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Synthesis and cytotoxic activities of organometallic Ru(II) diamine complexes.
Bioorganic Chemistry 99 (2020) 103793
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and cytotoxic activities of organometallic Ru(II) diamine
complexes
T
Serdar Batıkan Kavukcua, Onur Şahinb, Hafize Seda Vatanseverc,f, Feyzan Ozdal Kurtd,
⁎
Mehmet Korkmaze, Remziye Kendircic, Levent Pelita, Hayati Türkmena,
a
University of Ege, Faculty of Science, Department of Chemistry, 35100 Izmir, Turkey
University of Sinop, Scientific and Technological Research Application and Research Center, Sinop, Turkey
c
University of Manisa Celal Bayar, Faculty of Medicine, Department of Histology-Embryology, 45030 Manisa, Turkey
d
University of Manisa Celal Bayar, Faculty of Sciences and Letters, Department of Biology, 45030 Manisa, Turkey
e
University of Manisa Celal Bayar, Faculty of Medicine, Department of Medical Biology, 45030 Manisa, Turkey
f
Research Centre of Experimental Health Sciences (DESAM), Near East University, Mersin-10, Cyprus
b
ARTICLE INFO
ABSTRACT
Keywords:
Ruthenium(II) arene complex
Cancer
Cytotoxicity
Bimetallic complex
A series of mono and bimetallic ruthenium(II) arene complexes bearing diamine (Ru1-6) were prepared and fully
characterized by 1H, 13C, 19F, and 31P NMR spectroscopy and elemental analysis. The crystal structure of the
bimetallic complex (Ru5) was determined by X-ray crystallography. Monometallic analogues (Ru1-3) were
synthesized to investigate the contributions of ruthenium and the other organic groups (aren, ethylenediamine,
butyl) to the activity. The electrochemical behaviors of mono and bimetallic complexes were obtained from the
relationship between cyclic voltammetry (CV) and the biological activities of the compounds. The cytotoxic
activities of the complexes (Ru1-6) were tested against wide-scale cancer cell lines, namely HeLa, MDA-MB-231,
DU-145, LNCaP, Hep-G2, Saos-2, PC-3, and MCF-7, and normal cell lines 3T3-L1 and Vero. Diamine Ru(II) arene
complexes have unique biological characteristics and they are promising models for new anticancer drug development. MTT analysis reveals that each synthesized Ru complex showed cytotoxic activity towards the different cancer cells. In particular, three Ru complexes (Ru3, Ru5 and Ru6) showed less toxic effects on the cancer
cells than the others. These novel Ru complexes affected both cancer and normal cell lines. As they had a toxic
effect on the cells, the dosage applied should be tested before being used for in vivo applications. Cytotoxicity
tests have shown that the bimetallic complex Ru6 was effective on all cancer cells. The effect of bimetallic
enhancement on cancer cell lines, the systematic variation of the intermetallic distance and the ligand donor
properties of the mono and bimetallic complexes were explored based on the cytotoxic activity. The interaction
with FS-DNA and the stability/aquation of the complexes (Ru3 and Ru6) were investigated with 1H NMR
spectroscopy. The binding modes between the complexes (Ru3 and Ru6) and DNA were investigated via UV–Vis
spectroscopy.
1. Introduction
After the discovery of the anticancer activity of cis-platin by
Rosenborg in 1965 [1], some transition metal complexes, such as platinum and ruthenium, have been studied [2–8]. Carboplatin and oxaliplatin complexes have been the most widely used agents in chemotherapeutic treatment in recent years. The relationship between the
structure and activity of platinum-based chemotherapeutic agents was
revealed by Reedijk [9]. Because of the side effects of platinum-based
agents, interest in the other metal complexes has increased. The main
problems also include the toxicity and drug resistance of developed
⁎
platinum complexes on the cancer cells [10–12]. There are some negative effects of cis-platin, such as acute kidney problems, allergic reactions, reduced immunity to infections, gastrointestinal disorders,
hemorrhage, and hearing loss. For this reason, considerable efforts have
been made in this field to develop alternative metal-based anticancer
drugs. Ruthenium (Ru), which is the transition metal of the platinum
group that is potentially less toxic than platinum [2,13], can be found in
a range of oxidation states (II, III, and IV). The rate of ligand exchange
of metal complexes in aqueous solutions is important for the anticancer
activity. The rate of ligand exchange of ruthenium (II, III) complexes is
very similar to platinum(II) complexes [14]. Ru complexes can mimic
Corresponding author.
E-mail address: hayati.turkmen@ege.edu.tr (H. Türkmen).
https://doi.org/10.1016/j.bioorg.2020.103793
Received 5 August 2019; Received in revised form 16 March 2020; Accepted 23 March 2020
Available online 04 April 2020
0045-2068/ © 2020 Elsevier Inc. All rights reserved.
Bioorganic Chemistry 99 (2020) 103793
S.B. Kavukcu, et al.
Fig. 1. cis-platin and ruthenium complexes used in in vivo treatments.
iron binding to biological molecules [15–21], such as human serum
albumin and transferrin; therefore, some Ru compounds are highly
selective for cancer cells. The compound [RuCl3(NH3)3] synthesized by
Clarke was a pioneer for the anticancer activities of Ru complexes [22]
and also, other Ru(III) complexes such as NAMI-A and KP1019 were
able to enter the in vivo process with low toxicity [23–26]. According to
Clarke’s suggestion, the activity of Ru(III) complexes was based on the
reduction of Ru(II) complexes in vivo [27]. On the other hand, Sadler
discovered the cytotoxic activity of Ru(II) arene complexes [28] and
also half-sandwich Ru(II) arene complexes not only stood out as a result
of their catalytic properties, but also due to their anticancer activities.
Ru(II) arene complexes, which were attributed as piano stool complexes, can be classified into two main families; RAPTA ([Ru(η6-arene)
(PTA)X6] (PTA: 1,3,5-triaza-7-phosphoadamantane) [11,29] and RAED
([Ru(η6-arene)(en)Cl]+ (en: ethylenediamine) (Fig. 1) [28,30]. These
compounds include the hydrophobic arene group, a ligand containing
nitrogen or phosphorus, and halogen anion. The most important
property of Ru(II) arene complexes is their tunability. The arene group
provides a hydrophobic surface and makes Ru stable at +2 oxidation
state, which is a biologically active form. Promising results were obtained with RAED complexes against human ovarian cancer cells,
especially RAED-C. This study mainly focuses on the development of
RAED compounds for the treatment of common cancer tumors.
