Synthesis, characterization, DNA binding, DNA cleavage, antioxidant and in vitro cytotoxicity studies of ruthenium(II) complexes containing hydrazone ligands

Article abstract of DOI:10.1080/00958972.2016.1235700

The synthesis, spectral characterization, and biological studies of ruthenium(II) hydrazone complexes [RuCl(CO)(PPh₃)₂L] (where L?=?hydrazone ligands) have been carried out. The hydrazones are monobasic bidentate ligands with O and N

Full text of DOI:10.1080/00958972.2016.1235700

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: http://www.tandfonline.com/loi/gcoo20
Synthesis, characterization, dna binding, dna
cleavage, antioxidant and in vitro cytotoxicity
studies of ruthenium(II) complexes containing
hydrazine ligands
Maruthachalam Mohanraj, Ganesan Ayyannan, Gunasekaran Raja &
Chinnasamy Jayabalakrishnan
To cite this article: Maruthachalam Mohanraj, Ganesan Ayyannan, Gunasekaran Raja &
Chinnasamy Jayabalakrishnan (2016): Synthesis, characterization, dna binding, dna cleavage,
antioxidant and in vitro cytotoxicity studies of ruthenium(II) complexes containing hydrazine
ligands, Journal of Coordination Chemistry, DOI: 10.1080/00958972.2016.1235700
To link to this article: http://dx.doi.org/10.1080/00958972.2016.1235700
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Date: 13 September 2016, At: 03:25

Publisher: Taylor & Francis
Journal: Journal of Coordination Chemistry
DOI: http://dx.doi.org/10.1080/00958972.2016.1235700
Synthesis, characterization, dna binding, dna cleavage, antioxidant and
in vitro cytotoxicity studies of ruthenium(II) complexes
containing hydrazine ligands
MARUTHACHALAM MOHANRAJ, GANESAN AYYANNAN, GUNASEKARAN RAJA and
CHINNASAMY JAYABALAKRISHNAN1
Post-Graduate and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya College of
Arts and Science, Coimbatore-641 020, Tamil Nadu, India
The synthesis, spectral characterization and biological studies of ruthenium(II) hydrazone
complexes [RuCl(CO)(PPh3)2L] (where L = hydrazone ligands) have been carried out. The
hydrazones are monobasic bidentate ligands with O and N as the donors and are preferably found
in the enol form in all the complexes. The molecular structure of the ligands HL1, HL2 and HL3
were determined by single crystal X-ray diffraction. The DNA binding studies of the ligands and
complexes were carried out by absorption spectroscopic and viscosity measurements. The results
revealed that the ligands and complexes bind to DNA via intercalation. The DNA cleavage
activity of the complexes, evaluated by gel electrophoresis assay, revealed that the complexes are
good DNA cleaving agents. The antioxidant properties of the complexes were evaluated against
DPPH, OH and NO radicals, which showed that the complexes have strong radical-scavenging.
Further, the in vitro cytotoxic effect of the complexes examined on HeLa and MCF-7 cancer cell
lines showed that the complexes exhibited significant anticancer activity.
Keywords: Ru(II) hydrazone complexes; DNA-interaction; Antioxidant; Cytotoxicity
1Corresponding author. Email: drcjbstar@gmail.com

1. Introduction
In recent years, there has been interest in interactions between small molecules and
biomolecules, particularly DNA [1, 2]. DNA is an important cellular target; many compounds
exert their antitumor effects through binding to DNA, thereby changing its replication and
inhibiting cell growth [3]. Basically interaction of small molecules with DNA duplex is through
a variety of binding modes: intercalation, groove binding and electrostatic attractions [4]. Among
them, the intercalative mode is an important DNA-binding mode as it invariably leads to cellular
degradation.
This mode predominately occurs in compounds possessing aromatic ring systems, so that they
occupy the space between two adjacent DNA base pairs [5, 6]. Transition metal complexes have
attracted importance in nucleic acid chemistry for applications like foot printing, sequence-
specific binding to nucleic acids and as new structural probes and therapeutic agents [7]. The
development of sequence-specific DNA binding and cleavage agents for new potential DNA-
targeted antitumor drugs are essential for further applications in molecular biology and medicine
[8, 9]. Among the transition metal complexes, ruthenium complexes act as DNA–targeted guest
molecules. Ruthenium complexes have been widely studied because of low toxicity towards
normal cells and promising anticancer activity [10-12]. Some chemical properties such as rate of
ligand exchange, range of accessible oxidation states and ability to mimic iron in binding to
certain biological molecules make them fit for medicinal applications [13-15]. A number of
ruthenium complexes were recently shown to possess encouraging cytotoxic and antitumor
properties in preclinical models [16]. The entrance of two Ru based drugs, NAMI-A and
KP1019, into clinical trials for treatment of metastatic tumors increased interest in this metal
[17]. NAMI-A has been used as an antimetastatic drug and KP1019 has been employed as a
colon carcinoma drug [18, 19]. Schiff base hydrazone and benzaldehyde derivatives display a
wide range of pharmacological properties viz., antimicrobial, antituberculostatic, anticancer and
antioxidant activities [20, 21]. Incorporation of metal onto these Schiff base hydrazone ligands
results in alteration and enhancement of their biological activity [22, 23]. We now report the
synthesis and characterization of ruthenium(II) complexes containing hydrazone ligands derived
by condensing furan-2-carboxylic acid hydrazide with
2-nitrobenzaldehyde/2-bromobenzaldehyde/4-hydroxy benzaldehyde. The crystal structures of
the hydrazone ligands have been determined by X-ray crystallography. The biological properties
of the ligands and ruthenium(II) complexes focused on interaction with CT-DNA, antioxidant
properties against DPPH, OH and NO radicals and in vitro cytotoxic activity against HeLa and
MCF-7 cancer cell lines.

