Caenorhabditis elegans as a model for exploring the efficacy of synthesized organoruthenium complexes for aging and Alzheimer's disease a neurodegenerative disorder: A systematic approach

Article abstract of DOI:10.1016/j.jorganchem.2017.03.023

The current article deals with the preparation and characterisation of new organoruthenium(II) complexes, namely [RuCp(Dea-Sal-tsc)(PPh₃)] (1), [RuCp(Dea-Sal-mtsc)(PPh₃)] (2), [RuCp(Dea-Sal-etsc)(PPh₃)] (3) and [RuCp(Dea-Sal-ptsc)(PPh₃)] (4). The new ruthenium(II) complexes were characterized by various analytical, spectral techniques. The structure of the ligand [H₂-Dea-Sal-tsc] and the complex [RuCp(Dea-Sal-tsc)(PPh₃)] (1) were confirmed by X-ray crystallography. The complexes (1–4) were used to study the toxicity, stress resistance, aging and neuro-protective effects by taking Caenorhabditis elegans as model. In?vitro free radical scavenging activity was performed by DPPH free radical scavenging assay, the complexes (1–4) exhibited highest scavenging activity than standard Vitamin C (IC₅₀?=?5.28?±?0.10). The lifespan has increased over 22.4% in mev-1 mutant worms treated with complex 4. The complex 4 triggered the DAF-16 nuclear localization, increases sod-3 expression and reduced amyloid (Aβ) protein induced paralysis were observed. In the present study we confirmed that oxidative stress resistance of N2 and lifespan extension of mev-1 mutant which showed the potential ROS scavenging activity of complex 4. The results also confirmed the effective anti-aging potential of ruthenium complex 4 which may be developed as a therapeutic drug for the prevention of aging and age related neurodegenerative diseases. Further studies are required to find out the exact action of complex 4 on higher model.

Full text of DOI:10.1016/j.jorganchem.2017.03.023

Journal of Organometallic Chemistry 838 (2017) 12e23
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Caenorhabditis elegans as a model for exploring the efficacy of
synthesized organoruthenium complexes for aging and Alzheimer's
disease a neurodegenerative disorder: A systematic approach
,
G. Devagi a, G. Shanmugam b, A. Mohankumar b, P. Sundararaj b *, F. Dallemer c,
,
*
P. Kalaivani d, R. Prabhakaran a
a Department of Chemistry, Bharathiar University, Coimbatore 641 046, India
b Department of Zoology, Bharathiar University, Coimbatore 641 046, India
c
ꢀ
ꢀ ^
^
Laboratoire MADIREL CNRS UMR7246, Universite of Aix-Marseille, Centre de Saint-Jerome, bat. MADIREL, 13397 Marseille cedex 20, France
d Department of Chemistry, Nirmala College for Women, Bharathiar University, Coimbatore 641018, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The current article deals with the preparation and characterisation of new organoruthenium(II) com-
plexes, namely [RuCp(Dea-Sal-tsc)(PPh3)] (1), [RuCp(Dea-Sal-mtsc)(PPh3)] (2), [RuCp(Dea-Sal-
etsc)(PPh3)] (3) and [RuCp(Dea-Sal-ptsc)(PPh3)] (4). The new ruthenium(II) complexes were character-
ized by various analytical, spectral techniques. The structure of the ligand [H2-Dea-Sal-tsc] and the
complex [RuCp(Dea-Sal-tsc)(PPh3)] (1) were confirmed by X-ray crystallography. The complexes (1e4)
were used to study the toxicity, stress resistance, aging and neuro-protective effects by taking Caeno-
rhabditis elegans as model. In vitro free radical scavenging activity was performed by DPPH free radical
scavenging assay, the complexes (1e4) exhibited highest scavenging activity than standard Vitamin C
(IC50 ¼ 5.28 0.10). The lifespan has increased over 22.4% in mev-1 mutant worms treated with complex
4. The complex 4 triggered the DAF-16 nuclear localization, increases sod-3 expression and reduced
Received 6 January 2017
Received in revised form
8 March 2017
Accepted 9 March 2017
Available online 14 March 2017
Keywords:
Ruthenium(II) complexes
Spectroscopy
X-ray crystallography
CT-DNA/ BSA protein binding
Anti-oxidant activity
C. elegans
Toxicology
ROS
Alzheimer's diseases
amyloid (Ab) protein induced paralysis were observed. In the present study we confirmed that oxidative
stress resistance of N2 and lifespan extension of mev-1 mutant which showed the potential ROS scav-
enging activity of complex 4. The results also confirmed the effective anti-aging potential of ruthenium
complex 4 which may be developed as a therapeutic drug for the prevention of aging and age related
neurodegenerative diseases. Further studies are required to find out the exact action of complex 4 on
higher model.
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
normal oxidative metabolism, called reactive oxygen species (ROS).
The finding of the superoxide dismutase enzyme, which detoxifies
Aging study, with specific attention on solving the underlying
mechanisms of longevity, is becoming increased scientific interest.
Lifespan is influenced by genes controlling metabolic activity,
antioxidant systems, DNA repair, cellular senescence, and cell
death. Several lines of confirmation led to the broad acceptance
that oxidative stress and inflammation are main determinants of
the aging process [1e3]. Early, Harman [4] hypothesized in his free-
radical theory of aging (FRTA) that aging results from the aggre-
gation of molecular damage produced through by-products of the
the superoxide anion [5], additionally gave credibility to the free-
radical theory of aging. The extent of ROS formation and oxida-
tive damage to Mitochondrial DNA (mtDNA) is inversely correlated
with longevity across species [6] also causes damage to cellular
proteins, lipids, eventually contributing to various chronic diseases
including cancer, diabetes, Parkinson's and Alzheimer's disease,
cardiovascular disease and chronic inflammation [7].
If FRTA is correct, antioxidants should slow down aging and
prolong lifespan by interfering directly in the generation of radicals
or by scavenging them. Previously, Brown et al. showed that EGCG
reduces ROS levels via DAF-16 dependent manner [8]. Many re-
searchers have reported that a strong antioxidant, reduced the
oxidative stress and extend the life span of wild-type C. elegans
* Corresponding authors.
E-mail addresses: sunpalan@gmail.com (P. Sundararaj), rpnchemist@gmail.com
(R. Prabhakaran).
http://dx.doi.org/10.1016/j.jorganchem.2017.03.023
0022-328X/© 2017 Elsevier B.V. All rights reserved.

