Design, synthesis, and biological evaluation of 2-(phenoxyaryl)-3-urea derivatives as novel P2Y1 receptor antagonists

Article abstract of DOI:10.1016/j.ejmech.2018.09.014

A novel series of 2-(phenoxyaryl)-3-urea derivatives were designed, synthesized, and biologically evaluated for their anti-thrombotic activity. Most of compounds exhibited good inhibition against P2Y₁ receptor. Among them, three compounds 11, 12, and 13 demonstrated good P2Y₁ receptor antagonistic potency in vitro (IC₅₀ = 0.62 μM, 0.82 μM, and 0.21 μM, respectively). In antiplatelet aggregation study, four compounds 2, 3, 9, and 13 showed good antiplatelet activity. The possible binding modes of compounds with P2Y₁ receptor were also explored by molecular docking simulation. The docking studies demonstrated that compound 13 interacted well with Phe119 through hydrophobic interaction and modestly improved the P2Y₁ receptor antagonistic activity, making it justifiable for further investigation.

Full text of DOI:10.1016/j.ejmech.2018.09.014

European Journal of Medicinal Chemistry 158 (2018) 302e310
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis, and biological evaluation of 2-(phenoxyaryl)-3-urea
derivatives as novel P2Y1 receptor antagonists
,
b
,
,
,
,
, b
Jingjing Peng a 1, Lifen Zhao a 1, Lanlan Wang c 1, Hui Chen a b, Yunguang Qiu a
,
,
b
,
,
, b, **
Jiang Wang a 1, Huaiyu Yang d, Jun Liu c *, Hong Liu a
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai, 201203,
China
b University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing, 100049, China
c Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing, 210009, China
d Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences, School of Life Sciences, East China Normal University, Shanghai, 200241,
China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 2-(phenoxyaryl)-3-urea derivatives were designed, synthesized, and biologically eval-
uated for their anti-thrombotic activity. Most of compounds exhibited good inhibition against P2Y1 re-
ceptor. Among them, three compounds 11, 12, and 13 demonstrated good P2Y1 receptor antagonistic
Received 10 July 2018
Received in revised form
16 August 2018
Accepted 5 September 2018
Available online 6 September 2018
potency in vitro (IC50 ¼ 0.62
mM, 0.82 mM, and 0.21 mM, respectively). In antiplatelet aggregation study,
four compounds 2, 3, 9, and 13 showed good antiplatelet activity. The possible binding modes of com-
pounds with P2Y1 receptor were also explored by molecular docking simulation. The docking studies
demonstrated that compound 13 interacted well with Phe119 through hydrophobic interaction and
modestly improved the P2Y1 receptor antagonistic activity, making it justifiable for further investigation.
© 2018 Published by Elsevier Masson SAS.
Keywords:
P2Y1 receptor antagonist
2-(phenoxyaryl)-3-urea derivatives
Anti-Platelet
1. Introduction
has emerged as an attractive target for anti-thrombotic therapies
ꢀ
[15e19]. Leon et al. reported platelets from P2Y1 receptor deficient
Adenosine-50-diphosphate (ADP) is a key activator of platelets
and plays a vital role in platelet activation and thrombus formation
[1]. ADP activates platelets by simultaneously stimulating two G-
protein coupled receptors (GPCR) P2Y1 receptor and P2Y12 receptor.
P2Y1 and P2Y12 receptors are important in the process of hemo-
stasis and thrombosis regulation [2]. P2Y12 receptor inhibited
adenylyl cyclase through Gi-dependent pathway, while P2Y1 re-
ceptor facilitated platelet shape change and reversible aggregation
through Gq-dependent pathway [3,4]. P2Y12 receptor antagonists
are clinically well-validated drugs for antithrombotic therapy such
as clopidogrel [5e10], prasugrel [11], cangrelor [12], and ticagrelor
[13]. Although there are many launched antithrombotic drugs tar-
geting P2Y12 receptor, these P2Y12 receptor antagonists feature
more or less bleeding liability [14]. In recent years, P2Y1 receptor
mice were resistant to ADP-induced shape change and aggregation
[20,21]. It suggests that P2Y1 receptor antagonists could be a po-
tential treatment for a variety of thrombotic diseases and may
improve safety margins relative to P2Y12 receptor antagonists.
(10R,20S,40S,50S)-4-(2-iodo-6-methylaminopurin-9-yl)-1-[(phos-
phato)methyl]-2(phosphato)bicycle[3.1.0]-hexane (MRS2500) and
1-(2-(2-tert-butylphenoxy)pyridin-3-yl)-3e4-(trifluoromethoxy)
phenylurea (BPTU) are representative P2Y1 receptor antagonists
(Fig. 1). MRS2500 is a potent adenine nucleotide-based P2Y1 re-
ceptor antagonist (Ki ¼ 0.78 nM), which inhibited ADP activated
platelet aggregation in vitro and in vivo. Besides, MRS2500 could
also reduce arterial thrombosis with a moderate prolongation of
the bleeding time, with no spontaneous bleeding liability [22].
However, the chemical and enzymatic stabilities of MRS2500 and
other adenine nucleotide-based P2Y1 receptor antagonists are less
than desirable (mainly because of their poor pharmacokinetic
profiles and limited oral bioavailability), which would be expected
to limit their usefulness as oral drug candidate. Thus, the discovery
of novel P2Y1 receptor antagonists with improved pharmaceutical
characteristics could have significant utility in the treatment of a
variety of thromboembolic disorders [23]. Chao et al. reported a
* Corresponding author.
** Corresponding author. State Key Laboratory of Drug Research, Shanghai Insti-
tute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road,
Shanghai, 201203, China.
E-mail addresses: junliu@cpu.edu.cn (J. Liu), hliu@simm.ac.cn (H. Liu).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2018.09.014
0223-5234/© 2018 Published by Elsevier Masson SAS.

J. Peng et al. / European Journal of Medicinal Chemistry 158 (2018) 302e310
303
optimization of the phenoxy ring [36e38], however, there are few
reports about the optimization of ureido phenyl ring, which is very
important to form two aromatic edge-to-face interactions with
Phe62 and Phe66. According to the binding mode of BPTU with
P2Y1 receptor, ureido is important as a pharmacophore. As a result,
our work mainly focuses on the hydrophobic groups. Bioisosterism
strategy was applied to replace trifluoromethoxyphenyl with thi-
ophen ring. Using this strategy, compounds 1e7 were obtained. In
addition, there is no report about alkyl substitution in ureido. It is
assumed that alkyl group may enhance the hydrophobic interaction
with Leu102 and well occupy the binding site, which would
improve P2Y1 receptor binding affinity. As a result, different alkyl
groups were introduced to the ureido, and compounds 8e12 were
synthesized. Notably, there is a cavum in the binding pocket of
BPTU with P2Y1 receptor near Phe119. It was hypothesized that
introduction of a small group at the pyridine ring may accommo-
date well in the cavum, which therefore increases the hydrophobic
interaction with Phe119 (Fig. 2C). Based on this assumption, we
synthesized compounds 13e15. All 2-(phenoxyaryl)-3-urea de-
rivatives were evaluated for their inhibitory activity against P2Y1
receptor (Fig. 3).
