DNA binding, DNA cleavage, antioxidant and cytotoxicity studies on ruthenium(II) complexes of benzaldehyde 4-methyl-3-thiosemicarbazones

Article abstract of DOI:10.1016/j.saa.2012.12.064

Four new ruthenium(II) complexes with N(4)-methyl thiosemicarbazone ligands, (E)-2-(2-chlorobenzylidene)-N-methylhydrazinecarbothioamide (HL ¹) and (E)-N-methyl-2-(2-nitrobenzylidene)hydrazinecarbothioamide (HL²), were prepared and f

Full text of DOI:10.1016/j.saa.2012.12.064

Accepted Manuscript
DNA binding, DNA cleavage, antioxidant and cytotoxicity studies on rutheni‐
um(II) complexes of benzaldehyde 4-methyl-3-thiosemicarbazones
Krishnan Sampath, Subbaiyan Sathiyaraj, Chinnasamy Jayabalakrishnan
PII:
S1386-1425(12)01299-1
http://dx.doi.org/10.1016/j.saa.2012.12.064
SAA 10116
DOI:
Reference:
To appear in:
Spectrochimica Acta Part A: Molecular and Biomo‐
lecular Spectroscopy
Received Date:
Revised Date:
Accepted Date:
26 October 2012
7 December 2012
10 December 2012
Please cite this article as: K. Sampath, S. Sathiyaraj, C. Jayabalakrishnan, DNA binding, DNA cleavage, antioxidant
and cytotoxicity studies on ruthenium(II) complexes of benzaldehyde 4-methyl-3-thiosemicarbazones,
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2012), doi: http://dx.doi.org/10.1016/j.saa.
2012.12.064
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DNA binding, DNA cleavage, antioxidant and cytotoxicity studies on ruthenium(II)
complexes of benzaldehyde 4-methyl-3-thiosemicarbazones
Krishnan Sampath, Subbaiyan Sathiyaraj and Chinnasamy Jayabalakrishnan*
Post-Graduate and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya
College of Arts and Science, Coimbatore – 641 020, Tamil Nadu, India.
ABSTRACT
Four new ruthenium(II) complexes with N(4)-methyl thiosemicarbazone ligands,
(E)-2-(2-chlorobenzylidene)-N-methylhydrazinecarbothioamide (HL1) and (E)-N-methyl-2-
(2-nitrobenzylidene)hydrazinecarbothioamide (HL2), were prepared and fully characterized
by various spectro-analytical techniques. The Schiff bases act as bidentate, monobasic
chelating ligands with S and N as the donor sites and are preferably found in the thiol form in
all the complexes studied. The molecular structure of HL1 and HL2 were determined by
single crystal X-ray diffraction method. DNA binding of the compounds was investigated by
absorption spectroscopy which indicated that the complexes bind to DNA via intercalation.
The oxidative cleavage of the complexes with CT-DNA inferred that the effects of cleavage
are dose dependent. Antioxidant studies of the ligands and complexes showed the significant
antioxidant activity against DPPH radical. In addition, the in vitro cytotoxicity of the ligands
and complexes against MCF-7 cell line was assayed which showed higher cytotoxic activity
with the lower IC50 values indicating their efficiency in killing the cancer cells even at low
concentrations.
Keywords: Ruthenium(II) complexes, Thiosemicarbazones, DNA interaction, Antioxidant,
Cytotoxicity.
1. Introduction
Cancer is one of the fatal diseases, which claims over 6 million people each year
worldwide and still increasing. The majority of drugs used for the treatment of cancer today
are not ‘cancer cell-specific’ and potently cytotoxic against normal cells. So, the most rapidly
developing area of pharmaceutical research is the discovery of new drugs for cancer today.
To date, cisplatin is considered to be one of the most effective and widely used anticancer
drugs. Second generation platinum drugs including cisplatin and oxaliplatin have been
developed for clinical application [1]. The efficacy of these drugs, including cisplatin,
1

however, is reduced by increasing tumour resistance and in the case of cisplatin, high
toxicity. This in turn affects the administration of the drugs [2]. These limitations have
aroused interest towards the design and evaluation of transition metal complexes other than
platinum derivatives for therapeutic use. In this respect ruthenium complexes, due to their
favourable properties viz. kinetic ligand-exchange, variable oxidation states and lower
toxicity toward normal cells (due to similarity with iron), are in the forefront of these
investigations [3,4].
Thiosemicarbazones are versatile ligands as they possess a number of donor atoms
which may coordinate in various ways. In addition to this, thiosemicarbazones possess a
variety of biological properties including antiproliferative activity [5,6]. Studies have
demonstrated that thiosemicarbazones are potent inhibitors of the enzyme ribonucleotide
reductase and are capable of interrupting DNA synthesis and repair. Incorporation of metals
onto these thiosemicarbazone ligands can result in alteration or enhancement of their
biological activity [7,8]. In the case of pharmaceuticals, the binding capacity of
thiosemicarbazones is further increased by condensation of the thiosemicarbazide with an
aldehyde containing heteroatom [9,10]. This aroused our interest in the synthesis of
benzaldehyde thiosemicarbazones and their ruthenium(II) complexes with a viewpoint
towards evaluating biological properties such as DNA interaction, antioxidant and cytotoxic
activity. Herein, we report the synthesis, characterization, DNA binding, DNA cleavage,
antioxidative and cytotoxicity studies of ruthenium(II) complexes of 2-choloro/nitro
benzaldehyde 4-methyl-3-thiosemicarbazones. The crystal structure of the thiosemicarbazone
ligands have been determined by X-ray crystallography. The investigation of the biological
properties of the ligands and ruthenium(II) complexes has been focused on the binding
properties with CT-DNA performed by UV spectroscopy and cleavage properties of the
complexes performed by gel electrophoresis with CT-DNA. Finally, we have studied their
antioxidative property against DPPH radical and in vitro antitumor activity against MCF-7
cancer cell line.
2. Experimental
2.1. Materials and Methods
All the chemicals used were chemically pure and AR grade. Solvents were purified
and dried according to the standard procedure [11]. Calf-thymus (CT-DNA) was purchased
from Bangalore Genei, Bangalore, India. The metal precursors [RuHCl(CO)(PPh3)3]
2

