Structure-antiplatelet activity relationships of novel ruthenium (ii) complexes: Investigation of its molecular targets

Article abstract of DOI:10.3390/molecules23020477

The regulation of platelet function by pharmacological agents that modulate platelet signaling has proven to be a positive approach to the prevention of thrombosis. Ruthenium complexes are fascinating for the development of new drugs, as they possess numerous chemical and biological properties. The present study aims to evaluate the structure-activity relationship (SAR) of newly synthesized ruthenium (II) complexes, TQ-1, TQ-2 and TQ-3 in agonists-induced washed human platelets. Silica gel column chromatography, aggregometry, immunoblotting, NMR, and X-ray analyses were performed in this study. Of the three tested compounds, TQ-3 showed a concentration (1–5 μM) dependent inhibitory effect on platelet aggregation induced by collagen (1 μg/mL) and thrombin (0.01 U/mL) in washed human platelets; however, TQ-1 and TQ-2 had no response even at 250 μM of collagen and thrombin-induced aggregation. TQ-3 was effective with inhibiting collagen-induced ATP release, calcium mobilization ([Ca²⁺]i) and P-selectin expression without cytotoxicity. Moreover, TQ-3 significantly abolished collagen-induced Lyn-Fyn-Syk, Akt-JNK and p38 mitogen-activated protein kinases (p38 MAPKs) phosphorylation. The compound TQ-3 containing an electron donating amino group with two phenyl groups of the quinoline core could be accounted for by its hydrophobicity and this nature might be the reason for the noted antiplatelet effects of TQ-3. The present results provide a molecular basis for the inhibition by TQ-3 in collagen-induced platelet aggregation, through the suppression of multiple machineries of the signaling pathway. These results may suggest that TQ-3 can be considered a potential agent for the treatment of vascular diseases.

Full text of DOI:10.3390/molecules23020477

molecules
Article
Structure-Antiplatelet Activity Relationships of
Novel Ruthenium (II) Complexes: Investigation of Its
Molecular Targets
1
1
1
2
Chih-Hsuan Hsia , Thanasekaran Jayakumar , Joen-Rong Sheu , Shin-Yi Tsao ,
3
1
1
4
Marappan Velusamy , Chih-Wei Hsia , Duen-Suey Chou , Chao-Chien Chang ,
Chi-Li Chung 5 , Themmila Khamrang
ID
1,3,
1,6,
* and Kao-Chang Lin
*
1
Graduate Institute of Medical Sciences and Department of Pharmacology, College of Medicine,
Taipei Medical University, Taipei 110, Taiwan; d119102013@tmu.edu.tw (C.-H.H.);
tjaya_2002@yahoo.co.in (T.J.); sheujr@tmu.edu.tw (J.-R.S.); d119106003@tmu.edu.tw (C.-W.H.);
fird@tmu.edu.tw (D.-S.C.)
Division of Endocrinology & Metabolism, Department of Internal Medicine, Sijhih Cathay General Hospital,
New Taipei 22174, Taiwan; metaendocr@yahoo.com.tw
Department of Chemistry, North Eastern Hill University, Shillong 793022, India; mvelusamy@gmail.com
Department of Cardiology, Cathay General Hospital, Taipei 106, Taiwan; change@seed.net.tw
Division of Pulmonary Medicine, Department of Internal Medicine, Taipei Medical University Hospital,
Taipei 106, Taiwan; clchung@tmu.edu.tw
2
3
4
5
6
Department of Neurology, Chi Mei Medical Center, Tainan 710, Taiwan
*
Correspondence: gaujang@mail2000.com.tw (K.C.L); themmilakhamrang@gmail.com (T.K.);
Tel: +886-6-2812811-51792 (K.-C.L.); +91-364-2722601 (T.K.)
Received: 30 January 2018; Accepted: 19 February 2018; Published: 22 February 2018
Abstract: The regulation of platelet function by pharmacological agents that modulate platelet
signaling has proven to be a positive approach to the prevention of thrombosis. Ruthenium complexes
are fascinating for the development of new drugs, as they possess numerous chemical and biological
properties. The present study aims to evaluate the structure-activity relationship (SAR) of newly
synthesized ruthenium (II) complexes, TQ-1, TQ-2 and TQ-3 in agonists-induced washed human
platelets. Silica gel column chromatography, aggregometry, immunoblotting, NMR, and X-ray
analyses were performed in this study. Of the three tested compounds, TQ-3 showed a concentration
(
1–5
thrombin (0.01 U/mL) in washed human platelets; however, TQ-1 and TQ-2 had no response
even at 250 M of collagen and thrombin-induced aggregation. TQ-3 was effective with inhibiting
µM) dependent inhibitory effect on platelet aggregation induced by collagen (1 µg/mL) and
µ
2+
collagen-induced ATP release, calcium mobilization ([Ca ]i) and P-selectin expression without
cytotoxicity. Moreover, TQ-3 significantly abolished collagen-induced Lyn-Fyn-Syk, Akt-JNK and p38
mitogen-activated protein kinases (p38 MAPKs) phosphorylation. The compound TQ-3 containing
an electron donating amino group with two phenyl groups of the quinoline core could be accounted
for by its hydrophobicity and this nature might be the reason for the noted antiplatelet effects of TQ-3.
The present results provide a molecular basis for the inhibition by TQ-3 in collagen-induced platelet
aggregation, through the suppression of multiple machineries of the signaling pathway. These results
may suggest that TQ-3 can be considered a potential agent for the treatment of vascular diseases.
2+
Keywords: ruthenium complexes; platelets; ATP; [Ca ]i; Akt-JNK-p38; Lyn-Fyn-Syk; SAR
1
. Introduction
Cardiovascular diseases (CVDs) are the number one killer and thrombosis in particular
is accountable for the majority of CVD-related mortalities [1]. Platelets play crucial roles in
Molecules 2018, 23, 477; doi:10.3390/molecules23020477
www.mdpi.com/journal/molecules

