Impact of chelation on anticancer activities of organometallic ruthenium(II) complexes containing 2,5-di(1: H -pyrazol-1-yl)-1,4-benzoquinone: Synthesis, structure, DNA/protein binding, antioxidant activity and cytotoxicity

Article abstract of DOI:10.1039/c6ra05867a

To gain some insight into the influence of chelation on anticancer activities, two new bivalent organometallic ruthenium complexes, [Ru(L)Cl(CO)(PPh₃)₂] (3) and [Ru(L)Cl(CO)(AsPh₃)₂] (4), (HL = 2,5-di(1H-pyrazol-1-yl)-1,4-benzoquinone) were synthesized, structurally characterized comprehensively by elemental analysis, IR, electronic, ¹H/¹³C NMR spectral studies and their biological activities (DNA/protein interactions, antioxidant and cytotoxic activity studies) have been investigated and compared with that of appropriate precursor complexes [RuHCl(CO)(PPh₃)₃] (1), [RuHCl(CO)(AsPh₃)₃] (2) and the ligand LH. The crystal structures of both the new complexes 3 and 4 were determined by single crystal X-ray diffraction studies, which unveil that the central ruthenium ion adopted distorted octahedral geometry with L as a monobasic bidentate donor and the chelator was observed to undergo C-H activation at one of the ortho positions leading to the formation of a five-membered metallacycle. DNA/protein interactions of the complexes have been examined by photophysical studies, which demonstrated a non-intercalative binding mode of the complexes with DNA and the complexes strongly quench the fluorescence intensity of bovine serum albumin (BSA) through the mechanism of static quenching. The new complexes demonstrated better binding affinity with DNA/protein. The compounds were evaluated for their free radical scavenging ability involving DPPH radicals, hydroxyl radicals, nitric oxide radicals, superoxide anion radicals and hydrogen peroxide. The results showed that the new complexes 3 and 4 possess significant radical scavenging ability and outperform the standard antioxidants Vitamin C and BHT. The results of in vitro cytotoxicity of the complexes tested against B16F10, HeLa, A549, MCF7 and SKOV3 cells by SRB assay were compared with the standard drug cisplatin and that the new complexes 3 and 4 showed better activity in inhibiting the growth of the cancer cells. The influence of chelation was seen in all the biological activities studied and the new ruthenium chelates 3 and 4 were found to be superior to the precursor complexes 1, 2 and the ligand LH in DNA/protein binding properties, radical scavenging ability and cytotoxicity.

Full text of DOI:10.1039/c6ra05867a

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Page 1 of 37
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Impact of chelation on anticancer activities of organometallic
ruthenium(II) complexes containing 2,5ꢀdi(1Hꢀpyrazolꢀ1ꢀyl)ꢀ1,4ꢀ
benzoquinone: Synthesis, structure, DNA/protein binding,
antioxidant activity and cytotoxicity
Thangavel Sathiya Kamatchi,a,d Palaniappan Kalaivani,b Frank R. Fronczek,c Karuppannan
Natarajan*a, Rathinasabapathi Prabhakaran*a
a Department of Chemistry, Bharathiar University, Coimbatore 641046, India.
b Department of Chemistry, Nirmala College for Women, Bharathiar University, Coimbatore
641018, India.
c Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA.
d Department of Chemistry, Sakthi College of Arts and Science for Women, Oddanchatram,
Dindigul 624 624, India
ABSTRACT: To gain some insight into the influence of chelation on anticancer activities, two
new bivalent organometallic ruthenium complexes, [Ru(L)Cl(CO)(PPh3)2] (3) and
[Ru(L)Cl(CO)(AsPh3)2] (4), (HL = 2,5ꢀdi(1Hꢀpyrazolꢀ1ꢀyl)ꢀ1,4ꢀbenzoquinone) were synthesized,
1
structurally characterized comprehensively by elemental analysis, IR, electronic, H/13C NMR
spectral studies and their biological activities (DNA/protein interactions, antioxidant and
cytotoxic activity studies) have been investigated and compared with that of appropriate
precursor complexes [RuHCl(CO)(PPh3)3] (1), [RuHCl(CO)(AsPh3)3] (2) and the ligand LH.
The crystal structures of both the new complexes 3 and 4 were determined by single crystal
Xꢀray diffraction studies, which unveil that the central ruthenium ion adopted distorted
octahedral geometry with L as a monobasic bidentate donor and the chelator was observed to
undergo C–H activation at one of the ortho positions leading to the formation of fiveꢀmember
metallacycle. DNA/protein interactions of the complexes have been examined by photophysical
studies, which demonstrated nonꢀintercalative binding mode of the complexes with DNA and the
complexes strongly quench the fluorescence intensity of bovine serum albumin (BSA) through
the mechanism of static quenching. The new complexes demonstrated better binding affinity
with DNA/protein. The compounds were evaluated for their free radical scavenging ability
involving DPPH radical, hydroxyl radical, nitric oxide radical, superoxide anion radical and
hydrogen peroxide. The results showed that the new complexes 3 and 4 possess significant
radical scavenging ability and outperforming the standard antioxidants vitamin C and BHT.
The results of in vitro cytotoxicity of the complexes tested against B16F10, HeLa, A549, MCF7
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and SKOV3 cells by SRB assay were compared with the standard drug cisplatin and that the new
complexes 3 and 4 showed better activity in inhibiting the growth of the cancer cells. The
influence of chelation was seen in all the biological activities studied and the new ruthenium
chelates 3 and 4 were found to be superior to the precursor complexes 1, 2 and the ligand LH in
DNA/protein binding properties, radical scavenging ability and cytotoxicity.
……………………………
Corresponding authors: Eꢀmail: k_natraj6@yahoo.com; rpnchemist@gmail.com; Tel.: +91 422
2428319; Fax: +91 422 2422387.
2

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Introduction
Metal ions play a vital role in a vast number of biological processes1,2 and it is known
that chelation of metal ions with organic ligand acts synergistically to increase their biological
activities.3 Further, many organic compounds used in medicine do not have a purely organic
mode of action and require traces of metal ions directly or indirectly for activation or
biotransformation. Since antiquity, human beings are suffering from a variety of diseases out of
which cancer is the second most common cause of death after cardiovascular diseases. In the
search for more selective antiꢀtumour agents, there is considerable interest in finding metal
complexes as bioꢀreductive drugs. Bioꢀreductive drugs are designed to take advantage of some of
the unique features of solid tumours, in particular reduced oxygen tension and the overꢀ
expression of certain reductase enzymes,5 which are capable of reducing several quinoneꢀ
containing anticancer agents to DNAꢀdamaging species.6ꢀ8 The biological activity of the
quinones is attributed to their facile in vivo and in vitro reduction.9 It is well known that any
change in the substituent attached to the quinone ring alters its capability to accept electrons by
tuning its redox chemistry and consequently modifies its biological activity.10ꢀ12 In addition to
quinones, different pyrazole derivatives have also been tested for their antiproliferative activities
in vitro and antitumor activity in vivo, often resulting in promising lead compounds.13ꢀ17
Moreover, a variety of 1,4ꢀbenzoquinones and their nitrogen analogues have been reported for
their antiꢀtumour activities.18ꢀ20 The sensible to design the bioactive ligand was to combine in a
single molecule, the known pharmacophore benzoquinone ring with anchor pyrazole ring. On
moving to the bioꢀreductive metal fragment to chelate pyrazole bound benzoquinone ligand,
ruthenium scaffolds belong to a very promising class of antiꢀtumor agents, which have an
important role of the oxidation state that allows them to exist in the biological fluids in almost all
common oxidation states from II to IV.21 A number of ruthenium complexes containing
heterocyclic rings have previously been shown to display promising anticancer activities, and
two of them, NAMIꢀA and KP109, have been scheduled for clinical trials.22ꢀ24
With this back ground in mind, we attempted to synthesize new ruthenium chelates by
reacting [RuHCl(CO)(PPh3)3] / [RuHCl(CO)(AsPh3)3] and 2,5ꢀdi(1Hꢀpyrazolꢀ1ꢀyl)ꢀ1,4ꢀ
benzoquinone and obtained new organometallic ruthenium(II) complexes. Here in, we report the
synthesis, structure, DNA/protein binding, free radical scavenging and cytotoxicity of new novel
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organometallic ruthenium(II) complexes. Though 2,5ꢀdi(1Hꢀpyrazolꢀ1ꢀyl)ꢀ1,4ꢀbenzoquinone is
known to organic chemists as intermediates, it has not been used as a ligand for the synthesis of
any metal complexes.
