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Synthesis of Sorafenib-Ruthenium Complexes, Investigation of Biological Activities and Applications in Drug Delivery Systems as an Anticancer Agent.
Sorafenib, a multiple kinase inhibitor, is widely used
as a first-line
treatment for hepatocellular carcinoma. However, there is a need for
more effective alternatives when sorafenib proves insufficient. In
this study, we aimed to design a structure that surpasses sorafenib’s
efficacy, leading us to synthesize sorafenib–ruthenium complexes
for the first time and investigate their properties. Our results indicate
that the sorafenib–ruthenium complexes exhibit superior epidermal
growth factor receptor (EGFR) inhibition compared to sorafenib alone.
Interestingly, among these complexes, Ru3S demonstrated
high activity against various cancer cell lines including sorafenib-resistant
HepG2 cells while exhibiting significantly lower cytotoxicity than
sorafenib in healthy cell lines. Further evaluation of cell cycle,
cell apoptosis, and antiangiogenic effects, molecular docking, and
molecular dynamics studies revealed that Ru3S holds great
potential as a drug candidate. Additionally, when free Ru3S was encapsulated into polymeric micelles M1 , enhanced
cytotoxicity on HepG2 cells was observed. Collectively, these findings
position Ru3S as a promising candidate for EGFR inhibition
and warrant further exploration for drug development purposes.
## Introduction
1 Introduction The transition metals
exhibit the ability to bind to biomacromolecules
with high specificity and enable the synthesis of complexes in different
geometries thanks to their atomic properties. 1 Coordination complexes have recently attracted attention in the
scientific literature, with their unique geometries applicable to
organic small-molecule-based drugs and exhibiting modifiable reactivity. 2 Metallotherapeutics exert their effects on cancer
cells through different mechanisms, such as inhibiting cancer cell
division, inducing DNA damage, or disrupting the DNA repair process
and triggering cancer cell apoptosis. 3 Especially
after the discovery and development of platinum complexes, their use
against many tumor types has played an important role in the research
of metallotherapeutics. 4 , 5 Although cisplatin is one of the
most progressive and widely used drugs in clinical practice, complex
structures formed by metal ions other than platinum have become the
focus of further research due to the serious side effects, increased
drug resistance, and decreased effectiveness caused by platinum. 3 , 6 − 8 For this purpose, the ruthenium ion is an alternative
to platinum(II) because it is less toxic to healthy cells and has
ligand exchange kinetics similar to Pt(II) complexes. 9 − 11 In addition to these properties, ruthenium compounds are active
against some cisplatin-resistant cell lines and ruthenium can mimic
iron in binding to some biological molecules. 3 , 12 , 13 Six-coordinated Ru (II/III) polypyridyl
complexes exhibit good anticancer properties as they often contain
a planar aromatic ring that can intercalate with DNA using DNA as
a target. 14 , 15 Some Ru (II/III) complexes with these structures
( Figure 1 ), such as
KP1019, NAMI-A, TLD1433, and RAPTA-C, are in clinical trials. 4 , 16 , 17 Figure 1 Ru complex structures in clinical trials
and this work. Sorafenib is a multikinase inhibitor used against
renal cell carcinoma,
hepatocellular carcinoma, colorectal carcinoma, breast carcinoma,
lung carcinoma, and thyroid cancer. 18 Protein/tyrosine
kinases are key enzymes that play a role in the regulation of various
cellular processes that catalyze the transfer of a phosphate group
from the ATP to the hydroxyl group of the target protein. Examples
of tyrosine kinases (TKs) are epidermal growth factor receptor (EGFR),
platelet-derived growth factor receptor (PDGFR), vascular endothelial
growth factor receptor (VEGFR), fibroblast growth factor receptor
(FGFR), and Src, Abl, Lck proto-oncogenes. TKs function as components
of signal transduction pathways that play a central role in various
biological processes such as cell growth control, metabolism, differentiation,
and apoptosis. Therefore, protein kinases are important targets in
anticancer drug development. 19 Heffeter
et al. conducted the first study using sorafenib and ruthenium
complexes in 2013. 20 In this study, the
investigators explored the synergistic effect of KP1339 which is known
to have anticancer properties and sorafenib. According to the results,
when sorafenib and KP1339 were used together, they showed a cytotoxic
effect that was 10-fold better than when they were used alone. In
addition, this concomitant use increased apoptosis and further stopped
the cell cycle in the G0/G1 phase. There are many other studies in
the literature that show that ruthenium complexes can be protein kinase
inhibitors. 21 − 25 Also, Meggers and his group synthesized ruthenium complexes containing
many different ligands and investigated the protein kinase inhibitory
effects of these complexes. When the studies of the Heffeter and Meggers
groups are evaluated together, it is expected that the formation of
a direct Ruthenium complex, rather than a synergistic effect with
sorafenib, will have a higher potential for kinase activity. In this
respect, there is no study in the literature that has tested this
feature. In this study, sorafenib–ruthenium complexes
were prepared
for the first time using sorafenib and various ligands. Among these
ligands, only the bipyridyl structure was synthesized by Yidan Lai
et al. first time. 26 In this study, the
effectiveness of this complex on hepatocellular carcinoma through
the photocatalytic mechanism was investigated. In our study, the effects
of this and the original 7 complexes on hEGFR enzyme inhibition were
investigated. We then investigated its cytotoxic effects on five different
cancer cells. In order to comprehend the mechanism of enzyme inhibition,
we calculated the binding energies using molecular modeling. We investigated
apoptosis and the cell cycle to determine the mechanism of cell death.
In addition, we examined how complexes influence the expression of
tyrosine kinase pathway proteins and whether they have antiangiogenic
properties. To increase the bioavailability of the obtained complexes,
cytotoxic and apoptotic properties were investigated by forming micelles.
With drug release studies, we demonstrated the efficacy of micelles
as well. Thus, we evaluated the biological activity of our newly prepared
sorafenib–ruthenium complexes, on the one hand, through tyrosine
kinase enzyme inhibition, and on the other hand, we evaluated their
drug release properties.
## Result and Discussion
2 Result and Discussion 2.1 Chemistry Novel targeted ruthenium–sorafenib
complexes were synthesized as shown in Scheme 1 . Synthesis of sorafenib was carried out
according to the method in the literature in four steps. 27 − 29 The phenanthroline ring readily reacted with RuCl 3 and
LiCl in DMF 30 to obtain ruthenium–ligand
complexes ( RuL 2 Cl 2 ). Bipyridine and 4,4-dimethyl bipyridine
structures were successfully synthesized using this method. But for
substituents; when 6,6-dimethyl was changed to 4,4-dimethoxy and 4,4-di- t -butyl, the synthesis of these compounds by the same method
was not possible. Here, an ethylene glycol–water mixture was
used as a solvent, benefiting from the work of” Viala and Coudret. 31 In this method, in which glucose and ascorbic
acid are used as reducing agents, high concentrations of Cl ions were
used by using an ethylene glycol–water mixture, which enabled
the reaction to take place. In addition, removing the substance by
heating it with dichloromethane for purification has been an effective
method to remove the complex from many salts formed. At this stage,
the yields of the complexes were quite high with 55–78%. Scheme 1 Synthesis of Sorafenib–Ruthenium Complexes (i) SOCl 2 , 72 °C,
16 h, (ii) CH 3 NH 2 , MeOH, 0 °C, (iii) t -BuOK, DMF, K 2 CO 3 , 80 °C, p -aminophenol, (iv) THF, Et 3 N, 60 °C, (v)
Method A: ligand, DMF, 80 °C, N 2 , 18 h, Method B:
ligand, ethylene glycol, glucose, ascorbic acid, 110 °C, 1 h,
(vi) MeOH, 60 °C, 18 h. From the 1 H NMR spectra of the obtained RuL 2 Cl 2 complexes compared with the 1 H NMR spectra
of the ligands, it is seen that the aromatic ring signals shift to
a lower area in the 1 H NMR spectrum ( Figures S1–S8 ). For example, the signal seen in the
lowest area of the 1 H NMR spectrum of the bipyridine (bpy)
ligand is seen at 8.79 and 8.47 ppm. In the obtained Ru(bpy) 2 Cl 2 complex, the signals in the lowest area are seen at
9.98 and 8.64 ppm. Similarly, the signal seen in the lowest area of
the 1 H NMR spectrum of the other phenanthroline (phen)
ligand is at 9.77 and 8.46 ppm, while the peaks of the ruthenium complex
of this ligand [Ru(phen) 2 Cl 2 ] are seen at 10.28
and 8.72 ppm. In addition, in the 1 H NMR spectra of RuL 2 Cl 2 , the signals for aromatic protons were observed
between 6.92 and 10.35 ppm, while aliphatic proton signals were observed
about at 1.33–4.11 ppm. From the comparison of the 13 C NMR spectra of the RuL 2 Cl 2 complexes
with the spectra of the ligands,
it is seen that the aromatic ring carbons are similarly signaled in
the lower area. In the 13 C NMR spectrum of the bipyridine
(bpy) ligand, the signals seen in the lowest area are 155.8, 149.7,
and 137.7 ppm, while the 13 C NMR spectrum of the Ru(bpy) 2 Cl 2 complex is seen at 159.5, 157.5, 152.1, and
151.3 ppm. Similarly, in the 13 C NMR spectrum of the phenanthroline
(phen) ligand, these signals were seen at 150.4, 146.0, and 136.6
ppm, while for Ru(phen) 2 Cl 2 , these signals were
seen at 154.7, 153.4, 151.2, and 149.7 ppm. In addition, the signals
of the aliphatic and aromatic carbons were observed at 20–58
and 110–162 ppm, respectively ( Figures S1–S8 ). HRMS was used to measure the mass spectra of
the RuL 2 Cl 2 complexes ( Figures S9 and S10 ). The results obtained were measured in accordance
with the expected
mass values. For example, while the mass value calculated for Ru(bpy) 2 Cl 2 was 484.9874, the mass value found was 484.9806. Finally, the synthesis of ruthenium–sorafenib (RuL 2 S) complexes as a result of the reaction of RuL 2 Cl 2 and sorafenib molecule was carried out according to the method
given in the literature. 32 , 33 The complexes were
synthesized with yields varying between 18 and 80%. Structures were
characterized by 1 H NMR and 13 C NMR spectra.
From the 1 H NMR spectra of RuL 2 S ( Figures S11–S30 ), the signals for aromatic
protons were observed between 6.94 and 9.25 ppm, while aliphatic proton
signals were observed at about 1.73–4.09 ppm. While NH protons
belonging to the urea group and belonging to the amide group were
not observed in the NMR measurements taken with CD 3 OD,
NH protons were observed at 9.89, 10.23, and 10.92 ppm in the measurement
using DMSO- d 6 . The specific NH–CH 3 proton of sorafenib gives signals in the range of 2.80–2.95
ppm. From the 13 C NMR spectra, the signals of the
aliphatic
and aromatic carbons were observed at 19.8–55.9 and 113.3–172
ppm, respectively. The C=O group belonging to the amide group
signals around 170 ppm, while the signals belonging to the NH–CH 3 carbon are seen around 26.5 ppm ( Figures S13 and S24 ). The mass spectra of these structures also gave
signals in the expected range for the complexes obtained ( Figures S31–S38 ). For example, while the
mass value calculated for Ru1S (C 41 H 32 ClF 3 N 8 O 3 Ru [M – H] − ) was 877.1203, the mass value found was 877.1192. In addition, the
results of the elemental analysis confirm the structures of the complexes
( Table S1 ). It is seen that the proton
integral values of Ru7S and Ru8S complexes
within the RuL 2 Srf complexes
are exactly twice as much as they should be in the 1 H NMR
spectra. Although flash chromatography was applied to these complexes
many times, the complexes preserved the same 1 H NMR spectra.
When the elemental analysis results for these structures are examined,
it is seen that all structures are compatible with the theoretical
elemental analysis values. This shows that ruthenium is exactly twice
as high in Ru7S and Ru8S complexes. According
to these results, we can say that these two complexes contain two
molecules in the same structure. In general, cis structures are expected
to form in the synthesis of RuL 2 Cl 2 complexes,
but besides cis structures, trans structures can also occur. In the
other complexes obtained, these trans structures were either not formed
or could be removed by flash chromatography. However, it was not possible
to remove the trans complex in Ru7S and Ru8S complexes. It was concluded that both cis and trans forms exist
for the Ru7S and Ru8S complexes. We can
say that the complexes we obtained contain a 1:1 ratio of cis and
trans isomers according to 1 H NMR integral values. When
the elemental analysis results of the RuL 2 Srf complex structures
are examined, it is seen that the structures that are compatible with
the theoretically calculated results do not contain impurities. Based
on all of these data, we can say that the complexes were successfully
synthesized. Additionally, absorption spectra were followed
for 3 days to test
the stability of sorafenib–ruthenium complexes ( Figure S48 ). These results show that the sorafenib–ruthenium
ligand complexes remain stable, and the results are consistent with
the results found by Yidan Lai et al. 26 2.2 Micelle Formation 5 mg of PEG–PLGA
(50:50, MW = 5–10 kDa) and 10 mg of Ru3S were
dissolved in 1 mL of DMSO and allowed to stir at rt. The resulting
mixture was added to 4 mL ultrapure water at a rate of 0.2 mL/min
with a syringe pump. It was left in the mixture overnight at 300 rpm.
The next day, the unloaded drug and DMSO were removed by dialysis
(MWCO: 3500) method. 34 The mixture was
stirred overnight at rt. A similar procedure was repeated to obtain Ru4S -loaded polymeric micelles. The size and polydispersity
of micelles were determined by DLS analysis. The drug loading content
(DLC) and entrapment efficiency (EE) were measured by LC-HRMS (Orbitrap
Q-Exactive HRMS system (Thermo Fisher Scientific)) analysis. 35 LC-HRMS analyses of calibration samples ( Ru3S and Ru4S ) were carried out as follows: A=
water-1% formic acid, 0.1% ammonium formate; B= methanol-1% formic
acid, 0.1% ammonium formate. The gradient elution program for Ru3S and Ru4S calibration samples was optimized
and conducted as follows: 1 min. 50% B, 6 min. 100% B, 1 min. 50%
B, followed by a 7 min conditioning period using a Troyasil C18 column
(150 mm × 3.0 mm, 3.5 μm). The injection volume of the
samples was 1 μL. 2.3 Biological Activity 2.3.1 hEGFR Inhibition The hEGFR enzyme
inhibitions of the synthesized complexes were determined using the
Takara Universal Tyrosine Kinase Assay Kit. 36 The inhibition results obtained as a result of this experiment are
given in Table 1 as
the IC 50 value, and the enzyme inhibition graphs are given
in Figure S49 . Table 1 hEGFR Enzyme Inhibition Results of
Ru–Sorafenib Complexes maximum
inhibition complex structure IC 50 (nM) (%) [I] (μM) Ru1S Ru(bpy) 2 Srf 3.9 ± 0.08 99.74 0.3 Ru2S Ru(4,4-dimebpy) 2 Srf 8.1 ± 0.2 96.20 0.3 Ru3S Ru(6,6-dimebpy) 2 Srf 2.5 ± 0.07 96.92 0.3 Ru4S Ru(4,4-di- t -bu-bpy) 2 Srf 5.8 ± 0.15 96.30 0.3 Ru5S Ru(4,4-dimethoxybpy) 2 Srf 1.2 ± 0.02 97.17 0.3 Ru6S Ru(phen) 2 Srf 13 ± 0.1 85.41 0.3 Ru7S Ru(5-Clphen) 2 Srf 0.8 ± 0.02 97.07 0.3 Ru8S Ru(5-mephen) 2 Srf 1.4 ± 0.03 97.07 0.3 Sorafenib 88.7 ± 1.2 100 5 According to the EGFR enzyme inhibition results, all
of the synthesized
complexes showed higher inhibition than the sorafenib. Ru7S showed the best inhibition with an IC 50 value
of 0.8 nM, and Ru8S and Ru5S complexes
showed high inhibition with IC 50 values of 1.2 nm and 1.4
nM close to this value. Other complexes also showed very high inhibition
compared to the IC 50 values of 88.7 nM concentration shown
by sorafenib. Among the complexes, Ru6S showed the lowest
inhibition with an IC 50 value of 13 nM. 2.3.2 Cell Viability against Normal and Cancer
Cell Lines The effects of the synthesized complexes on cell
viability were tested by MTT assay in HepG2, Caco-2, HT-29, MCF-7,
A549, and HEK293T cell lines. The IC 50 values calculated
after 48 h of substance incubation in each cell are given in Table 2 . Concentration-%
viability plots of each compound are presented in Figures S50–S55 . According to these results, Ru3S was the most effective complex on HepG2 cells. Sorafenib
showed a cytotoxic effect on HepG2 cells with a value of IC 50 = 5.20 μM, while Ru3S exhibited a cytotoxic effect
with an IC 50 = 7.13 μM close to this value. According
to the cell viability results of complexes in Caco-2 cells, the five
complexes demonstrated higher cytotoxicity than sorafenib. Sorafenib
showed cytotoxicity with IC 50 = 13.80 μM, while the
most effective complex Ru4S showed cytotoxicity with
a value of IC 50 = 2.47 μM. In addition, Ru2S , Ru3S , Ru5S , and Ru6S showed
better cytotoxic effects than sorafenib in this cell line. Table 2 IC 50 (μM) Values
of Synthesized Complexes Calculated from Percent (%) Viability Values
in Different Cell Lines Comp HepG2 Caco-2 HT-29 MCF-7 A549 HEK293T Ru1S 26.51 ± 10.43 18.61 ± 4.96 64.0 ± 22.62 17.18 ± 3.84 131.48 ± 22.83 169.48 ± 97.97 Ru2S 78.84 ± 42.81 10.98 ± 2.47 19.41 ± 5.58 17.96 ± 5.55 94.15 ± 14.76 144.43 ± 158.6 Ru3S 7.13 ± 2.12 9.57 ± 2.33 6.93 ± 1.76 12.09 ± 3.36 25.24 ± 2.87 83.79 ± 58.05 Ru4S 23.76 ± 4.23 2.47 ± 0.42 6.24 ± 1.36 5.35 ± 0.78 29.90 ± 4.48 33.15 ± 9.17 Ru5S 27.36 ± 5.62 11.44 ± 3.76 21.88 ± 7.19 9.47 ± 1.96 34.72 ± 4.78 87.34 ± 35.95 Ru6S 54.79 ± 28.65 12.42 ± 2.75 41.77 ± 11.88 19.33 ± 4.09 65.03 ± 12.40 66.15 ± 18.46 Ru7S 30.01 ± 6.51 17.01 ± 4.25 14.60 ± 3.46 10.11 ± 2.65 172.97 ± 57.41 764.61 ± 188 Ru8S 25.65 ± 4.43 20.45 ± 4.80 33.56 ± 10.17 33.07 ± 12.50 92.36 ± 19.80 16.55 ± 18.32 Srf 5.20 ± 1.66 13.80 ± 3.89 7.89 ± 2.53 10.55 ± 2.60 7.48 ± 0.86 0.15 ± 0.16 The Ru4S complex exhibited the highest
cytotoxicity
on the HT-29 cell line. Sorafenib showed activity with IC 50 = 7.89 μM, while Ru4S was effective with IC 50 = 6.24 μM. Furthermore, in this cell line, Ru3S exhibited very close cytotoxicity (IC 50 = 6.93 μM)
to that of Ru4S ( IC 50 = 6.24 μM). On
the other hand, the Ru4S complex (IC 50 = 5.35
μM) showed a higher cytotoxic effect than sorafenib (IC 50 = 10.55 μM) in the MCF-7 cell line. In the A549 cell
line, none of the complexes demonstrated higher cytotoxicity than
sorafenib. However, when the complexes were compared within themselves, Ru3S and Ru4S showed the best cytotoxic properties. When the effects of the complexes on the healthy cell line (HEK293T)
were compared with sorafenib, the complexes showed low cytotoxicity
on healthy cells. Sorafenib showed higher cytotoxicity on the healthy
HEK293 cell line with IC 50 = 0.15 μM, while Ru3S showed cytotoxicity with IC 50 = 83.79 μM
and Ru4S IC 50 = 33.15 μM. The selectivity
of Sorafenib and Ru 3s and Ru4S over cancer
cells and healthy cells is a huge advantage for the potentials of
the complexes. Since sorafenib is the first-line drug in hepatocellular
carcinoma, we continued our studies with HepG2 cells. Cytotoxic
effects of Ru3S and Ru4S evaluated
in sorafenib-resistant HepG2 cell line HepG2-SR. IC 50 values
of Ru3S , Ru4S , and sorafenib increased compared
to the parental HepG2 cell line; however, the increment in the IC 50 of Ru3S was lower compared to that of sorafenib
( Figure 2 ). Figure 2 Effect of Ru3S and Ru4S on cell viability
in sorafenib-resistant HepG2-SR cells. 2.3.3 Cell Apoptosis and Cell Cycle The
apoptotic and cell cycle properties of the Ru3S and Ru4S complexes were determined on the HepG2 cell line in the
Muse Cell Analyzer. The selected Ru3S and Ru4S complexes were treated at 3 or 4 different concentrations for 48
h. At the end of this period, viable, early apoptotic, late apoptotic,
and dead cell profiles for apoptosis were determined in duplicate
for each compound and concentration. Cell profiles were plotted as
% and statistical calculations were performed. The resulting graphs
are given in Figure 3 A, and the apoptotic profiles of the complexes are given in Figure 3 B. Supplementary
apoptotic profiles are presented in Figure S56 . Figure 3 (A) Effect of concentration-dependent (0.78, 3.12, 12.5, and 50
μM) administration of Ru3S , Ru4S ,
and sorafenib on the percentages (%) of live, early apoptotic, late
apoptotic, and dead HepG2 cells. (B) Apoptotic profiles of concentration-dependent
administration of Ru3S , Ru4S , and sorafenib
in HepG2 cells. The analysis of the difference between groups was
conducted using a two-way analysis of variance (ANOVA) with a Dunnett
post hoc test in GraphPad Prism 9.00. A significance level of p < 0.05 was deemed as statistically significant. * p < 0.05; ** p < 0.01; *** p < 0.001; p < 0.0001. According to the apoptosis results, Ru3S decreased
the live cell percentage at all concentrations ( p < 0.01) and increased the rate of early apoptotic cells at 3.12
and 12.5 μM concentrations ( p < 0.05), the
rate of late apoptotic cells at 50 μM concentration ( p < 0.001). However, the rate of necrotic cells did not
change depending on the concentration ( p > 0.05). Ru4S showed a different profile than Ru3S . Ru4S decreased the live cell percentage at all concentrations
after 0.78 μM ( p < 0.05). The rate of early
apoptotic cells increased at lower concentrations (0.78 and 3.12 μM
( p < 0.0.5)), while at higher concentrations the
proportion of late apoptotic cells (3.12, 12.5, and 50 μM) and
dead cells (12.5 and 50 μM) increased compared to control ( p < 0.0.5). Apoptotic profiles of sorafenib and Ru3S were similar, sorafenib decreased the live cell percentage
at all concentrations ( p < 0.0001). However, different
from Ru3S , it increased the rate of early apoptotic cells
at all concentrations ( p < 0.0001) as well as
that of late apoptotic cells at higher concentrations (12.5 and 50
μM) ( p < 0.01). The necrotic cell percentage
did not change ( p > 0.05). On the other
hand, the percentage of cells in cell cycle phases
for cell cycle was determined by measuring DNA content. Sub G1 (DNA
content of cells in apoptotic phase), G0/G1 (Resting and protein synthesis),
S (DNA synthesis), and G2/M (Protein synthesis and mitosis) phases
were determined. DNA content was plotted as % and statistical calculation
was made. The resulting graphs are given in Figure 4 A, and the cell cycle profiles of the complexes
are given in Figure 4 B. Supplementary profiles are presented in Figure S57 . Figure 4 (A) Effect of concentration-dependent (0.78, 3.12, 12.5, and 50
μM) administered Ru3S , Ru4S , and sorafenib
on cell cycle phases in HepG2 cells. (B) Cell cycle profiles of concentration-dependent
administered Ru3S , Ru4S , and sorafenib in
HepG2 cells. The analysis of the difference between groups was conducted
using a two-way ANOVA with a Dunnett post hoc test in GraphPad Prism
9.00. A significance level of p < 0.05 was deemed
as statistically significant. * p < 0.05; ** p < 0.01; *** p < 0.001; p < 0.0001. Dose-dependent Ru3S administration
did not affect
cell cycle phases much in HepG2 cells. It increased Sub G1 population
at 12.5 μM. However, although there was a dose-dependent decrease
in G0/G1, S, and G2/M, it was not statistically significant ( p > 0.05). In contrast, Ru4S showed different
effects on cell cycle profiles at different doses. At low concentration
(0.78 μM), it increased Sub G1 ( p < 0.0001)
and decreased G0/G1, S, and G2/M percentages ( p <
0.05). At higher concentrations (3.12 and 12.5 μM) Sub G1 was
slightly increased ( p < 0.01), G0/G1 was slightly
decreased ( p < 0.01), S phase was not changed,
and G2/M was decreased ( p < 0.05). At the highest
50 μM dose, Ru4S increased Sub G1( p < 0.0001) and decreased S ( p < 0.01), and
did not affect the G0/G1 and G2/M phases. On the other hand, sorafenib
increased Sub G1 and decreased other phases similar to Ru3S . Sub G1 was decreased at all concentrations ( p <
0.05), G0/G1 was decreased at 50 μM ( p <
0.01), S phase was decreased at 12.5 and 50 μM ( p < 0.05), and G2/M was decreased; however, it was not statistically
significant ( p > 0.05). 2.3.4 Effect of Synthesized Complexes on Expression
of Tyrosine Kinase Pathway Proteins The effects of synthesized
complexes on the expression of proteins in the tyrosine kinase pathway
(EGFR, p-EGFR, Akt, p-Akt, Erk1/2 (p44/42 MAPK), p-Erk1/2 (p-p44/42
MAPK)) determined by the immunoblotting method in HepG2 cells. The
relative calculated densities of the band volumes for each sample
normalized to the reference protein (GAPDH) and representative bands
are given in Figure 5 A. The difference was evaluated statistically compared to the control
and between high and low concentrations. Figure 5 (A) Concentration (0.78
and/or 3.12 and/or 12.5 μM)-dependent
effect of Ru3S , Ru4S , and sorafenib on expression
of tyrosine kinase pathway proteins in HepG2 cells (B) Concentration
(0.78 and/or 3.12 and/or 12.5 μM)-dependent antiangiogenic effect
of Ru3S , Ru4S , and sorafenib on expression
of tyrosine kinase pathway proteins in HUVEC cells. The analysis of
the difference between groups was conducted using a one-way ANOVA
with a Tukey post hoc test in GraphPad Prism 9.00. A significance
level of p < 0.05 was deemed as statistically
significant. * p < 0.05; ** p <
0.01. n = 2–3. Total EGFR decreased compared to control after Ru4S administration ( p < 0.05), and p-EGFR
decreased
statistically in higher concentrations compared to control after Ru3S ( p < 0.01) and Ru4S ( p < 0.05) administration. Ru3S (12.5 μM; p < 0.05) and Ru4S (3.12 and 0.78; p < 0.01) decreased total Akt compared to control. No
compound has changed the phosphorylated Akt compared to control; however,
phosphorylated Akt decreased at higher concentrations of Ru3S compared to lower concentrations ( p < 0.05).
Total Erk1/2 decreased at lower and higher concentrations of Ru3S , Ru4S , and sorafenib compared to control
( p < 0.05), while p-Erk1/2 decreased at higher
concentrations compared to lower concentrations in Ru3S ( p < 0.01) and at all concentrations of Ru4S compared to control ( p < 0.05). 2.3.5 Antiangiogenic Effect The antiangiogenic
effects of synthesized complexes in HUVEC (Human umbilical vein endothelial
cell) cells were determined by evaluating protein expressions in the
angiogenesis pathway (VEGFR, Akt, p-Akt, Erk1/2 (p44/42 MAPK), p-Erk1/2
(p-p44/42 MAPK)) by Western blot method. The relative calculated densities
of the band volumes for each sample normalized to the reference protein
(GAPDH) and representative bands are given in Figure 5 B. The differences were evaluated statistically
compared to the control and between high and low concentrations. Total VEGFR decreased compared to control after higher concentrations
of Ru4S and sorafenib administration ( p < 0.05) and decreased in higher concentrations compared to lower
after Ru3S ( p < 0.05). Total Akt
decreased after high and low concentrations of Ru3S and
high concentrations of Ru4S compared to control ( p < 0.05). However, p-Akt decreased after high and low
concentrations of Ru3S and sorafenib, and high concentration
of Ru4S compared to control ( p <
0.05). Total Erk1/2 and p-Erk1/2 decreased after higher concentrations
of Ru3S and sorafenib compared to controls ( p < 0.05). Upon combining the outcomes of enzyme inhibition,
cell cytotoxicity,
and expression of tyrosine kinase pathway proteins, it was observed
that sorafenib–ruthenium ligand complexes increased enzyme
inhibition by at least 6-fold compared to sorafenib alone. In addition,
we have shown in this study that while sorafenib has a very high cytotoxic
effect on healthy cells, sorafenib–ruthenium ligand complexes
reduce this effect by at least 100-fold. We also demonstrated that
ruthenium complexes down-regulate both phospho- and total Erk1/2 (MAPK1/2)
in HepG2 and HUVEC cells, where Erk is up-regulated in resistant cell
lines. Additionally, ruthenium complexes inhibited VEGFR expression
in HUVEC cells. A study conducted by Morgillo et al. provided
evidence of a positive
interaction between sorafenib and anti-EGFR drugs. 37 , 38 Accordingly, they provided evidence that the combined use of sorafenib
and an EGFR inhibitor in EGFR and/or VEGFR inhibitor-resistant human
cancer cells is active in inhibiting tumor cell growth in
vitro and in vivo . Here, the effect of sorafenib
was found to be linked to its ability to block RAF signaling via the
RAS/RAF/MEK/MAPK pathway. These results are consistent with our study
findings. When the outcomes of our study and the study conducted by
Morgillo et al. are compared, it becomes evident that our study indirectly
validates the notion that the synergistic effect is amplified when
the inhibition effect is employed concurrently. Because the new sorafenib–ruthenium
ligand complexes we made in this study are such good at blocking EGFR,
they may have helped a mechanism develop that can do this job without
the need for other inhibitors. Although establishing a definitive
mechanism is challenging, additional ruthenium and ligand moieties
may have the effect of blocking different pathways. These mechanisms
may become apparent through subsequent comprehensive investigations
in the field. 2.3.6 Effect of Micelles Obtained from Synthesized
Complexes on Cell Viability When the enzyme inhibition and
cytotoxicity results are evaluated together, Ru3S and Ru4S showed potential as active drug substances. Therefore,
the effects of these complexes were investigated on apoptosis, cell
cycle, antiangiogenic effects, and proteins in the tyrosine kinase
pathway. In order to increase the bioavailability of the obtained
complexes we prepared micelle forms and investigated their drug release,
cytotoxic, and apoptotic properties. The effects of M1 and M2 and noncomplex M on cell viability,
respectively, obtained using Ru3S and Ru4S substances were tested by MTT assay in HepG2 cells. IC 50 values were calculated from the percent viability values calculated
according to the negative controls with the GraphPad Prism program.
IC 50 values calculated after 48 h of substance incubation
are presented graphically in Figure 6 A. According to the cytotoxicity results of the prepared Ru3S - and Ru4S -loaded micelles on the HepG2 cell
line, noncomplex micelle M showed the value of IC 50 = 175.2 μM, the Ru3S -loaded M1 micelles showed IC 50 = 1.07 μM, and the Ru4S -loaded M2 micelles showed IC 50 = 3.52 μM.
These values showed that the micelle-loaded forms exhibited higher
cytotoxicity when compared to the results of the complexes without
loading the micelle ( Ru3S ; IC 50 = 7.13 μM; Ru4S , IC 50 = 23.76 μM). Figure 6 (A) Concentration–viability
(%) graphics of noncomplex M , Ru3S -loaded M1 , and Ru4S -loaded M2 micelles
in HepG2 cell line. (B) Concentration-dependent
apoptotic cell levels of M1 and M2 in HepG2
cells. The apoptotic properties of M1 and M2 were determined on the HepG2 cell line in the Muse Cell
Analyzer.
Viable, early apoptotic, late apoptotic, and dead cell profiles were
determined for each compound at 2 concentrations and 2 replicates.
The resulting graphs are presented in Figure 6 B, and the profiles of the apoptotic properties
of the compounds are presented in Figure S19 . According to these results, it was determined that M1 prepared from Ru3S decreased the percentage
of viable
cells more than free Ru3S , despite the lower concentration.