Fig. 1. Representative P2Y1 receptor antagonists.
series of non-nucleotide small molecule 2-(phenoxypyridine)-3-
urea analogs as P2Y1 receptor antagonists [24e26]. Among them,
BPTU showed good binding affinity with P2Y1 receptor (Ki ¼ 6 nM)
and moderate antiplatelet activity in the ADP-induced platelet
aggregation assay in vitro. The pharmacokinetic profile of BPTU
revealed that this compound had moderate half-life (t1/2 ¼ 1.43 h)
and bioavailability (F ¼ 18%) when orally dosed at 30 mg/kg in rats
[24]. Other optimization and modification was reported subse-
quently to improve P2Y1 receptor binding affinity and physico-
chemical property [27e32].
2.2. Synthesis of 2-(phenoxyaryl)-3-urea derivatives
Zhao et al. reported the crystal structures of two different li-
gands (MRS2500 and BPTU) binding with P2Y1 receptor [33].
Nucleotide ligand MRS2500 occupied a pocket in extracellular part
of this receptor, which is orthosteric site of P2Y1 receptor, while
BPTU bound to P2Y1 receptor on the lipid interface of the trans-
membrane domain, instead of interacting within the seven-
transmembrane helical bundle (Fig. 2A and B). The location of
this ligand-binding site indicates that BPTU acts as an allosteric
modulator of P2Y1 receptor. In the absence of the endogenous
ligand, allosteric modulators do not exert any effect, thus preser-
ving the physiological effects of P2Y1 receptor [34]. Besides, allo-
steric ligands can act in a lower dosage exerting fewer side effects
while orthosteric antagonists usually act in a higher dosage because
their competition with endogenous ligands. Further, as allosteric
ligand binding sites are located in non-conserved regions of P2Y1
receptor, as opposed to the highly conserved orthosteric ligand
binding sites, it is possible to develop highly-selective ligands [35].
Therefore, BPTU may represent a promising candidate for the
development of new antiplatelet therapies. The relatively shallow
ligand-binding pocket which was formed by aromatic and hydro-
phobic residues accommodated BPTU predominantly through hy-
drophobic interactions. The only polar interactions are represented
by two hydrogen bonds between the nitrogen atoms of BPTU's urea
group and the mainchain carbonyl of Leu102 [10]. In addition, the
pyridyl group forms hydrophobic interactions with Ala106 and
Phe119. The ureido phenyl ring forms two aromatic edge-to-face
interactions with Phe62 and Phe66 (Fig. 2C and D).
The general synthetic route to 2-(phenoxyaryl)-3-urea de-
rivatives is outlined in Scheme 1. First, 5-nitrothiophene-2-
carboxylic acid (16), as the starting material, condensation with
different alcohols and amines in different conditions afforded cor-
responding esters and amides (17a-f). Subsequent reduction of the
nitro group with Zn/ammonium formate gave corresponding
amines 18a-f. Reacting 2-(tert-butyl)phenol (19) with 2-chloro-3-
nitropyridine in DMF at 80 ꢀC afforded the 2-(2-(tert-butyl)phe-
noxy)-3-nitropyridine (20). Subsequent reduction of the nitro
group with Zn/ammonium formate gave the 2-(2-(tert-butyl)phe-
noxy)pyridin-3-amine 21. Amine 21 was then reacted with
diphosgene in the presence of 1,8-bis(dimethylamino)-napthalene
to provide the key intermediate isocyanate 22. Isocyanate 22
reacted with different amines (18a-f) at 80 ꢀC under microwave
irradiation, affording compounds 1e6. Treatment of different
commercially available amine with key intermediate 22 gave the
corresponding target compounds 7e12.
The synthesis of target compounds 13e15 is shown in Scheme 2.
Briefly, condensation of commercially available 2-(tert-butyl)
phenol 19 with substituted 2-chloro-3-nitro compounds 23a-c
easily afforded compounds 24a-c, which was subsequently trans-
formed to amines 25a-c using Zn/ammonium formate. A nucleo-
philic substitution reaction of 1-isocyanato-4-(trifluoromethoxy)
benzene 26 with amines 25a-c provided compounds 13e15.
3. Results and discussion
In this paper, a novel series of 2-(phenoxyaryl)-3-urea de-
rivatives as P2Y1 receptor antagonists were designed and synthe-
sized. Most of compounds exhibited good P2Y1 receptor binding
affinities and good antiplatelet activity. Our structural optimization
was dedicated to improve both P2Y1 receptor binding and anti-
platelet activities aiming at discovery of potent compounds for
further development.
All the synthesized compounds were evaluated for inhibition
rate and IC50 test against P2Y1 receptor in vitro and anti-platelet
activity in rats. Antagonistic potency was measured by a competi-
tive binding assay with BPTU as the positive control, and the results
are reported as inhibition rate at 50 mM and concentration for 50%
inhibition (IC50). SAR for these compounds is summarized in
Tables 1 and 2.
We first focused on optimization of the ureido phenyl ring by
replacing the phenyl moiety with substituted thiophen rings and
alkyl groups. Initially, our SAR started from 5-substituted thiophen
derivatives. The effects of substitution on the thiophen ring were
explored. Compounds 1 and 2 were obtained by introduction of a
small ester groups at the 5-position of the thiophen ring. These
compounds displayed good inhibition activities against P2Y1
2. Chemistry
2.1. Design of 2-(phenoxyaryl)-3-urea derivatives
Although a number of BPTU analogs have been reported in the
past few years, most of the structure optimization focuses on

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J. Peng et al. / European Journal of Medicinal Chemistry 158 (2018) 302e310
Fig. 2. (A)The binding site of MRS2500 with P2Y1 receptor (PDB ID: 4XNW); (B)The binding site of BPTU with P2Y1 receptor (PDB ID: 4XNV); (C)The binding mode of BPTU
with P2Y1 receptor; (D) 2D interaction diagram between BPTU and P2Y1 receptor (hydrogen bond was presented by purple). The interaction was drawn using Chemdraw 12.0
Software.
