Ruthenium(II) carbonyl complexes containing S-methylisothiosemicarbazone based tetradentate ligand: Synthesis, characterization and biological applications

Article abstract of DOI:10.1007/s10534-013-9649-8

A series of hexa-coordinated ruthenium(II) complexes of the type [Ru(CO)(B)L ⁿ ] (n = 1-4; B = PPh₃, AsPh₃ or Py) have been synthesized by reacting dibasic quadridentate Schiff base ligands H₂L ⁿ (n = 1-4) with starting complexes [RuHCl(CO)(EPh₃)₂(B)] (E = P or As; B = PPh₃, AsPh₃ or Py). The synthesized complexes were characterized using elemental and various spectral studies including UV-Vis, FT-IR, NMR ( ¹H, ¹³C and ³¹P) and mass spectroscopy. An octahedral geometry was tentatively proposed for all the complexes based on the spectral data obtained. The experiments on antioxidant activity showed that the ruthenium(II) S-methylisothiosemicarbazone Schiff base complexes exhibited good scavenging activity against various free radicals (DPPH, OH and NO). The in vitro cytotoxicity of these complexes has been evaluated by MTT assay. The results demonstrate that the complexes have good anticancer activities against selected cancer cell line, human breast cancer cell line (MCF-7) and human skin carcinoma cell line (A431). The DNA cleavage studies showed that the complexes have better cleavage of pBR 322 DNA.

Full text of DOI:10.1007/s10534-013-9649-8

Biometals
DOI 10.1007/s10534-013-9649-8
Ruthenium(II) carbonyl complexes containing
S-methylisothiosemicarbazone based tetradentate ligand:
synthesis, characterization and biological applications
•
Sellappan Selvamurugan Rangasamy Ramachandran
•
Periasamy Viswanathamurthi
Received: 1 March 2013 / Accepted: 12 June 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of hexa-coordinated ruthenium(II)
complexes of the type [Ru(CO)(B)Ln] (n = 1–4;
B = PPh3, AsPh3 or Py) have been synthesized by
reacting dibasic quadridentate Schiff base ligands
H2Ln (n = 1–4) with starting complexes [RuHCl(CO)
(EPh3)2(B)] (E = P or As; B = PPh3, AsPh3 or Py).
The synthesized complexes were characterized using
elemental and various spectral studies including UV–
Vis, FT-IR, NMR (1H, 13C and 31P) and mass spectros-
copy. An octahedral geometry was tentatively
proposed for all the complexes based on the spectral
data obtained. The experiments on antioxidant activity
showed that the ruthenium(II) S-methylisothiosemi-
carbazone Schiff base complexes exhibited good
scavenging activity against various free radicals
(DPPH, OH and NO). The in vitro cytotoxicity of
these complexes has been evaluated by MTT assay.
The results demonstrate that the complexes have good
anticancer activities against selected cancer cell line,
human breast cancer cell line (MCF-7) and human skin
carcinoma cell line (A431). The DNA cleavage studies
showed that the complexes have better cleavage of
pBR 322 DNA.
Keywords Ruthenium(II) complexes ꢀ
Spectral studies ꢀ Antioxidant ꢀ
Anticancer activity ꢀ DNA cleavage
Introduction
Medicinal inorganic chemistry is continuously receiv-
ing interest in the field of biomedical applications (Ma
et al. 2012; Hsu et al. 2013). This interest is largely due
to the potential applications of metal containing com-
pounds as antibacterial, antiviral, antifungal, antima-
larial, and antitumor agents (Fricker 2007; Hartinger
and Dyson 2009; Fry and Mascharak 2011; Jouad et al.
2002). It is a well known fact that cisplatin is arguably
the most successful anticancer drug in the world. But, it
exhibits high toxicity to normal cells leading to
undesirable side-effects, although minimized by careful
administration protocols, and also it is inactive against
many cancer cell lines and metastasis (secondary)
cancers (Wang and Lippard 2005). Therefore, attempts
are being made to replace cisplatin with suitable
alternatives and hence numerous transition metal com-
plexes have been synthesized and tested for their
anticancer activities. Among the metal atoms used in
anticancer metal complexes, ruthenium is the most
unique. It is a rare noble metal unknown to living
systems and has strong complexation ability with
numerous ligands (Demoro et al. 2012; Sathyadevi
et al. 2012; Stringer et al. 2011).
S. Selvamurugan ꢀ R. Ramachandran ꢀ
P. Viswanathamurthi (&)
Department of Chemistry, Periyar University,
Salem 636011, India
e-mail: viswanathamurthi72@gmail.com
123

