the complexes are electroactive with respect
to the ligand and metal centre. The complexes show different
redox behaviors in the chosen potential window of 2 V at a
scan rate of 50 mV s-1. The complexes display two successive
one electron reductions and an oxidation. The redox process
is best described as reversible/quasireversible electron transfer
borne out by the fact that the peak-to-peak separation value
(Epa-Epc) ranges from 83–240 mV. There is little variation in
the cyclic voltammograms when triphenylphosphine is replaced
by triphenylarsine in the complexes. Hence, the redox behavior
of the complexes [RuII-hpa-P] and [RuIII-hpa-P] will be described
in detail here. The electrochemical data are given in Table 1. A
representative voltammogram is given in Fig. S2, ESI.†
For the above six assays, all the tests were run in triplicate and
the percentage activity was calculated by the following equation
Scavenging activity (%) = [(A0-A1) / A0] ¥ 100
where A0 is the absorbance of the control and A1 is the absorbance
of the complex/standard. When the percentage inhibition of the
tested compounds is 50%, the tested compound concentration is
the IC50 value.
In vitro anticancer activity evaluation by MTT assay
Cytotoxicity studies of the complexes and cisplatin were carried
out on human cervical cancer cells (HeLa), human laryngeal
epithelial carcinoma cells (HEp-2), human liver carcinoma cells
(HepG2) and mouse embryonic fibroblasts (NIH 3T3), all of which
were obtained from National Centre for Cell Science (NCCS),
Pune, India. Cell viability was carried out using the MTT assay
method.65 The HeLa, HepG2 and HEp-2 cells were grown in
Eagles minimum essential medium containing 10% fetal bovine
serum (FBS) and NIH 3T3 fibroblasts were grown in Dulbeccos
modified Eagles medium (DMEM) containing 10% FBS. For
Electrochemical behavior of [RuII-hpa-P]. [RuII-hpa-P] exhib-
ited a reversible reduction couple with E1/2 value -1.15 V and
peak-to-peak separation of 83 mV which can be assigned to
the one electron RuII/RuI redox process. The calculated E1/2
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Scheme 1 General scheme for the synthesis of new ruthenium complexes.
Table 1 Cyclic voltammetric data of the complexes and their estimated
redox potentials
Efa (V)/DEp (mV)
b
Complex
Reductionc ligand based reductiond Oxidatione
[RuII-hpa-P]
-1.15/083 -0.58/225
-1.17/117 -0.60/220
-1.17/210 -0.47/176
-1.17/240 -0.48/225
-0.52/147
1.45/161
1.45/142
1.46/147
1.47/195
[RuII-hpa-As]
[RuIII-hpa-P]
[RuIII-hpa-As]
Free Ligand (H2hpa)
a Ef = formal potential calculated as the average of anodic (Epa) and
cathodic (Epc) peak potentials. b DEp = Peak-to-Peak separation (Epa-Epc).
c RuI/RuII and RuII/RuIII d hpa/hpa∑- and H2hpa/H2hpa∑-. e RuIII/RuII
.
and RuIV/RuIII
.
values are concurrent with the literature reports.17,18 In addition,
a quasi-reversible cathodic process occurring at -0.58 V versus
Ag/Ag+ with peak-to-peak separation of 225 mV can be safely
assigned to a ligand based redox response on comparing with the
electrochemical data of H2hpa and it is attributed to the formation
of the hpa radical anion. An oxidation couple observed with
formal potential 1.45 V is quasi-reversible (DEp = 161 mV) and it
is assigned to the RuIII/RuII redox process. From this, it is inferred
that the RuII complex favors the reduction process to RuI rather
than the oxidation process to RuIII.
Fig.
2
X-ray crystal structure and atom numbering scheme for
[RuII-hpa-P] as thermal ellipsoids at a 50% probability level. The hydrogen
atoms have been omitted for clarity.
Electrochemical behavior of [RuIII-hpa-P]. [RuIII-hpa-P] dis-
played a well-defined one-electron quasi-reversible coupled redox
wave on either side of the reference electrode besides a quasi-
reversible redox response at less negative potentials with respect
to the coupled redox wave at the negative side of the Ag/AgCl
electrode. The complex gave a quasi-reversible cyclic voltammetric
response due to the RuIII–RuII couple at E1/2 = -1.17 V with
peak-to-peak separation (DEp) of 210 mV. This is attributed to
slow electron transfer and adsorption of the complexes onto the
electrode surface.19 A quasi-reversible ligand based reduction at
-0.47 V with DEp = 176 mV was also observed. In addition, metal
centered oxidation occurred at 1.46 V with DEp = 147 mV ascribed
to a quasi-reversible one electron RuIV/RuIII redox process.
hydrogen bond distances are given in Table 3 and Table S1, ESI†
respectively.
Crystal structure of [RuII-hpa-P]
Single crystals of [RuII-hpa-P] were obtained from slow evapora-
tion of a mixture of chloroform and ethanol at room temperature.