G. Devagi et al. / Journal of Organometallic Chemistry 838 (2017) 12e23
13
[9e11]. ROS also play important physiological roles, including in
cellular signaling pathways. For example, in mammals H2O2 is
generated to strengthen insulin signaling by inactivating redox-
sensitive signal-quenching phosphatases [12]. O2ꢀis also gener-
ated as an immune defense mechanism (the oxidative burst), which
may also occur in invertebrate ssuch as C. elegans [13].
Alzheimer's disease (AD) is a main and incurable neurodegen-
erative disorder with a very high social influence [14]. Its etiopa-
thogenesis is unclear, and no effective treatment is yet available
even to slow down disease progression. The “amyloid cascade hy-
pothesis” has encouraged to discover new drugs and methods to
discover substances suitable of blocking amyloid protein aggrega-
tion and preventing its neurotoxicity and a lot of compounds with
these properties were discovered in the past [15]. In this line, there
are few platinum-phenanthroline based metallo drugs were pre-
3p::GFP reporter constructs are used. Further, the neuroprotective
effect of all complexes against A
examined.
b toxic protein aggregation was
2. Results and discussion
The reaction of [RuCl(PPh3)2(h
5-C5H5)] with an equimolar
amount of various 4(N)-substituted thiosemicarbazones (H2L1
eH2L4) in methanol resulted in the formation of new complexes
(Scheme. 1), the analytical data of which confirmed the stoichi-
ometry of the complexes (1e4). The structure of the ligand (H2L1)
and complex 1 were confirmed by X-ray crystallographic studies
and attempts were made to grow single crystals of complex 2e4 in
various organic solvents were unsuccessful. The complexes are
soluble in common organic solvents such as dichloromethane,
chloroform, benzene, acetonitrile, ethanol, methanol, dime-
thylformamide and dimethylsulfoxide.
pared and investigated for their effectiveness in binding with A
b
1ꢀ42, and blocking the aggregation by conferring an appreciable
protection to cortical neurons [16].
Ruthenium compounds are, less toxic than platinum com-
pounds; furthermore, ruthenium compounds showed a prominent
reactivity toward histidines and selectively affect the conformation
2.1. Spectral investigation
The characteristic IR bands of the metal complexes (Fig. S1- S4)
compared with that of the free ligands provide significant infor-
mation regarding to the binding of the ligands to metal ions. In the
IR spectra of the ligands, a broad band observed around 3407-
3446 cmꢀ1 corresponding to nOH was observed around 3404-
3432 cmꢀ1 in the complexes indicating the non-participation of
phenolic oxygen in coordination [27]. A sharp band around 1631-
of A
portion [17]. Daniela Valensin et al., reported that ruthenium(II)
complex, fac-[Ru(CO)3Cl2-(N1-thz)] were (thz 1,3-thiazole),
forms a stable adduct with A
b
(1ꢀ42), bearing three histidine residues in its N-terminal
¼
b
(1ꢀ28) however the ruthenium(II)
centre is coordinated to histidines 13 and 14 simultaneously [18].
Messori et al., reported the promising in vitro anti-Alzheimer
properties for
showed to be highly effective in protecting cortical neurons against
(1ꢀ42) toxicity, moreover PMRU20 is devoid of any significant
toxicity in vitro at the applied concentrations, also it was neuro-
protective even against the toxicity induced by A
25ꢀ35 [19].
a
ruthenium(III) complex namely (PMRU20),
1634 cmꢀ1 in the ligands corresponding to nC
]N azomethine group
was shifted to lower frequency at 1501-1570 cmꢀ1 in the complexes
indicating the coordination of azomethine nitrogen to ruthenium
atom [28]. A sharp band observed around 782-796 cmꢀ1 corre-
A
b
b
sponding to nC]S in the ligands was completely disappeared in the
So far, the oxidative stress resistance and prolonged lifespan of
C. elegans was investigated only for organic compounds. To the best
of our knowledge, still now no one reported the oxidative stress
and prolonged lifespan of C. elegans by organoruthenium com-
plexes. Also only in vitro studies have been reported for metal
complexes and metal ions on Alzheimer's diseases [19e22] and
there is no report available on in vivo experimental implications for
Alzheimer's disease based on organoruthenium complexes. Hence,
we decided to study the impact of our newly synthesized organo-
ruthenium complexes on C. elegans as model to reveal the aging
and Alzheimer's disease.
The nematode C. elegans is an important animal model in
various fields such as neurobiology and genetics. Work with
C. elegans has led to seminal discoveries in the fields of neurosci-
ence, development, cell death, signal transduction, RNA interfer-
ence and aging [23]. Numerous physiological processes and stress
responses that are noticed in higher organisms (e.g., humans) are
preserved in C. elegans. According to bioinformatics approach,
C. elegans homologues have been identified for 60e80% of human
genes [24] and 12 out of 17 known signal transduction pathways
are conserved in C. elegans and human [25].
complexes and a new band appeared at 742-748 cmꢀ1 has been
assigned to nC-S indicating the enolisation of -NHeC]S and sub-
sequent deprotonation prior to the coordination [29]. In addition,
the characteristic absorption bands corresponding to the presence
of triphenylphosphine are also found in the expected region around
1425, 1085, 695 cmꢀ1 [30]. Electronic spectra of the new ruth-
enium(II) complexes were recorded in dichloromethane displayed
two to three bands in the region 233e403 nm (Fig. S5). The bands at
233 nm have been assigned to the intra ligand transition which take
place between the ligand orbitals [31]. However, the bands
observed at 351e389 nm was notified to LMCT (s/d) [33]. The
band observed at 403 nm in complex 4 was assigned to MLCT
transition [32]. The 1H-NMR spectra of the ligands and the com-
plexes (Fig. S6- S21) have been recorded in DMSO/CDCl3. The ligand
showed a singlet at
eOH group. However, a singlet appeared at
d
11.04e11.40 ppm corresponding to phenolic
11.61e11.62 ppm in
d
the complexes indicates the non-participation of phenolic oxygen
in coordination [35]. The remaining all other NMR descriptions are
given in supporting information. The 13C {1H} NMR spectra of the
complexes (1e4) contain resonances for the cyclopentadienyl ring
carbons around
around 160.44e169.14 ppm assigned to the azomethine (HC]N)
carbon of the ligands [36]. Thiolic carbon signal of the ligand
observed around 165.21e169.40 ppm [36]. The spectra also
showed resonance in the range of 119.60e134.40 ppm for the
aromatic carbons [36]. In all the complexes resonances observed
around 40.48e47.38 ppm and 12.57e25.46 ppm were due to
methylene and methyl carbons of diethylamino group of ligand. In
complex 2 a signal observed at 39.89 ppm has been assigned to
terminal HNeCH3 carbons. In complex 3, signals observed at
36.33 ppm and 18.86 ppm was corresponding to the presence of
d 79.91e83.76 ppm [34]. The resonance observed
Further, thiosemicarbazones are a class of Schiff base com-
pounds showed different molecular geometry with metal com-
plexes, and are familiar for their biological activity as antiparasitics,
antibacterial, antifungal, antiviral and antitumoral agents [26].
Considering the above facts, we synthesized and characterized
four new organoruthenium(II) complexes containing bio active
thiosemicarbazones and investigated their impact on lifespan and
d
d
d
d
d
A
b
toxicity of C. elegans. The complex 4 was found to have an
d
excellent antioxidant activity also extend the life span of mev-1
mutant strain compared to the rest of the complexes. In addition,
we examined their effects on pathways regulated by the DAF-16/
FOXO transcription factors. To confirm this a daf-16::GFP and sod-
d
d
methylene and methyl carbons of ethyl group. In order to confirm
the presence of triphenylphosphine, 31P NMR spectra were

