Molecular requirements for the expression of antiplatelet effects by synthetic structural optimized analogues of the anticancer drugs imatinib and nilotinib

Article abstract of DOI:10.2147/DDDT.S211907

Background: Platelets play important roles in cancer progression and metastasis, as well as in cancer-associated thrombosis (CAT). Tyrosine kinases are implicated in several intracellular signaling pathways involved in tumor biology, thus tyrosine kinase inhibitors (TKIs) represent an important class of anticancer drugs, based on the concept of targeted therapy. Purpose: The objective of this study is the design and synthesis of analogues of the TKIs imatinib and nilotinib in order to develop tyrosine kinase inhibitors, by investigating their molecular requirements, which would express antiplatelet properties. Methods: Based on a recently described by us improved approach in the preparation of imatinib and/or nilotinib analogues, we designed and synthesized in five-step reaction sequences, 8 analogues of imatinib (I–IV), nilotinib (V, VI) and imatinib/nilotinib (VII, VIII). Their inhibitory effects on platelet aggregation and P-selectin membrane expression induced by arachidonic acid (AA), adenosine diphosphate (ADP) and thrombin receptor activating peptide-6 (TRAP-6), in vitro, were studied. Molecular docking studies and calculations were also performed. Results: The novel analogues V–VIII were well established with the aid of spectroscopic methods. Imatinib and nilotinib inhibited AA-induced platelet aggregation, exhibiting IC₅₀ values of 13.30 μΜ and 3.91 μΜ, respectively. Analogues I and II exhibited an improved inhibitory activity compared with imatinib. Among the nilotinib analogues, V exhibited a 9-fold higher activity than nilotinib. All compounds were less efficient in inhibiting platelet aggregation towards ADP and TRAP-6. Similar results were obtained for the membrane expression of P-selectin. Molecular docking studies showed that the improved antiplatelet activity of nilotinib analogue V is primarily attributed to the number and the strength of hydrogen bonds. Conclusion: Our results show that there is considerable potential to develop synthetic analogues of imatinib and nilotinib, as TKIs with antiplatelet properties and therefore being suitable to target cancer progression and metastasis, as well as CAT by inhibiting platelet activation.

Full text of DOI:10.2147/DDDT.S211907

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O R I G I N A L R E S E A R C H
Molecular Requirements for the Expression of
Antiplatelet Effects by Synthetic Structural
Optimized Analogues of the Anticancer Drugs
Imatinib and Nilotinib
This article was published in the following Dove Press journal:
Drug Design, Development and Therapy
1
Background: Platelets play important roles in cancer progression and metastasis, as well as
Despoina Pantazi
Nikoleta Ntemou2
in cancer-associated thrombosis (CAT). Tyrosine kinases are implicated in several intracel-
Alexios Brentas2
lular signaling pathways involved in tumor biology, thus tyrosine kinase inhibitors (TKIs)
Dimitrios Alivertis3
represent an important class of anticancer drugs, based on the concept of targeted therapy.
Purpose: The objective of this study is the design and synthesis of analogues of the TKIs
Konstantinos Skobridis2
imatinib and nilotinib in order to develop tyrosine kinase inhibitors, by investigating their
Alexandros D Tselepis1
molecular requirements, which would express antiplatelet properties.
1Department of Chemistry,
Methods: Based on a recently described by us improved approach in the preparation of
Atherothrombosis Research Centre,
Laboratory of Biochemistry, University of
imatinib and/or nilotinib analogues, we designed and synthesized in five-step reaction
Ioannina, Ioannina 45110, Greece;
2Department of Chemistry, Section of
Organic Chemistry and Biochemistry,
University of Ioannina, Ioannina 45110,
Greece; 3Department of Biological
Applications and Technology, University
of Ioannina, Ioannina 45110, Greece
sequences, 8 analogues of imatinib (I–IV), nilotinib (V, VI) and imatinib/nilotinib (VII,
VIII). Their inhibitory effects on platelet aggregation and P-selectin membrane expression
induced by arachidonic acid (AA), adenosine diphosphate (ADP) and thrombin receptor
activating peptide-6 (TRAP-6), in vitro, were studied. Molecular docking studies and
calculations were also performed.
Results: The novel analogues V–VIII were well established with the aid of spectroscopic
methods. Imatinib and nilotinib inhibited AA-induced platelet aggregation, exhibiting IC50
values of 13.30 μΜ and 3.91 μΜ, respectively. Analogues I and II exhibited an improved
inhibitory activity compared with imatinib. Among the nilotinib analogues, V exhibited
a 9-fold higher activity than nilotinib. All compounds were less efficient in inhibiting platelet
aggregation towards ADP and TRAP-6. Similar results were obtained for the membrane
expression of P-selectin. Molecular docking studies showed that the improved antiplatelet
activity of nilotinib analogue V is primarily attributed to the number and the strength of
hydrogen bonds.
Conclusion: Our results show that there is considerable potential to develop synthetic
analogues of imatinib and nilotinib, as TKIs with antiplatelet properties and therefore
being suitable to target cancer progression and metastasis, as well as CAT by inhibiting
platelet activation.