Fig. 3. Design of 2-(phenoxyaryl)-3-urea derivatives.
receptor, with 91.41% and 91.78% inhibition rate at 50
duction of bigger ester groups gave compounds 3e5. All these three
compounds exhibited mild inhibition activities against P2Y1 re-
ceptor, with 53.07%, 52.84%, 27.97% inhibition rate at 50
m
M. Intro-
phenyl ring, compound 14 also displayed high potency (inhibition
rate ¼ 91.03%). However, introducing methyl group on the phenyl
ring, the potency decreased (inhibition rate ¼ 30.47%).
mM,
Furthermore, compounds with good inhibition rate at 50 mM
respectively. While introduction of amide group and cyano group
afforded compound 6 and 7, which displayed no inhibition activ-
were selected to test IC50. The ester thiophen derivatives demon-
strated the benzyl substitution was better than methyl and ethyl
ities toward P2Y1 receptor at the concentration of 50
m
M. The ef-
substitution (IC50 ¼ 12.92
mM, 12.52 mM, 7.19 mM, respectively). All
fects of alkyl substitution were also explored. As shown in Table 1,
most of the alkyl derivatives exhibited good in vitro potency against
P2Y1 receptor. Additionally, all of these antagonists occupied a
narrow potency range, indicating tolerability for a wide variety of
alkyl substituents. Basd on the crystal structure of BPTU, intro-
ducing small group at the ortho-position of the pyridine ring may
increase the hydrophobic interaction with Phe119. Compound 13
exhibited good potency against P2Y1 receptor, with 90.06% inhibi-
alkyl groups were tolerated for P2Y1 receptor inhibition. For
example, compounds 8 (n-butyl) and 9 (trans-4-methyl-cyclo-
hexan) exhibited mild inhibitory activities against P2Y1 receptor
with IC50 values of 9.98
containing a cyclopentyl substituent (10) displayed good P2Y1 re-
ceptor potency (IC50 ¼ 2.41 M). Introducing steric hindrance at the
urea group (11 and 12) also provided potent P2Y1 receptor inhibi-
tory activity with IC50 values of 0.62 M and 0.82 M, respectively.
Introduction of a small group like methyl group at the ortho-
mM and 9.05 mM, respectively. Compound
m
m
m
tion rate at 50 mM. While replacement the pyriding ring to the

J. Peng et al. / European Journal of Medicinal Chemistry 158 (2018) 302e310
305
Scheme 1. Reagents and conditions: (a) (i) for 17a and 17b thionyl chloride, reflux, MeOH; (ii) for 17c, 17d, and 17e thionyl chloride, TEA, CH2Cl2, 80 ꢀC; (iii) for 17f HATU, DIEA,
CH2Cl2; (b) ammonium formate, Zn, EtOH/EtOAc (4:1); (c) 2-chloro-3-nitropyridine, Cs2CO3, DMF, 80 ꢀC; (d) ammonium formate, Zn, EtOH/EtOAc (4:1); (e) diphosgene, 1,8-
bis(dimethylamino)-napthalene, CH2Cl2, 0 ꢀC; (f) toluene (dry), 80 ꢀC.
Scheme 2. Reagents and conditions: (a) Cs2CO3, DMF, 80 ꢀC; (b) ammonium formate, Zn, EtOH/EtOAc (4:1); (c) toluene (dry), 80 ꢀC.
position of pyridine (13) showed the best P2Y1 receptor potency
M), which is the same as
the positive control BPTU. While replacement the pyridine ring to
the phenyl ring, compound 14 exhibited mild P2Y1 receptor
compound 13 accommodates very well with the cavum in the
binding pocket, which formed hydrophobic interaction with
Phe119. Inspired by the docking results, we proposed that addition
of appropriate hindrance on the pyridine moiety might modestly
improve the inhibitory activity against P2Y1 receptor. Molecular
docking was used to investigate the binding modes of compounds
13 and 15 with ligand BPTU, which was illustrated in Fig. 4F. From
the molecular docking result, we noted that these two compounds
shared the similar binding modes with P2Y1 receptor. However,
there is a dihedral angle about 12e15ꢀ between the phenyl ring of
compound 15 and the pyridine ring of compound 13. As a result, the
methyl group in compound 15 could not accommodate well in the
cavum, compared with compound 13, which made its inhibition
rate decreased.
among all the compounds (IC50 ¼ 0.21
m
antagonistic potency (IC50 ¼ 7.53
mM).
Six 2-(phenoxyaryl)-3-urea derivatives were selected to eval-
uate their inhibitory effect on ADP-induced platelet aggregation in
rats when orally dosed of 3 mg/kg. BPTU was used as the positive
control (Table 3). Compounds 2 and 3 showed moderate anti-
platelet activity (inhibition rate ¼ 50.60% and 69.40%). However,
compounds 8 and 11 did not exhibit good anti-platelet activity,
indicating that increasing lipophilicity of P2Y1 receptor inhibitors
reduced the ex vivo potency. Compound 13, introducing the methyl
group at the ortho-position of pyridine displayed good anti-platelet
activity (inhibition rate ¼ 68.90%), which is the same as the positive
control BPTU.
4. Conclusion
To gain insight into the binding modes and interactions of these
compounds against P2Y1 receptor, molecular modeling was carried
out using Glide program. The crystal structure of P2Y1 receptor
(PDB ID: 4XNV) was used in molecular docking simulation (Fig. 4A).
It was observed that compounds 11, 12, and 13 occupied the same
binding pockets and shown similar binding modes as the positive
control BPTU. The alkyl moiety of compounds 11 and 12 formed
hydrophobic interactions with the residue Leu102 (Fig. 4B and C).
Compared to compounds 11 and 12, the 4-(trifluoromethoxy)
group of compound 13 and BPTU (Fig. 4D) formed a more favorable
In conclusion, we designed, synthesized, and evaluated a series
of 2-(phenoxyaryl)-3-urea derivatives as novel P2Y1 receptor in-
hibitors. In vitro evaluation found that most of the compounds
exhibited good in vitro potency against P2Y1 receptor in low
micromolar range. Among these, compounds 11 (IC50 ¼ 0.62
mM),
12 (IC50 ¼ 0.82
m
M), and 13 (IC50 ¼ 0.21
m
M) showed good P2Y1
receptor potency. Compounds 2, 3, 9, and 13 were found to display
the good anti-platelet activity in rats. Molecular docking results
reveal that both hydrophobic and hydrogen bonding interactions
are important for the inhibitory potency. These potent compounds
represent a novel scaffold for the development of P2Y1 receptor
p-
p
stacking interaction with Phe62 and Phe66. In addition, the
introduction of a methyl group at the ortho-position of pyridine,

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J. Peng et al. / European Journal of Medicinal Chemistry 158 (2018) 302e310
Table 1 (continued)
Table 1
Cellular binding assay results for 2-(phenoxyaryl)-3-urea derivatives.