14
G. Devagi et al. / Journal of Organometallic Chemistry 838 (2017) 12e23
Ph3P
N
Ru
N
OH
S
+
S
Ru
Cl
Methanol, 5 h
N
OH
Ph3P
PPh3
N
C
R
C
H
N
N
NH
H
H
R
N
1. R = H,
2. R = CH3,
3. R = C2H5,
4. R = C6H5
Scheme 1. Synthesis of new Ru(II) complexes.
recorded. The singlet observed at
29.40 ppm in complexes 1, 2, 3 and 4, respectively, suggested the
presence of a triphenylphosphine in each complexes [37].
d
29.28, 29.44, 29.36 and
O(1)ꢀH(1)$$$N(1) distance of 2.721 Å and oxygen atom of hydroxyl
group with the hydrogen atom of terminal nitrogen (N3) atom of
another molecule with O(1)ꢀH(1)$$$N(3) distance of 2.964 Å.
Further, the complex 1 contains an intramolecular hydrogen bond
(Table S2, Figs. S24 and S25) through the hydrogen atom of the
hydroxyl group with the hydrazinic nitrogen (N2) of thio-
semicarbazone moiety with O1eH(1)$$$N2 distance of 2.589 Å.
2.2. Single crystal X-ray crystallography
The suitable crystals of H2L1 and 1 suitable for X-ray diffraction
studies were obtained from dichloromethane/n-hexane (1:1)
mixture. The crystallographic data are listed in (Table 1). The ORTEP
diagrams of H2L1 and 1 shown in (Figs.1 and 2). In the ligand (H2L1),
C(8)ꢀS(1) bond, with the length of 1.6971(13) Å, is typical of double
bonds; that is, the thioamide tautomer is present [38]. In complex,
the ligand coordinated to ruthenium metal as NS chelating donor
by forming a stable five member ring with a bite angle N(1)-Ru(1)-
S(1) of 79.96(6)ꢁ and leaving the phenolic oxygen remains intact
without involving in bonding. The Ru(1)eN(1) and Ru(1)eS(1)
bond distances are found to be 2.096(2) Å and 2.3678 (9) Å
respectively. The remaining sites are occupied by phosphorus atom
of triphenylphosphine with RueP(1) distance of 2.2814 Å and five
carbons of cyclopentadienyl ligand with Ru(1)eC(13), Ru(1)eC(14),
Ru(1)eC(15), Ru(1)eC(16), Ru(1)eC(17) distance of 2.182 (4) Å,
2.205 (4) Å, 2.231 (3) Å, 2.183 (3) Å and 2.158 (4) Å respectively. The
Ru(1)eS(1), Ru(1)eN(1) and Ru(1)eP(1) are comparable with the
distances found in other octahedral ruthenium(II) complexes con-
taining triphenylphosphine [39].
3. DNA binding studies
UV absorption titration experiments were carried out to study
the DNA binding property of the new Ru(II) complexes (1e4). The
absorption spectra of the new complexes at constant concentration
(10
mM) in the presence of different concentration of CT-DNA
(5e50
mM) are given in Fig. 3. The absorption spectra of com-
plexes (1e4) mainly consist of two resolved bands [Intra ligand (IL)
and CT transitions]. Intra ligand transitions of complexes 1, 2, 3 and
4 centered at 249 nm, 250 nm, 248 nm and 249 nm respectively. As
the DNA concentration is increased, all the complexes (1e4)
showed hyperchromism around (A ¼ 0.1883e0.8562) with a red
shift of 8 nm, 7 nm, 9 nm and 8 nm respectively. However, CT band
of the complexes 1e4 were centered at 400 nm, 384 nm, 415 nm
and 383 nm respectively. All the complexes showed hypochromism
upon addition of CT-DNA around (A ¼ 0.1671e0.1808) without
any shift in the complexes (1e3) and 2 nm red shift in complex 4.
All the complexes exhibited isosbestic spectral change upon
addition of CT-DNA to complexes (1e4) at 340 nm, 329 nm, 383 nm
and 340 nm respectively. The observed hypochromic effect with
or without shift in the charge transfer band suggested that the
new ruthenium(II) complexes bind with adjacent base pairs of
DNA, possibly due to intercalative mode of binding. The intrinsic
binding constant Kb was determined by using Stern Volmer equa-
tion (1) [40].
The selected bond distances and bond angles are given in
(Table S1). The cis angles N(1)-Ru(1)-S(1) ¼ 79.96(6)ꢁ, C(13)-Ru(1)-
C(14) ¼ 37.75(17)ꢁ, C(14)-Ru(1)-C(15) ¼ 36.21(17)ꢁ, C(13)-Ru(1)-
C(15) ¼ 62.34(18)ꢁ, C(13)-Ru(1)-C(16) ¼ 62.55(18)ꢁ, C(16)-Ru(1)-
C(14) ¼ 61.41(16)ꢁ, C(16)-Ru(1)-C(15) ¼ 37.13(14)ꢁ, C(17)-Ru(1)-
C(13) ¼ 38.10(17)ꢁ, C(17)-Ru(1)-C(14) ¼ 62.35(16)ꢁ, C(17)-Ru(1)-
C(15) ¼ 61.97(15)ꢁ and C(17)-Ru(1)-C(16) ¼ 36.80(17)ꢁ are acute,
whereas the other cis angles P(1)-Ru(1)-S(1) ¼ 93.19(3)ꢁ, N(1)-
Ru(1)-P(1) ¼ 93.28(6)ꢁ, N(1)-Ru(1)-C(14) ¼ 94.62(12)ꢁ, C(15)-
Ru(1)-S(1) ¼ 97.93(13)ꢁ,
[DNA] ⁄ [εa-εf]) ¼ [DNA] ⁄ [εb-εf]þ1 ⁄ Kb[εb-εf]
(1)
C(16)-Ru(1)-S(1)
92.75(11)ꢁ are obtuse. The trans angles are N(1)-Ru(1)-C(13)
111.43(15)ꢁ, N(1)-Ru(1)-C(15) 111.97(12)ꢁ, N(1)-Ru(1)-
¼
98.71(14)ꢁ and C(17)-Ru(1)-P(1)
¼
From the binding constant Kb, the binding strength of the
complexes with CT-DNA can be determined by monitoring the
change in the absorbance in the intra ligand (IL) band with respect
to the increasing concentration of DNA. Kb is obtained from the
ratio of slope to the intercept in plots by DNA/(εa-εf) versus [DNA]
(Fig. 3 Inset). From the binding constant values (Table 2), it is
inferred that the complex 4 exhibited better binding than other
complexes; it may due to the electron withdrawing phenyl sub-
stitution present in the ligand moiety of the complex. Based on the
Kb value, we can arrange the complexes in the following order with
respect to the electron delocalization, 4 > 3>1 > 2.
¼
¼
C(16) ¼ 148.97(13)ꢁ, N(1)-Ru(1)-C(17) ¼ 149.41(15)ꢁ, C(13)-Ru(1)-
S(1) ¼ 159.52(13)ꢁ, C(13)-Ru(1)-P(1) ¼ 102.74(14)ꢁ, C(14)-Ru(1)-
S(1) ¼ 127.60(15)ꢁ, C(14)-Ru(1)-P(1) ¼ 139.20(15)ꢁ, C(15)-Ru(1)-
P(1) ¼ 153.78(10)ꢁ, C(16)-Ru(1)-P(1) ¼ 117.71(11)ꢁ and C(17)-Ru(1)-
S(1) ¼ 129.56(14)ꢁ were found similar to the reported values. The
variations in bond lengths and angles lead to a significant distortion
from an ideal octahedral geometry of the complex.
The ligand H2L1 showed one intra and inter molecular hydrogen
bonding through the hydrogen atom of the hydroxyl group with the
hydrazinic nitrogen (N2) of thiosemicarbazone moiety with an
The result obtained from the above experiments suggested that