2. Experimental details
2.1. Materials and instrumentation
The chemicals used were chemically pure and AR grade. Solvents were purified and dried
according to a standard procedure [24]. The metal precursor [RuHCl(CO)(PPh3)3] was prepared
by reported method [25]. Melting points were recorded with a Veego VMP-DS heating table and
elemental analyses (C, H and N) were performed on a Perkin Elmer 240C elemental analyzer at
the University of Hyderabad, Hyderabad, India. Infrared spectra were recorded as KBr pellets
from 400-4000 cm-1 using a Perkin Elmer FT-IR 8000 spectrophotometer. Electronic spectra
were recorded in DMSO solution with a systronics double beam UV-Vis spectrophotometer
2202 from 200-800 nm. 1H and 13C NMR spectra were recorded on a Bruker AV III 500 MHZ
instrument using TMS as an internal reference. 31P NMR spectra were recorded on a Bruker AV
III 500 MHZ instrument using ortho phosphoric acid as an internal reference. The EI mass
spectra of the complexes were recorded on a JEOL GCMATE II mass spectrometer. Calf-thymus
(CT-DNA) was purchased from Bangalore Genei, Bangalore, India. DNA cleavage studies were
carried out using Gelstan, Gel documentation system. Antioxidant and anticancer studies were
carried out at the Kovai Medical Centre and Hospital Pharmacy College, Coimbatore,
TamilNadu.
2.2. Preparation of hydrazone ligands
A methanolic solution (50 mL) of furan-2-carboxylic acid hydrazide (1.261 g, 0.01 mol) was
added to a methanol solution (25 mL) containing 2-nitrobenzaldehyde (1.511 g, 0.01 mol)/2-
bromobenzaldehyde (1.16 ml, 0.01 mol)/4-hydroxybenzaldehyde (1.221 g, 0.01 mol). The
mixture was refluxed for an hour, which results in formation of a precipitate. The reaction
mixture was cooled to room temperature and the solid compound was collected by filtration,
washed and recrystallized from ethanol. Needle-shaped crystals suitable for X-ray diffraction
analysis were formed (scheme 1).
2.2.1. Furan-2-carboxylic acid (2-nitrobenzylidene)-hydrazide (HL1) (1). Color: yellow;
Yield: 87%; M.P: 240 °C. Anal. calcd. for C12H9N3O4 (%): C, 55.60; H, 3.50; N, 16.21; O,
24.69. Found (%): C, 55.63; H, 3.48; N, 16.20; O, 24.65. IR (KBr, cm-1): 1643 ν(C=N); 1676
ν(C=O). 3202 ν(NH). UV–vis (DMSO), λmax (nm) ( , dm3 mol-1 cm-1): 266 (12,320), 378
(7,150) (π→π*, n→π*). 1H NMR (DMSO-d6); δ 9.97 (s, 1H, -NH); 8.51 (s, 1H, C6-CH); δ 6.48
(d, 1H, C1-CH); 6.69 (t, 1H, C2-CH); 6.33 (d, 1H, C3-CH); 7.90 (d, 1H, C9-CH); 7.41 (t, 1H, C10-
CH); 7.85 (t, 1H, C11-CH); 7.27 (d, 1H, C12-CH). 13C NMR (DMSO-d6); δ 165.4, 149.6, 142.3,
117.2, 114.7, 112.3, 109.5, 108.1, 107.2, 106.6, 103.4 and 102.3.

2.2.2. Furan-2-carboxylic acid (2-bromobenzylidene)-hydrazide (HL2) (2). Color: pale
yellow; Yield: 89%; M.P: 234 °C. Anal. calcd. for C12H9BrN2O2 (%): C, 49.17; H, 3.09; N, 9.56;
O, 10.92. Found (%): C, 49.15; H, 3.13; N, 9.59; O, 10.87. IR (KBr, cm-1): 1639 ν(C=N); 1670
ν(C=O), 3028 ν(NH). UV–vis (DMSO), λmax (nm) ( , dm3 mol-1 cm-1): 263 (12,770), 311
(9,320) (π→π*, n→π*). 1H NMR (DMSO-d6); δ 9.83 (s, 1H, -NH); 8.38 (s, 1H, C6-CH); δ 6.68
(d, 1H, C1-CH); 6.81 (t, 1H, C2-CH); 6.59 (d, 1H, C3-CH); 7.65 (d, 1H, C9-CH); 7.21 (t, 1H, C10-
CH); 7.42 (t, 1H, C11-CH); 6.97 (d, 1H, C12-CH). 13C NMR (DMSO-d6); δ 162.6, 143.7, 119.2,
117.3, 116.8, 114.9, 112.6, 111.3, 109.5, 107.3, 104.2 and 103.1.
2.2.3. Furan-2-carboxylic acid (4-hydroxybenzylidene)-hydrazide (HL3) (3). Color: creamy
white; Yield: 84%; M.P: 245 °C. Anal. calcd. for C12H10N2O3 (%): C, 62.60; H, 4.38; N, 12.17;
O, 20.85. Found (%): C, 62.59; H, 4.36; N, 12.15; O, 20.91. IR (KBr, cm-1): 1633 ν(C=N); 1660
ν(C=O), 3175 ν(NH), 3350 ν(Ph-OH). UV–vis (DMSO), λmax (nm) ( , dm3 mol-1 cm-1):
268(11,650), 369 (6,810) (π→π*, n→π*). 1H NMR (DMSO-d6); δ 9.92 (s, 1H, -NH); 8.33 (s,
1H, C6-CH); δ 6.83 (d, 1H, C1-CH); 6.67 (t, 1H, C2-CH); 6.75 (d, 1H, C3-CH); 7.68 (d, 1H,
C8-CH); 7.46 (d, 1H, C9-CH); 7.90 (d, 1H, C11-CH); 7.25 (d, 1H, C12-CH); δ 11.63 (s, 1H,
Ph-OH). 13C NMR (DMSO-d6); 164.5, 152.7, 148.3, 117.3, 116.2, 115.1, 112.6, 110.2, 108.3,
107.8, 107.1 and 106.3.
2.3. Preparation of ruthenium(II) complexes
The monobasic bidentate ligands HL1 (0.125 g, 0.5 mmol), HL2 (0.145 g, 0.5 mmol) and HL3
(0.115 g, 0.5 mmol) were added to a solution of [RuHCl(CO)(PPh3)3] (0.475 g, 0.5 mmol) in 1:1
molar ratio in ethanol-benzene (1:1) and the reaction mixture was refluxed for 6 h. The progress
of the reaction was monitored using TLC. The solution was concentrated to about 3 mL and
petroleum ether (60-80 °C) was added whereby solid separated. The obtained solids were
recrystallized from CH2Cl2/petroleum ether (1:1) and dried under vacuum (scheme 2).
2.3.1. [RuCl(CO)(PPh3)2L1] (4). Color: brown; Yield: 82%; M.P: 271–274 °C. Anal. calcd. for
C49H38ClN3O5P2Ru (%): C, 62.13; H, 4.04; N, 4.44; O, 8.44. Found (%): C, 62.18; H, 4.14; N,
4.38; O, 8.49. EI-MS: Found m/z = 947.3160 (M+) (calculated m/z = 947.3136). IR (KBr, cm-1):
1588 ν(C=N); 1344 ν(C-O); 1951 ν(C≡O) cm-1. UV–vis (DMSO), λmax (nm) ( , dm3 mol-1 cm-1):
273 (13,930), 327 (5,350) (ILCT), 401 (3,710) (MLCT). 1H NMR (DMSO-d6); δ 9.34 (s, 1H,
C6-CH); δ 6.57 (d, 1H, C1-CH); 6.68 (t, 1H, C2-CH); 6.45 (d, 1H, C3-CH); 6.79–7.62 (m, 30H,
PPh3); 7.97 (d, 1H, C9-CH); 7.75 (t, 1H, C10-CH); 7.87 (t, 1H, C11-CH); 7.68 (d, 1H, C12-CH).