An orange single crystal of approximate unit cell dimensions
0.25 ¥ 0.20 ¥ 0.17 mm was isolated and the single crystal X-ray
diffraction experiments were carried out at 90 K. From the unit
cell dimensions, it is clear that the crystal is triclinic belonging to
¯
the P1space group. The crystal was a solvate containing ethanol
and disordered chloroform.
The RuII ion exhibits hexa coordination with an octahedral
geometry where equatorial coordination comes from two carboxy-
late oxygen atoms, the ring nitrogen of the tridentate chelating
ligand H2hpa and a carbonyl carbon. A pair of triphenylphos-
phine ligands completes the axial coordination. The coordination
environment around the RuII ion can be considered as being a
distorted octahedron {RuNO2P2(CO)} because of distortions of
the bond angles (Table S3, ESI†) from the required 90◦and 180◦
at the basal plane which is reflected in the bonding parameters
X-ray crystallography
General. The structures of [RuII-hpa-P], [RuIII-hpa-P] and
[RuIII-hpa-As] have been established by single crystal X-ray analy-
sis. The ORTEP style drawings of [RuII-hpa-P] and [RuIII-hpa-P] are
displayed in Fig. 2 and 3. The details concerning the data collection
and structure refinement of the complexes are summarized in Table
2. The ORTEP diagram of [RuIII-hpa-As] (Fig. S3, ESI†), selected
geometrical parameters (interatomic distances and angles) and
2070 | Dalton Trans., 2012, 41, 2066–2077
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Table 2 Crystallographic data for [RuII-hpa-P], [RuIII-hpa-P] and [RuIII-hpa-As]
[RuII-hpa-P]
[RuIII-hpa-P]
[RuIII-hpa-As]
CCDC deposit number
Empirical formula
797111
797112
797113
C43H33As2ClNO5Ru·C2H3N
C44H33NO6P2Ru·2(C2H6O)·
C43H33ClNO5P2Ru·0.9(C2H3N)·
0.64(CHCl3)
1003.26
90
0.1(CH2Cl2)
887.61
Formula weight
971.1
90
0.71073
Triclinic
¯
P1
T (K)
90
˚
Wavelength (A)
0.71073
Triclinic
¯
P1
0.71073
Triclinic
¯
P1
Crystal system
Space group
Unit cell dimensions
˚
a (A)
12.0057(14)
14.0262(15)
15.237(2)
89.650(5)
81.378(5)
71.638(6)
2405.4(5)
12.0311(15)
13.5729(15)
13.996(2)
84.593(7)
71.947(6)
64.431(7)
1958.0(5)
12.3411(14)
13.6920(15)
14.2068(15)
82.983(6)
69.879(5)
63.268(5)
2011.6(4)
˚
b (A)
˚
c (A)
a (◦)
b (◦)
g (◦)
3
˚
Volume (A )
Z
2
2
2
Density (calculated) Mg m-3
Absorption coefficient mm-1
F(000)
1.385
0.551
1.505
0.614
1.603
2.138
1030
906
974
Theta range for data collection
Index ranges
2.7 to 34.3◦
1.5 to 32◦
2.8 to 34.9◦
-18< = h< = 18, -21< = k< = 21,
-23< = l< = 23
69 185/18 649, 0.042
0.931
-17< = h< = 17, -18< = k< = 20,
-20< = l< = 20
53 880/13 603, 0.039
0.997
-19< = h< = 19, -21< = k< = 21,
-22< = l< = 22
57 165/16 343, 0.042
0.932
Reflections collected/unique, Rint
Completeness to thetamax
Refinement method
Full-matrix least-squares on F2
18 649/0/545
Full-matrix least-squares on F2
13 603/0/530
Full-matrix least-squares on F2
16 343/0/508
Data/restraints/parameters
Goodness-of-fit on F2
1.091