Biometals
The chemistry of ruthenium is currently receiving a
lot of attention, primarily because of the fascinating
electron transfer and energy transfer properties dis-
played by the complexes of this metal (Hanson et al.
2013). Metal chelation is an important process useful to
afford new chemical features to metal complexes in
order to make them suitable for pharmacological
applications. Many useful drugs containing metal-
binding sites, which may alter the physiological profile
of the free species. Some of them increase their activity
by their ability to form chelates with specific metal ion.
Metal complexes of sulphur containing Schiff bases
have been the subject of current and growing interest
because it has been shown that many of these
complexes possess anticancer activity (Ramachandran
et al. 2012; Wang et al. 2009). In particular, thiosemi-
carbazones, as a class of compounds, exhibit several
interesting physical, chemical and wide range of
biological properties (Senthil Raja et al. 2011).
c
n
b
o
R
R
m
a
d
OH
N
OH
N
p
e
l
f
k
g
h
j
N
SCH3
i
Ligand
R
H2L1
H2L2
H2L3
H2L4
H
Cl
OCH3
C4H4
Fig. 1 Structure of S-methylisothiosemicarbazone Schiff base
ligands
First study on thiosemicarbazones activity against
Mycobacterium tuberculosis (Da et al. 2009) many
other biological activities of this type of molecules
have been described, for instance antitumoral, anti-
fungal and antiviral (Bal et al. 2007). Thiosemicarba-
zones, in their neutral or deprotonated form, behave as
ONS/NNS tridentate or tetradentate chelate towards
metal ions essential for life. Some thiosemicarba-
zones, such as marboran or triapine, are already used
in medical practice. Recent discovery of the anticancer
effects of metal complexes and their potential use in
cancer diseases have received increasing attention
(Alagesan et al. 2013; Su et al. 2013).
SAIF, Cochin, India. The IR spectra of the ligand and
their complexes were recorded as KBr pellets on a
Nicolet Avatar model spectrophotometer in 4,000–
400 cm-1 range. Electronic spectra of the ligand and
their complexes have been recorded in methanol using
a Shimadzu UV-1650 PC spectrophotometer in
1
800–200 nm range. H, 13C and 31P NMR spectra
were recorded in Jeol GSX-400 instrument using
DMSO as the solvent. The ESI–MS spectra were
recorded by using LC–MS Q-ToF Micro Analyzer
(Shimadzu) in the SAIF, Panjab University, and
Chandigarh. Melting points were recorded on a
Technico micro heating table and are uncorrected.
The above discussions have stimulated much
interest to investigate the biological studies of ruthe-
nium(II) complexes with aromatic 2-hydroxy alde-
hydes containing thiosemicarbazones. Hence, in the
present work, we describe the synthesis, characteriza-
tion, bioactivities of ruthenium(II) complexes con-
taining S-methylisothiosemicarbazone Schiff bases.
The general structure of the ligands used in this study
is given in Fig. 1.
Materials
All the reagents used were chemically pure and AR
grade. The solvents were purified and dried according
to standard procedures. RuCl3ꢀ3H2O was purchased
from Loba Chemie Pvt Ltd. The starting complexes
[RuHCl(CO)(PPh3)3], [RuHCl(CO)(AsPh3)3] and
[RuHCl(CO)(Py)(PPh3)2] were prepared according to
literature procedures (Narayana prabhu and Ramesh
2012; Anitha et al. 2012). S-methylthiosemicarbazide
and S-methylisothiosemicarbazone Schiff bases
(Fig. 1) were prepared according to the literature
procedures (Atasever et al. 2010).
Experimental
Physical measurements
Microanalyses of carbon, hydrogen and nitrogen were
carried out using Vario EL III Elemental analyzer at
123

