Ruthenium(II) carbonyl complexes containing thiourea ligand: Enhancing the biological assets through biomolecules interaction and enzyme mimetic activities

Article abstract of DOI:10.1007/s00706-019-2357-5

Abstract: The ligand H₂L (N-(N,N-diethylaminothiocarbonyl)benzimidoylchloride-2-aminoacetophenone-N-methylthiosemicarbazone) reacts with ruthenium(II) building blocks [RuHCl(CO)(EPh₃)₃] (E = P or As) to form new complexes

Full text of DOI:10.1007/s00706-019-2357-5

Monatshefte für Chemie - Chemical Monthly (2019) 150:1059–1071
https://doi.org/10.1007/s00706-019-2357-5
ORIGINAL PAPER
Ruthenium(II) carbonyl complexes containing thiourea ligand:
Enhancing the biological assets through biomolecules interaction
and enzyme mimetic activities
1
1
1
1
Paranthaman Vijayan · Subbarayan Vijayapritha · Chidambaram Ruba · Periasamy Viswanathamurthi ·
Wolfgang Linert2
Received: 25 June 2018 / Accepted: 8 January 2019 / Published online: 20 May 2019
©
The Author(s) 2019
Abstract
The ligand H L (N-(N,N-diethylaminothiocarbonyl)benzimidoylchloride-2-aminoacetophenone-N-methylthiosemicarbazone)
2
reacts with ruthenium(II) building blocks [RuHCl(CO)(EPh ) ] (E=P or As) to form new complexes [Ru(1,1-DT)(Cl )(CO)-
3
3
2
(
EPh ) ] (E=P or As; 1,1-DT=1,1-diethylthiourea). The ligand H L in these reactions undergoes C=N bond break and coor-
3
2
2
dinates through sulfur atom of C=S group. Analytical and spectral (IR, UV–Vis, NMR, ESI-MS) methods were used to char-
acterize the compounds. A distorted octahedral geometry for complexes has been furnished by X-ray crystallography, which
conꢀrmed the coordination mode of the ligand with metal precursor. The binding aꢁnity and mode of binding of the complex
towards some important biomolecules such as calf thymus DNA and bovine serum albumin protein were determined using
absorption and emission spectra and found intercalative binding with calf thymus DNA and static interaction with bovine serum
albumin. The in vitro cytotoxic activity of complex was assessed using human cervical cancer (HeLa), human hepatocellular
carcinoma (HepG2), and normal Vero cells. Furthermore, the complex was found to possess signiꢀcant enzyme mimic catalytic
activity in oxidation and hydrolysis reactions.
Graphical abstract
Keywords Thiourea · Ruthenium(II) complex · DNA/BSA binding · Enzyme kinetics
Introduction
In recent years, attention has been given for metallic
drug–DNA interaction in inorganic pharmaceutical research.
*
*
Periasamy Viswanathamurthi
viswanathamurthi@gmail.com
Due to the tunable coordination properties of the inorganic
drugs, the interaction of transition metal complexes with
molecular target DNA is of interest for understanding their
cytotoxic activities [1–3]. These compounds can bind with
DNA duplex through non-covalent interactions, viz inter-
calation between the bases, surface groove-binding, and
Wolfgang Linert
wlinert@mail.zserv.tuwien.ac.at
1
2
Department of Chemistry, Periyar University, Salem, India
Institute of Applied Synthetic Chemistry, Vienna University
of Technology, Vienna, Austria
Vol.:(0
1
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P. Vijayan et al.
electrostatic attraction with anionic sugar–phosphate back-
bones [4]. Based on the interaction of biomolecules and
metal complexes, search for new metal-based drugs for
cancer treatment has led to an increasing signiꢀcance for
the cytotoxic properties of metal complexes and their mecha-
nisms of action [5, 6]. One of the great accomplishments in
the domain of medicinal inorganic chemistry is the intro-
duction of cis-diamminedichloroplatinum(II) (cis-platin) and
its successors in the treatment of cancer. However, few of
their severe side eꢂects such as renal toxicity, neurotoxic-
ity, myelosuppression, immunosuppression, severe nausea,
vomiting, and ototoxicity impelled inorganic chemists to
explore new anticancer drugs based on transition metal com-
plexes [7]. Among the various transition metal complexes
scrutinized, the ruthenium complexes are the most favora-
ble alternatives in this regard due to their ease of synthesis,
advanced chemical and air stability, solubility, rich redox
chemistry, structural diversity, and kinetic aspects akin to
platinum [8, 9]. Presently, few ruthenium complexes were
entered in clinical trials as anticancer agents [10].
oxidation of a wide range of o-diphenols (catechol) to the
corresponding o-quinones [12, 13], a process known as
catecholase activity. Similarly, hydrolytic reactions, i.e.,
hydrolysis of phosphodiester bond (phosphodiester cleav-
age), a process called phosphatase activity, also involves
enzyme catalysis. Many research groups are working
towards synthesis of enzymes with diꢂerent materials
including metal complexes to imitate the biological func-
tions of above natural enzymes. Recently, our group has
reported metal complexes with various ligands [14], which
mimic catecholase and phosphatase activities.