Compd.
R1
X
R2
Inhibition Ratea
91.59 2.63
%
BPTU
H
N
a
The test was carried out in 1321N1/P2Y1 cell strain, MRS2365 as P2Y1 receptor
agonist.
Table 2
Compd.
R1
X
R2
Inhibition Ratea
91.41 1.71
%
Cellar binding assay of 2-(phenoxyaryl)-3-urea derivatives in vitro.
1
H
N
a
a
Compd.
IC50
(
m
M)
Compd.
IC50 (mM)
1
2
3
8
12.92 0.05
12.52 0.75
7.19 1.16
9.98 0.54
7.05 1.25
0.28 0.24
10
11
12
13
14
2.41 1.87
0.62 0.17
0.82 0.04
0.21 0.14
7.53 6.08
2
3
4
H
H
H
N
N
N
91.78 2.43
53.07 8.08
52.84 11.60
9
BPTU
a
The test was carried out in 1321N1/P2Y1 cell strain, MRS2365 as P2Y1 receptor
agonist.
Table 3
Inhibitory effect of 2-(phenoxyaryl)-3-urea derivatives on ADP-induced platelet
aggregation in rats at a dose of 3 mg/kg.
Compd.
Inhibition Ratea
%
Compd.
Inhibition Ratea
%
5
6
7
H
H
H
N
N
N
27.97 6.93
3.76 0.68
0.80 9.20
2
3
8
50.60%
69.40%
22.00%
67.40%
9
11
13
57.60%
26.67%
68.90%
BPTU
a
Assay details are described in Experimental Section. Aggregation data refer to
ex vivo measurements 2 h after intragastric administration.
inhibitors. Current efforts are aimed at further modifying these
compounds to achieve more potent and selective P2Y1 receptor
inhibitors.
8
9
H
H
N
N
91.09 2.32
84.77 7.74
5. Experimental section
5.1. General information
10
11
12
H
H
H
N
N
N
95.59 1.72
97.34 0.02
97.31 0.30
General procedures of the synthesis route were described as
below. The reagents (chemicals) were purchased from Alfa Aesar,
Sigma, Acros and Shanghai Chemical Reagent Company, and used
without further purification. Analytical thin-layer chromatography
(TLC) was performed on HSGF 254 (150e200 mm thickness, Yantai
Huiyou Company, China). Column chromatography was performed
with CombiFlash® Companion system (Teledyne Isco, Inc.). Nuclear
magnetic resonance (NMR) spectra were performed on a Brucker
AMX-300, AMX-400, AMX-500, and AMX-600 NMR (TMS as IS).
Chemical shifts were reported in parts per million (ppm,
d)
downfield from tetramethylsilane. Proton coupling patterns were
described as singlet (s), doublet (d), doublet of doublets (dd), triplet
(t), triplet of doublets (td), quartet (q), multiplet (m), and broad (br).
Low- and high-resolution mass spectra (MS and HRMS) were given
with electrospray ionization (ESI) and electric ionization (EI) pro-
duced by Finnigan MAT-95. Melting points (mp) were measured in
open capillary tubes, using an SGW X-4 melting point apparatus
(ꢁ50e400 ꢀC). All compounds were confirmed with over 90% pu-
rity which were determined by Agilent-1100 HPLC with binary
pump, photodiode array detector (DAD), using Agilent Extend-C18
13
14
15
CH3
H
N
C
C
90.06 2.43
91.03 4.52
30.47 23.62
CH3
column (150 ꢂ 4.6 mm, 5
mm). All compounds were analyzed by,
using MeOH/H2O ¼ 80:20 (v/v) (30 min, 1 mL/min) and calculated
the peak areas at 214 nm.

J. Peng et al. / European Journal of Medicinal Chemistry 158 (2018) 302e310
307
Fig. 4. (A) Superposition of compounds 11 (blue), 12 (pink), 13 (yellow), and 15 (purple) with ligand BPTU from 4XNV. Docking structures of compounds 11 (B), 12 (C), 13 (D) and 15
(E) in the active site of P2Y1. (F) Superposition of compounds 13 (yellow) and 15 (purple). Hydrogen atoms have been omitted for clarity. All figures were prepared using PyMol
(http://www.pymol.org).
5.2. General procedures
2.0 Hz, 1H), 7.58 (d, J ¼ 4.0 Hz, 1H), 7.48 (dd, J ¼ 8.0, 2.0 Hz, 1H),
7.25e7.12 (m, 2H), 7.09 (dd, J ¼ 8.0, 4.8 Hz, 1H), 6.86 (dd, J ¼ 8.0,
The preparation and data of key intermediate 20e22 have
already been reported [23]. The preparation intermediate 23e25
are the same as the synthetic route of 20e22.
1.6 Hz, 1H), 6.53 (d, J ¼ 4.4 Hz, 1H), 3.83 (s, 3H), 1.39 (s, 9H) ppm; 13
C
NMR (125 MHz, DMSO‑d6)
d 170.3, 162.5, 152.9, 152.4, 151.5, 147.6,
141.1, 139.8, 132.4, 127.1, 127.0, 124.7, 124.0, 123.8, 121.0, 118.9, 110.1,
51.7, 34.3, 30.5 ppm. LC-MS: 424.0 [M-H]-, 425.9 [MþH]þ; HRMS
(ESI-TOF) m/z: [M-H]- Calcd for C22H22N3O4S 424.1337; Found
424.1341. Purity: 97.9%.
Compounds 2e12 were prepared according to the procedures
described for compound 1.