Biometals
8.59(8.16). IR (KBr, cm-1): 3313 (OHa), 3414 (OHp),
Preparation of S-methylisothiosemicarbazone
1
1622 (C=Ng), 1651 (C=Nj). H NMR (DMSO-d6, d,
Schiff base ligands
ppm): 11.22 (s, 1Ha, OH), 10.76 (s, 1Hp, OH), 8.56
(s, 1Hg, –CH=N), 8.23 (s, 1Hj, –CH=N), 6.90–7.23
(m, aromatic), 2.32 (s, CH3). UV kmax: 220, 264, 338.
A mixture of thiosemicarbazide (0.910 g, 10 mmol)
and methyl iodide (1.505 g, 10.6 mmol) in absolute
ethanol (20 ml) was refluxed for 45 min and allowed to
cool at room temperature for 12 h. White crystals of S-
methylisothiosemicarbazide separated was filtered,
washed with ethanol and used for next step without
further purification. Yield: 68 %. In the second step, a
mixture of S-methylisothiosemicarbazide (0.525 g,
5 mmol) and corresponding aldehydes (salicylalde-
hyde/5-chloro salicylaldehyde/o-vanallin/2-hydroxy-
naphthaldehyde) (1.23, 1.57, 1.52, 1.72 g respectively,
10 mmol) in absolute ethanol was neutralized with
aqueous Na2CO3ꢀ10H2O solution. The pale yellow
solid of the ligands obtained were filtered off and
washed with water and finally with ethanol. Yield:
85–90 %.
Bis(2-hydroxynaphthaldehyde) S-
methylisothiosemicarbazone ligand
Yellow solid. Anal. Calc. (Found). (C24H19O2N3S): C,
69.71(61.46); H, 4.63(4.24); N, 10.16(10.10); S,
7.75(7.34). IR (KBr, cm-1): 3313 (OHa), 3414
(OHp), 1622 (C=Ng), 1651 (C=Nj). 1H NMR
(DMSO-d6, d, ppm): 12.99 (s, 1Ha, OH), 12.67 (s,
1Hp, OH), 9.28 (s, 1Hg, –CH=N), 9.19 (s, 1Hj,
–CH=N), 6.81–9.35 (m, aromatic), 2.31 (s, CH3).
UV kmax: 238, 262, 328, 367, 382.
Synthesis of new ruthenium(II)
S-methylisothiosemicarbazone Schiff base
complexes
Bis(salicylaldehyde)
S-methylisothiosemicarbazone ligand
All the new metal complexes were prepared according
to the following general procedure. To a benzene
solution of 1 mmol (0.769–1.084 g) [RuHCl(CO)
(EPh3)2(B)] (E = P or As; B = PPh3, AsPh3 or Py),
1 mmol (0.315–0.413 g) Schiff base ligand was added
(mole ratio of ruthenium starting complex and ligand
is 1:1 respectively) and the mixture was refluxed for
6 h and the reactions were monitored by TLC. The
reaction mixture was reduced to 2–3 ml and the
product was separated by the addition of small amount
of petroleum ether at room temperature. The resulting
complexes were recrystallized from CH2Cl2/petro-
leum ether and dried under vacuum. The overall yield
obtained for all the complexes were 70–79 %.
Yellow solid. Anal. Calc. (Found). (C16H15O2N3S): C,
61.32(61.46); H, 4.82(4.71); N, 13.41(13.21); S,
10.23(10.18). IR (KBr, cm-1): 3280 (OHa), 3459
(OHp), 1617 (C=Ng), 1635 (C=Nj). 1H NMR (DMSO-
d6, d, ppm): 11.56 (s, 1Ha, OH), 10.83 (s, 1Hp, OH),
8.44 (s, 1Hg, –CH=N), 8.31 (s, 1Hj, –CH=N),
6.78–7.61 (m, aromatic), 2.31 (s, CH3). UV kmax:
220, 298, 304, 332.
Bis(5-chloro salicylaldehyde)
S-methylisothiosemicarbazone ligand
Yellow solid. Anal. Calc. (Found). (C16H13O2
N3Cl2S): C, 50.27(50.12); H, 3.43(3.15); N,
10.99(10.74); S, 8.39(8.14). IR (KBr, cm-1): 3283
[Ru(CO)(PPh3)L1]
1
(OHa), 3450 (OHp), 1637 (C=Ng), 1661 (C=Nj). H
NMR (DMSO-d6, d, ppm): 11.63 (s, 1Ha, OH), 10.75
(s, 1Hp, OH), 8.42 (s, 1Hg, –CH=N), 8.32 (s, 1Hj,
–CH=N), 6.80–7.81 (m, aromatic), 2.33 (s, CH3). UV
kmax: 225, 276, 337.
Anal. Calc. (Found). (C35H28O3N3SPRu): C, 59.82
(59.62); H, 4.02(3.96); N, 5.98(5.53); S, 4.56(4.59). IR
(KBr, cm-1): 1956 (C:O), 1603 (C=Ng), 1621 (C=Nj),
737 (C–S). 1H NMR (DMSO-d6, d, ppm):8.23 (s, 1Hg,
–CH=N), 8.01 (s, 1Hj, –CH=N), 7.12–7.81 (m, aro-
matic), 2.32 (s, CH3). 13C NMR (DMSO-d6, d, ppm):
203.58 (C:O), 173.45 (C–S), 168.01 (C–O), 160.59
(C=Ng), 161.23 (C=Nj), 120.14–139.76 (Ar–C), 23.09
(CH3). 31P NMR (CDCl3, d, ppm): 31.49. UV kmax:
230, 250, 303, 323, 402. ESI–MS, m/e: 702.60.
Bis(o-vanallin) S-methylisothiosemicarbazone
ligand
Yellow solid. Anal. Calc. (Found). (C18H19O4N3S):
C, 57.89(57.66); H, 5.13(5.11); N, 11.25(11.07); S,
123