The interplay of coordination geometry, thermodynamics, and kinetic properties of the metal ions and the structural features of the ligands form a mononuclear or a set of well-defined polynuclear structures. To gain an insight into the effect of the basic nitrogen atom on the
diamine ligand, the number of aryl subunits on the central phenyl
scaffold and the number of metal atoms on cytotoxic activity, we synthesized a series of mono and bimetallic Ru(II) arene complexes (Ru2-6)
and characterized by 1H, 13C, 19F and 31P NMR spectroscopy and elemental analysis. Sadler et al. demonstrated that the Ru1 complex has
promising cytotoxic activity against ovarian cancer cells [31]. In this
study, we investigated the in vitro cytotoxic activities of mono and bimetallic complexes for different cancer cells. In addition, the Ru1
complex, which has notably cytotoxic activity, was used as a reference
for comparison with other complexes (Ru2-6). The cytotoxic activities of
mono and bimetallic Ru(II) complexes were investigated in ten cell
lines (HeLa, 3T3-L1, MDA-MB-231, DU-145, LNCaP, Hep-G2, Vero,
Saos-2, PC-3 and MCF-7). The chemical properties and cytotoxic activities of bimetallic Ru(II) complexes were compared with monometallic analogues.
2. Results and discussions
Sadler and co-workers reported the synthesis and cytotoxic activity
of Ru(II) p-cymene complex with o-phenylenediamine (o-pda). The opda Ru(II) complex exhibited promising cytotoxic activity against
A2780 human ovarian cancer cells, with an IC50 value of 10 μM [31].
In comparison with o-benzoquinone diimine (o-bqdi) obtained by oxidation of the o-pda, the importance of NH proton was understood by
better activity results [32]. However, the cytotoxic activity of the o-pda
in A549 human lung cancer cells was not good. The complex Ru1 has
been synthesized in order to investigate its cytotoxic activity in various
cancer cells and to compare with our mono and bimetallic complexes
due to promising results obtained in ovarian cancer cells. On the other
hand, the complex [(η6-C6H5Ru(en)Cl]+ showed anticancer efficacy
against A2870 cancer cells, with an IC50 value of 17 mM [28]. Recently, we have synthesized a series of mono-metallic Ru(II) complexes
and investigated their catalytic properties on the alpha(α)‐alkylation of
ketones with alcohols [33]. These complexes included both aryl and
butyl substituents. The aromatic ring on the complex provides a steric
effect and hydrophobic surface, while the hydrocarbon chain group
(butyl) provides solubility in oil. With these substituents, the aim was
that the complexes could easily pass through the cell wall. The purpose
of this study was to investigate the effects of the metal amount and
ligand properties (electronic, steric and NH effect and so forth.) on
cytotoxic activity (see Fig. 2).
The presence of monodentate or bidentate ligands on the complex
structure is known to affect pharmacological properties [34,35]. The
distance between metals on bimetallic complexes significantly affects
the cytotoxic activity, and the biological activity is generally enhanced
Fig. 2. Prepared complexes Ru1-3.
2
Bioorganic Chemistry 99 (2020) 103793
S.B. Kavukcu, et al.
as the amount of metal increases [36–38]. However, decreased solubility and diffusion into the cell are the main problems of the growing
molecule size. Sadler and co-workers prepared long and flexible bimetallic Ru (II) arene complexes by linking two {(η6-Bip)RuCl(en)}
units with a hexamethylene chain [39]. They revealed the importance
of the NH group and the bimetallic systems containing ethylenediamine. Also, four novel dinuclear ruthenium(II) arene complexes were
designed by Gou et al. and they demonstrated the importance of this
kind of dinuclear ruthenium(II) arene complex in drug development
[40] HYPERLINK "SPS:refid::bib41" .
Aird et al. investigated the interaction of arene Ru(II) complexes
with nucleic acids to gain further insight into the mechanism. In kinetic
studies, they examined the reaction rate of halogen with cGMP (3′,5′cyclic-GMP) according to different arene groups. As a result, the cytotoxic activity depends on the reaction rate, diamine NH2 groups, the
hydrophobic arene and the ancillary halogen. All played an important
role in the interaction with nucleic acids [41]. The dinuclear arene Ru
(II) complexes make these interactions more possible with increasing
multiple ligand-binding sites [42]. Mechanism studies showed that
DNA molecules were the main target of dinuclear ruthenium complexes
[43]. O. Novákova et al. designed a series of dinuclear arene Ru(II)
complexes with a straight-chain linker and these complexes were found
to bind to DNA by forming intrastrand and interstrand crosslinks in one
DNA molecule in the absence of proteins [44]. Keppler et al. investigated the effects of lipophilicity on cell toxicity with varying chain
lengths [36–37]. These studies showed that dinuclear arene Ru(II)
complexes have more significant biological effects than other multinuclear arene Ru(II) complexes [43]. The primary molecular target of
the Ru(II) arene complexes is DNA. The Ru(II) arene complexes bind to
DNA with the interaction of aromatic ligands by covalent or noncovalent interactions. These interactions induce apoptosis by causing
defects in the structure of DNA [43].
cymene)]2 and NH4PF6. The bimetallic Ru(II) complexes Ru5,6 were
highly soluble in polar solvents. They were air- and moisture-stable
orange solids. The bimetallic Ru(II) complexes (Ru4-6) were characterized by 1H, 13C, 19F, and 31P NMR spectroscopy, elemental analysis
and cyclic voltammetry. Single crystals for the solid-state structures
were obtained by the diffusion of diethyl ether into concentrated solutions of the complexes in dichloromethane. The molecular structure
of the complex (Ru5) was determined by single-crystal X-ray diffraction. The complex (Ru5) exhibited piano-stool type geometry.
In 1H NMR spectra, the p-cymene aromatic peaks of Ru5 and Ru6
appeared as four doublets with two protons each, while the p-cymene
aromatic peaks of Ru4 were observed as two doublets with four protons
each. For the bimetallic complexes Ru4, Ru5 and Ru6, the p-cymene
aromatic peaks were observed at between 5.77–5.53, 5.45–3.97 and
5.61–4.71 ppm, respectively. The chemical shifts of these protons
clearly indicated that the complex Ru4 contains the highest electronwithdrawing bridge-ligand and we can say that the electron density of
the metal is less in this complex. Also, the peaks of the NH protons were
observed in the range of 6.40–8.05 ppm for Ru4, 6.69–6.93 ppm for
Ru5 and 6.40–6.73 ppm for Ru6 (see the corresponding figures in the
SI).
2.1. Synthesis and characterization of bimetallic complexes
2.3. Electrochemical studies
We synthesized the bimetallic Ru(II) complex (Ru4) bridged by the
3,3-Diaminobenzidine (dab) as a analogue of the Ru1. The complex Ru4
was obtained as a result of the reaction of [Ru(p-cymene)Cl2]2 with
3,3′-diaminobenzidine and NH4PF6 in acetonitrile. The complex Ru4 is
an air- and moisture-stable orange solid and soluble in alcohols and
DMSO (see Scheme 1).