Molecules 2018, 23, 477
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hemostasis by preventing excessive blood loss during vascular damage through blood clotting.
Nevertheless, abnormal activation of platelets leads to thrombosis in pathological conditions of
rupturing atherosclerotic plaques [
resulting in heart attacks and strokes. Therefore, targeting platelets has been confirmed to be operative
in the prevention and treatment of CVDs [ ]. Despite the presently used anti-platelets aspirin and
clopidogrel exhibiting worthwhile results in many patients, they cause adverse side effects such as
bleeding complications [ ]. Thus, the progress of potential therapeutic strategies for the prevention
2]. Thrombosis decreases the blood supply to the heart and brain
3,4
5
and treatment of thrombotic diseases demands further study.
Organometallic complexes have been considered for therapeutic applications in various
pathological conditions [
FDA)-approved anti-cancer drug—increased the survival rate of patients with testicular cancer [
has been used to treat different types of cancer [ ]. The achievement of cisplatin and its dose-limiting side
5,
6]. For instance, cisplatin—a platinum-based Food and Drug Administration
(
7] and
8
effects inspired important investigation in this area. Ruthenium-based organometallics are an encouraging
class of therapeutic compound, as two ruthenium agents, NAMI-A and KP1019, have entered human
clinical trials [9,10]. Experimental studies established that numerous ruthenium-based compounds display
high cytotoxicity towards a wide range of cancer cells with reduced side effects [11]. It has been specified
that three specific properties make ruthenium an interesting metal for drug development: (i) its range of
oxidation states; (ii) its ability to mimic iron binding under physiological conditions and (iii) its low
toxicity compared to platinum [12]. Our recent study established that a novel ruthenium-based
compound TQ-5 suppressed platelet aggregation in vitro in washed human platelets via inhibiting the
phosphorylation of Akt and JNK1 and subsequently reducing the ATP release reaction and intracellular
calcium mobilization [13]. Another interesting study from our group also showed that the ruthenium
compound TQ-6 has a novel role in inhibiting platelet activation through the inhibition of the agonist
receptors-mediated inside-out signaling such as Src-Syk-PLCγ2 cascade and subsequent suppression
of granule secretion, leading to disturbing integrin 3-mediated outside-in signaling and ultimately
α
β
IIb
inhibiting platelet aggregation [14]. In this study, the structure activity relationship (SAR) of three
newly synthesized ruthenium-based compounds—TQ-1, TQ-2 and TQ-3—was performed in collagen
and thrombin-induced washed human platelet aggregation. The possible molecular target was
also examined.
2
. Results
2
.1. Molecular Structure of Complexes
The molecular structures of some of the complexes were investigated by single-crystal X-ray
studies. The crystallographic details and structure refinement details are summarized in Table S1.
The geometrical parameters around the metal atom involving ring centroid are listed in Table S2.
The Oak Ridge Thermal-Ellipsoid Plot (ORTEP) program views of the complexes are shown in
Figure 1B. The ruthenium complex TQ-1 crystallized in the monoclinic system with P21/n space group.
Complexes TQ-2 and TQ-3 are crystalized in the triclinic system with P-1 space group. The coordination
geometry around Ru(II) in the complexes is best described as pseudo-octahedral with the arene ring
6
occupying the three coordination sites in a
η
-fashion and the two nitrogen atoms of the bidentate
ligands and a chloride ion occupying the remaining coordination sites. The Ru-N bond lengths fall in
the range 2.072–2.109 Å (TQ-1: 2.072, 2.108; TQ-2: 2.081, 2.109; TQ-3: 2.078, 2.109 Å) and the Ru-Cl
bond length in the range 2.390–2.40 Å (TQ-1: 2.398, TQ-2: 2.40, TQ-3: 2.390 Å) [15]. The six Ru–C
bonds have almost comparable bond distances with an average Ru–C bond lengths of 2.202 (TQ-1);
2.212 (TQ-2); 2.193 (TQ-3) Å and the ruthenium-arene centroid ring distance (Ru-Ct, 1.684 Å) is almost
the same for all the complexes and is similar to those in related Ru(II)-arene complexes [16].

Molecules 2018, 23, 477
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Figure 1. Scheme of synthesis of ligands 2-pyridin-2-yl-quinoline (L1) and 2-pyridin-2-yl-quinoxaline
(
[
L2) and diphenyl-(2-pyridin-2-yl-quinolin-6-yl)-amine (L3) (A); Scheme of synthesis of complexes
6
6
6
Ru(η
-cymene)(L1)Cl]BF (TQ-1), [Ru(
η
-cymene)(L2)Cl]BF (TQ-2) and [Ru(
η
-cymene)(L3)Cl]BF4
4
4
(
TQ-3) (
B); ORTEP drawings of TQ-1, TQ-2 and TQ-3. Hydrogen atoms and BF anion are omitted for
4
clarity (C).
2
.2. Electronic Spectra
UV-Vis spectra of the ligands and complexes were recorded in acetonitrile and the resulting
data (Figures S1 and S2) are summarized in the experimental section. In the absorption spectra,
all the ligands display a band located in the range 335–396 nm, which possibly originates from
the π → π* transitions located in the chromophores such as quinoline and quinoxaline moieties [17].
The incorporation of diphenyl amine on the quinoline ring of 2-pyridin-2-yl-quinoline (L1) to obtain
diphenyl-(2-pyridin-2-yl-quinolin-6-yl)-amine (L3) leads to an additional band at 396 nm, which is
expected of the electron releasing amino group enhance the electron density on the quinoline ring.
The intense absorption bands were noticed in the ranges of 223–321 nm and were assigned to intra

Molecules 2018, 23, 477
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ligand n→π*, π→π* transitions. The UV-Vis spectra of all complexes exhibit intense absorption bands
in the range 240–370 nm, assigned to intraligand * transitions. The lowest-energy absorption in
each case (421–465 nm) is tentatively assigned as metal-to-ligand charge transfer (MLCT) transition
from the 4d orbitals of Ru(II) to the empty * ligand orbitals [18]. Further, the MLCT transition of
π–π
π
the complexes is following the order of 465 (TQ-3) > 445 (TQ-2) > 421 (TQ-1) nm. The incorporation
of amino group on the quinoline ring of TQ-1 to TQ-3 and additional nitrogen atom (TQ-2) causing
red-shift in the band position illustrating that the change in electronic nature of ligand by additional
nitrogen atoms enhances the electron density on the aromatic rings.
2
.3. Influence of TQ-1, 2 and 3 on Collagen and Thrombin-Induced Platelet Aggregation
The schematic diagram for the synthetic procedure of ligands, complex and ORTEP has been given
in Figure 1. The concentration of collagen and thrombin induced maximal human platelet aggregation,
that was approximately 1 g/mL and 0.01 U/mL, respectively, thus, these concentrations were used
µ
in this study. Platelets were stimulated with collagen and thrombin to analyze the effect of TQ-1,
TQ-2 and TQ-3 on washed human platelet aggregation. Off these three compounds, TQ-3 concentration
dependently (1–5
µM) inhibited the signal intensities, which we derived from aggregometer in collagen
(Figure 2A,c) and thrombin (Figure 2B,c)-induced platelet aggregation. Nevertheless, TQ-1 and
TQ-2 did not alter the signal on either collagen (Figure 2A,a,b) or thrombin (Figure 2B,a,b)-induced
aggregation at any tested concentrations.
Figure 2. Inhibitory activity of ruthenium complexes (TQ-1-TQ3) on (
platelet aggregation in washed human platelets. Washed human platelets (3.6
pre-incubated with the solvent control (0.1% DMSO) or TQ-1, TQ-2 (50–250
and then treated with 1 g/mL collagen and 0.01 U/mL thrombin to stimulate platelet aggregation.
standard errors of the means (n = 4). * p < 0.05, *** p < 0.001 compared
A
) collagen and (
B
) thrombin–induced
8
×
10 cells/mL) were
µ
M) and TQ-3 (1–5 µM)
µ
Data are presented as means
with the DMSO group.
±

Molecules 2018, 23, 477
5 of 15
2+
2
.4. Effect of TQ-1, 2 and 3 on Cytotoxicity, [Ca ]i Mobilization and ATP Release in Human Platelets
The effect of TQ-1, TQ-2 and TQ-3 compounds on cell toxicity was examined by measuring
extracellular activity of lactate dehydrogenase (LDH). LDH is a marker of cell injury or death, which is
released into the culture medium upon cell death due to the damage of plasma membrane. As shown
in Figure 3A, there were no significant differences in LDH level among the three tested compounds
(
TQ-1, 2 and 3, vehicle (DMSO) and tyrodes solution, indicating that tested compounds do not affect
platelet permeability or induce platelet cytolysis. A maximal value (MAX) of LDH was observed in
the sonicated platelets, which is used as a positive control.
Figure 3. Comparative effects of TQ-1-TQ-3 on collagen induced cytotoxicity, ATP release, relative [Ca2+]i
mobilization and on surface FITC-P-selectin expression in human platelets. Washed human platelets
8
(
(
3.6
×
10 cells/mL) were pre-incubated with TQ-1, TQ-2 (250
µ
M) and TQ-3 (5
µ
M) or a solvent control
g/mL of collagen to
); to induce the cytoplasmic influx of Ca from intracellular stores
); or to check the direct binding of FITC-P-selectin ( ) as described in the materials and methods
section. Data are presented as the means S.E.M. (n = 4). ** p < 0.01 and *** p < 0.001 compared with
the DMSO group. # p < 0.05 and ### p < 0.001 compared with the collagen induced group.
0.5% DMSO) to check the cytotoxicity (
stimulate ATP release reaction (
A
) and subsequently treated with 1 µ
2
+
B
(
C
D
±