Also, M1 increased the early apoptotic cells compared
to control and free Ru3S . Even though M2 decreased live cells with increasing concentrations, it increased
late apoptotic and dead cells instead of early apoptotic cells as
we have seen in free Ru4S . Both cytotoxicity and apoptosis
results show that micelles prepared from the complexes increase the
cytotoxic effect and the most prominent compound was Ru3S as well as its micelle M1 . 2.4 Molecular Docking Studies In this
study, to explain the inhibition mechanisms of sorafenib–ruthenium
complexes molecular docking studies of both sorafenib and its ruthenium
complexes were performed and the results were compared. Molecular
docking studies were performed with the induced fit docking (IFD)
technique, and MM-GBSA (molecular mechanics with generalized born
and surface area) Δ G binding free energies
and glide emodel scores of the compounds against the target protein
EGFR were also determined. The induced fit docking XP (extra
precision) glide scores, MM-GBSA Δ G binding
free energies, and glide emodel scores of the ruthenium complexes
as well as sorafenib against EGFR protein are given in Table 3 . Table 3 Molecular Docking IFD Scores and MM-GBSA
Δ G Binding Free Energies of Ruthenium Complexes
and Sorafenib Compounds IFD XP Gscore (kcal/mol) MM-GBSA Δ G bind (kcal/mol) glide emodel (kcal/mol) Ru1S –8.583 –66.17 –103.735 Ru2S –10.554 –64.54 –100.927 Ru3S –10.771 –78.51 –117.996 Ru4S –7.524 –60.41 –85.799 Ru5S –6.277 –69.27 –59.830 Ru6S –10.163 –65.67 –85.368 Ru7S –5.354 –67.30 –27.392 Ru8S –9.150 –67.00 –24.458 Sorafenib –9.812 –60.12 –90.643 According to the molecular docking studies, Ru3S exhibited
the best IFD XP Gscore with −10.771 kcal/mol, indicating strong
binding interaction, best MM-GBSA Δ G binding
free energy at −78.51 kcal/mol, implying a stable binding affinity,
and best Glide emodel score (−117.996 kcal/mol), suggesting
a potentially strong binding mode. All scores of Ru3S are better than sorafenib. The IFD score, MM-GBSA Δ G binding free energy, and glide emodel of sorafenib are
−9.812, −60.12, and −90.643 kcal/mol, respectively.
In addition to Ru3S , the IFD scores of Ru2S and Ru6S are −10.554 and −10.163 kcal/mol,
respectively, and also they are better than sorafenib. Other ruthenium
complexes showed moderate and comparable docking scores, and they
varied from −5.354 to −9.150 kcal/mol. According
to in vitro test results, the IC 50 values
of all of the ruthenium complexes against hEGFR enzyme
inhibitions are at the nanomolar level. According to the results obtained
from anticancer studies, the compound with the highest selectivity
against healthy and cancer cells is the Ru3S complex.
In the in vitro anticancer activity studies against
five different cancer cell lines, the inhibitions of sorafenib and Ru3S are very close to each other, but the fact that the toxicity
of Ru3S in healthy cells is much lower than sorafenib.
Since Ru3S has better scores than all other ruthenium
complexes and sorafenib in molecular docking studies, detailed molecular
docking analyses of sorafenib and Ru3S were evaluated
and the inhibition mechanisms were discussed and compared with each
other. Molecular docking two-dimensional (2D) and three-dimensional
(3D)
ligand–protein interactions of sorafenib–EGFR ligand–protein
complex are given in Figure 7 . As seen in Figure 7 a, the oxygen of urea carbonyl formed a hydrogen-bond
interaction
with Thr-854. Both nitrogen of urea group formed an additional hydrogen-bond
interaction with Gln-791. Finally, the nitrogen atom of the pyridyl
ring also formed a hydrogen-bond interaction with Cys-797. In Figure 7 b, the yellow dashes
show hydrogen-bond interactions. Hydrogen-bond lengths vary from 1.86
to 2.36 Å. Key amino acids in the Srf-EGFR ligand–protein
complex stand out as Gln-791, Thr-854, and Cys-797. Four different
hydrogen bonds occurring in the active site of the enzyme indicate
the stability of the Srf-EGFR ligand–protein complex
and its inhibition ability. Figure 7 Molecular docking ligand–protein interactions
between sorafenib
and the active site of EGFR (PDB: 5X2A ): (a) 2D ligand–protein interactions
and (b) 3D ligand–protein interactions. Molecular docking two-dimensional (2D) and three-dimensional
(3D)
ligand–protein interactions of Ru3S-EGFR ligand–protein
complex are given in Figure 8 . As can be seen from Figure 8 a, there are three different hydrogen-bond interactions,
two different π–π stacking interactions, and two
different π–cationic interactions between Ru3S and the amino acid residues in the active site of EGFR. Figure 8 Molecular docking
ligand–protein interactions between Ru3S and the
active site of EGFR (PDB: 5X2A ): (a) 2D ligand–protein
interactions, (b) 3D ligand–protein interactions. When we look at the ligand–protein interactions
of Ru3S - EGFR complex, it is seen that the
oxygen
of the urea carbonyl interacts with Thr-854, and the urea nitrogen
interacts with Gln-791, as in the Srf-EGFR complex. Since
the nitrogen atom in the pyridine ring of sorafenib is coordinated
with Ru, the interaction of this nitrogen atom with Cys-797 in sorafenib
is not seen in the Ru3S-EGFR complex. Instead, the pyridine
ring of sorafenib makes a π–cationic interaction with
Lys-728, while the oxygen of the amide carbonyl makes a hydrogen-bond
interaction (purple) with Lys-716. Crucially, the bipyridyl rings
attached to Ru engage in π–π stacking (green) and
π–cationic (red) interactions with Phe-795, His-805,
and Lys-716 amino acid residues in the EGFR active site. These interactions
likely contribute to stabilizing the Ru3S-EGFR complex
and disrupting the normal function of EGFR. Figure 8 b depicts
the 3D ligand–protein interactions of the Ru3S-EGFR complex. The yellow dashes represent the hydrogen-bond interactions,
the turquoise dashes represent the π–π stacking
interactions, and the red dashes represent the π–cationic
interactions. On the other hand, ruthenium (Ru) forms six coordination
bonds with six different nitrogen atoms provided by sorafenib and
the bipyridyl rings. In the Ru3S-EGFR complex, the hydrogen-bond
lengths range from 1.95 to 2.07 Å, which closely resemble the
hydrogen-bond lengths in the Srf-EGFR complex. Shorter
hydrogen bonds signify stronger binding and a more stable ligand–protein
complex. Additionally, the lengths of the Ru–N coordination
bonds correlate with normal bond lengths, measured at 2.1 Å.
As depicted in Figure 8 b, the bipyridyl rings are positioned perpendicular to each other
and to sorafenib. These ring orientations contribute to the π–cationic
and π–π stacking interactions observed in the ligand–protein
complex, enhancing both stability and inhibition. When the interactions
of sorafenib and Ru3S with EGFR
are compared, they share some common features such as hydrogen-bond
interactions with Thr-854 and Gln-791. However, Ru3S distinguishes
itself by contributing unique additional interactions, specifically
π–cationic and π–π stacking interactions.
These distinctive features contribute to the inhibitory potential
of Ru3S and highlight its prominence in ligand–protein
stability. The additional π–cationic and π–π
stacking interactions of Ru3S likely play a significant
role in enhancing its inhibitory capacity compared to sorafenib. Molecular docking two-dimensional (2D) and three-dimensional (3D)
ligand–protein interactions of the rest of the ruthenium complex
are given in the Supporting Information ( Figures S62–S75 ). 2.5 Molecular Dynamics Simulations In
this study, 200 ns MD simulation analyses were carried out for Srf-EGFR and Ru3S-EGFR complexes and compared
the obtained results. The findings obtained from the MD simulation
of the Srf-EGFR complex were compared with those derived
from the Ru3S-EGFR complex. This comparative analysis
aimed to highlight the differences between the inhibitory effects
demonstrated by sorafenib alone and the effects exhibited by Ru3S . The objective was to elucidate and outline the distinctions
in the inhibition mechanisms of sorafenib and Ru3S when
interacting with the EGFR complex. The 200 ns MD simulation
analysis of the Srf-EGFR complex is given in Figure 9 . Figure 9 a represents the 2D key ligand–protein
interactions with the percentage simulation time of Srf-EGFR MD simulations. Figure 9 200 ns MD simulation analysis of Sorafenib-EGFR complex.
(a) 2D key ligand–protein interactions, (b) RMSD graphics of
protein and ligand atoms, (c) RMSF graphic of protein Cα atoms,
(d) RMSF graphic of ligand atoms, and (e) fractional interaction histogram. As seen in Figure 9 a, the urea carbonyl formed a water-bridged hydrogen-bond
interaction
with Arg-841 (30% of the simulation time). The urea carbonyl also
formed a direct hydrogen-bond interaction with Lys-745 (27% of the
sim.). In addition, the urea carbonyl also formed additional two water-bridged
hydrogen-bond interactions with Lys-745 (14% of the sim.) and Asp-885
(12% of the sim.). In addition to these interactions, there are several
hydrogen bonds and water-bridged hydrogen-bond interactions with different
percentages of the simulation time. The other most important interactions
for the Srf-EGFR complex are the interactions occurring
between urea nitrogens and Gln-791 (9% of the sim.) and Asp-800 (25%
of the sim.). Although Gln-791 stood out as the key amino acid for
sorafenib in molecular docking studies, its interaction in MD simulations
continued for only 9% of the simulation time. In addition, the amino
acid residue Asp-800 showed multiple water-bridged hydrogen-bond interactions
throughout the simulation. Finally, Arg-841 shoved a π–cationic
interaction with the p -substituted phenyl ring during
13% of the simulation time. According to Figure 9 b, the average RMSD of the protein Cα
was calculated as 2.7
Å, the average RMSD of ligand atoms was calculated as 7.50 Å,
and the average ligand deviation from its original position was 2.4
Å. While the RMSD of the ligand atoms at 4.5 Å is almost
half of the simulation, it stabilized at 9 Å levels in the second
half of the simulation. Since 9 Å is a high value for the ligand
RMSD, the stability of the ligand–protein complex is thought
to be low. Figure 9 c,d represents the RMSF values of the protein Cα atoms and
ligand atoms, respectively. As seen in Figure 9 c,d, the average RMSF of the protein Cα
and ligand atoms were calculated as 1.8 and 4 Å, respectively.
In Figure 9 c, the vertical
green bars represent the ligand-amino acid contacts during the simulation
time. Finally, Figure 9 e represents the fractional interaction histograms of the Srf-EGFR complex. Throughout the simulation, interactions
like hydrogen bonds
and hydrophobic, ionic, and water bridges were continuously monitored
and categorized. The stacked bar charts in Figure 9 e efficiently show how often these interactions
occur, with values like 0.8 indicating an 80% presence. Values over
1.0 suggest instances where specific protein residues engage with
the ligand multiple times in the same category. The most abundant
fractional interactions were observed with Arg-841, Asp-800, Leu-718,
Lys-845, and Gln-791. These amino acid residues are the key amino
acids that showed multiple interactions during the simulation. The 200 ns MD simulation analysis of the Ru3S-EGFR complex
is given in Figure 10 . Figure 10 a represents
the 2D key ligand–protein interactions with the
percentage simulation time of Ru3S-EGFR MD simulations. Figure 10 200
ns MD simulation analysis of Ru3S-EGFR complex.
(a) 2D key ligand–protein interactions, (b) RMSD graphics of
protein and ligand atoms, (c) RMSF graphic of protein Cα atoms,
(d) RMSF graphic of ligand atoms, and (e) fractional interaction histogram. As seen in Figure 10 a, the urea nitrogens formed two hydrogen-bond interactions
with
Gln-791 (47 and 50% of the sim), and a water-bridged hydrogen-bond
interaction with Gln-791 (37% of the sim.). On the other hand, the
urea carbonyl formed two different water-bridged hydrogen-bond interactions
with Asp-855 (33% of the sim.) and Lys-745 (29% of the sim.). In addition,
the amid carbonyl formed an additional water-bridged hydrogen-bond
interaction with Val-742 (24% of the sim.) and the bipyridyl ring
of the Ru complex formed two π–cationic bond interactions
with Lys-728. For the Gln-791 key amino acid residue, which stands
out in molecular docking studies for both ligands, the interaction
time in the Srf-EGFR complex is 9% of the simulation,
while this time is 50% in the Ru3S-EGFR complex. This
result indicates that the Ru3S forms a stable complex
throughout the simulation because binding to a specific amino acid
residue and remaining bound for a long time is better than short-term
interactions with more than one amino acid residue. Moreover, π–cationic
interactions of bipyridyl rings attached to the ruthenium complex
contribute to the stability of the ligand–protein complex. Figure 10 b represents
the RMSD values of the Ru3S-EGFR complex. According to Figure 10 b, the average
RMSD of the protein Cα atoms was calculated as 2.8 Å (pale
blue), the average RMSD of the ligand atoms was calculated as 4.5
Å, and the average deviation of the ligand from its original
position is 1 Å. Comparing the RMSD values, the Ru3S-EGFR complex appears more stable than the Srf-EGFR complex.
In Ru3S-EGFR , the ligand shows a lower average RMSD (4.5
Å) and a smaller deviation from its original position (1 Å)
compared to Srf-EGFR (ligand RMSD of 7.50 Å and
a deviation of 2.4 Å). These results suggest that Ru3S maintains a more consistent position within the binding site, indicating
greater stability during the simulations. Figure 10 c,d represents
the RMSF plots of the protein Cα atoms and ligand atoms of Ru3S-EGRF complex, respectively. The average RMSF of the protein
Cα was calculated as 1.8 Å, and this value is the same
as the value in the Srf-EGFR complex. The protein backbones
have the same stability and fluctuation in both complexes. On the
other hand, the RMSF of the ligand atoms was calculated to be 1.5
Å on average, and this value is much lower than that of the Srf-EGFR complex. The ruthenium complex created a more stable
ligand–protein complex by restricting the fluctuation of the
molecule throughout the simulation. Finally, Figure 10 e represents the fractional interaction
histogram of the Ru3S-EGFR complex during the 200 ns
simulation time. The most abundant fractional histograms were observed
with Gln-791, Leu-792, Met-790, and Lys-728. Actually, the abundance
of the Gln-791 fractions is so high and Gln-791 stands out as the
key amino acid residue for the inhibition. In conclusion, the
200 ns MD simulations of Srf-EGFR and Ru3S-EGFR complexes revealed distinct dynamic behaviors.
In Srf-EGFR , diverse interactions were observed with
varying durations, resulting in ligand fluctuations (average RMSD
of 7.50 Å). Conversely, Ru3S-EGFR exhibited enhanced
stability, demonstrated by sustained hydrogen-bond interactions, lower
ligand RMSD (4.5 Å), and a smaller deviation (1 Å). The
π–cationic interactions of the bipyridyl ring in Ru3S contributed to its stability. Both complexes highlighted
Gln-791 as a key amino acid, but Ru3S exhibited more
abundant and consistent interactions, making it a potentially more
stable and promising candidate for drug development. The RMSF analysis
indicated a more rigid binding in Ru3S-EGFR , emphasizing
the advantages of Ru3S in forming a stable ligand–protein
complex. 2.6 Micelle Formation and Drug Release Studies The size of Ru3S - and Ru4S -loaded PEG–PLGA
polymeric micelles was performed with DLS analysis. M1 and M2 refer to Ru3S - and Ru4S -loaded polymeric micelles, respectively. The sizes of micelles were
99.7 ± 0.9 nm with a PDI value of 0.22 for M1 and
67.7 ± 0.7 nm with a PDI value of 0.23 for M2 . The
amount of Ru3S and Ru4S loaded into the
micelles was determined by LC-HRMS analysis. A certain amount of freeze-dried M1 and M2 micelles was dissolved in DMF, and
the LC-HRMS was carried out. The drug loading content (DLC) and entrapment
efficiency (EE) were determined according to the equations in Figure S60 . According to these equations, %DLC
of 7.81% and %EE of 15.6% were determined for Ru3S -loaded
micelles (M1). For Ru4S -loaded micelles (M2), %DLC and
%EE were determined as 31.25 and 62.5%, respectively. To investigate
the controlled release properties of polymeric micelles, the prepared M2 nanocarrier was dispersed in two buffer solutions at different
pH values (7.4 and 5.6) at 37 °C. The release profile of Ru4S from M2 was
determined by LC-HRMS analysis. As expected, only a very small amount
(less than 25%) of the Ru4S was released from M2 in a neutral medium (pH = 7.4) even after 24 h. However, in a weakly
acidic environment (pH = 5.5), the release was 80% as shown in Figure S61C . At pH values of 7.4 and 5.5, release
efficiencies of 34 and 93% were achieved after 96 h, respectively
( Figure S61 ).
## Chemistry
2.1 Chemistry Novel targeted ruthenium–sorafenib
complexes were synthesized as shown in Scheme 1 . Synthesis of sorafenib was carried out
according to the method in the literature in four steps. 27 − 29 The phenanthroline ring readily reacted with RuCl 3 and
LiCl in DMF 30 to obtain ruthenium–ligand
complexes ( RuL 2 Cl 2 ). Bipyridine and 4,4-dimethyl bipyridine
structures were successfully synthesized using this method. But for
substituents; when 6,6-dimethyl was changed to 4,4-dimethoxy and 4,4-di- t -butyl, the synthesis of these compounds by the same method
was not possible. Here, an ethylene glycol–water mixture was
used as a solvent, benefiting from the work of” Viala and Coudret. 31 In this method, in which glucose and ascorbic
acid are used as reducing agents, high concentrations of Cl ions were
used by using an ethylene glycol–water mixture, which enabled
the reaction to take place. In addition, removing the substance by
heating it with dichloromethane for purification has been an effective
method to remove the complex from many salts formed. At this stage,
the yields of the complexes were quite high with 55–78%. Scheme 1 Synthesis of Sorafenib–Ruthenium Complexes (i) SOCl 2 , 72 °C,
16 h, (ii) CH 3 NH 2 , MeOH, 0 °C, (iii) t -BuOK, DMF, K 2 CO 3 , 80 °C, p -aminophenol, (iv) THF, Et 3 N, 60 °C, (v)
Method A: ligand, DMF, 80 °C, N 2 , 18 h, Method B:
ligand, ethylene glycol, glucose, ascorbic acid, 110 °C, 1 h,
(vi) MeOH, 60 °C, 18 h. From the 1 H NMR spectra of the obtained RuL 2 Cl 2 complexes compared with the 1 H NMR spectra
of the ligands, it is seen that the aromatic ring signals shift to
a lower area in the 1 H NMR spectrum ( Figures S1–S8 ). For example, the signal seen in the
lowest area of the 1 H NMR spectrum of the bipyridine (bpy)
ligand is seen at 8.79 and 8.47 ppm. In the obtained Ru(bpy) 2 Cl 2 complex, the signals in the lowest area are seen at
9.98 and 8.64 ppm. Similarly, the signal seen in the lowest area of
the 1 H NMR spectrum of the other phenanthroline (phen)
ligand is at 9.77 and 8.46 ppm, while the peaks of the ruthenium complex
of this ligand [Ru(phen) 2 Cl 2 ] are seen at 10.28
and 8.72 ppm. In addition, in the 1 H NMR spectra of RuL 2 Cl 2 , the signals for aromatic protons were observed
between 6.92 and 10.35 ppm, while aliphatic proton signals were observed
about at 1.33–4.11 ppm. From the comparison of the 13 C NMR spectra of the RuL 2 Cl 2 complexes
with the spectra of the ligands,
it is seen that the aromatic ring carbons are similarly signaled in
the lower area. In the 13 C NMR spectrum of the bipyridine
(bpy) ligand, the signals seen in the lowest area are 155.8, 149.7,
and 137.7 ppm, while the 13 C NMR spectrum of the Ru(bpy) 2 Cl 2 complex is seen at 159.5, 157.5, 152.1, and
151.3 ppm. Similarly, in the 13 C NMR spectrum of the phenanthroline
(phen) ligand, these signals were seen at 150.4, 146.0, and 136.6
ppm, while for Ru(phen) 2 Cl 2 , these signals were
seen at 154.7, 153.4, 151.2, and 149.7 ppm. In addition, the signals
of the aliphatic and aromatic carbons were observed at 20–58
and 110–162 ppm, respectively ( Figures S1–S8 ). HRMS was used to measure the mass spectra of
the RuL 2 Cl 2 complexes ( Figures S9 and S10 ). The results obtained were measured in accordance
with the expected
mass values. For example, while the mass value calculated for Ru(bpy) 2 Cl 2 was 484.9874, the mass value found was 484.9806. Finally, the synthesis of ruthenium–sorafenib (RuL 2 S) complexes as a result of the reaction of RuL 2 Cl 2 and sorafenib molecule was carried out according to the method
given in the literature. 32 , 33 The complexes were
synthesized with yields varying between 18 and 80%. Structures were
characterized by 1 H NMR and 13 C NMR spectra.
From the 1 H NMR spectra of RuL 2 S ( Figures S11–S30 ), the signals for aromatic
protons were observed between 6.94 and 9.25 ppm, while aliphatic proton
signals were observed at about 1.73–4.09 ppm. While NH protons
belonging to the urea group and belonging to the amide group were
not observed in the NMR measurements taken with CD 3 OD,
NH protons were observed at 9.89, 10.23, and 10.92 ppm in the measurement
using DMSO- d 6 . The specific NH–CH 3 proton of sorafenib gives signals in the range of 2.80–2.95
ppm. From the 13 C NMR spectra, the signals of the
aliphatic
and aromatic carbons were observed at 19.8–55.9 and 113.3–172
ppm, respectively. The C=O group belonging to the amide group
signals around 170 ppm, while the signals belonging to the NH–CH 3 carbon are seen around 26.5 ppm ( Figures S13 and S24 ). The mass spectra of these structures also gave
signals in the expected range for the complexes obtained ( Figures S31–S38 ). For example, while the
mass value calculated for Ru1S (C 41 H 32 ClF 3 N 8 O 3 Ru [M – H] − ) was 877.1203, the mass value found was 877.1192. In addition, the
results of the elemental analysis confirm the structures of the complexes
( Table S1 ). It is seen that the proton
integral values of Ru7S and Ru8S complexes
within the RuL 2 Srf complexes
are exactly twice as much as they should be in the 1 H NMR
spectra. Although flash chromatography was applied to these complexes
many times, the complexes preserved the same 1 H NMR spectra.
When the elemental analysis results for these structures are examined,
it is seen that all structures are compatible with the theoretical
elemental analysis values. This shows that ruthenium is exactly twice
as high in Ru7S and Ru8S complexes. According
to these results, we can say that these two complexes contain two
molecules in the same structure. In general, cis structures are expected
to form in the synthesis of RuL 2 Cl 2 complexes,
but besides cis structures, trans structures can also occur. In the
other complexes obtained, these trans structures were either not formed
or could be removed by flash chromatography. However, it was not possible
to remove the trans complex in Ru7S and Ru8S complexes. It was concluded that both cis and trans forms exist
for the Ru7S and Ru8S complexes. We can
say that the complexes we obtained contain a 1:1 ratio of cis and
trans isomers according to 1 H NMR integral values. When
the elemental analysis results of the RuL 2 Srf complex structures
are examined, it is seen that the structures that are compatible with
the theoretically calculated results do not contain impurities. Based
on all of these data, we can say that the complexes were successfully
synthesized. Additionally, absorption spectra were followed
for 3 days to test
the stability of sorafenib–ruthenium complexes ( Figure S48 ). These results show that the sorafenib–ruthenium
ligand complexes remain stable, and the results are consistent with
the results found by Yidan Lai et al. 26
## Micelle Formation
2.2 Micelle Formation 5 mg of PEG–PLGA
(50:50, MW = 5–10 kDa) and 10 mg of Ru3S were
dissolved in 1 mL of DMSO and allowed to stir at rt. The resulting
mixture was added to 4 mL ultrapure water at a rate of 0.2 mL/min
with a syringe pump. It was left in the mixture overnight at 300 rpm.
The next day, the unloaded drug and DMSO were removed by dialysis
(MWCO: 3500) method. 34 The mixture was
stirred overnight at rt. A similar procedure was repeated to obtain Ru4S -loaded polymeric micelles. The size and polydispersity
of micelles were determined by DLS analysis. The drug loading content
(DLC) and entrapment efficiency (EE) were measured by LC-HRMS (Orbitrap
Q-Exactive HRMS system (Thermo Fisher Scientific)) analysis. 35 LC-HRMS analyses of calibration samples ( Ru3S and Ru4S ) were carried out as follows: A=
water-1% formic acid, 0.1% ammonium formate; B= methanol-1% formic
acid, 0.1% ammonium formate. The gradient elution program for Ru3S and Ru4S calibration samples was optimized
and conducted as follows: 1 min. 50% B, 6 min. 100% B, 1 min. 50%
B, followed by a 7 min conditioning period using a Troyasil C18 column
(150 mm × 3.0 mm, 3.5 μm). The injection volume of the
samples was 1 μL.
## Biological Activity
2.3 Biological Activity 2.3.1 hEGFR Inhibition The hEGFR enzyme
inhibitions of the synthesized complexes were determined using the
Takara Universal Tyrosine Kinase Assay Kit. 36 The inhibition results obtained as a result of this experiment are
given in Table 1 as
the IC 50 value, and the enzyme inhibition graphs are given
in Figure S49 . Table 1 hEGFR Enzyme Inhibition Results of
Ru–Sorafenib Complexes maximum
inhibition complex structure IC 50 (nM) (%) [I] (μM) Ru1S Ru(bpy) 2 Srf 3.9 ± 0.08 99.74 0.3 Ru2S Ru(4,4-dimebpy) 2 Srf 8.1 ± 0.2 96.20 0.3 Ru3S Ru(6,6-dimebpy) 2 Srf 2.5 ± 0.07 96.92 0.3 Ru4S Ru(4,4-di- t -bu-bpy) 2 Srf 5.8 ± 0.15 96.30 0.3 Ru5S Ru(4,4-dimethoxybpy) 2 Srf 1.2 ± 0.02 97.17 0.3 Ru6S Ru(phen) 2 Srf 13 ± 0.1 85.41 0.3 Ru7S Ru(5-Clphen) 2 Srf 0.8 ± 0.02 97.07 0.3 Ru8S Ru(5-mephen) 2 Srf 1.4 ± 0.03 97.07 0.3 Sorafenib 88.7 ± 1.2 100 5 According to the EGFR enzyme inhibition results, all
of the synthesized
complexes showed higher inhibition than the sorafenib. Ru7S showed the best inhibition with an IC 50 value
of 0.8 nM, and Ru8S and Ru5S complexes
showed high inhibition with IC 50 values of 1.2 nm and 1.4
nM close to this value. Other complexes also showed very high inhibition
compared to the IC 50 values of 88.7 nM concentration shown
by sorafenib. Among the complexes, Ru6S showed the lowest
inhibition with an IC 50 value of 13 nM. 2.3.2 Cell Viability against Normal and Cancer
Cell Lines The effects of the synthesized complexes on cell
viability were tested by MTT assay in HepG2, Caco-2, HT-29, MCF-7,
A549, and HEK293T cell lines. The IC 50 values calculated
after 48 h of substance incubation in each cell are given in Table 2 . Concentration-%
viability plots of each compound are presented in Figures S50–S55 . According to these results, Ru3S was the most effective complex on HepG2 cells. Sorafenib
showed a cytotoxic effect on HepG2 cells with a value of IC 50 = 5.20 μM, while Ru3S exhibited a cytotoxic effect
with an IC 50 = 7.13 μM close to this value. According
to the cell viability results of complexes in Caco-2 cells, the five
complexes demonstrated higher cytotoxicity than sorafenib. Sorafenib
showed cytotoxicity with IC 50 = 13.80 μM, while the
most effective complex Ru4S showed cytotoxicity with
a value of IC 50 = 2.47 μM. In addition, Ru2S , Ru3S , Ru5S , and Ru6S showed
better cytotoxic effects than sorafenib in this cell line. Table 2 IC 50 (μM) Values
of Synthesized Complexes Calculated from Percent (%) Viability Values
in Different Cell Lines Comp HepG2 Caco-2 HT-29 MCF-7 A549 HEK293T Ru1S 26.51 ± 10.43 18.61 ± 4.96 64.0 ± 22.62 17.18 ± 3.84 131.48 ± 22.83 169.48 ± 97.97 Ru2S 78.84 ± 42.81 10.98 ± 2.47 19.41 ± 5.58 17.96 ± 5.55 94.15 ± 14.76 144.43 ± 158.6 Ru3S 7.13 ± 2.12 9.57 ± 2.33 6.93 ± 1.76 12.09 ± 3.36 25.24 ± 2.87 83.79 ± 58.05 Ru4S 23.76 ± 4.23 2.47 ± 0.42 6.24 ± 1.36 5.35 ± 0.78 29.90 ± 4.48 33.15 ± 9.17 Ru5S 27.36 ± 5.62 11.44 ± 3.76 21.88 ± 7.19 9.47 ± 1.96 34.72 ± 4.78 87.34 ± 35.95 Ru6S 54.79 ± 28.65 12.42 ± 2.75 41.77 ± 11.88 19.33 ± 4.09 65.03 ± 12.40 66.15 ± 18.46 Ru7S 30.01 ± 6.51 17.01 ± 4.25 14.60 ± 3.46 10.11 ± 2.65 172.97 ± 57.41 764.61 ± 188 Ru8S 25.65 ± 4.43 20.45 ± 4.80 33.56 ± 10.17 33.07 ± 12.50 92.36 ± 19.80 16.55 ± 18.32 Srf 5.20 ± 1.66 13.80 ± 3.89 7.89 ± 2.53 10.55 ± 2.60 7.48 ± 0.86 0.15 ± 0.16 The Ru4S complex exhibited the highest
cytotoxicity
on the HT-29 cell line. Sorafenib showed activity with IC 50 = 7.89 μM, while Ru4S was effective with IC 50 = 6.24 μM. Furthermore, in this cell line, Ru3S exhibited very close cytotoxicity (IC 50 = 6.93 μM)
to that of Ru4S ( IC 50 = 6.24 μM). On
the other hand, the Ru4S complex (IC 50 = 5.35
μM) showed a higher cytotoxic effect than sorafenib (IC 50 = 10.55 μM) in the MCF-7 cell line. In the A549 cell
line, none of the complexes demonstrated higher cytotoxicity than
sorafenib. However, when the complexes were compared within themselves, Ru3S and Ru4S showed the best cytotoxic properties. When the effects of the complexes on the healthy cell line (HEK293T)
were compared with sorafenib, the complexes showed low cytotoxicity
on healthy cells. Sorafenib showed higher cytotoxicity on the healthy
HEK293 cell line with IC 50 = 0.15 μM, while Ru3S showed cytotoxicity with IC 50 = 83.79 μM
and Ru4S IC 50 = 33.15 μM. The selectivity
of Sorafenib and Ru 3s and Ru4S over cancer
cells and healthy cells is a huge advantage for the potentials of
the complexes. Since sorafenib is the first-line drug in hepatocellular
carcinoma, we continued our studies with HepG2 cells. Cytotoxic
effects of Ru3S and Ru4S evaluated
in sorafenib-resistant HepG2 cell line HepG2-SR. IC 50 values
of Ru3S , Ru4S , and sorafenib increased compared
to the parental HepG2 cell line; however, the increment in the IC 50 of Ru3S was lower compared to that of sorafenib
( Figure 2 ). Figure 2 Effect of Ru3S and Ru4S on cell viability
in sorafenib-resistant HepG2-SR cells. 2.3.3 Cell Apoptosis and Cell Cycle The
apoptotic and cell cycle properties of the Ru3S and Ru4S complexes were determined on the HepG2 cell line in the
Muse Cell Analyzer. The selected Ru3S and Ru4S complexes were treated at 3 or 4 different concentrations for 48
h. At the end of this period, viable, early apoptotic, late apoptotic,
and dead cell profiles for apoptosis were determined in duplicate
for each compound and concentration. Cell profiles were plotted as
% and statistical calculations were performed. The resulting graphs
are given in Figure 3 A, and the apoptotic profiles of the complexes are given in Figure 3 B. Supplementary
apoptotic profiles are presented in Figure S56 . Figure 3 (A) Effect of concentration-dependent (0.78, 3.12, 12.5, and 50
μM) administration of Ru3S , Ru4S ,
and sorafenib on the percentages (%) of live, early apoptotic, late
apoptotic, and dead HepG2 cells. (B) Apoptotic profiles of concentration-dependent
administration of Ru3S , Ru4S , and sorafenib
in HepG2 cells. The analysis of the difference between groups was
conducted using a two-way analysis of variance (ANOVA) with a Dunnett
post hoc test in GraphPad Prism 9.00. A significance level of p < 0.05 was deemed as statistically significant. * p < 0.05; ** p < 0.01; *** p < 0.001; p < 0.0001. According to the apoptosis results, Ru3S decreased
the live cell percentage at all concentrations ( p < 0.01) and increased the rate of early apoptotic cells at 3.12
and 12.5 μM concentrations ( p < 0.05), the
rate of late apoptotic cells at 50 μM concentration ( p < 0.001). However, the rate of necrotic cells did not
change depending on the concentration ( p > 0.05). Ru4S showed a different profile than Ru3S . Ru4S decreased the live cell percentage at all concentrations
after 0.78 μM ( p < 0.05). The rate of early
apoptotic cells increased at lower concentrations (0.78 and 3.12 μM
( p < 0.0.5)), while at higher concentrations the
proportion of late apoptotic cells (3.12, 12.5, and 50 μM) and
dead cells (12.5 and 50 μM) increased compared to control ( p < 0.0.5). Apoptotic profiles of sorafenib and Ru3S were similar, sorafenib decreased the live cell percentage
at all concentrations ( p < 0.0001). However, different
from Ru3S , it increased the rate of early apoptotic cells
at all concentrations ( p < 0.0001) as well as
that of late apoptotic cells at higher concentrations (12.5 and 50
μM) ( p < 0.01). The necrotic cell percentage
did not change ( p > 0.05). On the other
hand, the percentage of cells in cell cycle phases
for cell cycle was determined by measuring DNA content. Sub G1 (DNA
content of cells in apoptotic phase), G0/G1 (Resting and protein synthesis),
S (DNA synthesis), and G2/M (Protein synthesis and mitosis) phases
were determined. DNA content was plotted as % and statistical calculation
was made. The resulting graphs are given in Figure 4 A, and the cell cycle profiles of the complexes
are given in Figure 4 B. Supplementary profiles are presented in Figure S57 . Figure 4 (A) Effect of concentration-dependent (0.78, 3.12, 12.5, and 50
μM) administered Ru3S , Ru4S , and sorafenib
on cell cycle phases in HepG2 cells. (B) Cell cycle profiles of concentration-dependent
administered Ru3S , Ru4S , and sorafenib in
HepG2 cells. The analysis of the difference between groups was conducted
using a two-way ANOVA with a Dunnett post hoc test in GraphPad Prism
9.00. A significance level of p < 0.05 was deemed
as statistically significant. * p < 0.05; ** p < 0.01; *** p < 0.001; p < 0.0001. Dose-dependent Ru3S administration
did not affect
cell cycle phases much in HepG2 cells. It increased Sub G1 population
at 12.5 μM. However, although there was a dose-dependent decrease
in G0/G1, S, and G2/M, it was not statistically significant ( p > 0.05). In contrast, Ru4S showed different
effects on cell cycle profiles at different doses. At low concentration
(0.78 μM), it increased Sub G1 ( p < 0.0001)
and decreased G0/G1, S, and G2/M percentages ( p <
0.05). At higher concentrations (3.12 and 12.5 μM) Sub G1 was
slightly increased ( p < 0.01), G0/G1 was slightly
decreased ( p < 0.01), S phase was not changed,
and G2/M was decreased ( p < 0.05). At the highest
50 μM dose, Ru4S increased Sub G1( p < 0.0001) and decreased S ( p < 0.01), and
did not affect the G0/G1 and G2/M phases. On the other hand, sorafenib
increased Sub G1 and decreased other phases similar to Ru3S . Sub G1 was decreased at all concentrations ( p <
0.05), G0/G1 was decreased at 50 μM ( p <
0.01), S phase was decreased at 12.5 and 50 μM ( p < 0.05), and G2/M was decreased; however, it was not statistically
significant ( p > 0.05). 2.3.4 Effect of Synthesized Complexes on Expression
of Tyrosine Kinase Pathway Proteins The effects of synthesized
complexes on the expression of proteins in the tyrosine kinase pathway
(EGFR, p-EGFR, Akt, p-Akt, Erk1/2 (p44/42 MAPK), p-Erk1/2 (p-p44/42
MAPK)) determined by the immunoblotting method in HepG2 cells. The
relative calculated densities of the band volumes for each sample
normalized to the reference protein (GAPDH) and representative bands
are given in Figure 5 A. The difference was evaluated statistically compared to the control
and between high and low concentrations. Figure 5 (A) Concentration (0.78
and/or 3.12 and/or 12.5 μM)-dependent
effect of Ru3S , Ru4S , and sorafenib on expression
of tyrosine kinase pathway proteins in HepG2 cells (B) Concentration
(0.78 and/or 3.12 and/or 12.5 μM)-dependent antiangiogenic effect
of Ru3S , Ru4S , and sorafenib on expression
of tyrosine kinase pathway proteins in HUVEC cells. The analysis of
the difference between groups was conducted using a one-way ANOVA
with a Tukey post hoc test in GraphPad Prism 9.00. A significance
level of p < 0.05 was deemed as statistically
significant. * p < 0.05; ** p <
0.01. n = 2–3. Total EGFR decreased compared to control after Ru4S administration ( p < 0.05), and p-EGFR
decreased
statistically in higher concentrations compared to control after Ru3S ( p < 0.01) and Ru4S ( p < 0.05) administration. Ru3S (12.5 μM; p < 0.05) and Ru4S (3.12 and 0.78; p < 0.01) decreased total Akt compared to control. No
compound has changed the phosphorylated Akt compared to control; however,
phosphorylated Akt decreased at higher concentrations of Ru3S compared to lower concentrations ( p < 0.05).