and [RuHCl(CO)(AsPh3)3] were prepared by literature methods [12,13]. Micro analyses
(C, H, N & S) were performed on a Vario EL III CHNS analyser at STIC, Cochin University
of Science and Technology, Kerala, India. IR spectra were recorded as KBr pellets in the
400-4000 cm-1 region using a Perkin Elmer FT-IR 8000 spectrophotometer. Electronic
spectra were recorded in DMSO solution with a Systronics double beam UV-vis
1
spectrophotometer 2202 in the range 200-800 nm. H NMR 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. Antioxidant and anti cancer studies were
carried out at the Kovai Medical Centre and Hospital Pharmacy College, Coimbatore,
Tamilnadu. Melting points were recorded with Veego VMP-DS heating table.
2.2. Preparation of thiosemicarbazone ligands
2.2.1. (E)-2-(2-chlorobenzylidene)-N-methylhydrazinecarbothioamide (HL1) (1)
A methanolic solution (50 mL) of 4-methyl-3-thiosemicarbazide (1.051 g, 0.01 mol)
was added to a methanol solution (25 mL) containing 2-chlorobenzaldehyde (1.12 mL, 0.01
mol). The mixture was refluxed for an hour during which period a white color precipitate was
formed. The reaction was cooled to room temperature and the solid compound was filtered,
washed and recrystallized from methanol. White colored crystals, suitable for single crystal
X-ray diffraction analysis, were obtained by slow evaporation of its methanolic solution.
Yield: 89%; M.P: 200-201 °C. Anal. calcd. for C9H10ClN3S (%): C, 47.47; H, 4.43; N, 18.45;
S, 14.08. Found (%): C, 47.40; H, 4.45; N, 18.43; S, 14.01. IR (KBr, cm-1): 1585 ν(C=N);
951 ν(C=S). UV-vis (DMSO), λmax (nm) (ε M-1 cm-1): 305 (11400), 360 (14233) (π→π*,
1
n→π*). H NMR (DMSO-d6); δ 8.60 (s, 1H, hydrazine NH); δ 8.47 (s, 1H, methyl NH);
δ 11.67 (s, 1H, H–C=N); δ 3.03 (d, 3H, CH3); δ 7.39-8.30 (m, 4H, aromatic). 13C NMR
(DMSO-d6); δ 142 (C=S); δ 178 (C=N); δ 31 (CH3); δ 127-133 (aromatic).
2.2.2. (E)-N-methyl-2-(2-nitrobenzylidene)hydrazinecarbothioamide (HL2) (2)
It was prepared by using the same procedure as described for HL1 with
4-methyl-3-thiosemicarbazide (1.051 g, 0.01 mol) and 2-nitrobenzaldehyde (1.511 g,
0.01 mol). A yellow product was formed. The solid compound was filtered, washed and
recrystallized from methanol. Yellow colored crystals, suitable for single crystal X-ray
diffraction analysis, were obtained by slow evaporation of its methanolic solution.
3

Yield: 85%; M.P: 229-230 °C. Anal. calcd. for C9H10N4O2S (%): C, 45.37; H, 4.23; N, 23.51;
S, 13.46. Found (%): C, 45.39; H, 4.29; N, 23.52; S, 13.42. IR (KBr, cm-1): 1530 ν(C=N);
850 ν(C=S). UV-vis (DMSO), λmax (nm) (ε M-1 cm-1): 305 (11466), 368 (14744), 396
(20955) (π→π*, n→π*). 1H NMR (DMSO-d6); δ 8.64 (s, 1H, hydrazine NH); δ 8.48 (s, 1H,
methyl NH); δ 11.79 (s, 1H, H–C=N); δ 3.04 (d, 3H, CH3); δ 7.61-8.43 (m, 4H, aromatic).
13C NMR (DMSO-d6); δ 148 (C=S); δ 178 (C=N); δ 31 (CH3); δ 125-137 (aromatic).
2.2.3. Single crystal X-ray diffraction studies.
Single crystal X-ray diffraction data of 1 and 2 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 of ligands 1 and 2
were performed using a graphite-mono chromate Mo Kα (λ = 0.71073 Å) radiation by φ and
ω scans. The data collected were reduced SAINT program [14] and the empirical absorption
corrections were carried out using the SADABS program [15]. The structure of the
compounds 1 and 2 was solved by direct methods [16] using SHELXS-97, which revealed
the position of all non-hydrogen atoms, and was refined by full-matrix least squares on
F2 (SHELXL-97) [17]. All non-hydrogen atoms were refined anisotropically, while the
hydrogen atoms were placed in calculated positions and refined as riding atoms.
2.3. Preparation of ruthenium(II) complexes
2.3.1. Synthesis of [RuCl(CO)(PPh3)2L1] (3)
A methanolic solution (20 mL) containing HL1 (1) (0.110 g, 0.5 mmol) and
triethylamine (0.07 mL, 0.5 mmol) were added to [RuHCl(CO)(PPh3)3] (0.475 g, 0.5 mmol)
in benzene (20 mL). The resulting red color solution was refluxed for 8 h. The reaction
mixture was then cooled to room temperature, which results in the formation of orange color
precipitate. It was filtered off and then subjected to purification by TLC. This solid was
recrystallized from CH2Cl2/Hexane mixture. Our sincere effort to obtain single crystal of the
complexes went unsuccessful. Yield: 59%. M.P: 230-232 °C. Anal. calcd. for
C46H39Cl2N3OP2RuS (%): C, 60.33; H, 4.29; N, 4.59; S, 3.50. Found (%): C, 60.31; H, 4.21;
N, 4.55; S, 3.58. IR (KBr, cm-1): 1575 ν(C=N); 745 ν(C–S); 1948 ν(C≡O). UV-vis (DMSO),
1
λmax (nm) (ε M-1 cm-1): 305 (11455), 368 (14866) (ILCT), 404 (19866) (MLCT). H NMR
(DMSO-d6); δ 8.47 (s, 1H, methyl NH); δ 11.69 (s, 1H, H–C=N); δ 3.03 (d, 3H, CH3);
4