13C NMR (DMSO-d6); δ 191.6, 159.2, 153.5, 146.3, 139.6, 138.3, 137.5, 136.4, 135.8, 134.2–
132.3, 131.7, 130.3, 129.6 and 128.4. 31P NMR (DMSO-d6); δ 38.12.
2.3.2. [RuCl(CO)(PPh3)2L2] (5). Color: yellow; Yield: 87%; M.P: 251-256 °C. Anal. calcd. for
C49H38BrClN2O3P2Ru (%): C, 59.98; H, 3.90; N, 2.85; O, 4.89. Found (%): C, 59.93; H, 3.84; N,
2.88; O, 4.95. EI-MS: Found m/z = 981.2122 (M+) (calculated m/z = 981.2121). IR (KBr, cm-1):
1589 ν(C=N); 1359 ν(C-O); 1947 ν(C≡O) cm-1. UV–vis (DMSO), λmax (nm) ( , dm3 mol-1 cm-1):
270 (12,100), 323 (6,060) (ILCT), 410 (3,790) (MLCT). 1H NMR (DMSO-d6); δ 9.13 (s, 1H,
C6-CH); δ 6.94 (d, 1H, C1-CH); 6.83 (t, 1H, C2-CH); 6.67 (d, 1H, C3-CH); 7.19–7.60 (m, 30H,
PPh3); 7.98 (d, 1H, C9-CH); 7.71 (t, 1H, C10-CH); 7.77 (t, 1H, C11-CH); 7.63 (d, 1H, C12-CH).
13C NMR (DMSO-d6); δ 195.5, 153.9, 148.8, 134.8, 134.3, 134.2, 133.8, 133.4, 133.1, 132.4–
130.5, 129.8, 129.7, 129.2 and 128.8. 31P NMR (DMSO-d6); δ 39.25.
2.3.3. [RuCl(CO)(PPh3)2L3] (6). Color: brown; Yield: 84%; M.P: 276–279 °C. Anal. calcd. for
C49H39ClN2O4P2Ru (%): C, 64.09; H, 4.28; N, 3.05; O, 6.97. Found (%): C, 64.16; H, 4.21; N,
3.03; O, 6.91. EI-MS: Found m/z = 918.3129 (M+) (calculated m/z = 918.3155). IR (KBr, cm-1):
1599 ν(C=N); 1356 ν(C-O); 3399 ν(Ph-OH); 1944 ν(C≡O) cm-1. UV–vis (DMSO), λmax (nm) ( ,
dm3 mol-1 cm-1): 279 (11,510), 326 (6,260) (ILCT), 403(3,880) (MLCT). 1H NMR (DMSO-d6);
δ 9.25 (s, 1H, C6-CH); δ 6.83 (d, 1H, C1-CH); 6.63 (t, 1H, C2-CH); 6.76 (d, 1H, C3-CH); 7.07–
7.45 (m, 30H, PPh3); 7.86 (d, 1H, C8-CH); 7.75 (d, 1H, C9-CH); 7.98 (d, 1H, C11-CH); 7.66 (d,
1H, C12-CH); δ 11.82 (s, 1H, Ph-OH). 13C NMR (DMSO-d6); δ 191.8, 159.3, 155.3, 152.1,
134.2, 133.8, 133.5, 133.4, 133.2, 132.4-130.3, 129.4, 129.1, 128.5 and 127.9. 31P NMR
(DMSO-d6); δ 36.08.
2.4. Single crystal X-ray diffraction studies
Single crystal X-ray diffraction data of HL1, HL2 and HL3 were collected at room temperature on
a Bruker AXS KAPPA APEX2 CCD diffractometer equipped with a fine focused sealed tube.
The unit cell parameters were determined and the data collections for HL1, HL2 and HL3 were
performed using graphite monochromated Mo Kα (λ = 0.71073 Å) radiation by φ and ω scans.
The data collected were reduced by SAINT [26] and the empirical absorption corrections were
carried out using SADABS [27]. The structures of HL1, HL2 and HL3 were solved by direct
methods [28] using SHELXS-97, which revealed the position of all non-hydrogen atoms, and
was refined by full-matrix least squares on F2 (SHELXL-97) [29]. All non-hydrogen atoms were
refined anisotropically, while hydrogens were placed in calculated positions and refined as
riding.