Generally, the nature of ligands and chelation decides
the properties of the metal complexes. Nowadays, inter-
est is being given to develop thiourea-based ligands that
support highly reactive transition metal complexes. N,N-
[(Dialkylamino)(thiocarbonyl)]benzamidine has been
observed as a diverse selection of multifunctional thiourea
scaꢂolds that can support metal ions by providing a wide
variety of diꢂerent steric and electronic environments [15,
16]. A new class of tridentate thiocarbanoylbenzamidines
containing an additional thiosemicarbazone moiety and their
rhenium complexes have been reported recently together
with the biological activity of the compounds [17].
On the other hand, over the past few decades, atten-
tion has been made to artiꢀcial enzymes, which mimic the
structural and functional aspects of naturally occurring
enzymes, for utilizing oxidation of organic substrates in
the arena of industrial and synthetic processes [11]. In this
direction, many catalysts having bio-mimicking activity
for various enzymes have been synthesized by chemists.
Such artiꢀcial enzymes have the same catalytic function,
but they are more stable, cost-eꢁcient, and structurally
less complex than their natural counterparts. One of the
prominent natural catecholase enzymes, a less well-known
member of the type-III copper proteins that originate
in plant tissues and crustaceans, plays a key role in the
Motivated on the above contemplations and with the
aim to study transition metal complex interaction on bio-
molecules, we have synthesized ruthenium(II) complexes
containing thiourea ligands (Scheme 1). The interaction
studies of ruthenium(II) complex have been performed on
selected biomolecules such as calf thymus DNA (CT-DNA)
and bovine serum albumin (BSA) to determine the binding
mode. The cytotoxic as well as enzyme mimic activities of
the complex were also done to understand the biological
reactivity preferences.
Scheme 1
1
3

Ruthenium(II) carbonyl complexes containing thiourea ligand: Enhancing the biological assets…
1061
1
Results and discussion
The H NMR spectrum of ligand H2L shows three dif-
ferent NH resonances at 8.10, 8.47, and 12.72 ppm. But in
1 and 2, the peaks for NH groups were completely disap-
peared and a new peak appeared around 8.17–8.21 ppm
for –NH2 due to ligand C=N bond cleavage. The four
well separated multiplet resonances at 3.52–3.94 ppm
have been assigned to two methylene groups in ligand
and its complexes 1 and 2. Furthermore, appearance of
two triplet signals region at 1.15–1.62 ppm were assigned
to N(CH2CH3)2 methyl groups in the spectra of ligand
and its complexes [21, 22]. In addition, signals for aro-
Synthesis and characterization
The ligand H L was obtained by simple condensation
2
using our previously reported methodology [18] with
crystalline purity and suꢁcient yield to use without fur-
ther puriꢀcation for the complex synthesis. Successively,
ruthenium(II) complexes 1, 2 were synthesized in good
yields by the reaction of H L and stable prefabricated
2
ruthenium(II) precursors [RuHCl(CO)(EPh ) )] (E = P
3
3
1
3
or As) in CH OH/CHCl solvent mixture as shown in
matic protons appear in the expected region. In the
C
3
3
Scheme 1. Interestingly, the ligand undergoes hydrolytic
C=N bond cleavage and coordinates monodentate mode
via sulfur of C=S group with ruthenium metal.
NMR spectrum of uncoordinated ligand H2L, the C=N
and C=S signals appear around 149.36–155.41 and
187.08–189.61 ppm, respectively [23]. In the spectra of
Ru(II) complexes, the signals of the C=N carbon disap-
peared and C=S carbon signals shifted to upꢀeld region
(169.19–171.69 ppm). These are consistent with the sulfur
donor coordination for complexes 1 and 2 and break of
C=N bond in benzamidine ligands. The carbonyl carbon
resonates at 203.01–204.12 ppm, was comparable with
the literature reports. In addition, peaks for CH and CH
The new complexes are crystalline solids, non-hygro-
scopic, air-stable in solution and solid state at room tem-
perature. The complexes were characterized satisfactorily
by analytical, spectral (IR, UV–Vis, NMR, and ESI-MS)
as well as single crystal X-ray crystallography studies.
2
3
Spectroscopic analysis
carbon were observed region at 13.21–32.61 ppm. Fur-
thermore, the aromatic carbons in the complexes exhibit
3
1
The typical IR bands of the new ruthenium(II) complexes
were compared with those of the free ligand to ꢀnd the
coordination mode of ligand in complexes. The spectrum
of ligand shows three peaks in the range of 3236–3302/
cm due to νNH groups, whereas in the spectra of com-
plexes the νNH peaks disappeared indicates deprotona-
tion of these groups in ligand on complexation. However,
in complexes 1 and 2 a new broad band was observed
peaks in the region of 127.62–138.83 ppm. The P NMR
spectrum of complex 1 show a sharp singlet at 34.83 ppm
indicates the coordination of two magnetically equivalent
triphenylphosphine co ligands in trans position [24]. The
mass spectral analysis of complexes 1, 2 reveals a sig-
nal that corresponds to a molecular ion at m/z = 821.32,
+
909.22, respectively, which is assigned to [M−Cl] with
an experimental isotope pattern that matches the calcu-
lated values.