Molecules 2018, 23, 477
6 of 15
Stimulation of the collagen receptor GPVI leads to rapid intracellular mobilization of calcium,
which is essential for platelet secretion and aggregation [19]. To investigate the effect of tested
compounds on the intracellular mobilization of calcium stores, calcium levels were measured
fluorometrically using the calcium-sensitive dye, Fura-2 AM. Experiments were performed in the
presence of 2 mM EGTA to prevent the influx of extracellular calcium. Fura-2 AM-loaded platelets
were pre-incubated with tested compounds or solvent alone for 5 min and then stimulated with
collagen (1 µg/mL). Stimulation of platelets with collagen resulted in a rapid increase in intracellular
calcium concentration (Figure 3B). However, this increment was significantly decreased when platelets
pretreated with a maximal concentration of 5 µM TQ-3 (Figure 3B).
2+
Intracellular [Ca ] immobilization is known to be involved in the release of ATP from a dense
2+
body of platelets [20]. Because TQ-3 inhibited collagen-elevated [Ca ] immobilization as shown in
Figure 3B, we investigated whether this compound involves on the inhibition of ATP release. Figure 3C
shows that TQ-3 significantly inhibited collagen-induced ATP release. However, collagen induced
ATP release had not blocked by TQ-1 and TQ-2 even at their maximum concentration of 250 µM.
2
.5. TQ-1, 2 and 3 on Collagen-Induced P-Selectin Expression
P-selectin is a major platelet -granule protein that is highly expressed on the platelet
α
surface during activation and plays important role in platelet-leukocyte and platelet-endothelial
cell interactions [21]. Therefore, we examined the effects of TQ-1, 2 and 3 on collagen-induced
P-selectin expression. As shown in Figure 3D, stimulation with collagen (1
enhanced P-selectin expression in platelets, as compared to that in control untreated platelets. TQ-3 at
a concentration of 5 M significantly inhibits P-selectin expression in human washed platelets induced
by collagen but TQ-1 and 2 did not effective (Figure 3D).
µg/mL) significantly
µ
2
.6. The Kinase Activities of the Src-Family Kinases Fyn and Lyn and the Tyrosine Kinase Syk are Inhibited by TQ-3
Binding of collagen with GPVI is reported to be mediated by the Src-family kinases Fyn and
Lyn [22], resulting to activate the cytosolic tyrosine kinase Syk. In this study, these proteins were
investigated to test the inhibitory effect of TQ-1, 2 and 3 on tyrosine kinase activities. To this,
platelets were stimulated with collagen (1
assayed for kinase (Fyn, Lyn and Syk) phosphorylation in vitro. As shown in Figure 4A–C, the collagen
induced phosphorylation of Fyn, Lyn and Syk were inhibited by TQ-3 at a lower concentration of 5 M.
However, TQ-1 and TQ-2 was not seen to inhibit the kinase phosphorylation of Fyn, Lyn and Syk even
at 250 M concentration (Figure 4A–C). The representative histograms illustrating the effect of tested
compounds on SFK and Syk phosphorylation induced by collagen is shown in Figure 4, right panel.
µg/mL) in the presence and absence of TQ-1, 2 and 3 and
µ
µ
2
.7. TQ-3 Attenuated Collagen Stimulated Akt, JNK and p38 MAPK Phosphorylation in Platelets
Protein kinase B (Akt), JNK and p38 MAPK have been shown to play critical roles in platelet
activation. To investigate whether tested ruthenium compounds inhibit these signaling events during
platelet activation, we analyzed the phosphorylation of Akt, JNK and p38 MAPK. The immunoblotting
analysis exhibits collagen triggered visible Akt, JNK and p38 MAPK phosphorylation in human
platelets and TQ-3 more significantly (p < 0.01 for Akt and p < 0.001 for JNK and p38 than the other
compounds TQ-1 and TQ-2) suppressed these phosphorylation (Figure 4D–F). These data suggest that
Akt and MAPKs signaling molecules are highly implicated for the observed effects of TQ-3 against
collagen-stimulated human platelets.

Molecules 2018, 23, 477
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Figure 4. Effects of ruthenium complexes on the phosphorylation of Fyn/Lyn-Syk and MAPKs induced
9
by collagen in human platelets. Washed platelets (1.2
control (0.5% DMSO) or 250 M TQ-1 and TQ-2 and 5 M TQ-3 and then treated with 1
to induce platelet activation. The subcellular extracts were analyzed for the phosphorylation of Fyn
); Lyn ( ); Syk ( ); Akt ( ); JNK1( ) and p38 MAPK ( ) by western blotting. Data are presented
as the mean SEM (n = 4). * p < 0.01 and *** p < 0.001 compared with the DMSO group, # p < 0.05,
# p < 0.01 and ### p < 0.001 compared with the collagen induced group.
×
10 cells/ml) were incubated with solvent
µ
µ
µg/mL collagen
(
A
B
C
D
E
F
±
#
3
. Discussion
Recent our studies have established that novel ruthenium based compounds TQ-5 and TQ-6
possess in vitro antiplatelet and in vivo antithrombotic effects [13 14]. These outcomes provided us
,
with the impetus to further analyze the structure activity relationship of three (TQ-1, TQ-2 and
TQ-3) newly synthesized ruthenium derived compounds in agonists induced washed human
platelets. The results show that compound TQ-3 only inhibits collagen and thrombin-induced platelet
aggregation but TQ-1 and TQ-2 not effective even at their maximum tested concentration of 250 µM.
Moreover, TQ-3 significantly inhibited collagen-induced ATP release, Ca2+ mobilization and P-selectin
expression but these parameters were unaltered in TQ-1 and TQ-2 treated platelets. The noted
inhibitory effect of TQ-3 was attributable by inhibiting SFK-Syk-MAPKs-signaling molecules.
Platelets attach to sub-endothelial matrix protein collagen, altering their shape and leading
to the release of ATP, Ca2 and P-selectin. Thus, collagen acts as a platelet adhesion substrate as
+