Biometals
[Ru(CO)(PPh3)L2]
7.15–7.78 (m, aromatic), 2.32(s, CH3). UV kmax:
231, 270, 330, 382. ESI–MS, m/e: 745.72.
Anal. Calc. (Found). (C35H26O3N3Cl2SPRu): C,
54.48(54.24); H, 3.40(3.32); N, 5.45(5.14); S,
4.16(4.15). IR (KBr, cm-1): 1957 (C:O), 1603
[Ru(CO)(AsPh3)L2]
1
(C=Ng), 1622 (C=Nj), 737 (C–S). H NMR (DMSO-
Anal. Calc. (Found). (C35H26O3N3Cl2SAsRu): C,
51.54(51.42); H, 3.21(3.33); N, 5.15(5.02); S,
3.93(3.66). IR (KBr, cm-1): 1903 (C:O), 1600
d6, d, ppm): 8.12 (s, 1Hg, –CH=N), 7.99 (s, 1Hj,
–CH=N), 7.18–7.83 (m, aromatic), 2.30 (s, CH3). 13
C
(C=Ng), 1630 (C=Nj), 744 (C–S). H NMR (DMSO-
1
NMR (DMSO-d6, d, ppm): 202.82 (C:O), 175.02
(C–S), 168.91 (C–O), 162.72 (C=Ng), 163.23 (C=Nj),
121.28–137.76 (Ar–C), 25.87 (CH3). 31P NMR
(CDCl3, d, ppm): 31.95. UV kmax: 232, 264, 345,
384. ESI–MS, m/e: 770.60.
d6, d, ppm): 8.21(s, 1Hg, –CH=N), 8.05 (s, 1Hj,
–CH=N), 7.11–7.79 (m, aromatic), 2.31 (s, CH3). UV
kmax: 232, 264, 345, 384.231, 265, 360.
[Ru(CO)(AsPh3)L3]
[Ru(CO)(PPh3)L3]
Anal. Calc. (Found). (C37H32O5N3SAsRu): C, 55.09
(55.04); H, 4.00(3.76); N, 5.21(5.24); S, 3.97(3.87). IR
(KBr, cm-1): 1930 (C:O), 1568 (C=Ng), 1592
Anal. Calc. (Found). (C37H32O5N3SPRu): C, 58.26
(58.02); H, 4.23(4.11); N, 5.51(5.40); S, 4.20(4.03). IR
(KBr, cm-1): 1928 (C:O), 1575 (C=Ng), 1634
1
(C=Nj), 740 (C–S). H NMR (DMSO-d6, d, ppm):
8.19 (s, 1Hg, –CH=N), 8.01 (s, 1Hj, –CH=N),
7.09–7.85 (m, aromatic), 2.31 (s, CH3). UV kmax:
235, 273, 331, 380. ESI–MS, m/e: 806.49.
(C=Nj), 737 (C–S). H NMR (DMSO-d6, d, ppm):
1
8.24 (s, 1Hg, –CH=N), 8.03 (s, 1Hj, –CH=N),
7.10–7.79 (m, aromatic), 2.29 (s, CH3). 13C NMR
(DMSO-d6, d, ppm): 202.96 (C:O), 174.02 (C–S),
168.24 (C–O), 161.79 (C=Ng), 164.05 (C=Nj),
122.21–138.09 (Ar–C), 26.79 (CH3). 31P NMR
(CDCl3, d, ppm): 33.51. UV kmax: 232, 255, 301, 408.
[Ru(CO)(AsPh3)L4]
Anal. Calc. (Found). (C43H32O3N3SAsRu): C, 60.99
(60.91); H, 3.81(3.62); N, 4.96(4.78); S, 3.79(3.57). IR
(KBr, cm-1): 1951 (C:O), 1601 (C=Ng), 1616
[Ru(CO)(PPh3)L4]
1
(C=Nj), 745 (C–S). H NMR (DMSO-d6, d, ppm):
8.20 (s, 1Hg, –CH=N), 7.99 (s, 1Hj, –CH=N),
7.16–7.80 (m, aromatic), 2.29 (s, CH3). UV kmax:
232, 260, 327, 388, 460.
Anal. Calc. (Found). (C43H32O3N3SPRu): C, 64.33
(64.36); H, 4.02(3.98); N, 5.23(5.15); S, 3.99(4.10). IR
(KBr, cm-1): 1959 (C:O), 1601 (C=Ng), 1616
(C=Nj), 738 (C–S). H NMR (DMSO-d6, d, ppm):
1
[Ru(CO)(Py)L1]
8.20 (s, 1Hg, –CH=N), 8.09 (s, 1Hj, –CH=N),
7.13–7.80 (m, aromatic), 2.30 (s, CH3). 13C NMR
(DMSO-d6, d, ppm): 203.58 (C:O), 177.95 (C–S),
169.02 (C–O), 162.74 (C=Ng), 164.15 (C=Nj),
123.11–139.76 (Ar–C), 28.42(CH3). 31P NMR
(CDCl3, d, ppm): 34.74. UV kmax: 231, 254, 329,
382, 436. ESI–MS, m/e: 803.52.
Anal. Calc. (Found). (C22H18O3N4SRu): C,
50.86(50.91); H, 3.49(3.36); N, 10.78(10.71); S,
6.17(6.07). IR (KBr, cm-1): 1949 (C:O), 1602
1
(C=Ng), 1620 (C=Nj), 746 (C–S). H NMR (DMSO-
d6, d, ppm): 8.11 (s, 1Hg, –CH=N), 7.94 (s, 1Hj,
–CH=N), 7.10–7.79 (m, aromatic), 2.29 (s, CH3). UV
kmax: 230, 271, 330, 381.
[Ru(CO)(AsPh3)L1]
[Ru(CO)(Py)L2]
Anal. Calc. (Found). (C35H28O3N3SAsRu): C, 56.30
(56.25); H, 3.78(3.45); N, 5.63(5.56); S, 4.29(4.10). IR
(KBr, cm-1): 1948 (C:O), 1603 (C=Ng), 1621
Anal. Calc. (Found). (C22H16O3Cl2N4SRu): C, 44.91
(44.96); H, 2.74(2.71); N, 9.52(9.31); S, 5.45(5.38). IR
(KBr, cm-1): 1957 (C:O), 1601 (C=Ng), 1619
1
(C=Nj), 745 (C–S). H NMR (DMSO-d6, d, ppm):
8.15 (s, 1Hg, –CH=N), 7.95 (s, 1Hj, –CH=N),
1
(C=Nj), 745 (C–S). H NMR (DMSO-d6, d, ppm):
123