in the region at 3379–3381/cm specified to new NH
2
group formed due to C=N bond break in the benzamidine
ligands [19]. A sharp band appeared range at 820–843/cm
in the spectrum of ligand was assigned to ν C=S group
which shifted to lower wave number in complexes 1 and
X‑ray crystallographic analysis
X-ray crystallographic analysis of 1 and 2 also conꢀrms
the metal-induced hydrolytic C=N bond break in H2L and
formation of unpredicted complexes 1 and 2. The crystal
structure and parameters of complex 1 are similar to the
previous report of our group in which the ligand under-
goes similar hydrolytic cleavage [25]. Hence, here we
discuss only the crystal structure of complex 2 in detail
(Figs. 1, 2; Table 1). Similar to complex 1, complex 2
also displays an octahedral geometry. The coordination
sites are ꢀlled up by a C=S sulfur atom, a carbonyl group,
two chlorine atom and two triphenylarsine trans to each
other. The ruthenium atom is in a distorted octahedral
environment with trans angles of [As(2)–Ru(1)–As(1)]
176.61(4)° and [Cl(1)–Ru(1)–S(1)] 168.64(8)°. The other
two axial sites are occupied by a carbonyl group and one
chlorine atom with Ru(1)–C(1) and Ru(1)–Cl(1) distance
of 1.803(10) and 2.573(2) Å. The carbonyl group trans to
2
upon complex formation. The shift of ν C=S by 20–30
unit in complexes indicates that the ligand is coordinated
to the ruthenium(II) center via the sulfur atom. In addi-
tion, the ruthenium(II) complexes show strong vibra-
tions in the anticipated region conform the presence of
triphenylphosphine and triphenylarsine. The electronic
spectra of ligand and complexes show two–four bands in
the region 237–443 nm. The bands in the 237–272 nm
regions can be assigned to π → π* and n → π* transitions
[
20]. The absorption maxima located in the 332–339 nm
2
+
range were assigned to S(pπ) → M(dπ) (M = Ru ) LMCT
transition for complexes 1 and 2. Furthermore, a broad
band at 438–443 nm region in the spectra of complexes
have been assigned to d–d transition band of spin-paired
6
d species with a distorted octahedral structure.
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3

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P. Vijayan et al.
Table 1 Crystal data and structure reꢀnement for complex 2
2
Empirical formula
Formula weight
Temperature/K
Wavelength/Å
Crystal system
Space group
Unit cell dimensions
a/Å
C H As Cl N ORuS
4
2
42
2
2
2
944.64
293(2)
0.71073
Monoclinic
P 1 21/c 1
12.9337(7)
18.3880(8)
17.7344(7)
90
b/Å
c/Å
α/°
β/°
γ/°
Volume/Å
Z
Density (calcd)/Mg/M
Absorption coeꢁcient
F(000)
102.314(5)
90
4120.7(3)
4
1.523
2.189/mm
1904
3
Fig. 1 Perspective view (25% probability ellipsoids) of complex 2
3
with the atom numbering scheme. Phenyl rings showed as wire frame
ɵ Range for data collection/°
Index ranges
3.313–29.434
− 17≪ h≪ 17
−
−
18≪ k≪ 25
23≪ l≪ 18
Reꢃections collected
22,883
Independent reꢃections
Data/restraints/parameters
Goodness-of-ꢀt on F
Final R indices [I>2σ(I)]
9837 [R(int)=0.0613]
9837/0/462
1.011
2
R1=0.0843,
wR2=0.2185
R indices (all data)
R1=0.1791, wR2=0.2882
DNA interaction studies
Fig. 2 Weak hydrogen bonding interactions between Cl and NH atom
in complex 2
Fluorescence emissive titration
Coordination compound and DNA interaction is one of the
criterions for comprehending the molecular basis of therapeu-
tic activity or advanced clinical trials. Generally, molecules/
complexes bind non-covalently (intercalation, groove-binding
and external electrostatic mode) with DNA. The metal ion,
ligands and geometry of the complexes aꢂect the binding
eꢁciency [27]. As a basic testing method, emissive titra-
tion study is unanimously employed method to ꢀnd out the
binding mode of metal complexes with DNA which usually
involves the changes in emissive intensity and wavelength.
Emissive titration experiments were done by aliquot addi-
tion of DNA to the test compound and observe the changes in
emission intensity. The intercalative mode of aꢁnity between
metal complexes and DNA results in hypochromism with or
without red/blue shift; on the other hand, non-intercalative/
the coordinated Cl(2) atom [Cl(2)–Ru(1)–C(1)] with an
angle of 176.3(3)°. This is due to strong Ru(II) →CO back
donation as showed by the short Ru(1) − C(1) [1.803(10)
Å] bond and low CO stretching frequency (1952/cm),
which prefers σ or π weak donor groups occupying the site
opposite to CO to favor the d–π back bonding donation.
The second chloride ion attached to the ruthenium ion can
be furnished by chloroform solvent [25]. The metal ligand
bond distances (Table 2) in the complex 2 agree well with
previous reports containing similar coordination environ-
ment [26]. Moreover, the complex 2 exhibited weak hydro-
gen bonding interactions between Cl and NH atom.