2.5. DNA binding experiments
2.5.1. Electronic absorption titration. All experiments involving the binding of compounds
with CT-DNA were carried out in a doubly distilled water buffer with
tris(hydroxymethyl)aminomethane (Tris, 5 mM) and sodium chloride (50 mM) and adjusted to
pH 7.2 with hydrochloric acid. A solution of CT-DNA in the buffer gave a ratio of UV
absorbance of about 1.8-1.9 at 260 and 280 nm, indicating that the DNA was sufficiently free of
protein. The CT-DNA concentration per nucleotide was determined spectrophotometrically by
employing an extinction coefficient of 6600 dm3 mol-1cm-1 at 260 nm. The compounds were
dissolved in a mixed solvent of 5% DMSO and 95% Tris–HCl buffer. Stock solutions were
stored at 4 °C and used within 4 days. Absorption titration experiments were performed with
fixed concentrations of the complexes (25 µM) with varying concentration of DNA (0–50 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.
2.5.2. Viscosity measurements. The viscosity measurement was carried out using an Ubbelodhe
viscometer immersed in a thermostatic water bath maintained at 25 ( 0.l) °C. DNA samples with
approximately 200 base pairs in length were prepared by sonication in order to minimize the
complexities arising from DNA flexibility. Titrations were performed by addition of a small
volume of concentrated stock solutions of ligands and complexes to a solution of calf thymus
DNA in the viscometer. Flow times were observed with a digital stopwatch; each sample was
measured three times and average flow time was calculated. Relative viscosities for CT-DNA in
the presence and absence of the ligand and complex were calculated from the relation η =
(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 (η/η0)1/3 versus binding ratio (R =
[complex]/[DNA]=0.0–0.25), where η is the viscosity of CT-DNA in the presence of complex
and η0 is the viscosity of CT-DNA alone [30, 31].
2.6. Nuclease activity using gel electrophoresis
The DNA cleavage activities of the ruthenium(II) hydrazone complexes were monitored by
agarose gel electrophoresis on CT-DNA. The tests were performed under aerobic conditions.
Each reaction mixture contained 30 µM of CT-DNA, 30 and 60 µM of each complex in DMSO
in 50 mM Tris–HCl, (pH 7.1). The reaction was incubated at 37 °C for 2 h. After incubation, 1 µl
of loading buffer (0.25% bromophenol blue, 0.25% xylene cynol and 60% glycerol) was added
to the reaction mixture and loaded onto a 1% agarose gel containing 1.0l g/ml of ethidium

bromide. The electrophoresis was carried out for 2 h at 50 V in Tris–acetic acid EDTA buffer.
The bands were visualized under UV light and photographed.
2.7. Antioxidant assays
The 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity of the compounds was
measured according to the method of Kunchandy and Rao [32]. The DPPH radical is a stable
free radical having a λmax at 517 nm. A fixed concentration (100 µL) of samples were mixed with
1 mL of 0.1 mM methanol solution of DPPH and the final volume was made up to 4 mL with
double distilled water. The tubes were allowed to stand at room temperature for 30 min. The
control was prepared as above without any extract, and methanol was used for baseline
correction. Changes in the absorbance of the samples were measured at 517 nm.
The scavenging activity for hydroxyl radical recommended by Yu et al. was used with major
changes [33]. The reaction mixture contained 0.6 mL of 1.0 mM deoxyribose, 0.4 mL of 0.2 mM
phenylhydrazine, and 0.6 mL of 10 mM aqueous phosphate buffer (pH 7.4) and was incubated
for 1 h at room temperature. Then, 1 mL of 2-8% trichloroacetic acid, 1 mL of 1% thiobarbituric
acid and 0.4 mL of sample at various concentrations were added and kept in a water bath for 20
min. The absorbance of the mixture at 532 nm was measured with a spectrophotometer. The
hydroxyl radical-scavenging activity was calculated.
Nitric oxide scavenging activity was determined based on the reported method [34], where
sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric
oxide, which interacts with oxygen to produce nitrite ions that can be estimated using Greiss
reagent. Scavengers of nitric oxide compete in this process leading to reduced production of
nitrite ions. For the experiment, sodium nitroprusside (10 mM) in phosphate buffered saline was
mixed with 100 µL of test samples of variable concentrations and incubated at room temperature
for 150 min. The same reaction mixture without the sample, but the equivalent amount of the
solvent, served as the control. After the incubation period, 0.5 mL of Griess reagent containing
sulfanilamide (5.8 mM), H3PO4 (20 mM) and N-(1-naphthyl)ethylenediamine dihydrochloride
(0.39 mM) was added. The absorbance of the chromophore formed was measured at 546 nm.
For each assay, the tests were run in triplicate by varying the concentration. The percentage
inhibition of absorbance was calculated and plotted as a function of the concentration of standard
and sample to determine the antioxidant concentration. The percentage activity was calculated by
using the formula % activity = [(Ao - Ac)/Ao] × 100, where Ao and Ac represent the absorbance in
the absence and presence of the test compounds, respectively. The 50% activity (IC50) was
calculated from the result of percentage activity. Ascorbic acid was used as a standard for all the
above assays.

2.8. Cytotoxicity assay
Cytotoxicity studies of the ruthenium(II) hydrazone complexes were carried out on human
cervical cancer cells (HeLa) and human breast cancer cell line (MCF-7). Cell viability was
assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
method. The HeLa and MCF-7 cells were grown in Eagle’s minimum essential medium
containing 10% fetal bovine serum (FBS). For the screening experiment, the cells were seeded
into 96-well plates in 100 µL of the respective medium containing 10% FBS, at a plating density
of 10,000 cells/well, and incubated at 37 °C under conditions of 5% CO2, 95% air and 100%
relative humidity for 24 h prior to 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 under conditions of 5% CO2, 95% air and 100% relative
humidity for 48 h. Experiments were carried out in triplicate, and the medium not containing the
compounds served as the control. After 48 h, 10 µL of MTT (5 mg/mL) in phosphate buffered
saline (PBS) was added to each well and incubated at 37 °C for 4 h. The medium containing
MTT was removed and the formazan crystals were dissolved in 100 µL of DMSO. The
absorbance was then measured at 570 nm using a microplate reader. The % cell inhibition was
determined using the following formula, % growth inhibition = 100-Abs (sample)/Abs (control)
×100. Nonlinear regression graphs were plotted between % cell inhibition and Log10
concentration and used to obtain IC50 [35].
3. Results and discussion
Analytical and spectroscopic data for the ligands and their complexes indicate 1:1 metal–ligand
stoichiometry for all the complexes. The synthetic route of the ligands and the proposed
structures of the complexes are shown in schemes 1 and 2.
where HL1: R= 2-NO2, HL2: R= 2-Br, HL3: R= 4-OH.
Scheme 1. Synthetic route of the hydrazone ligands.