Experimental section
Materials and Instrumentation.
All the chemicals were reagent grade and were used as
received from commercial suppliers unless otherwise stated. All the solvents were degassed and
distilled according to the standard procedures.25 Commercially available RuCl3.3H2O (Himedia)
was used to prepare the precursor complexes. The starting complexes [RuHCl(CO)(PPh3)3],26
[RuHCl(CO)(AsPh3)3]27 were prepared as reported earlier. The ligand 2,5ꢀdi(1Hꢀpyrazolꢀ1ꢀyl)ꢀ
1,4ꢀbenzoquinone (LH) was prepared according to the literature procedure28 with necessary
modifications. Melting points were determined with Lab India instrument. Elemental analyses of
carbon, hydrogen and nitrogen were performed on Vario EL III Elementar elemental analyzer.
Electronic absorption spectra of the complexes were recorded using JASCO 600
spectrophotometer and emission measurements were carried out by using a JASCO FPꢀ6600
spectrofluorometer. Nicolet Avatar Model FTꢀIR spectrophotometer was used to record the IR
1
spectra (4000–400 cmꢀ1) of the free ligand and the complexes as KBr pellets. H NMR and
13C NMR spectra were recorded on Bruker AV 500 (500 MHz (1H) and 125 MHz (13C))
spectrometer using tetramethylsilane (TMS) as an internal reference. The chemical shifts are
expressed in parts per million (ppm). Protein free Herring sperm DNA obtained from SRL
chemicals was stored at 0ꢀ4 °C. Bovine serum albumin (BSA) and ethidium bromide (EB) were
obtained from SigmaꢀAldrich. Antioxidant activity measurements were done using UV
spectrophotometer (UVꢀ1800, Shimadzu).
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Xꢀray crystallography
Xꢀray diffraction measurements were performed on a Nonius Kappa CCD diffractometer
equipped with graphite monochromated Mo Kα radiation and an Oxford Cryostream cryostat.
The structure of the complexes was solved by direct methods and refinements were carried out
by Full matrix leastꢀsquares techniques. The hydrogen atoms were generally visible in difference
maps and were placed in idealized positions and treated as riding in the refinement. The
following computer programs were used: structure solution SIRꢀ97,29 refinement SHELXLꢀ97,30
molecular diagrams and ORTEPꢀ331 for Windows.
DNA interaction studies
All the experiments involving the binding of the compounds with HSꢀDNA were carried
out in deionised water with tris(hydroxymethyl)ꢀaminomethane (Tris, 5 mM) and sodium
chloride (50 mM) and adjusted to pH 7.2 with hydrochloric acid at room temperature. A solution
of herring sperm DNA (0.1 g) in TrisꢀHCl buffer (10 ml) gave a ratio of UV absorbance at 260
and 280 nm of ca. 1.7ꢀ1.8: 1 indicating that the DNA was sufficiently free of protein. The DNA
concentration per nucleotide was determined by absorption spectroscopy using the molar
absorption coefficient (6600 Mꢀ1 cmꢀ1) at 260 nm. The compounds were dissolved in a mixed
solvent of 5 % DMSO and 95 % TrisꢀHCl buffer. Absorption spectra were recorded after
equilibrium at 20 °C for 10 min. Absorption titration experiments were performed with a fixed
concentration of the compounds (10 µM) while gradually increasing the concentration of DNA
(0ꢀ50 µM). While measuring the absorption spectra, an equal amount of DNA was added to both
the test solution and the reference solution to eliminate the absorbance of DNA itself.
In order to know the exact mode of attachment of compounds with HSꢀDNA,
fluorescence quenching experiments of ethidium bromide (referred to as EB)ꢀDNA complex
were also carried out by increasing concentration of ruthenium(II) complexes (0ꢀ50 µM) to the
samples containing 10 µM EB, 10 µM DNA and Tris buffer. Before the measurements, the
system was shaken and incubated at room temperature for ~5 min. The emission was recorded at
530ꢀ750 nm. The fluorescence spectra were obtained with an excitation wavelength of 522 nm
and an emission wavelength of 620 nm.
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Protein binding studies
Binding of the compounds with bovine serum albumin (BSA) was studied from the
fluorescence spectra recorded with an excitation wavelength of at 280 nm and the corresponding
emission at 345 nm assignable to that of BSA. The excitation and emission slit widths and scan
rates were maintained constant for all of the experiments. A stock solution of BSA was prepared
in 50 mM phosphate buffer (pH = 7.2) and stored in the dark at 4 °C for further use.
A concentrated stock solution of the compounds was prepared as mentioned for the DNA
binding experiments, except that the phosphate buffer was used instead of a TrisꢀHCl buffer for
all of the experiments. Titrations were manually done by using a micropipette for the addition of
the compounds. For synchronous fluorescence spectra also, the same concentrations of BSA and
the compounds were used, and the spectra were measured at two different ꢁλ values (difference
between the excitation and emission wavelengths of BSA), such as 15 and 60 nm.
Antioxidant assays
The ability of the ligand and the new organometallic ruthenium complexes to act as
hydrogen donors or free radical scavengers was tested by conducting a series of in vitro
antioxidant assays involving DPPH radical, hydroxyl radical, nitric oxide radical, hydrogen
peroxide, superoxide anion radical and metal chelating assay. The results obtained were
compared with standard antioxidants including natural antioxidant Vitamin C and synthetic
antioxidant BHT (Butylated Hydroxy Toluene).
DPPH
˙
scavenging assay
The DPPH radical scavenging activity of the compounds was measured according to the
method of Blois.32 DPPH radical is a stable free radical and due to the presence of an odd
electron, it shows a strong absorption band at 517 nm in visible spectrum. If this electron
becomes paired off in the presence of a free radical scavenger, this absorption vanishes resulting
in decolorization stoichiometrically with respect to the number of electrons taken up. Various
concentrations of the experimental compounds were taken and the volumes were adjusted to 100
µL with methanol. About 5 mL of 0.1 mM methanolic solution of DPPH was added to the
portion of samples and standards (BHT and Vitamin C) and shaken vigorously. Negative control
was prepared by adding 100 µL of methanol in 5 mL of 0.1 mM methanolic solution DPPH. The
tubes were allowed to stand for 20 minutes at 27 °C. The absorbance of the sample was
measured at 517 nm against the blank (methanol).
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OH
˙
scavenging assay
The hydroxyl radical scavenging activity of the compounds has been investigated by
using the Nash method.33 In vitro hydroxyl radicals were generated by 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. In a typical experiment, a mixture of 1.0 mL of ironꢀ
EDTA solution (0.13 % ferrous ammonium sulfate 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 which contains fixed concentration of the test
compounds. The reaction was initiated by adding 0.5 mL of ascorbic acid (0.22 %) and was
incubated at 80ꢀ90 °C for 15 min in a water bath. After incubation, the reaction was terminated
by the addition of 1.0 mL of iceꢀcold 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 the reagent blank.