1.065
1.032
Final R indices [I > 2s(I)]
R indices (all data)
Largest diff. peak and hole
R1 = 0.044, wR2 = 0.107
R1 = 0.040, wR2 = 0.102
R1 = 0.039, wR2 = 0.080
R1 = 0.066, wR2 = 0.114
R1 = 0.065, wR2 = 0.129
R1 = 0.070, wR2 = 0.089
-3
-3
-3
˚
˚
˚
0.92 and -1.14 e. A
0.86 and -1.77e. A
1.14 and -1.08 e. A
Table 3 Selected geometrical parameters for [RuII-hpa-P], [RuIII-hpa-P] and [RuIII-hpa-As]
˚
Interatomic distances (A)
[RuII-hpa-P]
[RuIII-hpa-P]
[RuIII-hpa-As]
Ru1–C8
Ru(1)–O(3)
Ru(1)–O(4)
Ru(1)–N(1)
Ru(1)–P(1)
Ru(1)–P(2)
1.867(2)
2.117(1)
2.123(1)
2.034(1)
2.3825(6)
2.3704(6)
Ru1–Cl1
2.3402(6)
2.046(2)
2.080(1)
2.015(2)
2.399(6)
2.3848(7)
Ru1–Cl1
2.3381(6)
2.048(1)
2.075(1)
2.009(2)
2.4691(4)
2.4637(4)
Ru(1)–O(3)
Ru(1)–O(4)
Ru(1)–N(1)
Ru(1)–P(1)
Ru(1)–P(2)
Ru(1)–O(3)
Ru(1)–O(4)
Ru(1)–N(1)
Ru(1)–As(1)
Ru(1)–As (2)
Bond angles (◦)
C(8)–Ru(1)–N(1)
C(8)–Ru(1)–O(3)
C(8)–Ru(1)–O(4)
C(8)–Ru(1)–P(1)
C(8)–Ru(1)–P(2)
O(3)–Ru(1)–P(1)
O(3)–Ru(1)–P(2)
O(3)–Ru(1)–O(4)
O(4)–Ru(1)–P(1)
O(4)–Ru(1)–P(2)
N(1)–Ru(1)–O(3)
N(1)–Ru(1)–O(4)
N(1)–Ru(1)–P(1)
N(1)–Ru(1)–P(2)
P(1)–Ru(1)–P(2)
177.24(7)
101.39(7)
104.97(7)
91.23(6)
85.86(6)
93.22(4)
91.57(4)
153.64(5)
85.94(4)
90.66(4)
76.86(5)
76.82(5)
90.99(4)
92.04(4)
174.81(2)
Cl(1)–Ru(1)–N(1)
Cl(1)–Ru(1)–O(3)
Cl(1)–Ru(1)–O(4)
Cl(1)–Ru(1)–P(1)
Cl(1)–Ru(1)– P(2)
O(3)–Ru(1)–P(1)
O(3)–Ru(1)–P(2)
O(3)–Ru(1)–O(4)
O(4)–Ru(1)–P(1)
O(4)–Ru(1)–P(2)
N(1)–Ru(1)–O(3)
N(1)–Ru(1)–O(4)
N(1)–Ru(1)–P(1)
N(1)–Ru(1)–P(2)
P(1)–Ru(1)–P(2)
175.63(6)
98.44(5)
107.79(5)
88.95(2)
86.47(2)
94.23(5)
90.53(5)
153.75(7)
85.55(5)
91.91(5)
77.20(7)
76.57(7)
91.79(6)
93.09(6)
173.83(2)
Cl(1)–Ru(1)–N(1)
Cl(1)–Ru(1)–O(3)
Cl(1)–Ru(1)–O(4)
Cl(1)–Ru(1)–As(1)
Cl(1)–Ru(1)–As(2)
O(3)–Ru(1)–As(1)
O(3)–Ru(1)–As(2)
O(3)–Ru(1)–O(4)
O(4)–Ru(1)–As(1)
O(4)–Ru(1)–As(2)
N(1)–Ru(1)–O(3)
N(1)–Ru(1)–O(4)
N(1)–Ru(1)–As(1)
N(1)–Ru(1)–As(2)
As(1)–Ru(1)–As(2)
175.47(5)
98.19(5)
107.69(4)
88.88(2)
86.66(2)
94.45(4)
90.59(4)
154.10(6)
85.24(4)
91.88(4)
77.32(7)
76.80(7)
92.10(5)
92.68(5)
173.71(1)
II
˚
around the ruthenium atom. Though the PPh3 ligands usually
prefer to occupy mutually cis positions for better p-interaction,20
in this complex the presence of CO, a stronger p-acidic ligand,
might have forced the bulky PPh3 ligands to take up mutually
trans positions for steric reasons. The carbonyl group occupies
the site trans to the N1 (ring nitrogen from H2hpa (N1–Ru1–C8,
177.24(7) A)). This may be a consequence of strong Ru →CO
back donation as indicated by the short Ru–C bond (1.867(2)
A) and low CO stretching frequency (1917 cm ), which prefers
s or weak p donor groups occupying the site opposite to CO
to favor the d–p* back donation. The Ru–C bond length of
1.867(2) A in the Ru(CO) fragment is quite normal, similar to
-1
˚
˚
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distortion of the RuNO2P2Cl core from the ideal octahedral
geometry, which is reflected in the twelve cis and three trans angles.