Biometals
8.15 (s, 1Hg, –CH=N), 7.98 (s, 1Hj, –CH=N),
7.16–7.85 (m, aromatic), 2.31 (s, CH3). UV kmax:
235, 337, 370. ESI–MS, m/e: 588.33.
27 °C. The absorbance of the sample was measured at
517 nm against the blank (methanol).
OHꢀ scavenging assay
[Ru(CO)(Py)L3]
The hydroxyl radical scavenging activity of the
compounds has been investigated by using the Nash
method (Sathiya Kamatchi et al. 2012). In vitro
hydroxyl radicals were generated by a 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 spectrophoto-
metrically at 412 nm. A mixture of 1.0 ml of iron-
EDTA solution (0.13 % ferrous ammonium sulphate
and 0.26 % EDTA), 0.5 ml of EDTA solution
(0.018 %), and 1.0 ml of DMSO (0.85 % DMSO
(v/v) in 0.1 M phosphate buffer, pH 7.4) were
sequentially added in the test tubes. The reaction
was initiated by adding 0.5 ml of ascorbic acid
(0.22 %) and was incubated at 80–90 °C for 15 min
in a water bath. After incubation, the reaction was
terminated by the addition of 1.0 ml of ice-cold
trichloroacetic acid (17.5 % w/v). Subsequently,
3.0 ml of Nash reagent was added to each tube and
left at room temperature for 15 min. The intensity of
the colour formed was measured spectrophotometri-
cally at 412 nm against reagent blank.
Anal. Calc. (Found). (C24H22O5N4SRu): C, 49.73
(49.61); H, 3.83(3.39); N, 9.67(9.54); S, 5.53(5.69). IR
(KBr, cm-1): 1931 (C:O), 1588 (C=Ng), 1611
1
(C=Nj), 742 (C–S). H NMR (DMSO-d6, d, ppm):
8.13 (s, 1Hg, –CH=N), 8.00 (s, 1Hj, –CH=N),
7.12–7.89 (m, aromatic), 2.31 (s, CH3). UV kmax:
228, 269, 331, 380.
[Ru(CO)(Py)L4]
Anal. Calc. (Found). (C30H22O3N4SRu): C,
58.15(58.01); H, 3.58(3.53); N, 9.04(9.12); S,
5.17(5.04). IR (KBr, cm-1): 1949 (C:O), 1601
1
(C=Ng), 1616 (C=Nj), 744 (C–S). H NMR (DMSO-
d6, d, ppm): 8.16 (s, 1Hg, –CH=N), 7.95 (s, 1Hj,
–CH=N), 7.09–7.86 (m, aromatic), 2.29 (s, CH3). UV
kmax: 230, 262, 323, 383, 459.
Antioxidant assays
DPPHꢀ scavenging assay
NOꢀ scavenging assay
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical
scavenging activity of the compounds was measured
according to the method of Blios (Sathiya Kamatchi
et al. 2012). The DPPH radical is a stable free radical.
Because of the odd electron, DPPH shows a strong
absorption band at 517 nm in the visible spectrum. As
this electron becomes paired off in the presence of a
free radical scavenger, the absorption vanishes and the
resulting decolorization is stoichiometric with respect
to the number of electrons taken up. Various concen-
trations of the experimental complexes were taken and
the volume was adjusted to 100 ll with methanol.
About 5 ml of a 0.1 mM methanolic solution of DPPH
was added to the aliquots of samples and standards
(BHT and vitamin C) and shaken vigorously. A
negative control was prepared by adding 100 ll of
methanol in 5 ml of 0.1 mM methanolic solution of
DPPH. The tubes were allowed to stand for 20 min at
The assay of nitric oxide (NO) scavenging activity is
based on a method (Sathiya Kamatchi et al. 2012)
where sodium nitroprusside in aqueous solution at
physiological pH spontaneously generates nitric
oxide, which interacts with oxygen to produce nitrite
ions. These can be estimated using the Greiss reagent.
Scavengers of nitric oxide compete with oxygen
leading to reduced production of nitrite ions. For the
experiment, sodium nitroprusside (10 mM) in phos-
phate buffered saline was mixed with a fixed concen-
tration of the complex and standards and incubated at
room temperature for 150 min. After the incubation
period, 0.5 ml of Griess reagent containing 1 %
sulfanilamide, 2 % H3PO4 and 0.1 % N-(1-naphthyl)
ethylenediamine dihydrochloride was added. The
absorbance of the chromophore formed was measured
at 546 nm.
123