Molecules 2018, 23, 477
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well as an endogenous platelet activator. Upon collagen stimulation, a rapid rise in intracellular
2+
2+
Ca occurs due to PI3K activation [23]. Increased Ca consequently activates platelet granule
secretion [24]. Dense granules released ATP, which causes a rapid influx of calcium by sensitizing the
ionotropic receptor P2X1 [25]. In addition, P-selectin released from
α granule not only aid leukocyte
adhesion to endothelial cells but also acts as important for inter-platelet aggregation, stabilizing the
initial GPIIb/IIIa-fibrinogen interactions, thus allowing the formation of large and stable platelet
aggregates [24]. In line with these results, the effect of TQ-1, TQ-2 and TQ-3 in collagen-induced calcium
mobilization, ATP secretion and P-selectin expression was tested in human platelets. The results found
that among the three compounds tested, TQ-3 exerted inhibitory effects on collagen induced calcium
mobilization, ATP secretion and P-selectin expression, representing that changes of these granular
substances play major role in platelet activation.
Src family kinases (SFKs) are involved in the regulation of several cellular processes such as
proliferation, differentiation, motility and adhesion [15]. It has been proposed that there are nine
members of the Src family of tyrosine kinases, which include Src, Lck, Hck, Blk, Fyn, Lyn, Yes, Fgr and
Yrk [26,27]. Previous studies described that both human and rodent platelets contain high levels of
Src as well as Fyn, Lyn, Hck, Yes, Lck and Fgr [28]. Though platelets comprise elevated levels of
SFKs, proposing an essential role for these enzymes in platelet function, the role of SFKs in platelet
function has not been fully clarified. In platelets, SFKs, particularly Lyn and Fyn, play crucial role
−
/−
in downstream of collagen receptors [22]. Studies have suggested that platelets from Fyn
exhibit delayed spreading on immobilized fibrinogen [29], whereas platelets from Lyn
mice
/−
mice spread
−
poorly on von Willebrand factor [30]. Studies have also proven that SFKs play a role in thromboxane
generation, shape change, as well as regulation of phosphorylation of Akt [31] and ERK [32]. In this
study, TQ-3 visibly reduced collagen-induced phosphorylation of Fyn, Lyn and Syk; however, TQ-1 and
TQ-2 had no direct effects on these proteins, suggesting that TQ-3-mediated inhibition of platelet
activation involves SFKs and Syk downstream signaling.
The Akt pathway has been discovered in platelet activation by GPVI through the regulation
of the serine/threonine kinase, Akt [33]. Akt can be phosphorylated by protein kinase C and by
2+
Ca /calmodulin-dependent protein kinase independently of PI3K [34]. Activation by collagen is
found to be impaired in mouse platelets deficient in Akt [35]. In this study, it has been evidenced
that TQ-3 significantly inhibited collagen induced Akt phosphorylation but it did not effectively
obstruct by TQ-1 and TQ-2, intending that TQ-3-potentiated inhibition of platelet activation involve
the inhibition of Akt signaling pathways. Studies with inhibitors and/or genetic manipulations
have demonstrated that MAPKs contribute greatly to platelet responses in various agonists [36].
Various inflammatory cytokines and stress stimuli that lead to cellular apoptosis activate JNK1/2 and
−
/−
platelets are associated
p38 MAPK [37]. Previous studies have also demonstrated that JNK
with increased bleeding time, decreased integrin activation and severe granule secretion
α
β
3
IIb
impairment [36]. Therefore, it seems that inhibition of the Akt, JNK and p38 phosphorylation plays
important role in the platelet activation process. Consistent with this demonstration, we found that
TQ-3 markedly inhibited collagen-induced Akt, JNK and p38 phosphorylation but TQ-1 and TQ-2 did
not effective on these molecules.
As ruthenium is able to accomplish several oxidation states (II, III and IV) under physiological
conditions and interact with a range of biomolecules, it may expedite efficient interaction of the
small molecule with its molecular targets [5,38]. Besides, some ruthenium-based organometallic
complexes have displayed enhanced stability in water and air [38], which may be beneficial to exert
prolonged effects of the target small molecule. On studying the variation of N, N’-chelating ligands,
the addition of more phenyl ring enhanced the hydrophobicity of the Ru (II) complexes. The more
hydrophobic nature of the complex can be responsible for the activity toward the drug intrinsic
potential. In case of the complex TQ-3 containing electron donating amino group with two phenyl
group of the quinoline core could be accounted for the hydrophobicity of the complex and this might
be the reason for the noted effects of TQ-3. Generally, the complexes having higher rate of hydrolysis

Molecules 2018, 23, 477
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exhibit higher cytotoxicity than those that do not hydrolyze which are inactive or weakly active. For Ru
II) arene complexes, the hydrolysis increases with the increase in electron donating ability system.
(
A previous study performed to test the human platelet aggregation inhibitory activities of bovine,
ovine and caprine kappa-casein and their tryptic hydrolysates indicated that increased hydrolysis of
the compounds could enhance platelet inhibitory activities [39]. Another related study also found the
comparable results that carbamoylpiperidine and nipecotoylpiperazine derivatives enhance desired
antithrombotic effects with increased levels of hydrophobicity [40]. The pKa value of the quinoxaline
ligand (TQ-2) is 0.6 which is lower than that of quinoline ligand (TQ-1 and TQ-3) ~4.9 and this value
could be related to the rate of hydrolysis where the quinoline containing ligand has faster hydrolysis
rate as compared to that of quinoxaline ligand. This higher pKa value of the quinoxaline ligand of
TQ-3 might be accountable to its observed potent antiplatelet effects.
4
. Methods
4
.1. Reagents
Collagen (type I) and thrombin were purchased from Sigma (St. Louis, MO, USA).
183
185
The anti-phospho-c-Jun N-terminal kinase (JNK) (Thr /Tyr ), anti-JNK, anti-phospho-p38
mitogen-activated protein kinase (MAPK), anti-p38 MAPK and anti-phospho-Syk (Tyr5
monoclonal antibodies (mAbs) were purchased from Cell Signaling (Beverly, MA, USA).
The anti-phospho-Akt (Ser4 ) and anti-Akt mAbs were purchased from Biovision (Mountain View,
25/526
)
73
CA, USA). Anti-phospho-Fyn (Y5 ) mAb, anti-phospho-Lyn (Y ) mAb was obtained from Abcam
30
507
(
Cambridge, UK). The horseradish peroxidase (HRP) conjugated donkey anti-rabbit immunoglobulin
G (IgG), the Hybond-P polyvinylidene difluoride (PVDF) membrane, the sheep anti-mouse IgG and
the enhanced chemiluminescence western blotting detection reagent was purchased from Amersham
◦
(
Buckinghamshire, UK). The TQ1, TQ-2 and TQ-3 were dissolved in DMSO and stored at 4 C.
4
.2. Synthesis of Ligands (L1–L3)
All the solvents were purified according to the reported standard purification procedures.
The chemicals obtained commercially were used without further purification. The starting
material 5-bromo-2-nitro-benzaldehyde was prepared by following the reported procedure.
Ligands 2-pyridin-2-yl-quinoline (L1) and 2-pyridin-2-yl-quinoxaline (L2) were synthesized by
following the reported synthetic procedures [41]. Diphenyl-(2-pyridin-2-yl-quinolin-6-yl)-amine (L3)
was prepared by adopting Friedländer quinoline synthesis followed by employing Pd-catalyzed C-N
cross coupling of 6-bromo-2-pyridin-2-yl-quinoline with diphenyl amine. A representative synthetic
procedure of L3 is illustrated in the following.
Diphenyl-(2-pyridin-2-yl-quinolin-6-yl)-amine (L3). To a flask containing 25 ml ethanolic solution of
5
-bromo-2-nitro-benzaldehyde (1.15 g, 5 mM), iron powder (1.11 g, 20 mM) and 0.1N HCl (5 mL) were
◦
added and stirred vigorously at 100 C. After 1 h, 2-acetyl pyridine (0.6 g, 5 mM) and crushed KOH
(
0.33 g, 6 mM) were added in portion wise and allowed to continue stirring for 2 h. Finally, the reaction
mixture was cooled to RT, diluted with dichloromethane and filtered through a celite pad. The filtrate
collected was then poured into water and extracted from dichloromethane. The organic layer was
dried in Na SO , filtered and dried. The crude product was purified by column chromatography on
2
4
◦
silica gel (hexane:ethylacetate, 2:1) to obtain pale yellow solid. m.p. 118–120 C.
Mixture of 6-bromo-2-pyridin-2-yl-quinoline (0.5 g, 2 mM), diphenyl amine (0.4 g, 2.4 mM),
Pd (dba) (0.03 g, 0.04 mM), dppf (0.06 g, 0.12 mM) and sodium tert-butoxide (0.3 g, 2.8 mM)
2
3
were added to the freshly distilled toluene and the reaction mixture was refluxed under inert
atmosphere. After 48 h, the reaction was cooled to ambient temperature; solvent was removed,
washed with water and extracted from chloroform. The organic layer was dried over anhydrous
Na SO , filtered and dried. The desired product was obtained after purification in column
2
4

