Switching a Xanthine Oxidase Inhibitor to a Dual-Target Antagonist of P2Y1and P2Y12as an Oral Antiplatelet Agent with a Wider Therapeutic Window in Rats than Ticagrelor

Article abstract of DOI:10.1021/acs.jmedchem.0c01524

ADP-mediated platelet aggregation is signaled through G protein-coupled receptors P2Y1 and P2Y12 on the platelet. The clinical effectiveness of inhibiting P2Y12 has been well established, and preclinical studies indicated that the inhibition of P2Y1 could provide equivalent antithrombotic efficacy as P2Y12 antagonists and reduce bleeding risks. On the basis of the 2-phenyl-1H-imidazole scaffold of our previously reported xanthine oxidase inhibitor WSJ-557, we first achieved the transition from the xanthine oxidase inhibitors to dual-target antagonists against P2Y1 and P2Y12. We described the structure-activity relationships of the 2-phenyl-1H-imidazole compounds, which led to the identification of the most potent antiplatelet agents, 24w and 25w, both showing a rapid onset of action in pharmacokinetic study. Furthermore, the rat model suggested that 24w demonstrated a wider therapeutic window than ticagrelor, displaying equivalent and dose-dependent antithrombotic efficacy with lower blood loss compared to ticagrelor at same oral dose. These results supported that 24w and 25w could be promising drug candidates.

Full text of DOI:10.1021/acs.jmedchem.0c01524

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Article
Switching a Xanthine Oxidase Inhibitor to a Dual-Target Antagonist
of P2Y and P2Y as an Oral Antiplatelet Agent with a Wider
1
12
Therapeutic Window in Rats than Ticagrelor
∥
∥
Yu Lei, Bing Zhang, Dan Liu, Jian Zhao, Xiwen Dai, Jun Gao, Qing Mao, Yao Feng, Jiaxing Zhao,
Fengwei Lin, Yulin Duan, Yan Zhang, Ziyang Bao, Yuwei Yang, Yanhua Mou,* and Shaojie Wang*
Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c01524
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sı Supporting Information
ABSTRACT: ADP-mediated platelet aggregation is signaled through G protein-coupled receptors P2Y and P2Y12 on the platelet.
1
The clinical effectiveness of inhibiting P2Y12 has been well established, and preclinical studies indicated that the inhibition of P2Y1
could provide equivalent antithrombotic efficacy as P2Y12 antagonists and reduce bleeding risks. On the basis of the 2-phenyl-1H-
imidazole scaffold of our previously reported xanthine oxidase inhibitor WSJ-557, we first achieved the transition from the xanthine
oxidase inhibitors to dual-target antagonists against P2Y and P2Y . We described the structure−activity relationships of the 2-
1
12
phenyl-1H-imidazole compounds, which led to the identification of the most potent antiplatelet agents, 24w and 25w, both showing
a rapid onset of action in pharmacokinetic study. Furthermore, the rat model suggested that 24w demonstrated a wider therapeutic
window than ticagrelor, displaying equivalent and dose-dependent antithrombotic efficacy with lower blood loss compared to
ticagrelor at same oral dose. These results supported that 24w and 25w could be promising drug candidates.
6
,11−16
INRODUCTION
formation.
The inhibition of platelet aggregation
■
targeting the P2Y12 receptor has been recognized as an
important element in the short-term treatment as well as for
the long-term prevention of thrombotic events in patients with
Acute coronary syndromes (ACSs), including unstable angina
and acute myocardial infarction (AMI), are life-threatening
thrombotic disorders, which have been the most common
causes of morbidity and mortality over the past decade
2
,11−14,16
ACS.
P2Y is ubiquitously expressed, and mobilizes
1
2
+
1
−3
the transitory increases in intracellular free Ca ions leading to
platelet shape changes in response to ADP.
worldwide in cardiovascular patients.