Biometals
In vitro cytotoxicity activity evaluation by MTT
assay
(365 nm, 10 W). The samples were analyzed by
electrophoresis for 1.5 h at 80 V on a 0.8% agarose
gel in TBE (89 mM Tris–borate acid, 2 mM EDTA,
pH = 8.3). The gel was stained with 1 lg/ml ethidium
bromide and observes the bands under illuminator.
Cytotoxicity studies of the complexes were carried out
on human breast cancer cell line (MCF-7) and human
skin carcinoma cell line (A431) which was obtained
from National Centre for Cell Science (NCCS), Pune,
India. Cell viability was carried out using the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
\bromide) assay method (Blagosklonny and EI-diery
1996). The MCF-7 cells were grown in Eagles
minimum essential medium containing 10 % fetal
bovine serum (FBS) and A431 were grown in Dul-
becco’s Modified Eagles Medium (DMEM) containing
10 % fetal bovine serum (FBS). For screening exper-
iments, the cells were seeded into 96-well plates in
100 mm3 of the respective medium containing 10 %
FBS, at a plating density of 10,000 cells/well and
incubated at 37 °C, 5 % CO2, 95 % air and 100 %
relative humidity for 24 h prior to the addition of
compounds. The compounds were dissolved in DMSO
and diluted in the respective medium containing 1 %
FBS. After 24 h, the medium was replaced with the
respective medium with 1 % FBS containing the
compounds at various concentrations and incubated
at 37 °C, 5 % CO2, 95 % air and 100 % relative
humidity for 48 h. Experiments were performed in
triplicate and the medium without the compounds
served as control. After 48 h, 15 mm3 of MTT (5 mg/cm3)
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 mm3 of DMSO and the
absorbance measured at 570 nm using a micro plate
reader. The % cell inhibition was determined using the
following formula and a graph was plotted between %
of cell inhibition and concentration. From this plot, the
IC50 value was calculated.
Results and discussion
The reactions of [RuHCl(CO)(EPh3)2(B)] (E = P or
As; B = PPh3, AsPh3 or Py) with S-methylisothiose-
micarbazone Schiff base ligands in 1:1 molar ratio in
dry benzene afforded new hexa-coordinated low-spin
ruthenium(II) complexes (Scheme 1). The analytical
data are in good agreement with proposed molecular
formula of the complexes. In addition, ESI-Mass
spectra of complexes [Ru(CO)(PPh3)L1], [Ru(CO)
(AsPh3)L1], [Ru(CO)(PPh3)L2], [Ru(CO)(Py)L2],
[Ru(CO)(AsPh3)L3] and [Ru(CO)(PPh3)L4] have also
confirmed the molecular weights of their proposed
structures with m/e values 702.60, 745.72, 770.60,
588.33, 806.49 and 803.52 respectively. In all the
reactions, the Schiff base ligand behaves as dibasic
quadridentate ligands by replacing two molecules of
triphenylphosphine or triphenylarsine, one hydride
and one chloride ion from the precursors. All the
complexes are stable in air at room temperature,
brown in color, non-hygroscopic in nature and highly
soluble in common organic solvents such as dichlo-
romethane, methanol, acetonitrile and DMSO.
Infrared spectroscopic analysis
The IR spectra of the complexes have been examined in
comparison with that of the ligand. The spectra of free
ligands showed two intense bands in the region 1,635–
1,665 and 1,604–1,637 cm-1 characteristic of two tC=N
imine groups. These bands shifted towards lower
frequencies 1,592–1,634 and 1,568–1,603 cm-1 in
complexes indicated that the coordination of ligand with
metal through azomethine nitrogens (Pal 2002). The two
bands appeared in ligand spectra very close to each other
in the region 3,253-3,459 cm-1 were due to the stretch-
ing frequency of two phenolic OH groups. These bands
disappeared in complexes showed that the binding of
phenolic oxygen with metal via deprotonation. Further
the strong absorption around the 1,920–1,957 cm-1 has
been assigned to the terminally coordinated carbonyl
% inhibition ¼ ½mean OD of untreated cells ðcontrolÞ
=mean OD of treated cells ðcontrolÞꢁ ꢂ 100:
DNA cleavage experiments
For the gel electrophoresis experiment, supercoiled
pBR 322 DNA (0.1 lg) was treated with the ruthe-
nium(II) complexes with buffer (50 mM Tris–HCl,
18 mM NaCl, pH = 7.2) and the solution was then
irradiated at room temperature with a UV lamp
123

Biometals
c
n
b
o
R
R
m
B
a
n
c
b
g
o
d
OH
N
OH
N
R
R
m
p
a
d
O
O
N
e
l
p
j
f
k
benzene
reflux/6 h
Ru
[RuHCl(CO)(EPh3)2B]
e
g
h
j
f
l
k
N
N
h
q
CO
N
SCH3
i
SCH3
i
(E = P or As; B = PPh3, AsPh3 or Py)
Scheme 1 General scheme for the synthesis of new ruthenium(II) complexes
group in the new ruthenium complexes (Nareshkumar
and Ramesh 2005). In addition, the other characteristic
bands due to triphenylphosphine and triphenylarsine
(around 695 and 1,435 cm-1) were also present in the
spectra of all complexes (Ramachandran and Viswa-
nathamurthi 2013).
multiplets at 7.12–7.91 ppm in the spectra of com-
plexes are assigned to aromatic protons. In addition,
the methyl group protons appeared at 2.31 ppm.
13C NMR spectroscopic analysis
The 13C NMR spectra of the complexes have showed a
peak at 203.58–201.30 ppm region is due to C:O
carbon. The presence of a peak at 177.95–173.45 ppm
region is assigned to C–S carbon. The azomethine
([C=N) carbons exhibited its peak in the region of
164.15–160.59 ppm. In addition, the peak in the region
of 169.62–168.01 ppm is assigned to C–O carbons.
The multiplets appeared around 120.14–139.76 ppm
region are assigned to aromatic carbons. A sharp
singlet at 23.09–25.28.42 ppm is assigned to methyl
carbon.
Electronic spectroscopic analysis
The electronic spectra of all the complexes in meth-
anol showed three to five bands in the region
460–220 nm. The bands around 408–323 nm have
been assigned to charge transfer transitions arising
from the metal t2g level to the unfilled molecular
orbitals derived from the p* level of the ligands based
on their extinction coefficient values. The bands below
300 nm were characterized by intra-ligand charge
transfer. The electronic spectra are similar to those
observed for other octahedral ruthenium(II) com-
plexes (Muthu Tamizh et al. 2012).
31P NMR spectral studies
31P NMR spectra of some of the complexes were
recorded to confirm the presence of triphenylphos-
phine group in the complexes. A sharp singlet was
observed around 31.49–34.74 ppm due to presence of
triphenylphosphine ligand in the complexes.
1H NMR spectral studies
1
The H NMR spectra of the ligands and the corre-
sponding ruthenium(II) complexes were recorded in
DMSO to confirm the presence of coordinated ligand in
the complexes. The two singlets at 10.75–13.01 ppm
are assigned to two phenolic OH groups of the ligands.
The bands are not found in the spectra of complexes,
which is consistent with deprotonation of these ligands
upon metal complexation. The spectra of ligands have
shown two peaks for two azomethine proton (–CH=N–)
in the range of 8.31–9.28 ppm, which are shifted
slightly to the up field and appeared at 7.95–8.23 ppm
in complexes indicated that the coordination of
ligand with metal through azomethine nitrogens. The
From the above all spectral data, an octahedral
geometry was proposed tentatively for all the com-
plexes in which the two azomethine nitrogens and two
phenoxy groups of the ligands are in coordination with
ruthenium(II) metal ion (Fig. 2).
Antioxidant activity
Free radicals contain one or more unpaired electrons,
produced in normal or pathological cell metabolism.
Reactive oxygen species (ROS) react easily with these
123