NO
˙
scavenging assay
Assay of nitric oxide (NO˙) scavenging activity is based on the method,34 where sodium
nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide,
which interacts with oxygen to produce nitrite ions. This can be estimated using Griess 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 compounds, 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% Nꢀ(1ꢀnaphthyl)ethylenediaminedihydrochloride was added. The absorbance of
the chromophore formed was measured at 546 nm.
H2O2 scavenging assay
The ability of the compounds to scavenge hydrogen peroxide was determined using the
method of Ruch et al.35 In a typical experiment, a solution of hydrogen peroxide (2.0 mM) was
prepared in phosphate buffer (0.2 M, pHꢀ7.4) and its concentration was determined
spectrophotometrically from absorption at 230 nm with molar extinction coefficient 81 Mꢀ1cmꢀ1.
The compounds (100 ꢂg/mL), BHT and Vitamin C (100 µg/mL) were added to 3.4 mL of
phosphate buffer prepared above together with hydrogen peroxide solution (0.6 mL). An
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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 is based on the capacity of the
compounds to inhibit formazan formation by scavenging the superoxide radicals generated in
riboflavinꢀlightꢀNBT system.36 In a typical experiment, a 3 mL reaction mixture contained 50
mM sodium phosphate buffer (pH 7.6), 20 µg riboflavin, 12 mM EDTA, 0.1 mg NBT and 1 mL
complex solution (20ꢀ100 µg/mL). Reaction was started by illuminating the reaction mixture
with different concentrations of complex for 90 s and immediately, the absorbance was measured
at 590 nm. The entire reaction assembly was enclosed in a box lined with aluminium foil.
Identical tubes with reaction mixture kept in dark served as blanks.
For the above five assays, all the tests were run in triplicate and the percentage activity
was calculated using the following equation
Scavenging activity (%) = [(A0 – A1) / A0] × 100
Where A0 is the absorbance of the control, A1 is the absorbance of the complex/standard.
When the inhibition of the tested compounds is 50 %, the tested compound concentration is IC50.
In vitro anticancer activity evaluation by SRB assay
The cell lines were grown in RPMIꢀ1640 medium containing 10 % fetal bovine serum
and 2 mM Lꢀglutamine. For the screening experiment cells were inoculated into 96 well
microtiter plates in 90 µL at plating densities, depending on the doubling time of individual cell
lines. After cell inoculation, the microtiter plates were incubated at 37 °C, 5 % CO2, 95 % air and
100 % relative humidity for 24 h prior to addition of experimental compounds. After 24 h, one
plate of each cell line was fixed in situ with TCA, to represent a measurement of the cell
population for each cell line at the time of compound addition. Cisplatin and the test compounds
were dissolved in dimethyl sulphoxide and stored frozen prior to use. The cytotoxicity assay was
done against B16F10 (Mouse melanoma), HeLa (Human Cervix cancer), A549 (Human Lung
adenocarcinoma), SKOV3 (Human Ovarian adenocarcinoma) and MCF7 (Human Breast cancer)
cell lines using a colorimetric SRB assay method. Solutions of different concentration of the
complexes and cisplatin under test were added to the cell monolayer. Triplicate wells were
prepared for each individual concentration. After compound addition, plates were incubated at
standard conditions for 48 h and assay was terminated by the addition of cold trichloroacetic acid
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(TCA). Cells were fixed in situ by the gentle addition of 50 µL of cold 30 % (w/v) TCA (final
concentration, 10 % TCA) and incubated for 60 minutes at 4 °C. Sulforhodamine B (SRB)
solution (50 µL) at 0.4 % (w/v) in 1 % acetic acid was added to each of the wells, and plates
were incubated for 20 minutes at room temperature. After staining, unbound dye was recovered
and the residual dye was removed by washing five times with 1 % acetic acid. Bound stain was
subsequently eluted with 10 mM tris base (pH 10.5) for 5 min. The absorbance was read on an
Elisa plate reader at a wavelength of 540 nm with 690 nm as reference wavelength. The results
were expressed as the concentration at which there was 50 % inhibition (IC50).
SYNTHESIS OF LIGAND
Preparation of 2,5ꢀdi(1Hꢀpyrazolꢀ1ꢀyl)ꢀ1,4ꢀbenzoquinone, C12H8N4O2
It was prepared by the reaction of pyrazole (2.5 g, 37 mmol) and
pꢀbenzoquinone (3.97 g, 37 mmol) in dry dioxane (15 mL). The mixture was heated under reflux
for 6 h and the progress of the reaction was monitored by TLC. After completion of the reaction,
the crude mixture was poured into iceꢀcold water and the resulting semiꢀsolid that separated was
extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium
sulphate. It was then purified by column chromatography over silica gel using petroleum ether:
ethyl acetate (98:2) to yield 2,5ꢀdi(1Hꢀpyrazolꢀ1ꢀyl)ꢀ1,4ꢀbenzoquinone. The product obtained
was recrystallized from ethanol and compared with data reported.37
Yield: 42 %. Mp.: 206 ºC. Anal. Calcd. for C12H8N4O2: C, 60.00; H, 3.36; N, 23.32 %. Found: C,
60.55; H, 3.37; N, 23.29 %. FTꢀIR (cmꢀ1) in KBr: 1534 ν(C=O+C=C), 1412 ν(C=N). UVꢀvis
(DMSO), λmax: 257 (41333), 311 (33403), nm (dm3 molꢀ1cmꢀ1). 1H NMR (CDCl3, ppm): 7.07 (s,
2H, quinone =CꢀH; (Ha, Hb)), 7.67, 7.97 (2d, 4H, =CꢀH, J = 1.6 Hz; J = 2.4 Hz; (Hc, H′c), (He,
13
H′e)), 6.45 (t, 2H, pyrazole =CꢀH, J = 2.0 Hz; J = 2.4 Hz; (Hd, H′d)). C NMR (CDCl3, ppm):
183.32 (ring C=O), 156.01 (CꢀN), 139.20 (C=C–N) 107.79, 131.60, 142.15 (pyrazole).
Synthesis of [Ru(L)Cl(CO)(PPh3)2] [complex 3]
A solution of [RuHCl(CO)(PPh3)3] (0.100 g, 0.105 mmol) in dry ethanol (15 mL) was
refluxed for 15 minutes before adding the equimolar solution of the ligand 2,5ꢀdi(1Hꢀpyrazolꢀ1ꢀ
yl)ꢀ1,4ꢀbenzoquinone (LH) (0.026 g, 0.105 mmol) in ethanol. The mixture was heated under
reflux for 3 h, after which time the dark green solution was reduced in volume by rotary
evaporation to approximately 10 mL. Then it was purified using silica gel column
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chromatography. Elutions with (95:5) pet etherꢀethyl acetate gave the pure cyclometallated
complex 3.