δ 7.24-8.31 (m, 34H, aromatic). 13C NMR (DMSO-d6); δ 137 (C–S); δ 181 (C=N); δ 194
(C≡O); δ 31 (CH3); δ 125-134 (aromatic). 31P NMR (DMSO-d6); δ 36.3 (PPh3).
2.3.2. Synthesis of [RuCl(CO)(AsPh3)2L1] (4)
It was prepared by using the same procedure as described for 3 with HL1 (1) (0.110 g,
0.5 mmol) and [RuHCl(CO)(AsPh3)3] (0.540 g, 0.5 mmol). Yellow colored powder obtained.
Yield: 55%. M.P: 238-240 °C. Anal. calcd. for C46H39Cl2N3OAs2RuS (%): C, 55.05; H, 3.92;
N, 4.19; S, 3.19. Found (%): C, 55.15; H, 3.95; N, 4.15; S, 3.12. IR (KBr, cm-1):
1567 ν(C=N); 735 ν(C–S); 1939 ν(C≡O). UV-vis (DMSO), λmax (nm) (ε M-1 cm-1):
305 (11533), 368 (14877) (ILCT), 404 (19622) (MLCT). 1H NMR (DMSO-d6); δ 8.46 (s, 1H,
methyl NH); δ 11.68 (s, 1H, H–C=N); δ 3.03 (d, 3H, CH3); δ 7.24-8.30 (m, 34H, aromatic).
13C NMR (DMSO-d6); δ 138 (C–S); δ 182 (C=N); δ 191 (C≡O); δ 31 (CH3); δ 127-133
(aromatic).
2.3.3. Synthesis of [RuCl(CO)(PPh3)2L2] (5)
It was prepared by using the same procedure as described for 3 with HL2 (2) (0.138 g,
0.5 mmol) and [RuHCl(CO)(PPh3)3] (0.475 g, 0.5 mmol). Brown colored powder obtained.
Yield: 58%. M.P: 248-250 °C. Anal. calcd. for C46H39ClN4O3P2RuS (%): C, 59.64; H, 4.24;
N, 6.05; S, 3.46. Found (%): C, 59.69; H, 4.27; N, 6.00; S, 3.42. IR (KBr, cm-1):
1519 ν(C=N); 744 ν(C–S); 1947 ν(C≡O). UV-vis (DMSO), λmax (nm) (ε M-1 cm-1):
305 (11500), 368 (14566), 401 (18388) (ILCT), 431 (20855) (MLCT). 1H NMR (DMSO-d6);
δ 8.48 (s, 1H, methyl NH); δ 11.81 (s, 1H, H–C=N); δ 3.03 (d, 3H, CH3); δ 7.23-8.18 (m,
34H, aromatic). 13C NMR (DMSO-d6); δ 138 (C–S); δ 182 (C=N); δ 192 (C≡O); δ 31 (CH3);
δ 125-133 (aromatic). 31P NMR (DMSO-d6); δ 36.3 (PPh3).
2.3.4. Synthesis of [RuCl(CO)(AsPh3)2L2](6)
It was prepared by using the same procedure as described for 3 with HL2 (2) (0.138 g,
0.5 mmol) and [RuHCl(CO)(AsPh3)3] (0.54 g, 0.5 mmol). Orange colored powder obtained.
Yield: 56%. M.P: 263-265 °C. Anal. calcd. for C46H39ClN4O3As2RuS (%): C, 54.57; H, 3.88;
N, 5.52; S, 3.16. Found (%): C, 54.52; H, 3.80; N, 6.55; S, 3.19. IR (KBr, cm-1):
1520 ν(C=N); 737 ν(C–S); 1949 ν(C≡O). UV-vis (DMSO), λmax (nm) (ε M-1 cm-1):
305 (11577), 368 (14933), 404 (19977) (ILCT), 432 (20855) (MLCT). 1H NMR (DMSO-d6);
δ 8.49 (s, 1H, methyl NH); δ 11.81 (s, 1H, H–C=N); δ 3.03 (d, 3H, CH3); δ 7.27-8.19
5