As seen in Scheme 2, two novel bimetallic Ru(II) complexes (Ru5,6)
containing butyl and bridged by aryl group (1,5-naphthyl (5), 1,4phenyl (6)) were prepared. The compounds A5,6 were prepared by the
reaction of the aryl diamines (1,5-naphthyl (5), 1,4-phenyl (6)) with
ethyl oxalyl chloride. The compounds B5,6 were synthesized by the
reaction of compounds A5,6 with the n-butylamine. The new symmetrical pro-ligands C5,6 were prepared by the reduction of the compounds (B5,6) with LiAlH4. Finally, the new bimetallic Ru(II) complexes
(Ru5,6) were obtained by the reaction of ligands (C5,6) with [RuCl2(p-
Cyclic voltammetry (CV) results can explain the interaction of a ligand with the d orbitals of metal, and therefore reveal why the ligand is
more labile to the cancer cells [46]. All complexes (Ru1-6) were electroactive in the working range and showed quasi-reversible redox
properties. Electrochemical oxidation/reduction couples for the same
oxidation number such as Ru2+/3+ and its reverse reaction (Ru3+/2+)
were indicated with the same index numbers in Fig. 4 (i.e. Ox1/Red1).
The CV of Ru1 showed irreversible two-step oxidation waves
with +0.59 V (Ox1) for the Ru2+/3+ and +1.62 V (Ox2) for the Ru3+/
4+
redox reactions. In the reverse direction, a single step reduction
wave with −0.78 V for the Ru3+/2+ (Red2) was also observed. Ru2
complex exhibited three step oxidation/reduction waves in the working
potential. The oxidation wave with −1.41 V (Ox1) in forward scan and
reduction wave with −2.33 V (Red1) in reverse scan can be explained
by the irreversible oxidation/reduction reaction of the ligands in the
2.2. X-ray crystallography
The molecular structure of complex Ru5, with the atom numbering
scheme, is shown in Fig. 3. The asymmetric unit of complex Ru5 contains one Ru(II) ion, one ethylenediamine ligand, one hexafluorophosphate anion, one coordinated chlorine anion, and one p-cymene group.
The molecule has the center of symmetry at the mid-point of the central
CeC (C5eC5i) bond [(i) -x, -y + 1, -z + 1]. The bond distances of Ru-N
are 2.167 (4) and 2.203 (3) Å, respectively. The Ru-Cl bond length is
2.4157 (12) Å. The molecules of Ru5 are connected by intermolecular
NeH⋯F hydrogen bonds (see Table 1).
Scheme 1. Synthesis of complex Ru4.
3
Bioorganic Chemistry 99 (2020) 103793
S.B. Kavukcu, et al.
Scheme 2. Synthesis of ligands and complexes.
Table 1
Selected bond distance for complexes Ru5 (Å, °).
Ru5
Ru1-N1
Ru1-N2
Ru1-Cl1
2.164 (4)
2.203 (3)
2.4157 (12)
to the oxidation of Ru2+ to Ru3+. A high oxidation wave was also
observed after +1.0 V with undefined peak potential for Ru3+/4+
oxidation in Ru3 complex. In the reverse direction, non-distinguished
two step reduction waves with −0.91 V for the Ru4+/3+ (Red 1) and
−1.03 V for the Ru3+/2+ (Red2) were observed. Ru4 showed different
redox behavior from the others and three step irreversible oxidation
and reduction waves were observed. The oxidation waves
with + 0.18 V and + 1.41 V can be dedicated to two-step single-electro
oxidation of Ru2+ to Ru3+ (Ox1) and Ru3+ to Ru4+ (Ox3) respectively.
In the reverse direction, the reduction waves with + 0.91 V and
−1.34 V can be dedicated to the reduction of Ru4+ to Ru3+ (Red3) and
Ru3+ to Ru2+ (Red1) respectively. In addition, irreversible oxidation/
reduction waves with + 0.57 V (Ox2) and −0.83 V (Red2) can be
explained by the electrochemical reaction of different ruthenium species formed by the decomposition of unstable Ru4 complex. Ru5
Fig. 3. Molecular structure of Ru5.
complex Ru2 body. The oxidation wave with −0.41 V (Ox2) and reduction wave with −0.65 V (Red2) can be dedicated for the quasireversible redox reaction of Ru2+/3+ redox couples. A final very high
irreversible wave for Ru3+/4+ oxidation after +1.0 V with an undefined peak potential was observed. Ru3 complex showed similar
redox behavior to the Ru2 complex and the first oxidation wave with
−1.45 V (Ox1) can be explained by the oxidation of the impurities in
the solution. The oxidation wave with −0.20 V (Ox2) can be dedicated
4
Bioorganic Chemistry 99 (2020) 103793
S.B. Kavukcu, et al.
Fig. 4. Cyclic voltommogram of 5.0 × 10−4 M Ru1, Ru2, Ru3, Ru4, Ru5 and Ru6 complexes between +2.0 V and −2.5 V vs sat. Ag/AgCl.
Table 2
The redox behaviors of complexes.
Peak Potential for Redox Couples
2+/3+
Ru
Oxidation Peak (V)
Ru3+/2+ Reduction Peak (V)
Ru3+/4+ Oxidation Peak (V)
Ru4+/3+ Reduction Peak (V)
Ru1
Ru2
Ru3
Ru4
Ru5
Ru6
+0.59
−0.78
+1.62
–
−0.41
−0.65
+1.0 <
–
−0.20
−1.03
+1.0 <
−0.91
0.18
−1.34
+1.41
–
+0.39
−1.09
+1.38
–
+0.69
−1.96
+1.38
–
complex also has similar redox behavior to Ru1. Irreversible two successive oxidation waves at + 0.39 V (Ox1) and + 1.38 V (Ox2) can be
dedicated to oxidation reaction of Ru2+/3+ and Ru3+/4+,respectively.
In the reverse scan, a single step reduction wave with −1.09 V for the
Ru3+/2+ (Red2) reduction was observed. Two step irreversible oxidation waves were also observed for Ru6 complex. Two successive oxidation waves with + 0.69 V (Ox1) for the Ru2+/3+ oxidation
and + 1.38 V (Ox2) for the Ru3+/4+ oxidation reactions were observed. In the reverse scan, a single step reduction wave with −1.96 V
for the Ru3+//2+ (Red2) was observed. The redox behaviors of all
complexes have also been summarized in Table 2. The reduction potential of the complex is directly related to the load density of the
complex. When stronger electron donor ligands are used, a smaller
positive charge density will be observed in the metal ion and the reduction potential of the metal will shift to more negative potential
values [44]. There is no significant difference in the reduction potential
of the complex studied for Ru4+ to Ru3+ reduction. For this purpose,
the Ru3+ to Ru2+ reduction potential of complexes was compared. The
ease of reduction of Ru(III) to Ru(II) in the studied complexes were
Ru2 > Ru1 > Ru3 > Ru5 > Ru4 > Ru6. CV experiments showed
that Ru2 has the smallest and Ru6 has the highest charge density. The
highest electron density of complex Ru6 can be attributed to the close
location of metal centers in the structure.