Yield: 46 %. Mp.: 263 ºC. Anal. Calcd. for C49H37N4O3P2Ru: C, 63.40; H, 4.02; N, 6.09 %. Found:
C, 63.49; H, 4.02; N, 6.10 %. FTꢀIR (cmꢀ1) in KBr: 1495 ν(C=O+C=C), 1432 ν(C=N), 1936 ν
(C≡O), 693, 1074, 1320 ν (PPh3). UVꢀvis (DMSO), λmax: 232 (95740), 265 (65000), 302
1
(25820), nm (dm3molꢀ1cmꢀ1) (intraligand transition); 449 (6670), (MLCT π*(LH)←dπ(Ru)). H
NMR (CDCl3, ppm): 6.62 (s, 1H, quinone =CꢀHa), 7.67, 7.61 (2d, 2H, free pyrazole –N=CꢀH, J
= 1.6 Hz; J = 2.0 Hz; (Hf,Hh)), 6.01 (t, 1H, free pyrazole =CꢀHg, J = 2.8 Hz), 8.77 (d, 1H, Ru–
N=CꢀHc, J = 2.0 Hz), 8.13 (d, 1H, bound prazole =CꢀHe, J = 2.4 Hz), 6.36 (t, 1H, bound pyrazole
=CꢀHd, J = 2.4 Hz), 7.14ꢀ7.47 (m, 30 H, aromatic PPh3). 13C NMR (CDCl3, ppm): 204.01
(C19≡O), 190.36 (C2ꢀRu), 172.79 (ring C1=O), 183.04 (ring O=C4–CꢀRu), 140.42 (C=C6ꢀH),
141.03 (C5ꢀN(free pyrazole)), 142.01 (C2ꢀN(bound pyrazole)), 119.41 (C10=C–C(free
pyrazole)), 129.62 (C9=C–C(bound pyrazole)), 108.01 (C11–C=N(free pyrazole)),
108.33(C8=C–C(bound pyrazole)), 128.46 (C12=N–N(free pyrazole)), 140.80 (C7=N–N(bound
pyrazole)), 132.20, 132.86, 133.31, 133.40 (aromatic PPh3).
Synthesis of [Ru(L)Cl(CO)(AsPh3)2] [complex 4]
Treatment of 2,5ꢀdi(1Hꢀpyrazolꢀ1ꢀyl)ꢀ1,4ꢀbenzoquinone (LH) (0.026 g, 0.106 mmol) with
[RuHCl(CO)(AsPh3)3] (0.115 g, 0.106 mmol) under identical conditions to those used to prepare
complex 3 (Scheme 1) afforded 4.
Yield: 49 %. Mp.: 285 ºC. Anal. Calcd. for C49H37N4O3As2Ru: C, 57.80; H, 3.66; N, 5.50. Found: C,
57.89; H, 3.66; N, 5.49. FTꢀIR (cmꢀ1) in KBr: 1480 ν(C=O+C=C), 1430 ν(C=N), 1924 ν (C≡O),
695, 1089, 1316 ν (AsPh3). UVꢀvis (DMSO), λmax: 236 (134710), 261 (117342), 298 (61342),
1
nm (dm3molꢀ1cmꢀ1) (intraligand transition); 489 (8370), (MLCT π*(LH)←dπ(Ru)). H NMR
(CDCl3, ppm): 6.48 (s, 1H, quinone =CꢀHa), 7.64, 7.61 (2d, 2H, free pyrazole –N=CꢀH, J = 1.6
Hz; J = 2.8 Hz; (Hf,Hh)), 6.05 (t, 1H, free pyrazole =CꢀHg, J = 2.8 Hz), 8.73 (d, 1H, Ru–N=CꢀHc,
J = 2.0), 8.14 (d, 1H, bound prazole =CꢀHe, J = 2.4 Hz), 6.30 (t, 1H, bound pyrazole =CꢀHd, J =
2.4 Hz), 7.13ꢀ7.24 (m, 30 H, aromatic AsPh3). 13C NMR (CDCl3, ppm): 207.27 (C19≡O),
193.12 (C2ꢀRu), 173.51 (ring C1=O), 181.75 (ring ring O=C4–CꢀRu), 140.84 (C=C6ꢀH), 141.92
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(C5ꢀN(free pyrazole)), 143.27 (C2ꢀN(bound pyrazole)), 121.15 (C10=C–C(free pyrazole)),
128.54 (C9=C–C(bound pyrazole)), 103.94 (C11–C=N(free pyrazole)), 108.21 (C8=C–C(bound
pyrazole)), 127.90 (C12=N–N(free pyrazole)), 140.28 (C7=N–N(bound pyrazole)), 133.07,
133.44, 133.91, 135.75 (aromatic AsPh3).
RESULTS AND DISCUSSION
Synthesis and characterization of the complexes
Straightforward reactions of 2,5ꢀdi(1Hꢀpyrazolꢀ1ꢀyl)ꢀ1,4ꢀbenzoquinone (LH) with
[RuHCl(CO)(PPh3)3]/ [RuHCl(CO)(AsPh3)3] gave new cyclometallated complexes of the type
[RuLCl(CO)(EPh3)2] (where E= P/As), as depicted in Scheme 1. The complexes are diamagnetic
which correspond to the bivalent state of ruthenium (lowꢀspin d6, S = 0). The ligand was
observed to undergo CꢀH activation at one of the ortho positions leading to the formation of fiveꢀ
member metallacycle. The complexes were stable to air and light, nonꢀhygroscopic in nature and
were remarkably soluble in chloroform, dichloromethane, DMF and DMSO and insoluble in
1
water. The new complexes were characterized by elemental analysis, IR, H and 13C NMR
spectroscopic techniques. The complexes were analytically pure as their microanalytical data
confirmed the proposed molecular formula. It has been observed that a molecule of
2,5ꢀdi(1Hꢀpyrazolꢀ1ꢀyl)ꢀ1,4ꢀbenzoquinone replaced
a
hydride ion and one of the
triphenylphosphines/ triphenylarsines from the precursor complexes. The solid state structure of
both of the complexes (3 and 4) was determined by single crystal Xꢀray crystallographic studies.
It revealed that LH behaved as monobasic bidentate donor.
O
O
N
N
N
EPh3
Ru
N
EPh3
Cl
H
CO
C
N
N
EtOH
3h,
Ru
CO
O
N
N
Cl
EPh3
Ph3E
EPh3
O
E = P, 3
E = As, 4
E = P, 1
E = As, 2
LH
Scheme 1 General scheme for the synthesis of new organometallic ruthenium(II) complexes
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Spectroscopic studies
The IR spectrum of the free 2,5ꢀdi(1Hꢀpyrazolꢀ1ꢀyl)ꢀ1,4ꢀbenzoquinone (LH) showed
peaks of principal IR frequencies around 2855ꢀ3148, 1534 and 1412 cmꢀ1 for ν(CꢀH),
ν(C=O+C=C) and ν(C=N) respectively. The band due to ν(C=O+C=C) in the free ligand was
found to be shifted towards lower wave numbers and the band due to ν(C=N) moved to higher
wave numbers in the complexes. Thus, it is possible to assume that the ligand could give its
coordination to ruthenium ion through O, N or even carbon. The appearance of new band around
1920ꢀ1936 cmꢀ1 for terminal carbonyl vibrational responses in complexes 3 and 4 evidenced the
complexation of precursor complexes with the ligand LH. The electronic spectrum of complexes
3 and 4 displayed absorption maxima at the range 232ꢀ 489 nm. The intense absorptions at 232ꢀ
236 nm and 261ꢀ265 nm is most likely due to a transition involving only ligand orbitals (π→π*
and n→π*). The intense absorption observed in the visible region at 298ꢀ300 nm and at 449ꢀ489
nm are probably due to metalꢀtoꢀligand charge transfer transitions. These charge transfer
transitions may be taking place from the t2 orbital of ruthenium (Highest Occupied Molecular
Orbital) to the vacant π*(–C=C–, –C=N– or –C=O–) orbital of the 2,5ꢀdi(1Hꢀpyrazolꢀ1ꢀyl)ꢀ1,4ꢀ
benzoquinone (Lowest Unoccupied Molecular Orbital) or to the higher energy vacant orbitals of
other fragment of ligand. Complexation of ruthenium to the pyrazole nitrogen and to the quinone
carbon rather than to the quinone oxygen was confirmed by NMR data (experimental section).