1
3

Ruthenium(II) carbonyl complexes containing thiourea ligand: Enhancing the biological assets…
1063
Table 2 Selected bond lengths (Å) and bond angles (°) for complex 2
Table 3 Fluorescence spectral parameters of ruthenium complexes
bound with CT-DNA
2
Complex
Kb
Ksv
Kapp
C(1)–Ru(1)
As(2)–Ru(1)
As(1)–Ru(1)
S(1)–Ru(1)
Cl(1)–Ru(1)
Cl(2)–Ru(1)
As(1)–Ru(1)–Cl(1)
As(2)–Ru(1)–As(1)
As(2)–Ru(1)–Cl(1)
Cl(2)–Ru(1)–As(1)
Cl(2)–Ru(1)–As(2)
Cl(2)–Ru(1)–Cl(1)
S(1)–Ru(1)–As(1)
S(1)–Ru(1)–As(2)
S(1)–Ru(1)–Cl(1)
S(1)–Ru(1)–Cl(2)
C(1)–Ru(1)–As(1)
C(1)–Ru(1)–As(2)
1.803(10)
2.4750(12)
2.4844(11)
2.4096(16)
2.573(2)
2.466(2)
87.34(5)
176.61(4)
91.54(5)
88.14(8)
88.66(8)
90.29(8)
89.56(8)
92.14(8)
168.64(8)
100.53(8)
91.4(4)
1a
2
3.98×10
1.06×10
4
6
1.19×10
3.80×10
4
4
6.30×105
1.97×105
aData referred from Ref. [25]
[
29]. However, some more experiments require for proving the
binding mode.
Fluorescence quenching study
To gain support for the type of binding of the complex with
CT-DNA, the ꢃuorescence quenching studies were performed
using metal complex as a quencher. Ethidium bromide (EB) is
commonly used as sensitive ꢃuorescent and emits enhanced
emissive property in the presence of DNA [30]. From the
above info in mind, the quenching of EB bound DNA is cal-
culated with the successive addition of metal complex 2. The
emission band at 686 nm for EB displays hypochromism up
to 15.52% with a small red shift from the initial ꢃuorescence
intensity (Fig. 4). From this evidence, it is concluded that EB is
being released from EB-DNA compound due to the reduction
of the corresponding ruthenium complex. The quenching con-
91.7(4)
electrostatic interaction causes hyperchromism [28]. The
ꢃuorescence emission titration curves (Fig. 3) were used to
study the binding mode and propensity of complex 2 with
CT-DNA in Tris–HCl buꢂer (5 mM Tris–HCl/50 mM NaCl
buꢂer for pH=7.2) at 25 °C. Upon increasing concentra-
tion of CT-DNA, the emission spectral bands of complex 2
at 323 nm exhibit hypochromism of about 21.23% with very
small slight red shifts of 0.5 nm. The marked decreases in the
ꢃuorescence intensity indicate the intercalative binding mode
stant (K ) value was calculated using classical Stern–Volmer
sv
equation according to the previous literature [31], which was
listed in Table 3. In addition, the following equation is used to
calculate the apparent DNA-binding constant (K ) values:
app
ꢀ
ꢁ,
KEB[EB] = Kapp M50%
of complex 2 and DNA. The calculated binding constant (K )
b
−
7
where K =1.0×10 /M is the DNA-binding constant of
value (Table 3) is comparable to similar type of previously
EB
EB, [EB] is the concentration of EB (7.5 μM) and [M
]
reported complex [25] and also supports the intercalative mode
50%
Fig. 3 Emission titrations of complex 2 (25 µM) with CT-DNA (0–50 µM) and Scatchard plots of r/Cf vs r for complex 2
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P. Vijayan et al.
Fig. 4 Emission titrations of complex 2 (0–50 µM) with EB bound CT-DNA (7.5 µM) and Scatchard plots of I /I vs [Q] for complex 2
0
is the concentration of the compound used to obtain a 50%
reduction in ꢃuorescence intensity of DNA pretreated with
EB. The K and K values are found to be comparable
1.1
.09
1
sv
app
1.08
with our previous reported ruthenium(II) complex [25]. The
results support the intercalative binding mode of complex 2
with DNA and the results are consistent with the previous
emissive titrations.
1
1
1
1
1
1
1
.07
.06
.05
.04
.03
.02
.01
1
Viscosity measurements
To clarify the binding mode of the Ru(II) complex with
DNA, the viscosity measurements were carried out on CT-
DNA by varying the concentration of added complex. The
experiment involves the measurement of the ꢃow rate of
DNA solution through a capillary viscometer. For a small
molecule that binds by non-classical intercalation, causes
less pronounced or no change in the viscosity of DNA [32].
On the other hand, a classical intercalation mode into DNA
causes a signiꢀcant increase in the viscosity of DNA solu-
tion due to increase in the overall DNA molecular length.
-
0.01
0.01
0.03
0.05
0.07
0.09
0.11
0.13
r = [Compound] / [DNA]
0
1/3
Fig. 5 Relative viscosity (η/η ) of CT-DNA in Tris–HCl buꢂer
solution in the presence of complex 2 at increasing amounts
(r=0–0.12)
0
1/3
The plot of (η/η ) vs [complex]/[DNA] gives a measure
of the viscosity changes (Fig. 5). The obtained plot shows
increase in the relative viscosity, which clearly indicates
intercalate binding mode for complexes. Therefore, we can
conclude that the complex 2 binds to CT-DNA in the inter-
calate regions, which is in accordance with ꢃuorescence
spectral results.