˚
The equatorial Ru–N [2.015(2) A] Ru–O [2.046(2), 2.080(1)] Ru–Cl
˚
[2.0432(6) A] and axial Ru–P [2.3990(8), 2.3848(7)] bond lengths
indicate an axially elongated octahedral coordination environment
around the RuIII ion. The two triphenylphosphine ligands which
are mutually trans to each other are slightly bent towards the
chlorine atom due to the steric requirements of the somewhat bulky
chelating ligand, H2hpa causing a slight deviation from a linear
trans arrangement, which is evident from the P(1)–Ru(1)–Cl(1)
bond angle of 88.95(2)◦ [smaller than P(1)–Ru(1)–N(1) = 91.79(6)◦
and P(2)–Ru(1)–N(1) = 93.09(6)◦]. The N(1)–Ru(1)–Cl(1) bond
angle is 175.63(6)◦ showing that the Cl atom lies trans to the ring
nitrogen. The Ru–P, Ru–O, Ru–N and Ru–Cl bond lengths found
in the complex agree well with those reported for similar ruthenium
complexes.26–29 An acetonitrile and a dichloromethane molecule
share a disordered site. Though the hydroxyl group at the C1 atom
of H2hpa does not take part in metal coordination, it is involved in
intermolecular hydrogen bonding interactions with a neighboring
carboxylate carbonyl O5 (H–Acceptor) atom. In addition, the
complex possesses intermolecular interactions between nitrogen
(from a solvated acetonitrile molecule) and chlorine (from a
solvated dichloromethane molecule) and between chlorine and
oxygen.
Fig.
3
X-ray crystal structure and atom numbering scheme for
[RuIII-hpa-P] as thermal ellipsoids at a 50% probability level. The hydrogen
DNA binding studies
atoms have been omitted for clarity.
Electronic absorption titration. In order to elucidate the nature
of the interaction of the complexes with DNA, UV-vis absorption
spectra were obtained by titration of the complexes with increasing
concentrations of DNA (Fig. 4). The titration processes were
repeated until there was no change in the spectra indicating that
binding saturation had been achieved. The electronic spectra of
all the complexes showed an intense absorption peak around 259
nm in the UV region, which could be attributed to an intraligand
p–p* transition of the coordinated groups in the complex and a
lower energy absorption band around 330 nm. In addition to the
above two bands, the complex [RuIII-hpa-P] showed an additional
MLCT band at 357 nm. The absorbance spectra of [RuIII-hpa-P]
and [RuII-hpa-P] were considerably affected upon binding to calf
thymus DNA, as shown in Fig. 4 and subsequent changes are given
in Table 4.
Clear isosbestic points at 300 nm for [RuIII-hpa-P] and at 290
nm for [RuII-hpa-P] do reveal the presence of two spectroscopically
distinct chromophores (free and bound) in the solution which
indicate the occurrence of a single binding mode of the complexes
with DNA. It was anticipated that the binding of [RuIII-hpa-P]
and [RuII-hpa-P] with DNA could be either by intercalation or
by sitting within either the minor or major grooves of the DNA
structure. As the R ratio increases, no isosbestic point was observed
(data not shown). This is perhaps indicative of a heterogeneous
that observed in structurally characterized carbonyl complexes of
ruthenium.21–23 The two Ru–P bond lengths are similar and are
comparable to those reported for other PPh3 coordinated RuII
complexes.24,25
The molecule is stabilized by intermolecular hydrogen bonding
networks with lattice ethanol molecules and oxygen atoms of
H2hpa. Two ethanol molecules are solvated with the complex.
One ethanol molecule is hydrogen bonded with an uncoordinated
carboxylate oxygen atom O5 (H–Acceptor) of the H2hpa. The
hydroxyl group O1 (H–Donor), the carboxylate group O2 (H–
Acceptor) of the neighboring molecule form hydrogen bonds
to the other ethanol molecule. The oxygen atom of the other
ethanol molecule acts as a donor to the uncoordinated carboxylate
oxygen atom O2 of the neighboring molecule and as an acceptor
to oxygen atom O1 of the hydroxyl group at the C1 atom of
H2hpa.
Structure of [RuIII-hpa-P] and [RuIII-hpa-As]
The coordination geometry around the RuIII ion is a slightly
distorted octahedron, where the basal plane is constructed of
two oxygen atoms, the nitrogen of the tridendate dibasic ligand
H2hpa, and a chlorine atom. The remaining apical coordination
sites are filled up by two triphenylphosphines/triphenylarsines.
The coordination sphere is the same in the two RuIII complexes and
the general structural motifs are very similar with only very slight
differences in the geometrical parameters. Thus, the structure of
one of the RuIII complexes, [RuIII-hpa-P], will be described in detail
here.
Table 4 Changes observed in l for interaction of the complexes with
CT-DNA
Complex
l (nm)
Hypochromism
Red shift (nm)
[RuIII-hpa-P]
[RuII-hpa-P]
330
357
330
39%
36%
30%
11
9
—
The H2hpa ligand coordinated equatorially to the metal with
bite angles of 76.57(7)◦ and 77.20(7)◦. This results in significant
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in the fully DNA-bound form. Each set of data, when fitted to the
above equation, gave a straight line with a slope of 1/(eb-ef) and a
y-intercept of 1/Kb (eb-ef) and Kb was determined from the ratio
of the slope to intercept.