NMR Spectra of the ligand and new complexes are given in Figures S1ꢀS5 (Supporting
Information). (Labeling scheme can found in Figure 1).
Figure 1 Numbering scheme for the ligand and complexes 3 and 4
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1
The HꢀNMR spectrum of the free ligand showed two protons singlet, a pair of two
proton doublets and a two proton triplet assigned to (Ha, Hb), (Hc, H′c), (He, H′e) and (Hd, H′d)
protons respectively. Complex 3 and 4 showed a sharp one proton singlet at δ 6.48 and 6.62 ppm
which can be assigned to the Ha proton of the quinone ring. Generally complexation reduces the
residual electron density on the ligands, which has a deshielding effect on the resonance field.
Consistent with the above fact, the protons Hc, Hd, He of coordinated pyrazole resonated in down
fields in comparison to free pyrazole protons Hf, Hg, Hh. A low field doublets at δ = 8.77 ppm (J
= 2.0 Hz) for 3 and δ = 8.73 ppm (J = 2.0 Hz) and for 4 are assigned to the Hc proton that is
nearer to the coordinated nitrogen atom. It is more down fielded than any other pyrazole protons
of the ligand LH. Hf proton is observed as a doublet at δ 7.67 ppm (J=1.6 Hz) for 3 and δ = 7.64
ppm (J = 1.6 Hz) for 4 respectively. Hd and Hg protons of complex 3 appeared as triplets at δ
6.36 ppm (J=2.4 Hz) and δ 6.01 ppm (J = 2.8 Hz) respectively. However, in complex 4 they
were appeared at δ 6.30 and δ 6.05 ppm. He and Hh protons resonated as doublets at δ 8.13 ppm
(J=2.4 Hz) and δ 7.61 ppm (J=3.2 Hz). The aromatic protons of PPh3/ AsPh3 in complex 3 and 4
13
were observed as multiplet around δ 7.13ꢀ7.47 ppm. In the CꢀNMR spectra of the complex 3
and 4, a highly downfield sharp singlet at δ 204.01 and 207.27 ppm has been assigned to the
terminal carbonyl carbon C19. The next downfield singlet at δ 190.36 and 193.12 ppm is due to
benzoquinone carbon C3 which is directly bonded to ruthenium. It is more down fielded than any
other benzoquinone carbon atoms of LH ligand. C6 (sp2, low intensity band at δ 140.42 and
140.48 ppm respectively) is around 50 ppm up field when compared to that of C3. C1 and C4
carbons are appeared as singlets at δ 172.79 and 183.04 ppm respectively for 3 and for the same
carbon atoms in 4 is at δ 173.51 ppm and 181.75 ppm respectively. C4 carbon is deshielded
when compared to C1 because C4 is directly attached to C3, which is coordinated to ruthenium
ion. A couple of singlets at δ 142.01 ppm and δ 141.03 ppm (for 3) and δ 143.27 ppm and δ
141.92 ppm (for 4) are due to the C2 and C5 carbons which are coupled to pyrazole nitrogens.
C7 is resonated as singlet at δ 140.80 ppm and 140.28 ppm for 3 and 4 respectively. It is
deshielded than any other pyrazole carbon atoms, because it is adjacent to nitrogen through
which LH is bonded to ruthenium. Signals at δ 108.33 and δ 129.62 ppm for 3 and δ 108.21 ppm
and δ 128.54 ppm for 4 are the resonances of bound pyrazole carbons C8 and C9 and signals at δ
128.46 ppm, δ 119.41 ppm and δ 108.01 ppm are due to C12, C10 and C11 of free pyrazole
moiety respectively for complex 3. Whereas in complex 4 these resonances were observed at δ
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127.90 ppm, δ 121.15 ppm and δ 103.94 ppm respectively. All carbon atoms of the six phenyl
groups showed only 4 singlet signals in the range of 132.20ꢀ135.75 ppm. All the above points are
in agreement with the interpretation of IR spectra suggesting bidentate coordination of the ligand
to ruthenium and support the results of single crystal Xꢀray crystallographic studies.
Xꢀray crystallography
The molecular structures of 3 and 4 have been determined by single crystal Xꢀray
analysis. The ORTEP drawings of 3 and 4 are displayed in Figure 2 and 3 respectively along
with their atom labeling scheme. The unit cell packing diagrams and intermolecular interactions
of the complexes are given in Figure S6 and S7 (Supporting Information). The summary of
single crystal Xꢀray structures refinement is shown in Table 1. The selected geometrical
parameters (inter atomic distances and angles) and hydrogen bond distances are given in Table 2.
Single crystals of complexes 3 and 4 were obtained from slow evaporation of the mixture of
dichloromethane, ethanol and ethyl acetate at room temperature as blue wedges. From the unit
cell dimensions, it is clear that the crystal system of 3 and 4 is triclinic belonging to the space
group Pī. In the new complexes 3 and 4, the coordination geometry around the ruthenium(II) ion
is a slightly distorted octahedron C1N1(CO)Cl1E2, (E= P/As) where the ligand LH is coordinated
to the metal by utilizing its pyrazole nitrogen and quinone carbon atom. A chloride and a
carbonyl carbon complete the CN(CO)Cl square plane. As commonly observed for hexa
coordinated
complexes
containing
the
{Ru(EPh3)2}
unit,
the
two
bulky
triphenylphosphine/triphenylarsine molecules occupy the remaining two axial sites.38 The +2
oxidation state of the complexes is compensated by a chloride ion and a carbanion of
benzoquinone ring. The ligand coordinated equatorially to ruthenium having the bite angle of
C(2)–Ru(1)–N(1) = 77.40(6)° in complex 3 and 77.52(7)° in complex 4. This constrained bite
angle has resulted in significant distortion in {CN(CO)ClE2} core from the ideal octahedral
geometry, which is reflected in the twelve cis and three trans angles.