Scheme 2. Synthetic route of the ruthenium(II) hydrazone complexes. 4: R= 2-NO2, 5:R= 2-Br,
6: R= 4-OH.
3.1. X-ray crystallographic studies
The molecular structure of HL1, HL2 and HL3 along with the atom numbering scheme are
depicted in figures 1-3 and crystal packing diagrams are given in figures S1-S3. The relevant
crystallographic data along with selected bond lengths and angles are predicted in tables 1-4.
The molecular structures of HL1 and HL2 show a displacement ellipsoid plot of two independent
molecules A and B in the asymmetric unit, along with the atom labeling scheme, which have
slightly different conformations. HL1 crystallizes in a monoclinic system in space group P21/c
with Z=8 in the unit cell. The bond lengths of [C6-N2] 1.257(4) Å, [C5-O2] 1.224(3) Å in
molecule B and [C18-N5] 1.266(3) Å, [C17-O6] 1.227(3) Å in molecule A agree with the formal
C=N and C=O bond distances. HL2 crystallizes in an orthorhombic system in space group Pbca
with Z=16 in the unit cell. The bond lengths of [C6-N2] 1.278(9) Å, [C5-O2] 1.211(9) Å in
molecule A and [C18-N4] 1.245(9) Å, [C17-O4] 1.215 (9) Å in molecule B agree with the
formal C=N and C=O bond distances. HL3 crystallizes in the orthorhombic system in space
group Pbca with Z=8 in the unit cell. The lengthening of azomethine carbon and nitrogen bond
[C6-N2] 1.272(4) Å is closer to the formal C=N bond length, 1.28 Å. The bond length [C5-O2]
1.236(4) Å is shorter than the standard C=O bond length 1.24 Å. The distance between [C10-O3]
1.361(4) Å indicates formation of the phenolate hydroxyl.
3.2. Infrared spectral analysis
IR spectra of the hydrazone ligands exhibit a medium sharp band at 1643, 1639 and 1633 cm-1
due to the azomethine C=N stretch of HL1, HL2 and HL3, respectively. In the spectra of the
complexes these bands shift to lower wavenumbers at 1588, 1589 and 1599 cm-1, suggesting
coordination of the azomethine nitrogen to ruthenium [36]. The C=O stretches of HL1, HL2 and
HL3 are at 1676, 1670 and 1660 cm-1, respectively. In spectra of 4, 5 and 6 the C=O stretch
disappears and new bands for C–O appear at 1344, 1359 and 1356 cm-1, respectively, indicating
coordination through keto oxygen after enolization followed deprotonation [37, 38]. Strong
bands at 1951, 1947 and 1944 cm-1 are due to terminally coordinated ruthenium carbonyl.
Characteristic absorptions due to triphenylphosphine were also observed for all complexes in

their expected regions [39]. Overall, IR spectra of the complexes confirm coordination of the
hydrazone to ruthenium(II) via the azomethine nitrogen and the keto oxygen.
3.3. Electronic absorption spectra
Electronic absorption spectra of the ligands and complexes have been studied to obtain more
information on the configuration of the complexes. The absorption spectra of the hydrazone
ligands displayed two bands at 263-378 nm. The first band at 263-268 nm is assigned to π→π*
transition, while the second at 311-378 nm is attributed to an n→π* transition associated with the
azomethine and the keto function of the hydrazones. The spectra of the complexes observed
three bands from 270-410 nm. The bands at 270-279 and 323-327 nm are ligand centered
transitions occurring within the ligand orbitals. These bands are associated with intraligand
transitions. These bands are shifted when compared to ligands suggesting involvement of
azomethine nitrogen and keto oxygen in coordination with ruthenium [40]. An intense absorption
is observed at 401-410 nm due to the charge transfer transitions arising from excitation of an
electron from metal t2g level to an unfilled molecular orbital derived from the π* level of the
ligands. The pattern of the electronic spectra of all the complexes indicates the presence of an
octahedral environment around ruthenium ion, similar to that of other ruthenium(II) octahedral
complexes [41, 42].
3.4. Nuclear magnetic resonance spectral studies
NMR experiments confirm the structures of the synthesized ligands and complexes. The 1H, 13C
and 31P NMR spectra of 4, 5 and 6 are given in figures S4-S12. The 1H NMR spectra of HL1,
HL2 and HL3 show a singlet at δ 9.97, 9.83 and 9.92 due to hydrazine NH protons, which
disappears in complexes, indicating the enolization and deprotonation of the –NH–C=O group
prior to coordination of ligand to metal through keto oxygen [43]. In addition, HL1, HL2 and HL3
exhibit a sharp singlet for azomethine (HC=N) proton at δ 8.51, 8.38 and 8.33, respectively.
However, in the ruthenium complexes, the signal corresponding to the azomethine shifts slightly
downfield due to participation of azomethine nitrogen in coordination with the metal ion, at
δ 9.34, 9.13 and 9.25, respectively [44]. The aromatic protons of the ligands are multiplets from
δ 6.33-7.90; on complexation the protons on the phenyl ring remain more or less unchanged at
δ 6.45-7.98 due to delocalization of electron density in the system and these signals in the
complexes cannot be distinguished from the aromatic signals of PPh3 due to their extensive
overlap [45, 46].
The 13C NMR spectra of HL1, HL2 and HL3 exhibit signals at δ 165, 162 and 164, respectively,
due to the carbonyl carbon (C=O) which is absent in the complexes and appears as new signals at
δ 159, 153 and 159 (C–O), indicating coordination of oxygen via deprotonation. The spectra of