As previously stated, like all metal drugs, the biological activity of
the ruthenium compounds is mainly related to the oxidation state of the
metal center. The most biologically active form of ruthenium is
5
Bioorganic Chemistry 99 (2020) 103793
S.B. Kavukcu, et al.
proposed as the +2 oxidation step [14]. According to the CV results,
the Ru2+/3+ oxidation potential and Ru3+/2+ reduction potential values of the complex Ru6 demonstrated that the Ru (II) form of the
complex is more stable (Table 2). The tendency to remain in the +2
oxidation step according to the reduction/oxidation potential of the
complex Ru6 was reflected in the biological results, and this oxidation
state also provided more biological activity.
the toxicity of the complex Ru2 increased in a dose dependent manner
(p < 0.05) (Fig. 9). All the novel Ru complexes showed a similar effect
in Hep G2 cells, but the most effective ones were Ru1 and Ru5
(p < 0.01) (Fig. 10).
In Saos-2 cells, no dose-dependent effect was observed with the
bimetallic complexes (Ru4-6), but interestingly, the cytotoxic effect of
monometallic complexes (Ru1-3) increased in fewer dilutions
(p > 0.05) (Fig. 11). In addition, the complexes Ru5 and Ru6 also
showed cytotoxic effects in Vero cells.
While the effect of the bimetallic complex Ru5 was not significant
(p > 0.05), the effect of the bimetallic complex Ru6 was significant
(p < 0.05). However, the monometallic complexes Ru1 and Ru3 had
non-toxic effects on non-cancer cells; therefore, it was concluded that
they could be potential candidates as anti-cancer agents (Fig. 12).
In PC-3 cells, the cytotoxic effects of the monometallic complex Ru3
and the bimetallic analogue Ru6 were found to be more efficient than
the other complexes after the MTT analysis. However, the effect of Ru3
was significant compared with other Ru complexes (p < 0.05)
(Fig. 13).
While the cytotoxic effect of the bimetallic complexes Ru5 decreased according to its low dilutions, and the effects were detected to
be significant (p < 0.05), the monometallic complex Ru2 had less
effect in MCF-7 cells (Fig. 14). The efficiency difference between the
complexes could be explained by the effect of bimetallic enhancement
on cancer cell lines.
IC50 values after the MTT assay were calculated with Graphpad
Prism 8 and the results are given in Table 3. According to the IC50
values, the monometallic complex Ru1 displayed a toxic effect on the
control cells (3T3-L1 and Vero cells), and exhibited a moderate antiproliferative effect on HeLa, MDA-MB-231, LnCaP and MCF-7 cells. The
monometallic complex Ru2 was considerably cytotoxic on 3T3-L1 cells,
and an antiproliferative effect was observed, especially on LnCap, HepG2 and PC-3 cells. While the monometallic complex Ru3 exhibited a
strong toxic effect on Vero cells, there were no antiproliferative effects
on LnCap and Saos-2 cells. The bimetallic complex Ru4 was strongly
cytotoxic on HeLa, MDA-MB-231, LnCap and MCF-7 cells. Saos-2 and
PC-3 cells responded appropriately. The bimetallic complex Ru5 was
notably cytotoxic on Vero, except Du-145, Hep-G2 and Saos-2 cells, and
it had an antiproliferative effect for the rest of the cell lines. The bimetallic complex Ru6 showed a balanced toxic effect for the cancer cell
lines. Therefore, the novel Ru complexes showed different effects on
both cancer and normal cells.
2.4. In vitro Cytotoxicity
Cancer is a common problem because of its high prevalence and
mortality rates. Therapeutic strategies for cancer treatment may lead to
a reduction in the proliferation, dedifferentiation, and metastatic capability. Some treatment strategies are based on the exposure of cancer
cells to toxic compounds as it triggers cell death. When new compounds
are used in cancer treatment, their cytotoxic effects have to be tested.
The cytotoxic activities of the complexes (Ru1-6) were investigated towards HeLa, 3T3-L1, MDA-MB-231, DU-145, LNCaP, Hep-G2, Vero,
Saos-2, PC-3, and MCF-7 cell lines. The potential effect of Ru complexes
on cell viability was investigated by the colorimetric MTT assay. The
results showed that cell proliferation and viability decreased with Ru
treatment compared to control cells, although it was determined that
the complexes and their effective doses were different in each cell line
tested.
MTT analysis revealed that the monometallic complex Ru1 and the
bimetallic complex Ru6 were more effective than the other complexes
in HeLa cells and this effect was dose dependent (Fig. 5). When the
concentration of the complex was increased, the cytotoxic effect of the
complex on cancer cells was enhanced (p < 0.05). The toxic effects
were observed in the 3T3-L1 cells for both Ru2 and Ru6 complexes in
the MTT analysis. The numbers of cells were very low and Ru1, Ru3,
and Ru4 complexes were less toxic than the other complexes, and their
effects were significant in the 3T3-L1 cells (p < 0.05) (Fig. 6). It was
found that the monometallic complex Ru2 was the most effective
complex in DU-145 cells (p < 0.05). On the other hand, the bimetallic
complex Ru6 displayed a toxic effect at all applied concentrations in
DU-145 cells (Fig. 7). After statistical analysis, it was found to be significant (p < 0.001). All the novel Ru complexes showed a toxic effect
in MDA-MB-231 cells but the most effective ones were Ru1, Ru2 and
Ru4 (p < 0.05). However, their toxicities decreased in lower dosages
(p < 0.05) (Fig. 8). MTT analysis also revealed that Ru4 was the most
effective agent in the androgen-dependent LNCaP cells. Interestingly,
HeLa
Pure
0,35
1/2
1/4
1/8
1/16
1/32
0,3
ABSORBANCE
0,25
0,2
0,15
0,1
0,05
0
Ru1
Ru2
Ru3
Ru4
Ru5
COMPLEXES
Fig. 5. MTT results of the Ru complexes effect for HeLa cells.
6
Ru6
Bioorganic Chemistry 99 (2020) 103793
S.B. Kavukcu, et al.
3T3-L1
Pure
0,8
1/2
1/4
1/8
1/16
1/32
0,7
ABSORBANCE
0,6
0,5
0,4
0,3
0,2
0,1
0
Ru1
-0,1
Ru2
Ru3
Ru4
Ru5
Ru6
COMPLEXES
Fig. 6. MTT results of the Ru complexes effect for 3T3-L1 cells.
According to the data obtained from the MTT analysis, the novel Ru
complexes showed different effects at different concentrations on the
proliferation of both cancer and normal cells. All complexes had cytotoxic activity; however, high concentrations of the complexes resulted
as a reduction of their effects on MCF-7 and LNCaP cells. Therefore,
during in vivo studies, the medium concentration of Ru complexes
should be considered in the treatment stage. However, the bimetallic
complex Ru6 was affected in both cancer and normal cell lines.