Total Erk1/2 decreased at lower and higher concentrations of Ru3S , Ru4S , and sorafenib compared to control
( p < 0.05), while p-Erk1/2 decreased at higher
concentrations compared to lower concentrations in Ru3S ( p < 0.01) and at all concentrations of Ru4S compared to control ( p < 0.05). 2.3.5 Antiangiogenic Effect The antiangiogenic
effects of synthesized complexes in HUVEC (Human umbilical vein endothelial
cell) cells were determined by evaluating protein expressions in the
angiogenesis pathway (VEGFR, Akt, p-Akt, Erk1/2 (p44/42 MAPK), p-Erk1/2
(p-p44/42 MAPK)) by Western blot method. The relative calculated densities
of the band volumes for each sample normalized to the reference protein
(GAPDH) and representative bands are given in Figure 5 B. The differences were evaluated statistically
compared to the control and between high and low concentrations. Total VEGFR decreased compared to control after higher concentrations
of Ru4S and sorafenib administration ( p < 0.05) and decreased in higher concentrations compared to lower
after Ru3S ( p < 0.05). Total Akt
decreased after high and low concentrations of Ru3S and
high concentrations of Ru4S compared to control ( p < 0.05). However, p-Akt decreased after high and low
concentrations of Ru3S and sorafenib, and high concentration
of Ru4S compared to control ( p <
0.05). Total Erk1/2 and p-Erk1/2 decreased after higher concentrations
of Ru3S and sorafenib compared to controls ( p < 0.05). Upon combining the outcomes of enzyme inhibition,
cell cytotoxicity,
and expression of tyrosine kinase pathway proteins, it was observed
that sorafenib–ruthenium ligand complexes increased enzyme
inhibition by at least 6-fold compared to sorafenib alone. In addition,
we have shown in this study that while sorafenib has a very high cytotoxic
effect on healthy cells, sorafenib–ruthenium ligand complexes
reduce this effect by at least 100-fold. We also demonstrated that
ruthenium complexes down-regulate both phospho- and total Erk1/2 (MAPK1/2)
in HepG2 and HUVEC cells, where Erk is up-regulated in resistant cell
lines. Additionally, ruthenium complexes inhibited VEGFR expression
in HUVEC cells. A study conducted by Morgillo et al. provided
evidence of a positive
interaction between sorafenib and anti-EGFR drugs. 37 , 38 Accordingly, they provided evidence that the combined use of sorafenib
and an EGFR inhibitor in EGFR and/or VEGFR inhibitor-resistant human
cancer cells is active in inhibiting tumor cell growth in
vitro and in vivo . Here, the effect of sorafenib
was found to be linked to its ability to block RAF signaling via the
RAS/RAF/MEK/MAPK pathway. These results are consistent with our study
findings. When the outcomes of our study and the study conducted by
Morgillo et al. are compared, it becomes evident that our study indirectly
validates the notion that the synergistic effect is amplified when
the inhibition effect is employed concurrently. Because the new sorafenib–ruthenium
ligand complexes we made in this study are such good at blocking EGFR,
they may have helped a mechanism develop that can do this job without
the need for other inhibitors. Although establishing a definitive
mechanism is challenging, additional ruthenium and ligand moieties
may have the effect of blocking different pathways. These mechanisms
may become apparent through subsequent comprehensive investigations
in the field. 2.3.6 Effect of Micelles Obtained from Synthesized
Complexes on Cell Viability When the enzyme inhibition and
cytotoxicity results are evaluated together, Ru3S and Ru4S showed potential as active drug substances. Therefore,
the effects of these complexes were investigated on apoptosis, cell
cycle, antiangiogenic effects, and proteins in the tyrosine kinase
pathway. In order to increase the bioavailability of the obtained
complexes we prepared micelle forms and investigated their drug release,
cytotoxic, and apoptotic properties. The effects of M1 and M2 and noncomplex M on cell viability,
respectively, obtained using Ru3S and Ru4S substances were tested by MTT assay in HepG2 cells. IC 50 values were calculated from the percent viability values calculated
according to the negative controls with the GraphPad Prism program.
IC 50 values calculated after 48 h of substance incubation
are presented graphically in Figure 6 A. According to the cytotoxicity results of the prepared Ru3S - and Ru4S -loaded micelles on the HepG2 cell
line, noncomplex micelle M showed the value of IC 50 = 175.2 μM, the Ru3S -loaded M1 micelles showed IC 50 = 1.07 μM, and the Ru4S -loaded M2 micelles showed IC 50 = 3.52 μM.
These values showed that the micelle-loaded forms exhibited higher
cytotoxicity when compared to the results of the complexes without
loading the micelle ( Ru3S ; IC 50 = 7.13 μM; Ru4S , IC 50 = 23.76 μM). Figure 6 (A) Concentration–viability
(%) graphics of noncomplex M , Ru3S -loaded M1 , and Ru4S -loaded M2 micelles
in HepG2 cell line. (B) Concentration-dependent
apoptotic cell levels of M1 and M2 in HepG2
cells. The apoptotic properties of M1 and M2 were determined on the HepG2 cell line in the Muse Cell
Analyzer.
Viable, early apoptotic, late apoptotic, and dead cell profiles were
determined for each compound at 2 concentrations and 2 replicates.
The resulting graphs are presented in Figure 6 B, and the profiles of the apoptotic properties
of the compounds are presented in Figure S19 . According to these results, it was determined that M1 prepared from Ru3S decreased the percentage
of viable
cells more than free Ru3S , despite the lower concentration.
Also, M1 increased the early apoptotic cells compared
to control and free Ru3S . Even though M2 decreased live cells with increasing concentrations, it increased
late apoptotic and dead cells instead of early apoptotic cells as
we have seen in free Ru4S . Both cytotoxicity and apoptosis
results show that micelles prepared from the complexes increase the
cytotoxic effect and the most prominent compound was Ru3S as well as its micelle M1 .
## hEGFR Inhibition
2.3.1 hEGFR Inhibition The hEGFR enzyme
inhibitions of the synthesized complexes were determined using the
Takara Universal Tyrosine Kinase Assay Kit. 36 The inhibition results obtained as a result of this experiment are
given in Table 1 as
the IC 50 value, and the enzyme inhibition graphs are given
in Figure S49 . Table 1 hEGFR Enzyme Inhibition Results of
Ru–Sorafenib Complexes maximum
inhibition complex structure IC 50 (nM) (%) [I] (μM) Ru1S Ru(bpy) 2 Srf 3.9 ± 0.08 99.74 0.3 Ru2S Ru(4,4-dimebpy) 2 Srf 8.1 ± 0.2 96.20 0.3 Ru3S Ru(6,6-dimebpy) 2 Srf 2.5 ± 0.07 96.92 0.3 Ru4S Ru(4,4-di- t -bu-bpy) 2 Srf 5.8 ± 0.15 96.30 0.3 Ru5S Ru(4,4-dimethoxybpy) 2 Srf 1.2 ± 0.02 97.17 0.3 Ru6S Ru(phen) 2 Srf 13 ± 0.1 85.41 0.3 Ru7S Ru(5-Clphen) 2 Srf 0.8 ± 0.02 97.07 0.3 Ru8S Ru(5-mephen) 2 Srf 1.4 ± 0.03 97.07 0.3 Sorafenib 88.7 ± 1.2 100 5 According to the EGFR enzyme inhibition results, all
of the synthesized
complexes showed higher inhibition than the sorafenib. Ru7S showed the best inhibition with an IC 50 value
of 0.8 nM, and Ru8S and Ru5S complexes
showed high inhibition with IC 50 values of 1.2 nm and 1.4
nM close to this value. Other complexes also showed very high inhibition
compared to the IC 50 values of 88.7 nM concentration shown
by sorafenib. Among the complexes, Ru6S showed the lowest
inhibition with an IC 50 value of 13 nM.
## Cell Viability against Normal and Cancer
Cell Lines
2.3.2 Cell Viability against Normal and Cancer
Cell Lines The effects of the synthesized complexes on cell
viability were tested by MTT assay in HepG2, Caco-2, HT-29, MCF-7,
A549, and HEK293T cell lines. The IC 50 values calculated
after 48 h of substance incubation in each cell are given in Table 2 . Concentration-%
viability plots of each compound are presented in Figures S50–S55 . According to these results, Ru3S was the most effective complex on HepG2 cells. Sorafenib
showed a cytotoxic effect on HepG2 cells with a value of IC 50 = 5.20 μM, while Ru3S exhibited a cytotoxic effect
with an IC 50 = 7.13 μM close to this value. According
to the cell viability results of complexes in Caco-2 cells, the five
complexes demonstrated higher cytotoxicity than sorafenib. Sorafenib
showed cytotoxicity with IC 50 = 13.80 μM, while the
most effective complex Ru4S showed cytotoxicity with
a value of IC 50 = 2.47 μM. In addition, Ru2S , Ru3S , Ru5S , and Ru6S showed
better cytotoxic effects than sorafenib in this cell line. Table 2 IC 50 (μM) Values
of Synthesized Complexes Calculated from Percent (%) Viability Values
in Different Cell Lines Comp HepG2 Caco-2 HT-29 MCF-7 A549 HEK293T Ru1S 26.51 ± 10.43 18.61 ± 4.96 64.0 ± 22.62 17.18 ± 3.84 131.48 ± 22.83 169.48 ± 97.97 Ru2S 78.84 ± 42.81 10.98 ± 2.47 19.41 ± 5.58 17.96 ± 5.55 94.15 ± 14.76 144.43 ± 158.6 Ru3S 7.13 ± 2.12 9.57 ± 2.33 6.93 ± 1.76 12.09 ± 3.36 25.24 ± 2.87 83.79 ± 58.05 Ru4S 23.76 ± 4.23 2.47 ± 0.42 6.24 ± 1.36 5.35 ± 0.78 29.90 ± 4.48 33.15 ± 9.17 Ru5S 27.36 ± 5.62 11.44 ± 3.76 21.88 ± 7.19 9.47 ± 1.96 34.72 ± 4.78 87.34 ± 35.95 Ru6S 54.79 ± 28.65 12.42 ± 2.75 41.77 ± 11.88 19.33 ± 4.09 65.03 ± 12.40 66.15 ± 18.46 Ru7S 30.01 ± 6.51 17.01 ± 4.25 14.60 ± 3.46 10.11 ± 2.65 172.97 ± 57.41 764.61 ± 188 Ru8S 25.65 ± 4.43 20.45 ± 4.80 33.56 ± 10.17 33.07 ± 12.50 92.36 ± 19.80 16.55 ± 18.32 Srf 5.20 ± 1.66 13.80 ± 3.89 7.89 ± 2.53 10.55 ± 2.60 7.48 ± 0.86 0.15 ± 0.16 The Ru4S complex exhibited the highest
cytotoxicity
on the HT-29 cell line. Sorafenib showed activity with IC 50 = 7.89 μM, while Ru4S was effective with IC 50 = 6.24 μM. Furthermore, in this cell line, Ru3S exhibited very close cytotoxicity (IC 50 = 6.93 μM)
to that of Ru4S ( IC 50 = 6.24 μM). On
the other hand, the Ru4S complex (IC 50 = 5.35
μM) showed a higher cytotoxic effect than sorafenib (IC 50 = 10.55 μM) in the MCF-7 cell line. In the A549 cell
line, none of the complexes demonstrated higher cytotoxicity than
sorafenib. However, when the complexes were compared within themselves, Ru3S and Ru4S showed the best cytotoxic properties. When the effects of the complexes on the healthy cell line (HEK293T)
were compared with sorafenib, the complexes showed low cytotoxicity
on healthy cells. Sorafenib showed higher cytotoxicity on the healthy
HEK293 cell line with IC 50 = 0.15 μM, while Ru3S showed cytotoxicity with IC 50 = 83.79 μM
and Ru4S IC 50 = 33.15 μM. The selectivity
of Sorafenib and Ru 3s and Ru4S over cancer
cells and healthy cells is a huge advantage for the potentials of
the complexes. Since sorafenib is the first-line drug in hepatocellular
carcinoma, we continued our studies with HepG2 cells. Cytotoxic
effects of Ru3S and Ru4S evaluated
in sorafenib-resistant HepG2 cell line HepG2-SR. IC 50 values
of Ru3S , Ru4S , and sorafenib increased compared
to the parental HepG2 cell line; however, the increment in the IC 50 of Ru3S was lower compared to that of sorafenib
( Figure 2 ). Figure 2 Effect of Ru3S and Ru4S on cell viability
in sorafenib-resistant HepG2-SR cells.
## Cell Apoptosis and Cell Cycle
2.3.3 Cell Apoptosis and Cell Cycle The
apoptotic and cell cycle properties of the Ru3S and Ru4S complexes were determined on the HepG2 cell line in the
Muse Cell Analyzer. The selected Ru3S and Ru4S complexes were treated at 3 or 4 different concentrations for 48
h. At the end of this period, viable, early apoptotic, late apoptotic,
and dead cell profiles for apoptosis were determined in duplicate
for each compound and concentration. Cell profiles were plotted as
% and statistical calculations were performed. The resulting graphs
are given in Figure 3 A, and the apoptotic profiles of the complexes are given in Figure 3 B. Supplementary
apoptotic profiles are presented in Figure S56 . Figure 3 (A) Effect of concentration-dependent (0.78, 3.12, 12.5, and 50
μM) administration of Ru3S , Ru4S ,
and sorafenib on the percentages (%) of live, early apoptotic, late
apoptotic, and dead HepG2 cells. (B) Apoptotic profiles of concentration-dependent
administration of Ru3S , Ru4S , and sorafenib
in HepG2 cells. The analysis of the difference between groups was
conducted using a two-way analysis of variance (ANOVA) with a Dunnett
post hoc test in GraphPad Prism 9.00. A significance level of p < 0.05 was deemed as statistically significant. * p < 0.05; ** p < 0.01; *** p < 0.001; p < 0.0001. According to the apoptosis results, Ru3S decreased
the live cell percentage at all concentrations ( p < 0.01) and increased the rate of early apoptotic cells at 3.12
and 12.5 μM concentrations ( p < 0.05), the
rate of late apoptotic cells at 50 μM concentration ( p < 0.001). However, the rate of necrotic cells did not
change depending on the concentration ( p > 0.05). Ru4S showed a different profile than Ru3S . Ru4S decreased the live cell percentage at all concentrations
after 0.78 μM ( p < 0.05). The rate of early
apoptotic cells increased at lower concentrations (0.78 and 3.12 μM
( p < 0.0.5)), while at higher concentrations the
proportion of late apoptotic cells (3.12, 12.5, and 50 μM) and
dead cells (12.5 and 50 μM) increased compared to control ( p < 0.0.5). Apoptotic profiles of sorafenib and Ru3S were similar, sorafenib decreased the live cell percentage
at all concentrations ( p < 0.0001). However, different
from Ru3S , it increased the rate of early apoptotic cells
at all concentrations ( p < 0.0001) as well as
that of late apoptotic cells at higher concentrations (12.5 and 50
μM) ( p < 0.01). The necrotic cell percentage
did not change ( p > 0.05). On the other
hand, the percentage of cells in cell cycle phases
for cell cycle was determined by measuring DNA content. Sub G1 (DNA
content of cells in apoptotic phase), G0/G1 (Resting and protein synthesis),
S (DNA synthesis), and G2/M (Protein synthesis and mitosis) phases
were determined. DNA content was plotted as % and statistical calculation
was made. The resulting graphs are given in Figure 4 A, and the cell cycle profiles of the complexes
are given in Figure 4 B. Supplementary profiles are presented in Figure S57 . Figure 4 (A) Effect of concentration-dependent (0.78, 3.12, 12.5, and 50
μM) administered Ru3S , Ru4S , and sorafenib
on cell cycle phases in HepG2 cells. (B) Cell cycle profiles of concentration-dependent
administered Ru3S , Ru4S , and sorafenib in
HepG2 cells. The analysis of the difference between groups was conducted
using a two-way ANOVA with a Dunnett post hoc test in GraphPad Prism
9.00. A significance level of p < 0.05 was deemed
as statistically significant. * p < 0.05; ** p < 0.01; *** p < 0.001; p < 0.0001. Dose-dependent Ru3S administration
did not affect
cell cycle phases much in HepG2 cells. It increased Sub G1 population
at 12.5 μM. However, although there was a dose-dependent decrease
in G0/G1, S, and G2/M, it was not statistically significant ( p > 0.05). In contrast, Ru4S showed different
effects on cell cycle profiles at different doses. At low concentration
(0.78 μM), it increased Sub G1 ( p < 0.0001)
and decreased G0/G1, S, and G2/M percentages ( p <
0.05). At higher concentrations (3.12 and 12.5 μM) Sub G1 was
slightly increased ( p < 0.01), G0/G1 was slightly
decreased ( p < 0.01), S phase was not changed,
and G2/M was decreased ( p < 0.05). At the highest
50 μM dose, Ru4S increased Sub G1( p < 0.0001) and decreased S ( p < 0.01), and
did not affect the G0/G1 and G2/M phases. On the other hand, sorafenib
increased Sub G1 and decreased other phases similar to Ru3S . Sub G1 was decreased at all concentrations ( p <
0.05), G0/G1 was decreased at 50 μM ( p <
0.01), S phase was decreased at 12.5 and 50 μM ( p < 0.05), and G2/M was decreased; however, it was not statistically
significant ( p > 0.05).
## Effect of Synthesized Complexes on Expression
of Tyrosine Kinase Pathway Proteins
2.3.4 Effect of Synthesized Complexes on Expression
of Tyrosine Kinase Pathway Proteins The effects of synthesized
complexes on the expression of proteins in the tyrosine kinase pathway
(EGFR, p-EGFR, Akt, p-Akt, Erk1/2 (p44/42 MAPK), p-Erk1/2 (p-p44/42
MAPK)) determined by the immunoblotting method in HepG2 cells. The
relative calculated densities of the band volumes for each sample
normalized to the reference protein (GAPDH) and representative bands
are given in Figure 5 A. The difference was evaluated statistically compared to the control
and between high and low concentrations. Figure 5 (A) Concentration (0.78
and/or 3.12 and/or 12.5 μM)-dependent
effect of Ru3S , Ru4S , and sorafenib on expression
of tyrosine kinase pathway proteins in HepG2 cells (B) Concentration
(0.78 and/or 3.12 and/or 12.5 μM)-dependent antiangiogenic effect
of Ru3S , Ru4S , and sorafenib on expression
of tyrosine kinase pathway proteins in HUVEC cells. The analysis of
the difference between groups was conducted using a one-way ANOVA
with a Tukey post hoc test in GraphPad Prism 9.00. A significance
level of p < 0.05 was deemed as statistically
significant. * p < 0.05; ** p <
0.01. n = 2–3. Total EGFR decreased compared to control after Ru4S administration ( p < 0.05), and p-EGFR
decreased
statistically in higher concentrations compared to control after Ru3S ( p < 0.01) and Ru4S ( p < 0.05) administration. Ru3S (12.5 μM; p < 0.05) and Ru4S (3.12 and 0.78; p < 0.01) decreased total Akt compared to control. No
compound has changed the phosphorylated Akt compared to control; however,
phosphorylated Akt decreased at higher concentrations of Ru3S compared to lower concentrations ( p < 0.05).
Total Erk1/2 decreased at lower and higher concentrations of Ru3S , Ru4S , and sorafenib compared to control
( p < 0.05), while p-Erk1/2 decreased at higher
concentrations compared to lower concentrations in Ru3S ( p < 0.01) and at all concentrations of Ru4S compared to control ( p < 0.05).
## Antiangiogenic Effect
2.3.5 Antiangiogenic Effect The antiangiogenic
effects of synthesized complexes in HUVEC (Human umbilical vein endothelial
cell) cells were determined by evaluating protein expressions in the
angiogenesis pathway (VEGFR, Akt, p-Akt, Erk1/2 (p44/42 MAPK), p-Erk1/2
(p-p44/42 MAPK)) by Western blot method. The relative calculated densities
of the band volumes for each sample normalized to the reference protein
(GAPDH) and representative bands are given in Figure 5 B. The differences were evaluated statistically
compared to the control and between high and low concentrations. Total VEGFR decreased compared to control after higher concentrations
of Ru4S and sorafenib administration ( p < 0.05) and decreased in higher concentrations compared to lower
after Ru3S ( p < 0.05). Total Akt
decreased after high and low concentrations of Ru3S and
high concentrations of Ru4S compared to control ( p < 0.05). However, p-Akt decreased after high and low
concentrations of Ru3S and sorafenib, and high concentration
of Ru4S compared to control ( p <
0.05). Total Erk1/2 and p-Erk1/2 decreased after higher concentrations
of Ru3S and sorafenib compared to controls ( p < 0.05). Upon combining the outcomes of enzyme inhibition,
cell cytotoxicity,
and expression of tyrosine kinase pathway proteins, it was observed
that sorafenib–ruthenium ligand complexes increased enzyme
inhibition by at least 6-fold compared to sorafenib alone. In addition,
we have shown in this study that while sorafenib has a very high cytotoxic
effect on healthy cells, sorafenib–ruthenium ligand complexes
reduce this effect by at least 100-fold. We also demonstrated that
ruthenium complexes down-regulate both phospho- and total Erk1/2 (MAPK1/2)
in HepG2 and HUVEC cells, where Erk is up-regulated in resistant cell
lines. Additionally, ruthenium complexes inhibited VEGFR expression
in HUVEC cells. A study conducted by Morgillo et al. provided
evidence of a positive
interaction between sorafenib and anti-EGFR drugs. 37 , 38 Accordingly, they provided evidence that the combined use of sorafenib
and an EGFR inhibitor in EGFR and/or VEGFR inhibitor-resistant human
cancer cells is active in inhibiting tumor cell growth in
vitro and in vivo . Here, the effect of sorafenib
was found to be linked to its ability to block RAF signaling via the
RAS/RAF/MEK/MAPK pathway. These results are consistent with our study
findings. When the outcomes of our study and the study conducted by
Morgillo et al. are compared, it becomes evident that our study indirectly
validates the notion that the synergistic effect is amplified when
the inhibition effect is employed concurrently. Because the new sorafenib–ruthenium
ligand complexes we made in this study are such good at blocking EGFR,
they may have helped a mechanism develop that can do this job without
the need for other inhibitors. Although establishing a definitive
mechanism is challenging, additional ruthenium and ligand moieties
may have the effect of blocking different pathways. These mechanisms
may become apparent through subsequent comprehensive investigations
in the field.
## Effect of Micelles Obtained from Synthesized
Complexes on Cell Viability
2.3.6 Effect of Micelles Obtained from Synthesized
Complexes on Cell Viability When the enzyme inhibition and
cytotoxicity results are evaluated together, Ru3S and Ru4S showed potential as active drug substances. Therefore,
the effects of these complexes were investigated on apoptosis, cell
cycle, antiangiogenic effects, and proteins in the tyrosine kinase
pathway. In order to increase the bioavailability of the obtained
complexes we prepared micelle forms and investigated their drug release,
cytotoxic, and apoptotic properties. The effects of M1 and M2 and noncomplex M on cell viability,
respectively, obtained using Ru3S and Ru4S substances were tested by MTT assay in HepG2 cells. IC 50 values were calculated from the percent viability values calculated
according to the negative controls with the GraphPad Prism program.
IC 50 values calculated after 48 h of substance incubation
are presented graphically in Figure 6 A. According to the cytotoxicity results of the prepared Ru3S - and Ru4S -loaded micelles on the HepG2 cell
line, noncomplex micelle M showed the value of IC 50 = 175.2 μM, the Ru3S -loaded M1 micelles showed IC 50 = 1.07 μM, and the Ru4S -loaded M2 micelles showed IC 50 = 3.52 μM.
These values showed that the micelle-loaded forms exhibited higher
cytotoxicity when compared to the results of the complexes without
loading the micelle ( Ru3S ; IC 50 = 7.13 μM; Ru4S , IC 50 = 23.76 μM). Figure 6 (A) Concentration–viability
(%) graphics of noncomplex M , Ru3S -loaded M1 , and Ru4S -loaded M2 micelles
in HepG2 cell line. (B) Concentration-dependent
apoptotic cell levels of M1 and M2 in HepG2
cells. The apoptotic properties of M1 and M2 were determined on the HepG2 cell line in the Muse Cell
Analyzer.
Viable, early apoptotic, late apoptotic, and dead cell profiles were
determined for each compound at 2 concentrations and 2 replicates.
The resulting graphs are presented in Figure 6 B, and the profiles of the apoptotic properties
of the compounds are presented in Figure S19 . According to these results, it was determined that M1 prepared from Ru3S decreased the percentage
of viable
cells more than free Ru3S , despite the lower concentration.
Also, M1 increased the early apoptotic cells compared
to control and free Ru3S . Even though M2 decreased live cells with increasing concentrations, it increased
late apoptotic and dead cells instead of early apoptotic cells as
we have seen in free Ru4S . Both cytotoxicity and apoptosis
results show that micelles prepared from the complexes increase the
cytotoxic effect and the most prominent compound was Ru3S as well as its micelle M1 .
## Molecular Docking Studies
2.4 Molecular Docking Studies In this
study, to explain the inhibition mechanisms of sorafenib–ruthenium
complexes molecular docking studies of both sorafenib and its ruthenium
complexes were performed and the results were compared. Molecular
docking studies were performed with the induced fit docking (IFD)
technique, and MM-GBSA (molecular mechanics with generalized born
and surface area) Δ G binding free energies
and glide emodel scores of the compounds against the target protein
EGFR were also determined. The induced fit docking XP (extra
precision) glide scores, MM-GBSA Δ G binding
free energies, and glide emodel scores of the ruthenium complexes
as well as sorafenib against EGFR protein are given in Table 3 . Table 3 Molecular Docking IFD Scores and MM-GBSA
Δ G Binding Free Energies of Ruthenium Complexes
and Sorafenib Compounds IFD XP Gscore (kcal/mol) MM-GBSA Δ G bind (kcal/mol) glide emodel (kcal/mol) Ru1S –8.583 –66.17 –103.735 Ru2S –10.554 –64.54 –100.927 Ru3S –10.771 –78.51 –117.996 Ru4S –7.524 –60.41 –85.799 Ru5S –6.277 –69.27 –59.830 Ru6S –10.163 –65.67 –85.368 Ru7S –5.354 –67.30 –27.392 Ru8S –9.150 –67.00 –24.458 Sorafenib –9.812 –60.12 –90.643 According to the molecular docking studies, Ru3S exhibited
the best IFD XP Gscore with −10.771 kcal/mol, indicating strong
binding interaction, best MM-GBSA Δ G binding
free energy at −78.51 kcal/mol, implying a stable binding affinity,
and best Glide emodel score (−117.996 kcal/mol), suggesting
a potentially strong binding mode. All scores of Ru3S are better than sorafenib. The IFD score, MM-GBSA Δ G binding free energy, and glide emodel of sorafenib are
−9.812, −60.12, and −90.643 kcal/mol, respectively.
In addition to Ru3S , the IFD scores of Ru2S and Ru6S are −10.554 and −10.163 kcal/mol,
respectively, and also they are better than sorafenib. Other ruthenium
complexes showed moderate and comparable docking scores, and they
varied from −5.354 to −9.150 kcal/mol. According
to in vitro test results, the IC 50 values
of all of the ruthenium complexes against hEGFR enzyme
inhibitions are at the nanomolar level. According to the results obtained
from anticancer studies, the compound with the highest selectivity
against healthy and cancer cells is the Ru3S complex.
In the in vitro anticancer activity studies against
five different cancer cell lines, the inhibitions of sorafenib and Ru3S are very close to each other, but the fact that the toxicity
of Ru3S in healthy cells is much lower than sorafenib.
Since Ru3S has better scores than all other ruthenium
complexes and sorafenib in molecular docking studies, detailed molecular
docking analyses of sorafenib and Ru3S were evaluated
and the inhibition mechanisms were discussed and compared with each
other. Molecular docking two-dimensional (2D) and three-dimensional
(3D)
ligand–protein interactions of sorafenib–EGFR ligand–protein
complex are given in Figure 7 . As seen in Figure 7 a, the oxygen of urea carbonyl formed a hydrogen-bond
interaction
with Thr-854. Both nitrogen of urea group formed an additional hydrogen-bond
interaction with Gln-791. Finally, the nitrogen atom of the pyridyl
ring also formed a hydrogen-bond interaction with Cys-797. In Figure 7 b, the yellow dashes
show hydrogen-bond interactions. Hydrogen-bond lengths vary from 1.86
to 2.36 Å. Key amino acids in the Srf-EGFR ligand–protein
complex stand out as Gln-791, Thr-854, and Cys-797. Four different
hydrogen bonds occurring in the active site of the enzyme indicate
the stability of the Srf-EGFR ligand–protein complex
and its inhibition ability. Figure 7 Molecular docking ligand–protein interactions
between sorafenib
and the active site of EGFR (PDB: 5X2A ): (a) 2D ligand–protein interactions
and (b) 3D ligand–protein interactions. Molecular docking two-dimensional (2D) and three-dimensional
(3D)
ligand–protein interactions of Ru3S-EGFR ligand–protein
complex are given in Figure 8 . As can be seen from Figure 8 a, there are three different hydrogen-bond interactions,
two different π–π stacking interactions, and two
different π–cationic interactions between Ru3S and the amino acid residues in the active site of EGFR. Figure 8 Molecular docking
ligand–protein interactions between Ru3S and the
active site of EGFR (PDB: 5X2A ): (a) 2D ligand–protein
interactions, (b) 3D ligand–protein interactions. When we look at the ligand–protein interactions
of Ru3S - EGFR complex, it is seen that the
oxygen
of the urea carbonyl interacts with Thr-854, and the urea nitrogen
interacts with Gln-791, as in the Srf-EGFR complex. Since
the nitrogen atom in the pyridine ring of sorafenib is coordinated
with Ru, the interaction of this nitrogen atom with Cys-797 in sorafenib
is not seen in the Ru3S-EGFR complex. Instead, the pyridine
ring of sorafenib makes a π–cationic interaction with
Lys-728, while the oxygen of the amide carbonyl makes a hydrogen-bond
interaction (purple) with Lys-716. Crucially, the bipyridyl rings
attached to Ru engage in π–π stacking (green) and
π–cationic (red) interactions with Phe-795, His-805,
and Lys-716 amino acid residues in the EGFR active site. These interactions
likely contribute to stabilizing the Ru3S-EGFR complex
and disrupting the normal function of EGFR. Figure 8 b depicts
the 3D ligand–protein interactions of the Ru3S-EGFR complex. The yellow dashes represent the hydrogen-bond interactions,
the turquoise dashes represent the π–π stacking
interactions, and the red dashes represent the π–cationic
interactions. On the other hand, ruthenium (Ru) forms six coordination
bonds with six different nitrogen atoms provided by sorafenib and
the bipyridyl rings. In the Ru3S-EGFR complex, the hydrogen-bond
lengths range from 1.95 to 2.07 Å, which closely resemble the
hydrogen-bond lengths in the Srf-EGFR complex. Shorter
hydrogen bonds signify stronger binding and a more stable ligand–protein
complex. Additionally, the lengths of the Ru–N coordination
bonds correlate with normal bond lengths, measured at 2.1 Å.