Biometals
with the ruthenium ions. Among all free radicals, the
hydroxyl radical (OHꢀ) is by far the most potent and
therefore the most dangerous oxygen metabolite,
elimination of this radical is one of the major aims
of antioxidant administration (Udilova et al. 2003).
Further, it is significant to mention that the metal
complexes synthesized herein possess superior anti-
oxidant activity against the above said radicals than
that of the standard antioxidant Vitamin C and
butylated hydroxyl toluene (Sathiya Kamatchi et al.
2012). Moreover, the ruthenium(II) complexes
showed higher antioxidant activity when compared
to that of other metal complexes (Udilova et al. 2003).
B
c
b
g
o
R
a
d
m
O
O
N
p
j
Ru
e
f
l
k
N
h
q
CO
N
SCH3
i
B=PPh3 , Asph3 or Py
Fig. 2 Structure of new ruthenium(II) complexes
free radicals to become radicals themselves. ROS are
various forms of activated oxygen, which include free
radicals such as superoxide anion radicals (O-2 ꢀ) and
hydroxyl radicals (OHꢀ) as well as non-free radical
species (H2O2) and the singlet oxygen (1O2) (Gulcin
et al. 2004; Rice-Evans and Diplock 1993). They are
formed in living organisms in different ways, includ-
ing normal aerobic respiration, stimulated polymor-
phonuclear leukocytes and macrophages and
peroxisomes. They are natural by-products of our
body’s metabolism. The free radical can induce DNA
damage in humans. The damage to DNA has been
suggested to contribute to aging and various diseases
including cancer and chronic inflammation (Tsai et al.
2001).
Table 1 Antioxidant activity of ligands and ruthenium(II)
complexes against various radicals
Compound
IC50(lM)
DPPHꢀ
OHꢀ
22.56
NOꢀ
27.75
H2L1
H2L2
H2L4
[Ru(CO)(PPh3)L1]
[Ru(CO)(PPh3)L2]
[Ru(CO)(PPh3)L3]
[Ru(CO)(PPh3)L4]
[Ru(CO)(AsPh3)L1]
[Ru(CO)(Py)L1]
Vitamin C
65.17
61.80
67.87
42.88
48.55
50.23
50.25
35.11
42.92
145.80
86.92
11.86
21.49
5.58
35.27
32.79
12.63
18.33
11.78
13.96
13.53
13.51
216.85
152.82
6.31
Hence, we carried out experiments to explore the
free radical scavenging ability of the ligands and
complexes, with the hope of developing potential
antioxidants and therapeutic reagents for respiratory
diseases such as asthma emphysema and asbestosis.
The antioxidant potential of free ligands and corre-
sponding ruthenium(II) complexes against DPPH
radical, OH radical and NO radical assay were
investigated with respect to different concentrations
of the test compounds varying from 0 to 50 lM and
the results were shown in Table 1 (Fig. 3). It was
observed that, the 50% inhibitory concentration (IC50)
value of ligands and complexes varies from 11.86 to
22.56 lM and from 5.58 to 8.30 lM respectively,
against OH radical. The ligands showed their IC50
values against NO and DPPH radicals up to
27.75–35.27 and 61.80–67.87 lM respectively,
whereas the complexes showed their IC50 values up
to 11.78–18.33 and 35.11–50.25 lM respectively.
From the above results, it can be concluded that less
scavenging activity was exhibited by the free ligands
when compared to that of their corresponding ruthe-
nium complexes which is due to the chelation of them
8.30
6.17
5.89
5.72
234.21
162.35
BHT
H2L1
H2L2
H2L4
250
200
150
100
50
1
[Ru(CO)(PPh3 )L ]
[Ru(CO)(PPh3 )L ]
[Ru(CO)(PPh3)L ]
[Ru(CO)(PPh3 )L ]
[Ru(CO)(AsPh3 )L ]
[Ru(CO)(Py)L]
Vitamin C
BHT
2
3
4
1
1
0
DPPH
OH
NO
Radical
Fig. 3 Scavenging effect of the ligands and ruthenium(II)
complexes on various radicals compared with standard vitamin
C and BHT
123

Biometals
In vitro cytotoxic activity evaluation by MTT
assay
was expressed by median growth inhibitory concen-
tration (IC50), which required producing 50% cyto-
toxic effect against MCF-7 and A431 cells after 48 h
exposure to the tested complexes. The cytotoxicities of
the complexes were found to be concentration depen-
dent. The cell viability decreased with increasing
concentrations of complexes (Figs. 4, 5, 6, 7). The
MTT assay was performed on human breast cancer
cell line (MCF-7) and human skin carcinoma cell line
(A431) to check the anticancer activity of the
complexes. The cytotoxicity of the tested complexes
Fig. 4 The % growth
inhibition against log10
concentrations of different
complexes on breast cancer
cell line (MCF-7)
Fig. 5 The % growth
inhibition against log10
concentrations of four
different complexes on skin
carcinoma cell line (A431)
123