Therefore, the dosage of the complex Ru6 should be well controlled in
both in vitro and in vivo studies. The high electron density on the metal
center of complex Ru6 also resulted in higher cytotoxic activity.
NMR spectroscopy. The complexes (Ru3 and Ru6) were hydrolyzed
quickly and the equilibrium was reached when they were just prepared.
The stability of the complexes was also examined with 19F- and 31P
NMR spectroscopies. The complexes were pure in the solution according to 1H-, 19F- and 31P NMR spectrums for 45 days (Fig. S14 and
S15 in the SI) (see Figs. 15 and 16).
2.6. Interaction of the complexes Ru3 and Ru6 with FS-DNA
Inorganic metal-based drugs show their therapeutic effects through
coordination to DNA or proteins. This coordination can be via intra- or
inter-DNA–DNA, DNA–protein, and protein–protein crosslinks. This is
called a fragment-based approach and its application to metal-based
drugs is becoming widespread [45]. This approach is considered to be
an important way of designing bioactive compounds. The interaction of
the monometallic complex Ru3 and the bimetallic complex Ru6 with
FS-DNA (fish sperm-DNA) was investigated by 1H NMR spectroscopy in
DMSO‑d6 at ambient temperature (see the corresponding figures in the
SI). The addition of an equal of FS-DNA to an equilibrated solution of
Ru3 or Ru6 in DMSO‑d6 induced relatively fast changes in the 1H NMR
spectrums (Figure S11 and S12 in the SI). Binding of a Ru(II) center to N
atom of FS-DNA moieties typically induces a downfield shift of the
aromatic region resonances in the 1H NMR spectrums compared to the
free FS-DNA.
2.5. Stability/aquation of the complexes Ru3 and Ru6
The stability or aquation of metal-based anticancer drugs directly
affects their biological properties. To study the biological properties of
metal-based anticancer drugs with low or no solubility in water, stock
solutions are usually prepared in DMSO. In such cases, the chloride ion
in the complex structure is exchanged by water or any solvent’s molecules like DMSO. In this mechanism, the aquation step is important for
the interaction with DNA base pairs or with proteins to form adducts.
Thus, the time-dependent hydrolysis was monitored to reveal the
aquation and stability of the monometallic complex Ru3 and the bimetallic complex Ru6 in a D2O/DMSO‑d6 (20:80) solvent system by 1H
Pure
DU145
1/2
1/4
1/8
1/16
1/32
1,4
1,2
ABSORBANCE
1
0,8
0,6
0,4
0,2
0
Ru1
Ru2
Ru3
COMPLEXES
Ru4
Ru5
Fig. 7. MTT results of the Ru complexes effect for DU-145 cells.
7
Ru6
Bioorganic Chemistry 99 (2020) 103793
S.B. Kavukcu, et al.
MDA-MB-231
Pure
1/2
1/4
1/8
1/16
1/32
0,6
ABSORBANCE
0,5
0,4
0,3
0,2
0,1
0
Ru1
Ru2
Ru3
Ru4
Ru5
Ru6
COMPLEXES
Fig. 8. MTT results of the Ru complexes effect for MDA-MB-231 cells.
LNCaP
Pure
0,2
1/2
1/4
1/8
1/16
1/32
0,18
ABSORBANCE
0,16
0,14
0,12
0,1
0,08
0,06
0,04
0,02
0
Ru1
Ru2
Ru3
COMPLEXES
Ru4
Ru5
Ru6
Fig. 9. MTT results of the Ru complexes effect for LNCaP cells.
Fig. 10. MTT results of the Ru complexes effect for Hep-G2 cells.
2.7. DNA binding studies
binding, electrostatic or intercalation) interactions [43]. Therefore, it is
important to determine the DNA binding properties of Ru(II) arene
complexes. The UV–Vis technique, which is an electronic absorption
spectroscopy, is a good application for the investigation of the binding
Ru(II) arene complexes can bind to DNA through both covalent
(replacement of an ancillary halogen ligand) and non-covalent (groove
8
Bioorganic Chemistry 99 (2020) 103793
S.B. Kavukcu, et al.
Saos-2
Pure
1/2
1/4
1/8
1/16
1/32
0,16
0,14
ABSORBANCE
0,12
0,1
0,08
0,06
0,04
0,02
0
Ru1
Ru2
Ru3
COMPLEXES
Ru4
Ru5
Ru6
Fig. 11. MTT results of the Ru complexes effect for Saos-2 cells.
Vero
Pure
1,4
1/2
1/4
1/8
1/16
1/32
1,2
ABSORBANCE
1
0,8
0,6
0,4
0,2
0
Ru1
Ru2
Ru3
COMPLEXES
Ru4
Ru5
Ru6
Fig. 12. MTT results of the Ru complexes effect for Vero cells.
PC-3
Pure
0,9
1/2
1/4
1/8
1/16
1/32
0,8
AxBSORBANCE
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
Ru1
Ru2
Ru3
COMPLEXES
Ru4
Ru5
Fig. 13. MTT results of the Ru complexes effect for PC-3 cells.
9
Ru6
Bioorganic Chemistry 99 (2020) 103793
S.B. Kavukcu, et al.
Pure
0,35
MCF-7
1/2
1/4
1/8
1/16
1/32
0,3
ABSORBANCE
0,25
0,2
0,15
0,1
0,05
0
Ru1
Ru2
Ru3
Ru4
Ru5
Ru6
COMPLEXES
Fig. 14. MTT results of the Ru complexes effect for MCF-7 cells.
properties with DNA [50]. In order to investigate these interactions, we
performed UV–Vis analyses of the complexes Ru3 and Ru6 (0–15 µM) in
20 mM Tris-HCl/NaCl (pH: 7.0) with 0.1 mM FS-DNA and the absorption spectra were given in Fig. 17. The absorption differences in
hypochromic and hyperchromic values indicated non-covalent interactions between DNA and the complexes. The hyperchromism in the
absorption intensity of FS-DNA at λmax = 260 nm for both complexes
was related with the non-covalent intercalation between DNA and the
complexes. The intrinsic binding constants Kb of the complexes (Ru3
and Ru6) were (1.7 ± 0.1) × 105 M−1 and (7.6 ± 0.1) × 104 M−1,
respectively. The Kb values suggested intense binding of the complexes
to FS-DNA.
manner. These new synthesized Ru(II) arene complexes could have a
greater trigger effect on cell death pathways with good cellular tolerance. Therefore, when performing them in in vivo application, proper
doses need to be determined. Among the complexes, the highest cytotoxic activity and also the highest electron density on metal centers was
observed in the bimetallic complex Ru6. The complexes (Ru3 and Ru6)
were stable for 45 days in a D2O/DMSO‑d6 (20:80) solvent system. The
interaction of the complexes (Ru3 and Ru6) with FS-DNA was also
examined. When the reactivity of the mono and the bimetallic complexes was examined, it was observed that the introduction of a second
metal fragment increased the reactivity and stability with biomolecular
targets and led to an antimetastatic increase; potentially, a lower dose
of the drug may be used, which could reduce side effects.