(m, 34H, aromatic). 13C NMR (DMSO-d6); δ 139 (C–S); δ 181 (C=N); δ 192 (C≡O);
δ 31 (CH3); δ 128-133 (aromatic).
2.4. DNA binding - Titration experiments
All the 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 M-1 cm-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 (0-40 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. Nuclease activity using gel electrophoresis
The DNA cleavage activity of the ruthenium(II) complexes was monitored by agarose
gel electrophoresis on CT DNA. The tests were performed in the absence and presence of
activating agent, H2O2 under aerobic condition. Hydrolytic cleavage was monitored by
30 µM of CT DNA and 40 µM of each complex (3-6) in 5 % DMSO and 95 % Tris-HCl
buffer (5 mM, pH 7.2) with 50 mM NaCl.
For oxidative cleavage, each reaction mixture contained 30 µM of CT DNA, 30; 60;
90 µM of each complex (3-6) in 5 % DMSO and 95 % Tris-HCl buffer (5 mM, pH 7.2) with
50 mM NaCl and 60 µM of H2O2.
o
The samples with sufficient buffer were incubated for 2 h at 37 C. 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 1.0 µg/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.
6

2.6. Antioxidant activity
The 2,2-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity of the
compounds was measured according to the method of Elizabeth [18]. The DPPH radical is a
stable free radical having a λ at 517 nm. A fixed concentration of the experimental
max
compound (100 µL) was added to a solution of DPPH in methanol (0.3 mM, 1 mL) and the
final volume was made up to 4 mL with double distilled water. DPPH solution with methanol
was used as a positive control and methanol alone acted as a blank. The solution was
incubated at 37 °C for 30 min in dark. The decrease in absorbance of DPPH was measured at
517 nm. The tests were run in triplicate, and various concentrations (20-100 µg/ mL) of the
compounds used to fix a concentration at which the compounds showed 50 % of activity. In
addition, the percentage of activity was calculated using the formula, % of suppression ratio =
[(A0 – Ac)/A0] X 100. A0 and Ac are the absorbance in the absence and presence of the tested
compounds respectively. The 50 % activity (IC50) can be calculated using the percentage of
activity.
2.7. In vitro anticancer activity assay
Cytotoxicity of the compounds were carried out on human breast cancer cell line
(MCF-7) was obtained from National Centre for Cell Science (NCCS), Pune, India. Cell
viability was carried out using the MTT assay method [19]. MCF-7 cell was grown in eagles
minimum essential medium (EMEM) containing 10 % fetal bovine serum (FBS). For the
screening experiment, the cells were seeded onto 96-well plates at plating density of
10,000 cells/ well and incubated to allow for cell attachment at 37 °C, 5 % CO2, 95 % air and
100 % relative humidity for 24 h prior to the addition of the 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. Triplication was maintained and the medium
not containing the compounds served as control. After 48 h, 15 µL of MTT (5 mg mL-1) in
phosphate buffered saline (PBS) was added to each well and incubated at 37 °C for 4 h. The
medium with MTT was then removed and the formed formazan crystals were dissolved in
100 µL of DMSO. The absorbance was then measured at 570 nm using micro plate reader.
The % cell inhibition was determined using the following formula and a graph was plotted
with the percentage of cell inhibition versus concentration. From this, the IC50 was
7

calculated: % cell Inhibition = [mean OD of untreated cells (control)/mean OD of treated cells
(control)] x100.
3. Results and Discussion
Analytical and spectroscopic data for the ligands and its complexes indicate a 1:1
metal-ligand stoichiometry for all the complexes. The synthetic route of the complexes and
the proposed structure of the complexes are shown in Scheme 1. The complexes are soluble
in most common organic solvents like CH2Cl2, CHCl3, DMF, DMSO, etc.
R
R
Ligand
R
H
C
N
EPh3
CH
N
[RuHCl(CO)(EPh3)3]
HL1
HL1
Cl
Cl
HN
C
N
C
Cl
Ru
Methanol/Benzene, TEA
HN
S
CO
HN
S
22
NO
NO22
HHLL
CH3
EPh3
CH3
Scheme 1 Synthetic route of the ruthenium(II) thiosemicarbazone complexes, where E=P/As.
3.1. X-ray crystallography
The ORTEP representation of compounds 1 and 2 is shown in Fig. 1 and 2
respectively. Relevant data collection and details of the structure refinement are summarized
in Table 1. The selected bond lengths, bond angles and torsion angles for compounds 1 and 2
are given in tables 2, 3 and 4 respectively.
The ligand 1 crystallizes in a monoclinic space group C2/c with eight
crystallographically independent molecules in the unit cell (Fig 3). Its existence in the thione
form is confirmed by the C=S bond length of 1.6884(14) Å. The molecule exists in the
E conformation about N2-N3 bond as evidenced by the C2-N2-N3-C3 torsion angle of
175.6(1)°. The azomethine bond length, C3-N3 (1.276(2) Å) is in conformity with a formed
C=N double bond length (1.28 Å). The ligand 2 crystallizes in a triclinic space group P-1
with two crystallographically independent molecules in the unit cell (Fig. 4). The azomethine
bond length, C7-N2 (1.276(1) Å) is in conformity with a formal C=N double bond length
(1.28 Å) and C8-S1 bond distance of 1.683(1) Å is very close to a formal C=S bond length
(1.60 Å). It confirms the existence of the thiosemicarbazone in the thione form in the solid
8