Keywords: cancer, kinase inhibitors, platelets, synthesis, thrombosis
Correspondence: Konstantinos Skobridis
Department of Chemistry, Section of
Organic Chemistry and Biochemistry,
University of Ioannina, Ioannina 45110,
Greece
Tel + 30 26510 08598
Fax + 30 26510 08682
Introduction
Platelets play a crucial role in hemostasis having as a major function to prevent
bleeding and reduce blood loss following vascular injury.1 Platelets are also key
regulators of thrombotic complications in various disease states including cancer.2–5
Email kskobrid@uoi.gr
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http://doi.org/10.2147/DDDT.S211907

Pantazi et al
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Indeed, many studies have demonstrated that the develop-
Tyrosine kinases play important roles in the intracellu-
ment of malignancy is associated with a plethora of altera- lar signaling pathways. At present, around 40 drugs with
tions in the coagulation pathways, characterized by TKI activity have been approved by the FDA or are under
activation of clotting mechanisms to a different extent, investigation in clinical trials for anti-cancer
depending on the tumor type.3,6,7 The pathogenesis of treatment.24,25 Because of the comparable structure of the
thrombosis in cancer is multifactorial, including various catalytic pocket of tyrosine kinases, TKIs often target
platelet alterations, which may significantly contribute to multiple kinases that play a role in several different signal-
cancer-associated thrombosis (CAT).6–8 In this regard, ing pathways.26,27 Although the approved kinase inhibitors
P-selectin and platelet count are among the CAT risk are active against more than one type of cancer, however
factors identified and may also represent predictive bio- only a few of them have been used for the treatment of
markers of venous thromboembolism (VTE) in cancer non-oncological diseases.
patients.8,9
Platelets display high tyrosine kinase activities in com-
Besides their important role in coagulation, platelets parison to other cell types and tyrosine phosphorylation is
may significantly contribute to inflammation, cancer pro- one of the key signal transduction mechanisms of platelet
gression, metastasis and angiogenesis.10–12 Activated pla- activation.28 Therefore, it is expected that several TKIs used
telets secrete an array of biologically active molecules by to treat malignancies may interfere with platelet activation
which they can modulate tumor growth and processes thus exhibiting off-target antiplatelet effects.26,29,30
metastasis.10,11,13 Within the blood compartment, tumor
The ATP antagonists/mimetics imatinib and nilotinib
cells bind to platelets, thus escaping natural killer cell- represent privileged scaffolds in drug discovery. The non-
mediated cytotoxicity.11,14,15 Therefore, tumor cell adhe- receptor tyrosine kinase c-Src is involved in the intracel-
sion to platelets is a crucial step for tumor cell survival lular signaling pathways leading to platelet activation and
within the blood circulation.11,16,17 An important role in therefore represents a potential target for developing new
platelet adhesion to cancer cells is played by their antiplatelet therapies.31 Upon platelet activation, multiple
P-selectin which binds to primarily to the receptor intracellular proteins become phosphorylated at tyrosine
PSGL-1 (P-selectin glycoprotein ligand 1).18 Platelets residues.32 This process is mediated by at least four
can also promote an epithelial–mesenchymal transition in families of tyrosine kinases, Src, Syk, FAK, and JAK.33
cancer cells, an essential step in cancer invasion and Src-family kinases (SFKs) play a central role in mediating
metastasis.19
the rapid response of platelets to vascular injury, transmit-
Given the crucial roles of platelets in cancer progres- ting activation signals from a diverse repertoire of platelet
sion and metastasis, as well as in CAT, inhibiting platelet surface receptors.33
activation is considered as a promising strategy to reduce
Moreover, the crystal structure of regulated Abl closely
both cancer progression and CAT.20,21 Among the antipla- resembles that seen in structures of regulated Src-family
telet drugs, the effect of low aspirin dose (around 75 mg/ kinases.34 Structural differences between the inactive con-
day) on cancer progression has attracted considerable formations of tyrosine kinase domains of Abl, identical in
interest.22,23 In this regard, population and clinical studies sequence with the kinase domain of Bcr-Abl, and its close
have demonstrated that aspirin significantly reduces the relative c-Src suggest why imatinib can only inhibit the
risk of colon cancer development and inhibits cancer kinase domain of Abl but not that of c-Src.35
growth and invasiveness.22,23 A number of clinical trials
are currently ongoing to evaluate the therapeutic effect of been established that insubstantial structural modifications
aspirin treatment in cancer patients.20,21
of the molecule/compound resulted in increased inhibitory
During optimization of the imatinib structure, it has
Therefore, to limit platelet activation in cancer patients and selectivity properties. In particular, it was shown that
is a relevant aspect to take into account when designing the introduction of only a “flag-methyl” at the imatinib
new therapeutic approaches to treat cancer. However, such phenyl ring C (Figure 1) would completely abolish the
approaches would have to be carefully designed to avoid inhibitory effect of imatinib against protein kinase
risk for bleedings, considering the key role of platelets in C alpha (PKCα), while the inhibitory effect against Bcr-
hemostasis. Nevertheless, the available evidence suggests Abl was retained or even enhanced.31 The dramatic loss of
that targeting platelets in cancers known to have a high activity against PKCα was explained by a forced change of
risk of thrombotic events is therapeutically beneficial.20–23 the preferred conformation of imatinib upon the
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Pantazi et al
introduction of this “flag-methyl” group.36 Furthermore, in of new antiplatelet agents.38 The synthetic analogues were
our previous study, we had established that the elimination designed in order to increase spatial flexibility and to explore
potentially favorable halogen bond interactions and strong
interactions of the nitro groups, incorporating at the final
phenyl ring, with residues typically positioned at the ATP-
binding pocket of the c-Src, especially with positively
of the N-methylpiperazine ring and the incorporation of
different groups at the final imatinib phenyl ring
D (Figure 1) had greater activity against other kinase
family members and poorer activity against Abl.37
In light of the above developments, we hypothesized that charged residues. To further explore the Structure–Activity
slight changes in the imatinib and nilotinib structure might
exert an inhibitory activity on these enzymes and therefore
screened the three-dimensional architecture of Src-family
Relationship (SAR), the modifications involve, except the
removal of the N-methylpiperazine group in ring D of ima-
tinib and the 4-methylimidazolyl group on the final phenyl
kinases (SFKs), which are involved in platelet activation, ring D of nilotinib and replacement of the amide function
and are considered as potential targets for the development with the urea (Figure 1).