3. Conclusions
4. Experimental section
In summary, we have reported a series of mono and bimetallic Ru
(II) p-cymene complexes carrying the diamine ligand group. These
complexes were characterized by spectroscopic methods and analyzed
by CV. The complex Ru1, known to be cytotoxically active in the literature, was used as a reference in this study. The effects of monometallic (Ru1-3) and bimetallic (Ru4-6) complexes, which were analogues and had different dilution rates, on cytotoxic activity were
investigated. Subtle differences in the coordination of the ligand may
produce important differences in its electron donation, thus producing
an important impact on the cytotoxic efficacy. Subtle ligand modifications on Ru(II) arene complexes can lead to different mechanisms of
action and result in significant changes in the cytotoxic efficacy. In
conclusion, after MTT analysis, new synthesized Ru complexes have
different effects on different cancer cell lines depending on the dosage
4.1. General information
Reactions involving air-sensitive components were performed by
using a Schlenk-type flask under argon atmosphere and high vacuumline techniques. The glass equipment was heated under vacuum in
order to remove oxygen and moisture and then it was filled with argon.
The solvents were analytical grade and distilled under argon atmosphere from sodium (tetrahydrofuran, toluene, diethylether, pentane),
P2O5 (dichloromethane). Reagents were purchased from Aldrich or
Merck and were used as received. 1H-, 13C-, 19F- and 31P NMR spectra
were recorded on a Varian 400 MHz spectrometer. J values were given
in Hz. X-Ray diffraction analysis was performed on a D8-QUEST diffractometer equipped with graphite-monochromatic Mo-Ka radiation.
Table 3
IC50 values of the Ru complexes after MTT assay (μM).a
Cells
HeLa
3T3-L1
DU-145
MDA-MB-231
LNCaP
Hep-G2
Saos-2
Vero
PC-3
MCF-7
a
IC50 (μM)
Ru1
Ru2
Ru3
Ru4
Ru5
Ru6
16.3 ± 1.2
11.0 ± 1.0
34.5 ± 1.5
26.0 ± 1.4
20.0 ± 1.3
54.9 ± 1.7
55.6 ± 2.8
10.8 ± 1.0
41.8 ± 1.6
34.7 ± 1.5
41.3 ± 1.6
2.0 ± 0.3
12.2 ± 1.0
11.2 ± 1.0
27.6 ± 1.4
29.2 ± 1.4
39.3 ± 1.5
17.6 ± 1.2
20.0 ± 1.3
38.4 ± 1.8
24.2 ± 1.3
28.9 ± 1.4
14.6 ± 1.3
37.9 ± 1.5
82.8 ± 1.9
59.6 ± 1.7
> 100
7.2 ± 0.8
21.9 ± 1.3
31.0 ± 1.4
9.4 ± 0.9
46.5 ± 1.6
30.3 ± 1.4
5.4 ± 0.7
5.2 ± 0.7
30.6 ± 1.4
15.6 ± 1.1
20.3 ± 1.3
23.4 ± 1.2
4.4 ± 0.6
8.2 ± 0.9
13.3 ± 0.9
55.9 ± 1.7
12.7 ± 1.1
32.2 ± 1.6
55.9 ± 1.7
70.6 ± 1.8
0.19 ± 0.2
8.8 ± 0.9
25.0 ± 1.3
9.7 ± 6.2
17.6 ± 1.2
15.7 ± 1.1
11.8 ± 1.0
27.5 ± 1.4
14.9 ± 1.1
22.7 ± 1.3
14.7 ± 1.1
20.8 ± 1.3
14.5 ± 1.1
IC50 values are presented as mean ± SD (standard error of the mean) from three repeating experiments.
10
Bioorganic Chemistry 99 (2020) 103793
S.B. Kavukcu, et al.
Fig. 15. The stability of Ru3 was monitored via 1H NMR spectroscopy in 20% D2O/DMSO‑d6 over 45 days.
Fig. 16. The stability of Ru6 was monitored via 1H NMR spectroscopy in 20% D2O/DMSO‑d6 over 45 days.
11
Bioorganic Chemistry 99 (2020) 103793
2
1/[A-A0]
1/[A-A0]
S.B. Kavukcu, et al.
1
0
0,06
1/Q x106
2
1
0
1
0,06
1/Q x106
1
Fig. 17. Absorption spectra of FS-DNA with increasing concentrations of the complexes Ru3 (left) and Ru6 (right) (arrow shows the increase of the intensity on
increasing complex concentration). Kb was calculated by the ratio of intercept and slope of plot between 1/(A0 − A) and 1/[Q].
Melting points were measured on Gallenkamp electrothermal melting
point apparatus without correction. The cyclic voltammetry (CV) studies were carried out with an Autolab 204 electrochemical system with
three electrode assemblies. RuCl2(p-cymene)]2 was prepared according
to the method reported by Bennett and Smith through the reaction of
ruthenium(III) chloride with α–terpinene [47]. Ru1 was synthesized
according to the procedure published by Sadler [31]. All synthesis
procedures are available in the paper previously published by our group
[33].
4.4. Synthesis and characterization of compounds C
Compound C5: The compound C5 was prepared in the same manner
as our published procedure using B5 (1.00 g, 2.76 mmol.) and LiAlH4
(1.05 g, 27.62 mmol). Yield: 0.59 g (69%). Yellow oil. 1H NMR
(400 MHz, 303 K, CDCl3): δ 7.25 (s, 2H, NH), 6.58 (s, 4H, Ar-H), 3.16
(m, 4H, CH2), 2.85 (m, 4H, NCH2CH2N), 2.62 (m, 4H, NCH2CH2N),
1.48 (m, 4H, CH2), 1.36 (m, 4H, CH2), 0.91 (t, JH-H = 4.0 Hz, 6H, CH3).
13
C NMR (100 MHz, 303 K, CDCl3): δ 140.9, 114.9, 49.3, 48.8, 44.8,
32.0, 20.4, 13.9.
Compound C6: The compound C6 was prepared in the same manner
as our published procedure using B6 (1.00 g, 2.42 mmol.) and LiAlH4
(0.92 g, 24.24 mmol). Yellow oil. 1H NMR (400 MHz, CDCl3): δ 7.32
(m, 4H, Ar-H), 6.62 (d, JH-H = 7.6 Hz, 2H, Ar-H), 3.35 (m, 4H, CH2),
3.02 (m, 4H, NCH2CH2N), 2.67 (m, 4H, NCH2CH2N), 1.52 (m, 4H,
CH2), 1.41 (m, 4H, CH2), 0.93 (t, JH-H = 7.2 Hz, 6H, CH3). 13C NMR
(100 MHz, CDCl3): δ 144.3, 125.3, 124.2, 109.0, 104.5, 49.2, 48.4,
43.6, 32.3, 20.4, 14.0.