Platelets display a
16−19
It is reported
major role in these thrombotic complications. They adhere to
the subendothelial matrix following endothelial damage due to
the rupture of an atherosclerotic plaque and then aggregate to
cause thrombus formation. This activation process involves
several platelet-activating agonists, such as adenosine diphos-
phate (ADP), thrombin, and thromboxane A2. Among them,
ADP is a key mediator of activation as well as the aggregation
of platelets. It could bind to two purinergic receptors P2Y and
that the inhibition of P2Y1 could provide an equivalent
antithrombotic efficacy to P2Y12 in terms of blocking
aggregation and reducing thrombus weight, whereas P2Y1
antagonists may offer safety advantages in terms of a reduced
4
−
1
8−21
6,7
bleeding liability.
This demonstrates that the P2Y1
receptor could also represent a promising target for the
18−21
development of new antiplatelet therapies.
Therefore,
1
P2Y , which both further activate glycoprotein IIb/IIIa on
1
2
Received: September 1, 2020
platelets and lead to sustained platelet aggregation as well as
4
−8
thrombus growth. Specifically, P2Y is mainly expressed on
12
the membrane of human thrombocytes, and the binding of
ADP to the P2Y12 receptor results in a reduction of cAMP
(
cyclic adenosine monophosphate) levels, which is required to
amplify and sustain the responses leading to a stable thrombus
©
XXXX American Chemical Society
https://dx.doi.org/10.1021/acs.jmedchem.0c01524
A
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Figure 1. (A) Structures of P2Y12 antagonists: ticlopidine (1), clopidogrel (2), prasugrel (3), vicagrel (4), ticagrelor (5), cangrelor (6), AZD1283
7), elinogrel (8), selatogrel (9), BX048 (10), SAR216471 (11), and PSB-0739 (12). (B) Structures of P2Y antagonists: MRS2179 (13),
(
1
MRS2500 (14), MRS2279 (15), BPTU (16), and 4-aryl-7-hydroxylindoline derivative (17).
specifically inhibiting either of the two receptors or inhibiting
both P2Y1 and P2Y12 could halt ADP-induced platelet
aggregation for the treatment of arterial thrombosis and
2
2−24
related diseases.
B
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Figure 2. Design and structural modification of novel antiplatelet agents.
P2Y receptor antagonists can be broadly classified into two
4
7
clinical development: vicagrel (4, Figure 1) and selatog-
rel
1
2
36,48
classes on the basis of their chemical structures, namely,
(9, Figure 1). Vicagrel (Figure 1), an irreversible
thienopyridine oral P2Y12 receptor antagonist, may show
stronger platelet inhibition and a faster onset of action
compared to clopidogrel. However, it still retains the same
activation mechanism as prasugrel.
1
6,25
thienopyridines and nonthienopyridines.
The thienopyr-
(1, approved in 1978,
(2, approved in 1997, Figure 1), and
16,25
idine drugs, such as ticlopidine
2
,26
Figure 1), clopidogrel
2
7
2,16,47
prasugrel (3, approved in 2009, Figure 1), are irreversible
Selatogrel (2-phenyl-
oral P2Y receptor prodrugs and have proved to be successful
pyrimidine-4-carboxamide derivative, Figure 1) could be
rapidly absorbed and shows a potentially lower risk of
bleeding. However, it is only developed for subcutaneous not
1
2
in reducing the morbidity and mortality for cardiovascular
2
,16,28
patients.
In fact, the dual antiplatelet therapy of
36,48
clopidogrel and acetylsalicylic acid has long been the gold
oral administration.
Besides, P2Y antagonists, such as
1
20
16,29
49
standard of treatment.
However, they all require hepatic
MRS21795 (13, Figure 1), MRS2500 (14, Figure 1),
0 21
metabolic bioactivation for their active metabolites to
covalently bind to the P2Y12 receptor, which results in a
MRS2279 (15, Figure 1), BPTU (16, Figure 1), and 4-aryl-
7-hydroxylindoline derivative (17, Figure 1), are in preclinical
development. Therefore, it is still necessary to explore an oral
antiplatelet agent targeting P2Y and P2Y for the treatment
2
,16,30,31
slow onset of their pharmacological action.