G. Devagi et al. / Journal of Organometallic Chemistry 838 (2017) 12e23
15
Table 1
Crystallographic data of H2L1 and new Ru(II) thiosemicarbazone complexes.
[H2-DeaSal-tsc] (H2L1)
[RuCp(Dea-tsc)(PPh3)](1)
CCDC No.
1500357
1500358
Empirical formula
Formula weight
Temperature
C12H18N4OS
266.36
293 K
C35H37N4OPRuS
693.78
293 K
Wavelength
Crystal system
Space group
1.54184 Å
Monoclinic
P 21/c
0.71073 Å
Triclinic
P -1
a
b
c
a
b
g
11.4268(8) Å
11.0574 (2) Å
12.2568 (2) Å
12.2843 (3) Å
92.6273 (19)ꢁ
95.153 (2)ꢁ
99.6015(17)ꢁ
1631.77(7) Å3
2
10.54035(17)Å
18.7023(14)Å
90.0ꢁ
141.678(16)ꢁ
90.0ꢁ
Volume
Z
Calculated density
Absorption coefficient
F(000)
1396.76(15)Å3
4
1.267 Mg/m3
1.412 Mg/m3
0.627 mmꢀ1
716
2.021 mmꢀ1
568
Crystal size
0.18 ꢃ 0.12 x 0.03
4.736 to 73.674
ꢀ14 ¼ h<¼10, ꢀ12 ¼ k<¼13, ꢀ20 ¼ l<¼22
5184/2740 [R(int) ¼ 0.0258]
66.97ꢁ 100%
Semi-empirical from equivalents
1.00000 and 0.86588
Full-matrix least-squares on F2
2740/0/166
0.12 ꢃ 0.1 ꢃ 0.05 mm
Theta range for data collection
Limiting indices
Reflections collected/unique
Completeness to theta
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2sigma(I)]
R indices (all data)
Largest diff. peak and hole
3.350e28.909ꢁ
ꢀ14 ¼ h<¼14, ꢀ16 ¼ k<¼16, ꢀ16 ¼ l<¼16
32781/7581 [R(int) ¼ 0.0326]
25.30ꢁ 99.78%
Semi-empirical from equivalents
1.00000 and 0.94103
Full-matrix least-squares on F2
7581/6/392
1.059
1.049
R1 ¼ 0.0371, wR2 ¼ 0.1051
R1 ¼ 0.0408, wR2 ¼ 0.1106
0.295 and ꢀ0.176 e.Aꢀ3
R1 ¼ 0.0426, wR2 ¼ 0.0957
R1 ¼ 0.0561, wR2 ¼ 0.1043
0.788 and ꢀ0.754 e.Aꢀ3
all the compounds can bind with CT-DNA. Further, the binding af-
finity of the complexes with the CT-DNA has been confirmed by
Ethidium bromide (EB) displacement studies. Ethidium bromide
competitive binding studies using new ruthenium(II) complexes
1e4 as quenchers may give the further information about the
binding of these complexes to DNA. EB emits strong fluorescence in
the presence of DNA, due to its strong intercalation with DNA base
pairs. The quenching extent of fluorescence of EB bound to DNA is
used to determine the extent of binding of metal complexes to DNA.
When complexes 1e4 were added to DNA pretreated with EB, the
DNA induced emission intensity at 602e606 nm was decreased
(Fig. S27). This indicated that the complexes could replace EB from
the DNA-EB system. Based on the classical Stern-Volmer equation
(2) the quenching constant has been analyzed.
KEB½EBꢂ ¼ Kapp½complexꢂ
(3)
where [complex] is the value at 50% reduction in the fluorescence
intensity of EB, KEB (1.0 ꢃ 107 Mꢀ1) is the DNA binding constant of
EB, [EB] is the concentration of Ethidium Bromide (EB) (10 mM). Kapp
values are found (Table 2). From these experimental data, it is
inferred that the complex 4 replaces the EB more effectively than
other complexes. From this observation, it may conclude that all the
complexes can bind to DNA through the intercalation mode [36].
3.1. DNA cleavage study
Agarose gel electrophoresis assay is a valuable method to
examine various binding modes of small molecules to plasmid
DNA. Natural derived plasmid DNA mainly contains closed-circle
supercoiled form (Form I), nicked form (Form II) and linear form
I0=I ¼ Ksv½Qꢂ þ 1
(2)
The Ksv values have been obtained as a slope from the plot of I0/I
vs [Q] (Fig. S28) were evaluated for the complexes. Further, the
apparent DNA binding constants (Kapp) were calculated using
equation (3):
Fig. 1. ORTEP for [H2-DeaSal-tsc] (H2L1).
Fig. 2. ORTEP for [RuCp(DeaSal-tsc)(PPh3)](1).

16
G. Devagi et al. / Journal of Organometallic Chemistry 838 (2017) 12e23
Fig. 3. Absorption titration spectra of complexes (10 mM) 1e4 with increasing concentrations (0e50 mM) of CT-DNA (trisHCl buffer, pH 7.2); the inset shows binding isotherms with
CT-DNA.
(Form III). Intercalation of small molecules to plasmid DNA can
cleave the supercoiled form of DNA, which decreases its mobility
rate and can be distinctly visualized by agarose gel electrophoresis
method. Morever, simple electrostatic contact of small molecules to
DNA does not considerably influence the supercoiled form of
plasmid DNA, thus does not change the mobility of supercoiled
DNA. To identify the DNA cleavage ability of the free ligands and
ruthenium(II) complexes, supercoiled (SC) pBR322 DNA was incu-
bated with complexes and ligands in 5 mM Tris HCl/50 mM NaCl
buffer at pH 7.2 for 2 h without reductant addition. Upon gel
electrophoresis of the reaction mixture, DNA cleavage was
observed (Fig. S29). The relatively fast migration is the supercoil
form (Form I) and the slower migration is the open circular form
(Form II), which was produced from supercoiled when scission
occurred on its one strand [41]. The ligands and complexes at same
concentration is able to perform cleavage of pBR322 plasmid DNA.
The intensity of supercoiled SC (Form I) diminished and partly
converted to nicked form NC (Form II) in ligands and in complexes
(2 and 3) whereas in complexes (1 and 4), the intensity of super-
coiled SC (Form I) increased and partly converted to nicked form NC
(Form II). The intensity of the NC (Form II) band increases, whereas
the production of linear form LC (Form III) of DNA diminished. It is
obvious that the ligands and ruthenium(II) complexes have the
ability to cleave the supercoiled plasmid DNA and this cleavage
system does not require addition of any external agents.
3.2. Absorption spectra of BSA
Table 2
The Kb(binding constant), Ksv(Quenching constant) and Kapp(apparent with EB)
values for the interactions of compounds with CT-DNA.
The general method to distinguish between static and dynamic
quenching is by examining the absorption spectra of BSA (10 mM) in
Complex
Kb/Mꢀ1
Ksv/Mꢀ1
Kapp/Mꢀ1
the presence of complexes. The absorption intensity of BSA was
increased with the addition of the complexes (1e4) (Fig. S30). The
increasing intensity in the absorbance spectra for BSA þ complexes
indicate the interaction of complexes with BSA. It is well known
that dynamic quenching only disturbs the excited state of
1
2
3
4
2.21 ꢃ 105 0.23
0.40 ꢃ 105 0.09
3.82 ꢃ 105 0.12
4.08 ꢃ 105 0.14
1.45 ꢃ 104 0.01
1.07 ꢃ 104 0.07
2.03 ꢃ 104 0.04
2.94 ꢃ 104 0.03
6.77 ꢃ 106
6.32 ꢃ 106
5.83 ꢃ 106
6.87 ꢃ 106