5.2.1. Methyl 5-(3-(2-(2-(tert-butyl)phenoxy)pyridin-3-yl)ureido)
thiophene-2-carboxylate 1
To a solution of isocyanate 22 (100 mg, 0.81 mmol) in toluene
(1 mL) in microwave tube was added methyl 5-aminothiophene-2-
carboxylate (191 mg, 1.21 mmol). The resulting mixture was heated
with shaking at 80 ꢀC for 1.5 h. The solvent was removed by vac-
uum, extracted with ethyl acetate and water, the combined organic
layer was washed with brine, dried over Na2SO4, filtered, and
concentrated to give the crude product, which was purified by
column chromatography (petroleum ether/ethyl acetate 4:1 v/v) to
yield as a white solid (200 mg, 63%). m.p. 109e110 ꢀC. 1H NMR
5.2.2. Ethyl 5-(3-(2-(2-(tert-butyl)phenoxy)pyridin-3-yl)ureido)
thiophene-2-carboxylate 2
m.p. 205e206 ꢀC. 1H NMR (400 MHz, CD3OD)
d
8.59 (dd, J ¼ 8.0,
2.0 Hz, 1H), 7.71 (dd, J ¼ 4.8, 2.0 Hz, 1H), 7.58 (d, J ¼ 4.4 Hz, 1H), 7.48
(dd, J ¼ 8.0, 2.0 Hz, 1H), 7.27e7.12 (m, 2H), 7.09 (dd, J ¼ 8.0, 4.8 Hz,
1H), 6.86 (dd, J ¼ 8.0, 1.6 Hz, 1H), 6.52 (d, J ¼ 4.4 Hz, 1H), 4.29 (q,
J ¼ 7.2 Hz, 2H),1.39 (s, 9H), 1.35 (t, J ¼ 7.2 Hz, 3H) ppm; 13C NMR
(400 MHz, CD3OD)
d
8.59 (dd, J ¼ 8.0, 2.0 Hz, 1H), 7.71 (dd, J ¼ 4.8,

308
J. Peng et al. / European Journal of Medicinal Chemistry 158 (2018) 302e310
(125 MHz, DMSO‑d6)
d
162.1, 152.9, 152.4, 151.5, 147.4, 141.1, 139.7,
1.4 Hz, 1H), 6.66 (d, J ¼ 4.2 Hz, 1H), 1.32 (s, 9H) ppm; 13C NMR
132.2, 127.1, 126.9, 124.7, 124.0, 123.8, 121.3, 118.9, 110.0, 60.2, 34.3,
30.5, 14.3 ppm. LC-MS: 438.0 [M-H]-, 440.0 [MþH]þ; HRMS (ESI-
TOF) m/z: [M-H]- Calcd for C23H24N3O4S 438.1493; Found 438.1497.
Purity: 97.7%.
(125 MHz, DMSO‑d6) d 153.1, 152.4, 151.7, 147.1, 141.1, 140.1, 137.3,
127.3, 127.1, 124.7, 123.8, 119.0, 115.7, 109.6, 96.8, 34.3, 30.5 ppm. LC-
MS: 391.0 [M-H]-, 392.9 [MþH]þ; HRMS (ESI-TOF) m/z: [M-H]-
Calcd for C21H19N4O2S 391.1234; Found 391.1236. Purity: 95.3%.
5.2.3. Benzyl 5-(3-(2-(2-(tert-butyl)phenoxy)pyridin-3-yl)ureido)
thiophene-2-carboxylate 3
5.2.8. 1-Butyl-3-(2-(2-(tert-butyl)phenoxy)pyridin-3-yl)urea 8
m.p. 123e124 ꢀC. 1H NMR (400 MHz, CDCl3)
d
8.58 (dd, J ¼ 8.0,
m.p. 197e198 ꢀC. 1H NMR (400 MHz, CD3OD)
d
8.58 (dd, J ¼ 8.0,
1.7 Hz, 1H), 7.81 (dd, J ¼ 4.9, 1.7 Hz, 1H), 7.50 (dd, J ¼ 7.9, 1.8 Hz, 1H),
7.29e7.17 (m, 2H), 7.01 (dd, J ¼ 7.8, 4.9 Hz, 1H), 6.93 (dd, J ¼ 8.0,
1.4 Hz, 2H), 4.70 (s, 1H), 3.36e3.27 (m, 2H), 1.60e1.55 (m, 2H),
1.48e1.38 (m, 11H), 0.98 (t, J ¼ 7.3 Hz, 3H) ppm. 13C NMR (125 MHz,
1.6 Hz, 1H), 7.70 (dd, J ¼ 4.8, 1.6 Hz, 1H), 7.62 (d, J ¼ 4.4 Hz, 1H), 7.48
(dd, J ¼ 8.0, 2.0 Hz, 1H), 7.46e7.29 (m, 5H), 7.25e7.13 (m, 2H), 7.08
(dd, J ¼ 8.0, 4.8 Hz, 1H), 6.86 (dd, J ¼ 8.0, 1.6 Hz, 1H), 6.53 (d,
J ¼ 4.4 Hz, 1H), 5.29 (s, 2H), 1.38 (s, 9H) ppm; 13C NMR (125 MHz,
DMSO‑d6) d 155.2, 152.6, 152.2, 141.1, 137.7, 127.1, 127.0, 125.8, 125.7,
DMSO‑d6)
d
161.9, 152.9, 152.4, 151.5, 147.8, 141.1, 139.8, 136.3, 132.7,
124.4, 123.8, 118.7, 34.3, 31.6, 30.5, 19.5, 13.6 ppm. LC-MS: 340.1[M-
128.5, 128.0, 127.9, 127.1, 127.0, 124.7, 124.0, 123.8, 120.8, 118.9, 110.1,
65.6, 34.3, 30.5 ppm. LC-MS: 500.0 [M-H]-, 501.9 [MþH]þ; HRMS
(ESI-TOF) m/z: [M-H]- Calcd for C28H26N3O4S 500.1650; Found
500.1656. Purity: 96.0%.
H]-, 342.0[MþH]þ; HRMS (ESI-TOF) m/z: [M-H]- Calcd for
C20H26N3O2 340.2031; Found 340.2036. Purity: 95.5%.