Figure 1 Modifications of prepared imatinib and nilotinib derivatives. Upper panel left: Following removal of the piperazinyl ring of imatinib, modifications were made to the
final phenyl moiety as shown (I–IV). Upper panel right: Removal of the imidazolyl ring, modifications were made to the final phenyl ring (V, VI). Lower panel: Structure of
the imatinib or nilotinib analogues by replacement of the amide bond with the urea moiety (VII, VIII).
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In an effort to investigate the molecular requirements equiv, 0.39 mmol). After 30 min, a solution of 4-chloro-
for these drugs to influence the platelet functionality, we 3-nitroaniline (1.20 equiv, 0.39 mmol) in dry DMF (2 mL)
specifically focused on their effects on platelet activation was added. The mixture was stirred vigorously at room
induced by the platelet agonists, arachidonic acid (AA) temperature for 24 hrs and at 90°C for 3 hrs. The reaction
and adenosine diphosphate (ADP), which activate platelets mixture was cooled to room temperature, concentrated in
through the main pathways targeted by the established vacuo, and then portioned between water and CH2Cl2. The
antiplatelet drugs (aspirin and ADP receptor P2Y12
antagonists)39,40 as well by thrombin receptor activating
peptide-6 (TRAP-6), which activates platelets through pro-
tease-activated receptor-1 (PAR-1).41
aqueous layer was extracted with CH2Cl2, and the com-
bined organic layers were dried (Na2SO4). The solvent
was evaporated in vacuo and the residue was purified by
flash chromatography on silica gel (dichloromethane/
methanol 15:1) to give the product as a pale yellow solid
1
(36% yield). H NMR (400 MHz, DMSO-d6): δ 2.35 (s,
Materials and Methods
3H, CH3), 7.38 (d, J = 8.00 Hz, 1H), 7.44 (d, J = 8.00 Hz,
1H), 7.47–7.55 (m, 4H), 7.65 (d, J = 7.20 Hz, 1H), 7.75
(m, 1H), 8.08 (d, J = 8.80 Hz, 1H), 8.31 (s, 1H), 8.41–8.45
(m, 2H), 8.55 (t, J = 5.60 Hz, 1H), 8.60 (s, 1H), 8.69 (m,
2H), 9.05 (s, 1H), 9.15 (s, 1H), 9.27 (s, 1H), 9.31 (s, 1H),
10.67 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 18.14,
107.87, 108.06, 116.49, 123.48, 123.72, 124.19, 124.40,
124.52, 124.99, 130.31, 130.95, 131.77, 134.16, 137.73,
138.45, 139.26, 148.03, 151.36, 159.48, 161.00, 161.55,
167.31; HRMS (ESI): Calcd for C23H17ClN6O3 [M+H]+
m/z 461.1129, found [M+H]+ m/z 461.1127; Anal. calcd
for C23H17ClN6O3 (460.87): C 59.94, H 3.72, N 18.24,
found: C 59.71, H 3.56, N 18.31.
General
Solvents used were puriss, with the exception of THF,
which was purified by fresh distillation over Na/benzophe-
none. Commercially available reagents were used as
received, without any further purification. Nilotinib was
purchased from Selleck Chemicals, Munich, Germany.
Analytical TLC was performed on commercial Merck
silica gel 60 F254. Flash chromatography was carried out
using silica gel 60 (230–400 mesh). HPLC experiments
were performed using a Shimadzu system, consisting of
a DGU-20A controller, an LC-20AD pump, an SPD-
M20A photodiode-array detector and a CTO-10AS col-
umn oven. H and 13C NMR spectra were recorded on
1
a Brucker AMX (400/100 MHz 1H/13C) spectrometer
using tetramethyl silane (TMS) as an internal standard.
Chemical shifts are reported in ppm (δ) referenced to
TMS, coupling constants J in Hz. High-resolution ESI
mass spectra were measured on a Thermo Fisher
Scientific LTQ ORBITRAP/LC−MS system. Elemental
analyses were performed on a Heraeus CHN-Rapid
Analyser.
4-Methyl-N-(4-amino-3-trifluoromethyl)-3-[(4-pyridin-
3-ylpyrimidin-2-yl) amino]benzamide (VI)
To an ice-cold solution of acid 10 (1.00 equiv, 0.48 mmol)
in dry THF (5 mL) was added Et3N (0.5 mL) under argon,
followed by the addition of TBTU (1.10 equiv, 0.53
mmoL). After 30 min, a solution of 4-(trifluoromethyl)
benzene-1,3-diamine (1.20 equiv, 0.57 mmoL) in dry
THF (2 mL) was added. The reaction mixture was stirred
at 0°C for 30 min and at room temperature for 24 h. After
completion monitored by TLC (CH2Cl2/MeOH 15:1, v/v),
the mixture was concentrated in vacuo, and then portioned
between water and CH2Cl2. The aqueous layer was
extracted with CH2Cl2, and the combined organic layers
were dried (Na2SO4). The solvent was evaporated in vacuo
and the residue was purified by flash chromatography on
silica gel (CH2Cl2/MeOH 15:1, v/v) to give the product as
a pale yellow solid (93% yield). 1H NMR (400 MHz,
DMSO-d6): δ 2.33 (s, 3H, CH3), 6.82 (d, J = 8.80 Hz,
1H), 7.38 (d, J = 8.00 Hz, 1H), 7.46 (d, J = 5.20 Hz, 1H),
Chemistry
The synthesis of the intermediates 2, 4–6 and 8–10, as well
as of the final compounds, imatinib analogues I–IV, were
based on a recently described optimized approach in the
synthesis of imatinib intermediates and analogues and its
spectroscopic data are consistent with the reported ones.42
The experimental procedure for the final step of the target
compounds, V–VIII, and specific details are given below.