The intrinsic binding constants Kb of [RuIII-hpa-P] and [RuII-
hpa-P] were found to be 1.3 ¥ 104 and 4.6 ¥ 103 M-1 respectively.
Fig. S4, ESI† indicates that [RuIII-hpa-P] binds more strongly
than [RuII-hpa-P] to the double helix. The observed Kb values for
[RuIII-hpa-P] and [RuII-hpa-P] are lower than that of the classical
intercalators (ethidium bromide, Kb, 1.8 ¥ 106 M-1 in 25 mM
Tris-HCl/40 mM NaCl buffer, pH 7.9) and partial intercalating
metal complexes ([Ru(phen)2(dppz)]2+, Kb > 106 M-1) bound to
CT-DNA.30 Hence, it is inferred that the interaction of the two
complexes with DNA is nonintercalative.
Competitive studies with ethidium bromide
The new complexes show no fluorescence at room temperature
in solution or in the presence of DNA, and their binding to
DNA cannot be directly predicted through the emission spectra.
The extent of quenching of the fluorescence of ethidium bromide
(EB) bound to DNA is used to determine the binding of a
second molecule to DNA.31 This fluorescence based competition
technique can provide indirect evidence for the DNA binding
mode.32 Fig. S5, ESI† shows the emission spectra of the EB-
DNA system in the absence and presence of the complexes
[RuIII-hpa-P] and [RuII-hpa-P]. The fluorescence intensity of EB
shows a remarkable decrease with increasing concentration of the
complexes [RuIII-hpa-P] and [RuII-hpa-P], which is a consequence
of the displacement of some EB molecules from the DNA. The
addition of [RuIII-hpa-P] to DNA pretreated with EB caused an
appreciable reduction in emission intensity compared with that
observed in the complex [RuII-hpa-P] at a lower mixing ratio (R <
0.16). This indicated that the complex [RuIII-hpa-P] could strongly
compete with EB in binding to DNA. As the R ratio (R > 0.16)
increases, there is no appreciable change in the emission intensity
of the EB-DNA system (data not shown). The complexes [RuII-
hpa-As] and [RuIII-hpa-As] do not provoke any significant changes
in the intensity of the emission band at 592 nm of the DNA-EB
system indicating that they cannot displace EB from the DNA-EB
complex. This trend indicates that there is only weak binding of
complexes [RuII-hpa-As] and [RuIII-hpa-As] with the CT-DNA. The
binding constant Kb of complexes [RuII-hpa-P] and [RuII-hpa-P] to
DNA can be analyzed through the Stern–Volmer equation
Fig. 4 UV spectra of ruthenium complexes in the absence (- - -) and in the
presence of CT-DNA in increasing amounts, [Complex] = 10 mM, [DNA] =
0–130 mM.
binding of complexes for [RuIII-hpa-P] and [RuII-hpa-P] at higher
mixing ratios (R > 0.16).
In contrast, at various DNA concentrations, the absorp-
tion spectra of [RuII-hpa-As] and [RuIII-hpa-As] exhibited hyper-
chromicity without a clear isosbestic point. These compounds may
assume a different DNA binding mode from that of the other two.
In other words, strong interaction may be ruled out as a major
binding mode of [RuII-hpa-As] and [RuIII-hpa-As] with the double
helical DNA. The lack of an isosbestic point may suggest that its
chromophore environment was not altered after the interaction
with DNA. The observed hyperchromic effect for [RuII-hpa-As]
and [RuIII-hpa-As] suggested the binding could be via external
contact (electrostatic binding).
I0 / I = 1 + Ksqr
where I0 and I represent the fluorescence intensities in the
absence and presence of the complex, respectively, and r is the
concentration ratio of the complex to DNA. Ksq is a linear
Stern–Volmer quenching constant dependent on the ratio of the
bound concentration of EB to the concentration of DNA (Fig.
S6, ESI†). In particular, the high Ksq values of the complexes
show that they can be bound tightly to the DNA. The Kb values
for [RuIII-hpa-P] and [RuII-hpa-P] are 1.2 0.1 and 0.97 0.03
respectively, suggesting that [RuIII-hpa-P] binds with CT-DNA
more strongly than [RuII-hpa-P]. At lower concentrations, the
Ru complex competitively inhibits the EB interaction whereas
at higher concentrations, the complexes are noncompeting with
the EB interaction. The above results suggest that the complexes
Based on the above observations, we presume that there are
some interactions between complexes [RuIII-hpa-P], [RuII-hpa-P]
and DNA. The binding strength of the complex with DNA was
further mirrored in the value of the intrinsic binding constant (Kb).