wave length 290–430 nm (λ =280 nm) and their emission
ex
spectra recorded. The ꢃuorescence spectrum of BSA exhib-
its a broad band with a maximum at~340 nm. The outcome
of increasing concentration of 2 on the emission spectra of
BSA at room temperature is shown in Fig. 6a. The value
of Stern–Volmer constant (K ) calculated from following
q
Stern–Volmer equation [33] is used to express the amount
of quenching. Quenching constant (K ) was found from the
q
Protein binding studies
plot of log (I − I)/I vs log[Q] (Fig. 6a) which is much higher
0
than the molecular ꢃuorescence diꢂusing constant as shown
in Table 4. The result reveals that the ꢃuorescence quench-
ing mechanism of the compound-BSA system will not be
dynamic quenching. It may be static quenching. However,
The emission studies have also been used to explore the
interaction of complex 2 with BSA. BSA (1 μM) was titrated
with various increasing concentrations of 2 (0–50 μM) in the
1
3

Ruthenium(II) carbonyl complexes containing thiourea ligand: Enhancing the biological assets…
1065
Fig. 6 Emission titrations of complex 2 (0–50 μM) with BSA (1 μM)
tra of the complex 2 with BSA [dashed red line—BSA alone; green
line—BSA+complex 2] (lower B)
and Stern–Volmer plots of (I − I)/I vs. [Q] and Scatchard plots of
0
log [(I − I)/I] vs. log [Q] for complex 2 (upper A). Absorbance spec-
0
this conclusion needs to be conꢀrmed through other experi-
ments. The binding constant K was calculated using the
b
Scatchard equation:
Table 4 Quenching parameters of ruthenium complexes bound with
BSA
ꢀ
ꢁ
log (I − I)∕I = log K + n log [Q],
0
b
Complex
Ksv
Kq
Kbin
n
where K is the binding constant of the complex with BSA
b
1a
2
1.18×10
3.62×10
5
4
1.18×10
3.62×10
13
12
1.03×10
1.12×10
10
10
2.0
1.3
and n is the number of binding sites. The binding constant
(
K ) has been obtained as per Fig. 6a and its value is given in
b
Table 4 along with n. The value of n indicates the availability
aData referred from Ref. [25]
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3

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P. Vijayan et al.
Table 5 In vitro cytotoxicity of ruthenium complexes in normal and
Scheme 2
cancer cell lines
3
a
Complex
IC /µg/cm
50
Vero
HeLa
HepG2
1c
50.60± 0.85
55.12± 0.47
ND
28.69± 1.18
20.16± 1.10
12.29± 1.27
33.65± 1.75
25.39± 0.83
11.78± 1.52
2
b
Cis-platin
a
5
0% inhibitory concentration after exposure for 24 h in the MTT
shows marginally less activity than cis-platin and compara-
ble activity with previously reported ruthenium complexes
[10, 23, 25, 26]. It is noteworthy that the complex 2 is less
toxic towards the normal Vero cell line as is evident from
the IC values.
assay
b
c
No data
Data referred from Ref. [25]
5
0
of one binding site in BSA for complex 2. The higher val-
ues of K and K signify a strong interaction between the
Enzyme mimicking studies
q
b
BSA proteins. The quenching parameters for complex 2
are comparable to similar type of previously reported com-
plex [25]. Furthermore, the static quenching ꢃuorescence
mechanism of the compound is proved by UV–Vis absorp-
tion spectral studies. Generally, quenching happens either
by dynamic or static ways. When the ꢃuorophore and the
quencher come into contact during the transient existence
of the excited state, dynamic quenching occurs [34]. On
the other hand, static quenching represents to the forma-
tion of ꢃuorophore–quencher compound in the ground state.
UV–Vis absorption spectra of BSA have been measured in
the absence and presence of 2. Figure 6b shows that addition
of 2 to a ꢀxed concentration of BSA leads to an increase in
the absorbance of the BSA absorption peak at 280 nm with
a small blue shift for free BSA, indicating that the complex
Catecholase activity
3,5-Di-tert-butylcatachol (3,5-DTBC) was used as substrate
to study the catecholase mimic activity of the complex 2.
Due to the presence of two bulky tert-butyl substituents, the
substrate shows a low quinine–catechol reduction potential
and hence eꢂortless oxidation occurs to the corresponding
o-quinone (Scheme 2).
The stable oxidation product 3,5-di-tert-butylquinone
(3,5-DTBQ) displays a maximum emission at 390 nm.
Before proceeding to a thorough kinetic study, the complex
−
4
2 (1×10 M) were examined by addition of complex 2 to
−
2
a 100 equivalents DMF solution of 3,5-DTBC (1×10 M)
under oxygen atmosphere and the progress of the reaction
was monitored for every 15 min interval up to 2 h. Owing
to addition of the catecholic substrate, a new band (Fig. 7)
appeared at 434 nm and its intensity steadily increased may
be due to ligand to metal charge transfer band of the oxi-
dized product 3,5-di-tert-butylbenzoquinone (3,5-DTBQ).
Therefore, the experiment obviously demonstrates that the
oxidation of 3,5-DTBC to 3,5-DTBQ was catalyzed by 2,
as it is well known that 3,5-DTBQ displays a maximum at
2
interact with BSA by the static quenching which could be
attributed to the formation of the ground state complex as
reported earlier [35].