As depicted in Figure 8 b, the bipyridyl rings are positioned perpendicular to each other
and to sorafenib. These ring orientations contribute to the π–cationic
and π–π stacking interactions observed in the ligand–protein
complex, enhancing both stability and inhibition. When the interactions
of sorafenib and Ru3S with EGFR
are compared, they share some common features such as hydrogen-bond
interactions with Thr-854 and Gln-791. However, Ru3S distinguishes
itself by contributing unique additional interactions, specifically
π–cationic and π–π stacking interactions.
These distinctive features contribute to the inhibitory potential
of Ru3S and highlight its prominence in ligand–protein
stability. The additional π–cationic and π–π
stacking interactions of Ru3S likely play a significant
role in enhancing its inhibitory capacity compared to sorafenib. Molecular docking two-dimensional (2D) and three-dimensional (3D)
ligand–protein interactions of the rest of the ruthenium complex
are given in the Supporting Information ( Figures S62–S75 ).
## Molecular Dynamics Simulations
2.5 Molecular Dynamics Simulations In
this study, 200 ns MD simulation analyses were carried out for Srf-EGFR and Ru3S-EGFR complexes and compared
the obtained results. The findings obtained from the MD simulation
of the Srf-EGFR complex were compared with those derived
from the Ru3S-EGFR complex. This comparative analysis
aimed to highlight the differences between the inhibitory effects
demonstrated by sorafenib alone and the effects exhibited by Ru3S . The objective was to elucidate and outline the distinctions
in the inhibition mechanisms of sorafenib and Ru3S when
interacting with the EGFR complex. The 200 ns MD simulation
analysis of the Srf-EGFR complex is given in Figure 9 . Figure 9 a represents the 2D key ligand–protein
interactions with the percentage simulation time of Srf-EGFR MD simulations. Figure 9 200 ns MD simulation analysis of Sorafenib-EGFR complex.
(a) 2D key ligand–protein interactions, (b) RMSD graphics of
protein and ligand atoms, (c) RMSF graphic of protein Cα atoms,
(d) RMSF graphic of ligand atoms, and (e) fractional interaction histogram. As seen in Figure 9 a, the urea carbonyl formed a water-bridged hydrogen-bond
interaction
with Arg-841 (30% of the simulation time). The urea carbonyl also
formed a direct hydrogen-bond interaction with Lys-745 (27% of the
sim.). In addition, the urea carbonyl also formed additional two water-bridged
hydrogen-bond interactions with Lys-745 (14% of the sim.) and Asp-885
(12% of the sim.). In addition to these interactions, there are several
hydrogen bonds and water-bridged hydrogen-bond interactions with different
percentages of the simulation time. The other most important interactions
for the Srf-EGFR complex are the interactions occurring
between urea nitrogens and Gln-791 (9% of the sim.) and Asp-800 (25%
of the sim.). Although Gln-791 stood out as the key amino acid for
sorafenib in molecular docking studies, its interaction in MD simulations
continued for only 9% of the simulation time. In addition, the amino
acid residue Asp-800 showed multiple water-bridged hydrogen-bond interactions
throughout the simulation. Finally, Arg-841 shoved a π–cationic
interaction with the p -substituted phenyl ring during
13% of the simulation time. According to Figure 9 b, the average RMSD of the protein Cα
was calculated as 2.7
Å, the average RMSD of ligand atoms was calculated as 7.50 Å,
and the average ligand deviation from its original position was 2.4
Å. While the RMSD of the ligand atoms at 4.5 Å is almost
half of the simulation, it stabilized at 9 Å levels in the second
half of the simulation. Since 9 Å is a high value for the ligand
RMSD, the stability of the ligand–protein complex is thought
to be low. Figure 9 c,d represents the RMSF values of the protein Cα atoms and
ligand atoms, respectively. As seen in Figure 9 c,d, the average RMSF of the protein Cα
and ligand atoms were calculated as 1.8 and 4 Å, respectively.
In Figure 9 c, the vertical
green bars represent the ligand-amino acid contacts during the simulation
time. Finally, Figure 9 e represents the fractional interaction histograms of the Srf-EGFR complex. Throughout the simulation, interactions
like hydrogen bonds
and hydrophobic, ionic, and water bridges were continuously monitored
and categorized. The stacked bar charts in Figure 9 e efficiently show how often these interactions
occur, with values like 0.8 indicating an 80% presence. Values over
1.0 suggest instances where specific protein residues engage with
the ligand multiple times in the same category. The most abundant
fractional interactions were observed with Arg-841, Asp-800, Leu-718,
Lys-845, and Gln-791. These amino acid residues are the key amino
acids that showed multiple interactions during the simulation. The 200 ns MD simulation analysis of the Ru3S-EGFR complex
is given in Figure 10 . Figure 10 a represents
the 2D key ligand–protein interactions with the
percentage simulation time of Ru3S-EGFR MD simulations. Figure 10 200
ns MD simulation analysis of Ru3S-EGFR complex.
(a) 2D key ligand–protein interactions, (b) RMSD graphics of
protein and ligand atoms, (c) RMSF graphic of protein Cα atoms,
(d) RMSF graphic of ligand atoms, and (e) fractional interaction histogram. As seen in Figure 10 a, the urea nitrogens formed two hydrogen-bond interactions
with
Gln-791 (47 and 50% of the sim), and a water-bridged hydrogen-bond
interaction with Gln-791 (37% of the sim.). On the other hand, the
urea carbonyl formed two different water-bridged hydrogen-bond interactions
with Asp-855 (33% of the sim.) and Lys-745 (29% of the sim.). In addition,
the amid carbonyl formed an additional water-bridged hydrogen-bond
interaction with Val-742 (24% of the sim.) and the bipyridyl ring
of the Ru complex formed two π–cationic bond interactions
with Lys-728. For the Gln-791 key amino acid residue, which stands
out in molecular docking studies for both ligands, the interaction
time in the Srf-EGFR complex is 9% of the simulation,
while this time is 50% in the Ru3S-EGFR complex. This
result indicates that the Ru3S forms a stable complex
throughout the simulation because binding to a specific amino acid
residue and remaining bound for a long time is better than short-term
interactions with more than one amino acid residue. Moreover, π–cationic
interactions of bipyridyl rings attached to the ruthenium complex
contribute to the stability of the ligand–protein complex. Figure 10 b represents
the RMSD values of the Ru3S-EGFR complex. According to Figure 10 b, the average
RMSD of the protein Cα atoms was calculated as 2.8 Å (pale
blue), the average RMSD of the ligand atoms was calculated as 4.5
Å, and the average deviation of the ligand from its original
position is 1 Å. Comparing the RMSD values, the Ru3S-EGFR complex appears more stable than the Srf-EGFR complex.
In Ru3S-EGFR , the ligand shows a lower average RMSD (4.5
Å) and a smaller deviation from its original position (1 Å)
compared to Srf-EGFR (ligand RMSD of 7.50 Å and
a deviation of 2.4 Å). These results suggest that Ru3S maintains a more consistent position within the binding site, indicating
greater stability during the simulations. Figure 10 c,d represents
the RMSF plots of the protein Cα atoms and ligand atoms of Ru3S-EGRF complex, respectively. The average RMSF of the protein
Cα was calculated as 1.8 Å, and this value is the same
as the value in the Srf-EGFR complex. The protein backbones
have the same stability and fluctuation in both complexes. On the
other hand, the RMSF of the ligand atoms was calculated to be 1.5
Å on average, and this value is much lower than that of the Srf-EGFR complex. The ruthenium complex created a more stable
ligand–protein complex by restricting the fluctuation of the
molecule throughout the simulation. Finally, Figure 10 e represents the fractional interaction
histogram of the Ru3S-EGFR complex during the 200 ns
simulation time. The most abundant fractional histograms were observed
with Gln-791, Leu-792, Met-790, and Lys-728. Actually, the abundance
of the Gln-791 fractions is so high and Gln-791 stands out as the
key amino acid residue for the inhibition. In conclusion, the
200 ns MD simulations of Srf-EGFR and Ru3S-EGFR complexes revealed distinct dynamic behaviors.
In Srf-EGFR , diverse interactions were observed with
varying durations, resulting in ligand fluctuations (average RMSD
of 7.50 Å). Conversely, Ru3S-EGFR exhibited enhanced
stability, demonstrated by sustained hydrogen-bond interactions, lower
ligand RMSD (4.5 Å), and a smaller deviation (1 Å). The
π–cationic interactions of the bipyridyl ring in Ru3S contributed to its stability. Both complexes highlighted
Gln-791 as a key amino acid, but Ru3S exhibited more
abundant and consistent interactions, making it a potentially more
stable and promising candidate for drug development. The RMSF analysis
indicated a more rigid binding in Ru3S-EGFR , emphasizing
the advantages of Ru3S in forming a stable ligand–protein
complex.
## Micelle Formation and Drug Release Studies
2.6 Micelle Formation and Drug Release Studies The size of Ru3S - and Ru4S -loaded PEG–PLGA
polymeric micelles was performed with DLS analysis. M1 and M2 refer to Ru3S - and Ru4S -loaded polymeric micelles, respectively. The sizes of micelles were
99.7 ± 0.9 nm with a PDI value of 0.22 for M1 and
67.7 ± 0.7 nm with a PDI value of 0.23 for M2 . The
amount of Ru3S and Ru4S loaded into the
micelles was determined by LC-HRMS analysis. A certain amount of freeze-dried M1 and M2 micelles was dissolved in DMF, and
the LC-HRMS was carried out. The drug loading content (DLC) and entrapment
efficiency (EE) were determined according to the equations in Figure S60 . According to these equations, %DLC
of 7.81% and %EE of 15.6% were determined for Ru3S -loaded
micelles (M1). For Ru4S -loaded micelles (M2), %DLC and
%EE were determined as 31.25 and 62.5%, respectively. To investigate
the controlled release properties of polymeric micelles, the prepared M2 nanocarrier was dispersed in two buffer solutions at different
pH values (7.4 and 5.6) at 37 °C. The release profile of Ru4S from M2 was
determined by LC-HRMS analysis. As expected, only a very small amount
(less than 25%) of the Ru4S was released from M2 in a neutral medium (pH = 7.4) even after 24 h. However, in a weakly
acidic environment (pH = 5.5), the release was 80% as shown in Figure S61C . At pH values of 7.4 and 5.5, release
efficiencies of 34 and 93% were achieved after 96 h, respectively
( Figure S61 ).
## Conclusions
3 Conclusions Sorafenib is a well-known
tyrosine kinase inhibitor, but the need
for the discovery of new molecules still remains. This study demonstrates
that these new sorafenib–ruthenium complexes inhibit the cellular
proliferation of hepatocellular carcinoma cells and show EGFR enzyme
inhibition. Eight new sorafenib–ruthenium complexes were synthesized
and EGFR enzyme inhibition was investigated. All complexes showed
better nanomolar activity than sorafenib. Ru6S demonstrated
weaker inhibition compared to other complexes with IC 50 values of 13 nM, whereas Ru7S showed the highest inhibitory
effect against hEGFR with IC 50 values of 0.8 nM. When compared
to sorafenib (IC 50 = 88 nM), all complexes displayed extremely
strong inhibition. HepG2, Caco-2, HT-29, MCF-7, and A549 are
five distinct cancer
cell lines used to test the cytotoxicity effects of the complexes.
According to the cytotoxicity results of the complexes, it was found
that they exhibit different cytotoxicity for each cell. Considering
its selective effects on healthy and cancer lines, further research
on HepG2 cells was continued. Among all of the complexes, Ru3S demonstrated a higher antiproliferative effect on the HepG2 cell
line as well as sorafenib-resistant HepG2-SR cells. Additionally, Ru3S showed lower cytotoxicity on the healthy HEK293T cell
line compared to sorafenib, which is a desirable feature for a drug
candidate. The effects of Ru3S and Ru4S complexes
on the apoptotic and cell cycle properties of the HepG2 cell line
were distinct. Ru3S and sorafenib increased Sub G1 and
decreased other phases of the cell cycle, whereas various doses of Ru4S had varying effects on the cell cycle profiles. In HepG2
cells, immunoblotting revealed that the complexes decreased the expression
of total EGFR, p-EGFR, total Akt, p-Akt, total Erk1/2, and p-Erk1/2
relative to the control, with higher concentrations having a greater
effect than lower concentrations. The synthesized complexes had antiangiogenic
effects on HUVEC cells, as indicated by a decrease in angiogenesis
pathway protein expression. VEGFR, Akt, p-Akt, Erk1/2, and p-Erk1/2
all decreased following administration of Ru3S , Ru4S , and sorafenib compared to the control and lower concentrations.
These findings indicate that the Ru3S and Ru4S complexes have the potential to be used as anticancer agents. The encapsulation of free drugs ( Ru3S and Ru4S ) into polymeric micelles ( M1 and M2 ) resulted
in an increase in cytotoxicity of the drug on the HepG2 cells. Thus,
polymeric micelles have the potential to be carriers of the hydrophobic
drugs of Ru3S and Ru4S . In conclusion,
the cytotoxicity and apoptosis experiments revealed
that the micelles prepared from the complexes increased the cytotoxic
effect, with Ru3S and its micelle M1 being
the most prominent compound. Finally, to determine ligand–protein
interactions and complex
stability as well as to evaluate the inhibition mechanism of the ruthenium
complexes, molecular docking and dynamics studies were carried out
and compared with sorafenib. Docking studies revealed that Ru3S shows the best docking score, MM-GBSA Δ G binding
free energy, and glide emodel scores. In addition, MD simulations
showed that the Ru3S-EGFR complex is more stable than
the Srf-EGFR complex. Molecular docking and dynamics
studies confirmed Ru3S as a promising candidate, revealing
favorable binding interactions and enhanced stability in comparison
to sorafenib against EGFR.
## Experimental Section
4 Experimental Section 4.1 Materials The chemicals and solvents
were bought from Fluka Chemie, Merck, Alfa Aesar, and Sigma-Aldrich.
Melting points were determined on a STUART SMP40. IR spectra were
measured on an Alfa Bruker spectrometer. 1 H and 13 C NMR spectra were acquired on a Bruker spectrometer at 500 and 125
Hz, respectively. Mass spectra were obtained using a Thermo Fisher
Scientific LC-HRMS spectrometer. UV measurements were determined with
a HITACHI U-2900 brand spectrometer. Spectrophotometric analyses were
performed by a BioTek Power Wave XS (BioTek). The cell line was purchased
from American Type Culture Collection (ATCC). Dulbecco’s modified
Eagle’s medium-F12, RPMI medium, fetal calf serum, and PBS
were bought from GIBCO BRL, Invitrogen (Carlsbad, CA). All compounds
used for biological assays were >95% pure, as determined by HPLC
analysis
( Figures S39–S47 ). 4.2 Methods 4.2.1 General Procedures and Spectral Data 4.2.1.1 Synthesis of Sorafenib 4-Chloropicolinoyl
chloride ( 2 ): 0.08 mol of thionyl chloride and 1 mL of
DMF were mixed under nitrogen at 40 °C for 10 min. 0.4 mol of
picolinic acid ( 1 ) was added in half an hour piece by
piece. When the addition was complete, the temperature was increased
to 72 °C and stirred at this temperature for 16 h. A yellow precipitate
formed at the end of the reaction. The resulting solid was purified
with the help of toluene and ether to obtain 4-chloropicolinoyl chloride
( 2 ) in 60% yield. 27 1 H NMR (DMSO- d 6 , 300 MHz) δ/ppm:
8.62 (1H, d, J = 7.4 Hz), 8.10 (1H, s), 7.46 (1H,
d, J = 7.4 Hz). 4-Chloro- N -methylpicolinamide ( 3 ): 4-Chloropicolinoyl chloride
( 2 ) and methyl amine were mixed in cold methanol. The
solid part formed at the end of the reaction was dried by filtration
through the gooch crucible. 4-Chloro- N -methylpicolinamide
( 3 ) obtained in 20% yield was used for the next step. 27 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm: 8.72 (1H, d, J =
7.3 Hz), 8.14 (1H, s), 7.52 (1H, d, J = 7.3 Hz),
2.59 (3H, d, J = 4.5 Hz). 4-(4-Aminophenoxy)- N -methylpicolinamide ( 4 ): First, p -aminophenol and t -BuOK were mixed in DMF for 2
h under nitrogen, then compound 3 and K 2 CO 3 were added and mixed at
80 °C for 16 h. At the end of the reaction, DMF was evaporated
in the evaporator. 4-(4-Aminophenoxy)- N -methylpicolinamide
( 4 ) was obtained by column chromatography with hexane-ethyl
acetate. 28 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm: 2.79 (3H, d, J = 4.8 Hz), 5.22 (2H, s, NH 2 ), 6.65 (2H, d, J = 8.7 Hz), 6.87 (2H, d, J = 8.7 Hz),
7.08 (1H, dd, J = 5.6, 2.6 Hz), 7.35 (1H, d, J = 2.5 Hz), 8.46 (1H, d, J = 5.62 Hz),
8.78 (1H, q, J = 4.5 Hz); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm: 26.4, 108.7, 114.1,
115.3, 122.0, 143.2, 147.3, 150.6, 152.7, 164.3, 167.2. 4-(4-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)- N -methylpicolinamide ( Sorafenib ): 1 mmol of
4-(4-aminophenoxy)- N -methylpicolinamide ( 4 ), 1.1 mmol of 4-chloro-3-trifluoromethyl phenyl isocyanate, and
1.1 mmol of Et 3 N were dissolved in THF together and stirred
overnight at 60 °C. At the end of the reaction, THF was evaporated
in the evaporator. The solid was purified by washing with ether. 29 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm 2.81 (3H, d, J = 4.8
Hz), 7.29–7.06 (3H m), 7.41 (1H, d, J = 2.5
Hz), 7.78–7.56 (4H, m), 8.15 (1H, d, J = 2.5
Hz), 8.52 (1H, d, J = 5.6 Hz), 8.82 (1H, q, J = 4.7, 4.6 Hz), 9.04 (1H, s), 9.26 (1H, s); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm:
26.4, 109.0, 114.4, 117.2, 120.9, 121.9, 122.8, 123.5, 124.3, 127.0,
132.4, 137.5, 139.7, 148.2, 150.8, 152.8, 152.9, 164.2, 166.4. 4.2.1.2 Synthesis of RuL 2 Cl 2 Complexes Method A: The mixture of RuCl 3 ·(H 2 O) n (1 mmol), ligand (L) (2 mmol), LiCl (6
mmol), and DMF (2 mL) was stirred under nitrogen at 80 °C under
reflux for 18 h. At the end of the reaction, acetone was added after
cooling and the mixture was kept in a refrigerator for 24 h. The precipitate
formed was filtered, washed with cold water and diethyl ether, and
then dried. The resulting complex was purified by column chromatography. 30 The following complexes were synthesized according
to this method. Ru(bpy) 2 Cl 2 : Dark purple
powder, 60% yield, 360 °C (decomposition) mp. 1 H NMR
(DMSO- d 6 , 500 MHz) δ/ppm; 9.98 (d, J = 5.4 Hz, 1H), 8.64 (d, J = 8.0 Hz, 1H),
8.48 (d, J = 8.0 Hz, 1H), 8.12–8.01 (m, 1H),
7.77 (t, J = 6.4 Hz, 1H), 7.74–7.63 (m, 1H),
7.51 (d, J = 5.5 Hz, 1H), 7.10 (dd, J = 9.6, 3.4 Hz, 1H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 159.5, 157.5, 152.5, 151.3, 133.9,
132.6, 124.7, 124.6, 122.2, 121.8. HRMS; calcd for C 20 H 16 Cl 2 N 4 Ru [M + H] + ; 484.9874,
found; 484.9806. Ru(4,4-dimebpy) 2 Cl 2 :
Dark purple powder,
46% yield, 360 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 9.77 (d, J
= 5.7 Hz, 1H), 8.46 (s, 1H), 8.31 (s, 1H), 7.58 (d, J = 5.0 Hz, 1H), 7.31 (d, J = 5.9 Hz, 1H),
6.92 (dd, J = 5.9, 1.1 Hz, 1H), 2.62 (s, 3H), 2.34
(s, 3H). 13 C NMR (DMSO- d 6 ,
125 MHz) δ/ppm; 160.2, 158.4, 152.9, 151.6, 145.9, 144.7, 126.5,
126.5, 123.8, 123.4, 21.1, 20.7. HRMS; calcd for C 24 H 24 Cl 2 N 4 Ru [M + H] + ; 541.0500,
found; 541.1046. Ru(phen) 2 Cl 2 : Dark purple
powder, 56% yield,
370 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.28 (dd, J = 5.2, 1.2 Hz, 1H), 8.72 (dd, J = 8.1, 1.2 Hz, 1H),
8.29 (d, J = 8.8 Hz, 1H), 8.26–8.20 (m, 2H),
8.14 (d, J = 8.8 Hz, 1H), 7.75 (dd, J = 5.4, 1.0 Hz, 1H), 7.33 (dd, J = 8.0, 5.4 Hz, 1H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm;
154.7, 153.4, 151.2, 149.7, 134.0, 132.5, 130.0, 127.9, 127.5, 125.2,
124.9, 123.9. HRMS; calcd for C 24 H 16 Cl 2 N 4 Ru [M + H] + ; 532.9874, found; 532.1806. Ru(5-Clphen) 2 Cl 2 : Dark purple powder, 29%
yield, 375 °C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.35 (dt, J = 5.2,
1.1 Hz, 1H), 10.26 (td, J = 5.2, 1.2 Hz, 1H), 8.83
(dd, J = 8.4, 2.5 Hz, 1H), 8.68 (dt, J = 7.6, 1.3 Hz, 1H), 8.62 (d, J = 2.01 Hz, 1H), 8.47
(s, 1H), 8.40–8.28 (m, 2H), 8.28–8.20 (m, 2H), 7.98–7.88
(m, 1H), 7.85 (dd, J = 5.4, 1.0 Hz, 1H), 7.47–7.43
(m, 1H), 7.38–7.34 (m, 1H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 161.7, 154.5, 154.0,
153.7, 150.6, 149.2, 149.2, 147.9, 132.5, 131.1, 129.9, 128.9, 128.4,
127.1, 126.0, 125.7, 125.0, 124.8, 124.6, 124.4. HRMS; calcd for C 24 H 14 Cl 4 N 4 Ru [M + H] + ; 600.9094, found; 600.9550. Ru(5-mephen) 2 Cl 2 : Dark purple powder, 55%
yield, 373 °C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.29 (d, J = 4.0
Hz, 1H), 10.20 (s, 1H), 8.74 (d, J = 8.2 Hz, 1H),
8.58 (d, J = 7.9 Hz, 1H), 8.24–8.20 (m, 2H),
8.17–8.13 (m, 1H), 8.09 (d, J = 4.4 Hz, 2H),
7.95 (d, J = 6.7 Hz, 1H), 7.76–7.68 (m, 1H),
7.67–7.60 (m, 1H), 7.35 (dd, J = 8.1, 5.4
Hz, 1H), 7.28 (dd, J = 7.8, 5.4 Hz, 1H), 2.76 (s,
3H), 2.89 (s, 3H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 171.4, 153.1, 152.5, 149.6, 148.8,
147.9, 138.2, 134.4, 134.1, 132.0, 130.8, 130.1, 129.1, 129.0, 128.6,
128.5, 125.4, 125.1, 124.2, 123.9, 123.7, 123.5, 114.0, 21.4, 20.4.
HRMS; calcd for C 26 H 20 Cl 2 N 4 Ru [M + H] + ; 561.0187, found; 561.0657. Method B:
RuCl 3 ·(H 2 O) n (1 mmol)
and LiCl (40 mmol) were mixed in ethylene glycol:water
mixture at 110 °C for 15 min. After 15 min, ligand (2.2 mmol)
was added and mixing was continued. Then, glucose (2 mmol) was added
and mixed for another 15 min at 110 °C. Ascorbic acid was added
to it and mixing was continued for 30 min. After 30 min, 10 mL of
saturated NaCl solution was added and stirred at 0 °C for 1 h.