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Figure 2 Xꢀray crystal structure with atom numbering scheme for 3 as thermal ellipsoids at 50 %
probability level. The hydrogen atoms have been omitted for clarity
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Table 1 Crystallographic data for complexes 3 and 4
CCDC deposit number
797114
797115
Empirical formula
C50.23H40.69ClN4O3.62P2Ru C49.77H42.31As2ClN4O3.385Ru
Formula weight
956.62
1036.95
90.0(5)
Temperature/K
90.0(5)
Crystal system
Triclinic
Pī
Triclinic
Pī
Space group
a/Å
12.8966(10)
13.2531(12)
13.3050(14)
87.876(5)
80.577(6)
70.558(6)
2115.1(3)
2
12.9529(9)
13.5239(10)
13.2161(10)
88.945(6)
81.494(5)
69.810(5)
2147.6(3)
2
b/Å
c/Å
α/°
β/
γ/°
Volume/Å3
Z
ρ
calcmg/mm3
1.502
1.604
m/mmꢀ1
0.561
2.006
F(000)
980.0
1046.0
Crystal size/mm3
0.20 × 0.18 × 0.07
0.23 × 0.22 × 0.10
5.8 to 66.2°
2θ range for data collection 5.4 to 65°
ꢀ19 ≤ h ≤ 19,
ꢀ20 ≤ k ≤ 20,
ꢀ20 ≤ l ≤ 20
ꢀ19 ≤ h ≤ 19,
ꢀ20 ≤ k ≤ 20,
ꢀ20 ≤ l ≤ 20
Index ranges
Reflections collected
62665
61539
Independent reflections
Data/restraints/parameters
Goodnessꢀofꢀfit on F2
15231[R(int) = 0.044]
15231/0/542
1.069
16353[R(int) = 0.036]
16353/0/541
1.037
Final R indexes [I>=2σ (I)] R1 = 0.0390, wR2 = 0.0930 R1 = 0.0360, wR2 = 0.0840
Final R indexes [all data] R1 = 0.0570, wR2 = 0.0980 R1 = 0.0620, wR2 = 0.0910
Largest diff. peak/hole / e Åꢀ3 0.67/ꢀ0.89
1.47/ꢀ0.96
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Table 2 Selected bond lengths (Å) and angles (°) for complexes 3 and 4
Inter atomic distances (Å )
Complex 3
Ru(1)ꢀN(1)
Ru(1)–C(2)
Ru(1)–C(1)
Ru(1)–Cl(1)
Ru(1)–P(1)
Ru(1)–P(2)
Complex 4
Ru(1)ꢀCl(1)
Ru(1)–C(1)
Ru(1)–C(2)
Ru(1)–N(1)
Ru(1)–As(1)
Ru(1)–As(2)
2.126(1)
2.029(2)
1.857(1)
2.4870(5)
2.3941(5)
2.4045(6)
2.4930(6)
1.853(2)
2.024(2)
2.120(1)
2.4645(3)
2.4759(3)
Bond angles(°)
Complex 3
Complex 4
C(2)–Ru(1)–P(1)
C(2)–Ru(1)–C(1)
C(2)–Ru(1)–Cl(1) 161.93(5)
C(2)–Ru(1)–N(1)
C(2)–Ru(1)–P(2)
C(1)–Ru(1)–Cl(1) 100.46(6)
C(1)–Ru(1)–P(1)
C(1)–Ru(1)–N(1)
C(1)–Ru(1)–P(2)
N(1)–Ru(1)–Cl(1) 84.53(4)
N(1)–Ru(1)–P(1)
N(1)–Ru(1)–P(2)
89.78(5)
97.61(7)
C(1)–Ru(1)–Cl(1)
C(1)–Ru(1)–C(2)
C(1)–Ru(1)–N(1)
C(1)–Ru(1)–As(1)
C(1)–Ru(1)–As(2)
C(2)–Ru(1)–As(1)
C(2)–Ru(1)–As(2)
C(2)–Ru(1)–N(1)
C(2)–Ru(1)–Cl(1)
N(1)–Ru(1)–Cl(1)
N(1)–Ru(1)–As(1)
N(1)–Ru(1)–As(2)
Cl(1)–Ru(1)–As(1)
Cl(1)–Ru(1)–As(2)
As(1)–Ru(1)–As′(2)
98.99(7)
97.53(8)
175.00(8)
86.91(6)
89.48(6)
88.83(6)
92.65(6)
77.52(7)
163.45(6)
85.95(5)
92.29(5)
91.40(5)
91.10(1)
88.46(1)
176.24(1)
77.40(6)
92.53(5)
85.75(6)
174.91(7)
89.32(6)
93.17(4)
91.89(4)
Cl(1)–Ru(1)–P(1) 91.11(2)
Cl(1)–Ru(1)–P(2) 88.14(2)
P(1)–Ru(1)–P′(2)
174.79(2)
Inter and intra molecular interactions
Complex 3 O3…….O1(intra)
2.988(2)
O3…….O1(inter)
2.992(2)
Complex 4 O3…….O1
2.974(2)
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Figure 3 Xꢀray crystal structure with atom numbering scheme for 4 as thermal ellipsoids at
50 % probability level. The hydrogen atoms have been omitted for clarity.
The cis bond angles spread over the range 84.53(4)–100.46(6)°, while the trans bond
angles are within the range 161.93(5)– 176.24(1)°. The carbonyl group occupies a site trans
to the N1 (pyrazole ring nitrogen from LH, N1–Ru1–C1, 174.91(7) Å in 3 175.00(8) Å in 4).
This may be a consequence of strong Ru(II)→CO back donation as indicated by the short
Ru–C bond (1.857(1) Å) and low CO stretching frequency (1936 cmꢀ1), which prefers σ or
weak π donor groups occupying the site opposite to CO to favor the d–π* back donation.
Though the triphenylphosphine/triphenylarsine ligands usually prefer to occupy mutually cis
position for better πꢀinteraction,39 in this complex the presence of CO, a stronger πꢀacidic
ligand, might have forced the bulky triphenylphosphine/triphenylarsine ligands to take trans
position for steric reasons. The C(2)–Ru(1)–Cl(1) bond angle is 161.93(5) Å and 163.45(6) Å
in complexes 3 and 4 respectively showing that Cl atom lies trans to ring carbon. From the
covalent radii values, the Ru(II)–C(sp2) length is estimated to be 2.06 Å40 and the observed
values extent the range 1.96–2.16 Å.41 In the new complexes, the Ru1ꢀC2 distance is 2.024ꢀ
2.029 Å, which is identical to that of the other structurally characterized cyclometallated
ruthenium(II) complexes.42 The Ru1ꢀC1 bond distance (1.857 Å in complex 3 and 1.853 Å in
complex 4) is in accordance with those found in another ruthenium(II) complex.43 The RuꢀC2
(sp2) bond is 0.17 Å longer than the Ru1ꢀC1 (sp) distance in complexes 3 and 4, due to
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RuꢀCO back bonding. Among six Ruꢀligand distances, the Ru1ꢀCl distance of 2.487 Å is
significantly longer than other bond distances. This lengthening of the RuꢀCl bond distance
can be explained by the trans influence of the soft aryl carbon atom which is present trans to
the chloride. The Ru–P, Ru–As, Ru–O, Ru–N and Ru–Cl bond lengths found in the
complexes agree well with those reported for similar ruthenium complexes having same
coordination sphere.44ꢀ46,43 The absence of any solvent of crystallization in the crystal lattice
of the new complexes indicates possible existence of nonꢀcovalent interaction(s) between the
individual complex molecules. A closer look at the packing pattern of the crystal revealed
that the carbonyl oxygen in each complex molecule exhibited inter and intra molecular
interactions with benzoquinone oxygen atom.
DNA binding studies
Since DNA is an important cellular receptor, interaction with DNA, an elementary
mechanism of action responsible for anticancer activity which has been suggested for some
of the ruthenium complexes. Thus, the mode and propensity for binding of the ruthenium(II)
precursor complexes 1, 2 and their corresponding new ruthenium(II) complexes 3, 4 to
HSꢀDNA were studied with the aid of electronic absorption and luminescence quenching
techniques. The ligand LH was also interacted with DNA to find out the impact of chelation
on binding with biomolecules.
Electronic absorption titration
The application of electronic absorption spectroscopy is one of the simplest and most
useful techniques used to study the DNAꢀbinding properties of the metal complexes. In the
UVꢀvis spectra of the ligand and complexes, absorption bands were observed at 230 and 270
nm. In addition to these bands, the new complexes 3 and 4 showed additional low intense
MLCT bands at 449 and 489 nm respectively. In order to study the binding of the test
compounds (LH, ruthenium(II) starting materials 1, 2 and their corresponding new
ruthenium(II) organometallic complexes 3, 4) with DNA, the change in the electronic
spectrum was observed by titrating increasing concentrations of HSꢀDNA (0ꢀ50 µM). As the
concentration of DNA was increased, the intensity of the absorption bands at 257, 267, 259,
265 and 261 nm corresponding to LH and complexes 1ꢀ4 respectively were affected by
resulting hyperchromism with red shifts in the IL (intraꢀligand) bands. The spectral changes
of LH and complexes 1ꢀ4 upon addition of DNA are shown in Figure 4. The observed
spectroscopic variation i.e. hyperchromic effect with red shifts suggested that the test
compounds bind to HSꢀDNA by external contact, possibly due to electrostatic binding.47
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4
3
2
1
0
1
LH
4
2
0
300
300
Wavelength (nm)
Wavelength (nm)
4
3
2
1
0
3
2
3
2
1
0
300
300
Wavelength (nm)
Wavelength (nm)
4
3
2
1
300
Wavelength (nm)
Figure 4 Absorption titration spectra of fixed concentration (10 ꢂM) of LH and 1ꢀ4 with
increasing concentrations (0 ꢀ 50 ꢂM) of HSꢀDNA (phosphate buffer, pH 7)
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To compare quantitatively the binding affinity of the test compounds to DNA, the
intrinsic binding constants Kb of the compounds with DNA were calculated by monitoring
the changes in absorbance around 258ꢀ267 nm with increasing concentration of DNA using
the following equation.