In vitro cytotoxicity assay
The ruthenium complex was evaluated for its cytotoxicity
vs a pair of human tumor cell lines such as human cervical
cancer (HeLa), human hepatocellular carcinoma (HepG2),
and normal Vero cells by means of a colorimetric assay
λ
=434 nm in DMF. The catalytic performance exhibits
emis
saturation kinetics based on the Michaelis–Menten model
(
MTT assay) that measures mitochondrial enzyme succi-
appeared to be suitable under excess substrate conditions.
nate dehydrogenase activity as an indication of cell viability.
Cis-platin was used as the reference compound to evalu-
ate the cytotoxic activity. The results were analyzed by cell
viability curves and expressed with IC values of complex
Plots of k vs [3,5-DTBC] provided non-linear curve of
i
decreasing slope (Fig. 7) which are best designated kinetic
equation by our earlier report [25]. The Michaelis–Menten
constant (K ) and maximum initial rate (V ) were deter-
5
0
m
max
2
in Table 5. The amount of cell proliferation signiꢀcantly
mined by linearization using Lineweaver–Burk plots (Fig. 7).
3
decreases in various concentration ranges (10–100 µg/cm )
on supplementation with complex, as observed within 24 h
of incubation with respective of above cell lines.
The turnover number values (K ) were obtained by dividing
cat
the V values by the concentration of the complex and all
max
these parameters are listed in Table 6. The results unambigu-
ously demonstrate that the complex 2 is active towards the
oxidation of 3,5-DTBC. When compared to similar reported
complex, the activity of the present complex is slightly lower
From IC values, the complex demonstrates notable
5
0
activity in human cervical cancer (HeLa), human hepatocel-
lular carcinoma (HepG2) cell lines. However, the complex
1
3

Ruthenium(II) carbonyl complexes containing thiourea ligand: Enhancing the biological assets…
1067
Fig. 7 Oxidation of 3,5-DTBC by complex 2 monitored by emission spectroscopy and Lineweaver–Burk plot for complex 2
Table 6 Kinetic parameters for catecholase and phosphatase activity
λemis =486 nm (λ =435 nm) over 15 min break up to 2 h
ex
of ruthenium complexes
with 40 equivalents of the substrate (Scheme 3).
Vmax/M m1
Kcat/h−1
The dependence of the initial rate on the concentration of
the substrate was monitored at the respective wavelength by
emission spectroscopy, which corresponds to the increase
in 4-nitrophenolate (4-NP) concentration (Fig. 8). The rate
vs concentration of substrate data were analyzed based on
Michaelis–Menten approach of enzymatic kinetics to get the
Lineweaver–Burk (double reciprocal) plot as well as the val-
ues of variety of kinetic parameters V , K and K . Plot
Catalyst
K /M
m
a
−1
−3
3
−2
1
1
2
2
(3,5-DTBC)
(4-NPP)
9.88×10
4.26×10
2.85×10
1.01×10
1.58×10
1.23×10
988
85
374
569
a
−3
3.74×10−
5.69×10−
−3
(3,5-DTBC)
(4-NPP)
4
−4
aData referred from Ref. [25]
max
m
cat
of k vs [4-NPP] gave non-linear curve of decreasing slope
i
although it possesses appreciable catecholase mimic activ-
ity [25]. From all the data, it is concluded that the catalytic
oxidation follows the mechanism suggested by Chyn and
Urbach [36].
which is best designated by the ꢀrst-order kinetic equation.
The turnover number value (K ) was obtained by divid-
cat
ing the V value by the concentration of the complex and
max
all the parameters are listed in Table 6. The results show that
the complex 2 owns superior activity and eꢂectively catalyze
the hydrolysis with appreciable turnover rate [25]. Moreover,
the kinetic data reveal that the hydrolysis of 4-NPP followed
the mechanism described earlier [25].
Phosphatase activity
Based on the promising catecholase activity result, the com-
plex was further investigated for its phosphatase activity.
Phosphate esters play several important functions in bio-
logical systems such as information storage (DNA/RNA),
cellular signaling (cAMP), and energy transduction (ATP)
Conclusions
[
11]. The phosphodiester bonds in these molecules are
In conclusion, the ruthenium complexes 1 and 2 bearing
thiourea ligand have been synthesized and characterized by
analytical, spectral, and single crystal X-ray crystallographic
techniques. The characteristic results reveal the C=N bond
cleavage in ligands and monodentate coordination via C=S
group with the ruthenium metal ion. The results of single
crystal XRD analysis of complexes conꢀrm ligand cleav-
age, monodentate coordination mode and octahedral geom-
etry. The binding properties of the ruthenium complex with
extremely resistant towards hydrolysis because of the repul-
sion between the negatively charged backbone and poten-
tial nucleophiles. Since the compound 4-nitrophenyl phos-
phate disodium salt hexahydrate (4-NPP) ester is negatively
charged and under neutral conditions they are very resistant
to hydrolysis, was selected as substrate. The 4-NPP hydroly-
sis was monitored using complex 2 as catalyst by emissive
titrations (Fig. 8) on the time evolution of 4-NP in DMF at
1
3

1068
P. Vijayan et al.
Fig. 8 Hydrolysis of 4-NPP by the complex 2 monitored by emission spectroscopy and Lineweaver–Burk plot for complex 2
Scheme 3
III CHNS analyzer was used for elemental analysis measure-
ment. Bruker FT IR spectrophotometer equipped with an
ATR sampling accessory was employed for recording IR
spectra in the range 4000–450/cm. UV–Vis spectra were
recorded on Shimadzu UV-1650 PC spectrophotometer over
1
13
31
8
00–200 nm range. H, C, P NMR spectra were per-
formed on a Bruker Ultra Shield using DMSO-d and CDCl
6
3
as solvent. The chemical shifts are given in ppm relative to
SiMe and orthophosphoric acid. ESI-MS were recorded on
4
a Q-TOF micro™ mass spectrometer operating in ESI mode.