4.2. Synthesis and characterization of compounds A
Compound A5: The compound A5 was prepared in the same manner
as our published procedure using 1,5-diaminonaphthalene (1.00 g,
6.32 mmol), ethyl oxalyl chloride (2.07 mL, 18.50 mmol). Yield: 1.63 g
(72%). White solid. m.p.: 215 °C. 1H NMR (400 MHz, DMSO): δ 10.94
(s, 2H, NH), 7.94 (t, JH-H = 4.4 Hz, 2H, Ar-H), 7.60 (s, 2H, Ar-H), 7.59
(s, 2H, Ar-H), 4.37 (q, 4H, OCH2CH3), 1.36 (t, JH-H = 7.2 Hz, 3H,
OCH2CH3). 13C NMR (400 MHz, DMSO): δ 161.2, 157.4, 132.9, 129.7,
126.1, 124.3, 122.5, 62.7, 14.3.
Compound A6: The compound A6 was prepared in the same manner
as our published procedure using p-phenylenediamine (1.00 g,
9.25 mmol), ethyl oxalyl chloride (2.07 mL, 18.50 mmol). Yield: 2.17 g
(76%). White solid. m.p.: 208 °C. 1H NMR (400 MHz, DMSO): δ 10.70
(s, 2H, NH), 7.70 (s, 4H, Ar-H), 4.30 (m, 4H, OCH2CH3), 1.30 (t, JH13
C NMR (400 MHz, DMSO): δ 161.1,
H = 7.2 Hz, 6H, OCH2CH3).
155.8, 134.5, 121.2, 62.8, 14.2.
4.5. Synthesis and characterization of Ru complexes
Complex Ru4: The complex Ru4 was prepared in the same manner
as our published procedure using 3,3′-Diaminobenzidine (1.00 g,
4.67 mmol), [RuCl2(p-cymene)]2 (2.86 g, 4.67 mmol) and NH4PF6
(1.51 g, 9.34 mmol). 3.58 g (72%). Brown powder. m.p.: 165 °C.
Elemental analysis: calcd (%) for C32H42Cl2F12N4P2Ru2 (MW: 1045,68):
C, 36.76; H, 4.05; N, 5.36%. Found: C, 36.80; H, 4.03; N, 5.34%. 1H
NMR (400 MHz, DMSO): δ 8.07 (br, 2H, NH), 7.36 (m, 3H, Ar-H), 6.42
(m, 2H, NH), 5.76 (d, JH-H = 6.4 Hz, 2H, p-cymene-Ar-H), 5.54 (d, JHH = 5.2 Hz, 2H, p-cymene-Ar-H), 2.89 (m, 1H, CH2), 2.22 (s, 3H, pcymene-CH3), 1.19 (d, JH-H = 6.8 Hz, 6H, p-cymene- CH(CH3)2). 13C
NMR (100 MHz, DMSO): δ 141.0, 140.0, 137.6, 127.0, 125.4, 124.1,
103.1, 98.5, 82.5, 80.6, 30.5, 22.6, 18.4. 19F NMR (376 MHz, DMSO): δ
−69.1, −71.0 (d, JF-F = 711.0 Hz, PF6). 31P NMR (166 MHz, DMSO): δ
−144.0 (septed, JP-P = 713.5 Hz, PF6).
Complex Ru5: The complex Ru5 was prepared in the same manner
as our published procedure using ligand C5 (1.00 g, 2.80 mmol),
[RuCl2(p-cymene)]2 (1.72 g, 2.80 mmol) and NH4PF6 (0.92 g,
5.60 mmol). Yield: 2.03 g (61%). Yellow powder. m.p.: 243–245 0C.
Elemental analysis: calcd (%) for C42H64Cl2F12N4P2Ru2 (MW: 1187,96):
C, 42.46; H, 5.43; N, 4.72%. Found: C, 42.44; H, 5.45; N, 4.73%. 1H
NMR (400 MHz, DMSO): δ 8.80 (d, J = 8.0 Hz, 2H, Ar-H), 7.80 (m, 2H,
Ar-H), 6.93 (br, 1H, NH), 6.70 (br, 1H, NH), 5.45 (d, J = 5.6 Hz, 2H, pcymene-H), 5.11 (d, J = 5.6 Hz, 2H, p-cymene-H), 4.52 (d, J = 5.6 Hz,
2H, p-cymene-H), 3.98 (d, J = 5.6 Hz, 2H, p-cymene-H), 3.00 (m, 6H,
4.3. Synthesis and characterization of compounds B
Compound B5: The compound B5 was prepared in the same manner
as our published procedure using A5 (1.00 g, 3.24 mmol) and n-butylamine (0.64 mL, 6.48 mmol). White solid. m.p.: 285 °C. 1H NMR
(400 MHz, DMSO): δ 10.55 (s, 2H, NH), 8.91 (t, 2H, NH), 7.75 (s, 4H,
Ar-H), 3.17 (m, 4H, CH2), 1.46 (s, 4H, CH2), 1.26 (s, 4H, CH2), 0.87 (t,
JH-H = 7.6 Hz, 6H, CH3).
Compound B6: The compound B6 was prepared in the same manner
as our published procedure using A6 (1.00 g, 2.79 mmol) and n-butylamine (0.56 mL, 5.58 mmol). Yield: 1.03 g (90%). White solid. m.p.:
274 °C. 1H NMR (400 MHz, DMSO): δ 10.73 (s, 2H, NH), 8.93 (t, 2H,
NH), 7.78 (d, JH-H = 8.0 Hz, 2H, Ar-H), 7.60 (m, 4H, Ar-H), 3.23 (m,
2H, CH2), 1.52 (s, 4H, CH2), 1.31 (s, 4H, CH2), 0.91 (t, JH-H = 7.2 Hz,
6H, CH3).
12
Bioorganic Chemistry 99 (2020) 103793
S.B. Kavukcu, et al.