The [RuIII-hpa-P] and [RuII-hpa-P] exhibited a regular decrease
in intensity of the absorption band from which the value of the
intrinsic equilibrium binding constant (Kb) was calculated using
[DNA] / (ea-ef) = [DNA] / (eb-ef) + 1/Kb (eb-ef)
where [DNA] is the concentration of the DNA in base pairs,
ea = Aobsd/[compound], ef is the extinction coefficient for the free
compound and eb is the extinction coefficient for the compound
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[RuIII-hpa-P] and [RuII-hpa-P] efficiently bind with CT-DNA at
lower concentrations only. This insight is in accordance with the
electronic absorption spectral information.
of denaturation of CT-DNA were measured by plotting the UV
maximum absorption of DNA at 260 nm versus the temperature, in
the absence and presence of ruthenium complexes at two different
molar ratios R = [Ru]/[DNA] = 0, 0.2, and 0.07 (Fig. 6). The
results revealed that the Tm of the free DNA duplex was 60.0
0.1 ◦C, which was increased by 2 and 3 ◦C in the presence
of complexes [RuII-hpa-P] and [RuIII-hpa-P] respectively at low R
ratios. This implies that they are non-intercalators because the
relative absorbance is not so high compared to that of the pure
DNA sample. At a high R ratio, no obvious change in the melting
profile of DNA can be observed under the same measurement
conditions. This scenario may indicate the interaction of the [RuII-
hpa-P] and [RuIII-hpa-P] with DNA is stronger at low mixing ratios
(R < 0.16) than at higher ratios (R > 0.16), which is in consonance
with the conclusions derived from the other studies given above.
Under the same set of conditions, the addition of [RuII-hpa-As]
and [RuIII-hpa-As] to the double helical DNA had no appreciable
effect on Tm of free DNA (data not shown). It is revealed that they
did not interact substantially with DNA.
Viscometric studies
The structural changes of DNA by binding with small molecules
have been the authentic indication of the binding mode. Viscosity
measurements are one of the traditional and still one of the
most unambiguous methods to scrutinize DNA structural changes
by the length changes of rod-like DNA. It has been proved
that intercalative DNA binding with small molecules leads to
an increase in viscosity, since the intercalative binding causes
elongation of the DNA polymer by separation of the DNA base
pairs.33 The effect of the complexes on the viscosity of DNA
is shown in Fig. 5. Complexes [RuII-hpa-As] and [RuIII-hpa-As]
actually have no effect on the viscosity of DNA, while complexes
[RuIII-hpa-P] and [RuII-hpa-P] decreased the relative viscosity of the
DNA at a low mixing ratios (R < 0.16). The observed significant
decreases in viscosity of DNA with the complexes [RuIII-hpa-P]
and [RuII-hpa-P] are similar to that of D-[Ru(phen)3]2+34,35 which
may be explained by a binding mode that produces bends or
kinks in the DNA helix and thus reduces its effective length and,
concomitantly, its viscosity. The viscosity results suggested that
complexes [RuIII-hpa-P] and [RuII-hpa-P] bind with DNA in a non-
intercalative manner, possibly a groove binding mode.
Fig. 5 Effect of increasing amounts of [RuIII-hpa-P] ( ) and [RuII-hpa-P]
᭹
(ꢀ) on the relative viscosities of CT-DNA in 5 mM Tris-HCl buffer (pH
7.0).
Thermal denaturation experiments
Optical photophysical probes commonly provide basic, but not
sufficient clues to ascertain the binding mode. The viscosity
measurement was introduced as further support to elucidate the
interaction between the metal complex and DNA. Additional
strong evidence for binding of the ruthenium complexes to the
double helix of DNA is a thermal denaturation experiment. The
melting temperature (Tm) is defined as the temperature at which
half of the DNA strands are in the double-helical state and half
are in the “random-coil” state. A large change in the Tm of DNA is
indicative of a strong interaction with DNA.36 The melting profiles
Fig. 6 Thermal denaturation plots of 100 mM CT-DNA alone ( ) and
in the presence of complexes at R = [Ru]/[DNA] = 0.2 (᭡), 0.07 (ꢁ).
᭹
The planarity area (S) of a chelating ligand of the metal
complex is surely an important factor because the larger the
planarity area of the ligand, the greater the stacking interaction
between the ligand and base pairs of DNA. From the above DNA
binding results, it is obvious that the present ligand system lacks
extended p-systems, ruling out the possibility of any intercalative
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Table 5 Antioxidant activity of all the complexes, vitamin C and BHT against various radicals
IC50(mM)
∑
DPPH∑
OH∑
NO∑
O2
H2O2
Metal chelating
-
Compounds
[RuII-hpa-P]
[RuII-hpa-As]
[RuIII-hpa-P]
[RuIII-hpa-As]
Vitamin C
BHT
55
42
53
39.6
57.2
13.8
24.4
221.4
131.6
55.5
66
23.9
31
238.5
149.8
59.6
73.7
32.7
38.8
—
62.7
19.6
26.5
147.6
86.2
53.6
11.5
17.1
232.8
163.4
60.9
18.2
25.2
215.8
154.3
—
interaction, but has the potential to bind to DNA. The ex-
perimental results suggest that the complexes could bind DNA
in two different modes. The binding behavior observed for the
triphenylphosphine analog with double helical CT-DNA differs
from triphenylarsine complexes. The [RuII-hpa-P] and [RuIII-hpa-P]
bind with double helical DNA in the groove binding mode whereas
[RuII-hpa-As] and [RuIII-hpa-As] bind by means of the electrostatic
mode. This is related to the molecular structures of the complexes.