Buꢂer solutions were prepared using doubly distilled water.
Stock solution of complex (1 mM in DMSO) was stored
at 4 °C and used whenever required. Data were expressed
as the mean± the standard deviation from three independ-
ent experiments. The ruthenium precursors [RuHCl(CO)
(EPh ) ] (E=As or P) and the ligand (N-(N,N-diethylami-
CT-DNA were evaluated by emissive titrations, ꢃuorescence
quenching, viscosity measurements, and the results show
that the complex interacts with CT-DNA through interca-
lative mode. Furthermore, the complex BSA protein inter-
actions were examined by UV–Vis and emissive titrations
reveal that complex has shown high binding aꢁnity and acts
as static quencher. The in vitro cytotoxic study demonstrates
the substantial cytotoxicity of the complex. Moreover, the
complex was eꢂectively used for aerial oxidation of catechol
and phosphate hydrolysis. On the basis of Michaelis–Men-
ton approach of enzyme kinetics, the complex shows good
catalytic eꢁciency in catechol-oxidase functional mimic in
the conversion of 3,5-DTBC to 3,5-DTBQ and ester hydroly-
sis functional mimic in the hydrolysis of 4-NPP to 4-NP in
aerobic conditions.
3
3
nothiocarbonyl)benzimidoylchloride-2-aminoacetophenone-
N-methylthiosemicarbazone) (H L) were prepared according
2
to literature methods [18, 38].
Synthesis of complexes 1 and 2
The metal complexes were prepared as per reported proce-
dure with minor alteration [39]. [RuHCl(CO)(EPh ) ] (E=P
3
3
or As) (1 mmol) and the ligand H L (1 mmol) were dissolved
2
3
in 10 cm of methanol and chloroform solvent mixture (1:1
ratio). The solution mixture was mildly heated under reꢃux
for 12 h. The progress of the reaction was monitored by thin
layer chromatography (TLC) using 9:1 mixture of petroleum
ether–ethyl acetate mixture as mobile phase. After comple-
tion of the reaction, the resulting solution was ꢀltered and
the ꢀltrate was left untroubled for slow evaporation of the
solvent. After 5 days, brown colored crystals suitable for
X-ray diꢂraction were obtained.
Experimental
All chemicals were procured commercially and used as
received. Solvents were dried and distilled following the
literature procedures [37] and reactions carried out under
room temperature. Microanalyses were performed using an
apparatus designed by Technico Instuments, India. Vario EL
1
3

Ruthenium(II) carbonyl complexes containing thiourea ligand: Enhancing the biological assets…
1069
Carbonyldichlorido(1,1‑diethylthiourea)‑bis(triphenylphos‑
phine)ruthenium(II) (1) The analytical and spectral data are
identical to the previously reported complex [25].
recorded for complexes in the range of 300–600 nm. Titra-
tions were manually done by a micropipette for the addition
of CT-DNA and reference solution to eliminate the absorb-
ance of CT-DNA itself. Further support for the relative bind-
ing property of complexes binding to DNA via intercalation
is given through emission quenching experiments. DNA was
pretreated with ethidium bromide (EB) for 30 min. Then
the test solutions were added to the mixture of EB-DNA,
and the change in the ꢃuorescence intensity was measured.
The excitation and the emission wavelength were 510 nm
and 604 nm, respectively. EB was non-emissive in buꢂer
solution (pH 7.2) due to ꢃuorescence quenching of the free
EB by solvent. In the ꢃuorescence quenching spectra, the
reduction in emission intensity measured the binding mode
of complexes to CT-DNA.
Carbonyldichlorido(1,1‑diethylthiourea)bis(triphenylarsine)‑
ruthenium(II) (2, C H As Cl N O RuS) Yield: 54%; color:
4
2
42
2
2 2 2
brown; IR (KBr):=3380 (NH ), 1939 (C≡O), 829 (C=S)/
2
1
cm; H NMR (300.13 MHz, CDCl ): δ = 8.21 (s, NH ),
3
2
7
3
1
.21–7.80 (m, aromatic), 3.11 (q, J = 7.0 Hz, 2H, CH ),
2
.02 (q, J=7.0 Hz, 2H, CH ), 1.40 (t, J=7.0 Hz, 3H, CH ),
2
3
1
3
.37 (t, J=7.0 Hz, 3H, CH ) ppm; C NMR (300.13 MHz,
3
CDCl ): δ = 202.12 (C≡O), 172.14 (C=S), 126.64 (Ar
3
C), 119.24 (Ar C), 118.71 (Ar C), 112.41 (Ar C), 46.95
(
CH ), 45.92 (CH ), 14.32 (CH ), 13.94 (CH ) ppm; UV–
2
2
3
3
Vis (CHCl ): λ
(ε) = 238 (44,200), 266 (27,000), 339
3
max
3
(
12,540), 443(5240) nm (dm /mol/cm); ESI-MS: m/z calcd.