The precipitate was then filtered off and purified by column chromatography. 31 , 39 The following complexes were synthesized according to this method. Ru(6,6-dimebpy) 2 Cl 2 : Dark purple powder,
78% yield, 370 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 8.58 (d, J
= 7.8 Hz, 1H), 8.44 (d, J = 7.7 Hz, 2H),
8.36 (d, J = 7.7 Hz, 1H), 8.06 (t, J = 7.5 Hz, 1H), 7.95 (t, J = 7.6 Hz, 1H), 7.85 (t, J = 7.6 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H),
7.55 (d, J = 7.4 Hz, 1H), 7.48 (d, J = 7.4 Hz, 1H), 7.28–7.23 (m, 2H), 2.91 (s, 3H), 2.53 (s, 3H),
1.86 (s, 3H), 1.65 (s, 3H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 167.9, 166.8, 166.5, 164.9, 163.9,
161.9, 161.1, 160.5, 137.1, 136.5, 135.8, 135.2, 124.6, 124.6, 123.9,
123.675, 121.6, 121.0, 120.7, 120.6, 27.2, 24.7, 24.6, 23.7. HRMS;
calcd for C 24 H 24 Cl 2 N 4 Ru
[M + H] + ; 541.0500, found; 541.1334. Ru(4,4-dimethoxybpy) 2 Cl 2 : Dark purple powder,
50% yield, 370 °C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 9.83 (d, J = 6.6 Hz, 1H), 9.20 (d, J = 6.5 Hz, 1H), 8.46 (d, J = 2.7 Hz, 1H), 8.36 (d, J = 2.6 Hz, 1H),
8.30 (dd, J = 8.2, 2.6 Hz, 2H), 7.59 (dd, J = 6.6, 2.6 Hz, 1H), 7.51 (d, J = 6.6
Hz, 1H), 7.47 (dd, J = 6.6, 2.7 Hz, 1H), 7.07 (dd, J = 6.5, 2.5 Hz, 1H), 7.03–7.00 (m, 2H), 4.11 (s,
3H), 4.08 (s, 3H), 3.94 (s, 3H), 3.92 (s, 3H); 13 C NMR
(DMSO- d 6 , 125 MHz) δ/ppm; 167.5,
167.3, 166.9, 166.7, 159.2, 159.0, 158.8, 157.5, 156.4, 154.0, 153.4,
150.1, 114.6, 114.1, 113.5, 113.2, 112.0, 111.6, 111.2, 110.4, 57.4,
57.2, 57.2, 57.1. HRMS; calcd for C 24 H 24 Cl 2 N 4 O 4 Ru [M + H] + ; 605.0296,
found; 605.0755. Ru(4,4-di- t -bu-bpy) 2 Cl 2 :
Dark purple powder, 60% yield, 370 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm;
8.55 (s, 4H), 7.59 (s, 4H), 7.26 (s, 4H), 1.50 (s, 18H), 1.33 (s,
18H); 13 C NMR (DMSO- d 6 , 125
MHz) δ/ppm; 163.2, 162.7, 161.9, 161.8, 158.0, 157.5, 157.4,
156.3, 155.1, 152.6, 125.0, 124.6, 124.2, 124.1, 122.4, 122.1, 122.0,
121.5, 121.1, 120.1, 36.1, 35.8, 35.7, 30.7, 30.6, 30.5, 30.4. HRMS;
calcd for C 36 H 48 Cl 2 N 4 Ru
[M + H] + ; 709.2378, found; 709.2895. 4.2.1.3 Synthesis of RuL 2 S Complexes Ru–ligand complex (RuL 2 Cl 2 ) (1 mmol)
and sorafenib (Srf) (1 mmol) were stirred in methanol overnight at
60 °C. At the end of the reaction, methanol was removed. The
solid was dried and purified by flash chromatography. 32 , 33 Ru(bpy) 2 Srf (Ru1S): Red powder, 30% yield, 196
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
8.73 (d, J = 8.1 Hz, 1H), 8.70 (d, J = 7.6 Hz, 2H), 8.63 (d, J = 8.0 Hz, 1H), 8.60 (d, J = 7.9 Hz, 1H), 8.23–8.17 (m, 2H), 8.15–8.13
(m, 1H), 8.07 (d, J = 2.6 Hz, 1H), 8.03–7.99
(m, 2H), 7.95 (td, J = 8.0, 1.4 Hz, 1H), 7.81 (d, J = 5.7 Hz, 1H), 7.78–7.74 (m, 1H), 7.71 (d, J = 5.6, 1H), 7.68–7.65 (m, 1H), 7.70–7.50
(m, 4H), 7.48 (d, J = 8.7 Hz, 1H), 7.37 (td, J = 7.3, 1.2 Hz, 1H), 7.32 (td, J = 7.3,
1.3 Hz, 1H), 7.16–7.12 (m, 2H), 7.11 (dd, J = 6.4, 2.6 Hz, 1H), 2.95 (s, 3H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 172.0, 166.8, 159.1, 158.2, 157.9, 157.4,
153.4, 153.1, 152.8, 151.9, 151.6, 151.4, 150.5, 148.0, 138.7, 137.5,
137.3, 137.0, 136.2, 131.6, 127.5, 127.4, 126.8, 126.5, 124.1, 123.8,
123.7, 123.5, 122.8, 121.2, 121.0, 117.3, 117.2, 114.1, 26.5. HRMS;
calcd for C 41 H 32 ClF 3 N 8 O 3 Ru [M – H] − ; 877.1203, found;
877.1192. Ru(4,4-dimebpy) 2 Srf (Ru2S): Red powder,
52% yield, 215
°C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.92 (s, 1H), 10.23 (s, 1H), 9.89 (s, 1H),
8.72 (d, J = 13.7 Hz, 2H), 8.64 (d, J = 15.9 Hz, 2H), 8.50 (s, 1H), 8.44 (d, J = 5.7 Hz,
1H), 8.11 (d, J = 1.6 Hz, 1H), 7.88 (d, J
= 5.7 Hz, 1H), 7.64–7.61 (m, 2H), 7.61–7.57
(m, 3H), 7.52–7.50 (m, 2H), 7.48 (d, J = 6.4
Hz, 1H), 7.45 (d, J = 5.8 Hz, 1H), 7.25 (dd, J = 5.8, 1.0 Hz, 1H), 7.20–7.19 (m, 2H), 7.18–7.17
(m, 1H), 7.09 (dd, J = 6.4, 2.6 Hz, 1H), 2.84 (d, J = 4.6 Hz, 3H), 2.47 (s, 3H), 2.44 (s, 3H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm;
171.3, 170.7, 164.8, 157.4, 156.6, 156.2, 155.8, 152.2, 152.0, 151.4,
150.6, 150.3, 150.2, 149.2, 148.4, 148.3, 148.1, 147.1, 146.4, 138.8,
137.1, 131.4, 127.8, 127.7, 127.3, 127.0, 124.4, 124.0, 123.8, 123.2,
121.9, 121.4, 121.1, 120.6, 119.2, 116.6, 115.5, 115.5, 114.1, 26.7,
20.2, 20.1, 19.9, 19.8. HRMS; calcd for C 45 H 40 ClF 3 N 8 O 3 Ru [M – H] − ; 933.1829, found; 933.1818. Ru(6,6-dimebpy) 2 Srf
(Ru3S): Red powder, 25% yield, 220
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
8.60–8.54 (m, 3H), 8.51 (d, J = 8.0 Hz, 1H),
8.07–7.99 (m, 4H), 7.90 (t, J = 7.8 Hz, 1H),
7.72 (d, J = 2.3 Hz, 1H), 7.68 (d, J = 6.5 Hz, 1H), 7.62 (dd, J = 8.7, 2.3 Hz, 1H), 7.57
(d, J = 8.8 Hz, 2H), 7.5–7.39 (m, 5H), 7.08
(dd, J = 6.5, 2.6 Hz, 1H), 7.04 (d, J = 8.8 Hz, 2H), 2.69 (s, 3H), 2.36 (s, 3H), 2.06 (s, 3H), 1.99 (s,
3H), 1.73 (s, 3H); 13 C NMR (CD 3 OD, 125 MHz)
δ/ppm; 170.7, 167.5, 167.0, 166.2, 165.0, 164.7, 160.6, 159.5,
159.2, 158.3, 153.3, 151.6, 147.7, 138.7, 137.7, 137.6, 137.6, 137.0,
136.9, 131.6, 126.4, 126.2, 126.1, 126.0, 124.0, 123.9, 122.8, 122.5,
122.0, 121.3, 121.2, 121.1, 120.9, 117.2, 117.2, 116.4, 113.4, 26.1,
24.5, 24.3, 23.1, 22.7. HRMS; calcd for C 45 H 40 ClF 3 N 8 O 3 Ru [M – H] − ; 933.1829, found; 933.1814. Ru(4,4-di- t -bu-bpy) 2 Srf (Ru4S): Red
powder, 65% yield, 207 °C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm; 8.73 (dd, J = 6.9, 1.7
Hz, 2H), 8.63 (dd, J = 9.9, 1.8 Hz, 2H), 8.57 (d, J = 5.9 Hz, 1H), 8.06 (d, J = 2.6 Hz, 1H),
8.05 (d, J = 2.5 Hz, 1H), 8.01 (d, J = 5.9 Hz, 1H), 7.80 (dd, J = 5.9, 1.6 Hz, 1H), 7.69
(dd, J = 5.9, 1.7 Hz, 1H), 7.66 (d, J = 6.1 Hz, 1H), 7.64–7.54 (m, 5H), 7.46 (d, J = 8.7 Hz, 1H), 7.41 (dd, J = 6.1, 1.9 Hz, 1H), 7.36
(dd, J = 6.1, 2.0 Hz, 1H), 7.14–7.10 (m, 3H),
2.96 (s, 3H), 1.52 (s, 9H), 1.51 (s, 9H), 1.41 (s, 9H), 1.39 (s, 9H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 172.0, 166.5,
162.5, 162.3, 162.0, 161.2, 158.8, 158.0, 157.8, 157.2, 153.4, 152.7,
152.4, 151.6, 151.2, 150.9, 149.9, 148.0, 138.8, 137.5, 131.5, 128.0,
127.7, 124.6, 124.6, 124.2, 124.0, 123.9, 123.8, 122.8, 121.3, 121.1,
121.0, 120.9, 120.8, 120.6, 117.2, 115.2, 114.0, 113.3, 35.3, 35.2,
35.0, 35.0, 33.5, 31.6, 30.7, 29.3, 29.3, 29.2, 29.0, 28.8, 28.7,
26.5, 22.3. HRMS; calcd for C 57 H 64 ClF 3 N 8 O 3 Ru [M – H] − ; 1101.3707,
found; 1101.3700. Ru(4,4-dimethoxybpy) 2 Srf (Ru5S):
Red powder, 80% yield,
235 °C (decomposition) mp. 1 H NMR (CD 3 OD,
500 MHz) δ/ppm; 8.39 (d, J = 6.4 Hz, 1H), 8.28
(d, J = 2.6 Hz, 1H), 8.25 (d, J = 2.6 Hz, 1H), 8.19 (dd, J = 5.2, 2.7 Hz, 2H), 8.03
(d, J = 2.5 Hz, 1H), 8.01 (d, J = 2.6 Hz, 1H), 7.85 (d, J = 6.4 Hz, 1H), 7.65 (d, J = 6.4 Hz, 1H), 7.61–7.52 (m, 5H), 7.48 (d, J = 8.7 Hz, 1H), 7.32 (dd, J = 6.4, 2.6
Hz, 1H), 7.22 (dd, J = 6.4, 2.6 Hz, 1H), 7.16–7.12
(m, 2H), 7.10 (dd, J = 6.4, 2.6 Hz, 1H), 6.98 (dd, J = 6.5, 2.7 Hz, 1H), 6.94 (dd, J = 6.6,
2.7 Hz, 1H), 4.09 (s, 3H), 4.08 (s, 3H), 3.99 (s, 3H), 3.98 (s, 3H),
2.95 (s, 3H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm;
172.3, 167.2, 167.1, 166.9, 166.4, 166.1, 160.0, 159.3, 158.7, 153.4,
153.1, 152.3, 152.1, 151.9, 151.2, 148.1, 138.7, 137.5, 131.6, 122.8,
121.2, 120.9, 117.3, 117.2, 117.0, 113.9, 113.6, 113.5, 113.4, 112.7,
110.8, 110.4, 110.4, 110.2, 55.9, 55.8, 26.4. HRMS; calcd for C 45 H 40 ClF 3 N 8 O 7 Ru
[M – H] − ; 997.1626, found; 997.1596. Ru(phen) 2 Srf (Ru6S): Red powder, 37% yield, 205 °C
mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm; 9.18
(dd, J = 5.1, 1.1 Hz, 1H), 8.81 (dd, J = 8.3, 1.1 Hz, 2H), 8.61 (dd, J = 5.1, 1.1 Hz, 1H),
8.53 (dd, J = 8.2, 1.1 Hz, 1H), 8.48 (dd, J = 8.2, 1.0 Hz, 1H), 8.33 (dd, J = 8.9,
4.9 Hz, 2H), 8.24 (t, J = 8.7 Hz, 2H), 8.15 (dd, J = 8.2, 5.1 Hz, 1H), 8.10 (d, J = 2.6
Hz, 1H), 8.05 (dd, J = 8.2, 5.1 Hz, 1H), 8.03–7.98
(m, 2H), 7.91 (dd, J = 5.3, 1.1 Hz, 1H), 7.65–7.51
(m, 6H), 7.48 (d, J = 8.7 Hz, 1H), 7.15–7.09
(m, 2H), 7.01 (dd, J = 6.4, 2.6 Hz, 1H), 2.94 (s,
3H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 172.3,
166.8, 154.1, 153.4, 153.2, 152.8, 151.8, 149.5, 148.7, 148.6, 148.0,
138.7, 137.5, 136.6, 136.4, 136.0, 135.2, 131.5, 131.0, 130.8, 130.8,
130.6, 127.8, 127.8, 127.7, 126.0, 125.4, 125.1, 122.8, 121.2, 120.9,
117.3, 117.2, 117.1, 114.1, 26.5. HRMS; calcd for C 45 H 32 ClF 3 N 8 O 3 Ru [M – H] − ; 925.1203, found; 925.1192. Ru(5-Clphen) 2 Srf (Ru7S): Red powder, 51% yield, 208
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
9.25 (td, J = 3.5, 1.2 Hz, 1H), 9.18 (td, J = 3.5, 1.3 Hz, 1H), 9.07–9.01 (m, 2H), 8.79–8.73
(m, 3H), 8.7 (d, J = 8.7 Hz, 1H), 8.68 (t, J = 4.1 Hz, 1H), 8.60 (t, J = 5.1 Hz, 1H),
8.53 (d, J = 1.4 Hz, 2H), 8.49 (d, J = 8.2 Hz, 1H), 8.44–8.42 (m, 3H), 8.29–8.24 (m, 1H),
8.19–8.11 (m, 5H), 8.06–8.03 (m, 3H), 7.96–7.95
(m, 3H), 7.69–7.62 (m, 2H), 7.61–7.52 (m, 10H), 7.41
(d, J = 8.7 Hz, 2H), 7.09 (dd, J = 8.8, 1.3 Hz, 4H), 7.02–6.99 (m, 2H), 2.94 (d, J = 3.3 Hz, 6H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm;
173.8, 172.3, 167.8, 166.9, 155.1, 154.6, 153.9, 153.7, 153.4, 153.3,
153.3, 153.2, 152.1, 151.7, 149.1, 148.6, 147.9, 147.2, 138.7, 137.6,
136.0, 135.8, 135.4, 133.7, 133.5, 132.3, 132.1, 131.7, 131.5, 131.5,
130.9, 130.2, 130.0, 129.3, 129.1, 128.4, 127.0, 126.9, 126.9, 126.6,
126.5, 126.0, 125.7, 124.0, 122.8, 122.7, 121.1, 120.9, 117.2, 117.1,
114.2, 113.3, 29.3, 28.7. HRMS; calcd for C 45 H 30 Cl 3 F 3 N 8 O 3 Ru [M + H] + ; 995.0580, found; 995.0386. Ru(5-mephen) 2 Srf (Ru8S): Red powder, 18% yield, 195
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
9.19 (t, J = 5.0 Hz,1H), 9.12 (t, J = 5.0 Hz, 1H), 8.95–8.91 (m, 2H), 8.73–8.69 (m, 2H),
8.66–8.61 (m, 2H), 8.58 (d, J = 8.3 Hz, 1H),
8.54 (t, J = 5.2 Hz, 1H), 8.42 (d, J = 8.1 Hz, 1H), 8.37 (d, J = 8.1 Hz, 1H), 8.23–8.18
(m, 1H), 8.18–8.14 (m, 4H), 8.06 (d, J = 4.1
Hz, 2H), 8.02–7.99 (m, 4H), 7.93 (d, J = 5.0
Hz, 2H), 7.86–7.83 (m, 1H), 7.64–7.46 (m, 14H), 7.43
(d, J = 8.7 Hz, 2H), 7.12 (td, J = 8.9, 1.6 Hz, 4H), 7.06–7.00 (m, 2H), 2.99 (s, 3H), 2.97 (s,
6H), 2.96 (s, 3H), 2.90 (d, J = 5.0 Hz, 6H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 173.7, 172.2,
167.8, 166.7, 153.6, 153.3, 153.2, 153.1, 152.3, 151.8, 151.7, 151.3,
150.8, 149.8, 148.8, 148.0, 147.9, 147.9, 138.7, 138.7, 137.5, 136.4,
136.2, 136.1, 135.7, 135.5, 135.1, 134.3, 133.6, 133.4, 133.0, 132.3,
132.1, 131.5, 131.2, 130.9, 130.8, 130.6, 130.4, 129.1, 128.4, 128.0,
127.7, 126.6, 126.5, 126.5, 126.0, 125.8, 125.4, 125.2, 125.1, 124.9,
124.0, 123.9, 122.9, 122.8, 122.7, 121.8, 121.1, 120.9, 117.2, 117.2,
117.1, 115.2, 114.1, 113.3, 29.3, 28.7, 24.1, 23.5, 22.6, 22.3. HRMS;
calcd for C 47 H 36 ClF 3 N 8 O 3 Ru [M] − ; 953.1516, found; 953.1502. 4.3 Biological Activity 4.3.1 Stability Studies Absorption spectra
of freshly prepared solutions of the samples were acquired at 25 °C
in the range of 200–800 nm with a Shimadzu UV-1280 spectrophotometer,
taking into account the solvent cutoff. Samples were dissolved in
the proper solvents, and the resulting solutions were placed in QS
quartz cuvettes (path length 1 cm). 40 4.3.2 EGFR Inhibition Assay Enzyme activity
was performed using the TAKARA Universal Tyrosine Kinase Assay and
Wash and Stop Solution for Sulfuric Acid Free ELISA according to Ölgen
et al. 36 The enzyme activity reaction was
performed on a precoated 96-well plate. To determine the activity,
40 μL of hEGFR and 10 μL of ATP-2Na solution were added
to each well on the plate and incubated at 37 °C for 30 min.
Then, the solution was removed from the well and the wells were washed
4 times with wash buffer. After this process, 100 μL of blocking
solution was added and incubated for another 30 min at 37 °C.
The blocking solution was discarded and the wells were washed again
with wash buffer. Subsequently, 50 μL of antiphosphotyrosine
(PY20-HRP) solution was added to each well and incubated at 37 °C
for 30 min. Afterward, the antibody solution was removed and the wells
were washed 4 times with the washing solution. 100 μL of HRP
substrate solution (TMBZ) was added to the wells and the plate was
incubated at 37 °C for 15 min. In addition to this mixture, 100
μL of stop solution was added to the wells in the same order
that the substrate solution was added. Activity was measured at 450
nm. In the studies to determine the inhibition potentials of
the synthesized molecules on hEGFR, the solutions of different concentrations
were prepared by dissolving the molecules with ethanol. The volumes
were adjusted so that the solvent amount in the reaction medium was
3% and preincubated with the enzyme (Copeland 2005) (ISBN: 978-1-118-48813-3).
The reaction was repeated only in the presence of this solvent to
determine the effect of the solvent on the enzyme. Using the results
obtained as a result of the inhibition studies, % relative activity–inhibitor
concentration graph was drawn for each molecule and the inhibitory
concentration at which the activity decreased by 50% was determined
as the IC 50 value. 4.3.3 Cytotoxicity Assay HepG2 (hepatocellular
carcinoma; ATCC HB-8065), Caco-2 (Colon carcinoma; ATCC HTB-37), HT-29
(colon carcinoma; ATCC HTB-37), MCF-7 (Breast cancer; ATCC HTB-22),
A549 (lung carcinoma; ATCC CCL-185), and healthy HEK293T (embryonic
kidney epithelial; ATCC CRL-3216) cell lines were cultured in DMEM-F12
medium containing 10% fetal bovine serum (FBS) and antibiotic (100
U/mL penicillin and 100 μg/mL streptomycin). The cells were
incubated at 37 °C under an atmosphere of 5% CO 2 and
95% humidity. HepG2-SR cells were selected according to the short-term
sorafenib exposure protocol. 41 The cells
were exposed to sorafenib at concentrations of 1, 2, and 4 μM
for 72 h in gradual increments. At each stage, surviving cells were
plated in sorafenib-free medium. They were exposed to the next higher
concentration of sorafenib by passaging 3 times at each concentration.
Resistant cells were maintained in medium containing 4 μM sorafenib. Effects of synthesized compounds on cell viability of HepG2, Caco-2,
HT-29, MCF-7, A549, and HEK293T cell lines tested with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay. The cells were passaged as 5 × 10 3 per well in basal medium and incubated for adherence in 96-well
plates. Sorafenib and synthesized sorafenib–ruthenium complexes
were added at 10 decreasing concentrations between 100 and 0.2 μM
and incubated for 48 h. At the end of 48 h, 10 μL of yellow-colored
MTT agent was added and plates were incubated at 37 °C for 4
h. Formazan crystals formed after 4 h were dissolved with 100 μL
of DMSO, and absorbance was measured at 540 nm. Viable cells produced
darker color and nonviable cells produced lighter color because they
could not convert MTT to formazan. The % viability was calculated
relative to the negative controls. Concentration–viability
graphs were drawn for each test substance in the GraphPad Prism 9.00,
and the IC 50 values of the substances were calculated for
each cell line. 42 4.3.4 Apoptosis Assay The apoptotic properties
of the synthesized compounds were determined in HepG2 cell line using
Muse Annexin V & Dead Cell reagent in Muse Cell Analyzer. HepG2
cells were seeded in 24-well plates and synthesized substances were
added at 3 or 4 different concentrations (0.78, 3.125, 12.5, and 50
μM). After 48 h of incubation, cells were harvested with trypsin
and transferred to PBS. 100 μL of Muse Annexin V & Dead
Cell reagent was added to the cell suspension in 100 μL of PBS.
The cells were allowed to stain for 30 min at room temperature in
the dark. The apoptotic profiles of the substances were determined
by flowing the samples in the Muse Cell Analyzer. Results are given
as a percentage (%). 43 There are
two different dyes in the reagent: Annexin V (apoptosis marker) and
7-AAD (dead cell marker). The profiles of the cells after staining
with these dyes according to their viability are as follows: Bottom-left: Living cells [Annexin V (−) and 7-AAD (−)] Bottom-right: Early apoptotic cells [Annexin V (+) and 7-AAD (−)] Top-right: Late apoptotic or dead cells by apoptosis [Annexin V
(+) and 7-AAD (+)] Top-left: Necrotic cells [Annexin V (−)
and 7-AAD (+)] 4.3.5 Cell Cycle Assay The effects of
the synthesized compounds on the cell cycle in the HepG2 cell line
were determined using the Muse Cell Cycle reagent in Muse Cell Analyzer.
HepG2 cells were seeded in 24-well plates and synthesized substances
were added at 3 or 4 different concentrations (0.78, 3.125, 12.5,
and 50 μM). After 48 h of incubation, cells were harvested with
trypsin and washed with PBS. The cell pellet was fixed in cold 70%
ethanol for 3 h at 20 °C. The fixed cells were washed again with
PBS, the supernatant was discarded, and 200 μL of Muse Cell
Cycle reagent was added. The cells were allowed to stain for 30 min
at room temperature in the dark. The effects of the substances on
the cell cycle were determined by flowing the samples in the Muse
Cell Analyzer device. Results are given as a percentage (%). The assay uses PI-based staining of DNA content to distinguish and
quantify the percentage of cells in each cell cycle phase (G0/G1,
S, and G2/M). Cells in G0/G1 phase are selected in blue, the S phase
in purple, and the G2/M phase in green. 44 4.3.6 Western Blot Assay Effects of synthesized
compounds on the expression of proteins in the tyrosine kinase pathway
(Egfr, p-Egfr, Akt, p-Akt, Erk1/2 (p44/42 MAPK), p-Erk1/2 (p-p44/42
MAPK)) in HepG2 cell line was determined by the immunoblotting method.
HepG2 cells were seeded in 6-well plates and synthesized substances
were added at 2 different concentrations (12.5 μM and/or 3.125
μM and/or 0.78 μM). After 48 h of incubation, cells were
harvested with trypsin. The cells were washed with cold PBS and were
transferred to Ripa cell lysis buffer (Santa Cruz). They were centrifuged
at 14000 rpm at +4 °C for 20 min. Protein concentration was measured
with Qubit 2.0 Fluorometer. Samples were boiled at 95 °C for
5 min in Laemmli sample buffer (Bio-Rad). Samples were run on 4–10%
polyacrylamide gels. Samples were transferred to the PVDF membrane
by a cold transfer device (Bio-Rad) and blocked with 5% skim milk
or 1% bovine serum albumin (BSA). Membranes were incubated with primary
antibodies overnight for EGFR (Abclonal A2909), p-EGFR (Abclonal AP0301),
Akt (CST 4691), p-Akt (CST 4060), Erk1/2 (CST 4695), p-Erk1/2 (CST
9101) and 1 h at RT for GAPDH (Abclonal). After secondary antibody
incubation for 1 h at RT, images were taken on Bio-Rad Chemidoc imaging
device using LumiGLO (CST) chemiluminescence substrate. Bands were
calculated with the Image Lab (Bio-Rad) and were analyzed with GraphPad
Prism 9.00. 42 4.3.7 Antiangiogenic Activity The antiangiogenic
effects of the synthesized compounds were evaluated by the Western
blot method on the expression of proteins in the angiogenesis pathway
(VEGFR, Akt, p-Akt, Erk1/2 (p44/42 MAPK), p-Erk1/2 (p-p44/42 MAPK)
in the HUVEC (Human umbilical vein endothelial) cell line). HUVEC
cells were seeded in 6-well plates and synthesized substances were
added at 2 different concentrations (12.5 μM and/or 3.125 μM
and/or 0.78 μM). After 48 h of incubation, cells were induced
with 100 ng/mL VEGFR for 15 min and harvested with trypsin. The same
procedures were applied as 4.3.5. VEGFR (CST 9698) antibody incubation
was made overnight. 42 4.4 Computational Studies Molecular Docking
studies were carried out by Schrödinger Molecular Modeling
Software (2023-1) with Maestro (13.5) interface. MD simulations were
carried out using Desmond (D. E. Shaw Research) with Maestro (13.1)
interface. 4.4.1 Preparation of Ligands and Proteins Two-dimensional structures of the ligands were drawn with Chem-Draw.
Initially, the coordination bonds of the ruthenium complex were drawn
as normal bonds and the molecule was transferred to Schrödinger.
By selecting the molecule on Schrödinger’s workspace,
the covalent bonds between the metal and the ligand were set as coordination
interactions using the “create zero-order bonds to metal”
parameter under the build tab. Then, the energy minimization of the
molecule was achieved by using the minimize parameter under the edit
tab of Schrödinger Maestro and the preparation of the ligands
was completed. The verification of correct bond orders has been accomplished
by assigning bond orders through the Edit → Assign →
Bond Orders menu. Additionally, to ensure a reasonable coordination
geometry and prevent distorted structures, the “Perform postdocking
minimization” setting in the Output tab of the Glide ligand
docking application has been unchecked. 45 The 3D X-ray crystallographic structures of the target proteins
were obtained from the Protein Data Bank, accessible via the RCSB
Web site ( https://www.rcsb.org ). In this study, epidermal growth factor receptor (EGFR) protein
(PDB ID: 5X2A , the crystal resolution: 1.85 Å, origin: Homo sapiens ) was used to carry out all in silico studies. Careful
preparation and refinement of these protein structure was carried
out using the Protein Preparation Wizard module within Schrödinger
Maestro 13.5. 46 4.4.2 Glide Docking and Induced Fit Docking The methods previously published by our research group were used
to perform glide docking and induced fit docking. 47 , 48 The ligands that were prepared underwent docking with the receptors
using the Induced Fit Docking (IFD) protocol in Schrödinger
Release 2023-1. The IFD protocol, integrated into the Schrödinger
suite, employs Glide (Schrödinger, LLC, New York, NY). The
centroid of the native ligand served as the center for the Glide grid,
with the inner box side set to 20 Å and the outer box side automatically
determined. The binding energy, represented by the IFD Score, was
computed for each generated pose, and the resulting poses for each
protein–ligand complex underwent thorough visual examination. 49 4.4.3 Prime MM-GBSA Analysis The methods
previously published by our research group were used to perform prime
MM-GBSA analysis. 50 The calculation of
the free binding energy for the docked poses were carried out utilizing
the Prime MM/GBSA (Molecular Mechanics Generalized Born Surface Area)
module within the Schrödinger suite 2023. Default parameters
were applied in the MM/GBSA module, including the VSGB solvation model
and the Minimize sampling method. To account for flexibility, constraints
on flexible residues were implemented, designating the residues around
the receptor pocket as having flexible conformations during the energy
calculation. 51 4.4.4 Molecular Dynamics Simulations The methods previously published by our research group were used
to perform molecular dynamics simulations. 52 The Desmond System Builder module was employed to configure the
complex within a solvent-soaked orthorhombic periodic box, with a
selected orthorhombic box size of 10 Å as boundary conditions.
To achieve a neutral system charge, Na + and Cl – ions were added, resulting in a 0.15 M NaCl salt concentration.
The constructed solvated system underwent minimization using the OPLS3e
force field. System equilibration occurred at 300 K and 1.01325 bar
in the NPT (normal pressure and temperature) ensemble. Simulations
were conducted over a 200 ns time frame, with data recorded at 200
ps intervals. Molecular dynamic trajectories were generated and analyzed
utilizing Desmond’s Simulation Interaction Diagram (SID). 47 4.4.5 Molecular Docking Validations The
methods previously published by our research group were used to perform
molecular docking validations. 53 4.5 Formation of Micelles PEG–PLGA
polymeric micelles containing Ru3S or Ru4S complexes, which showed the best results in the cytotoxicity test,
were formed by the coprecipitation method. For this purpose, 1 mg
of either Ru3S or Ru4S complex and poly(ethylene
glycol) methyl ether- block -poly(lactide- co -glycolide) (PEG–PLGA, PEG average M n 5000, PLGA average M n 10,000,
lactide:glycolide 50:50, 10 mg) polymer were mixed overnight in a
vial containing 1 mL of DMSO. The next day, micelle (“self-assembly”)
structures were formed by adding dropwise into a vial containing 4
mL of water and the solution was stirred for 24 h. Then, DMSO was
removed by dialysis. 54 The micelle formation
process of the amphiphilic polymer without any complexes was followed
by using a similar procedure. M refers to PEG–PLGA polymeric
micelles without any complexes. M1 and M2 refer to Ru3S - and Ru4S -loaded polymeric
micelles. 4.6 Release Studies The release profile
of Ru4S from M2 was evaluated by the dialysis
method in two different pH buffer solutions (pH = 5.5 and pH 7.4).
2 mL of M2 was placed into a dialysis bag with a molecular
weight cutoff of 3.50 kDa and dialyzed against 50 mL of phosphate-buffered
saline (PBS) in an orbital shaker with a stirring speed of 100 rpm
at 37 °C. 2 mL of samples was taken at a certain time point and
fresh buffer solution was replaced in the same volume instead. To
quantitatively determine the amount of drug released, the taken samples
at each time interval were analyzed by LC-HRMS. 55 − 57
## Materials
4.1 Materials The chemicals and solvents
were bought from Fluka Chemie, Merck, Alfa Aesar, and Sigma-Aldrich.
Melting points were determined on a STUART SMP40. IR spectra were
measured on an Alfa Bruker spectrometer. 1 H and 13 C NMR spectra were acquired on a Bruker spectrometer at 500 and 125
Hz, respectively. Mass spectra were obtained using a Thermo Fisher
Scientific LC-HRMS spectrometer. UV measurements were determined with
a HITACHI U-2900 brand spectrometer. Spectrophotometric analyses were
performed by a BioTek Power Wave XS (BioTek). The cell line was purchased
from American Type Culture Collection (ATCC). Dulbecco’s modified
Eagle’s medium-F12, RPMI medium, fetal calf serum, and PBS
were bought from GIBCO BRL, Invitrogen (Carlsbad, CA). All compounds
used for biological assays were >95% pure, as determined by HPLC
analysis
( Figures S39–S47 ).
## Methods
4.2 Methods 4.2.1 General Procedures and Spectral Data 4.2.1.1 Synthesis of Sorafenib 4-Chloropicolinoyl
chloride ( 2 ): 0.08 mol of thionyl chloride and 1 mL of
DMF were mixed under nitrogen at 40 °C for 10 min. 0.4 mol of
picolinic acid ( 1 ) was added in half an hour piece by
piece. When the addition was complete, the temperature was increased
to 72 °C and stirred at this temperature for 16 h. A yellow precipitate
formed at the end of the reaction. The resulting solid was purified
with the help of toluene and ether to obtain 4-chloropicolinoyl chloride
( 2 ) in 60% yield. 27 1 H NMR (DMSO- d 6 , 300 MHz) δ/ppm:
8.62 (1H, d, J = 7.4 Hz), 8.10 (1H, s), 7.46 (1H,
d, J = 7.4 Hz). 4-Chloro- N -methylpicolinamide ( 3 ): 4-Chloropicolinoyl chloride
( 2 ) and methyl amine were mixed in cold methanol. The
solid part formed at the end of the reaction was dried by filtration
through the gooch crucible. 4-Chloro- N -methylpicolinamide
( 3 ) obtained in 20% yield was used for the next step. 27 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm: 8.72 (1H, d, J =
7.3 Hz), 8.14 (1H, s), 7.52 (1H, d, J = 7.3 Hz),
2.59 (3H, d, J = 4.5 Hz). 4-(4-Aminophenoxy)- N -methylpicolinamide ( 4 ): First, p -aminophenol and t -BuOK were mixed in DMF for 2
h under nitrogen, then compound 3 and K 2 CO 3 were added and mixed at
80 °C for 16 h. At the end of the reaction, DMF was evaporated
in the evaporator. 4-(4-Aminophenoxy)- N -methylpicolinamide
( 4 ) was obtained by column chromatography with hexane-ethyl
acetate. 28 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm: 2.79 (3H, d, J = 4.8 Hz), 5.22 (2H, s, NH 2 ), 6.65 (2H, d, J = 8.7 Hz), 6.87 (2H, d, J = 8.7 Hz),
7.08 (1H, dd, J = 5.6, 2.6 Hz), 7.35 (1H, d, J = 2.5 Hz), 8.46 (1H, d, J = 5.62 Hz),
8.78 (1H, q, J = 4.5 Hz); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm: 26.4, 108.7, 114.1,
115.3, 122.0, 143.2, 147.3, 150.6, 152.7, 164.3, 167.2. 4-(4-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)- N -methylpicolinamide ( Sorafenib ): 1 mmol of
4-(4-aminophenoxy)- N -methylpicolinamide ( 4 ), 1.1 mmol of 4-chloro-3-trifluoromethyl phenyl isocyanate, and
1.1 mmol of Et 3 N were dissolved in THF together and stirred
overnight at 60 °C. At the end of the reaction, THF was evaporated
in the evaporator. The solid was purified by washing with ether. 29 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm 2.81 (3H, d, J = 4.8
Hz), 7.29–7.06 (3H m), 7.41 (1H, d, J = 2.5
Hz), 7.78–7.56 (4H, m), 8.15 (1H, d, J = 2.5
Hz), 8.52 (1H, d, J = 5.6 Hz), 8.82 (1H, q, J = 4.7, 4.6 Hz), 9.04 (1H, s), 9.26 (1H, s); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm:
26.4, 109.0, 114.4, 117.2, 120.9, 121.9, 122.8, 123.5, 124.3, 127.0,
132.4, 137.5, 139.7, 148.2, 150.8, 152.8, 152.9, 164.2, 166.4. 4.2.1.2 Synthesis of RuL 2 Cl 2 Complexes Method A: The mixture of RuCl 3 ·(H 2 O) n (1 mmol), ligand (L) (2 mmol), LiCl (6
mmol), and DMF (2 mL) was stirred under nitrogen at 80 °C under
reflux for 18 h. At the end of the reaction, acetone was added after
cooling and the mixture was kept in a refrigerator for 24 h. The precipitate
formed was filtered, washed with cold water and diethyl ether, and
then dried. The resulting complex was purified by column chromatography. 30 The following complexes were synthesized according
to this method. Ru(bpy) 2 Cl 2 : Dark purple
powder, 60% yield, 360 °C (decomposition) mp. 1 H NMR
(DMSO- d 6 , 500 MHz) δ/ppm; 9.98 (d, J = 5.4 Hz, 1H), 8.64 (d, J = 8.0 Hz, 1H),
8.48 (d, J = 8.0 Hz, 1H), 8.12–8.01 (m, 1H),
7.77 (t, J = 6.4 Hz, 1H), 7.74–7.63 (m, 1H),
7.51 (d, J = 5.5 Hz, 1H), 7.10 (dd, J = 9.6, 3.4 Hz, 1H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 159.5, 157.5, 152.5, 151.3, 133.9,
132.6, 124.7, 124.6, 122.2, 121.8. HRMS; calcd for C 20 H 16 Cl 2 N 4 Ru [M + H] + ; 484.9874,
found; 484.9806. Ru(4,4-dimebpy) 2 Cl 2 :
Dark purple powder,
46% yield, 360 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 9.77 (d, J
= 5.7 Hz, 1H), 8.46 (s, 1H), 8.31 (s, 1H), 7.58 (d, J = 5.0 Hz, 1H), 7.31 (d, J = 5.9 Hz, 1H),
6.92 (dd, J = 5.9, 1.1 Hz, 1H), 2.62 (s, 3H), 2.34
(s, 3H). 13 C NMR (DMSO- d 6 ,
125 MHz) δ/ppm; 160.2, 158.4, 152.9, 151.6, 145.9, 144.7, 126.5,
126.5, 123.8, 123.4, 21.1, 20.7. HRMS; calcd for C 24 H 24 Cl 2 N 4 Ru [M + H] + ; 541.0500,
found; 541.1046. Ru(phen) 2 Cl 2 : Dark purple
powder, 56% yield,
370 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.28 (dd, J = 5.2, 1.2 Hz, 1H), 8.72 (dd, J = 8.1, 1.2 Hz, 1H),
8.29 (d, J = 8.8 Hz, 1H), 8.26–8.20 (m, 2H),
8.14 (d, J = 8.8 Hz, 1H), 7.75 (dd, J = 5.4, 1.0 Hz, 1H), 7.33 (dd, J = 8.0, 5.4 Hz, 1H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm;
154.7, 153.4, 151.2, 149.7, 134.0, 132.5, 130.0, 127.9, 127.5, 125.2,
124.9, 123.9. HRMS; calcd for C 24 H 16 Cl 2 N 4 Ru [M + H] + ; 532.9874, found; 532.1806. Ru(5-Clphen) 2 Cl 2 : Dark purple powder, 29%
yield, 375 °C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.35 (dt, J = 5.2,
1.1 Hz, 1H), 10.26 (td, J = 5.2, 1.2 Hz, 1H), 8.83
(dd, J = 8.4, 2.5 Hz, 1H), 8.68 (dt, J = 7.6, 1.3 Hz, 1H), 8.62 (d, J = 2.01 Hz, 1H), 8.47
(s, 1H), 8.40–8.28 (m, 2H), 8.28–8.20 (m, 2H), 7.98–7.88
(m, 1H), 7.85 (dd, J = 5.4, 1.0 Hz, 1H), 7.47–7.43
(m, 1H), 7.38–7.34 (m, 1H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 161.7, 154.5, 154.0,
153.7, 150.6, 149.2, 149.2, 147.9, 132.5, 131.1, 129.9, 128.9, 128.4,
127.1, 126.0, 125.7, 125.0, 124.8, 124.6, 124.4. HRMS; calcd for C 24 H 14 Cl 4 N 4 Ru [M + H] + ; 600.9094, found; 600.9550. Ru(5-mephen) 2 Cl 2 : Dark purple powder, 55%
yield, 373 °C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.29 (d, J = 4.0
Hz, 1H), 10.20 (s, 1H), 8.74 (d, J = 8.2 Hz, 1H),
8.58 (d, J = 7.9 Hz, 1H), 8.24–8.20 (m, 2H),
8.17–8.13 (m, 1H), 8.09 (d, J = 4.4 Hz, 2H),
7.95 (d, J = 6.7 Hz, 1H), 7.76–7.68 (m, 1H),
7.67–7.60 (m, 1H), 7.35 (dd, J = 8.1, 5.4
Hz, 1H), 7.28 (dd, J = 7.8, 5.4 Hz, 1H), 2.76 (s,
3H), 2.89 (s, 3H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 171.4, 153.1, 152.5, 149.6, 148.8,
147.9, 138.2, 134.4, 134.1, 132.0, 130.8, 130.1, 129.1, 129.0, 128.6,
128.5, 125.4, 125.1, 124.2, 123.9, 123.7, 123.5, 114.0, 21.4, 20.4.