Molecules 2018, 23, 477
10 of 15
1
chromatography through silica gel in hexane:ethylacetate (1:1); H NMR (400 MHz, CDCl )
δ
8.64–8.63
3
(d, 1H, J = 4 Hz), 8.52–8.50 (d, 1H, J = 8 Hz), 8.37–8.35 (d, 1H, J = 8 Hz), 7.94–7.89 (t, 2H, J = 10 Hz),
7
.78–7.75 (t, 1H, J = 12 Hz), 7.45–7.43 (d, 1H, J = 8 Hz), 7.25–7.21 (t, 6H, J = 8 Hz), 7.11–7.09 (d, 4H,
13
J = 8 Hz), 7.03–7.00 (t, 2H, J = 6 Hz); C NMR (400 MHz, CDCl3)
δ: 156.0, 153.8, 148.6, 146.9, 145.8,
+
144.0, 136.4, 134.9, 130.1, 129.0, 128.8, 126.6, 124.5, 123.2, 121.0, 118.7, 117.2; ESI-MS m/z: 374 (M );
Anal. found (calcd.) for C H N : C, 83.58 (83.62); H, 5.20 (5.13); N, 11.35 (11.25); λmax, nm (CH CN),
26
19
3
3
3
96, 347, 293, 240.
1
2
-Pyridin-2-yl-quinoline (L1). H NMR ( 400 MHz, CDCl )
δ
8.67–8.66 (d, 1H, J = 4 Hz), 8.61–8.59
3
(
7
d, 1H, J = 8 Hz), 8.50–8.48 (d, 1H, J = 8 Hz), 8.22-8.20 (d, 1H, J = 8 Hz), 8.14–8.12 (d, 1H, J = 8 Hz),
.82–7.76 (m, 2H), 7.68–7.64 (t, 1H, J = 8 Hz), 7.49–7.45 (t, 1H, J = 8 Hz), 7.30–7.27 (t, 1H, J = 6 Hz); ESI-MS
m/z: 207.16 (M+H); Anal. found (calcd.) for C H N : C, 81.45 (81.53); H, 4.92 (4.89); N, 13.63 (13.58);
14
10
2
λmax, nm (CH CN), 335, 321, 253, 247(sh), 227.
3
1
2
-Pyridin-2-yl-quinoxaline (L2). H NMR ( 400 MHz, CDCl )
δ
9.90 (s, 1H), 8.73–8.72 (d, 1H, J = 4 Hz),
3
8.55–8.53 (d, 1H, J = 8 Hz), 8.11–8.09 (d, 2H, J = 8 Hz), 7.86–7.82 (t, 1H, J = 8 Hz), 7.74–7.72 (t, 1H,
J = 6 Hz), 7.37–7.34 (t, 1H, J = 6 Hz), 6.97–6.94 (d, 1H, J = 12 Hz); ESI-MS m/z: 208.09 (M + H); Anal.
found (calcd.) for C H N : C, 75.48 (75.35); H, 4.47 (4.38); N, 20.23 (20.28); λmax, nm (CH CN), 335,
13
9
3
3
2
75, 252, 223.
4
.3. Synthesis of TQ1, TQ2 and TQ3
The dimeric compound [Ru(p-cymene)(Cl)2]2 was synthesized following the reported
procedures [42]. The complexes of TQ-1, TQ-2 and TQ-3 were prepared by following the reported
methods [43]. Illustrative synthetic procedure of complexes is listed in Figure 1.
[
Ru( 6
dissolved in methanol into the solution of L1 (0.08 g, 0.1 mM) in methanol and stirred for 2 h at room
temperature. NH BF was then added and the mixture was allowed to continue stirring overnight.
η -cymene)(L1)Cl]BF (TQ-1). TQ-1 was prepared by adding [Ru(p-cymene)(Cl) ] (0.1 g, 0.2 mM)
4 2 2
4
4
Finally, the solvent was removed and the solid residue obtained was dissolved in dichloromethane.
Excess NH BF was filtered off and the filtrate was concentrated. The solid product obtained was then
4
4
wash with diethyl ether and recrystallized from chloroform and hexane yielding orange microcrystals.
1
Yield: 82%; H NMR (400 MHz, DMSO-d )
δ 9.57-9.56 (d, 1H, J = 4 Hz), 8.93–8.91 (d, 1H, J = 8 Hz),
6
8.86–8.84 (d, 1H, J = 8 Hz), 8.76–8.74 (t, 2H, J = 8 Hz), 8.39–8.35 (t, 1H, J = 4 Hz), 8.27–8.25 (d, 1H,
J = 8 Hz), 8.12–8.09 (t, 1H, J = 6 Hz), 7.96–7.93 (t, 1H, J = 6 Hz), 7.90–7.87 (t, 1H, J = 6 Hz), 6.16–6.12 (t, 2H,
J = 8 Hz), 6.02–6.01 (d, 1H, J = 4 Hz), 5.96–5.94 (d, 1H, J = 4 Hz), 2.49 (s, 6H), 2.21 (s, 3H), 0.77–0.70
+
(
m, 1H); ESI-MS: m/z = 477 [M -BF ]; Anal. found (calcd) for C H BClF N Ru: C, 51.65 (51.13);
4
24 24
4
2
2
H, 4.36 (4.29); N, 5.03 (4.97). λmax, nm (CH CN, ε × 10 ) 421 (122), 350 (600), 335 (536), 290 (930),
3
2
65 (976), 240 (1120).
Ru( 6
d, 1H, J = 8 Hz), 9.13–9.11 (d, 1H, J = 8 Hz), 8.76–8.75 (d, 1H, J = 4 Hz), 8.53–8.43 (m, 2H),
.27–8.26 (d, 2H, J = 4 Hz), 8.03–8.00 (t, 1H, J = 6 Hz), 6.38–6.36 (d, 1H, J = 8 Hz), 6.32–6.30 (d, 1H,
1
[
(
8
η -cymene)(L2)Cl]BF (TQ-2). Yield: 87%; H NMR (400 MHz, DMSO-d ) δ 10.14 (s, 1H), 9.70–9.68
4 6
J = 8 Hz), 6.19–6.18 (d, 2H, J = 4 Hz), 2.56 (s, 6H), 2.41–2.38 (m, 1H), 2.28 (s, 3H); ESI-MS: m/z = 478
+
[
M -BF ]. Anal. found (calcd) for C H BClF N Ru: C, 49.02 (48.91); H, 4.16 (4.10); N, 7.52 (7.44).
4
23 23
4
3
2
λmax, nm (CH CN, ε × 10 ), 445 (100), 370 (527), 250 (437), 290 (452), 270 (583), 250 (704).
Ru(
3
6
1
[
η -cymene)(L3)Cl]BF (TQ-3). Yield: 84%; H NMR (400 MHz, DMSO-d ) δ 9.49–9.48 (d, 1H,
4 6
J = 4 Hz), 8.70–8.68 (d, 1H, J = 8 Hz), 8.59–8.50 (m, 3H), 8.31–8.27 (t, 1H, J = 8 Hz), 7.79 (s, 1H), 7.66–7.64
(
d, 1H, J = 8 Hz), 7.47–7.28 (m, 11H), 6.11 (s, 2H), 5.97 (s, 2H), 2.18 (s, 3H), 1.12–0.99 (m, 1H), 0.76 (s, 6H);
+
ESI-MS: m/z = 644 [M -BF ]. Anal. found (calcd) for C H BClF N Ru: C, 59.25 (59.15); H, 4.48 (4.55);
N, 5.63 (5.75). λmax, nm (CH CN, ε × 10 ), 465 (187), 360 (128), 320 (354), 305 (385), 240 (316).
4
36 33
4
3
2
3