HL1, HL2 and HL3 show a signal at δ 149, 143 and 148 which is assigned to azomethine carbon.
On complexation, this signal shifts downfield at δ 153, 148 and 152, indicating coordination of
the azomethine carbon. The aromatic carbons of the free ligands and ruthenium complexes show
signals from δ 102-146. For all the complexes, the ruthenium carbonyl carbon (C≡O) appears at
δ 191-195 [47, 48].
In order to confirm the presence of triphenylphosphine, 31P NMR spectra were recorded for the
complexes. A sharp singlet was observed at δ 38.12, 39.25 and 36.08, respectively, for 4, 5 and 6
due to the presence of magnetically equivalent phosphorus atoms, suggesting the presence of two
triphenylphosphine groups in a position trans to each other [49].
3.5. Mass spectral analysis
The EI-mass spectra of C49H38ClN3O5P2Ru (4), C49H38BrClN2O3P2Ru (5) and
C49H39ClN2O4P2Ru (6) have been recorded. The molecular ion peaks (M+) of 4, 5 and 6 are at
947.3160, 981.2122 and 918.3129, respectively, agreeing well with the proposed molecular
formulas of the complexes. The mass spectra of 4, 5 and 6 are shown in figures S13-S15.
Complex 4 gives a spectrum with several intensive peaks. One at 899.1381 is assigned for [M-
C2H8O]+, confirmed by comparison of experimental and calculated isotope distribution patterns
(figure S16). Similarly, 5 and 6 give a spectra with several intensive peaks. One peak matches
with [M-C4H3O]+ 914.1296 for 5 and 851.2694 [M-C4H3O]+ for 6. This is confirmed by
comparison of experimental and calculated isotope distribution patterns (figures S17 and S18)
[50, 51].
3.6. DNA binding experiments
3.6.1. Electronic absorption titration. Electronic absorption spectroscopy is a useful method
for DNA-binding studies. A compound binding to DNA through intercalation usually results in
hypochromism with or without a small red or blue shift, involving a strong stacking interaction
between the aromatic chromophore and DNA base pairs [52, 53]. The extent of hypochromism is
consistent with the strength of the intercalative binding interaction. Electronic absorption spectra
of the ligands and complexes are given in figures 4 and S19. The binding of the ligands and
complexes have been characterized through absorbance and a shift in the wavelength as a
function of added concentration of DNA. Upon incremental addition of DNA, the absorption
band of HL1 exhibits hypochromism of 48%, with red shifts of 2 nm at 282 nm. The absorption
bands of HL2 and HL3 exhibit hypochromism of 60% and 62% with blue shifts of 2 nm at 260
and 256 nm, respectively. However, the absorption bands of 4, 5 and 6 exhibit hypochromism of

66% at 259 nm, 68% at 253 nm and 73% at 255 nm with blue shifts of 3-4 nm, respectively.
These results indicate a strong interaction between DNA and compound, likely via intercalation.
In order to illustrate quantitatively the affinity of the compounds binding to DNA, the intrinsic
binding constant (Kb) has been evaluated. From the absorption data, Kb was determined using the
following equation [54],
[DNA]/ (εa – εf) = [DNA]/ (εb – εf) + 1/Kb (εb – εf)
where [DNA] is the concentration of DNA in base pairs and the apparent absorption coefficients
εa, εf and εb correspond to Aobs / [compound], the extinction coefficient of the free compound and
the extinction coefficient of the compound when fully bound to DNA, respectively. The plot of
[DNA]/(εa – εf) versus [DNA] gave a slope and intercept which are equal to 1/(εb – εf) and 1/Kb
(εb – εf), respectively; Kb is the ratio of the slope to the intercept. The magnitudes of the intrinsic
binding constants (Kb) were calculated to be 2.1 0.1×103 M-1, 2.5 0.1×103 M-1 and
4.2 0.2×103 M-1 for HL1, HL2 and HL3, respectively, and for 4, 5 and 6 the values are
3.0 0.1×104 M-1, 6.6 0.1×104 M-1 and 2.5 0.1×105 M-1, respectively. The observed binding
constant values are comparable with the classical intercalator ethidium bromide 1.4 0.1×106 M-1
[55]. The observed values of Kb reveal that the ruthenium complexes bind more strongly to DNA
than their corresponding ligands.
3.6.2. Viscosity measurements. The nature of the interaction between compounds and DNA is
further explored by viscosity measurements. A classical intercalative mode causes a significant
increase in viscosity of DNA solution [56]. Viscosity measurements of the ligands and
complexes were carried out as shown in figure 5. Upon increasing the concentration of the
compounds, the relative viscosity of DNA solutions increased steadily following the order of
6>5>4>3>2>1. It can be concluded that the ligands and complexes bind to DNA via
intercalation. The increase in viscosity of the DNA solution is a result of the DNA helix
lengthening, as base pairs are separated to accommodate the intercalating compound [57].
3.7. DNA cleavage study by gel electrophoresis
The interaction of ruthenium(II) hydrazone complexes with DNA was monitored using agarose
gel electrophoresis. A control experiment using DNA alone does not show any significant
cleavage of CT-DNA even on long exposure time (figure S20). DNA is allowed to interact with
4, 5 and 6 at two different concentrations (30 and 60 µM); a significant decrease in the intensities
of the bands for the metal bound DNA as compared to untreated control DNA was observed,