HRMS; calcd for C 26 H 20 Cl 2 N 4 Ru [M + H] + ; 561.0187, found; 561.0657. Method B:
RuCl 3 ·(H 2 O) n (1 mmol)
and LiCl (40 mmol) were mixed in ethylene glycol:water
mixture at 110 °C for 15 min. After 15 min, ligand (2.2 mmol)
was added and mixing was continued. Then, glucose (2 mmol) was added
and mixed for another 15 min at 110 °C. Ascorbic acid was added
to it and mixing was continued for 30 min. After 30 min, 10 mL of
saturated NaCl solution was added and stirred at 0 °C for 1 h.
The precipitate was then filtered off and purified by column chromatography. 31 , 39 The following complexes were synthesized according to this method. Ru(6,6-dimebpy) 2 Cl 2 : Dark purple powder,
78% yield, 370 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 8.58 (d, J
= 7.8 Hz, 1H), 8.44 (d, J = 7.7 Hz, 2H),
8.36 (d, J = 7.7 Hz, 1H), 8.06 (t, J = 7.5 Hz, 1H), 7.95 (t, J = 7.6 Hz, 1H), 7.85 (t, J = 7.6 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H),
7.55 (d, J = 7.4 Hz, 1H), 7.48 (d, J = 7.4 Hz, 1H), 7.28–7.23 (m, 2H), 2.91 (s, 3H), 2.53 (s, 3H),
1.86 (s, 3H), 1.65 (s, 3H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 167.9, 166.8, 166.5, 164.9, 163.9,
161.9, 161.1, 160.5, 137.1, 136.5, 135.8, 135.2, 124.6, 124.6, 123.9,
123.675, 121.6, 121.0, 120.7, 120.6, 27.2, 24.7, 24.6, 23.7. HRMS;
calcd for C 24 H 24 Cl 2 N 4 Ru
[M + H] + ; 541.0500, found; 541.1334. Ru(4,4-dimethoxybpy) 2 Cl 2 : Dark purple powder,
50% yield, 370 °C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 9.83 (d, J = 6.6 Hz, 1H), 9.20 (d, J = 6.5 Hz, 1H), 8.46 (d, J = 2.7 Hz, 1H), 8.36 (d, J = 2.6 Hz, 1H),
8.30 (dd, J = 8.2, 2.6 Hz, 2H), 7.59 (dd, J = 6.6, 2.6 Hz, 1H), 7.51 (d, J = 6.6
Hz, 1H), 7.47 (dd, J = 6.6, 2.7 Hz, 1H), 7.07 (dd, J = 6.5, 2.5 Hz, 1H), 7.03–7.00 (m, 2H), 4.11 (s,
3H), 4.08 (s, 3H), 3.94 (s, 3H), 3.92 (s, 3H); 13 C NMR
(DMSO- d 6 , 125 MHz) δ/ppm; 167.5,
167.3, 166.9, 166.7, 159.2, 159.0, 158.8, 157.5, 156.4, 154.0, 153.4,
150.1, 114.6, 114.1, 113.5, 113.2, 112.0, 111.6, 111.2, 110.4, 57.4,
57.2, 57.2, 57.1. HRMS; calcd for C 24 H 24 Cl 2 N 4 O 4 Ru [M + H] + ; 605.0296,
found; 605.0755. Ru(4,4-di- t -bu-bpy) 2 Cl 2 :
Dark purple powder, 60% yield, 370 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm;
8.55 (s, 4H), 7.59 (s, 4H), 7.26 (s, 4H), 1.50 (s, 18H), 1.33 (s,
18H); 13 C NMR (DMSO- d 6 , 125
MHz) δ/ppm; 163.2, 162.7, 161.9, 161.8, 158.0, 157.5, 157.4,
156.3, 155.1, 152.6, 125.0, 124.6, 124.2, 124.1, 122.4, 122.1, 122.0,
121.5, 121.1, 120.1, 36.1, 35.8, 35.7, 30.7, 30.6, 30.5, 30.4. HRMS;
calcd for C 36 H 48 Cl 2 N 4 Ru
[M + H] + ; 709.2378, found; 709.2895. 4.2.1.3 Synthesis of RuL 2 S Complexes Ru–ligand complex (RuL 2 Cl 2 ) (1 mmol)
and sorafenib (Srf) (1 mmol) were stirred in methanol overnight at
60 °C. At the end of the reaction, methanol was removed. The
solid was dried and purified by flash chromatography. 32 , 33 Ru(bpy) 2 Srf (Ru1S): Red powder, 30% yield, 196
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
8.73 (d, J = 8.1 Hz, 1H), 8.70 (d, J = 7.6 Hz, 2H), 8.63 (d, J = 8.0 Hz, 1H), 8.60 (d, J = 7.9 Hz, 1H), 8.23–8.17 (m, 2H), 8.15–8.13
(m, 1H), 8.07 (d, J = 2.6 Hz, 1H), 8.03–7.99
(m, 2H), 7.95 (td, J = 8.0, 1.4 Hz, 1H), 7.81 (d, J = 5.7 Hz, 1H), 7.78–7.74 (m, 1H), 7.71 (d, J = 5.6, 1H), 7.68–7.65 (m, 1H), 7.70–7.50
(m, 4H), 7.48 (d, J = 8.7 Hz, 1H), 7.37 (td, J = 7.3, 1.2 Hz, 1H), 7.32 (td, J = 7.3,
1.3 Hz, 1H), 7.16–7.12 (m, 2H), 7.11 (dd, J = 6.4, 2.6 Hz, 1H), 2.95 (s, 3H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 172.0, 166.8, 159.1, 158.2, 157.9, 157.4,
153.4, 153.1, 152.8, 151.9, 151.6, 151.4, 150.5, 148.0, 138.7, 137.5,
137.3, 137.0, 136.2, 131.6, 127.5, 127.4, 126.8, 126.5, 124.1, 123.8,
123.7, 123.5, 122.8, 121.2, 121.0, 117.3, 117.2, 114.1, 26.5. HRMS;
calcd for C 41 H 32 ClF 3 N 8 O 3 Ru [M – H] − ; 877.1203, found;
877.1192. Ru(4,4-dimebpy) 2 Srf (Ru2S): Red powder,
52% yield, 215
°C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.92 (s, 1H), 10.23 (s, 1H), 9.89 (s, 1H),
8.72 (d, J = 13.7 Hz, 2H), 8.64 (d, J = 15.9 Hz, 2H), 8.50 (s, 1H), 8.44 (d, J = 5.7 Hz,
1H), 8.11 (d, J = 1.6 Hz, 1H), 7.88 (d, J
= 5.7 Hz, 1H), 7.64–7.61 (m, 2H), 7.61–7.57
(m, 3H), 7.52–7.50 (m, 2H), 7.48 (d, J = 6.4
Hz, 1H), 7.45 (d, J = 5.8 Hz, 1H), 7.25 (dd, J = 5.8, 1.0 Hz, 1H), 7.20–7.19 (m, 2H), 7.18–7.17
(m, 1H), 7.09 (dd, J = 6.4, 2.6 Hz, 1H), 2.84 (d, J = 4.6 Hz, 3H), 2.47 (s, 3H), 2.44 (s, 3H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm;
171.3, 170.7, 164.8, 157.4, 156.6, 156.2, 155.8, 152.2, 152.0, 151.4,
150.6, 150.3, 150.2, 149.2, 148.4, 148.3, 148.1, 147.1, 146.4, 138.8,
137.1, 131.4, 127.8, 127.7, 127.3, 127.0, 124.4, 124.0, 123.8, 123.2,
121.9, 121.4, 121.1, 120.6, 119.2, 116.6, 115.5, 115.5, 114.1, 26.7,
20.2, 20.1, 19.9, 19.8. HRMS; calcd for C 45 H 40 ClF 3 N 8 O 3 Ru [M – H] − ; 933.1829, found; 933.1818. Ru(6,6-dimebpy) 2 Srf
(Ru3S): Red powder, 25% yield, 220
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
8.60–8.54 (m, 3H), 8.51 (d, J = 8.0 Hz, 1H),
8.07–7.99 (m, 4H), 7.90 (t, J = 7.8 Hz, 1H),
7.72 (d, J = 2.3 Hz, 1H), 7.68 (d, J = 6.5 Hz, 1H), 7.62 (dd, J = 8.7, 2.3 Hz, 1H), 7.57
(d, J = 8.8 Hz, 2H), 7.5–7.39 (m, 5H), 7.08
(dd, J = 6.5, 2.6 Hz, 1H), 7.04 (d, J = 8.8 Hz, 2H), 2.69 (s, 3H), 2.36 (s, 3H), 2.06 (s, 3H), 1.99 (s,
3H), 1.73 (s, 3H); 13 C NMR (CD 3 OD, 125 MHz)
δ/ppm; 170.7, 167.5, 167.0, 166.2, 165.0, 164.7, 160.6, 159.5,
159.2, 158.3, 153.3, 151.6, 147.7, 138.7, 137.7, 137.6, 137.6, 137.0,
136.9, 131.6, 126.4, 126.2, 126.1, 126.0, 124.0, 123.9, 122.8, 122.5,
122.0, 121.3, 121.2, 121.1, 120.9, 117.2, 117.2, 116.4, 113.4, 26.1,
24.5, 24.3, 23.1, 22.7. HRMS; calcd for C 45 H 40 ClF 3 N 8 O 3 Ru [M – H] − ; 933.1829, found; 933.1814. Ru(4,4-di- t -bu-bpy) 2 Srf (Ru4S): Red
powder, 65% yield, 207 °C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm; 8.73 (dd, J = 6.9, 1.7
Hz, 2H), 8.63 (dd, J = 9.9, 1.8 Hz, 2H), 8.57 (d, J = 5.9 Hz, 1H), 8.06 (d, J = 2.6 Hz, 1H),
8.05 (d, J = 2.5 Hz, 1H), 8.01 (d, J = 5.9 Hz, 1H), 7.80 (dd, J = 5.9, 1.6 Hz, 1H), 7.69
(dd, J = 5.9, 1.7 Hz, 1H), 7.66 (d, J = 6.1 Hz, 1H), 7.64–7.54 (m, 5H), 7.46 (d, J = 8.7 Hz, 1H), 7.41 (dd, J = 6.1, 1.9 Hz, 1H), 7.36
(dd, J = 6.1, 2.0 Hz, 1H), 7.14–7.10 (m, 3H),
2.96 (s, 3H), 1.52 (s, 9H), 1.51 (s, 9H), 1.41 (s, 9H), 1.39 (s, 9H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 172.0, 166.5,
162.5, 162.3, 162.0, 161.2, 158.8, 158.0, 157.8, 157.2, 153.4, 152.7,
152.4, 151.6, 151.2, 150.9, 149.9, 148.0, 138.8, 137.5, 131.5, 128.0,
127.7, 124.6, 124.6, 124.2, 124.0, 123.9, 123.8, 122.8, 121.3, 121.1,
121.0, 120.9, 120.8, 120.6, 117.2, 115.2, 114.0, 113.3, 35.3, 35.2,
35.0, 35.0, 33.5, 31.6, 30.7, 29.3, 29.3, 29.2, 29.0, 28.8, 28.7,
26.5, 22.3. HRMS; calcd for C 57 H 64 ClF 3 N 8 O 3 Ru [M – H] − ; 1101.3707,
found; 1101.3700. Ru(4,4-dimethoxybpy) 2 Srf (Ru5S):
Red powder, 80% yield,
235 °C (decomposition) mp. 1 H NMR (CD 3 OD,
500 MHz) δ/ppm; 8.39 (d, J = 6.4 Hz, 1H), 8.28
(d, J = 2.6 Hz, 1H), 8.25 (d, J = 2.6 Hz, 1H), 8.19 (dd, J = 5.2, 2.7 Hz, 2H), 8.03
(d, J = 2.5 Hz, 1H), 8.01 (d, J = 2.6 Hz, 1H), 7.85 (d, J = 6.4 Hz, 1H), 7.65 (d, J = 6.4 Hz, 1H), 7.61–7.52 (m, 5H), 7.48 (d, J = 8.7 Hz, 1H), 7.32 (dd, J = 6.4, 2.6
Hz, 1H), 7.22 (dd, J = 6.4, 2.6 Hz, 1H), 7.16–7.12
(m, 2H), 7.10 (dd, J = 6.4, 2.6 Hz, 1H), 6.98 (dd, J = 6.5, 2.7 Hz, 1H), 6.94 (dd, J = 6.6,
2.7 Hz, 1H), 4.09 (s, 3H), 4.08 (s, 3H), 3.99 (s, 3H), 3.98 (s, 3H),
2.95 (s, 3H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm;
172.3, 167.2, 167.1, 166.9, 166.4, 166.1, 160.0, 159.3, 158.7, 153.4,
153.1, 152.3, 152.1, 151.9, 151.2, 148.1, 138.7, 137.5, 131.6, 122.8,
121.2, 120.9, 117.3, 117.2, 117.0, 113.9, 113.6, 113.5, 113.4, 112.7,
110.8, 110.4, 110.4, 110.2, 55.9, 55.8, 26.4. HRMS; calcd for C 45 H 40 ClF 3 N 8 O 7 Ru
[M – H] − ; 997.1626, found; 997.1596. Ru(phen) 2 Srf (Ru6S): Red powder, 37% yield, 205 °C
mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm; 9.18
(dd, J = 5.1, 1.1 Hz, 1H), 8.81 (dd, J = 8.3, 1.1 Hz, 2H), 8.61 (dd, J = 5.1, 1.1 Hz, 1H),
8.53 (dd, J = 8.2, 1.1 Hz, 1H), 8.48 (dd, J = 8.2, 1.0 Hz, 1H), 8.33 (dd, J = 8.9,
4.9 Hz, 2H), 8.24 (t, J = 8.7 Hz, 2H), 8.15 (dd, J = 8.2, 5.1 Hz, 1H), 8.10 (d, J = 2.6
Hz, 1H), 8.05 (dd, J = 8.2, 5.1 Hz, 1H), 8.03–7.98
(m, 2H), 7.91 (dd, J = 5.3, 1.1 Hz, 1H), 7.65–7.51
(m, 6H), 7.48 (d, J = 8.7 Hz, 1H), 7.15–7.09
(m, 2H), 7.01 (dd, J = 6.4, 2.6 Hz, 1H), 2.94 (s,
3H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 172.3,
166.8, 154.1, 153.4, 153.2, 152.8, 151.8, 149.5, 148.7, 148.6, 148.0,
138.7, 137.5, 136.6, 136.4, 136.0, 135.2, 131.5, 131.0, 130.8, 130.8,
130.6, 127.8, 127.8, 127.7, 126.0, 125.4, 125.1, 122.8, 121.2, 120.9,
117.3, 117.2, 117.1, 114.1, 26.5. HRMS; calcd for C 45 H 32 ClF 3 N 8 O 3 Ru [M – H] − ; 925.1203, found; 925.1192. Ru(5-Clphen) 2 Srf (Ru7S): Red powder, 51% yield, 208
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
9.25 (td, J = 3.5, 1.2 Hz, 1H), 9.18 (td, J = 3.5, 1.3 Hz, 1H), 9.07–9.01 (m, 2H), 8.79–8.73
(m, 3H), 8.7 (d, J = 8.7 Hz, 1H), 8.68 (t, J = 4.1 Hz, 1H), 8.60 (t, J = 5.1 Hz, 1H),
8.53 (d, J = 1.4 Hz, 2H), 8.49 (d, J = 8.2 Hz, 1H), 8.44–8.42 (m, 3H), 8.29–8.24 (m, 1H),
8.19–8.11 (m, 5H), 8.06–8.03 (m, 3H), 7.96–7.95
(m, 3H), 7.69–7.62 (m, 2H), 7.61–7.52 (m, 10H), 7.41
(d, J = 8.7 Hz, 2H), 7.09 (dd, J = 8.8, 1.3 Hz, 4H), 7.02–6.99 (m, 2H), 2.94 (d, J = 3.3 Hz, 6H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm;
173.8, 172.3, 167.8, 166.9, 155.1, 154.6, 153.9, 153.7, 153.4, 153.3,
153.3, 153.2, 152.1, 151.7, 149.1, 148.6, 147.9, 147.2, 138.7, 137.6,
136.0, 135.8, 135.4, 133.7, 133.5, 132.3, 132.1, 131.7, 131.5, 131.5,
130.9, 130.2, 130.0, 129.3, 129.1, 128.4, 127.0, 126.9, 126.9, 126.6,
126.5, 126.0, 125.7, 124.0, 122.8, 122.7, 121.1, 120.9, 117.2, 117.1,
114.2, 113.3, 29.3, 28.7. HRMS; calcd for C 45 H 30 Cl 3 F 3 N 8 O 3 Ru [M + H] + ; 995.0580, found; 995.0386. Ru(5-mephen) 2 Srf (Ru8S): Red powder, 18% yield, 195
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
9.19 (t, J = 5.0 Hz,1H), 9.12 (t, J = 5.0 Hz, 1H), 8.95–8.91 (m, 2H), 8.73–8.69 (m, 2H),
8.66–8.61 (m, 2H), 8.58 (d, J = 8.3 Hz, 1H),
8.54 (t, J = 5.2 Hz, 1H), 8.42 (d, J = 8.1 Hz, 1H), 8.37 (d, J = 8.1 Hz, 1H), 8.23–8.18
(m, 1H), 8.18–8.14 (m, 4H), 8.06 (d, J = 4.1
Hz, 2H), 8.02–7.99 (m, 4H), 7.93 (d, J = 5.0
Hz, 2H), 7.86–7.83 (m, 1H), 7.64–7.46 (m, 14H), 7.43
(d, J = 8.7 Hz, 2H), 7.12 (td, J = 8.9, 1.6 Hz, 4H), 7.06–7.00 (m, 2H), 2.99 (s, 3H), 2.97 (s,
6H), 2.96 (s, 3H), 2.90 (d, J = 5.0 Hz, 6H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 173.7, 172.2,
167.8, 166.7, 153.6, 153.3, 153.2, 153.1, 152.3, 151.8, 151.7, 151.3,
150.8, 149.8, 148.8, 148.0, 147.9, 147.9, 138.7, 138.7, 137.5, 136.4,
136.2, 136.1, 135.7, 135.5, 135.1, 134.3, 133.6, 133.4, 133.0, 132.3,
132.1, 131.5, 131.2, 130.9, 130.8, 130.6, 130.4, 129.1, 128.4, 128.0,
127.7, 126.6, 126.5, 126.5, 126.0, 125.8, 125.4, 125.2, 125.1, 124.9,
124.0, 123.9, 122.9, 122.8, 122.7, 121.8, 121.1, 120.9, 117.2, 117.2,
117.1, 115.2, 114.1, 113.3, 29.3, 28.7, 24.1, 23.5, 22.6, 22.3. HRMS;
calcd for C 47 H 36 ClF 3 N 8 O 3 Ru [M] − ; 953.1516, found; 953.1502.
## General Procedures and Spectral Data
4.2.1 General Procedures and Spectral Data 4.2.1.1 Synthesis of Sorafenib 4-Chloropicolinoyl
chloride ( 2 ): 0.08 mol of thionyl chloride and 1 mL of
DMF were mixed under nitrogen at 40 °C for 10 min. 0.4 mol of
picolinic acid ( 1 ) was added in half an hour piece by
piece. When the addition was complete, the temperature was increased
to 72 °C and stirred at this temperature for 16 h. A yellow precipitate
formed at the end of the reaction. The resulting solid was purified
with the help of toluene and ether to obtain 4-chloropicolinoyl chloride
( 2 ) in 60% yield. 27 1 H NMR (DMSO- d 6 , 300 MHz) δ/ppm:
8.62 (1H, d, J = 7.4 Hz), 8.10 (1H, s), 7.46 (1H,
d, J = 7.4 Hz). 4-Chloro- N -methylpicolinamide ( 3 ): 4-Chloropicolinoyl chloride
( 2 ) and methyl amine were mixed in cold methanol. The
solid part formed at the end of the reaction was dried by filtration
through the gooch crucible. 4-Chloro- N -methylpicolinamide
( 3 ) obtained in 20% yield was used for the next step. 27 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm: 8.72 (1H, d, J =
7.3 Hz), 8.14 (1H, s), 7.52 (1H, d, J = 7.3 Hz),
2.59 (3H, d, J = 4.5 Hz). 4-(4-Aminophenoxy)- N -methylpicolinamide ( 4 ): First, p -aminophenol and t -BuOK were mixed in DMF for 2
h under nitrogen, then compound 3 and K 2 CO 3 were added and mixed at
80 °C for 16 h. At the end of the reaction, DMF was evaporated
in the evaporator. 4-(4-Aminophenoxy)- N -methylpicolinamide
( 4 ) was obtained by column chromatography with hexane-ethyl
acetate. 28 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm: 2.79 (3H, d, J = 4.8 Hz), 5.22 (2H, s, NH 2 ), 6.65 (2H, d, J = 8.7 Hz), 6.87 (2H, d, J = 8.7 Hz),
7.08 (1H, dd, J = 5.6, 2.6 Hz), 7.35 (1H, d, J = 2.5 Hz), 8.46 (1H, d, J = 5.62 Hz),
8.78 (1H, q, J = 4.5 Hz); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm: 26.4, 108.7, 114.1,
115.3, 122.0, 143.2, 147.3, 150.6, 152.7, 164.3, 167.2. 4-(4-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)- N -methylpicolinamide ( Sorafenib ): 1 mmol of
4-(4-aminophenoxy)- N -methylpicolinamide ( 4 ), 1.1 mmol of 4-chloro-3-trifluoromethyl phenyl isocyanate, and
1.1 mmol of Et 3 N were dissolved in THF together and stirred
overnight at 60 °C. At the end of the reaction, THF was evaporated
in the evaporator. The solid was purified by washing with ether. 29 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm 2.81 (3H, d, J = 4.8
Hz), 7.29–7.06 (3H m), 7.41 (1H, d, J = 2.5
Hz), 7.78–7.56 (4H, m), 8.15 (1H, d, J = 2.5
Hz), 8.52 (1H, d, J = 5.6 Hz), 8.82 (1H, q, J = 4.7, 4.6 Hz), 9.04 (1H, s), 9.26 (1H, s); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm:
26.4, 109.0, 114.4, 117.2, 120.9, 121.9, 122.8, 123.5, 124.3, 127.0,
132.4, 137.5, 139.7, 148.2, 150.8, 152.8, 152.9, 164.2, 166.4. 4.2.1.2 Synthesis of RuL 2 Cl 2 Complexes Method A: The mixture of RuCl 3 ·(H 2 O) n (1 mmol), ligand (L) (2 mmol), LiCl (6
mmol), and DMF (2 mL) was stirred under nitrogen at 80 °C under
reflux for 18 h. At the end of the reaction, acetone was added after
cooling and the mixture was kept in a refrigerator for 24 h. The precipitate
formed was filtered, washed with cold water and diethyl ether, and
then dried. The resulting complex was purified by column chromatography. 30 The following complexes were synthesized according
to this method. Ru(bpy) 2 Cl 2 : Dark purple
powder, 60% yield, 360 °C (decomposition) mp. 1 H NMR
(DMSO- d 6 , 500 MHz) δ/ppm; 9.98 (d, J = 5.4 Hz, 1H), 8.64 (d, J = 8.0 Hz, 1H),
8.48 (d, J = 8.0 Hz, 1H), 8.12–8.01 (m, 1H),
7.77 (t, J = 6.4 Hz, 1H), 7.74–7.63 (m, 1H),
7.51 (d, J = 5.5 Hz, 1H), 7.10 (dd, J = 9.6, 3.4 Hz, 1H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 159.5, 157.5, 152.5, 151.3, 133.9,
132.6, 124.7, 124.6, 122.2, 121.8. HRMS; calcd for C 20 H 16 Cl 2 N 4 Ru [M + H] + ; 484.9874,
found; 484.9806. Ru(4,4-dimebpy) 2 Cl 2 :
Dark purple powder,
46% yield, 360 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 9.77 (d, J
= 5.7 Hz, 1H), 8.46 (s, 1H), 8.31 (s, 1H), 7.58 (d, J = 5.0 Hz, 1H), 7.31 (d, J = 5.9 Hz, 1H),
6.92 (dd, J = 5.9, 1.1 Hz, 1H), 2.62 (s, 3H), 2.34
(s, 3H). 13 C NMR (DMSO- d 6 ,
125 MHz) δ/ppm; 160.2, 158.4, 152.9, 151.6, 145.9, 144.7, 126.5,
126.5, 123.8, 123.4, 21.1, 20.7. HRMS; calcd for C 24 H 24 Cl 2 N 4 Ru [M + H] + ; 541.0500,
found; 541.1046. Ru(phen) 2 Cl 2 : Dark purple
powder, 56% yield,
370 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.28 (dd, J = 5.2, 1.2 Hz, 1H), 8.72 (dd, J = 8.1, 1.2 Hz, 1H),
8.29 (d, J = 8.8 Hz, 1H), 8.26–8.20 (m, 2H),
8.14 (d, J = 8.8 Hz, 1H), 7.75 (dd, J = 5.4, 1.0 Hz, 1H), 7.33 (dd, J = 8.0, 5.4 Hz, 1H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm;
154.7, 153.4, 151.2, 149.7, 134.0, 132.5, 130.0, 127.9, 127.5, 125.2,
124.9, 123.9. HRMS; calcd for C 24 H 16 Cl 2 N 4 Ru [M + H] + ; 532.9874, found; 532.1806. Ru(5-Clphen) 2 Cl 2 : Dark purple powder, 29%
yield, 375 °C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.35 (dt, J = 5.2,
1.1 Hz, 1H), 10.26 (td, J = 5.2, 1.2 Hz, 1H), 8.83
(dd, J = 8.4, 2.5 Hz, 1H), 8.68 (dt, J = 7.6, 1.3 Hz, 1H), 8.62 (d, J = 2.01 Hz, 1H), 8.47
(s, 1H), 8.40–8.28 (m, 2H), 8.28–8.20 (m, 2H), 7.98–7.88
(m, 1H), 7.85 (dd, J = 5.4, 1.0 Hz, 1H), 7.47–7.43
(m, 1H), 7.38–7.34 (m, 1H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 161.7, 154.5, 154.0,
153.7, 150.6, 149.2, 149.2, 147.9, 132.5, 131.1, 129.9, 128.9, 128.4,
127.1, 126.0, 125.7, 125.0, 124.8, 124.6, 124.4. HRMS; calcd for C 24 H 14 Cl 4 N 4 Ru [M + H] + ; 600.9094, found; 600.9550. Ru(5-mephen) 2 Cl 2 : Dark purple powder, 55%
yield, 373 °C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.29 (d, J = 4.0
Hz, 1H), 10.20 (s, 1H), 8.74 (d, J = 8.2 Hz, 1H),
8.58 (d, J = 7.9 Hz, 1H), 8.24–8.20 (m, 2H),
8.17–8.13 (m, 1H), 8.09 (d, J = 4.4 Hz, 2H),
7.95 (d, J = 6.7 Hz, 1H), 7.76–7.68 (m, 1H),
7.67–7.60 (m, 1H), 7.35 (dd, J = 8.1, 5.4
Hz, 1H), 7.28 (dd, J = 7.8, 5.4 Hz, 1H), 2.76 (s,
3H), 2.89 (s, 3H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 171.4, 153.1, 152.5, 149.6, 148.8,
147.9, 138.2, 134.4, 134.1, 132.0, 130.8, 130.1, 129.1, 129.0, 128.6,
128.5, 125.4, 125.1, 124.2, 123.9, 123.7, 123.5, 114.0, 21.4, 20.4.
HRMS; calcd for C 26 H 20 Cl 2 N 4 Ru [M + H] + ; 561.0187, found; 561.0657. Method B:
RuCl 3 ·(H 2 O) n (1 mmol)
and LiCl (40 mmol) were mixed in ethylene glycol:water
mixture at 110 °C for 15 min. After 15 min, ligand (2.2 mmol)
was added and mixing was continued. Then, glucose (2 mmol) was added
and mixed for another 15 min at 110 °C. Ascorbic acid was added
to it and mixing was continued for 30 min. After 30 min, 10 mL of
saturated NaCl solution was added and stirred at 0 °C for 1 h.