NCH2CH2N and Bu-CH2), 2.52 (m, 1H, p-cymene-CH), 1.92 (s, 3H, pcymene-CH3), 1.71 (m, 2H, Bu-CH2), 1.36 (m, 2H, Bu-CH2), 1.10 (d,
J = 6.4 Hz, 3H, p-cymene-CH(CH3)2), 1.03 (d, J = 6.4 Hz, 3H, pcymene-CH(CH3)2), 0.96 (t, J = 7.2 Hz, 3H, Bu-CH3). 13C NMR
(100 MHz, DMSO): δ 146.8, 146.7, 126.5, 126.4, 125.9, 125.7, 122.3,
116.7, 116.3, 108.4, 107.8, 93.9, 93.3, 88.0, 87.2, 84.1, 83.8, 79.4,
78.7, 78.1, 77.4, 56.2, 56.2, 53.3, 52.9, 47.9, 47.8, 30.6, 30.5, 30.2,
22.7, 21.4, 21.2, 20.3, 17.0, 16.8, 14.2. 19F NMR (376.2 MHz, DMSO): δ
−69.2, −71.1 (d, JF-F = 711.3 Hz, PF6). 31P NMR (161.8 MHz, DMSO):
δ −144.2 (septed, JP-P = 714.0 Hz, PF6).
Complex Ru6: The complex Ru6 was prepared in the same manner
as our published procedure using ligand C6 (1.00 g, 3.26 mmol,),
[RuCl2(p-cymene)]2 (1.99 g, 3.26 mmol) and NH4PF6 (1.06 g,
6.52 mmol). Yield: 2.30 g (62%). Yellow powder. m. p.: 237–239 °C.
Elemental analysis: calcd (%) for C38H62Cl2F12N4P2Ru2 (MW: 1137,90):
C, 40.11; H, 5.49; N, 4.92%. Found: C, 40.13; H, 5.50; N, 4.90%. 1H
NMR (400 MHz, DMSO): δ 7.49 (s, 2H, Ar-H), 6.73 (br, 1H, NH), 6.41
(br, 1H, NH), 5.61 (d, J = 6.4 Hz, 2H, p-cymene-H), 5.40 (d,
J = 6.0 Hz, 2H, p-cymene-H), 4.85 (d, J = 6.0 Hz, 2H, p-cymene-H),
4.72 (d, J = 6.0 Hz, 2H, p-cymene-H), 2.94 (m, 5H, NCH2CH2N and BuCH2), 2.65 (m, 1H, p-cymene-CH), 2.14 (s, 3H, p-cymene-CH3), 2.00 (m,
1H, Bu-CH2), 1.66 (m, 2H, Bu-CH2), 1.35 (m, 2H, Bu-CH2), 1.19 (d,
J = 6.8 Hz, 3H, p-cymene-CH(CH3)2), 1.13 (d, J = 6.8 Hz, 3H, pcymene-CH(CH3)2), 0.94 (t, J = 7.2 Hz, 3H, Bu-CH3). 13C NMR
(100 MHz, DMSO): δ 147.9, 106.7, 94.6, 85.8, 83.2, 80.8, 80.0, 56.3,
53.0, 48.6, 30.5, 30.3, 22.7, 21.6, 20.2, 17.3, 14.2. 19F NMR
(376.2 MHz, DMSO): δ −69.2, −71.1 (d, JF-F = 711.0 Hz, PF6). 31P
NMR (161.8 MHz, DMSO): δ −144.1 (septed, JP-P = 713.8 Hz, PF6).
possible binding modes to FS-DNA and to calculate the binding constants (Kb). The intrinsic binding constants (Kb) were calculated according to the Benesi-Hildebrand equation HYPERLINK "SPS:refid::bib52" [51].
1/(A
A 0) = 1/{Kb (Amax
A 0)[Q]} + 1/[Amax
A 0]
where A0 is the absorption intensity of DNA at 260 nm in the absence of
complex, Amax is the saturated absorption intensity of the DNA-metal
complex adduct, A is the absorption intensity of DNA interacted with a
metal complex and [Q] is the concentration of the metal complex. The
binding constant (Kb) was graphically evaluated by plotting 1/[A − A0]
versus 1/[Q].
4.9. Application of Ru complexes
A stock solution of Ru complexes was prepared at a concentration of
10 mM in the cell culture medium. The stock solution was diluted 1/2,
1/4, 1/8, 1/16 and 1/32 with the medium solution. Then, 100 μL of
cancer cell was added to each well from all dilutions prepared, including stock solution, and cells were incubated for 48 h in 37 °C and
5% CO2.
4.10. Cell viability analyses with MTT assay
The cell viability and proliferation rate were analyzed the tetrazolium reduction assayin all cells. The stock solution of 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was prepared in PBS (final concentration was 5000 mg/mL) and stored in 4+
0
C. All types of cells were cultured in 96-well plates for 24 h and the
density of cells in each well was 104 in 200 μL of culture medium. The
culture medium from all wells was discarded and 200 µL per well of 1/
10 diluted MTT solution was added and then incubated for 3 h at 37 0C
after incubation. 200 µL of MTT solvent (DMSO) was added to each well
and it was then shaken on an orbital shaker for 15 min. Absorbance was
measured at 570 nm with a 690 nm reference filter [48,49].
4.6. CV experiments
The three electrode system consisted of a platinum disk as the
working electrode, platinum wire as the counter electrode and saturated Ag/AgCl as the reference electrodes. The voltommograms were
recorded at DMSO in the presence of 0.1 M of tetrabutylammonium
hexaflouroborate solution. Potential scan was performed between −2.5
and +2.0 V.
Acknowledgements
The authors thank Ege University (Project Number: 17-FEN-042)
and the Scientific and Technological Research Council of Turkey
(TUBITAK, Project Number: 214Z098) for their financial support. The
authors acknowledge the Scientific and Technological Research
Application and Research Center, Sinop University, Turkey, for the use
of the Bruker D8 QUEST diffractometer.
4.7. Cell culture
Human cell lines such as cervix adenocarcinoma HeLa, androgendependent lymph node metastasis prostate adenocarcinoma LNCaP,
androgen-independent bone metastasis prostate adenocarcinoma PC-3,
androgen-independent brain metastasis prostate adenocarcinoma
DU145, estrogen-responsive primary breast adenocarcinoma MCF-7,
estrogen-unresponsive metastatic breast adenocarcinoma cell line
MDA-MB-231, liver hepatocellular carcinoma HepG2, colorectal adenocarcinoma HT-29, bone osteosarcoma Saos-2 as well as normal fibroblast 3T3-L1 and Vero (African green monkey kidney) were purchased from ATCC (American type culture collection). LNCaP, MCF-7,
MDA-MB-231, Saos-2 and HepG2 cell lines were maintained in RPMI1640 while PC-3, DU145, HeLa, 3T3 and Vero cell lines were maintained in DMEM-F12 supplemented with 10% FBS (Fetal bovine Serum)
(Invitrogen, UK), 1% L–glutamine (Invitrogen, UK), and 1% penicillin–streptomycin (Invitrogen, UK) in a humidified incubator at 37 °C
and 5% CO2. The morphology of the cells was examined every second
day using a phase-contrast inverted microscope (CK40-F200; Olympus,
Tokyo, Japan) and photographed. When the cells were confluent, they
were routinely sub-cultured using 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) solution.
Declaration of Competing Interest
The authors declared that there is no conflict of interest.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103793.
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