4-Methyl-N-(4-chloro-3-nitrophenyl)-3-[(4-pyridin-
3-ylpyrimidin-2-yl) amino]benzamide (V)
To an ice-cold solution of acid 10 (1.00 equiv, 0.326 7.50 (dd, J = 7.20, 4.80 Hz, 1H), 7.64 (d, J = 8.80 Hz, 1H),
mmol) in dry DMF (3 mL) was added DIPEA (0.6 mL) 7.69 (d, J = 7.60 Hz, 1H), 7.81 (s, 1H), 8.22 (s, 1H), 8.43
under argon, followed by the addition of HATU (1.20 (d, J = 8.00 Hz, 1H), 8.53 (d, J = 5.20 Hz, 1H), 8.68 (d,
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Pantazi et al
J = 4.80 Hz, 1H), 9.10 (s, 1H), 9.26 (s, 1H), 10.01 (s, 1H); were dried (Na2SO4). The solvent was evaporated in vacuo
13C NMR (100 MHz, DMSO-d6): δ 18.06, 107.73, 116.99, and the residue precipitated by the addition of 2–3 mL
118.16, 118.21, 123.25, 123.69, 124.11, 126.21, 127.84, CH2Cl2. The precipitated product was filtered off and
130.09, 132.04, 132.47, 134.17, 135.96, 137.88, 142.45, dried to yield a first crop of the product. A second crop
148.04, 151.34, 159.46, 160.99, 161.49, 164.54; HRMS of product was obtained from the filtrate, which was con-
(ESI): Calcd for C24H19F3N6O [M+H]+ m/z 465.1651, centrated in vacuo and the residue purified by flash chro-
found [M+H]+ m/z 465.1647; Anal. calcd for C24H19F3N6 matography on silica gel (CH2Cl2/MeOH 20:1) to give the
1
O (464.4425): C 62.07, H 4.12, N 18.09, found: C 62.22, product as a pale yellow solid (71% total yield). H NMR
H 4.03, N 18.31.
(400 MHz, DMSO-d6): δ 2.21 (s, 3H, CH3), 7.15 (m, 2H),
7.44 (d, J = 5.20 Hz, 1H), 7.52 (m, 1H), 7.61–7.66 (m,
2H), 7.89 (d, J = 1.20 Hz, 1H), 8.35 (d, J = 2.4 Hz, 1H),
8.50 (dt, J = 8.20, 2.40 Hz,1H), 8.52 (d, J = 5.20 Hz, 1H),
8.68 (dd, J = 4.80, 1.60 Hz, 1H), 8.87 (s, 1H), 8.93 (s, 1H),
9.32 (t, J = 2.00, 1H); 13C NMR (100 MHz, DMSO-d6): δ
25.06, 115.22, 121.68, 122.36, 122.52, 123.92, 130.50,
131.30, 133.25, 137.82, 139.24, 139.72, 141.98, 144.57,
145.55, 147.59, 155.15, 155.81, 158.92, 159.85, 167.01,
168.55, 169.16; HRMS (ESI): Calcd for C23H18ClN7O3
[M+H]+ m/z 476.1238, found [M+H]+ m/z 476.1262; Anal.
calcd for C23H18ClN7O3 (475.8871): C 58.05, H 3.81,
N 20.60, found: C 58.27, H 3.66, N 20.52.
1-(4-Methyl-3-[(4-pyridin-3-yl)pyrimidin-2-ylamino)
phenyl]-3-(Phenyl)urea (VII)
In a solution of amine 6 (1.00 equiv, 0.25 mmol) and Et3
N (0.5 mL) in dry CH2Cl2 (10 mL) solution of phenyl
isocyanate (1.10 equiv, 0.28 mmol) in dry CH2Cl2 (5 mL)
was added dropwise under stirring at 5°C. The resulting
mixture was stirred at 5°C for 30 mins and at room
temperature for 24 hrs. The mixture was concentrated in
vacuo, and then portioned between water and CH2Cl2. The
aqueous layer was extracted with CH2Cl2, and the com-
bined organic layers were dried (Na2SO4). The solvent
was evaporated in vacuo and the residue was purified by
flash chromatography on silica gel (CH2Cl2/MeOH 10:1)
to give the product as a pale yellow solid (78% yield).
1H NMR (400 MHz, DMSO-d6): δ 2.20 (s, 3H, CH3), 6.96
(d, J = 7.60 Hz, 1H), 7.14 (s, 2H), 7.27 (t, J = 7.60 Hz,
2H), 7.44 (m, 3H), 7.52 (m, 1H), 7.80 (s, 1H), 8.49 (m,
1H), 8.52 (d, J = 5.20 Hz, 1H), 8.60 (s, 1H), 8.61 (s, 1H),
8.70 (dd, J = 4.80, 1.20 Hz, 1H), 8.88 (s, 1H), 9.29 (d, J =
5.20 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ 25.04,
115.12, 122.06, 122.30, 125.67, 129.25, 131.33, 132.88,
136.30, 137.79, 139.79, 141.99, 145.17, 145.49, 147.34,
155.74, 158.93, 160.09, 166.99, 168.64, 169.15; HRMS
(ESI): Calcd for C23H20N6O [M+H]+ m/z 397.1777, found
[M+H]+ m/z 397.1757; Anal. calcd for C23H20N6
O (396.4445): C 69.68, H 5.08, N 21.20, found: C 69.81,
H 5.02, N 21.33.