state. The thione sulphur atom S(1) is trans to the azomethine nitrogen atom N(2) which
confirmed by a torsion angle of 179.68(8) for S(1)-C(8)-N(3)-N(2) bond.
3.2. Infrared spectra
The IR spectra of the free ligands were compared with those of the metal complexes
in order to study the binding mode(s) of the thiosemicarbazone ligand to metal. A medium
sharp band at 1530 and 1585 cm-1 due to the azomethine C=N stretching frequency of the free
ligands 1 and 2 respectively was shifted to lower frequency in the spectra of the complexes at
1519-1575 cm-1 indicating that the coordination through N atom [20]. A band appeared at
951 cm-1 for 1 and 850 cm-1 for 2 due to vibration of the C=S double bond which disappeared
in the spectra of the complexes and a new band, C–S appeared at 735-745 cm-1 indicating that
the other coordination is through thiolate sulphur after enolization followed by deprotonation
on sulphur [21]. Further, the replacement of hydride ion in the metal precursors by the
ligands has been confirmed by the absence of a band around 2020 cm-1 in all the complexes
[22]. In all the complexes, the strong band in the region 1939-1949 cm-1 is due to terminally
coordinated carbonyl group. Overall, the complexes contain monobasic NS coordinated
thiosemicarbazones. In addition, the characteristic absorption bands due to
triphenylphosphine and triphenylarsine were also observed for all the complexes in their
expected regions.
3.3. Electronic spectra
The electronic spectra of the ligands show bands at 305, 360-396 nm. The first band
at 305 nm, is assigned to a ligand π→π* transition, while the band at 360-396 nm is assigned
to an n→π* transition associated with the imine and thioamide function of the
thiosemicarbazones. The spectra of the complexes showed three to four bands in the region
303-432 nm. The bands at 303-308 and 368 nm were associated with intra ligand transitions.
These bands are shifted when compared to ligands indicating the involvement of imine
nitrogen and thionyl sulphur in coordination with ruthenium atom [21]. The bands appearing
in the region 401-432 nm have been assigned to charge transfer transitions arising from the
excitation of an electron from metal t2g level to an unfilled molecular orbital derived from the
π* level of the ligands [22]. The pattern of the electronic spectra for the complexes are
similar to other ruthenium(II) octahedral complexes [23,24].
9

3.4. NMR spectra
The NMR spectra of the compounds were recorded in DMSO-d6 solution for
1
confirming the binding mode of the ligands to the ruthenium ion and the H, 13C and
1
31P NMR spectra of the complex 3 are shown in Fig. 5. The H NMR spectra of the free
ligands showed a singlet in the region δ 8.60-8.64 for the hydrazine NH proton, which is
absent in the spectra of the complexes indicating the enolization and deprotonation of the
–NH–C=S group prior to coordination of ligand to metal through thiolate sulphur. The
–CH=N proton observed at δ 11.67-11.79 in the spectra of the ligands, is shifted slightly
downfield in the spectra of the complexes in the region δ 11.68-11.81. This observation
supports the involvement of –C=N chromophore in coordination. The aromatic protons,
observed as multiplets in the region δ 7.39-8.43 in the spectra of the ligands, are remain more
or less unchanged in the complexes in the region δ 7.23-8.43, due to the delocalisation of
electron density in the system [25] and these signals in the complexes cannot be distinguished
from the aromatic signals of PPh3/ AsPh3 due to their extensive overlap [23]. For ligands and
complexes, the chemical shift at δ 3.03-3.04 and δ 8.46-8.49 are due to methyl proton and
methyl NH proton respectively.
In the 13C NMR spectra of the ligands, the signal at δ 142-148 corresponds to the
thioamide carbon (C=S), which disappears in the spectra of the complexes and a new signal,
(C–S) at δ 137-139 indicates the coordination of sulphur via deprotonation [24]. The spectra
of the ligands displayed a single resonance at δ 178 due to the azomethine carbon atom,
downfield shift of this signal at δ 181-182 clearly indicates the coordination of C=N carbon
atom. The aromatic carbons of the free ligands and complexes show signal in the region
δ 125-137. The signal due to methyl carbon of ligands and the complexes appear at δ 31. For
all the complexes, the terminal carbonyl group C≡O appeared at δ 191-194 [26].
31
In order to confirm the presence of triphenylphosphine group, P NMR spectra were
recorded for the complexes, 3 and 5. A sharp singlet was observed at δ 36.3 ppm for the
complexes, 3 and 5 due to presence of magnetically equivalent phosphorous atoms
suggesting the presence of two triphenylphosphine groups in a position trans to each
other [27].
10