[DNA] ꢃ [εaꢀεf]) = [DNA] ꢃ [εbꢀεf]+1 ꢃ Kb[εbꢀεf]
→ (1)
Wherein the present case, [DNA] is the concentration of DNA in base pairs, εa, εf, and
εb are corresponding respectively to the extinction coefficient for each addition of DNA to the
test compound, the extinction coefficient of free compound and the extinction coefficient of
the test compound in the fully bound form. Kb is the equilibrium binding constant of the test
compound binding to HSꢀDNA. Each set of data, when fitted to the above equation generate
a straight line with a slope of 1/(εbꢀεf) and a yꢀintercept of 1/Kb (εbꢀεf) and Kb was determined
from the ratio of the slope to intercept (Table 3, Figure 5).
3.5
LH
1
3.0
2
2.5
3
4
2.0
1.5
1.0
0.5
0.0
0.0
0.1
0.2
0.3
0.4
0.5
[DNA]
Figure 5 Binding isotherms of the LH and 1ꢀ4 with HSꢀDNA
Table 3 Binding constant for the interaction of compounds with HSꢀDNA
System
Kb (Mꢀ1)
1.74 ×105
1.21 ×104
1.08 ×104
2.18 ×106
6.95 ×105
HSꢀDNA + LH
HSꢀDNA + 1
HSꢀDNA + 2
HSꢀDNA + 3
HSꢀDNA + 4
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From the binding constant values (Table 3), it is inferred that the new organometallic
complexes 3, 4 showed better binding affinity than the ligand LH and their parent complexes
1 and 2. Complex 3 exhibited better DNA binding than complex 4 and this may be due to the
relatively small size of phosphine as compare with arsine.
Competitive binding Studies with Ethidium Bromide
Though absorption spectral studies point out the electrostatic binding of the test
compounds with DNA, it has to be reconfirmed by other methods and hence, steadyꢀstate
competitive binding experiments using compounds LH, 1, 2, 3 and 4 as quenchers were
undertaken to get final proof for the mode of binding of the compounds to DNA. For that
Ethidium Bromide (EB) was employed as a fluorescent probe. EB is a classical intercalator
exhibits a dramatic enhancement of its emission intensity when intercalated to DNA. This
enhanced fluorescence of the EBꢀDNA complex can be quenched by the addition of a second
DNA binder which is either an intercalator or a groove binder and such a decrease in the
emission intensity of the preꢀtreated EB is the basis of the fluorescence intercalator
displacement assay. In our experiment, the EBꢀDNA system exhibits a strong emission band
at wavelength around 620 nm. Emission spectra of EB bound to HSꢀDNA in the absence and
presence of each compound have been recorded for [EB] = 10 ꢂM, [DNA] = 10 ꢂM for
increasing amounts of each compound (0–50 µM). Upon the addition of LH and complexes
1ꢀ4, the fluorescence emission of the EBꢀDNA system was enhanced rather than quenched in
the emission maximum shown in Figure S8 (Supporting Information), indicating that they
could not displace EB from the EBꢀDNA complex. This observation also indicates that the
compounds bind weakly to the DNA probably by electrostatic interaction which is supported
by the absorption titration experiment.
BSA binding studies
Since the mode of action of ruthenium(II)ꢀbased metallo drugs in chemotherapy, still
a subject of debate. The investigation of interaction between metabolism products such as
protein and ruthenium complexes is essential for a better understanding of their
pharmacological profile. It has also been proved that organometallic ruthenium complexes are
reactive towards a number of proteins and play a significant role in their biological activity49
and hence, investigations on the interactions of our compounds with protein were undertaken.
Among the proteins, since bovine serum albumin is one of the most extensively studied
proteins, particularly because of its medical importance, stability and structural homology
with human serum albumin (HSA) and thus our model protein. The nature of interaction of
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our compounds (LH and complexes 1ꢀ4) with BSA has been explored by means of UVꢀvis
and fluorescence spectroscopy.
UV absorption spectra of BSA in the presence of compounds
UV–vis spectra of BSA in the absence and presence of the test compounds are shown
in Figure 6. It is seen from the figure that a progressive decrease in the absorbance intensity
of BSA was observed with the addition of these test compounds and there was a small blue
shift of 1 nm for 1 and red shift of about 1 nm for the compounds LH, 2, 3 and 4 respectively.
This result suggested not only the complex formation between BSA and the test compounds
but also confirms a static quenching process because the dynamic quenching mechanism does
not change the electronic absorption spectrum of the compounds.50
0.20
LH
1
0.15
2
0.10
3
4
0.05
BSA
0.00
-0.05
-0.10
-0.15
-0.20
250
300
350
400
Wavelength (nm)
Figure 6 UV absorption spectra of BSA (1µM) in the absence and presence of LH and 1ꢀ4
(10 µM).
Fluorescence quenching studies of BSA
To get more information on the binding of the compounds with BSA, fluorescence
spectrum of BSA was studied in the presence of the test compounds. BSA contains three
fluorophores, namely, tryptophan, tyrosine and phenylalanine, the intrinsic fluorescence of
BSA is mainly due to tryptophan alone51 and changes in the emission spectra of tryptophan
are common in response to protein conformational transitions, subunit associations, substrate
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binding or denaturation. Therefore, the intrinsic fluorescence of BSA can provide
considerable information on their structure and dynamics and is often employed in the study
of protein folding and association reactions. Hence, the interaction of BSA with our ligand
LH and complexes (1ꢀ4) was studied by fluorescence measurement at room temperature and
from which the binding constants of the complexes were calculated. In a typical experiment,
a solution of BSA (1 µM) was titrated with various concentrations of the complexes (0–35
µM). Fluorescence spectra were recorded in the range of 290–500 nm upon excitation at 280
nm. The changes observed on the fluorescence emission spectra of a solution of BSA on the
addition of increasing amounts of test compounds are shown in Figure 7. Addition of
compounds to BSA produced a dramatic modification on the emission profile. The
fluorescence of BSA was quenched effectively around 345 nm accompanied with the blue
shift in emission maximum of LH, complexes 3, 4 and with the red shift in starting
complexes 1, 2 and this may be due to the binding of compounds with the active site in
BSA.52
1
800
LH
700
700
600
600
500
500
400
400
300
300
200
200
100
0
100
0
300
350
400
450
500
300
350
400
450
Wavelength (nm)
Wavelength (nm)
900
3
2
800
700
600
500
400
300
200
100
0
800
600
400
200
0
300
350
400
450
500
300
350
400
450
Wavelength (nm)
Wavelength (nm)
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DOI: 10.1039/C6RA05867A
1000
800
600
400
200
0
4
300
350
400
450
Wavelength (nm)
Figure 7 The emission spectrum of BSA (1 µM; λexc = 280 nm; λemi = 346 nm) in the
presence of increasing amounts of LH and 1ꢀ4 (0 – 35 µM). The arrow shows the emission
intensity changes upon increasing complex concentration.