G. Devagi et al. / Journal of Organometallic Chemistry 838 (2017) 12e23
17
fluorophore, thus shows no change the absorption spectrum. But,
3.6. In vitro antioxidant study
the formation of non-fluorescence ground-state complex induced
the change in absorption spectrum of fluorophore. Thus, possible
quenching mechanism of fluorophore-quencher (BSA) by the
complexes was found as static quenching [42].
Since the experiments conducted so far revealed that the Ru(II)
complexes exhibit good DNA and protein binding affinity, it is
considered worthwhile to study the antioxidant activity of these
complexes (Fig. 4). Antioxidants that exhibit radical scavenging
activity are receiving increased attention since they present inter-
esting anticancer, anti-aging and anti-inflammatory activities [47].
The radical scavenging activities of our compounds along with
standards, ascorbic acid in a cell free system, have been examined
with reference to 2,2-diphenyl-1-picrylhydrazyl (DPPH). and their
corresponding IC50 values have been tabulated in Table S4. The
power of the complexes ability to scavenge DPPH radicals was
found to be higher than that of the standard antioxidant (ascorbic
acid), Thus the scavenging ability of the Ru(II) complexes was most
appreciable for DPPH radicals when compared to the standards.
The IC50 values indicated that the activities of the complexes are in
the order of 4 > 3>1 > 2.
3.3. Fluorescence quenching studies of BSA
In order to infer more on the binding of the complexes with
BSA (10 mM), fluorescence emission spectrum of BSA was studied
upon addition of the complexes (1e4). BSA has three fluo-
rophores, namely, tryptophan, tyrosine, and phenylalanine, but
the intrinsic emission intensity of BSA is due to tryptophan alone,
because phenylalanine has low quantum yield and the fluores-
cence of tyrosine is nearly quenched when it becomes ionized or
near to an amino group, a carbonyl group, or a tryptophan res-
idue [43]. Changes in the emission spectra of tryptophan are
common in protein conformational transitions, substrate binding,
subunit associations, or denaturation. Whereas, the interaction of
BSA with our complexes (1e4) was studied at room temperature
and the binding constants (1e4) were calculated. The fluores-
cence spectra were recorded in the range of 290e500 nm upon
excitation at 280 nm. When the concentration of complexes
increased (1e4), a gradual decrease in the fluorescence intensity
was detected with a blue shift (Fig. S31). The blue shift was due
to the binding of the complexes (1e4) with the active site in BSA
[44]. The binding constant KSV value was obtained from the plot
of I0/Icorr versus [Q] in SterneVolmer equation (4) (Fig. S32 and
Table S3).
3.7. Toxicity testing assay
Dose dependent studies on a beneficial and adverse effect of the
synthetic antioxidant such as BHA on animal models were inves-
tigated [48]. The preliminary assay was conducted at 20 ꢁC in 48
well plates according to the previous method [49]. The dose
dependent toxicity at 100 mM and 200 mM in all the complexes were
found after 24 h treatment. The Fig. 5 showed the percentage sur-
vival of adulthood worms at selected molarities. The results
showed that the increase in the concentration of the complexes, the
toxicity increases. The concentration 100 and 200
to the worms and hence the remaining concentrations viz., 10, 30
and 50 M were taken for further studies.
mM showed toxic
I0=Icorr ¼ 1 þ KSV½Qꢂ
(4)
m
The observed linearity in the plots indicated the ability of
complexes to quench the BSA emission intensity. From KSV values, it
is seen that the new complexes (1e4) showed strong protein-
binding ability with greater hydrophobicity.
3.8. Oxidative stress resistance assay
In general oxidative stress resistance is proportional to the su-
peroxide dismutase and catalase activity in C. elegans [50]. The
oxidative stress resistance on wild type (N2) strain were presented
in the (Fig. 6). Two days old adult N2 worms were pre-treated with
3.4. Binding constants and the number of binding sites
10, 30 and 50 mM of four complexes and transferred into 240 mM
For the static quenching interaction, if it is considered that
there are similar and independent binding sites in the biomole-
cule, the binding constant (Kb) and the number of binding sites (n)
can be determined according to the Scatchard equation (5) [45].
log [(I0ꢀI)/I] ¼ log Kb þ n log [Q] (5) The values of n at room
temperature are approximately equal to 1, which indicates that
the complexes bind with BSA in one site only (Fig. S33 and
Table S3).
Juglone plates for 5 h. Worms were considered as dead if they are
not responding to gentle touch. Juglone is a redox cycling pro-
oxidant that generate O2ꢀ, H2O2 and OH leads to death [51]. The
survival percentage against the oxidative stress was observed
34.54, 50.76 and 62.46 % in complex 1; 33.28, 45.59 and 53.19 % in
complex 2; 33.51, 48.54 and 59.26 % in complex 3; 38.69, 55.78 and
65.05 % in complex 4 at 10, 30 and 50 mM concentrations respec-
tively. The complex 4 holds a higher scavenging activity and
attenuate oxidative damage in C. elegans. The results of oxidative
3.5. Synchronous fluorescence spectroscopic studies of BSA
Synchronous fluorescence spectral study was used to obtain
information about the molecular environment in the vicinity of the
fluorophore moieties of BSA [46]. Synchronous fluorescence
spectra showed tyrosine residues of BSA only at the wavelength
interval Dl of 15 nm whereas tryptophan residues of BSA at Dl of
60 nm. While increase the concentration of complexes (0e50
mM)
added to BSA (10 M), a decrease in the fluorescence intensity with
m
a blue shift in the tryptophan emission maximum is observed for all
the complexes (Fig. S34). In contrast, the emission intensity of
tyrosine residue increases without any change in the wavelength of
emission. These observations indicate that the test compounds did
not affect the microenvironment of tyrosine residues during the
binding process significantly but the tryptophan microenviron-
ment to a larger extent.
Fig. 4. In vitro free radical scavenging activity of complexes (1e4) with control ascorbic
acid. DPPH Scavenging activity in the sample is denoted by IC50
(mg/ml) value. Three
independent experiments were carried and bar represents the mean S.E.M.

18
G. Devagi et al. / Journal of Organometallic Chemistry 838 (2017) 12e23
experiment the complex 4 has significant increase in longevity of
C. elegans than others. At 50 M the complex 4 extent the mean
m
lifespan of mev-1mutant into 22.4%, (p < 0.0001***) and this final
concentration was used for analyse the sod-3 expression and DAF-
16 nuclear localization using GFP tagged strains.
The mev-1 mutant worms were treated with all four complexes
with different concentrations from L4 (young adult) stage until the
completion of lifespan. The complex 4 significantly increased the
mean lifespan on a dose dependent manner. As the highest anti-
oxidant and longevity was found for complex 4, we further used
complex 4 alone to confirm daf-16 mediated activity by applying on
strain TJ356 and CF1553.
Fig. 5. The toxicity assay was conducted in two day adult worms at 20 ꢁC for 24 h. The
survival rate was significantly reduced proportional to increasing concentrations in all
four derivatives. An approximately ꢄ30 worm per group was taken and three inde-
pendent biological experiment were conducted. The means of control and treatment
were compare with one way analysis of variance (ANOVA) using statistical software
SPSS v. 16.0. The error bar represents the mean S.E.M. **p ¼ 0.0001 and *p ¼ 0.001.
3.10. DAF-16 nuclear localization and SOD-3 expression analysis
The daf-16 is a key activator for resistance and extension of
lifespan under stress condition which activate pro-longevity genes
and it is negatively regulated by Insulin/Insulin like growth
factor ꢀ1(IIS) [56e58]. Activated DAF-16 transcription factor into to
nucleus and stimulates stress resistant genes, which confer resis-
tance to oxidative damage. In this study we observed that DAF-16
stress resistance clearly shown that the complexes having a great
intracellular ROS scavenging activity. The antioxidants like 4-
hydroxybenzoic acid and curcumin are intracellular free radical
scavengers that increase mev-1 mutant lifespan and resistance to
oxidative stress in wild type (N2) worms confirm that oxidative
stress resistance in wild type is correlated with mev-1 mutant
lifespan extension in C. elegans [52]. However, one form of catechin,
epigallocatechingallate ([(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydr-
oxyphenyl) chroman-3-yl] 3,4,5-trihydroxybenzoate) increases
nuclear translocation is induced by 4 at 50
mM suggesting that
DAF-16/FOXO-dependent longevity is mediated by expression of
multiple target genes [57]. The (Fig. 8) showing the nuclear local-
ization of DAF-16::GFP in the transgenic worm TJ356. We further
analyzed the DAF-16 target gene sod-3 expression using SOD-
3::GFP tagged transgenic strain CF1553. Superoxide dismutase
and heat shock protein are direct target genes of DAF-16 tran-
scription factor. The (Fig. 9) showing the expression of sod-3 treated
oxidative stress resistance and decreases Ab peptide aggregation in
muscles by either direct or indirect activation of DAF-16 nuclear
localization [53].
with complex 4 at 50
day adult after immediately transferred into new plates exposed
with 40 M juglone for 24 h and visualized under at excitation
mM and 0.2% DMSO for 72 h to reach second
m
3.9. Lifespan assay
emission of 450e520 nm in fluorescent microscope. Our result
confirmed that the expression of SOD-3 is higher than control
suggesting that the complex 4 partially or indirectly activate DAF-
16 and extend the lifespan and resistance to oxidative stress
through the DAF-16/FOXO dependent pathway in C. elegans. Pre-
sent study suggest that the test complex 4 might extend the mean
lifespan as previously reported with antioxidant kaempferol
[59e62], (Fig. 10). In this study we conclude that complex 4 have
the potential anti-aging activity in two parallel ways by stimulate
DAF-16 nuclear movement and free radical scavenging activity
in vivo.
The in vivo antioxidant activities of complexes (1e4) were
investigated with the mutant strain mev-1 (Fig. 7). Mutation in
succinate dehydrogenase cytochrome b encoding gene mev-1(kn1)
in C. elegans cause overproduction of superoxide anions which
leads to short lifespan [54,55]. Previous studies with the antioxi-
dant 4-hydroxybenzoic acid extend the lifespan of mev-1 mutants
worms. This indicates the antioxidant potential of the compound
that could reduce the oxidative damage which could lead to in-
crease in lifespan of C. elegans [52]. In the present study we
observed mean lifespan of C. elegans increased overall in four de-
rivatives. The Table S5 showed the mean lifespan and percentage
increase in mev-1 mutant lifespan at 20 ꢁC. In the present
3.11. Amyloid e
b induced paralysis assay
Paralysis assay was performed with transgenic strain CL4176 a
temperature inducible strain expressing amyloid beta (A ) in its
muscle cells. While increasing the temperature from 16 ꢁC to 25 ꢁC
induces the human A expression in body wall muscle cells
b
b
resulting paralysis phenotype [63]. To examine whether the com-
plexes has protective effects on Alzheimer's disease (AD), the syn-
chronized L1 larvae of CL4176 were cultured on NGM plates
previously spotted with 50
50 h at 16 ꢁC. After that the temperature were raised to 25 ꢁC to
induce the A toxic peptide for muscle paralysis (Fig. 11). Paralysis
was noticed in the worms fed with complexes (1e4) at 50
mM of complexes (1e4) and maintained
b
m
M
concentration compared to control group worms (p; < 0.0001). The
lowest paralysis was found in the complex 4 than rest of the
complexes. Also the result suggested that all the complexes had
protective role against Ab toxicity in CL4176 transgenic C. elegans.
Oxidative stress plays a vital role in the pathogenesis of Alz-
heimer's disease which significantly affects the nervous system and
Fig. 6. Synchronized L1 larvae (as described in lifespan assay) of N2 worms were
transferred to NGM plates previously supplemented with respective dosage of all
complexes at 20 ꢁC. Subsequently day 2 adult N2 worms were transferred to treatment
plates contains acute lethal concentration of juglone (5-Hydroxy-1,4-napthoquinone)
an intracellular ROS generator at 240 mM concentration. Survival was calculated after
5 h of exposure. The means of control and treatment were compare with one way
analysis of variance (ANOVA) using statistical software SPSS v. 16.0. The error bar
represents them mean S.E.M. **p ¼ 0.0001 and *p ¼ 0.001.
their functions. The inhibitory effects of A
b toxic peptide accumu-
lation by anti-oxidants have been demonstrated through both