Biological Assays
Platelet Aggregation Studies
Platelet aggregation studies were performed in platelet-
rich plasma (PRP) prepared from peripheral venous
blood of apparently healthy normolipidemic volunteers,
using as anticoagulant citric acid solution (ACD) as we
have previously described.43 The study protocol was
approved by the ethics committee of the University
Hospital of Ioannina and adheres to the principles of the
declaration of Helsinki. All participants gave their written
informed consent before peripheral venous blood was
drawn. Isolated platelets in PRP were enumerated and
their number was adjusted to 250×106/mL with homolo-
gous platelet-poor plasma (PPP). Platelets were pre-
incubated at 37°C with various concentrations of imatinib,
nilotinib and their synthetic analogues solubilized in
DMSO (or its vehicle; DMSO, considering as control)
1-(4-Methyl-3-[(4-pyridin-3-yl)pyrimidin-2-ylamino)
phenyl]-3-(4-chloro-3-nitrophenyl)urea (VIII)
To a stirred solution of amine 6 (1.00 equiv, 0.25 mmol) for 1 min or 5 min. The maximum concentration of each
and Et3N (0.7 mL) in dry THF (10 mL) was added tested compound was 100 μΜ. Light transmittance aggre-
a solution of 4-chloro-3-nitrophenyl isocyanate (1.10 gometry (LTA) was performed in 0.5 mL of PRP in the
equiv, 0.28 mmoL) in dry THF (5 mL) dropwise with presence of the platelet agonists AA (500 μM), ADP (5
stirring at 5°C. The resulting mixture was stirred at 5°C μΜ) and TRAP-6 (10 μΜ) in an aggregometer (Model
for 30 mins and at room temperature for 24 hrs. The 700-4DR, Chrono-Log, Havertown, PA, USA), using
mixture was concentrated in vacuo, and then portioned 0.5 mL of corresponding platelet-poor plasma (PPP) as
between water and AcOEt. The aqueous layer was a blank. In all studies, the concentration of DMSO added
extracted with AcOEt, and the combined organic layers in PRP was 0.5% (v/v).44 Aggregation was monitored with
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the use of the software AggroLink (ver. 8.0). The inhibi- t-test was performed using the Statistical Package for the
tory efficacy of imatinib, nilotinib and their synthetic Social Sciences (SPSS), version 25 (IBM). A p-value of
analogues was expressed as IC50 values, in μΜ (the con-
centration that induces 50% inhibition of platelet aggrega-
tion). In parallel experiments, we incubated platelets with
imatinib, nilotinib and related synthetic analogues at
a concentration of 100 μΜ for up to 10 min at 37°C to
investigate the possible existence of agonistic activity. All
aggregation assays were conducted within 3 h after blood
venipuncture.43,45
<0.05 was considered significant.
Results and Discussion
Chemistry
The synthesis of imatinib and all imatinib (I-IV), nilotinib
(V, VI) and imatinib/nilotinib (VII, VIII) analogues
(Figure 1), as mentioned above, were based on a recently
described by us improved and efficiently optimized
approach in the preparation of imatinib and/or nilotinib
analogues.42 The imatinib analogues, compounds I-IV, and
the imatinib/nilotinib analogues, compounds VII and VIII,
containing the urea moiety, were prepared as shown in
Scheme 1. The nilotinib analogues, compounds V and
VI, were prepared as shown in Scheme 2. Briefly, 3-acetyl
pyridine 1 was converted into the corresponding enami-
none 2 by the use of N,N-dimethylformamide-diethyl
acetal which was subsequently reacted with N-(2-methyl-
5-nitrophenyl)-guanidinium hydrochloride 4, prepared
from aniline hydrochloride 3 with excess of molten cya-
namide, to the corresponding phenylamino-pyrimidine 5.
Catalytic hydrogenation of the nitro group of compound 5
in the presence of PtO2 (Adam’s catalyst) led to the corre-
sponding aniline 6 quantitatively, which, subsequently,
was coupled with the appropriate benzoyl chloride to
afford the desired imatinib analogues I-IV (Scheme 1).
The novel imatinib or nilotinib analogues VII and VIII,
N,N’-diphenyl urea derivatives, were prepared in high
yields and high purity from the pharmacophore 6 and the
appropriate phenylisocyanate in THF or CH2Cl2 under
basic conditions (Scheme 1).
P-Selectin Membrane Expression
The surface expression of P-selectin was studied in
a FACSCalibur flow cytometer (Becton-Dickinson, San
Jose, CA, USA) using the fluorescently labeled monoclo-
nal antibody (anti-CD62P-PE) as we previously described
with slight modifications.45 Platelets in PRP were incu-
bated for 5 min at 37°C with imatinib, nilotinib or their
synthetic analogues at various concentrations up to 100
μΜ. Platelets were then activated with AA (800 μΜ), ADP
(50 μΜ), or TRAP-6 (50 μΜ) for 5 min at 37°C without
stirring. After incubation, a small portion of the reaction
mixture was incubated with the fluorescently labeled
monoclonal antibody towards P-selectin, anti-CD62P-PE,
for 20 min at room temperature in the dark, diluted 1:5 (v/
v) with 10 mM PBS, pH=7.4 and immediately analyzed by
flow cytometry, as previously described.45,46 Platelets were
gated according to staining for the platelet antigen CD61,
using the fluorescently labeled monoclonal antibody anti-
CD61-PerCP. Analyses included the percentage of positive
events facilitated by evaluating the mean fluorescence
intensity (MFI) values.45,46
Following the above-described route, we prepared the
nilotinib analogues V and VI. The intermediate key com-
pound phenylaminopyrimidine 9, analogue to the previous
imatinib intermediate, bearing the ethyl ester group instead of
the nitro group, was prepared by the reaction of the enam-
inone 2 and the corresponding guanidinium hydrochloride 8.