Among the complexes examined, the triphenylarsine analog shows
the lowest binding strength (electrostatic) to double-helical DNA.
The difference in the binding modes between triphenylphosphine
and triphenylarsine analogs may be due to the presence of bulky
AsPh3 groups trans to each other, which causes steric hindrance.
It reveals that the effective binding strength of the chelating ligand
(H2hpa) towards the double helical DNA would be prevented by
the steric clashes from the six phenyl rings of the trans AsPh3 with
the DNA surface. The steric clash involving co ligands and the
DNA polymer would hinder the placement of the chelating rings
in between the DNA helix. These steric clashes also appeared in
the triphenylphosphine complexes but the potency is small when
compared to the triphenylarsine analogs. These observations also
indicate that the planarity of the chelating ligand in the complex
has a significant effect on DNA binding affinity, which decreases
in the order [RuIII-hpa-P] > [RuII-hpa-P] > [RuII-hpa-As] ~ [RuIII-
hpa-As]. The difference in DNA-binding properties of complexes
is due to the different co-ligands and different oxidation states of
the metal ion.
evaluate the antioxidant activity. Hydrogen peroxide itself is not
very reactive, but sometimes it is toxic to cells because it may
give rise to hydroxyl radicals in the cells.39 The free radical
scavenger action is very important, especially in the case of the
superoxide anion, since it prevents or attenuates the formation
of peroxynitrite and hydroxyl radicals. The overproduction of
peroxynitrite and hydroxyl radicals constitutes a very important
feature regarding tissue damage mechanisms during pathological
processes. Numerous biological reactions generate the superoxide
radical, which is a highly toxic species, along with other reactive
molecules that tend to react with cellular components such as
membrane lipids and proteins, thereby disturbing their function
and consequently cellular homeostasis.40,41 Thus, the study of
scavenging of this radical is important.42 Nitric oxide or reactive
nitrogen species, formed during their reaction with oxygen or
-
+
with superoxide, such as NO2, N2O4, N3O4, NO3 and NO2
are very reactive. These species are responsible for altering the
structural and functional behavior of many cellular components.
Further, the scavenging activity may also help to arrest the chain of
reactions initiated by excess generation of NO that is detrimental
to human health. Among all free radicals, the hydroxyl radical
is a major product arising from the high-energy ionization of
water, the most important biological sources of which are the
Haber–Weiss and Fenton reactions, involving the superoxide
anion, hydrogen peroxide and transition metals.43 This is the
most aggressive oxidant known and therefore the most dangerous
oxygen metabolite, which is capable of attacking any biological
molecule and can be implicated in the tissue damage associated
with inflammation, therefore elimination of this radical is one of
the major aims of antioxidant administration.44
Antioxidative activity
The two RuII complexes and the two RuIII complexes with similar
structures showed almost comparable antioxidant activities. In
general, the RuIII complexes showed better activity than the
RuII complexes. This might be due to the d5 low spin electronic
configuration and the availability of an odd electron in RuIII
complexes, which increases the capacity to stabilize the unpaired
electrons and thereby scavenge the free radicals. The electron
deficient metal centers in RuIII complexes might increase the acidity
of the available ligand proton thereby increasing the antioxidant
activity in terms of H+ donors when compared to RuII complexes.
The hydroxyl radical scavenging power of the tested complexes
was the greatest, and the metal chelating ability was the lowest.
Being a potent scavenger of hydroxyl radicals, the complexes
could capture superoxide anion radicals and hydrogen peroxide,
as evident from the IC50 values. The metal chelating ability of
the tested complexes is poor owing to the lack of active groups
for chelation, and might be the weak interaction between the
Since the complexes exhibit reasonable DNA-binding affinity, it
is considered worthwhile to study other potential aspects, such
as antioxidant and antiradical activity. Hence, we carried out
experiments to explore the free radical scavenging ability of the
complexes, with the hope of developing potential antioxidants
and therapeutic reagents for respiratory diseases, such as asthma,
emphysema and asbestosis.37 The IC50 values of all the complexes
(Table 5 and Fig. S7, ESI†) obtained from different types of
assay experiments strongly supports that they possess excellent
antioxidant activities, which are better than those of standard
antioxidants including the natural antioxidant vitamin C and the
synthetic antioxidant BHT (butylated hydroxytoluene).
Free radicals can induce DNA damage in humans. The dam-
age to DNA has been suggested to contribute to aging and
various diseases including cancer and chronic inflammation.38
The DPPH radical has been widely used to test the ability of
compounds as free radical scavengers or hydrogen donors to
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Scheme 2 Proposed mode of action of ruthenium anticancer agents.
complexes and Fe2+. Although the radical scavenging mechanism
of the complexes under study remains unclear, the experimental
results are helpful in designing more effective antioxidant agents
against free radicals.