Molecules 2018, 23, 477
11 of 15
4
.4. Platelet Aggregation and ATP Release
The methods described by Sheu et al. [44] and Lin et al. [45] were followed for the preparation
of human platelet suspensions. Blood was collected from healthy human volunteers who did not
take medication during the preceding 2 weeks and was mixed with acid-citrate–dextrose solution
(
1:9). The blood samples were subjected to centrifugation at 120
×
g for 10 min and platelet-rich
M) and heparin (6.4 IU/mL)
g for 10 min, the platelet pellets
plasma (PRP) was collected. PRP was supplemented with PGE (0.5
and then incubated for 10 min at 37 C. After centrifugation at 500
µ
1
◦
×
−
1
were suspended in Tyrode’s solution containing 3.5 mg mL bovine serum albumin (BSA), pH 7.35
NaCl 137 mM, KCl 2.7 mM, MgCl 1 mM, NaH PO 0.2 mM, NaHCO 12 mM and glucose 5.5 mM).
(
2
2
4
3
Then, PGE (0.5
µ
M), apyrase (1.0 U/mL) and heparin (6.4 IU/mL) were added and the mixture was
1
◦
incubated for 10 min at 37 C. The mixtures were centrifuged at 500
the repeated washing procedure. Finally, the platelet pellets were resuspended by Tyrode’s solution
×
g for 10 min and subjected for
2+
and then CaCl was added to platelet suspensions in which the concentration of Ca was 1 mM.
2
This study was approved by the Institutional Review Board of Taipei Medical University (TMU-JIRB
No. 201401020) and conformed to the directives of the Helsinki Declaration.
As previously described [44,45], platelet aggregation was measured according to the turbidity
of platelet suspensions and recorded by a Lumi-Aggregometer (Payton Associates, Scarborough,
ON, Canada). Before the addition of collagen and thrombin to induce platelet aggregation, the platelet
8
suspensions (3.6
TQ-2 and 1–5
unit was used to present the extent of platelet aggregation. For the measurement of ATP release,
a 20 L of luciferin-luciferase mixture was added 1 min before adding collagen and the relative amount
of ATP release was compared to the solvent control.
×
10 cells/mL) were pretreated with various concentrations (50–250
µ
M) of TQ1 and
µ
M TQ-3 or an isovolumetric solvent control (0.5% DMSO) for 3 min. Light-transmission
µ
4
.5. Detection of Lactate Dehydrogenase
8
Washed platelets (3.6
TQ-3 (5
supernatant (50
×
10 cells/mL) were pretreated with TQ-1 (250
µM), TQ-2 (250
µM),
◦
µ
M) or solvent control (0.1% DMSO) for 10 min at 37 C. After centrifugation, an aliquot of the
µ
L) was measured the levels of LDH by using CytoTox 96 non-radioactive cytotoxicity
assay kit from Promega (Madison, WI, USA) and the absorbance wavelength was read at 500 nm using
a Synergy H1 microplate reader (BioTek, VT, USA).
4
.6. Measurement of Relative Ca2+ Mobilization by Fura 2-AM Fluorescence
Citrated whole blood was centrifuged at 120 g for 10 min. The supernatant was incubated with
Fura 2-AM (5 M) for 1 h. Human platelets were then prepared as described above. Finally, the external
×
µ
2+
2+
Ca concentration of the platelet suspensions was adjusted to 1 mM. The relative Ca mobilization
was measured as described previously [46].
4
.7. Immunoblotting
9
Washed platelets (1.2
×
10 cells/mL) were pre-incubated with 250 µM TQ-1and TQ-2 and
5
µ
M TQ-3 or 0.5% DMSO for 3 min, followed by the addition of collagen to trigger platelet
L of
g of protein were separated on a 12% acrylamide gel using
activation. The reaction was stopped and the platelets were immediately re-suspended in 200
a lysis buffer. Samples containing 80
µ
µ
sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the proteins were electro
transferred to the PVDF membranes by using a Bio-Rad semidry transfer unit (Hercules, CA, USA).
Blots were blocked with TBST (10 mM Tris-base, 100 mM NaCl and 0.01% Tween 20) containing 5% BSA
for 1 h and probed with various primary antibodies. The membranes were incubated with the HRP-linked
anti-mouse IgG or anti-rabbit IgG (diluted 1:3000 in TBST) for 1 h. Immunoreactive bands were detected
using an enhanced chemiluminescence system. Ratios of the semi quantitative results were obtained

Molecules 2018, 23, 477
12 of 15
by scanning the reactive bands and quantifying the optical density by using a video densitometer and
Bio-profil Biolight software, Version V2000.01 (Vilber Lourmat, Marne-la-Vallée, France).
4
.8. Statistical Analysis
The experimental results are expressed as the means
±
SEM and are accompanied by the number
of observations (n). Values of n refer to the number of experiments and each experiment was conducted
using different blood donors. The paired Student s t test was used to determine significant differences
'
in the occlusion time in mice. Differences between groups in other experiments were assessed using
an analysis of variance. When this analysis indicated significant differences among group means,
the groups were compared using the Student-Newman-Keuls method. p < 0.05 indicated statistical
significance. Statistical analyses were performed using SAS Version 9.2 (SAS Inc., Cary, NC, USA).
5
. Conclusions
This study reports that among the three compounds tested, TQ-3 has potently inhibited
platelet aggregation in vitro. The inhibitory effect of TQ-3 on platelet function was attributed to
the suppression of Syk-Lyn-Fyn cascade and subsequent destruction of Akt, JNK and p38 MAPKs
activation. These alterations reduce the level of ATP, surface P-selectin expression and [Ca2 i and
ultimately inhibit platelet aggregation. The increased hydrophobicity of TQ-3 containing electron
donating amino group with two phenyl group of the quinoline might be the reason for the noted
effects of TQ-3. Together with the numerous advantages of organometallic complexes, the results of
this study demonstrate the importance of using ruthenium-based organometallic complexes in the
development of novel anti-platelet agents for the prevention and treatment of thrombotic diseases.
+]
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1:
Absorption spectra of ligands L1–L3 in acetonitrile, Figure S2: Absorption spectra of complexes TQ1–TQ3
in acetonitrile Table S1: Crystallographic data and structure refinement parameters for TQ1–TQ3, Table S2:
◦
Selected bond lengths (Å) and angles ( ) for TQ1–TQ3.
Acknowledgments: This work was supported by grants from the Ministry of Science and Technology of
Taiwan (MOST 104-2622-B-038-003, MOST 104-2320-B-038-045-MY2, MOST 106-2320-B-038-012), Cathay General
Hospital–Taipei Medical University (104CGH-TMU-01-3), Chi Mei Medical Center, Taipei Medical University
(
105CM-TMU-07) and the University Grants Commission, India (MRP-MAJOR-CHEM-2013-5144; 69/2014 F.
No. 10-11/12UGC).
Author Contributions: Joen-Rong Sheu and Kao-Chang Lin conceived and designed the experiments. Chih-Hsuan
Hsia, Thanasekaran Jayakumar and Joen-Rong Sheu conducted the experiments. Thanasekaran Jayakumar
and Joen-Rong Sheu wrote the paper. Marappan Velusamy and Themmila Khamrang contributed to the
compounds synthesis and analysis. Shin-Yi Tsao, Marappan Velusamy, Chih-Wei Hsia, Duen-Suey Chou,
Chao-Chien Chang and Chi-Li Chung contributed interpretations and assistance on the manuscript. Chih-Hsuan
Hsia, Thanasekaran Jayakumar and Joen-Rong Sheu contributed equally to this work. All authors contributed
clarifications and guidance on the manuscript. All authors were involved in editing the manuscript. All authors
read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
2
3
4
.
.
.
.
Davi, G.; Patrono, C. Platelet activation and atherothrombosis. N. Engl. J. Med. 2007, 357, 2482–2494.
CrossRef] [PubMed]
Gibbins, J.M. Platelet adhesion signalling and the regulation of thrombus formation. J. Cell Sci.
004, 117, 3415–3425. [CrossRef] [PubMed]
Antithrombotic Trialists’ Collaboration. Collaborative meta-analysis of randomised trials of antiplatelet
therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ 2002, 324, 71–86.
Barrett, N.E.; Holbrook, L.; Jones, S.; Kaiser, W.J.; Moraes, L.A.; Rana, R.; Sage, T.; Stanley, R.G.; Tucker, K.L.;
Wright, B.; et al. Future innovations in anti-platelet therapies. Br. J. Pharmacol. 2008, 154, 918–939. [CrossRef]
[
2
[PubMed]