Biometals
Fig. 6 Cytotoxic effect of ruthenium(II) complexes against
MCF-7 at different concentrations (0.1, 1.0, 10, 100 lM). Cell
viability decreased with increasing concentrations of
complexes.
(1-[Ru(CO)(PPh3)L1];
2-[Ru(CO)(PPh3)L2];
3-[Ru(CO)(PPh3)L3]; 4-[Ru(CO)(AsPh3)L4])
DNA cleavage studies by gel electrophoresis
screening results are summarized in Tables 2 and 3. It
is evident that all of the tested complexes showed
anticancer activity, with IC50 values ranging from
19.18 to 31.10 lM for MCF-7 and 9.30-42.64 for
A431 cell lines. From the IC50 values of complexes, it
is observed that the cytotoxic activity of complexes
decreasing in the order [Ru(CO)(PPh3)L1] [ [Ru(CO)
(PPh3)L3] [[Ru(CO)(PPh3)L2] [[Ru(CO)(AsPh3)L4]
against MCF-7 and A431 cell lines. The lower toxicity
of ruthenium complexes compared to platinum drugs
has been attributed to the ability of ruthenium
compounds to specifically accumulate in cancer
tissues. Cisplatin and their derivatives mainly target
DNA where as ruthenium pharmaceuticals bind to
multiple biological targets (Beckford et al. 2009;
Kalaivani et al. 2012; Sathyadevi et al. 2012).
The cleavage reaction on plasmid DNA with ruthe-
nium(II) complexes can be monitored by agarose gel
electrophoresis. When circular plasmid DNA is subject
to electrophoresis, relatively fast migration will be
observed for the intact supercoil form (form-I). If
scission occurs on one strand (nicking), the supercoil
will relax to generate a slower moving open circular
form (form-II). If both strands are cleaved, a linear
form (form-III) that migrates between form-I and
form-II will be generated (Xie et al. 2013). Figure 8
showed the gel electrophoresis separation of pBR 322
DNA after incubation with the ruthenium(II) com-
plexes and irradiation at 365 nm for 30 min. No
obvious DNA cleavage was observed for controls in
123

Biometals
Fig. 7 Cytotoxic effect of ruthenium(II) complexes against
A431 at different concentrations (0.1, 1.0, 10, 100 lM). Cell
viability decreased with increasing concentrations of
complexes.
3-[Ru(CO)(PPh3)L3]; 4-[Ru(CO)(AsPh3)L4])
(1-[Ru(CO)(PPh3)L1];
2-[Ru(CO)(PPh3)L2];
Table 2 IC50 (lM) value
Complex
% Cell inhibition
IC50
of ruthenium(II) complexes
against breast cancer cell
line (MCF-7)
0.1 lM
1 lM
10 lM
100 lM
[Ru(CO)(PPh3)L1]
2.24
1.92
0.89
1.28
4.37
2.56
2.91
4.37
8.64
8.75
99.79
98.93
98.77
94.34
19.18
21.98
21.19
31.10
[Ru(CO)(PPh3)L2]
[Ru(CO)(PPh3)L3]
[Ru(CO)(AsPh3)L4]
10.85
6.08
Table 3 IC50 (lM) value
Complex
% Cell inhibition
IC50
of ruthenium(II) complexes
against human skin
0.1 lM
1 lM
10 lM
100 lM
carcinoma cell line (A431)
[Ru(CO)(PPh3)L1]
1.05
1.70
3.89
7.56
4.61
1.27
52.89
15.20
10.04
2.98
100
9.30
26.70
16.87
42.64
[Ru(CO)(PPh3)L2]
[Ru(CO)(PPh3)L3]
[Ru(CO)(AsPh3)L4]
90.94
100
-0.03
0.03
88.54
123

Biometals
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[Ru(CO)(PPh3)L4] at different concentrations(5, 10, 20 lg/ml)
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Conclusion
New ruthenium(II) complexes were synthesized and
characterized using spectral and elemental analyses.
An octahedral geometry was tentatively proposed for
all the complexes from the spectral data. Radical
scavenging ability of the complexes was found to be
higher when compared to their corresponding ligands.
This is due to the chelation of ligands with ruthenium
metal. In vitro cytotoxicity of few complexes has been
evaluated by MTT assay. The results showed that the
complexes displayed good anticancer activity against
human breast cancer (MCF-7) and human skin carci-
noma cell line (A431). In addition, the ruthenium(II)
complexes are efficiently cleaved the pBR 322 DNA.
Acknowledgments We are thankful to the IISC Bangalore,
STIC Cochin, SAIF Panjab University, Chandigarh for
providing instrumental facilities. We also thankful to KMCH,
Coimbatore and Biogenics Research and training center in
Biotechnology, Hubli, Karnataka for anticancer and DNA
cleavage studies.
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Hsu CW, Kuo CF, Chuang SM, Hou MH (2013) Elucidation of
the DNA-interacting properties and anticancer activity of a
Ni (II)-coordinated mithramycin dimer complex. Biomet-
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