The precipitate was then filtered off and purified by column chromatography. 31 , 39 The following complexes were synthesized according to this method. Ru(6,6-dimebpy) 2 Cl 2 : Dark purple powder,
78% yield, 370 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 8.58 (d, J
= 7.8 Hz, 1H), 8.44 (d, J = 7.7 Hz, 2H),
8.36 (d, J = 7.7 Hz, 1H), 8.06 (t, J = 7.5 Hz, 1H), 7.95 (t, J = 7.6 Hz, 1H), 7.85 (t, J = 7.6 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H),
7.55 (d, J = 7.4 Hz, 1H), 7.48 (d, J = 7.4 Hz, 1H), 7.28–7.23 (m, 2H), 2.91 (s, 3H), 2.53 (s, 3H),
1.86 (s, 3H), 1.65 (s, 3H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 167.9, 166.8, 166.5, 164.9, 163.9,
161.9, 161.1, 160.5, 137.1, 136.5, 135.8, 135.2, 124.6, 124.6, 123.9,
123.675, 121.6, 121.0, 120.7, 120.6, 27.2, 24.7, 24.6, 23.7. HRMS;
calcd for C 24 H 24 Cl 2 N 4 Ru
[M + H] + ; 541.0500, found; 541.1334. Ru(4,4-dimethoxybpy) 2 Cl 2 : Dark purple powder,
50% yield, 370 °C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 9.83 (d, J = 6.6 Hz, 1H), 9.20 (d, J = 6.5 Hz, 1H), 8.46 (d, J = 2.7 Hz, 1H), 8.36 (d, J = 2.6 Hz, 1H),
8.30 (dd, J = 8.2, 2.6 Hz, 2H), 7.59 (dd, J = 6.6, 2.6 Hz, 1H), 7.51 (d, J = 6.6
Hz, 1H), 7.47 (dd, J = 6.6, 2.7 Hz, 1H), 7.07 (dd, J = 6.5, 2.5 Hz, 1H), 7.03–7.00 (m, 2H), 4.11 (s,
3H), 4.08 (s, 3H), 3.94 (s, 3H), 3.92 (s, 3H); 13 C NMR
(DMSO- d 6 , 125 MHz) δ/ppm; 167.5,
167.3, 166.9, 166.7, 159.2, 159.0, 158.8, 157.5, 156.4, 154.0, 153.4,
150.1, 114.6, 114.1, 113.5, 113.2, 112.0, 111.6, 111.2, 110.4, 57.4,
57.2, 57.2, 57.1. HRMS; calcd for C 24 H 24 Cl 2 N 4 O 4 Ru [M + H] + ; 605.0296,
found; 605.0755. Ru(4,4-di- t -bu-bpy) 2 Cl 2 :
Dark purple powder, 60% yield, 370 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm;
8.55 (s, 4H), 7.59 (s, 4H), 7.26 (s, 4H), 1.50 (s, 18H), 1.33 (s,
18H); 13 C NMR (DMSO- d 6 , 125
MHz) δ/ppm; 163.2, 162.7, 161.9, 161.8, 158.0, 157.5, 157.4,
156.3, 155.1, 152.6, 125.0, 124.6, 124.2, 124.1, 122.4, 122.1, 122.0,
121.5, 121.1, 120.1, 36.1, 35.8, 35.7, 30.7, 30.6, 30.5, 30.4. HRMS;
calcd for C 36 H 48 Cl 2 N 4 Ru
[M + H] + ; 709.2378, found; 709.2895. 4.2.1.3 Synthesis of RuL 2 S Complexes Ru–ligand complex (RuL 2 Cl 2 ) (1 mmol)
and sorafenib (Srf) (1 mmol) were stirred in methanol overnight at
60 °C. At the end of the reaction, methanol was removed. The
solid was dried and purified by flash chromatography. 32 , 33 Ru(bpy) 2 Srf (Ru1S): Red powder, 30% yield, 196
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
8.73 (d, J = 8.1 Hz, 1H), 8.70 (d, J = 7.6 Hz, 2H), 8.63 (d, J = 8.0 Hz, 1H), 8.60 (d, J = 7.9 Hz, 1H), 8.23–8.17 (m, 2H), 8.15–8.13
(m, 1H), 8.07 (d, J = 2.6 Hz, 1H), 8.03–7.99
(m, 2H), 7.95 (td, J = 8.0, 1.4 Hz, 1H), 7.81 (d, J = 5.7 Hz, 1H), 7.78–7.74 (m, 1H), 7.71 (d, J = 5.6, 1H), 7.68–7.65 (m, 1H), 7.70–7.50
(m, 4H), 7.48 (d, J = 8.7 Hz, 1H), 7.37 (td, J = 7.3, 1.2 Hz, 1H), 7.32 (td, J = 7.3,
1.3 Hz, 1H), 7.16–7.12 (m, 2H), 7.11 (dd, J = 6.4, 2.6 Hz, 1H), 2.95 (s, 3H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 172.0, 166.8, 159.1, 158.2, 157.9, 157.4,
153.4, 153.1, 152.8, 151.9, 151.6, 151.4, 150.5, 148.0, 138.7, 137.5,
137.3, 137.0, 136.2, 131.6, 127.5, 127.4, 126.8, 126.5, 124.1, 123.8,
123.7, 123.5, 122.8, 121.2, 121.0, 117.3, 117.2, 114.1, 26.5. HRMS;
calcd for C 41 H 32 ClF 3 N 8 O 3 Ru [M – H] − ; 877.1203, found;
877.1192. Ru(4,4-dimebpy) 2 Srf (Ru2S): Red powder,
52% yield, 215
°C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.92 (s, 1H), 10.23 (s, 1H), 9.89 (s, 1H),
8.72 (d, J = 13.7 Hz, 2H), 8.64 (d, J = 15.9 Hz, 2H), 8.50 (s, 1H), 8.44 (d, J = 5.7 Hz,
1H), 8.11 (d, J = 1.6 Hz, 1H), 7.88 (d, J
= 5.7 Hz, 1H), 7.64–7.61 (m, 2H), 7.61–7.57
(m, 3H), 7.52–7.50 (m, 2H), 7.48 (d, J = 6.4
Hz, 1H), 7.45 (d, J = 5.8 Hz, 1H), 7.25 (dd, J = 5.8, 1.0 Hz, 1H), 7.20–7.19 (m, 2H), 7.18–7.17
(m, 1H), 7.09 (dd, J = 6.4, 2.6 Hz, 1H), 2.84 (d, J = 4.6 Hz, 3H), 2.47 (s, 3H), 2.44 (s, 3H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm;
171.3, 170.7, 164.8, 157.4, 156.6, 156.2, 155.8, 152.2, 152.0, 151.4,
150.6, 150.3, 150.2, 149.2, 148.4, 148.3, 148.1, 147.1, 146.4, 138.8,
137.1, 131.4, 127.8, 127.7, 127.3, 127.0, 124.4, 124.0, 123.8, 123.2,
121.9, 121.4, 121.1, 120.6, 119.2, 116.6, 115.5, 115.5, 114.1, 26.7,
20.2, 20.1, 19.9, 19.8. HRMS; calcd for C 45 H 40 ClF 3 N 8 O 3 Ru [M – H] − ; 933.1829, found; 933.1818. Ru(6,6-dimebpy) 2 Srf
(Ru3S): Red powder, 25% yield, 220
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
8.60–8.54 (m, 3H), 8.51 (d, J = 8.0 Hz, 1H),
8.07–7.99 (m, 4H), 7.90 (t, J = 7.8 Hz, 1H),
7.72 (d, J = 2.3 Hz, 1H), 7.68 (d, J = 6.5 Hz, 1H), 7.62 (dd, J = 8.7, 2.3 Hz, 1H), 7.57
(d, J = 8.8 Hz, 2H), 7.5–7.39 (m, 5H), 7.08
(dd, J = 6.5, 2.6 Hz, 1H), 7.04 (d, J = 8.8 Hz, 2H), 2.69 (s, 3H), 2.36 (s, 3H), 2.06 (s, 3H), 1.99 (s,
3H), 1.73 (s, 3H); 13 C NMR (CD 3 OD, 125 MHz)
δ/ppm; 170.7, 167.5, 167.0, 166.2, 165.0, 164.7, 160.6, 159.5,
159.2, 158.3, 153.3, 151.6, 147.7, 138.7, 137.7, 137.6, 137.6, 137.0,
136.9, 131.6, 126.4, 126.2, 126.1, 126.0, 124.0, 123.9, 122.8, 122.5,
122.0, 121.3, 121.2, 121.1, 120.9, 117.2, 117.2, 116.4, 113.4, 26.1,
24.5, 24.3, 23.1, 22.7. HRMS; calcd for C 45 H 40 ClF 3 N 8 O 3 Ru [M – H] − ; 933.1829, found; 933.1814. Ru(4,4-di- t -bu-bpy) 2 Srf (Ru4S): Red
powder, 65% yield, 207 °C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm; 8.73 (dd, J = 6.9, 1.7
Hz, 2H), 8.63 (dd, J = 9.9, 1.8 Hz, 2H), 8.57 (d, J = 5.9 Hz, 1H), 8.06 (d, J = 2.6 Hz, 1H),
8.05 (d, J = 2.5 Hz, 1H), 8.01 (d, J = 5.9 Hz, 1H), 7.80 (dd, J = 5.9, 1.6 Hz, 1H), 7.69
(dd, J = 5.9, 1.7 Hz, 1H), 7.66 (d, J = 6.1 Hz, 1H), 7.64–7.54 (m, 5H), 7.46 (d, J = 8.7 Hz, 1H), 7.41 (dd, J = 6.1, 1.9 Hz, 1H), 7.36
(dd, J = 6.1, 2.0 Hz, 1H), 7.14–7.10 (m, 3H),
2.96 (s, 3H), 1.52 (s, 9H), 1.51 (s, 9H), 1.41 (s, 9H), 1.39 (s, 9H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 172.0, 166.5,
162.5, 162.3, 162.0, 161.2, 158.8, 158.0, 157.8, 157.2, 153.4, 152.7,
152.4, 151.6, 151.2, 150.9, 149.9, 148.0, 138.8, 137.5, 131.5, 128.0,
127.7, 124.6, 124.6, 124.2, 124.0, 123.9, 123.8, 122.8, 121.3, 121.1,
121.0, 120.9, 120.8, 120.6, 117.2, 115.2, 114.0, 113.3, 35.3, 35.2,
35.0, 35.0, 33.5, 31.6, 30.7, 29.3, 29.3, 29.2, 29.0, 28.8, 28.7,
26.5, 22.3. HRMS; calcd for C 57 H 64 ClF 3 N 8 O 3 Ru [M – H] − ; 1101.3707,
found; 1101.3700. Ru(4,4-dimethoxybpy) 2 Srf (Ru5S):
Red powder, 80% yield,
235 °C (decomposition) mp. 1 H NMR (CD 3 OD,
500 MHz) δ/ppm; 8.39 (d, J = 6.4 Hz, 1H), 8.28
(d, J = 2.6 Hz, 1H), 8.25 (d, J = 2.6 Hz, 1H), 8.19 (dd, J = 5.2, 2.7 Hz, 2H), 8.03
(d, J = 2.5 Hz, 1H), 8.01 (d, J = 2.6 Hz, 1H), 7.85 (d, J = 6.4 Hz, 1H), 7.65 (d, J = 6.4 Hz, 1H), 7.61–7.52 (m, 5H), 7.48 (d, J = 8.7 Hz, 1H), 7.32 (dd, J = 6.4, 2.6
Hz, 1H), 7.22 (dd, J = 6.4, 2.6 Hz, 1H), 7.16–7.12
(m, 2H), 7.10 (dd, J = 6.4, 2.6 Hz, 1H), 6.98 (dd, J = 6.5, 2.7 Hz, 1H), 6.94 (dd, J = 6.6,
2.7 Hz, 1H), 4.09 (s, 3H), 4.08 (s, 3H), 3.99 (s, 3H), 3.98 (s, 3H),
2.95 (s, 3H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm;
172.3, 167.2, 167.1, 166.9, 166.4, 166.1, 160.0, 159.3, 158.7, 153.4,
153.1, 152.3, 152.1, 151.9, 151.2, 148.1, 138.7, 137.5, 131.6, 122.8,
121.2, 120.9, 117.3, 117.2, 117.0, 113.9, 113.6, 113.5, 113.4, 112.7,
110.8, 110.4, 110.4, 110.2, 55.9, 55.8, 26.4. HRMS; calcd for C 45 H 40 ClF 3 N 8 O 7 Ru
[M – H] − ; 997.1626, found; 997.1596. Ru(phen) 2 Srf (Ru6S): Red powder, 37% yield, 205 °C
mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm; 9.18
(dd, J = 5.1, 1.1 Hz, 1H), 8.81 (dd, J = 8.3, 1.1 Hz, 2H), 8.61 (dd, J = 5.1, 1.1 Hz, 1H),
8.53 (dd, J = 8.2, 1.1 Hz, 1H), 8.48 (dd, J = 8.2, 1.0 Hz, 1H), 8.33 (dd, J = 8.9,
4.9 Hz, 2H), 8.24 (t, J = 8.7 Hz, 2H), 8.15 (dd, J = 8.2, 5.1 Hz, 1H), 8.10 (d, J = 2.6
Hz, 1H), 8.05 (dd, J = 8.2, 5.1 Hz, 1H), 8.03–7.98
(m, 2H), 7.91 (dd, J = 5.3, 1.1 Hz, 1H), 7.65–7.51
(m, 6H), 7.48 (d, J = 8.7 Hz, 1H), 7.15–7.09
(m, 2H), 7.01 (dd, J = 6.4, 2.6 Hz, 1H), 2.94 (s,
3H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 172.3,
166.8, 154.1, 153.4, 153.2, 152.8, 151.8, 149.5, 148.7, 148.6, 148.0,
138.7, 137.5, 136.6, 136.4, 136.0, 135.2, 131.5, 131.0, 130.8, 130.8,
130.6, 127.8, 127.8, 127.7, 126.0, 125.4, 125.1, 122.8, 121.2, 120.9,
117.3, 117.2, 117.1, 114.1, 26.5. HRMS; calcd for C 45 H 32 ClF 3 N 8 O 3 Ru [M – H] − ; 925.1203, found; 925.1192. Ru(5-Clphen) 2 Srf (Ru7S): Red powder, 51% yield, 208
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
9.25 (td, J = 3.5, 1.2 Hz, 1H), 9.18 (td, J = 3.5, 1.3 Hz, 1H), 9.07–9.01 (m, 2H), 8.79–8.73
(m, 3H), 8.7 (d, J = 8.7 Hz, 1H), 8.68 (t, J = 4.1 Hz, 1H), 8.60 (t, J = 5.1 Hz, 1H),
8.53 (d, J = 1.4 Hz, 2H), 8.49 (d, J = 8.2 Hz, 1H), 8.44–8.42 (m, 3H), 8.29–8.24 (m, 1H),
8.19–8.11 (m, 5H), 8.06–8.03 (m, 3H), 7.96–7.95
(m, 3H), 7.69–7.62 (m, 2H), 7.61–7.52 (m, 10H), 7.41
(d, J = 8.7 Hz, 2H), 7.09 (dd, J = 8.8, 1.3 Hz, 4H), 7.02–6.99 (m, 2H), 2.94 (d, J = 3.3 Hz, 6H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm;
173.8, 172.3, 167.8, 166.9, 155.1, 154.6, 153.9, 153.7, 153.4, 153.3,
153.3, 153.2, 152.1, 151.7, 149.1, 148.6, 147.9, 147.2, 138.7, 137.6,
136.0, 135.8, 135.4, 133.7, 133.5, 132.3, 132.1, 131.7, 131.5, 131.5,
130.9, 130.2, 130.0, 129.3, 129.1, 128.4, 127.0, 126.9, 126.9, 126.6,
126.5, 126.0, 125.7, 124.0, 122.8, 122.7, 121.1, 120.9, 117.2, 117.1,
114.2, 113.3, 29.3, 28.7. HRMS; calcd for C 45 H 30 Cl 3 F 3 N 8 O 3 Ru [M + H] + ; 995.0580, found; 995.0386. Ru(5-mephen) 2 Srf (Ru8S): Red powder, 18% yield, 195
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
9.19 (t, J = 5.0 Hz,1H), 9.12 (t, J = 5.0 Hz, 1H), 8.95–8.91 (m, 2H), 8.73–8.69 (m, 2H),
8.66–8.61 (m, 2H), 8.58 (d, J = 8.3 Hz, 1H),
8.54 (t, J = 5.2 Hz, 1H), 8.42 (d, J = 8.1 Hz, 1H), 8.37 (d, J = 8.1 Hz, 1H), 8.23–8.18
(m, 1H), 8.18–8.14 (m, 4H), 8.06 (d, J = 4.1
Hz, 2H), 8.02–7.99 (m, 4H), 7.93 (d, J = 5.0
Hz, 2H), 7.86–7.83 (m, 1H), 7.64–7.46 (m, 14H), 7.43
(d, J = 8.7 Hz, 2H), 7.12 (td, J = 8.9, 1.6 Hz, 4H), 7.06–7.00 (m, 2H), 2.99 (s, 3H), 2.97 (s,
6H), 2.96 (s, 3H), 2.90 (d, J = 5.0 Hz, 6H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 173.7, 172.2,
167.8, 166.7, 153.6, 153.3, 153.2, 153.1, 152.3, 151.8, 151.7, 151.3,
150.8, 149.8, 148.8, 148.0, 147.9, 147.9, 138.7, 138.7, 137.5, 136.4,
136.2, 136.1, 135.7, 135.5, 135.1, 134.3, 133.6, 133.4, 133.0, 132.3,
132.1, 131.5, 131.2, 130.9, 130.8, 130.6, 130.4, 129.1, 128.4, 128.0,
127.7, 126.6, 126.5, 126.5, 126.0, 125.8, 125.4, 125.2, 125.1, 124.9,
124.0, 123.9, 122.9, 122.8, 122.7, 121.8, 121.1, 120.9, 117.2, 117.2,
117.1, 115.2, 114.1, 113.3, 29.3, 28.7, 24.1, 23.5, 22.6, 22.3. HRMS;
calcd for C 47 H 36 ClF 3 N 8 O 3 Ru [M] − ; 953.1516, found; 953.1502.
## Synthesis of Sorafenib
4.2.1.1 Synthesis of Sorafenib 4-Chloropicolinoyl
chloride ( 2 ): 0.08 mol of thionyl chloride and 1 mL of
DMF were mixed under nitrogen at 40 °C for 10 min. 0.4 mol of
picolinic acid ( 1 ) was added in half an hour piece by
piece. When the addition was complete, the temperature was increased
to 72 °C and stirred at this temperature for 16 h. A yellow precipitate
formed at the end of the reaction. The resulting solid was purified
with the help of toluene and ether to obtain 4-chloropicolinoyl chloride
( 2 ) in 60% yield. 27 1 H NMR (DMSO- d 6 , 300 MHz) δ/ppm:
8.62 (1H, d, J = 7.4 Hz), 8.10 (1H, s), 7.46 (1H,
d, J = 7.4 Hz). 4-Chloro- N -methylpicolinamide ( 3 ): 4-Chloropicolinoyl chloride
( 2 ) and methyl amine were mixed in cold methanol. The
solid part formed at the end of the reaction was dried by filtration
through the gooch crucible. 4-Chloro- N -methylpicolinamide
( 3 ) obtained in 20% yield was used for the next step. 27 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm: 8.72 (1H, d, J =
7.3 Hz), 8.14 (1H, s), 7.52 (1H, d, J = 7.3 Hz),
2.59 (3H, d, J = 4.5 Hz). 4-(4-Aminophenoxy)- N -methylpicolinamide ( 4 ): First, p -aminophenol and t -BuOK were mixed in DMF for 2
h under nitrogen, then compound 3 and K 2 CO 3 were added and mixed at
80 °C for 16 h. At the end of the reaction, DMF was evaporated
in the evaporator. 4-(4-Aminophenoxy)- N -methylpicolinamide
( 4 ) was obtained by column chromatography with hexane-ethyl
acetate. 28 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm: 2.79 (3H, d, J = 4.8 Hz), 5.22 (2H, s, NH 2 ), 6.65 (2H, d, J = 8.7 Hz), 6.87 (2H, d, J = 8.7 Hz),
7.08 (1H, dd, J = 5.6, 2.6 Hz), 7.35 (1H, d, J = 2.5 Hz), 8.46 (1H, d, J = 5.62 Hz),
8.78 (1H, q, J = 4.5 Hz); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm: 26.4, 108.7, 114.1,
115.3, 122.0, 143.2, 147.3, 150.6, 152.7, 164.3, 167.2. 4-(4-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)- N -methylpicolinamide ( Sorafenib ): 1 mmol of
4-(4-aminophenoxy)- N -methylpicolinamide ( 4 ), 1.1 mmol of 4-chloro-3-trifluoromethyl phenyl isocyanate, and
1.1 mmol of Et 3 N were dissolved in THF together and stirred
overnight at 60 °C. At the end of the reaction, THF was evaporated
in the evaporator. The solid was purified by washing with ether. 29 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm 2.81 (3H, d, J = 4.8
Hz), 7.29–7.06 (3H m), 7.41 (1H, d, J = 2.5
Hz), 7.78–7.56 (4H, m), 8.15 (1H, d, J = 2.5
Hz), 8.52 (1H, d, J = 5.6 Hz), 8.82 (1H, q, J = 4.7, 4.6 Hz), 9.04 (1H, s), 9.26 (1H, s); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm:
26.4, 109.0, 114.4, 117.2, 120.9, 121.9, 122.8, 123.5, 124.3, 127.0,
132.4, 137.5, 139.7, 148.2, 150.8, 152.8, 152.9, 164.2, 166.4.
## Synthesis of RuL
4.2.1.2 Synthesis of RuL 2 Cl 2 Complexes Method A: The mixture of RuCl 3 ·(H 2 O) n (1 mmol), ligand (L) (2 mmol), LiCl (6
mmol), and DMF (2 mL) was stirred under nitrogen at 80 °C under
reflux for 18 h. At the end of the reaction, acetone was added after
cooling and the mixture was kept in a refrigerator for 24 h. The precipitate
formed was filtered, washed with cold water and diethyl ether, and
then dried. The resulting complex was purified by column chromatography. 30 The following complexes were synthesized according
to this method. Ru(bpy) 2 Cl 2 : Dark purple
powder, 60% yield, 360 °C (decomposition) mp. 1 H NMR
(DMSO- d 6 , 500 MHz) δ/ppm; 9.98 (d, J = 5.4 Hz, 1H), 8.64 (d, J = 8.0 Hz, 1H),
8.48 (d, J = 8.0 Hz, 1H), 8.12–8.01 (m, 1H),
7.77 (t, J = 6.4 Hz, 1H), 7.74–7.63 (m, 1H),
7.51 (d, J = 5.5 Hz, 1H), 7.10 (dd, J = 9.6, 3.4 Hz, 1H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 159.5, 157.5, 152.5, 151.3, 133.9,
132.6, 124.7, 124.6, 122.2, 121.8. HRMS; calcd for C 20 H 16 Cl 2 N 4 Ru [M + H] + ; 484.9874,
found; 484.9806. Ru(4,4-dimebpy) 2 Cl 2 :
Dark purple powder,
46% yield, 360 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 9.77 (d, J
= 5.7 Hz, 1H), 8.46 (s, 1H), 8.31 (s, 1H), 7.58 (d, J = 5.0 Hz, 1H), 7.31 (d, J = 5.9 Hz, 1H),
6.92 (dd, J = 5.9, 1.1 Hz, 1H), 2.62 (s, 3H), 2.34
(s, 3H). 13 C NMR (DMSO- d 6 ,
125 MHz) δ/ppm; 160.2, 158.4, 152.9, 151.6, 145.9, 144.7, 126.5,
126.5, 123.8, 123.4, 21.1, 20.7. HRMS; calcd for C 24 H 24 Cl 2 N 4 Ru [M + H] + ; 541.0500,
found; 541.1046. Ru(phen) 2 Cl 2 : Dark purple
powder, 56% yield,
370 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.28 (dd, J = 5.2, 1.2 Hz, 1H), 8.72 (dd, J = 8.1, 1.2 Hz, 1H),
8.29 (d, J = 8.8 Hz, 1H), 8.26–8.20 (m, 2H),
8.14 (d, J = 8.8 Hz, 1H), 7.75 (dd, J = 5.4, 1.0 Hz, 1H), 7.33 (dd, J = 8.0, 5.4 Hz, 1H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm;
154.7, 153.4, 151.2, 149.7, 134.0, 132.5, 130.0, 127.9, 127.5, 125.2,
124.9, 123.9. HRMS; calcd for C 24 H 16 Cl 2 N 4 Ru [M + H] + ; 532.9874, found; 532.1806. Ru(5-Clphen) 2 Cl 2 : Dark purple powder, 29%
yield, 375 °C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.35 (dt, J = 5.2,
1.1 Hz, 1H), 10.26 (td, J = 5.2, 1.2 Hz, 1H), 8.83
(dd, J = 8.4, 2.5 Hz, 1H), 8.68 (dt, J = 7.6, 1.3 Hz, 1H), 8.62 (d, J = 2.01 Hz, 1H), 8.47
(s, 1H), 8.40–8.28 (m, 2H), 8.28–8.20 (m, 2H), 7.98–7.88
(m, 1H), 7.85 (dd, J = 5.4, 1.0 Hz, 1H), 7.47–7.43
(m, 1H), 7.38–7.34 (m, 1H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 161.7, 154.5, 154.0,
153.7, 150.6, 149.2, 149.2, 147.9, 132.5, 131.1, 129.9, 128.9, 128.4,
127.1, 126.0, 125.7, 125.0, 124.8, 124.6, 124.4. HRMS; calcd for C 24 H 14 Cl 4 N 4 Ru [M + H] + ; 600.9094, found; 600.9550. Ru(5-mephen) 2 Cl 2 : Dark purple powder, 55%
yield, 373 °C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.29 (d, J = 4.0
Hz, 1H), 10.20 (s, 1H), 8.74 (d, J = 8.2 Hz, 1H),
8.58 (d, J = 7.9 Hz, 1H), 8.24–8.20 (m, 2H),
8.17–8.13 (m, 1H), 8.09 (d, J = 4.4 Hz, 2H),
7.95 (d, J = 6.7 Hz, 1H), 7.76–7.68 (m, 1H),
7.67–7.60 (m, 1H), 7.35 (dd, J = 8.1, 5.4
Hz, 1H), 7.28 (dd, J = 7.8, 5.4 Hz, 1H), 2.76 (s,
3H), 2.89 (s, 3H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 171.4, 153.1, 152.5, 149.6, 148.8,
147.9, 138.2, 134.4, 134.1, 132.0, 130.8, 130.1, 129.1, 129.0, 128.6,
128.5, 125.4, 125.1, 124.2, 123.9, 123.7, 123.5, 114.0, 21.4, 20.4.
HRMS; calcd for C 26 H 20 Cl 2 N 4 Ru [M + H] + ; 561.0187, found; 561.0657. Method B:
RuCl 3 ·(H 2 O) n (1 mmol)
and LiCl (40 mmol) were mixed in ethylene glycol:water
mixture at 110 °C for 15 min. After 15 min, ligand (2.2 mmol)
was added and mixing was continued. Then, glucose (2 mmol) was added
and mixed for another 15 min at 110 °C. Ascorbic acid was added
to it and mixing was continued for 30 min. After 30 min, 10 mL of
saturated NaCl solution was added and stirred at 0 °C for 1 h.
The precipitate was then filtered off and purified by column chromatography. 31 , 39 The following complexes were synthesized according to this method. Ru(6,6-dimebpy) 2 Cl 2 : Dark purple powder,
78% yield, 370 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 8.58 (d, J
= 7.8 Hz, 1H), 8.44 (d, J = 7.7 Hz, 2H),
8.36 (d, J = 7.7 Hz, 1H), 8.06 (t, J = 7.5 Hz, 1H), 7.95 (t, J = 7.6 Hz, 1H), 7.85 (t, J = 7.6 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H),
7.55 (d, J = 7.4 Hz, 1H), 7.48 (d, J = 7.4 Hz, 1H), 7.28–7.23 (m, 2H), 2.91 (s, 3H), 2.53 (s, 3H),
1.86 (s, 3H), 1.65 (s, 3H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm; 167.9, 166.8, 166.5, 164.9, 163.9,
161.9, 161.1, 160.5, 137.1, 136.5, 135.8, 135.2, 124.6, 124.6, 123.9,
123.675, 121.6, 121.0, 120.7, 120.6, 27.2, 24.7, 24.6, 23.7. HRMS;
calcd for C 24 H 24 Cl 2 N 4 Ru
[M + H] + ; 541.0500, found; 541.1334. Ru(4,4-dimethoxybpy) 2 Cl 2 : Dark purple powder,
50% yield, 370 °C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 9.83 (d, J = 6.6 Hz, 1H), 9.20 (d, J = 6.5 Hz, 1H), 8.46 (d, J = 2.7 Hz, 1H), 8.36 (d, J = 2.6 Hz, 1H),
8.30 (dd, J = 8.2, 2.6 Hz, 2H), 7.59 (dd, J = 6.6, 2.6 Hz, 1H), 7.51 (d, J = 6.6
Hz, 1H), 7.47 (dd, J = 6.6, 2.7 Hz, 1H), 7.07 (dd, J = 6.5, 2.5 Hz, 1H), 7.03–7.00 (m, 2H), 4.11 (s,
3H), 4.08 (s, 3H), 3.94 (s, 3H), 3.92 (s, 3H); 13 C NMR
(DMSO- d 6 , 125 MHz) δ/ppm; 167.5,
167.3, 166.9, 166.7, 159.2, 159.0, 158.8, 157.5, 156.4, 154.0, 153.4,
150.1, 114.6, 114.1, 113.5, 113.2, 112.0, 111.6, 111.2, 110.4, 57.4,
57.2, 57.2, 57.1. HRMS; calcd for C 24 H 24 Cl 2 N 4 O 4 Ru [M + H] + ; 605.0296,
found; 605.0755. Ru(4,4-di- t -bu-bpy) 2 Cl 2 :
Dark purple powder, 60% yield, 370 °C (decomposition) mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm;
8.55 (s, 4H), 7.59 (s, 4H), 7.26 (s, 4H), 1.50 (s, 18H), 1.33 (s,
18H); 13 C NMR (DMSO- d 6 , 125
MHz) δ/ppm; 163.2, 162.7, 161.9, 161.8, 158.0, 157.5, 157.4,
156.3, 155.1, 152.6, 125.0, 124.6, 124.2, 124.1, 122.4, 122.1, 122.0,
121.5, 121.1, 120.1, 36.1, 35.8, 35.7, 30.7, 30.6, 30.5, 30.4. HRMS;
calcd for C 36 H 48 Cl 2 N 4 Ru
[M + H] + ; 709.2378, found; 709.2895.
## Synthesis of RuL
4.2.1.3 Synthesis of RuL 2 S Complexes Ru–ligand complex (RuL 2 Cl 2 ) (1 mmol)
and sorafenib (Srf) (1 mmol) were stirred in methanol overnight at
60 °C. At the end of the reaction, methanol was removed. The
solid was dried and purified by flash chromatography. 32 , 33 Ru(bpy) 2 Srf (Ru1S): Red powder, 30% yield, 196
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
8.73 (d, J = 8.1 Hz, 1H), 8.70 (d, J = 7.6 Hz, 2H), 8.63 (d, J = 8.0 Hz, 1H), 8.60 (d, J = 7.9 Hz, 1H), 8.23–8.17 (m, 2H), 8.15–8.13
(m, 1H), 8.07 (d, J = 2.6 Hz, 1H), 8.03–7.99
(m, 2H), 7.95 (td, J = 8.0, 1.4 Hz, 1H), 7.81 (d, J = 5.7 Hz, 1H), 7.78–7.74 (m, 1H), 7.71 (d, J = 5.6, 1H), 7.68–7.65 (m, 1H), 7.70–7.50
(m, 4H), 7.48 (d, J = 8.7 Hz, 1H), 7.37 (td, J = 7.3, 1.2 Hz, 1H), 7.32 (td, J = 7.3,
1.3 Hz, 1H), 7.16–7.12 (m, 2H), 7.11 (dd, J = 6.4, 2.6 Hz, 1H), 2.95 (s, 3H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 172.0, 166.8, 159.1, 158.2, 157.9, 157.4,
153.4, 153.1, 152.8, 151.9, 151.6, 151.4, 150.5, 148.0, 138.7, 137.5,
137.3, 137.0, 136.2, 131.6, 127.5, 127.4, 126.8, 126.5, 124.1, 123.8,
123.7, 123.5, 122.8, 121.2, 121.0, 117.3, 117.2, 114.1, 26.5. HRMS;
calcd for C 41 H 32 ClF 3 N 8 O 3 Ru [M – H] − ; 877.1203, found;
877.1192. Ru(4,4-dimebpy) 2 Srf (Ru2S): Red powder,
52% yield, 215
°C mp. 1 H NMR (DMSO- d 6 , 500 MHz) δ/ppm; 10.92 (s, 1H), 10.23 (s, 1H), 9.89 (s, 1H),
8.72 (d, J = 13.7 Hz, 2H), 8.64 (d, J = 15.9 Hz, 2H), 8.50 (s, 1H), 8.44 (d, J = 5.7 Hz,
1H), 8.11 (d, J = 1.6 Hz, 1H), 7.88 (d, J
= 5.7 Hz, 1H), 7.64–7.61 (m, 2H), 7.61–7.57
(m, 3H), 7.52–7.50 (m, 2H), 7.48 (d, J = 6.4
Hz, 1H), 7.45 (d, J = 5.8 Hz, 1H), 7.25 (dd, J = 5.8, 1.0 Hz, 1H), 7.20–7.19 (m, 2H), 7.18–7.17
(m, 1H), 7.09 (dd, J = 6.4, 2.6 Hz, 1H), 2.84 (d, J = 4.6 Hz, 3H), 2.47 (s, 3H), 2.44 (s, 3H); 13 C NMR (DMSO- d 6 , 125 MHz) δ/ppm;
171.3, 170.7, 164.8, 157.4, 156.6, 156.2, 155.8, 152.2, 152.0, 151.4,
150.6, 150.3, 150.2, 149.2, 148.4, 148.3, 148.1, 147.1, 146.4, 138.8,
137.1, 131.4, 127.8, 127.7, 127.3, 127.0, 124.4, 124.0, 123.8, 123.2,
121.9, 121.4, 121.1, 120.6, 119.2, 116.6, 115.5, 115.5, 114.1, 26.7,
20.2, 20.1, 19.9, 19.8. HRMS; calcd for C 45 H 40 ClF 3 N 8 O 3 Ru [M – H] − ; 933.1829, found; 933.1818. Ru(6,6-dimebpy) 2 Srf
(Ru3S): Red powder, 25% yield, 220
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
8.60–8.54 (m, 3H), 8.51 (d, J = 8.0 Hz, 1H),
8.07–7.99 (m, 4H), 7.90 (t, J = 7.8 Hz, 1H),
7.72 (d, J = 2.3 Hz, 1H), 7.68 (d, J = 6.5 Hz, 1H), 7.62 (dd, J = 8.7, 2.3 Hz, 1H), 7.57
(d, J = 8.8 Hz, 2H), 7.5–7.39 (m, 5H), 7.08
(dd, J = 6.5, 2.6 Hz, 1H), 7.04 (d, J = 8.8 Hz, 2H), 2.69 (s, 3H), 2.36 (s, 3H), 2.06 (s, 3H), 1.99 (s,
3H), 1.73 (s, 3H); 13 C NMR (CD 3 OD, 125 MHz)
δ/ppm; 170.7, 167.5, 167.0, 166.2, 165.0, 164.7, 160.6, 159.5,
159.2, 158.3, 153.3, 151.6, 147.7, 138.7, 137.7, 137.6, 137.6, 137.0,
136.9, 131.6, 126.4, 126.2, 126.1, 126.0, 124.0, 123.9, 122.8, 122.5,
122.0, 121.3, 121.2, 121.1, 120.9, 117.2, 117.2, 116.4, 113.4, 26.1,
24.5, 24.3, 23.1, 22.7. HRMS; calcd for C 45 H 40 ClF 3 N 8 O 3 Ru [M – H] − ; 933.1829, found; 933.1814. Ru(4,4-di- t -bu-bpy) 2 Srf (Ru4S): Red
powder, 65% yield, 207 °C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm; 8.73 (dd, J = 6.9, 1.7
Hz, 2H), 8.63 (dd, J = 9.9, 1.8 Hz, 2H), 8.57 (d, J = 5.9 Hz, 1H), 8.06 (d, J = 2.6 Hz, 1H),
8.05 (d, J = 2.5 Hz, 1H), 8.01 (d, J = 5.9 Hz, 1H), 7.80 (dd, J = 5.9, 1.6 Hz, 1H), 7.69
(dd, J = 5.9, 1.7 Hz, 1H), 7.66 (d, J = 6.1 Hz, 1H), 7.64–7.54 (m, 5H), 7.46 (d, J = 8.7 Hz, 1H), 7.41 (dd, J = 6.1, 1.9 Hz, 1H), 7.36
(dd, J = 6.1, 2.0 Hz, 1H), 7.14–7.10 (m, 3H),
2.96 (s, 3H), 1.52 (s, 9H), 1.51 (s, 9H), 1.41 (s, 9H), 1.39 (s, 9H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 172.0, 166.5,
162.5, 162.3, 162.0, 161.2, 158.8, 158.0, 157.8, 157.2, 153.4, 152.7,
152.4, 151.6, 151.2, 150.9, 149.9, 148.0, 138.8, 137.5, 131.5, 128.0,
127.7, 124.6, 124.6, 124.2, 124.0, 123.9, 123.8, 122.8, 121.3, 121.1,
121.0, 120.9, 120.8, 120.6, 117.2, 115.2, 114.0, 113.3, 35.3, 35.2,
35.0, 35.0, 33.5, 31.6, 30.7, 29.3, 29.3, 29.2, 29.0, 28.8, 28.7,
26.5, 22.3. HRMS; calcd for C 57 H 64 ClF 3 N 8 O 3 Ru [M – H] − ; 1101.3707,
found; 1101.3700. Ru(4,4-dimethoxybpy) 2 Srf (Ru5S):
Red powder, 80% yield,
235 °C (decomposition) mp. 1 H NMR (CD 3 OD,
500 MHz) δ/ppm; 8.39 (d, J = 6.4 Hz, 1H), 8.28
(d, J = 2.6 Hz, 1H), 8.25 (d, J = 2.6 Hz, 1H), 8.19 (dd, J = 5.2, 2.7 Hz, 2H), 8.03
(d, J = 2.5 Hz, 1H), 8.01 (d, J = 2.6 Hz, 1H), 7.85 (d, J = 6.4 Hz, 1H), 7.65 (d, J = 6.4 Hz, 1H), 7.61–7.52 (m, 5H), 7.48 (d, J = 8.7 Hz, 1H), 7.32 (dd, J = 6.4, 2.6
Hz, 1H), 7.22 (dd, J = 6.4, 2.6 Hz, 1H), 7.16–7.12
(m, 2H), 7.10 (dd, J = 6.4, 2.6 Hz, 1H), 6.98 (dd, J = 6.5, 2.7 Hz, 1H), 6.94 (dd, J = 6.6,
2.7 Hz, 1H), 4.09 (s, 3H), 4.08 (s, 3H), 3.99 (s, 3H), 3.98 (s, 3H),
2.95 (s, 3H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm;
172.3, 167.2, 167.1, 166.9, 166.4, 166.1, 160.0, 159.3, 158.7, 153.4,
153.1, 152.3, 152.1, 151.9, 151.2, 148.1, 138.7, 137.5, 131.6, 122.8,
121.2, 120.9, 117.3, 117.2, 117.0, 113.9, 113.6, 113.5, 113.4, 112.7,
110.8, 110.4, 110.4, 110.2, 55.9, 55.8, 26.4. HRMS; calcd for C 45 H 40 ClF 3 N 8 O 7 Ru
[M – H] − ; 997.1626, found; 997.1596. Ru(phen) 2 Srf (Ru6S): Red powder, 37% yield, 205 °C
mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm; 9.18
(dd, J = 5.1, 1.1 Hz, 1H), 8.81 (dd, J = 8.3, 1.1 Hz, 2H), 8.61 (dd, J = 5.1, 1.1 Hz, 1H),
8.53 (dd, J = 8.2, 1.1 Hz, 1H), 8.48 (dd, J = 8.2, 1.0 Hz, 1H), 8.33 (dd, J = 8.9,
4.9 Hz, 2H), 8.24 (t, J = 8.7 Hz, 2H), 8.15 (dd, J = 8.2, 5.1 Hz, 1H), 8.10 (d, J = 2.6
Hz, 1H), 8.05 (dd, J = 8.2, 5.1 Hz, 1H), 8.03–7.98
(m, 2H), 7.91 (dd, J = 5.3, 1.1 Hz, 1H), 7.65–7.51
(m, 6H), 7.48 (d, J = 8.7 Hz, 1H), 7.15–7.09
(m, 2H), 7.01 (dd, J = 6.4, 2.6 Hz, 1H), 2.94 (s,
3H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 172.3,
166.8, 154.1, 153.4, 153.2, 152.8, 151.8, 149.5, 148.7, 148.6, 148.0,
138.7, 137.5, 136.6, 136.4, 136.0, 135.2, 131.5, 131.0, 130.8, 130.8,
130.6, 127.8, 127.8, 127.7, 126.0, 125.4, 125.1, 122.8, 121.2, 120.9,
117.3, 117.2, 117.1, 114.1, 26.5. HRMS; calcd for C 45 H 32 ClF 3 N 8 O 3 Ru [M – H] − ; 925.1203, found; 925.1192. Ru(5-Clphen) 2 Srf (Ru7S): Red powder, 51% yield, 208
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
9.25 (td, J = 3.5, 1.2 Hz, 1H), 9.18 (td, J = 3.5, 1.3 Hz, 1H), 9.07–9.01 (m, 2H), 8.79–8.73
(m, 3H), 8.7 (d, J = 8.7 Hz, 1H), 8.68 (t, J = 4.1 Hz, 1H), 8.60 (t, J = 5.1 Hz, 1H),
8.53 (d, J = 1.4 Hz, 2H), 8.49 (d, J = 8.2 Hz, 1H), 8.44–8.42 (m, 3H), 8.29–8.24 (m, 1H),
8.19–8.11 (m, 5H), 8.06–8.03 (m, 3H), 7.96–7.95
(m, 3H), 7.69–7.62 (m, 2H), 7.61–7.52 (m, 10H), 7.41
(d, J = 8.7 Hz, 2H), 7.09 (dd, J = 8.8, 1.3 Hz, 4H), 7.02–6.99 (m, 2H), 2.94 (d, J = 3.3 Hz, 6H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm;
173.8, 172.3, 167.8, 166.9, 155.1, 154.6, 153.9, 153.7, 153.4, 153.3,
153.3, 153.2, 152.1, 151.7, 149.1, 148.6, 147.9, 147.2, 138.7, 137.6,
136.0, 135.8, 135.4, 133.7, 133.5, 132.3, 132.1, 131.7, 131.5, 131.5,
130.9, 130.2, 130.0, 129.3, 129.1, 128.4, 127.0, 126.9, 126.9, 126.6,
126.5, 126.0, 125.7, 124.0, 122.8, 122.7, 121.1, 120.9, 117.2, 117.1,
114.2, 113.3, 29.3, 28.7. HRMS; calcd for C 45 H 30 Cl 3 F 3 N 8 O 3 Ru [M + H] + ; 995.0580, found; 995.0386. Ru(5-mephen) 2 Srf (Ru8S): Red powder, 18% yield, 195
°C mp. 1 H NMR (CD 3 OD, 500 MHz) δ/ppm;
9.19 (t, J = 5.0 Hz,1H), 9.12 (t, J = 5.0 Hz, 1H), 8.95–8.91 (m, 2H), 8.73–8.69 (m, 2H),
8.66–8.61 (m, 2H), 8.58 (d, J = 8.3 Hz, 1H),
8.54 (t, J = 5.2 Hz, 1H), 8.42 (d, J = 8.1 Hz, 1H), 8.37 (d, J = 8.1 Hz, 1H), 8.23–8.18
(m, 1H), 8.18–8.14 (m, 4H), 8.06 (d, J = 4.1
Hz, 2H), 8.02–7.99 (m, 4H), 7.93 (d, J = 5.0
Hz, 2H), 7.86–7.83 (m, 1H), 7.64–7.46 (m, 14H), 7.43
(d, J = 8.7 Hz, 2H), 7.12 (td, J = 8.9, 1.6 Hz, 4H), 7.06–7.00 (m, 2H), 2.99 (s, 3H), 2.97 (s,
6H), 2.96 (s, 3H), 2.90 (d, J = 5.0 Hz, 6H); 13 C NMR (CD 3 OD, 125 MHz) δ/ppm; 173.7, 172.2,
167.8, 166.7, 153.6, 153.3, 153.2, 153.1, 152.3, 151.8, 151.7, 151.3,
150.8, 149.8, 148.8, 148.0, 147.9, 147.9, 138.7, 138.7, 137.5, 136.4,
136.2, 136.1, 135.7, 135.5, 135.1, 134.3, 133.6, 133.4, 133.0, 132.3,
132.1, 131.5, 131.2, 130.9, 130.8, 130.6, 130.4, 129.1, 128.4, 128.0,
127.7, 126.6, 126.5, 126.5, 126.0, 125.8, 125.4, 125.2, 125.1, 124.9,
124.0, 123.9, 122.9, 122.8, 122.7, 121.8, 121.1, 120.9, 117.2, 117.2,
117.1, 115.2, 114.1, 113.3, 29.3, 28.7, 24.1, 23.5, 22.6, 22.3. HRMS;
calcd for C 47 H 36 ClF 3 N 8 O 3 Ru [M] − ; 953.1516, found; 953.1502.