3.5. Pharmacology
3.5.1. DNA binding - Titration experiments
The study of non-covalent interactions of transition metal complexes with DNA is an
area of intense current interest. Because DNA is an important cellular receptor, many
compounds exert their anticancer effects through binding to DNA, thereby changing the
replication of DNA and inhibiting the growth of the tumor cells, which is the basis of
designing new and more efficient anticancer drugs, where their effectiveness depends on the
mode and affinity of the binding [28,29]. Therefore, the binding studies of metal complexes
to DNA are considered to be highly important in the development of new anticancer drugs.
Electronic absorption spectroscopy is an effective method to examine the binding
modes of metal complexes with DNA. Compounds binding through intercalation usually
results in hypochromism with or without small red or blue shift, since the intercalative mode
involves a strong interaction between the planar aromatic chromophore and the base pairs of
DNA [30]. The results of absorption spectra of the compounds in the absence and presence of
CT-DNA are given in Fig. 6. Upon increasing the concentration of DNA to the test
compounds, the absorption bands of the ligands 1 and 2 exhibited hypochromism of 5.65 %
and 17.34 % with blue shifts of 2 nm at 310 and 280 nm, whereas the absorption bands of
complexes 3 and 4 exhibited a hypochromism of about 17.34 % at 366 nm and 24.92 % at
363 nm with blue shifts of 3 nm, respectively. However, complexes 5 at 356 nm and 6 at 366
nm exhibited a hypochromism of about 8.55 % and 3.71 % with red shifts of 4 and 2 nm,
respectively. These results suggested an intimate association of the test compounds with
CT-DNA, and it is also likely that these compounds bind to the DNA helix via intercalation.
After the compounds intercalate to the base pairs of DNA, the π* orbital of the intercalated
compounds could couple with π orbitals of the base pairs, thus decreasing the π→π*
transition energies, hence resulting in hypochromism [31]. In order to compare quantitatively
the binding strength of the compounds, the intrinsic binding constants (Kb) of them with
CT-DNA were determined from the following equation.
[DNA]/(ε – ε ) = [DNA]/(ε – ε ) + 1/Kb (ε – ε )
a
f
b
f
b
f
where [DNA] is the concentration of DNA in base pairs and the apparent absorption
coefficient ε , ε and ε correspond to Aobs/[compound], the extinction coefficient of the free
a
f
b
compound and the extinction coefficient of the compound when fully bound to DNA,
respectively. The plot of [DNA]/(ε – ε ) versus [DNA] gave a slope and the intercept
a
f
11

which are equal to 1/(ε – ε ) and 1/Kb (ε – ε ), respectively; Kb is the ratio of the slope to
b
f
b
f
the intercept. The magnitudes of intrinsic binding constants (Kb) were calculated to be
1.7( 0.08) X 104 M-1, 4.6( 0.12) X 103 M-1, 3.4( 0.17) X 105 M-1, 3.0( 0.09) X 105 M-1,
6.3( 0.10) X 104 M-1 and 2.6( 0.16) X 104 M-1 for compounds 1, 2, 3, 4, 5 and 6
respectively. The observed values of Kb revealed that the ruthenium(II) complexes (3 - 6)
bind strongly than the respective ligands (1 and 2) to DNA via intercalative mode. From the
results obtained, it has been found that complex 3 strongly bound with CT-DNA relative to
that with 4, 5 and 6, and the order of binding affinity is 3 > 4 > 5 > 6 > 1 > 2.
3.5.2. DNA cleavage study by gel electrophoresis
Suitably designed metal complexes, after binding to DNA, can induce several changes
in the DNA conformation, such as bending, ‘local denaturation’ (overwinding and
underwinding), intercalation, micro loop formation and subsequent DNA shorting lead to
decrease in molecular weight of DNA [32]. The ability of metal complexes to perform DNA
cleavage is generally monitored by agarose gel electrophoresis and in the present work
CT DNA was chosen to investigate its cleavage. The cleavage experiments were carried out
in the absence and presence of activating agent, H2O2 under aerobic conditions and are shown
in Fig 7 and 8 respectively. Fig. 7, shows the hydrolytic cleavage activity of the complexes,
3-6 at 40 µM. A control experiment using DNA alone does not show any significant cleavage
of DNA (Lane 1). Further, when DNA is allowed to interact with the complexes, no
considerable difference in the intensity of the bands for metal bound DNA as compared to
control DNA was observed. This result suggests that the nuclease activity of the complexes
does not involve by hydrolytic pathway. These experimental facts demonstrated that a
combination of both the ruthenium complexes and activating agent, H2O2 are required to
show effective cleavage of CT DNA. Fig. 8 reveals that the oxidative cleavage of CT DNA
induced by complexes, 3-6 in the presence of H2O2. Control experiment using DNA alone
(Lane 1 & 9) and DNA with H2O2 (Lane 2 and 10) does not show any significant cleavage of
CT-DNA even on longer exposure time. When the DNA is allowed to interact with the
complexes (3-6) at various concentrations (30, 60 and 90 µM), a substantial decrease in the
intensities of the bands for the metal bound DNA as compared to untreated control DNA was
observed, which suggests the cleavage of DNA by metal complexes [33]. As can be seen
from Lanes 5, 8, 13 and 16 of complexes 3, 4, 5 and 6 respectively, the bands were
completely disappeared, indicating the sufficient cleavage of DNA by ruthenium(II)
12