G. Devagi et al. / Journal of Organometallic Chemistry 838 (2017) 12e23
19
100
80
60
40
20
0
1 - mev-1
Control
100
80
60
40
20
0
2 - mev-1
Control
10μM
30μM
50μM
10μM
30μM
50μM
2
4
6
8
10 12 14 16 18 20 22 24 26
Days
2
4
6
8
10 12 14 16 18 20 22 24 26
Days
100
80
60
40
20
0
100
4 - mev-1
Control
10μM
3 - mev-1
Control
10μM
80
60
40
20
0
30μM
50μM
30μM
50μM
2
4
6
8
10 12 14 16 18 20 22 24 26
Days
2
4
6
8
10 12 14 16 18 20 22 24 26
Days
Fig. 7. Effect of complexes on the C. elegans mev-1 (kn1) mutants which overproduce ROS. The complex 1 at 50
(12.14 0.33, p < 0.0001***) (1). Complex 2 at 50 M significantly extent mean lifespan 14.28 0.34 days than control (12.61 0.34, p < 0.0001***). (2). Complex 3 at 50
significantly extent mean lifespan 14.84 0.32 days than control (12.67 0.35, p < 0.0010**). (3). Complex 4 at 50 M significantly extend the mean lifespan 15.49 0.42days than
mM significantly extend mean lifespan 14.47 0.45 days than control
m
mM
m
control (12.64 0.33, p < 0.0001***) (4). Three independent experiment were conducted and the statistical significance of the survival curve were processed using log rank test
using the KaplaneMeir survival analysis in Med Calc software version 14.8.1.0.
Fig. 8. Effects of thermal stress and complex 4 on nuclear translocation of DAF-16 in TJ356 transgenic worms. A. Negative control (without any treatment); B. Thermal stress at 37 ꢁ
for 15 min (positive control); C. Partial nuclear translocation of DAF-16 induced by 50 M complex 4. At least 20 worms were performed. RED colour arrow indicates the nuclear
C
m
localization and YELLOW colour arrow indicates the cytoplasmic localization. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

20
G. Devagi et al. / Journal of Organometallic Chemistry 838 (2017) 12e23
Fig. 9. Complex 4 up-regulates the expression of stress response gene sod-3 in CF1553 (SOD-3::GFP). A. Control worms (without any treatment); B. Worms treated with 50
complex 4.
mM of
in vitro and in vivo models [64]. Free radical scavengers have healing
effects with respect to chronic and degenerative diseases.
Furthermore, it is believed that the anti-amyloid accumulation ef-
fects of complexes are considerably associated to their anti-
oxidative abilities.
Based on the results we confirmed, complexes 4 have linked with
DAF-16/FOXO mediated lifespan extension and antioxidant prop-
erties of complexes.
5. Experimental section
All the reagents used were analar grade, were purified and dried
according to the standard procedure [65]. The ligands (H2L1¡4) [66]
4. Conclusion
and the ruthenium complex [RuCl(PPh3)2(h
5-C5H5)] [67] were
The four new complexes, (1e4) were synthesized and charac-
terized by elemental analyses, various spectroscopic techniques
(NMR, UVꢀVis, IR) and X-ray crystallography. In all the complexes,
the ligand coordinated in a bidentate monobasic fashion by form-
ing a stable NS five-member ring in 65e69% yield. Comparative
biological studies such as DNA, protein binding have been carried
out for new ruthenium(II) complexes (1e4). From the DNA binding
studies, it is inferred that all the complexes exhibited electrostatic
binding modes. The complex 4 showed better binding which is
supported by further biological studies. The protein binding studies
have been monitored by quenching of tryptophan and tyrosine
residues in the presence of compounds by taking bovine serum
albumin (BSA) as a model protein and the mechanism of quenching
was found as static. The biological activity concludes that the
complexes 1e4 increase the protection from oxidative damage and
up regulating antioxidant gene sod-3 in C. elegans. Our further
study in strain mev-1(kn1) and the worm expressing human amy-
loid protein Ab3-42 (CL4176) confirm that the complex 4 increase
the maximum lifespan through direct scavenging or up regulating
synthesized according to the standard literature procedures.
5.1. Measurements
Melting points were measured in a Lab India apparatus. Infrared
spectra were measured as KBr pellets on a Jasco FT-IR 400-
4100 cmꢀ1 range. Elemental analyses of carbon, hydrogen, nitrogen,
and sulphur were determined using Vario EL III CHNS at the
Department of chemistry, Bharathiar University, Coimbatore, India.
Electronic absorption spectra of the compounds were recorded
using JASCO 600 spectrophotometer and emission measurements
were carried out by using a JASCO FP-6600 spectrofluorometer.
1H, 13C and 31P NMR spectra were recorded in CDCl3 and DMSO
at room temperature with a Bruker 400 MHz instrument, chemical
shift relative to tetramethylsilane. The chemical shifts are
expressed in parts per million (ppm). CT-DNA, pBR322 plasmid
DNA, BSA and ethidium bromide (EB) were obtained from Hi media.
Single crystal data collections and corrections for the ligand (H2L1)
and new Ru(II) complexes 1 were done at 293 K with CCD Kappa
Diffractometer using graphite mono chromated Mo Ka (k ¼ 0.71073
A) radiation [68]. The structural solution were done by using
SHELXS-97 [69] and refined by full matrix least square on F2 using
SHELXL-2014 [70].
antioxidant responsible genes and inhibit Ab deposits respectively.
5.2. Synthesis of new ruthenium(II) complexes
5.2.1. Synthesis of [RuCp(DeaSal-tsc)(PPh3)] (1)
To a metanolic solution (10 cm3) of [RuCl(PPh3)2( 5-C5H5)]
h
(0.05 g, 0.0688 mmol), 4(N,N0)-diethylaminosalicylaldehydethiose-
micarbazone [H2-DeaSal-tsc] (0.018 g, 0.0688 mmol) in methanol
(10 cm3) was added. The reaction mixture was then refluxed for 5 h.
The reddish orange suspension gradually turned orange colour. The
solvent was removed under reduced pressure. The orange solid was
washed with petroleum ether (60-80 ꢁC) 2e3 times to remove
traces of triphenylphosphine. The complex was dissolved in CH2Cl2/
n-hexane and kept for crystallisation. On slow evaporation orange
crystals were obtained. Yield: 67%. Mp 132 ꢁC. Anal. calcd for
Fig. 10. SOD-3 expression by treatment with complex 4 at 50
juglone on CF1553. Young adult N2 worms treated two days with complex 4 then
transferred into 40 M juglone for 1 day. The nematode were then transferred into
30 M sodium azide agar pad for anesthetized. The florescence intensity of GFP was
quantified in the worm's pharynx from at least 30 worms. Error bar represent
mM and control 40 mM
m
m
mean
S.E.M, **p; < 0.001. The florescence intensity were quantified by Image J
software.
C35H37N4OPRuS: C, 60.59; H, 5.38; N, 8.08; S, 4.62. Found: C, 60.55;