Alkaline hydrolysis of the carboxyl ester 9 with our protocol
Molecular Docking
The X-ray structure of tyrosine kinase protein c-Src was
obtained from the RSCB Protein Data Bank (PDB ID:
1Υ57). We used the known c-Src kinase domains to
model the synthetic drug complexes through flexible dock-
ing calculations. Before docking, the inhibitors were opti-
mized and docked onto the active site of enzyme used. The
docking calculations were performed using discovery stu- in non-aqueous conditions, under very mild conditions, such
dio visualizer 2016 and Chimera 1.11c. with imatinib, as short time, room temperature and low concentration of
nilotinib and their synthetic analogues in the c-Src kinase alkali, by the use of dichloromethane/methanol (9:1, v/v) as
solvent47 was then performed to produce the corresponding
domain (5 dockings for each complex).
carboxylic acid 10 which subsequently coupled with the
aniline fragment 4-chloro-3-nitroaniline, using 1-[Bis
Statistics
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridi-
All data are presented as mean SD for at least three
independent experiments. Independent–samples Student’s nium-3-oxide hexafluorophosphate (HATU) as coupling
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Pantazi et al
Scheme 1 Reactions and conditions for the synthesis of the imatinib I–IV and of the imatinib/nilotinib analogues VII and VIII.
agent and hydroxybenzotriazole (HOBt) as an additive well as by high-resolution mass spectra and elemental ana-
lysis (Supplementary materials).
in DMF in the presence of N,N-diisopropylethylamine
(DIPEA), to give the nilotinib analogue V. The coupling of
carboxylic acid 10 with 4-(trifluoromethyl)benzene-1,3-dia-
mine, using 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyla-
minium tetrafluoroborate (TBTU) in THF in the presence of
triethylamine (Et3N) proceeded readily and afforded the cor-
responding analogue VI in high yields (Scheme 2).
Biological Assays
Effect on Human Platelet Aggregation in PRP
All compounds were subsequently screened for their effect
on human platelet aggregation induced by 3 different
agonists, AA, ADP and TRAP-6. In preliminary experi-
ments, we evaluated whether any of the studied com-
pounds exhibited a platelet aggregating activity. None of
these compounds at a concentration of 100 μΜ exhibited
any agonistic effect (data not shown). The inhibitory effect
All the final compounds were purified by flash chromato-
graphy on a silica gel column. The purity of these synthetic
analogues ranged from 99.0% to 99.5% as estimated by RP-
HPLC and 1H NMR. The novel analogues V-VIII, reported
herein, were well established by their 1H NMR, 13C NMR, as of imatinib/nilotinib and their synthetic analogues were
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Scheme 2 Reactions and conditions for the synthesis of the nilotinib analogues V and VI.
subsequently studied on platelet aggregation. Among the 3 1 min with PRP instead of 5 min, before the initiation of
agonists evaluated, the strongest dose-dependent inhibitory the aggregation with AA, the compounds I to IV, VI and
effect of imatinib and nilotinib (incubated for 5 min in the VII being inactive (Table 1). This suggests that the
aggregometer at 37°C, under stirring), was observed
towards AA-induced platelet aggregation, exhibiting IC50
Table 1 The Inhibitory Effect of Imatinib, Nilotinib and Their
values of 13.30 μΜ and 3.91 μΜ, respectively, indicating
that nilotinib is about 4-fold more active than imatinib
(Table 1). Among the imatinib synthetic analogues, com-
pounds I and II exhibited an approximately 3- and 2-fold
higher inhibitory activity, respectively, whereas com-
pounds III and IV were significantly less active compared
with imatinib (Table 1). Among the nilotinib analogues,
compound V exhibited a 9-fold higher activity than niloti-
nib, being the most active among all compounds evalu-
ated, whereas VI exhibited a slightly higher inhibitory
activity compared with nilotinib (Table 1). Finally, the
imatinib/nilotinib analogue VII was a very weak inhibitor,
whereas VIII exhibited an inhibitory activity similar to
that of nilotinib (Table 1). The inhibitory activity of ima-
tinib, nilotinib and their analogues was significantly
reduced when these compounds were incubated for
Synthetic Analogues on AA-Induced Platelet Aggregation in PRP
Tyrosine
Kinase
IC50-Values (μΜ) (5 IC50-Values (μΜ) (1
min Incubation)
min Incubation)
Inhibitors
Imatinib
13.30
4.30
21.71
n.d.
I
II
6.88
n.d.
III
IV
30.39
23.77
n.d.
n.d.
Nilotinib
3.91
0.41
4.67
31.90
3.47
11.75
2.94
n.d.
V
VI
VII
VIII
n.d.
n.d.
Notes: Platelet aggregation in PRP was performed in the presence of 500 μΜ AA.
IC50 values (concentrations that induce 50% inhibition of platelet aggregation) are
the mean from at least 3 different experiments.