5.2.9. 1-(2-(2-(tert-butyl)phenoxy)pyridin-3-yl)-3-((1r,4r)-4-
methylcyclohexyl)urea 9
5.2.4. 2-Morpholinoethyl 5-(3-(2-(2-(tert-butyl)phenoxy)pyridin-
m.p. 197e199 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 8.52 (dd,
3-yl)ureido)thiophene-2-carboxylate 4
J ¼ 8.0, 1.7 Hz, 1H), 8.18 (s, 1H), 7.59 (dd, J ¼ 4.8, 1.7 Hz, 1H), 7.42 (dd,
J ¼ 7.9, 1.7 Hz, 1H), 7.18 (dtd, J ¼ 15.1, 7.3, 1.6 Hz, 2H), 7.04e6.92 (m,
2H), 6.84 (dd, J ¼ 7.9, 1.4 Hz, 1H), 1.89 (d, J ¼ 9.7 Hz, 2H), 1.67 (d,
J ¼ 11.4 Hz, 2H), 1.32 (s, 10H), 1.17 (dd, J ¼ 9.2, 5.1 Hz, 1H), 1.14e0.94
(m, 4H), 0.87 (d, J ¼ 6.5 Hz, 3H) ppm; 13C NMR (125 MHz, DMSO‑d6)
m.p. 166e168 ꢀC. 1H NMR (400 MHz, CD3OD)
d
8.58 (dd, J ¼ 8.0,
1.6 Hz, 1H), 7.71 (dd, J ¼ 4.8, 1.6 Hz, 1H), 7.61 (d, J ¼ 4.0 Hz, 1H), 7.48
(dd, J ¼ 8.0, 1.6 Hz, 1H), 7.25e7.13 (m, 2H), 7.12e7.06 (dd,
J ¼ 8.0,4.8 Hz, 1H), 6.86 (dd, J ¼ 8.0, 1.6 Hz, 1H), 6.53 (d, J ¼ 4.0 Hz,
1H), 4.41 (t, J ¼ 1.6 Hz, 2H), 3.70 (t, J ¼ 4.8 Hz, 4H), 2.77 (t, J ¼ 1.6 Hz,
2H), 2.60 (t, J ¼ 4.8 Hz, 4H), 1.39 (s, 9H) ppm; 13C NMR (125 MHz,
d
154.5, 152.7, 152.2, 141.1, 137.6, 127.1, 125.9, 125.5, 124.4, 123.7,
118.8, 59.7, 48.3, 34.3, 33.6, 32.9, 31.4, 30.5, 22.1, 20.6, 14.1 ppm. LC-
DMSO‑d6)
d
162.0, 152.9, 152.4, 151.5, 147.6, 141.1, 139.8, 132.4, 127.1,
MS: 380.1[M-H]-, 382.0[MþH]þ; HRMS (ESI-TOF) m/z: [M-H]- Calcd
127.0, 124.7, 124.0, 123.8, 121.1, 118.9, 110.1, 66.2, 61.7, 56.6, 53.4,
34.3, 30.5 ppm. LC-MS: 523.0 [M-H]-, 524.9 [MþH]þ; HRMS (ESI-
TOF) m/z: [M-H]- Calcd for C27H31N4O5S 523.2021; Found 523.2030.
Purity: 97.1%.
for C23H30N3O2 380.2344; Found 380.2349. [
EtOH). Purity: 95.3%.
a
]D25 ¼ þ1.33 (c ¼ 0.1,
5.2.10. 1-(2-(2-(tert-butyl)phenoxy)pyridin-3-yl)-3-
cyclopentylurea 10
5.2.5. 2-(Piperidin-1-yl)ethyl 5-(3-(2-(2-(tert-butyl)phenoxy)
pyridin-3-yl)ureido)thiophene-2-carboxylate 5
m.p. 217e219 ꢀC, 1H NMR (500 MHz, CDCl3)
d
8.55 (dd, J ¼ 8.0,
1.7 Hz, 1H), 7.76 (dd, J ¼ 4.9, 1.7 Hz, 1H), 7.45 (dd, J ¼ 7.9, 1.7 Hz, 1H),
7.19 (dtd, J ¼ 31.6, 7.4, 1.6 Hz, 2H), 6.97 (dd, J ¼ 7.9, 4.9 Hz, 1H), 6.90
(s, 1H), 6.88 (dd, J ¼ 8.0, 1.4 Hz, 1H), 4.61 (d, J ¼ 6.8 Hz, 1H), 4.06 (m,
1H), 2.07e1.99 (m, 2H), 1.74e1.66 (m, 2H), 1.62 (m, 2H), 1.50e1.42
m.p. 180e182 ꢀC. 1H NMR (400 MHz, CD3OD)
d
8.58 (dd, J ¼ 8.0,
1.6 Hz, 1H), 7.71 (dd, J ¼ 4.8, 1.6 Hz, 1H), 7.64 (d, J ¼ 4.4 Hz, 1H), 7.48
(dd, J ¼ 8.0, 1.6 Hz, 1H), 7.24e7.14 (m, 2H), 7.09 (dd, J ¼ 8.0, 5.2 Hz,
1H), 6.86 (dd, J ¼ 8.0, 1.6 Hz, 1H), 6.54 (d, J ¼ 4.0 Hz, 1H), 4.47 (t,
J ¼ 5.6 Hz, 2H), 2.98 (brs, 2H), 2.81 (brs, 4H), 1.74e1.68 (m, 4H),
1.57e1.52 (m, 2H), 1.39 (s, 9H) ppm. 13C NMR (125 MHz, DMSO‑d6)
(m, 2H), 1.39 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3)
d 154.8, 152.7,
152.5, 141.5, 139.8, 127.7, 127.4, 127.1, 125.3, 125.1, 123.2, 119.3, 52.8,
34.9, 33.8, 30.8, 23.8 ppm. LC-MS: 352.1[M-H]-, 354.0[MþH]þ;
HRMS (ESI-TOF) m/z: [M-H]- Calcd for C21H26N3O2 352.2031; Found
352.2030. Purity: 97.8%.
d
161.8,153.0,152.4,151.5,141.1,139.8,127.1,127.1,127.0,124.7,124.0,
123.8, 118.9, 110.1, 59.7, 34.3, 30.5, 20.7, 14.1 ppm. LC-MS:521.0 [M-
H]-, 523.0 [MþH]þ; HRMS (ESI-TOF) m/z: [M-H]- Calcd for
C28H33N4O4S 521.2228; Found 521.2238. Purity: 96.6%.