complexes at 90 µM concentration. Interestingly, the complex, 3 shows potential nuclease
activity against DNA at low concentration, 30 µM itself, indicates a superior DNA cleavage
than the other complexes. The cleavage activity may be due to the intercalative interaction of
the complexes with the DNA strands in the presence of H2O2 induces oxidative DNA
cleavage [33].
3.5.2. Antioxidant activity
Since the DNA interaction experiments conducted so far revealed that the compounds
exhibit good DNA binding affinity and good cleavage activity, it is considered worthwhile to
study the antioxidant activity of these compounds. 2,2-Diphenyl-2-picryl-hydrazyl (DPPH)
assay is widely used for assessing the ability of radical scavenging activity and it is measured
in terms of IC50 values. Because of the presence of odd electron, DPPH shows a strong
absorption band at 517 nm in the visible spectrum. As this electron becomes paired off in the
presence of a free radical scavenger, this absorption vanishes, and the resulting
decolourization is stoichiometric with respect to the number of electrons taken up. The DPPH
assay of the tested compounds is shown in Fig. 9, it is seen from the results that the all the
complexes exhibited moderate activity compared to the standard Ascorbic acid (Aca).
The IC50 values indicated that the compounds showed antioxidant activity in the order of
3 > 4 > 5 > 6 > 1 > 2. Complex 3 showed a higher antioxidant activity compared to other
three complexes. From the above results, the scavenging effect of the free ligands is
significantly less when compared to that of their corresponding ruthenium(II) complexes
which is due to the chelation of the organic ligands with the ruthenium(II) ion.
3.5.3. In vitro anticancer activity evaluation
Cancer is a complex disease that is normally associated with a wide range of
escalating effects both at the molecular and cellular levels. Synthesized metal complexes
have been successfully used as anticancer drugs with high selectivity against malignant cells
and with the ability to repress tumor metastasis. As candidates for such drugs, cytotoxic,
antitumor or anticancer natural products have been often sought and the synthesized
complexes have been used as therapeutics. The ligands and complexes were evaluated for
their cytotoxicity against human breast cancer cell line (MCF-7) by means of MTT assay
method that measures mitochondrial dehydrogenase activity as an indication of cell viability.
The results were analyzed by means of cell viability curves and expressed with IC50 values in
13

the studied concentration range from 0.1–100 µM. The activity that corresponds to the
inhibition of cancer cell growth at a maximum level is shown in Fig. 10, Table 5. Upon
increasing the concentration of complexes, the results of MTT assays revealed that the
complex 3 showed a higher cytotoxic effect followed by 4. Complexes, 5 and 6 showed
significant IC50 values but the cytotoxic effect of these complexes are less when compared to
the other two complexes 3 and 4. It is to be noted that the ligands does not show any
significant activity (IC50 100 µM), which confirmed that the chelation of the ligands with
the ruthenium(II) ion is the only responsible factor for the observed cytotoxic property of the
complexes. Comparison of the anticancer activity of the ruthenium(II) complexes reveal that
upon changing the thiosemicarbazone moieties and ancillary ligands the activity of the
complexes gets varied. The complexes of chlorobenzaldehyde thiosemicarbazone moiety
exhibited higher cytotoxic effects than the complexes of nitrobenzaldehyde
thiosemicarbazone. Moreover, the complexes containing triphenylphosphine as a co-ligand
showed better cytotoxic effects than the complexes containing triphenylarsine. For clinical
purposes, the compounds that show cytotoxicity in shorter time periods are preferred. The
data obtained for our compounds showed cytotoxicity with short incubation period (48 h) and
hence the data are highly significant. The findings of the in vitro cytotoxic activities confirm
the binding of the complexes to DNA, which consequently leads to cell death.
4. Conclusion
Four ruthenium(II) complexes bearing NS-thiosemicarbazone ligands and the PPh3/
AsPh3 co-ligands are synthesized and characterized by various spectroscopic techniques.
Thiosemicarbazone ligands have also been characterized by crystallographically. The
complexes are tentatively assigned an octahedral geometry. All the newly synthesized
compounds have been subjected to examine their biological property like DNA binding,
DNA cleavage, antioxidant and cytotoxicity under in vitro experimental conditions. The
DNA binding ability of the ligands and complexes has been assessed by absorption spectra
which inferred an intercalative mode of binding with binding constants ranging from
103-105 M-1. The DNA cleavage capabilities of complexes in the presence of H2O2 revealed
their potential nuclease activity to cleave CT DNA. The complex 3 showed superior cleavage
activity at low concentration than the other complexes. The antioxidant activity showed that
all the ruthenium(II) complexes can serve as potential antioxidants than the ligands against
DPPH radical in the order 3 > 4 > 5 > 6 > 1 > 2. In addition, the in vitro cytotoxicity of the
14

complexes possesses significant activity against MCF-7 cell line. Complex 3 exhibited good
cytotoxic effect than the other complexes and ligands. At this juncture, it is notable to
mention that the major chemical and biological findings of this study throw some light on the
potential of these complexes in a reasonable range of concentrations under in vitro
conditions. In our opinion, the significant outcome of the present investigation regarding the
abilities of ruthenium organometallic thiosemicarbazone complexes towards various
biological evaluations is that the substitution of chlorobenzaldehyde in the thiosemicarbazone
moiety with triphenylphosphine as co ligand led to an increased interaction with DNA, free
radical and tumor cell line than the rest of the complexes.
Acknowledgements
Financial assistance received from the Council of Scientific and Industrial Research,
New Delhi, India [Grant No. 01(2308)/09/EMR-II] is gratefully acknowledged.
Appendix A. Supplementary data
CCDC 893840 and 893999 contains the crystallographic data for compounds 1 and 2
respectively.
These
data
can
be
obtained
free
of
charge
via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:
deposit@ccdc.cam.ac.uk.
15

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16

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17

Fig. 1. ORTEP diagram of 1 with thermal ellipsoid at 50% probability
Fig. 2. ORTEP diagram of 2 with thermal ellipsoid at 50% probability.
18

Fig. 3. Crystal packing diagram of 1
Fig. 4. Crystal packing diagram of 2
19

Fig. 5. 1H (A), 13C (B) and 31P (C) NMR spectrum of the complex 3
20

21

22

23

Fig. 6. Electronic spectra of compounds 1 (A), 2 (B), 3 (C), 4 (D), 5 (E) and 6 (F) in Tris-HCl
buffer upon addition of CT-DNA. [Complex] = 25 µM, [DNA] = 0-40 µM. Arrow shows the
absorption intensities decrease upon increasing DNA concentrations
(Inset: Plot between [DNA] and [DNA]/ [ε -ε ] X 10-8).
a
f
Fig. 7. Gel electrophoresis diagram showing the hydrolytic cleavage of CT-DNA (30 µM)
incubated at 37 °C for a period of 2 h at a concentration of 40 µM of complex 3-6 in 5%
DMSO and 95% 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2. Lane 1: Control,
Lanes 2-5: DNA + complexes 3-6, respectively.
24