## Biological Activity
4.3 Biological Activity 4.3.1 Stability Studies Absorption spectra
of freshly prepared solutions of the samples were acquired at 25 °C
in the range of 200–800 nm with a Shimadzu UV-1280 spectrophotometer,
taking into account the solvent cutoff. Samples were dissolved in
the proper solvents, and the resulting solutions were placed in QS
quartz cuvettes (path length 1 cm). 40 4.3.2 EGFR Inhibition Assay Enzyme activity
was performed using the TAKARA Universal Tyrosine Kinase Assay and
Wash and Stop Solution for Sulfuric Acid Free ELISA according to Ölgen
et al. 36 The enzyme activity reaction was
performed on a precoated 96-well plate. To determine the activity,
40 μL of hEGFR and 10 μL of ATP-2Na solution were added
to each well on the plate and incubated at 37 °C for 30 min.
Then, the solution was removed from the well and the wells were washed
4 times with wash buffer. After this process, 100 μL of blocking
solution was added and incubated for another 30 min at 37 °C.
The blocking solution was discarded and the wells were washed again
with wash buffer. Subsequently, 50 μL of antiphosphotyrosine
(PY20-HRP) solution was added to each well and incubated at 37 °C
for 30 min. Afterward, the antibody solution was removed and the wells
were washed 4 times with the washing solution. 100 μL of HRP
substrate solution (TMBZ) was added to the wells and the plate was
incubated at 37 °C for 15 min. In addition to this mixture, 100
μL of stop solution was added to the wells in the same order
that the substrate solution was added. Activity was measured at 450
nm. In the studies to determine the inhibition potentials of
the synthesized molecules on hEGFR, the solutions of different concentrations
were prepared by dissolving the molecules with ethanol. The volumes
were adjusted so that the solvent amount in the reaction medium was
3% and preincubated with the enzyme (Copeland 2005) (ISBN: 978-1-118-48813-3).
The reaction was repeated only in the presence of this solvent to
determine the effect of the solvent on the enzyme. Using the results
obtained as a result of the inhibition studies, % relative activity–inhibitor
concentration graph was drawn for each molecule and the inhibitory
concentration at which the activity decreased by 50% was determined
as the IC 50 value. 4.3.3 Cytotoxicity Assay HepG2 (hepatocellular
carcinoma; ATCC HB-8065), Caco-2 (Colon carcinoma; ATCC HTB-37), HT-29
(colon carcinoma; ATCC HTB-37), MCF-7 (Breast cancer; ATCC HTB-22),
A549 (lung carcinoma; ATCC CCL-185), and healthy HEK293T (embryonic
kidney epithelial; ATCC CRL-3216) cell lines were cultured in DMEM-F12
medium containing 10% fetal bovine serum (FBS) and antibiotic (100
U/mL penicillin and 100 μg/mL streptomycin). The cells were
incubated at 37 °C under an atmosphere of 5% CO 2 and
95% humidity. HepG2-SR cells were selected according to the short-term
sorafenib exposure protocol. 41 The cells
were exposed to sorafenib at concentrations of 1, 2, and 4 μM
for 72 h in gradual increments. At each stage, surviving cells were
plated in sorafenib-free medium. They were exposed to the next higher
concentration of sorafenib by passaging 3 times at each concentration.
Resistant cells were maintained in medium containing 4 μM sorafenib. Effects of synthesized compounds on cell viability of HepG2, Caco-2,
HT-29, MCF-7, A549, and HEK293T cell lines tested with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay. The cells were passaged as 5 × 10 3 per well in basal medium and incubated for adherence in 96-well
plates. Sorafenib and synthesized sorafenib–ruthenium complexes
were added at 10 decreasing concentrations between 100 and 0.2 μM
and incubated for 48 h. At the end of 48 h, 10 μL of yellow-colored
MTT agent was added and plates were incubated at 37 °C for 4
h. Formazan crystals formed after 4 h were dissolved with 100 μL
of DMSO, and absorbance was measured at 540 nm. Viable cells produced
darker color and nonviable cells produced lighter color because they
could not convert MTT to formazan. The % viability was calculated
relative to the negative controls. Concentration–viability
graphs were drawn for each test substance in the GraphPad Prism 9.00,
and the IC 50 values of the substances were calculated for
each cell line. 42 4.3.4 Apoptosis Assay The apoptotic properties
of the synthesized compounds were determined in HepG2 cell line using
Muse Annexin V & Dead Cell reagent in Muse Cell Analyzer. HepG2
cells were seeded in 24-well plates and synthesized substances were
added at 3 or 4 different concentrations (0.78, 3.125, 12.5, and 50
μM). After 48 h of incubation, cells were harvested with trypsin
and transferred to PBS. 100 μL of Muse Annexin V & Dead
Cell reagent was added to the cell suspension in 100 μL of PBS.
The cells were allowed to stain for 30 min at room temperature in
the dark. The apoptotic profiles of the substances were determined
by flowing the samples in the Muse Cell Analyzer. Results are given
as a percentage (%). 43 There are
two different dyes in the reagent: Annexin V (apoptosis marker) and
7-AAD (dead cell marker). The profiles of the cells after staining
with these dyes according to their viability are as follows: Bottom-left: Living cells [Annexin V (−) and 7-AAD (−)] Bottom-right: Early apoptotic cells [Annexin V (+) and 7-AAD (−)] Top-right: Late apoptotic or dead cells by apoptosis [Annexin V
(+) and 7-AAD (+)] Top-left: Necrotic cells [Annexin V (−)
and 7-AAD (+)] 4.3.5 Cell Cycle Assay The effects of
the synthesized compounds on the cell cycle in the HepG2 cell line
were determined using the Muse Cell Cycle reagent in Muse Cell Analyzer.
HepG2 cells were seeded in 24-well plates and synthesized substances
were added at 3 or 4 different concentrations (0.78, 3.125, 12.5,
and 50 μM). After 48 h of incubation, cells were harvested with
trypsin and washed with PBS. The cell pellet was fixed in cold 70%
ethanol for 3 h at 20 °C. The fixed cells were washed again with
PBS, the supernatant was discarded, and 200 μL of Muse Cell
Cycle reagent was added. The cells were allowed to stain for 30 min
at room temperature in the dark. The effects of the substances on
the cell cycle were determined by flowing the samples in the Muse
Cell Analyzer device. Results are given as a percentage (%). The assay uses PI-based staining of DNA content to distinguish and
quantify the percentage of cells in each cell cycle phase (G0/G1,
S, and G2/M). Cells in G0/G1 phase are selected in blue, the S phase
in purple, and the G2/M phase in green. 44 4.3.6 Western Blot Assay Effects of synthesized
compounds on the expression of proteins in the tyrosine kinase pathway
(Egfr, p-Egfr, Akt, p-Akt, Erk1/2 (p44/42 MAPK), p-Erk1/2 (p-p44/42
MAPK)) in HepG2 cell line was determined by the immunoblotting method.
HepG2 cells were seeded in 6-well plates and synthesized substances
were added at 2 different concentrations (12.5 μM and/or 3.125
μM and/or 0.78 μM). After 48 h of incubation, cells were
harvested with trypsin. The cells were washed with cold PBS and were
transferred to Ripa cell lysis buffer (Santa Cruz). They were centrifuged
at 14000 rpm at +4 °C for 20 min. Protein concentration was measured
with Qubit 2.0 Fluorometer. Samples were boiled at 95 °C for
5 min in Laemmli sample buffer (Bio-Rad). Samples were run on 4–10%
polyacrylamide gels. Samples were transferred to the PVDF membrane
by a cold transfer device (Bio-Rad) and blocked with 5% skim milk
or 1% bovine serum albumin (BSA). Membranes were incubated with primary
antibodies overnight for EGFR (Abclonal A2909), p-EGFR (Abclonal AP0301),
Akt (CST 4691), p-Akt (CST 4060), Erk1/2 (CST 4695), p-Erk1/2 (CST
9101) and 1 h at RT for GAPDH (Abclonal). After secondary antibody
incubation for 1 h at RT, images were taken on Bio-Rad Chemidoc imaging
device using LumiGLO (CST) chemiluminescence substrate. Bands were
calculated with the Image Lab (Bio-Rad) and were analyzed with GraphPad
Prism 9.00. 42 4.3.7 Antiangiogenic Activity The antiangiogenic
effects of the synthesized compounds were evaluated by the Western
blot method on the expression of proteins in the angiogenesis pathway
(VEGFR, Akt, p-Akt, Erk1/2 (p44/42 MAPK), p-Erk1/2 (p-p44/42 MAPK)
in the HUVEC (Human umbilical vein endothelial) cell line). HUVEC
cells were seeded in 6-well plates and synthesized substances were
added at 2 different concentrations (12.5 μM and/or 3.125 μM
and/or 0.78 μM). After 48 h of incubation, cells were induced
with 100 ng/mL VEGFR for 15 min and harvested with trypsin. The same
procedures were applied as 4.3.5. VEGFR (CST 9698) antibody incubation
was made overnight. 42
## Stability Studies
4.3.1 Stability Studies Absorption spectra
of freshly prepared solutions of the samples were acquired at 25 °C
in the range of 200–800 nm with a Shimadzu UV-1280 spectrophotometer,
taking into account the solvent cutoff. Samples were dissolved in
the proper solvents, and the resulting solutions were placed in QS
quartz cuvettes (path length 1 cm). 40
## EGFR Inhibition Assay
4.3.2 EGFR Inhibition Assay Enzyme activity
was performed using the TAKARA Universal Tyrosine Kinase Assay and
Wash and Stop Solution for Sulfuric Acid Free ELISA according to Ölgen
et al. 36 The enzyme activity reaction was
performed on a precoated 96-well plate. To determine the activity,
40 μL of hEGFR and 10 μL of ATP-2Na solution were added
to each well on the plate and incubated at 37 °C for 30 min.
Then, the solution was removed from the well and the wells were washed
4 times with wash buffer. After this process, 100 μL of blocking
solution was added and incubated for another 30 min at 37 °C.
The blocking solution was discarded and the wells were washed again
with wash buffer. Subsequently, 50 μL of antiphosphotyrosine
(PY20-HRP) solution was added to each well and incubated at 37 °C
for 30 min. Afterward, the antibody solution was removed and the wells
were washed 4 times with the washing solution. 100 μL of HRP
substrate solution (TMBZ) was added to the wells and the plate was
incubated at 37 °C for 15 min. In addition to this mixture, 100
μL of stop solution was added to the wells in the same order
that the substrate solution was added. Activity was measured at 450
nm. In the studies to determine the inhibition potentials of
the synthesized molecules on hEGFR, the solutions of different concentrations
were prepared by dissolving the molecules with ethanol. The volumes
were adjusted so that the solvent amount in the reaction medium was
3% and preincubated with the enzyme (Copeland 2005) (ISBN: 978-1-118-48813-3).
The reaction was repeated only in the presence of this solvent to
determine the effect of the solvent on the enzyme. Using the results
obtained as a result of the inhibition studies, % relative activity–inhibitor
concentration graph was drawn for each molecule and the inhibitory
concentration at which the activity decreased by 50% was determined
as the IC 50 value.
## Cytotoxicity Assay
4.3.3 Cytotoxicity Assay HepG2 (hepatocellular
carcinoma; ATCC HB-8065), Caco-2 (Colon carcinoma; ATCC HTB-37), HT-29
(colon carcinoma; ATCC HTB-37), MCF-7 (Breast cancer; ATCC HTB-22),
A549 (lung carcinoma; ATCC CCL-185), and healthy HEK293T (embryonic
kidney epithelial; ATCC CRL-3216) cell lines were cultured in DMEM-F12
medium containing 10% fetal bovine serum (FBS) and antibiotic (100
U/mL penicillin and 100 μg/mL streptomycin). The cells were
incubated at 37 °C under an atmosphere of 5% CO 2 and
95% humidity. HepG2-SR cells were selected according to the short-term
sorafenib exposure protocol. 41 The cells
were exposed to sorafenib at concentrations of 1, 2, and 4 μM
for 72 h in gradual increments. At each stage, surviving cells were
plated in sorafenib-free medium. They were exposed to the next higher
concentration of sorafenib by passaging 3 times at each concentration.
Resistant cells were maintained in medium containing 4 μM sorafenib. Effects of synthesized compounds on cell viability of HepG2, Caco-2,
HT-29, MCF-7, A549, and HEK293T cell lines tested with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay. The cells were passaged as 5 × 10 3 per well in basal medium and incubated for adherence in 96-well
plates. Sorafenib and synthesized sorafenib–ruthenium complexes
were added at 10 decreasing concentrations between 100 and 0.2 μM
and incubated for 48 h. At the end of 48 h, 10 μL of yellow-colored
MTT agent was added and plates were incubated at 37 °C for 4
h. Formazan crystals formed after 4 h were dissolved with 100 μL
of DMSO, and absorbance was measured at 540 nm. Viable cells produced
darker color and nonviable cells produced lighter color because they
could not convert MTT to formazan. The % viability was calculated
relative to the negative controls. Concentration–viability
graphs were drawn for each test substance in the GraphPad Prism 9.00,
and the IC 50 values of the substances were calculated for
each cell line. 42
## Apoptosis Assay
4.3.4 Apoptosis Assay The apoptotic properties
of the synthesized compounds were determined in HepG2 cell line using
Muse Annexin V & Dead Cell reagent in Muse Cell Analyzer. HepG2
cells were seeded in 24-well plates and synthesized substances were
added at 3 or 4 different concentrations (0.78, 3.125, 12.5, and 50
μM). After 48 h of incubation, cells were harvested with trypsin
and transferred to PBS. 100 μL of Muse Annexin V & Dead
Cell reagent was added to the cell suspension in 100 μL of PBS.
The cells were allowed to stain for 30 min at room temperature in
the dark. The apoptotic profiles of the substances were determined
by flowing the samples in the Muse Cell Analyzer. Results are given
as a percentage (%). 43 There are
two different dyes in the reagent: Annexin V (apoptosis marker) and
7-AAD (dead cell marker). The profiles of the cells after staining
with these dyes according to their viability are as follows: Bottom-left: Living cells [Annexin V (−) and 7-AAD (−)] Bottom-right: Early apoptotic cells [Annexin V (+) and 7-AAD (−)] Top-right: Late apoptotic or dead cells by apoptosis [Annexin V
(+) and 7-AAD (+)] Top-left: Necrotic cells [Annexin V (−)
and 7-AAD (+)]
## Cell Cycle Assay
4.3.5 Cell Cycle Assay The effects of
the synthesized compounds on the cell cycle in the HepG2 cell line
were determined using the Muse Cell Cycle reagent in Muse Cell Analyzer.
HepG2 cells were seeded in 24-well plates and synthesized substances
were added at 3 or 4 different concentrations (0.78, 3.125, 12.5,
and 50 μM). After 48 h of incubation, cells were harvested with
trypsin and washed with PBS. The cell pellet was fixed in cold 70%
ethanol for 3 h at 20 °C. The fixed cells were washed again with
PBS, the supernatant was discarded, and 200 μL of Muse Cell
Cycle reagent was added. The cells were allowed to stain for 30 min
at room temperature in the dark. The effects of the substances on
the cell cycle were determined by flowing the samples in the Muse
Cell Analyzer device. Results are given as a percentage (%). The assay uses PI-based staining of DNA content to distinguish and
quantify the percentage of cells in each cell cycle phase (G0/G1,
S, and G2/M). Cells in G0/G1 phase are selected in blue, the S phase
in purple, and the G2/M phase in green. 44
## Western Blot Assay
4.3.6 Western Blot Assay Effects of synthesized
compounds on the expression of proteins in the tyrosine kinase pathway
(Egfr, p-Egfr, Akt, p-Akt, Erk1/2 (p44/42 MAPK), p-Erk1/2 (p-p44/42
MAPK)) in HepG2 cell line was determined by the immunoblotting method.
HepG2 cells were seeded in 6-well plates and synthesized substances
were added at 2 different concentrations (12.5 μM and/or 3.125
μM and/or 0.78 μM). After 48 h of incubation, cells were
harvested with trypsin. The cells were washed with cold PBS and were
transferred to Ripa cell lysis buffer (Santa Cruz). They were centrifuged
at 14000 rpm at +4 °C for 20 min. Protein concentration was measured
with Qubit 2.0 Fluorometer. Samples were boiled at 95 °C for
5 min in Laemmli sample buffer (Bio-Rad). Samples were run on 4–10%
polyacrylamide gels. Samples were transferred to the PVDF membrane
by a cold transfer device (Bio-Rad) and blocked with 5% skim milk
or 1% bovine serum albumin (BSA). Membranes were incubated with primary
antibodies overnight for EGFR (Abclonal A2909), p-EGFR (Abclonal AP0301),
Akt (CST 4691), p-Akt (CST 4060), Erk1/2 (CST 4695), p-Erk1/2 (CST
9101) and 1 h at RT for GAPDH (Abclonal). After secondary antibody
incubation for 1 h at RT, images were taken on Bio-Rad Chemidoc imaging
device using LumiGLO (CST) chemiluminescence substrate. Bands were
calculated with the Image Lab (Bio-Rad) and were analyzed with GraphPad
Prism 9.00. 42
## Antiangiogenic Activity
4.3.7 Antiangiogenic Activity The antiangiogenic
effects of the synthesized compounds were evaluated by the Western
blot method on the expression of proteins in the angiogenesis pathway
(VEGFR, Akt, p-Akt, Erk1/2 (p44/42 MAPK), p-Erk1/2 (p-p44/42 MAPK)
in the HUVEC (Human umbilical vein endothelial) cell line). HUVEC
cells were seeded in 6-well plates and synthesized substances were
added at 2 different concentrations (12.5 μM and/or 3.125 μM
and/or 0.78 μM). After 48 h of incubation, cells were induced
with 100 ng/mL VEGFR for 15 min and harvested with trypsin. The same
procedures were applied as 4.3.5. VEGFR (CST 9698) antibody incubation
was made overnight. 42
## Computational Studies
4.4 Computational Studies Molecular Docking
studies were carried out by Schrödinger Molecular Modeling
Software (2023-1) with Maestro (13.5) interface. MD simulations were
carried out using Desmond (D. E. Shaw Research) with Maestro (13.1)
interface. 4.4.1 Preparation of Ligands and Proteins Two-dimensional structures of the ligands were drawn with Chem-Draw.
Initially, the coordination bonds of the ruthenium complex were drawn
as normal bonds and the molecule was transferred to Schrödinger.
By selecting the molecule on Schrödinger’s workspace,
the covalent bonds between the metal and the ligand were set as coordination
interactions using the “create zero-order bonds to metal”
parameter under the build tab. Then, the energy minimization of the
molecule was achieved by using the minimize parameter under the edit
tab of Schrödinger Maestro and the preparation of the ligands
was completed. The verification of correct bond orders has been accomplished
by assigning bond orders through the Edit → Assign →
Bond Orders menu. Additionally, to ensure a reasonable coordination
geometry and prevent distorted structures, the “Perform postdocking
minimization” setting in the Output tab of the Glide ligand
docking application has been unchecked. 45 The 3D X-ray crystallographic structures of the target proteins
were obtained from the Protein Data Bank, accessible via the RCSB
Web site ( https://www.rcsb.org ). In this study, epidermal growth factor receptor (EGFR) protein
(PDB ID: 5X2A , the crystal resolution: 1.85 Å, origin: Homo sapiens ) was used to carry out all in silico studies. Careful
preparation and refinement of these protein structure was carried
out using the Protein Preparation Wizard module within Schrödinger
Maestro 13.5. 46 4.4.2 Glide Docking and Induced Fit Docking The methods previously published by our research group were used
to perform glide docking and induced fit docking. 47 , 48 The ligands that were prepared underwent docking with the receptors
using the Induced Fit Docking (IFD) protocol in Schrödinger
Release 2023-1. The IFD protocol, integrated into the Schrödinger
suite, employs Glide (Schrödinger, LLC, New York, NY). The
centroid of the native ligand served as the center for the Glide grid,
with the inner box side set to 20 Å and the outer box side automatically
determined. The binding energy, represented by the IFD Score, was
computed for each generated pose, and the resulting poses for each
protein–ligand complex underwent thorough visual examination. 49 4.4.3 Prime MM-GBSA Analysis The methods
previously published by our research group were used to perform prime
MM-GBSA analysis. 50 The calculation of
the free binding energy for the docked poses were carried out utilizing
the Prime MM/GBSA (Molecular Mechanics Generalized Born Surface Area)
module within the Schrödinger suite 2023. Default parameters
were applied in the MM/GBSA module, including the VSGB solvation model
and the Minimize sampling method. To account for flexibility, constraints
on flexible residues were implemented, designating the residues around
the receptor pocket as having flexible conformations during the energy
calculation. 51 4.4.4 Molecular Dynamics Simulations The methods previously published by our research group were used
to perform molecular dynamics simulations. 52 The Desmond System Builder module was employed to configure the
complex within a solvent-soaked orthorhombic periodic box, with a
selected orthorhombic box size of 10 Å as boundary conditions.
To achieve a neutral system charge, Na + and Cl – ions were added, resulting in a 0.15 M NaCl salt concentration.
The constructed solvated system underwent minimization using the OPLS3e
force field. System equilibration occurred at 300 K and 1.01325 bar
in the NPT (normal pressure and temperature) ensemble. Simulations
were conducted over a 200 ns time frame, with data recorded at 200
ps intervals. Molecular dynamic trajectories were generated and analyzed
utilizing Desmond’s Simulation Interaction Diagram (SID). 47 4.4.5 Molecular Docking Validations The
methods previously published by our research group were used to perform
molecular docking validations. 53
## Preparation of Ligands and Proteins
4.4.1 Preparation of Ligands and Proteins Two-dimensional structures of the ligands were drawn with Chem-Draw.
Initially, the coordination bonds of the ruthenium complex were drawn
as normal bonds and the molecule was transferred to Schrödinger.
By selecting the molecule on Schrödinger’s workspace,
the covalent bonds between the metal and the ligand were set as coordination
interactions using the “create zero-order bonds to metal”
parameter under the build tab. Then, the energy minimization of the
molecule was achieved by using the minimize parameter under the edit
tab of Schrödinger Maestro and the preparation of the ligands
was completed. The verification of correct bond orders has been accomplished
by assigning bond orders through the Edit → Assign →
Bond Orders menu. Additionally, to ensure a reasonable coordination
geometry and prevent distorted structures, the “Perform postdocking
minimization” setting in the Output tab of the Glide ligand
docking application has been unchecked. 45 The 3D X-ray crystallographic structures of the target proteins
were obtained from the Protein Data Bank, accessible via the RCSB
Web site ( https://www.rcsb.org ). In this study, epidermal growth factor receptor (EGFR) protein
(PDB ID: 5X2A , the crystal resolution: 1.85 Å, origin: Homo sapiens ) was used to carry out all in silico studies. Careful
preparation and refinement of these protein structure was carried
out using the Protein Preparation Wizard module within Schrödinger
Maestro 13.5. 46
## Glide Docking and Induced Fit Docking
4.4.2 Glide Docking and Induced Fit Docking The methods previously published by our research group were used
to perform glide docking and induced fit docking. 47 , 48 The ligands that were prepared underwent docking with the receptors
using the Induced Fit Docking (IFD) protocol in Schrödinger
Release 2023-1. The IFD protocol, integrated into the Schrödinger
suite, employs Glide (Schrödinger, LLC, New York, NY). The
centroid of the native ligand served as the center for the Glide grid,
with the inner box side set to 20 Å and the outer box side automatically
determined. The binding energy, represented by the IFD Score, was
computed for each generated pose, and the resulting poses for each
protein–ligand complex underwent thorough visual examination. 49
## Prime MM-GBSA Analysis
4.4.3 Prime MM-GBSA Analysis The methods
previously published by our research group were used to perform prime
MM-GBSA analysis. 50 The calculation of
the free binding energy for the docked poses were carried out utilizing
the Prime MM/GBSA (Molecular Mechanics Generalized Born Surface Area)
module within the Schrödinger suite 2023. Default parameters
were applied in the MM/GBSA module, including the VSGB solvation model
and the Minimize sampling method. To account for flexibility, constraints
on flexible residues were implemented, designating the residues around
the receptor pocket as having flexible conformations during the energy
calculation. 51
## Molecular Dynamics Simulations
4.4.4 Molecular Dynamics Simulations The methods previously published by our research group were used
to perform molecular dynamics simulations. 52 The Desmond System Builder module was employed to configure the
complex within a solvent-soaked orthorhombic periodic box, with a
selected orthorhombic box size of 10 Å as boundary conditions.
To achieve a neutral system charge, Na + and Cl – ions were added, resulting in a 0.15 M NaCl salt concentration.
The constructed solvated system underwent minimization using the OPLS3e
force field. System equilibration occurred at 300 K and 1.01325 bar
in the NPT (normal pressure and temperature) ensemble. Simulations
were conducted over a 200 ns time frame, with data recorded at 200
ps intervals. Molecular dynamic trajectories were generated and analyzed
utilizing Desmond’s Simulation Interaction Diagram (SID). 47
## Molecular Docking Validations
4.4.5 Molecular Docking Validations The
methods previously published by our research group were used to perform
molecular docking validations. 53
## Formation of Micelles
4.5 Formation of Micelles PEG–PLGA
polymeric micelles containing Ru3S or Ru4S complexes, which showed the best results in the cytotoxicity test,
were formed by the coprecipitation method. For this purpose, 1 mg
of either Ru3S or Ru4S complex and poly(ethylene
glycol) methyl ether- block -poly(lactide- co -glycolide) (PEG–PLGA, PEG average M n 5000, PLGA average M n 10,000,
lactide:glycolide 50:50, 10 mg) polymer were mixed overnight in a
vial containing 1 mL of DMSO. The next day, micelle (“self-assembly”)
structures were formed by adding dropwise into a vial containing 4
mL of water and the solution was stirred for 24 h. Then, DMSO was
removed by dialysis. 54 The micelle formation
process of the amphiphilic polymer without any complexes was followed
by using a similar procedure. M refers to PEG–PLGA polymeric
micelles without any complexes. M1 and M2 refer to Ru3S - and Ru4S -loaded polymeric
micelles.
## Release Studies
4.6 Release Studies The release profile
of Ru4S from M2 was evaluated by the dialysis
method in two different pH buffer solutions (pH = 5.5 and pH 7.4).
2 mL of M2 was placed into a dialysis bag with a molecular
weight cutoff of 3.50 kDa and dialyzed against 50 mL of phosphate-buffered
saline (PBS) in an orbital shaker with a stirring speed of 100 rpm
at 37 °C. 2 mL of samples was taken at a certain time point and
fresh buffer solution was replaced in the same volume instead. To
quantitatively determine the amount of drug released, the taken samples
at each time interval were analyzed by LC-HRMS. 55 − 57