Abbreviations: AA, Arachidonic Acid; n.d., not determined; PRP, Platelet-Rich Plasma.
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Pantazi et al
inhibitory effect of all compounds is time dependent. All Leu273 (one hydrogen bond), as well as van de Waals,
compounds tested towards ADP- and TRAP-6 were much carbon-hydrogen bonds (C-H bonds), pi-alkyl, pi-sigma
less efficient in inhibiting platelet aggregation compared and alkyl with Gly276, Gly274, Val281, Lys295, Tyr340,
with their effect towards AA (Figure 2A). Representative Leu293, Ala293 and Glu339 with a docking score of –
aggregation curves illustrating the dose-dependent inhibi- 12.80 kcal/mol.
tory effect of the nilotinib analogue V on AA-, ADP- and
The docking pose of the more active nilotinib analogue
TRAP-6-induced platelet aggregation are presented in V is shown in Figure 4D. Important hydrogen-bonding and
Figure 2B. Finally, no inhibitory effect of all compounds hydrophobic interactions with the c-Src have been identi-
tested was observed towards platelet aggregation induced fied for the analogue V. The presence of the NO2 group at
by ADP or TRAP-6 when they were incubated for 1 min the final phenyl ring at the 3-position was a decisive part
with PRP instead of 5 min prior to platelet activation (data of the pharmacophore due to formation of strong interac-
not shown).
tions. On the contrary, it develops two pairs of hydrogen
bonds with the residues Asp404 and Met341, two and one
more hydrogen bonds than those of imatinib and nilotinib,
respectively. Furthermore, compound V develops van de
Waals, carbon-hydrogen bonds (C-H bonds), pi-alkyl and
alkyl bonding interactions with Leu273, Tyr340, Leu293,
Ala293, Glu339, Lys295 and Val281 with a docking score
of – 14.30 kcal/mol, indicating high binding affinity
(Figure 4D).
Ιnhibition of P-Selectin Exposure
The effect of imatinib, nilotinib and their synthetic analo-
gues on platelet secretion, were also studied by determin-
ing the membrane expression of P-selectin on platelets
activated by AA, ADP and TRAP-6. The results obtained
were similar to those of the aggregation experiments,
indicating that among the 3 agonists evaluated, the stron-
gest inhibitory effect of imatinib, nilotinib and analogues,
was observed towards AA-induced platelet aggregation.
Furthermore, nilotinib was more active than imatinib,
whereas among the synthetic compounds tested the most
active was the nilotinib analogue V. Figure 3A illustrates
the inhibitory effect of imatinib, nilotinib and the most
active analogue V. Compounds tested towards ADP and
TRAP-6 were much less efficient in inhibiting P-selectin
membrane expression compared with their effect towards
AA, with some compounds being inactive. Figure 3B
and C illustrate the effects of imatinib, nilotinib and com-
pound V.
According to our results, the parent compounds imati-
nib and nilotinib and all their synthetic analogues inhibited
platelet aggregation and secretion induced by AA, exhibit-
ing a wide variability in their potency, the most active
compound being V. Although clear structure relationships
cannot conclusively be delineated, the improvements of
3.1 kcal/mol and 1.5 kcal/mol compared to imatinib and
nilotinib, respectively, based on the docking score, suggest
that the above variability could be at least partially attrib-
uted to the number and the strength of hydrogen bonds.
The nitro oxygens of the nitro moiety of V, as strong
proton acceptors, are involved in two additional hydrogen
bonds with the Asp404 side chain residue of c-Src
(Figure 4D), explaining probably its higher inhibitory
Docking Calculations
The superposition of the model of the V-c-Src (in green) activity.
and the structures of nilotinib (in brown), imatinib (in pink)
In addition, we tested how the replacement of the
at the active site of the kinase c-Src are illustrated in amide bond with the urea bond (analogues VII and VIII)
Figure 4A. The differences are focused on the final phenyl and the substituents at the final benzene ring affects plate-
moiety. Imatinib docked at the active site of the kinase c-Src let activation. Absence of the substituents at the final
(Figure 4B) develops hydrogen-bonding interactions with benzene ring (3-NO2, 4-Cl) resulted in a 9-fold weaker
the residues Met341, via a pair of hydrogen bonds, as well antiplatelet activity of VII compared with analogue VIII,
as van de Waals, carbon-hydrogen bonds (C-H bonds), pi- making the impact of these substituents on antiplatelet
alkyl and alkyl with Asp386, Asp404, Gly276, Gly274, potency apparent.
Leu273, Val281, Lys295, Leu393 and Glu339. The docking
However, we should highlight that the nilotinib analogue
score (maestro glide) is – 11.22 kcal/mol. As shown in V has the same molecular scaffold and the same substituents
Figure 4C, nilotinib docked at the active site of the c-Src, at the same positions at the final benzene ring, namely 3-NO2
developing hydrogen-bonding interactions with the residues and 4-Cl, with the much less (approximately 70-fold) active
Met341, via a pair of hydrogen bonds, and with the residue analogue III, the only difference between the 2 molecules
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Figure 2 (A). Bar graph illustrating the % inhibition induced by imatinib (Im), nilotinib (N) and related synthetic analogues (at a concentration of 100 μΜ) on platelet
aggregation induced by ADP or TRAP-6. Values represent the Mean SD from three different platelet preparations. (B). Representative aggregation curves illustrating the
inhibitory effect of compound V, at different concentrations, on platelet aggregation induced by i. AA ii. ADP, iii. TRAP-6.
Abbreviations: ADP, Adenosine Diphosphate; SD, Standard Deviation; TRAP-6, Thrombin Receptor Activating Peptide-6.