5.2.11. 1-(Adamantan-2-yl)-3-(2-(2-(tert-butyl)phenoxy)pyridin-
3-yl)urea 11
5.2.6. 5-(3-(2-(2-(tert-butyl)phenoxy)pyridin-3-yl)ureido)-N-(2-
morpholinoethyl)thiophene-2-carboxamide 6
m.p. 228e230 ꢀC, 1H NMR (500 MHz, CDCl3)
d
8.50 (dd, J ¼ 8.0,
1.7 Hz, 1H), 7.74 (dd, J ¼ 4.9, 1.7 Hz, 1H), 7.47e7.43 (m, 1H), 7.18 (dtd,
J ¼ 15.1, 7.3,1.6 Hz, 2H), 6.95 (dd, J ¼ 8.0, 4.9 Hz,1H), 6.87 (dd, J ¼ 7.9,
1.4 Hz, 1H), 6.72 (s, 1H), 4.38 (s, 1H), 2.11 (s, 3H), 2.04 (m, 6H), 1.70
m.p. 140e141 ꢀC. 1H NMR (400 MHz, CD3OD)
d
7.78 (dd, J ¼ 8.0,
1.6 Hz, 1H), 6.90 (dd, J ¼ 4.8, 1.6 Hz, 1H), 6.68 (dd, J ¼ 8.0, 1.6 Hz, 1H),
6.64 (d, J ¼ 4.4 Hz, 1H), 6.44e6.34 (m, 2H), 6.28 (dd, J ¼ 8.0, 5.2 Hz,
1H), 6.06 (dd, J ¼ 8.0, 1.2 Hz, 1H), 5.72 (d, J ¼ 4.0 Hz, 1H), 2.91 (t,
J ¼ 4.8 Hz, 4H), 2.69 (t, J ¼ 6.8 Hz, 2H), 1.77 (m, 6H), 0.59 (s, 9H)
(m, 6H), 1.39 (s, 9H) ppm. 13C NMR (125 MHz, CDCl3)
d 153.8, 152.9,
152.5, 141.5, 139.5, 127.8, 127.4, 127.0, 125.6, 125.1, 123.1, 119.3, 52.0,
42.5, 36.6, 34.9, 30.9, 29.8 ppm. LC-MS: 418.1[M-H]-, 420.0[MþH]þ;
HRMS (ESI-TOF) m/z: [M-H]- Calcd for C26H32N3O2 418.2500; Found
418.2503. Purity: 98.6%.
ppm; 13C NMR (125 MHz, DMSO‑d6)
d 170.3, 161.8, 152.8, 152.4,
151.4, 144.7, 141.1, 139.4, 128.6, 127.1, 126.6, 126.10, 124.7, 124.3,
123.8, 118.9, 109.5, 66.2, 59.7, 57.6, 53.3, 36.3, 34.3, 30.5 ppm. LC-
MS: 522.0 [M-H]-, 524.0 [MþH]þ; HRMS (ESI-TOF) m/z: [M-H]-
Calcd for C27H32N5O4S 522.2180; Found 522.2191. Purity: 97.7%.
5.2.12. 1-(2-(2-(tert-butyl)phenoxy)pyridin-3-yl)-3-(2-
methylcyclohexyl)urea 12
m.p. 209e210 ꢀC, 1H NMR (500 MHz, CDCl3)
d
8.53 (d, J ¼ 7.8 Hz,
5.2.7. 1-(2-(2-(tert-butyl)phenoxy)pyridin-3-yl)-3-(5-
1H), 7.76 (d, J ¼ 3.4 Hz, 1H), 7.46 (d, J ¼ 7.9 Hz, 1H), 7.19 (dt, J ¼ 33.1,
7.4 Hz, 2H), 6.97 (dd, J ¼ 7.8, 5.0 Hz, 1H), 6.91 (t, J ¼ 7.9 Hz, 1H), 6.83
(s, 1H), 4.38 (s, 1H), 3.29 (s, 1H), 2.11e2.01 (m, 1H), 1.76 (m, 2H), 1.67
(m, 1H), 1.46e1.31 (s, 9H), 1.29e1.06 (m, 4H), 1.03e0.91 (d,
J ¼ 6.5 Hz, 3H), 0.90e0.82 (m, 1H) ppm. 13C NMR (125 MHz, CDCl3)
cyanothiophen-2-yl)urea 7
m.p. 238e240 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 11.08 (s, 1H),
8.90 (s, 1H), 8.48 (dd, J ¼ 7.9, 1.7 Hz, 1H), 7.76 (dd, J ¼ 4.8, 1.7 Hz, 1H),
7.72 (dd, J ¼ 4.2, 2.7 Hz, 1H), 7.44 (dd, J ¼ 7.9, 1.7 Hz, 1H), 7.21 (dtd,
J ¼ 15.1, 7.3, 1.6 Hz, 2H), 7.11 (dd, J ¼ 7.9, 4.8 Hz, 1H), 6.91 (dd, J ¼ 7.9,
d
154.7, 152.7, 152.5, 141.5, 139.7, 127.7, 127.4, 127.2, 125.4, 125.1,

J. Peng et al. / European Journal of Medicinal Chemistry 158 (2018) 302e310
309
123.2,119.3, 39.1, 34.9, 34.6, 30.8, 25.9, 25.8, 19.4 ppm. LC-MS: 380.1
485 nm and emission at 525 nm.
[M-H]-, 382.0[MþH]þ; HRMS (ESI-TOF) m/z: [M-H]- Calcd for
C
23H30N3O2 380.2344; Found 380.2346. Purity: 98.7%.
5.3.2. Reagents and chemicals
Citric acid monohydrate (>99.5% purity, determined by HPLC)
and Trisdium citrate dehydrate (>99% purity, determined by HPLC)
were purchased from Nanjing Chemical Reagent CO., LTD.
Adenosine diphosphate (ADP) was purchased from Amresco
(USA). Vicagrel was purchased from Jiangsu Vcare Pharmatech CO.,
LTD.
5.2.13. 1-(2-(2-(tert-butyl)phenoxy)-6-methylpyridin-3-yl)-3-(4-
(trifluoromethoxy)phenyl)urea 13
To a solution of 1-isocyanato-4-(trifluoromethoxy)benzene 25
(215 mg, 1.06 mmol) in oluene (2 mL) in microwave tube was added
2-(2-(tert-butyl)phenoxy)pyridin-3-amine (170 mg, 0.70 mmol).
The resulting mixture was heated with shaking at 80 ꢀC for 1.5 h.
The solvent was removed by vacuum, extracted with ethyl acetate
and water, the combined organic layer was washed with brine,
dried over Na2SO4, filtered, and concentrated to give the crude
product, which was purified by column chromatography (petro-
leum ether/ethyl acetate 4:1 v/v) to yield as a white solid (120 mg,
5.3.3. Animals
Experiments were performed in adult male Sprague-Dawley
(SD) rats weighting 180e220 g. Rats were purchased from Qin-
glongshan Animmal Central (Nanjing, Jiangsu, China). The rats were
housed in a room with a 12: 12 h light: dark cycle at an ambient
temperature of 22e25 ꢀC and a relative 60% humidity. Animals
were allowed free access to water. Animals used in this study were
maintained in accordance with the guide for care and use of Lab-
oratory Animal published by the U.S. National institutes of Health
(NIH publication NO.85-23, revised 1996) and the experiment was
approved by the Animal Ethics Committee of China Pharmaceutical
University (Jiangsu, China).
Blood sample was collected from rats with 3.8% sodium citrate
solution (9:1, v/v). Then, the sample was centrifuged for 10 min at
1000 rpm to obtain platelet-rich plasma (PRP). The remaining
material was centrifuged again for 10 min at 3000 rpm to get
platelet-poor plasma (PPP).