G. Devagi et al. / Journal of Organometallic Chemistry 838 (2017) 12e23
21
100
80
60
40
20
0
Cl4176
1
2
3
4
Control
30
32
34
36
38
40
Time (h)
42
44
46
48
50
Fig. 11. Effects of complexes (1e4) on C. elegans transgenic strain CL4176 expressing a temperature inducible expression of amyloid beta (A
larvae of strain CL4176 were transferred to NGM with E. coli OP50 and 50
M concentration of each complex for 50 h at 16 ꢁC. After that the temperature was up-shift to 25 ꢁC to
induce the expression of muscle specific A transgene expression. The paralysis was scored at every 2 h intervals until the last worm become paralyzed. Data are expressed as
b) in its body wall muscles. Synchronized
m
b
percentage of non-paralyzed worms from three independent biological replicates with 100 worms in each set of experiments. Statistical significance of the dissimilarities between
the treated and untreated control was validated by log-rank test using the Kaplan-Meier survival analysis using Med Calc v.14. Differences at the p < 0.05 level were considered as
significantly different.
H, 5.37; N, 8.00; S, 4.59%. FT-IR (cmꢀ1) in KBr: 3432(ʋO-H), 1577
(ʋC]N), 1351 (ʋCeO), 747 (ʋCeS), 1421, 1023, 695 cmꢀ1 (for PPh3);
UV-Vis (CH2Cl2), lmax: 233 (65,890) nm (dm3 molꢀ1cmꢀ1) (intra-
ligand transition); 351 nm (21,353) (dm3 molꢀ1cmꢀ1) (LMCT); 1H
NMR (CDCl3, ppm): 11.61 (s, H, eOH), 4.33 (s, 5H, C5H5), mixed with
aromatic region (s, 1H, - CH]N), 9.48 (s, 2H, terminal eNH2),
3.08e3.12 (q, 2H, J ¼ 7 Hz, eCH2N), 1.13e1.16 (t, 3H, J ¼ 7 Hz,-CH3),
7.29e7.69 (m, aromatic protons); 13C NMR (CDCl3, ppm):
165.21(CꢀS), 159.94 (C]N),79.91 (Cp),158.20 (CeO), 140.77 (CeN),
127.82e134.40 (aromatic C);31P NMR (CDCl3 ppm): 29.28 (s).
A similar method was followed to synthesize other complexes.
4.44. Found: C, 61.51; H, 5.68; N, 7.74; S, 4.41%. FT-IR (cmꢀ1) in KBr:
3404(ʋO-H), 1563 (ʋC]N), 1267 (ʋCeO), 743 (ʋCeS), 1417, 1026,
694 cmꢀ1 (for PPh3); UV-Vis (CH2Cl2), lmax: 234 nm (66,639) (dm3
molꢀ1cmꢀ1) (intra-ligand transition); 335 (27,780), 390 (37,662)
(dm3 molꢀ1cmꢀ1) nm (LMCT); 1H NMR (CDCl3, ppm): 11.62 (s, 1H,
eOH), 4.34 (s, 5H, C5H5), 7.00e7.02 (d, 1H, J ¼ 9 Hz,- CH]N), 9.71 (s,
1H, NHC2H5), 3.09e3.13 (q, 2H,J ¼ 7 Hz,-NCH2e), 1.39e1.42 (t,
3H,J ¼ 7.5 Hz, eCH3), 1.04e1.07 (t, 3H,J ¼ 7 Hz, terminal CH3),
3.71e3.77 (p, 2H, J ¼ 7 Hz, terminal CH2) 6.05e7.89 (m, aromatic
protons);13C NMR (CDCl3, ppm): 167.41(CꢀS), 162.67 (C]N),83.35
(Cp),136.31 (CeO), 142.84 (CeN), 119.60e133.95 (aromatic PPh3);
31P NMR (CDCl3 ppm): 29.36 (s).
5.2.2. Synthesis of [RuCp(DeaSal-mtsc)(PPh3)] (2)
Complex 2 was prepared by the procedure as described for (1)
with 4(N,N0)-diethylaminosalicylaldehyde-4(N)-methylthiosemica-
rbazone [H2-DeaSal-mtsc] (0.019 g, 0.0688 mmol). Orange red solid
formed was filtered, washed with petroleum ether (60e80 ꢁC) and
recrystallized from dichloromethane and hexane. Yield: 64%. Mp:
118 ꢁC. Anal. Calcd for C36H39N4OPRuS: C, 61.09; H, 5.55; N, 7.92; S,
4.53. Found: C, 61.12; H, 5.50; N, 7.89; S, 4.50%. FT-IR (cmꢀ1) in KBr:
3432(ʋO-H), 1562 (ʋC]N), 1273 (ʋCeO), 749 (ʋCeS), 1422, 1022,
695 cmꢀ1 (for PPh3); UV-Vis (CH2Cl2), lmax: 233 (62,223) nm (dm3
5.2.4. Synthesis of [RuCp(DeaSal-ptsc)(PPh3)] (4)
Complex 4 was prepared by the procedure as described for (1)
with 4(N,N0)-diethylamino salicylaldehyde-4(N)-phenylthiosemic-
arbazone [H2-DeaSal-ptsc] (0.020 g, 0.0688 mmol). Orange solid
formed was filtered, washed with petroleum ether (60e80 ꢁC) and
recrystallized from dichloromethane and hexane. Yield: 68%. Mp.
125 ꢁC. Anal. calcd for C41H41N4OPRuS: C, 63.96; H, 5.37; N, 7.28; S,
4.16. Found: C, 64.01; H, 5.35; N, 7.22; S, 4.13%. FT-IR (cmꢀ1) in KBr:
3426(ʋO-H), 1501 (ʋC]N), 1228 (ʋCeO), 745 (ʋCeS), 1425, 1085, 691
cmꢀ1 (for PPh3); UV-Vis (CH2Cl2), lmax: 234, 281 nm (dm3
molꢀ1cmꢀ1
)
(intra-ligand transition); 383 (28,668) nm (dm3
molꢀ1cmꢀ1) (LMCT); 1H NMR (CDCl3, ppm): 11.62 (s, 1H -OH), 4.34
(s, 5H, C5H5), 7.88 (s, 1H, - CH]N), 9.60 (s, 1H, terminal NH),
3.09e3.14 (q, 2H,J ¼ 7.5 Hz,-CH2N-), 1.39e1.42 (t, 3H,J ¼ 7.5 Hz,-
CH3), 2.78e2.79 (d, 3H,J ¼ 5 Hz, terminal CH3), 6.02e7.88 (m, aro-
matic protons;13C NMR (CDCl3, ppm): 169.40(CꢀS), 162.16 (C]N),
80.19 (Cp), 145.19 (CeO), 139.56 (CeN), 128.28e133.31 (aromatic
PPh3); 31P NMR (CDCl3 ppm): 29.44 (s).
molꢀ1cmꢀ1) (intra-ligand transition); 403 nm (dm3 molꢀ1cmꢀ1
)
(MLCT); 1H NMR (CDCl3, ppm): 11.62 (s, 1H, eOH), 4.30 (s, 5H,
C5H5), 8.00 (1H, s, - CH]N), 9.48 (s, 1H, eNHPh), 3.09e3.13 (q, 2H,
J ¼ 7 Hz, eCH2e), 1.39e1.42 (t, 3H,J ¼ 7.5 Hz, eCH3), 6.19e7.61 (m,
aromatic protons);13C NMR (CDCl3, ppm): 165.21(CꢀS), 159.94 (C]
N), 79.91 (Cp),144.87 (CeO), 159.02 (CeN), 127.82e134.40 (aro-
matic PPh3);31P NMR (CDCl3 ppm): 29.40 (s).
5.2.3. Synthesis of [RuCp(DeaSal-etsc)(PPh3)] (3)
6. Biological study
Complex 3 was prepared by the procedure as described for (1)
with 4(N,N0)-diethylamino salicylaldehyde-4(N)-ethylthiosemic-
arbazone [H2-DeaSal-etsc] (0.019 g, 0.0688 mmol). Orange red solid
formed was filtered, washed with petroleum ether (60e80 ꢁC) and
recrystallized from dichloromethane and hexane. Yield: 66%. Mp.
151 ꢁC. Anal. calcd for C37H41N4OPRuS: C, 61.56; H, 5.72; N, 7.76; S,
6.1. DNA binding study
The DNA binding experiments of the compounds with CT DNA
were carried out according to the standard literature procedure
[40].