To study the quenching process further, fluorescence quenching data were analyzed
with the SternꢀVolmer equation (2) and Scatchard equation (3). The ratio of the fluorescence
intensity in the absence of (I0) and in the presence of (I) the quencher is related to the
concentration of the quencher [Q] by a coefficient Ksv.
I0/I = 1+ KSV [Q]
→ (2)
Ksv value obtained from the plot of I0/I vs [Q] was found to be 5.87 × 103 Mꢀ1, 1.39 ×
105 Mꢀ1, 5.10 × 103 Mꢀ1, 5.14 × 105 Mꢀ1 and 3.44 × 105 Mꢀ1 corresponding to LH and
complexes 1ꢀ4 respectively. The observed linearity in the plots (Figure 8 and Table 4)
indicated the ability of the test compounds to quench the emission intensity of BSA. From
KSV values, it is seen that the complexes 3 and 4 exhibited strong proteinꢀbinding ability with
enhanced hydrophobicity than LH and their corresponding starting materials 1 and 2.
For the static quenching, when small molecules bind independently to a set of equivalent
sites on a macromolecule, the following Scatchard equation (3) can be used to determine the
binding constant and the number of binding sites.
F0ꢀF
log
= log K + n log[Q]
F
→
(3)
Where K is the binding constant of quencher with BSA, n is the number of binding
sites per BSA, F0 and F are the fluorescence intensity in the absence and presence of the
quencher. The values of K can be determined from the slope of log [(F0 − F)/F] versus log
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[Q] as shown in Figure 9. The calculated binding constant (K) values and the number of
binding sites (n) are listed in Table 5. The values of n at room temperature are approximately
equal to 1 except for LH and complex 2, which indicates that there is just one single binding
site in BSA for the complexes 1, 3 and 4.
16
15
LH
1
2
14
13
12
3
11
4
10
9
8
7
6
5
4
3
2
1
0
5
10
15
20
25
30
[Q]
Figure 8 Plot of I0/I vs log [Q]
LH
1
1.0
2
0.5
3
4
0.0
-0.5
-1.0
-1.5
-5.4
-5.2
-5.0
-4.8
-4.6
-4.4
log Q
Figure 9 Plot of log [(F ꢀF)/F] vs log [Q]
0
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Table 4 Binding constant and number of binding sites for interaction of compounds with
BSA
System
BSA + LH
BSA + 1
BSA + 2
BSA + 3
BSA + 4
KSV × Mꢀ1
5.87 × 103
1.39 × 105
5.10 × 103
5.14 × 105
3.44 × 105
K × Mꢀ1
2.44 ×104
1.26 ×105
1.00 ×105
4.24 ×106
2.47 ×106
n
0.67
0.96
0.59
1.06
1.43
Synchronous fluorescence spectra
After having obtained the binding constant and number of binding sites of the test
compounds with BSA, it is essential to know about the conformational change of the protein
molecular environment in the vicinity of the fluorophore functional groups.53This can be
derived from synchronous fluorescence spectral studies and hence, the synchronous
fluorescence spectra of BSA with the compounds were carried out. According to Miller,54 in
synchronous fluorescence spectroscopy, the difference between excitation and emission
wavelength (ꢁλ= λemi ꢀ λexc) reflects the spectra of a different nature of chromophores. If the
ꢁλ value is 15 nm, the synchronous fluorescence of BSA is characteristic of tyrosine residue,
whereas a larger ꢁλ value of 60 nm is characteristic of tryptophan.55The variation in the
tryptophan emission is the consequence of the protein conformational changes. Therefore, to
explore the structural change of BSA, we measured synchronous fluorescence spectra of BSA
with various concentration of LH and 1ꢀ4 at ꢀλ= 15 nm and ꢀλ= 60 nm and the changes are
shown in Figure S9 (Supporting Information) and Figure 10 respectively.
800
700
600
500
400
300
200
100
0
900
800
700
600
500
400
300
200
100
0
1
LH
300
350
400
300
350
400
Wavelength (nm)
Wavelength (nm)
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DOI: 10.1039/C6RA05867A
900
800
700
600
500
400
300
200
100
0
3
900
800
700
600
500
400
300
200
100
0
2
Wavelengt3h50(nm)
300
350
Wavelength (nm)
900
4
800
700
600
500
400
300
200
100
0
300
350
Wavelength (nm)
Figure 10 Synchronous spectra of BSA (1 µM) in the presence of increasing amounts of the
LH and 1ꢀ4 for a wavelength difference of ꢀλ = 60 nm. The arrow shows the emission
intensity changes upon increasing concentration of compound
Results showed that the fluorescence of tyrosine residue was hyperchromic and that of
tryptophan residue was hypochromic. However, the fluorescence intensities of tryptophan
residues changed significantly with red shift by addition of increasing amount of the
compounds especially in case of the new complexes 3 and 4. It indicates that the
hydrophobicity of microenvironment around tryptophan residues decreases in the presence of
the added compounds56 and that the complexes bind to active site in the protein, which makes
them potential molecules for biological applications. This result has prompted us to explore
more on the anticancer activities of the new organometallic ruthenium(II) complexes.
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Antioxidant activity
Free radicals that are generated in many bioorganic redox processes may induce
oxidative damage in various components of the body (lipids, DNA and proteins) and have
been implicated in aging and a number of lifeꢀlimiting chronic diseases such as cancer,
hypertension, parkinson disease, alzheimer, cardiac infarction, atherosclerosis, rheumatism,
cataracts etc.57,58 Efforts to counteract the damage caused by these species are gaining
acceptance as a basis for novel therapeutic approaches and the field of preventive medicine is
experiencing an upsurge of interest in medically useful antioxidants. Since the experiments
conducted so far revealed that the complexes exhibit reasonable DNA and protein binding
affinity, it is considered worthwhile to study their ability to quench the free radicals and
antioxidant properties. Further, the inꢀvitro antioxidant properties of ruthenium complexes
have attracted a lot of interest, but the radical scavenging activity is limited to hydroxyl
radical.59 Hence, we carried out experiments to investigate the free radical scavenging ability
of the new ruthenium complexes against a group of free radicals. The ligand and the metallic
precursors were also subjected to radical scavenging experiments. The IC50 value of all test
compounds (Table 5) obtained from different types of assay experiments strongly supports
that the complexes presented in this work possess excellent antioxidant activities, which are
better than that of the standard antioxidants Vitamin C and BHT.
Table 5 Antioxidant activity of the ligand, precursors, new complexes, Vitamin C and BHT
IC50(µM)[a]
NO
208.49 ±0.7 122.52 ±2.3 165.26 ±1.9 181.1± 0.8
H2O2
223.8± 0.5
Compounds DPPH
˙
OH˙
˙
O2‾˙
LH
1
199.7 ± 3.4 234.9 ± 1.5 212.2 ± 4.8 267.4 ± 3.6 288.5 ± 3.2
243.5 ± 4.5 289.8 ± 5.8 256.3 ± 5.9 294.2 ± 4.8 302.2 ± 2.7
21.27 ± 2.8 50.65 ± 6.1 65.96 ± 5.6 33.75 ± 1.3 157.1± 0.9
19.40 ± 1.1 57.88 ± 4.6 71.00 ± 0.9 47.06 ± 0.4 169.4 ± 1.5
147.6 ± 4.2 232.8 ± 1.9 215.8 ± 2.7 221.4 ± 1.2 238.5 ± 3.6
2
3
4
Vitamin C
BHT
86.2 ± 1.8
163.4 ± 0.7 154.3 ± 2.4 131.6 ± 1.5 149.8 ± 4.3
[a] : fifty percent inhibitory concentration of the test compounds against free radicals
29