38.3%) m.p. 161e163 ꢀC. 1H NMR (400 MHz, CD3OD)
d 8.35 (d,
J ¼ 8.4 Hz, 1H), 7.55e7.49 (m, 2H), 7.45 (dd, J ¼ 7.6, 2.0 Hz, 1H),
7.22e7.15 (m, 3H), 7.14e7.10 (m, 1H), 6.91 (d, J ¼ 8.0 Hz, 1H), 6.81
(dd, J ¼ 8.0, 1.2 Hz, 1H), 2.25 (s, 3H), 1.40 (s, 9H) ppm.; 13C NMR
(125 MHz, DMSO‑d6)
d 152.8, 152.6, 151.9, 148.0, 142.7, 140.9, 138.8,
127.7, 127.1, 127.0, 124.3, 123.3, 122.1, 121.8, 119.2, 117.9, 34.3, 30.5,
23.0.ppm. LC-MS: 458.1 [M-H]-, 459.9 [MþH]þ; HRMS (ESI-TOF) m/
z: [M-H]- Calcd for C24H23F3N3O3 458.1697; Found 458.1696. Purity:
96.9%.
Compounds 14e15 were prepared according to the procedures
described for compound 13.
5.2.14. 1-(2-(2-(tert-butyl)phenoxy)phenyl)-3-(4-
(trifluoromethoxy)phenyl)urea 14
5.3.4. Rat anti-platelet aggregation assay
The antiplatelet aggregation study was performed by measuring
platelet aggregation using platelet aggregometer (LG-PABER-I).
m.p. 143e145 ꢀC. 1H NMR (400 MHz, CDCl3)
d
8.20 (dd, J ¼ 8.0,
1.6 Hz, 1H), 7.39 (dd, J ¼ 8.0, 2.0 Hz, 1H), 7.31e7.25 (m, 3H),
7.15e7.04 (m, 5H), 7.01e6.96 (m, 1H), 6.76 (dd, J ¼ 8.0, 1.6 Hz, 1H),
6.72e6.67 (m, 2H), 1.36 (s, 9H) ppm; 13C NMR (125 MHz, DMSO‑d6)
Platelet aggregation agonist ADP (100 mmol/L) was prepared with
0.9% saline solution. Forty SD rats were randomly divided into 8
group containing 5 rats in each group. The rats were treated with
chemical compounds (3.0 mg/kg) and vehicle. Two hours after
intragastric administration of chemical compounds and vehicle,
d
155.2, 152.4, 146.8, 140.6, 138.9, 130.6, 127.7, 127.3, 124.0, 122.9,
122.7, 121.8, 120.6, 120.0, 119.2, 117.1, 34.5, 30.3.ppm. LC-MS: 443.0
[M-H]-, 444.9 [MþH]þ; HRMS (ESI-TOF) m/z: [M-H]- Calcd for
C
24H22F3N2O3 443.1588; Found 443.1588. Purity: 98.1%.
blood samples were obtained. Then, 300
correction while 270 L PRP were added into a curvette and incu-
bated at 37 ꢀC for 60 s before the addition of 30
L of ADP with
mL PPP was as blank
m
5.2.15. 1-(2-(2-(tert-butyl)phenoxy)-4-methylphenyl)-3-(4-
m
(trifluoromethoxy)phenyl)urea 15
stirring. Platelet aggregation was estimated after stirring for 240 s.
m.p. 161e162 ꢀC. 1H NMR (400 MHz, CDCl3)
d
7.96 (d, J ¼ 8.0 Hz,
1H), 7.38 (dd, J ¼ 7.6, 2.0 Hz, 1H), 7.24e7.19 (m, 2H), 7.12e7.02 (m,
5H), 6.89 (dd, J ¼ 8.4,1.2 Hz, 1H), 6.84 (s, 1H), 6.67 (dd, J ¼ 8.0, 1.6 Hz,
1H), 6.60e6.57 (m, 1H), 2.22 (s, 3H), 1.35 (s, 9H) ppm.; 13C NMR
Abbreviations
ADP, adenosine-50-diphosphate; MRS2500, (10R,20S,40S,50S)-4-(2-
iodo-6-methylaminopurin-9-yl)-1-[(phosphato)methyl]-2(phos-
phato)bicycle[3.1.0]-hexane; BPTU, 1-(2-(2-tert-butylphenoxy)pyr-
(125 MHz, DMSO‑d6)
d 155.4, 152.5, 146.7, 142.5, 140.4, 139.0, 132.1,
128.1, 127.7, 127.3, 123.8, 123.6, 122.3, 121.8, 121.4, 120.3, 120.3, 119.1,
117.7, 34.4, 30.3, 20.4. ppm. LC-MS: 457.0 [M-H]-, 458.9 [MþH]þ;
HRMS (ESI-TOF) m/z: [M-H]- Calcd for C25H24F3N2O3 457.1745;
Found 457.1750. Purity: 95.0%.
idin-3-yl)-3-(4-
(trifluoromethoxy)phenyl)urea;
DMF,
N,N-
Dimethylformamide; HATU, 1-[Bis(dimethylamino)methylene]-1H-
1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; TEA,
Triethylamine; DIEA, N,N-Diisopropylethylamine; SAR, structure-
activity relationship.
5.3. Biological experiment
5.3.1. Materials and methods
5.3.1.1. Cell culture. 1321N1 cells stably expressing P2Y1 were
cultured in DMEM supplemented with 10% fetal bovine serum, 100
Acknowledgments
We gratefully acknowledge financial support from the
National Natural Science Foundation of China (81620108027,
21632008, 21672231, and 21472209), the Major Project of Chinese
National Programs for Fundamental Research and Development
(2015CB910304).
U/mL penicillin G, and 100 g/mL streptomycin sulfate, 275 mg/mL
G418 at 37 ꢀC in a humidified atmosphere of 95% air and 5% CO2.
5.3.1.2. Intracellular calcium assay. 1321N1/P2Y1 cells were
cultured in 96-well plates for 24 h, the cells were incubated with
2 m
M Fluo-4-AM diluted in HBSS buffer at 37 ꢀC for 50 min. After
loading, cells were treated with the compounds of interest, then
calcium responses were subsequently measured by MRS2365 (se-
lective P2Y1 receptor agonist) stimulation using Flexstation 3
(Molecular Device, USA) with fluorescence excition made at
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.09.014.

310
J. Peng et al. / European Journal of Medicinal Chemistry 158 (2018) 302e310
M. LeMeur, J.P. Cazenave, C. Gachet, Defective platelet aggregation and
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