Molecules 2018, 23, 477
13 of 15
5
6
7
8
.
.
.
.
Leung, C.H.; Lin, S.; Zhong, H.J.; Ma, D.L. Metal complexes as potential modulators of inflammatory and
autoimmune responses. Chem. Sci. 2015, 6, 871–884. [CrossRef] [PubMed]
Wang, X.; Wang, X.; Guo, Z. Functionalization of platinum complexes for biomedical applications.
Acc. Chem. Res. 2015, 48, 2622–2631. [CrossRef] [PubMed]
Feldman, D.R.; Bosl, G.J.; Sheinfeld, J.; Motzer, R.J. Medical treatment of advanced testicular cancer. JAMA
2
008, 299, 672–684. [CrossRef] [PubMed]
Dorcier, A.; Ang, W.H.; Bolano, S.; Gonsalvi, L.; Jeannerat, L.J.; Laurenczy, G.; Peruzzini, M.; Phillips, A.D.;
Zanobini, F.; Dyson, P.J. In Vitro Evaluation of rhodium and osmium RAPTA analogues: The case for
organometallic anticancer drugs not based on ruthenium. Organometallics 2006, 25, 4090–4096. [CrossRef]
Bergamo, A.; Sava, G. Ruthenium anticancer compounds: Myths and realities of the emerging metal-based
drugs. Dalton Trans. 2011, 40, 7817–7823. [CrossRef] [PubMed]
9
.
1
0. Lentz, F.; Drescher, A.; Lindauer, A.; Henke, M.; Hilger, R.A.; Hartinger, C.G.; Scheulen, M.E.; Dittrich, C.;
Keppler, B.K.; Jaehde, U. Central European Society for Anticancer Drug Research-EWIV. Pharmacokinetics of
a novel anticancer ruthenium complex (KP1019, FFC14A) in a phase I dose-escalation study. Anticancer Drugs
2
009, 20, 97–103. [CrossRef] [PubMed]
1
1. Scolaro, C.; Bergamo, A.; Brescacin, L.; Delfino, R.; Cocchietto, M.; Laurenczy, G.; Geldbach, T.J.; Sava, G.;
Dyson, P.J. In vitro and in vivo evaluation of ruthenium(II)-arene PTA c complexes. J. Med. Chem.
2
005, 48, 4161–4171. [CrossRef] [PubMed]
1
1
2. Strohfeldt, K.A. Essentials of Inorganic Chemistry; Wiley: Hoboken, NJ, USA, 2015.
3. Khamrang, T.; Hung, K.C.; Hsia, C.H.; Hsieh, C.Y.; Velusamy, M.; Jayakumar, T.; Sheu, J.R. Antiplatelet
activity of a newly synthesized novel ruthenium (II): A potential role for Akt/JNK signaling. Int. J. Mol. Sci.
2
017, 18, 916. [CrossRef] [PubMed]
1
1
4. Hsia, C.H.; Velusamy, M.; Sheu, J.R.; Khamrang, T.; Jayakumar, T.; Lu, W.J.; Lin, K.H.; Chang, C.C. A novel
ruthenium (II)-derived organometallic compound, TQ-6, potently inhibits platelet aggregation: Ex vivo and
in vivo studies. Sci. Rep. 2017, 7, 9556. [CrossRef] [PubMed]
5. Morris, R.E.; Aird, R.E.; Murdoch, P.S.; Chen, H.; Cummings, J.; Hughes, N.D.; Parsons, S.; Parkin, A.;
Boyd, G.; Jodrell, D.I.; Sadler, P.J. Inhibition of cancer cell growth by ruthenium(II) arene complexes.
J. Med. Chem. 2001, 44, 3616–3621. [CrossRef] [PubMed]
1
6. Türkmen, H.; Kani, I.; Çetinkaya, B. Transfer Hydrogenation of Aryl Ketones with Half-Sandwich Ru(II)
Complexes That Contain Chelating Diamines. Eur. J. Inorg. Chem. 2012, 2012, 4494–4499. [CrossRef]
7. Muthuramalingam, S.; Khamrang, T.; Velusamy, M.; Mayilmurugan, M. Catalytic fixation of atmospheric
carbon dioxide by copper(II) complexes of bidentate ligands. Dalton Trans. 2017, 46, 16065–16076. [CrossRef]
1
[
PubMed]
8. Tsolis, T.; Manos, M.J.; Karkabounas, S.; Zelovitis, I.; Garoufis, A. Synthesis, X-ray structure determination,
cytotoxicity and interactions with 9-methylguanine, of ruthenium(II) 6-arene complexes. J. Organomet. Chem.
014, 768, 1–9. [CrossRef]
1
η
2
1
2
2
2
9. Smith, J.B.; Selak, M.A.; Dangelmaier, C.; Daniei, J.L. Cytosolic calcium as a second messenger for
collagen-induced platelet responses. Biochem. J. 1992, 288, 925–929. [CrossRef] [PubMed]
0. Charo, I.F.; Feinman, R.D.; Detwiler, T.C. Inhibition of platelet secretion by an antagonist of intracellular
calcium. Biochem. Biophys. Res. Commun. 1976, 72, 1462–1467. [CrossRef]
1. von Hundelshausen, P.; Weber, C. Platelets as immune cells: Bridging inflammation and cardiovascular
disease. Circ. Res. 2007, 100, 27–40. [CrossRef] [PubMed]
2. Quek, L.S.; Pasquet, J.M.; Hers, I.; Cornall, R.; Knight, G.; Barnes, M.; Hibbs, M.L.; Dunn, A.R.; Lowell, C.A.;
Watson, S.P. Fyn and Lyn phosphorylate the Fc receptor gamma chain downstream of glycoprotein VI in
murine platelets, and Lyn regulates a novel feedback pathway. Blood 2000, 96, 4246–4253. [PubMed]
3. Guidetti, G.F.; Lova, P.; Bernardi, B.; Campus, F.; Baldanzi, G.; Graziani, A.; Balduini, C.; Torti, M.
The Gi-coupled P2Y12 receptor regulates diacylglycerol-mediated signaling in human platelets. J. Biol. Chem.
2
2
008, 283, 28795–28805. [CrossRef] [PubMed]
4. Flaumenhaft, R. Molecular basis of platelet granule secretion. Arterioscler. Thromb. Vasc. Biol. 2003, 23, 1152–1160.
CrossRef] [PubMed]
2
2
[
5. Mahaut-Smith, M.P.; Ennion, S.J.; Rolf, M.G.; Evans, R.J. ADP is not an agonist at P2X(1) receptors: Evidence
for separate receptors stimulated by ATP and ADP on human platelets. Br. J. Pharmacol. 2000, 131, 108–114.
[CrossRef] [PubMed]