Table 6 IC50 (mM) ruthenium complexes and cisplatin against various
cancer cell lines
IC50(mM)a
Complexes
HeLa
HepG2
HEp-2
NIH 3T3
[RuII-hpa-P]
[RuII-hpa-As]
[RuIII-hpa-P]
[RuIII-hpa-As]
Cisplatin
27
31
75
128
16
22
32
73
353
20
23
35
50
63
09
248
187
124
109
177
Cytotoxic activity evaluation by MTT assay
Kinetic lability/inertness toward ligand substitution is a major
determinant that controls the covalent interactions of a metal com-
plex with biological target molecules. RuIII complexes probably act
as prodrugs that are relatively inert toward ligand substitution and
therefore their anticancer activity depends on the ease of reduction
to more labile plus kinetically more reactive RuII complexes. The
resulting RuII species are generally less inert, have a high propensity
for ligand exchange reactions and may therefore interact with
target molecules more rapidly.45–47
a fifty percent inhibitory concentration after exposure for 48 h in the MTT
assay
bind DNA bases) in inhibiting the growth of the cancer cell.
Despite this potency, [RuII-hpa-P] and [RuII-hpa-As] were much
less toxic toward normal cells, with IC50 values at 248 and 187 mM
respectively, which are significantly higher than those of cisplatin
(177 mM). These results suggest that they possess great selectivity
between cancer and normal cells and display application potential
in cancer chemoprevention and chemotherapy. Cytotoxic effects
of [RuIII-hpa-P] and [RuIII-hpa-As] are only low to moderate on the
tested cell lines and the complex [RuIII-hpa-As] did not show any
significant activity even up to 128–353 mM concentration on HeLa
and HepG2 cell lines. This might be due to the need to reduce them
to RuII before they can exert their action.
We intended to verify this bioreductive activation mechanism
(Scheme 2) under in vitro conditions, and hence, cytotoxicity
studies were carried out. Moreover, as the balance between the
therapeutic potential and toxic side effects of a compound is
very important when evaluating its usefulness as a pharmaco-
logical drug, experiments were designed to investigate the in
vitro cytotoxicity of synthesized ruthenium complexes and a
benchmark compound cisplatin, against three human cancer lines
including HeLa, HepG2, HEp-2 and the normal cell line NIH 3T3.
Cytotoxicity was determined by means of a colorimetric micro-
culture MTT assay, which measures mitochondrial dehydrogenase
activity as an indication of cell viability. The IC50 values of four
ruthenium complexes and cisplatin for selected cell lines which
show interesting results are presented in Table 6. It is evident from
the graphs (Fig. S8–S11, ESI†) that the number of cells decreased
with an increase in the concentration of the complexes. With regard
to the hypothesized bioreductive activation mechanism, the RuII
complexes showed higher potential antineoplastic activity than
did the RuIII complexes, as evidenced by the lower IC50 values.
The low O2 content and lower pH in tumor cells should favor
RuII, which is generally supposed to coordinate more readily with
biomolecules than RuIII,48 and thus provide for selective toxicity.
Some promising cytotoxic effects were observed for RuII complexes
toward the HepG2 and HeLa cell lines as reported in Table 6,
even though they were slightly lower than cisplatin. These results
are much better than those previously reported for other RuII
complexes.49 With HepG2, the [RuII-hpa-P] appears to satisfy the
conditions for carrying out the “cisplatin magic” (differential
reactivity in inter and intracellular media and its capacity to
Conclusion
A series of RuII and RuIII complexes containing 4-hydroxy-
pyridine-2,6-dicarboxylic acid and PPh3/AsPh3 have been pre-
pared. The structures were established by X-Ray crystallography
and spectroscopic methods. Furthermore, they were characterized
with regard to drug-like properties such as DNA interactions,
radical scavenging ability and tumor-inhibiting potential in a
cancer cell line panel. The influence of nature and size of the co-
ligands in the complexes on binding with DNA was demonstrated.
The fine points obtained from various antioxidant assays showed
that the RuIII complexes exhibited excellent radical scavenging
activities against hydroxyl radical, superoxide anion radical, nitric
oxide radical, DPPH radical and hydrogen peroxide. Moreover,
the cytotoxic studies showed that RuII complexes displayed less
toxicity on normal cells and significant cytotoxic activity against
HepG2 and HeLa cell lines, different from that of RuIII complexes.
The findings described should be considered in the future design of
ruthenium pharmaceuticals to successfully predict the role of the
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ligand and the RuIII/RuII redox properties on anticancer activities,
although the generality of some of the above conclusions has still
to be tested with a wider variety of ruthenium complexes, which
is in progress in our laboratory.
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©