which suggests cleavage of DNA by metal complexes [58]. As can be seen from Lanes 3, 5 and 7
of 4, 5 and 6, respectively, the bands disappeared, indicating cleavage of DNA by ruthenium(II)
complexes at 60 µM concentration. The DNA cleavage results showed that the amount of
cleavage was enhanced with increasing concentration of the complexes, showing their potential
chemical nuclease activity.
3.8. Antioxidant activity
Transition metal hydrazone complexes exhibit significant antioxidant activity. Therefore, we
investigated the antioxidant potential of 4, 5 and 6 against 2,2-diphenyl-2-picrylhydrazyl
(DPPH), hydroxyl (OH) and nitric oxide (NO) radicals. The IC50 value of the complexes against
DPPH, OH and NO are shown in table 5. All the complexes exhibit significant scavenging
activity compared to the standard ascorbic acid (Aca). The IC50 values demonstrated that the
ruthenium(II) hydrazone complexes show antioxidant activity in the order 6>4>5. Complex 6 is
more effective to arrest the formation of DPPH than OH and NO radicals. The lower IC50 values
observed in antioxidant assay demonstrate that the complexes have significant potential to be
applied as scavengers to eliminate the radicals [59-61].
3.9. Cytotoxic activity evaluation
The in vitro cytotoxicity studies of the ruthenium(II) hydrazone complexes were investigated on
two cancer cell lines, HeLa (human cervical cancer cells) and MCF-7 (human breast cancer cell
line), by the MTT assay method and the results are expressed in terms of IC50 values as shown in
table 6. The results of MTT assays indicated reasonable cytotoxic activity against the two cell
lines. This activity may be due to the extended planar structure induced by the π→π*
conjugation, resulting from the chelation of the ligand with the ruthenium ion [62]. Moreover, 6
shows higher cytotoxic effect than 5 and 4 for both cell lines. Complex 6 containing
4-hydroxybenzaldehyde hydrazone exhibit higher toxicity due to its intercalative binding with
DNA [63, 64]. The ruthenium(II) complexes are more toxic towards the HeLa cell line than
MCF-7. Also, the IC50 values obtained for both cell lines were compared with those of similar
Ru(II) complexes [65, 66]. The comparative study reveals that 4-6 possess better cytotoxic
activity than reported complexes, but do reach the potency of cisplatin [67].
4. Conclusion
Three new ruthenium(II) complexes bearing hydrazone ligands HL1, HL2 and HL3 have been
synthesized and characterized using various spectroscopic methods. The molecular structures of
the ligands were determined by X-ray crystallography. Based on spectroscopic studies, an

octahedral geometry has been assigned to all the complexes. The DNA binding ability of the
ligands and complexes were assessed by absorption spectra and viscosity measurements,
indicating that the ligands and complexes bind to DNA via intercalation. The complexes also
promote cleavage of calf thymus DNA by hydrolytic mechanism. The radical scavenging activity
showed that the complexes possess greater activity against DPPH radical than OH and NO
radicals. Further, the ruthenium complexes displayed significant cytotoxic activity against HeLa
and MCF-7 cancer cell lines. In particular, 6 showed excellent cytotoxic activity against the
HeLa cell line. These results help to understand the mechanism of interactions of the compounds
binding to DNA and helpful in the development of their potential biological, pharmaceutical and
physiological implications in the future.
Supplementary data
Crystallographic data for the structural analysis have been deposited with the Cambridge
Crystallographic Data Centre, CCDC 1416586, 1416502 and 1416503. Copy of this information
may be obtained free of charge from the Director, CCDC, 12 Union Road, Cambridge, CB21 EZ,
UK (Fax: +44 1223 336033; E-mail: deposit@ccdc.cam.ac.uk or www.ccdc.cam.ac.uk/deposi).
Acknowledgements
The authors gratefully acknowledge the School of Chemistry, University of Hyderabad,
Hyderabad for their instrumental facilities. One of the authors, M. Mohanraj, thank Dr. K.
Muralidharan, Assistant Professor and UGC-Networking Centre, School of Chemistry,
University of Hyderabad, for awarding the visiting research fellowship to use the facilities at
School of Chemistry, University of Hyderabad, Hyderabad. One of the authors, G. Raja, thanks
University Grants Commission (UGC), Hyderabad, India for financial assistance (NO.F MRP-
5925/15 (SERO/UGC)).
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Figure 1. ORTEP diagram of HL1 with thermal ellipsoids at 50% probability.

Figure 2. ORTEP diagram of HL2 with thermal ellipsoids at 50% probability.

Figure 3. ORTEP diagram of HL3 with thermal ellipsoids at 50% probability.

Figure 4. Electronic absorption spectra of
6 (25 µM) in the absence and presence of increasing
amounts of CT-DNA (10, 20, 30, 40 and 50 µM). Arrow shows the changes in absorbance with
respect to an increase in the DNA concentrations (Inset: plot between [DNA] and [DNA]/(εa –
εf)×10-8).

Figure 5. Plot of relative viscosity (η/ηo)1/3vs [Compounds]/[DNA]. Effect of increasing amounts
of ), ), ), ), ) and ) on the viscosity of DNA.
1
(
2
(
3
(
4
(
5
(
6 (

Table 1. Crystal and structure refinement data for HL1, HL2 and HL3.
Compounds
HL1
HL2
HL3
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
C12H9N3O4
259.22
C12H9BrN2O2
293.11
C12H10N2O3
230.22
293(2)
293(2)
293(2)
0.71073
Monoclinic
P 21/c
0.71073
0.71073
Orthorhombic
P b c a
Orthorhombic
P b c a
Unit cell dimensions
a (Å)
17.3441(17)
9.1287(9)
15.5631(11)
2385.0(4)
8
8.9762(16)
15.495(2)
33.668(5)
4682.9(12)
16
11.602(2)
7.6513(13)
24.314(6)
2158.3(8)
8
b (Å)
c (Å)
Volume (Å3)
Z
Density (calculated) Mg/m3
Absorption coefficient mm-1
F(000)
1.444
1.663
1.417
0.112
3.501
0.104
1072
3762
880
θ range for data collection (°)
Index ranges
3.02 to 24.99
2.69 to 25.00
3.30 to 24.99
-16<=h<=20,
-7<=k<=10,
-18<=l<=14
-10<=h<=10,
-18<=k<=18,
-38<=l<=40
-13<=h<=10,
-8<=k<=9,
-28<=l<=21
Reflections collected
9569
14235
5450
Independent reflections
4196
4125
1892
[R(int) = 0.0356]
[R(int) = 0.1209]
[R(int) = 0.0717]
Refinement method
Full-matrix least-
squares on F2
Full-matrix least-
squares on F2
Full-matrix least-
squares on F2
Data / restraints / parameters
Goodness-of-fit on F2
R indices (all data)
4196 / 0 / 352
1.002
4125 / 0 / 307
0.974
1892 / 0 / 155
0.972
R1 = 0.1030,
wR2 = 0.1578
R1 = 0.1594,
wR2 = 0.2581
R1 = 0.1509,
wR2 = 0.0849
Largest diff. peak and hole e.Å-3
0.426 and -0.315
0.724 and -0.444
0.202 and -0.215