22
G. Devagi et al. / Journal of Organometallic Chemistry 838 (2017) 12e23
6.2. DNA cleavage study
1(cc546) I; dvls27 and E. coli OP50 were obtained from Caeno-
rhabditis Genetic Centre (CGC, University of Minnesota, Minneap-
olis, MN, USA). For all the experiments, age synchronized
populations were obtained by treating the gravid adult animals
with 5% NaOCl and 5 N NaOH.
The cleavage of DNA was checked using agarose gel electro-
phoresis. Supercoiled pBR322 DNA (100 ng) in 5% DMSO and 95%
Tris buffer (5 mM, pH 7.2) with 50 mM NaCl was incubated at 37 ꢁC
in the absence and presence of compounds (50
mM). The DNA,
compound, and sufficient buffer were premixed in a vial, and the
reaction was allowed to proceed for 2 h at 37 ꢁC before the addition
of ethylene glycol and loading onto an agarose gel. Agarose gel
electrophoresis of plasmid DNA was performed at 50 V in 1% slab
6.7. Toxicity testing assay
Toxicity assay was performed to eliminate the toxic concentra-
tion of worms as described previously [51]. In brief, age sorted late
L4 stage wild type worms (n ¼ 30) were treated with complex
gels containing 0.5 m
g mLꢀ1ethidium bromide in Tris buffer. DNA
was visualized by photographing the fluorescence of intercalated
ethidium bromide under a UV illuminator. The cleavage efficiency
was measured by determining the ability of the compounds to
convert the supercoiled (SC) DNA to the nicked circular (NC) form
and linear circular (LC) form. After that the gel was documented on
a Digital Gel Documentation system unit (Syngene, USA).
1e4 at five test concentrations (10, 30, 50, 100 and 200
m
M) in K-
medium previously seeded with E. coli OP50 (6
mg/ml) as a sole food
source and maintained at 20 ꢁC for 24 h. Afterwards, the worms
observed for inactivity under stereo zoom microscope. Both 100
and 200 mM test concentrations were found to be toxic to worms,
hence we excluded these concentrations for further experiments.
6.3. Competitive binding with ethidium bromide
6.8. Oxidative stress tolerance
To observe the mode of attachment of CT DNA to the complexes
fluorescence quenching experiments of EB-DNA were carried out
by adding 0e50 mM compounds containing 10 mM EB, 10 mM DNA
and tris-buffer (pH ¼ 7.2). Before measurements, the system was
shooked and incubated at room temperature for approximately
5 min. The emission was recorded at 530e750 nm. The quenching
extents of complexes were calculated qualitatively by employing
SterneVolmer equation (5).
For oxidative stress tolerance assay, age synchronized N2 worms
were grown from L1 to L4 stage on the nematode growth medium
(NGM) supplemented with complex 1e4 or 0.3% DMSO as a solvent
control. At late L4 stage (6th day adulthood), the worms were
subsequently transferred to new NGM plates consists of 240 mM
juglone, an intracellular ROS generator for 5 h. After the exposure
period the survival rate of the worms were confirmed by pharyn-
geal pumping and gentle physical touch with loop. Three inde-
pendent biological replicates were performed.
I0=I ¼ Ksv½Qꢂ þ 1
(5)
6.9. Lifespan assay
where I0 is the emission intensity in the absence of compound, I is
the emission intensity in the presence of compound, Ksv is the
quenching constant, and [Q] is the concentration of the compound.
The Ksv values have been obtained as a slope from the plot of I0/Ivs
[Q].
Lifespan assay was conducted at 20 ꢁC with mitochondrial
mutant strain TK-22 (mev-1) which is sensitive to oxidative stress
due to the over production of ROS. Synchronized L1 stage worms
were placed on the NGM plates seeded with E. coli OP50 contains
three concentrations (10, 30 and 50
mM) of complexes 1e4 and 0.3%
6.4. Protein binding study
DMSO as control. 50
m
M of 5-Fluoro-20deoxyuridine (FUdR) was
also added to NGM plates to prevent the progeny development
until the reproduction becomes ceased. Then the worms were
counted and transferred to new treatment plates on every other
day until the last worm become marked as dead or censored. The
mean lifespan was calculated as the average number of days the
worms survived in each test concentrations. The statistical signif-
icance of the survival curve were processed using KaplaneMeir
survival analysis (log rank test) in Med Calc software version 14.8.
The protein binding study of complexes with BSA was per-
formed according to the standard literature method [40].
6.5. Antioxidant study
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
activity of the compounds was measured according to the Braca
method [71]. Different concentration of complexes (0.5e2.5 mg/ml)
were prepared and subjected to antioxidant tests. Briefly 0.1 ml of
complexes from final concentration of the solution was added with
0.9 ml distilled water and 3 ml of 0.1 mM DPPH in methanol to
made for 4 ml, followed by incubation at 27 ꢁC, for 30 min following
the absorbance were determined at 517 nm. The percentage inhi-
bition activity was calculated according to the formula [(A0eA1)/
A0] ꢃ100, where A0 is the absorbance of the control, and A1 is the
absorbance of the extract/standard. Inhibition activity and IC50
values were obtained. Ascorbic acid was used for positive control
and methanol used as negative control. The mean values were
obtained from three independent experiments.
6.10. DAF-16 nuclear localization and SOD-3 expression
The nuclear localization of DAF-16/FoxO transcription factor and
SOD-3 expression was visualized using transgenic reporter strain
TJ356 and CF1553 which express DAF-16::GFP and SOD-3::GFP
fusion protein that is an ideal tool to assess the activity of com-
plexes respectively. Late L4 stage worms of both strains were
cultured on NGM plates contains complex 4 at 50
as a negative control for 4 days. A short thermal stress at 37 ꢁC for
15 min and 40 M juglone served as a positive control for TJ356 and
mM or 0.3% DMSO
m
CF1553 worms correspondingly. After that the worms were anes-
thetized with 30 mM sodium azide and mounted on agar pads. The
DAF-16 nuclear localization and SOD-3 expression was visualized
under Olympus fluorescence microscope adopted with Nikon
camera. The fluorescence intensity of photo micrographs was
measured by determining the mean pixel intensity using Image J
software.
6.6. Strains and maintenance
Nematode culture and maintenance was performed according
to the previously described method [72]. Strains used for this study
viz., Bristol N2, wild type; TK22,mev-1 (kn1); CF1553, muIs84 (sod-
3p::GFP þ rol-6); TJ356, zIs356 (daf-16::GFP þ rol-6); CL4176, smg-

G. Devagi et al. / Journal of Organometallic Chemistry 838 (2017) 12e23
23
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Acknowledgments
The author G. D. sincerely acknowledges Bharathiar University
for University Research Fellowship No: C2/1531/2013 Dated
(26.09.2013). The author P.K. gratefully acknowledges Department
of Science and Technology (DST-SERB), New Delhi, India (No. SB/FT/
CS-056-/2014 dated 12.08.2015) for the financial support and Dr.
MuthuTamizh, Assistant Professor, Department of Chemistry, SRM
University is acknowledged for his help in NMR studies. The au-
thors acknowledge for some strains that were provided by the CGC,
USA which is funded by NIH Office of Research Infrastructure Pro-
grams (P40 OD010440).
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
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