Viscosity experiments were carried out using an Ubbe-
lodhe viscometer at a constant temperature (30.0± 0.1 °C)
in a thermostat and the relative parameters were calculated
from the relation η=(t − t )/t , where t is the observed ꢃow
+
9
44.68, found 909.22 ([M−Cl] ).
X‑ray structure determination
o
o
time of DNA-containing solution and t is the ꢃow time
o
Crystals of complexes suitable for single crystal XRD were
grown from slow evaporation of methanol–chloroform sol-
vent mixture (CCDC reference number 1584536). Suitable
single crystal of compounds with accurate dimensions was
mounted on a glass ꢀber containing epoxy cement. Crystal
data were collected on Gemini Ultra diꢂractometer. Struc-
ture solutions and reꢀnements of the compounds were done
using the programs SHELXS-14 [40]. Reꢀnement and all
further calculations were carried out using SHELXL. All
non-solvent heavy atoms were reꢀned anisotropically. All
non-solvent hydrogen atoms were idealized using the stand-
ard SHELXL idealization methods [41]. When the non-
hydrogen atoms will be reꢀned by anisotropically, using
weighted full-matrix least squares on F2. Atomic scattering
factors were incorporated in the computer programs.
of Tris–HCl/NaCl buꢂer alone. Data were presented as (η/
0
1/3
η ) vs binding ratio (r=[Compounds]/[DNA]=0.0–0.1),
where η is the viscosity of CT-DNA in the presence of the
0
compound, and η is the viscosity of CT-DNA alone.
Protein binding studies
The excitation wavelength of BSA at 280 nm and the emis-
sion at 345 nm were monitored for the protein binding stud-
ies using ꢃuorescence spectra recorded with synthesized
compounds. The excitation wavelength of BSA at 280 nm
and the quenching of the emission intensity of tryptophan
residues of BSA at 346 nm were monitored using the com-
plexes as quenchers with increasing concentrations. The
excitation and emission slit widths (each 5 nm) remained
constant for all the experiments. A scan rate of 200 nm/min
was used. Samples were carefully degassed using pure nitro-
gen gas for 15 min. Stock solution of BSA was prepared in
50 µM phosphate buꢂer (pH 7.2) and stored in the dark at
4 °C for further use. Furthermore, the type of quenching
mechanism of complexes was determined from the UV–Vis
absorption spectra in the range of 200–600 nm.
DNA‑binding studies
The binding interaction experiments of complex with CT-
DNA in double distilled water containing 95% Tris/NaCl
buꢂer (5 mM Tris–HCl/50 mM NaCl buꢂer, pH 7.2) were
carried out using a Fluoromax spectroꢃuorometer with a rec-
tangular quartz cuvette of 1 cm path length. Stock solutions
of CT-DNA used in the studies not ever exceeded 5% DMSO
In vitro anticancer activity:
3‑[4,5‑dimethylthiazol‑2‑yl]2,5‑diphenyltetrazolium
bromide (MTT) assay
(
v/v) in the ꢀnal volume. A stock solution of CT-DNA was
stored at 277 K and used after no more than 4 days. Tris–HCl
buꢂer solution was used to base line correction. The excita-
tion wavelength was ꢀxed by the emission range and attuned
before measurements. The emissive titration experiments
were performed using a ꢀxed concentration (25 µM) of test
compounds to which increments of the (0–10 µM) DNA
stock solution were added. The emission intensities were
The human cervical cancer (HeLa), human hepatocellular
carcinoma (HepG2) were obtained from National Centre
for Cell Science (NCCS), Pune and grown in Eagles Mini-
mum Essential Medium containing 10% fetal bovine serum
(FBS). The cells were maintained at 37 °C, 95% air and
1
00% relative humidiꢀed atmosphere. The cytotoxicity of
1
3

1070
P. Vijayan et al.
the investigated ruthenium(II) complex in comparison to cis-
platin was determined using the MTT assay. The MTT color-
imetric assay is based on the measurement of mitochondrial
enzyme succinate dehydrogenase activity, as an indication
of cell viability. MTT is a yellow water-soluble tetrazolium
salt. A mitochondrial enzyme in living cells, succinate dehy-
drogenase, cleaves the tetrazolium ring, converting the MTT
to an insoluble purple formazan. Therefore, the amount of
formazan produced is directly proportional to the number
respectively (the peak corresponding to the band maxima),
as a function of time.
Acknowledgements Open access funding provided by TU Wien
(
TUW). The authors thank UGC, India for ꢀnancial assistance to
Department of Chemistry, Periyar University, Salem under SAP (no.
40/20/DRS-I/2016(SAP-I)).
5
Open Access This article is distributed under the terms of the Crea-
tive Commons Attribution 4.0 International License (http://creativeco
mmons.org/licenses/by/4.0/), which permits unrestricted use, distribu-
tion, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
of viable cells. After 48 h of incubation, 15 µM of MTT
3
(
5 mg/cm ) in phosphate buꢂered saline (PBS) was added to
each well and incubated at 37 °C for 4 h. The medium with
MTT was then ꢃicked oꢂ and the formed formazan crystals
were solubilized in 100 µM of DMSO and then measured
the absorbance at 570 nm using microplate reader. One hun-
dred microliters per well of cell suspension were seeded into
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