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Pantazi et al
Figure 3 Bar graphs illustrating the effect of imatinib, nilotinib and synthetic compound V on P-selectin membrane expression induced by the platelet agonists (A) AA, (B)
ADP and (C) TRAP-6. Platelets in PRP were labeled with anti-CD62P-PE monoclonal antibody and analyzed by flow cytometry to determine the membrane expression of
P-selectin on activated platelets. The compounds imatinib, nilotinib and V were used at the concentration of 100 μΜ.
Notes: Results are the mean SD from at least three independent experiments. *p<0.01 compared with activated platelets, $p<0.05 compared with imatinib, #p<0.01
compared with nilotinib. **p<0.05 compared with respective activated platelets, &p<0.05 compared with nilotinib.
Abbreviations: AA, Arachidonic Acid; ADP, Adenosine Diphosphate; MFI, Mean Fluorescence Intensity; PRP, Platelet-Rich Plasma; SD, Standard Deviation; TRAP-6,
Thrombin Receptor Activating Peptide-6.
being in the amide bond position (retro-amides) between the rings, C and D, such as in nilotinib, as well as the incorpora-
two phenyl rings C and D (Figure 1). Thus, it is confirmed tion of a strong electron-withdrawing substituent, specially
that even insubstantial structural modifications, as hypothe- NO2 group and of a halogen atom (F, Cl, Br) or CF3 sub-
sized, on imatinib and nilotinib (i.e. amide bond like nilotinib stituent at the 2-, 3-, or 4-positions at the terminal phenyl ring,
in the analogue V) can have a strong affinity impact to the could be a promising method for the discovery of novel
ATP-binding pocket and therefore a strong impact on their molecules with increased antiplatelet activity.
Our data further demonstrated that imatinib, nilotinib and
their synthetic structural analogues were weak inhibitors of
platelet activation induced by ADP and TRAP-6 compared
with AA. It has been established that SFKs associate with
G-protein-coupled receptors (GPCRs) and contribute to
downstream signaling in platelets induced by the soluble
antiplatelet potency. Except the above, compound V fulfil
Lipinski’s rule of five: molecular weight 460.87 (≤ 500Da),
logPo/w (iLOGP) 2.77 (≤ 5), hydrogen bond donors 2 (≤ 5),
hydrogen bond acceptors 6 (≤ 10), rotatable bonds 7 (≤ 10)
and polar surface area (TPSA) 125.62 Å2 (≤ 140 Å2).
The results of the present study show that there is sig-
nificant potential to develop tyrosine kinase inhibitors (syn- ligands of these receptors.38 Consequently, SFKs are
thetic imatinib and nilotinib analogues), which would express involved in the intracellular signalling resulting in platelet
antiplatelet properties. Lack of the 4-methylimidazolyl group activation induced not only by Thromboxane A2 (TxA2), the
of nilotinib, with the amide bond between the two final phenyl metabolite of AA that mediates the AA-induced platelet
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Figure 4 Models of imatinib, nilotinib and of the most active analogue V bound to c-Src enzyme based on PDB (ID: 1Υ57): (A) Superposition of the model of the V-c-Src (in
green) and the structures of nilotinib (in brown), imatinib (in pink) at the active site of the kinase c-Src. The differentiations are focused mainly on the final phenyl moiety. (B,
C, D) Modeled binding modes presented as 2D ligand-interaction ligand (LID) of imatinib, nilotinib and compound V bound, respectively, positioned at the active site of the
kinase c-Src, as derived from flexible docking calculations.
Note: Protein residues forming hydrogen bonds (dotted green lines) are labelled.
activation, but also by ADP and TRAP-6 via their GPCRs, the synthetic analog of nilotinib V demonstrated the stron-
P2Y12 and PAR-1, respectively.38 Therefore, the higher inhi-
gest antiplatelet action. Consequently, this analogue could
be considered as a new lead compound for the develop-
ment of inhibitors with improved antiplatelet properties
and studies are underway to further optimize its activity.
bitory activity of our compounds towards AA-induced plate-
let activation compared with ADP and TRAP-6 may not be
only due to the c-Src inhibition. Hence, the mechanisms
underlying the antiplatelet effects of the compounds tested
in the present study beyond the c-Src inhibition should be
further investigated.
Supporting Materials
1H NMR, 13C NMR and high-resolution ESI-MS spectra
(Figures S1–12) of the nilotinib (V, VI) and imatinib/niloti-
nib (VII, VIII) analogues are provided as Supplementary
materials.
Conclusion
Platelets play important roles in cancer progression and
metastasis, as well as in CAT, therefore inhibiting platelet
activation would be a promising strategy to reduce both
cancer progression and CAT. The present study indicates
that based on the structure of the TKIs imatinib and
nilotinib there is a significant potential to develop syn-
thetic analogues, expressing improved antiplatelet effects,
therefore being suitable to target cancer progression and
Acknowledgments
The authors thank the Atherothrombosis Research Center
of the University of Ioannina for providing access to the
laboratory equipment and facilities. We appreciate the use
of NMR Unit and the Unit of Environmental, Organic and
Biochemical high-resolution analysis – ORBITRAP-LC-
metastasis, as well as CAT. Among all compounds tested, MS of the University of Ioannina for providing access to
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Pantazi et al
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Supporting Laboratories of the University of Ioannina.
This research has been financially supported by General
Secretariat for Research and Technology (GSRT) and the
Hellenic Foundation for Research and Innovation (HFRI)
(Scholarship Codes: 2000 and 82195).
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Disclosure
The authors report no conflicts of interest in this work.
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