Molecules 2018, 23, 477
14 of 15
2
2
2
2
3
6. Thomas, S.M.; Brugge, J.S. Cellular functions regulated by Src family kinases. Annu. Rev. Cell Dev. Biol.
997, 13, 513–609. [CrossRef] [PubMed]
7. Boggon, T.J.; Eck, M.J. Structure and regulation of Src family kinases. Oncogene 2004, 23, 7918–7927.
CrossRef] [PubMed]
8. Stenberg, P.E.; Pestina, T.I.; Barrie, R.J.; Jackson, C.W. The Src family kinases, Fgr, Fyn, Lck, and Lyn,
colocalize with coated membranes in platelets. Blood 1997, 89, 2384–2393. [PubMed]
9. Reddy, K.B.; Smith, D.M.; Plow, E.F. Analysis of Fyn function in hemostasis and alphaIIbbeta3-integrin
signaling. J. Cell Sci. 2008, 121, 1641–1648. [CrossRef] [PubMed]
0. Yin, H.; Liu, J.; Li, Z.; Berndt, M.C.; Lowell, C.A.; Du, X. Src family tyrosine kinase Lyn mediates
VWF/GPIb-IX-induced platelet activation via the cGMP signaling pathway. Blood 2008, 112, 1139–1146.
1
[
[CrossRef] [PubMed]
3
3
3
3
1. Kim, S.; Jin, J.; Kunapuli, S.P. Relative contribution of G-protein-coupled pathways to protease-activated
receptor-mediated Akt phosphorylation in platelets. Blood 2006, 107, 947–954. [CrossRef] [PubMed]
2. Garcia, A.; Shankar, H.; Murugappan, S.; Kim, S.; Kunapuli, S.P. Regulation and functional consequences of
ADP receptor-mediated ERK2 activation in platelets. Biochem. J. 2007, 404, 299–308. [CrossRef] [PubMed]
3. Jackson, S.P.; Yap, C.L.; Anderson, K.E. Phosphoinositide 3-kinases and the regulation of platelet function.
Biochem. Soc. Trans. 2004, 32, 387–392. [CrossRef] [PubMed]
4. Deb, T.B.; Coticchia, C.M.; Dickson, R.B. Calmodulin-mediated activation of Akt regulates survival of
c-Myc-overexpressing mouse mammary carcinoma cells. J. Biol. Chem. 2004, 279, 38903–38911. [CrossRef]
[PubMed]
3
3
3
5. Chen, J.; De, S.; Damron, D.S.; Chen, W.S.; Hay, N.; Byzova, T.V. Impaired platelet responses to thrombin and
collagen in AKT-1-deficient mice. Blood 2004, 104, 1703–1710. [CrossRef] [PubMed]
6. Adam, F.; Kauskot, A.; Nurden, P.; Sulpice, E.; Hoylaerts, M.F.; Davis, R.J.; Rosa, J.P.; Bryckaert, M. Platelet
JNK1 is involved in secretion and thrombus formation. Blood 2010, 115, 4083–4092. [CrossRef] [PubMed]
7. Chang, L.; Karin, M. Mammalian MAP kinase signalling cascades. Nature 2001, 410, 37–40. [CrossRef]
[PubMed]
3
8. Page, S. Ruthenium compounds as anticancer agents. Educ. Chem. 2012, 49, 26–29.
3
9. Manso, A.; Escudero, C.; Alijo, M.; López-Fandiño, R. Platelet aggregation inhibitory activity of bovine, ovine,
and caprine kappa-casein macropeptides and their tryptic hydrolysates. J. Food Prot. 2002, 65, 1992–1996.
[CrossRef] [PubMed]
4
4
0. Alevriadou, B.R.; McIntire, L.V.; Lasslo, A. Inhibition of platelet adhesion and thrombus formation on
a collagen-coated surface by novel carbamoylpiperidine antiplatelet agents. Biochim. Biophys. Acta.–Mol.
Cell Res. 1992, 1137, 279–286. [CrossRef]
1. Li, A.H.; Ahmed, E.; Chen, X.; Cox, M.; Crew, A.P.; Dong, H.O.; Jin, M.; Ma, L.; Panicker, B.; Siu, K.W.;
et al. A highly effective one-pot synthesis of quinolines from o-nitroarylcarbaldehydes. Org. Biomol. Chem.
2
007, 5, 61–64. [CrossRef] [PubMed]
4
2. Bennelt, M.A.; Smith, A.K. Arene ruthenium(II) complexes formed by dehydrogenation of cyclohexadienes
with ruthenium(III) trichloride. J. Chem. Soc. Dalton Trans. 1974, 0, 233–241. [CrossRef]
4
3. Bugarcic, T.; Habtemariam, A.; Deeth, R.J.; Fabbiani, F.P.A.; Parsons, S.; Sadler, P.J. Ruthenium(II) arene
anticancer complexes with redox-active diamine ligands. Inorg. Chem. 2009, 48, 9444–9453. [CrossRef]
[PubMed]
4
4. Sheu, J.R.; Lee, C.R.; Lin, C.H.; Hsiao, G.; Ko, W.C.; Chen, Y.C.; Yen, M.H. Mechanisms involved in the
antiplatelet activity of Staphylococcus aureus lipoteichoic acid in human platelets. Thromb. Haemost. 2000, 83,
7
77–784. [PubMed]

Molecules 2018, 23, 477
15 of 15
4
4
5. Lin, K.H.; Kuo, J.R.; Lu, W.J.; Chung, C.L.; Chou, D.S.; Huang, S.Y.; Lee, H.C.; Sheu, J.R. Hinokitiol inhibits
platelet activation ex vivo and thrombus formation in vivo. Biochem. Pharmacol. 2013, 85, 1478–1485.
CrossRef] [PubMed]
[
6. Hsiao, G.; Lin, K.H.; Chang, Y.; Chen, T.L.; Tzu, N.H.; Chou, D.S.; Sheu, J.R. Protective mechanisms of inosine
in platelet activation and cerebral ischemic damage. Arterioscler. Thromb. Vasc. Biol. 2005, 2, 1998–2004.
[CrossRef] [PubMed]
Sample Availability: Samples of the compounds TQ-1, TQ-2 and TQ-3 are available from the authors.
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