Table 2. Selected bond lengths [Å] and angles [°] for HL1.
Bond lengths [Å]
Bond angles [°]
O(5)-C(16)
O(5)-C(13)
O(6)-C(17)
O(2)-C(5)
N(4)-C(17)
N(4)-N(5)
N(5)-C(18)
N(2)-C(6)
N(2)-N(1)
N(1)-C(5)
O(8)-N(6)
O(1)-C(4)
O(1)-C(1)
O(7)-N(6)
C(24)-N(6)
C(8)-N(3)
N(3)-O(4)
N(3)-O(3)
1.357(3)
1.362(3)
1.227(3)
1.224(3)
1.339(3)
1.384(3)
1.266(3)
1.257(4)
1.378(3)
1.347(4)
1.217(3)
1.353(4)
1.361(4)
1.229(4)
1.467(4)
1.466(5)
1.159(4)
1.178(4)
C(17)-N(4)-N(5)
C(18)-N(5)-N(4)
C(6)-N(2)-N(1)
C(5)-N(1)-N(2)
C(4)-O(1)-C(1)
O(6)-C(17)-N(4)
O(6)-C(17)-C(16)
N(4)-C(17)-C(16)
O(2)-C(5)-N(1)
O(2)-C(5)-C(4)
N(1)-C(5)-C(4)
O(5)-C(16)-C(17)
C(3)-C(4)-O(1)
O(1)-C(4)-C(5)
O(8)-N(6)-O(7)
O(8)-N(6)-C(24)
N(5)-C(18)-C(19)
N(2)-C(6)-C(7)
119.8(2)
114.0(2)
115.9(2)
118.9(2)
106.6(3)
124.4(3)
120.6(2)
115.0(2)
123.8(3)
121.7(3)
114.5(2)
118.3(2)
109.0(3)
116.1(2)
123.9(3)
118.2(3)
120.3(2)
118.8(3)

Table 3. Selected bond lengths [Å] and angles [°] for HL2.
Bond lengths [Å]
Bond angles [°]
N(1)-C(5)
1.342(10)
1.365(8)
1.215(9)
1.278(9)
1.245(9)
1.378(8)
1.344(10)
1.211(9)
1.335(9)
1.348(10)
1.345(9)
1.356(11)
1.325(12)
1.358(13)
1.875(9)
1.862(9)
1.423(12)
1.352(13)
C(5)-N(1)-N(2)
C(6)-N(2)-N(1)
C(18)-N(4)-N(3)
C(17)-N(3)-N(4)
O(2)-C(5)-N(1)
O(2)-C(5)-C(4
116.6(6)
117.6(7)
116.1(7)
121.1(6)
124.2(7)
122.0(8)
113.8(7)
107.1(7)
120.4(8)
109.6(8)
116.9(7)
119.8(7)
120.4(8)
119.8(7)
122.8(7)
115.7(7)
109.0(8)
104.8(7)
N(1)-N(2)
O(4)-C(17)
N(2)-C(6)
N(4)-C(18)
N(4)-N(3)
N(3)-C(17)
C(5)-O(2)
N(1)-C(5)-C(4)
C(4)-O(1)-C(1)
N(4)-C(18)-C(19)
C(3)-C(4)-O(1)
O(1)-C(4)-C(5)
N(2)-C(6)-C(7)
O(3)-C(16)-C(17)
N(2)-C(6)-C(7)
O(4)-C(17)-N(3)
N(3)-C(17)-C(16)
C(2)-C(1)-O(1)
C(16)-O(3)-C(13)
O(3)-C(16)
O(3)-C(13)
O(1)-C(4)
O(1)-C(1)
C(10)-C(9)
C(10)-C(11)
Br(2)-C(24)
Br(1)-C(12)
C(20)-C(21)
C(21)-C(22)

Table 4. Selected bond lengths [Å] and angles [°] for HL3.
Bond lengths [Å]
Bond angles [°]
N(2)-C(6)
N(2)-N(1)
O(3)-C(10)
O(1)-C(1)
O(1)-C(4)
O(2)-C(5)
N(1)-C(5)
C(2)-C(1)
C(2)-C(3)
1.272(4)
1.387(4)
1.361(4)
1.359(5)
1.365(4)
1.236(4)
1.347(4)
1.333(5)
1.411(5)
C(6)-N(2)-N(1)
C(5)-N(1)-N(2)
O(3)-C(10)-C(11)
O(3)-C(10)-C(9)
O(2)-C(5)-N(1)
O(2)-C(5)-C(4)
O(1)-C(4)-C(5)
N(1)-C(5)-C(4)
N(2)-C(6)-C(7)
116.9(3)
117.8(3)
123.0(4)
117.5(4)
122.7(4)
120.9(3)
119.7(3)
116.4(3)
121.0(3)

Table 5. The radical scavenging activities of the compounds.
IC50 µM
Compounds
DPPH
OH
NO
23.26 1.1
20.47 2.1
27.98 1.7
29.94 1.1
26.05 1.9 21.37 2.5
39.12 2.6 14.11 1.7
8.54 2.2 30.29 3.2
5.43 1.8 37.26 1.7
4
5
6
Aca

Table 6. The cytotoxic activities of the compounds.
IC50 µM
Compounds
HeLa
MCF-7
93.17 2.2
77.13 1.2
48.00 1.4
12.52 0.7
97.23 2.1
81.38 2.5
62.75 2.8
12.75 0.5
4
5
6
Cisplatin