Fig. 8. Gel electrophoresis diagram showing the oxidative cleavage of CT-DNA (30 µM) by
complexes 3-6 in 5% DMSO and 95% 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2 in the
presence of H2O2 (60 µM) as a co-oxidant incubated at 37 °C for a period of 2 h. Lane 1
and 9: DNA. Lanes 2 and 10: DNA + H2O2. Lane 3-5: 3 (30, 60 and 90 µM) + DNA + H2O2.
Lane 6-8: 4 (30, 60 and 90 µM) + DNA + H2O2. Lane 11-13: 5 (30, 60 and 90 µM) + DNA +
H2O2. Lane 14-16: 6 (30, 60 and 90 µM) + DNA + H2O2.
25

Fig. 9. Antioxidant activity of compounds
26

Fig. 10. % Growth inhibition of MCF-7 cell line as a function of concentration of the
compounds 1-6
27

Table 1 Experimental Data for Crystallographic Analyses.
Compound
1
2
CCDC Number
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
b (Å)
c (Å)
α (°)
β (°)
893840
C9H10ClN3S
227.71
893999
C9H10N4O2S
238.27
296(2)
0.71073
Triclinic
P-1
296(2)
0.71073
Monoclinic
C2/c
14.3409(11)
8.0487(7)
18.6839(14)
90
90.868(2)
90
2156.4(3)
8
1.403
7.3403(4)
8.0726(4)
10.1536(5)
87.194(2)
70.544(2)
72.861(2)
541.30(5)
2
1.462
0.290
248
0.40 x 0.30 x 0.20
2.64 to 28.16
γ (°)
Volume (Å3)
Z
Density (cal.) (mg/m3)
Abs. coefficient (mm-1)
F(000)
0.511
944
0.40 x 0.35 x 0.30
Crystal size (mm3)
θ range for data collection (°) 2.18 to 28.29
Index ranges
-18<=h<=17,
-10<=k<=10,
-24<=l<=24
9667
-9<=h<=9,
-10<=k<=10,
-13<=l<=13
8930
Reflections collected
Independent reflections
2652 [R(int) = 0.0315]
2585 [R(int) = 0.0196]
Absorption correction
Semi-empirical from
equivalents
Semi-empirical from
equivalents
Max. and min. transmission
Refinement method
0.8617 and 0.8216
Full-matrix least-squares on F2 Full-matrix least-squares on F2
0.9443 and 0.8928
Data/restraints/parameters
Goodness-of-fit on F2
2652 / 0 / 129
1.130
2585 / 0 / 146
1.028
Final R indices [I>2σ(I)]
R1 = 0.0333, wR2 = 0.1010
R1 = 0.0337, wR2 = 0.0947
R indices (all data)
R1 = 0.0400, wR2 = 0.1164
R1 = 0.0377, wR2 = 0.0992
28

Table 2 Selected bond lengths [Å]
1
2
Cl(1)-C(5)
S(1)-C(2)
N(1)-C(2)
N(1)-C(1)
N(1)-H(1)
N(2)-C(2)
N(2)-N(3)
N(2)-H(2)
N(3)-C(3)
1.7357(17)
1.6884(14)
1.321(2)
1.4474(19)
0.8600
1.3540(17)
1.3696(17)
0.8600
C(1)-C(2)
C(1)-C(6)
C(1)-H(1)
C(2)-C(3)
C(2)-H(2)
C(3)-C(4)
C(3)-H(3)
C(4)-C(5)
C(4)-H(4)
C(5)-C(6)
C(5)-N(1)
C(6)-C(7)
C(7)-N(2)
C(7)-H(7)
C(8)-N(4)
C(8)-N(3)
C(8)-S(1)
C(9)-N(4)
C(9)-H(9A)
C(9)-H(9B)
C(9)-H(9C)
N(1)-O(1)
N(1)-O(2)
N(2)-N(3)
N(3)-H(3N3)
N(4)-H(4N4)
1.3783(18)
1.3960(17)
0.9300
1.384(2)
0.9300
1.377(2)
0.9300
1.3893(17)
0.9300
1.3999(15)
1.4656(16)
1.4700(15)
1.2761(15)
0.9300
1.3224(16)
1.3572(15)
1.6834(11)
1.4522(16)
0.9600
1.2764(18)
C(1)-H(1A) 0.9600
C(1)-H(1B) 0.9600
C(1)-H(1C) 0.9600
C(3)-C(4)
C(3)-H(3)
C(4)-C(5)
C(4)-C(9)
C(5)-C(6)
C(6)-C(7)
C(6)-H(6)
C(7)-C(8)
C(7)-H(7)
C(8)-C(9)
C(8)-H(8)
C(9)-H(9)
1.4616(19)
0.9300
1.3951(19)
1.396(2)
1.381(2)
1.373(3)
0.9300
1.382(3)
0.9300
1.380(2)
0.9300
0.9600
0.9600
1.2232(16)
1.2256(15)
1.3664(13)
0.8600
0.9300
0.8600
29