This leads
to the development of several reversible nonthienopyridine
1
12
P2Y12 receptor antagonists based on the nucleotide scaffold,
of ACS on the basis of new chemical scaffolds to overcome the
1
such as ticagrelor (5, approved in 2011, Figure 1) and
drawbacks of these clinical drugs and achieve a fast onset of
3
0−32
1
11,14,16,35,36
cangrelor
(6, approved in 2015, Figure 1). Ticagrelor
action with less bleeding risk.
(
Figure 1) was the first drug developed and is administered
In our previous reports of the nonpurine xanthine oxidase
(XO) inhibitor, WSJ-557 (2-(3-cyano-4-isobutoxyphenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxylic acid, Figure 2)
demonstrated a stronger XO inhibitory potency (IC50 = 0.003
μM for XO) than that of febuxostat (IC = 0.01 μM for
orally. It reversibly binds the P2Y12 receptor and has a faster
onset of action than clopidogrel, but it has been shown to
increase the rate of nonprocedure-related bleeding compared
3
3,34
30−32
to clopidogrel.
Cangrelor
(Figure 1) is another
5
0
51,52
nucleotide-derived reversible P2Y12 antagonist with a rapid
onset of action. However, it must be administered by a
continuous intravenous infusion and is only used at the time of
percutaneous coronary intervention (PCI) in patients not
preloaded with an oral P2Y12 receptor antagonist.
Consequently, there is still room for improvement in currently
approved oral antiplatelet agents due to the drawbacks of slow
XO).
The remarkable XO inhibitory potency of WSJ-557
encouraged us to investigate its pharmacokinetic (PK) profiles
52
to further assess its development potential. In this procedure,
it was found that WSJ-557 could delay blood coagulation,
which drove us to investigate its action mechanism. The
subsequent antiplatelet aggregation assay induced by ADP
showed that the compound could display an apparent
antiplatelet potency (IC50 = 15.727 μM).
2
,16
14,16,35,36
onset of action and high bleeding risk.
25,37,38
38,39
Recently, AZD1283
(7, Figure 1) and elinogrel
These interesting results suggested that the WSJ-557 with a
2-phenyl-1H-imidazole scaffold could be considered as an
initial compound for the development of P2Y and P2Y dual
(
8, Figure 1), based on the ethyl 6-aminonicotinate acyl
sulfonamide and quinazoline-2,4-dione scaffolds, respectively,
both showed potent antithrombotic efficacy and reduced
1
12
antagonists.
11,40
bleeding effects in animal models.
Unfortunately, they
We described the procedure for switching the XO inhibitor
were discontinued in clinical trials due to low metabolic
WSJ-557 to dual-target antagonists of P2Y and P2Y through
1
12
11,39
stability and elevated liver transaminases.
Apart from these,
structure−activity relationships (SAR) investigation of the 2-
phenyl-1H-imidazole compounds, which led to the identi-
fication of compounds 24w and 25w as the most potential
antiplatelet agents in ADP-induced rabbit platelet-rich plasma
(rPRP) aggregation test in vitro (Figure 2). P2Y and P2Y
other P2Y12 receptor antagonists based on various chemical
4
1,42
scaffolds, including BX048
(10, 7-methylquinoline-2-
43,44
carboxamide derivative, Figure 1), SAR216471
(11, indole
(12, anthraquinone
derivative, Figure 1), are reported to exhibit more effective
45,46
derivative, Figure 1), and PSB-0739
1
12
binding assays were also performed to investigate the effect of
compound 25w on platelet P2Y and P2Y receptors by the
43−46
platelet inhibition, but they are still in preclinical research.
1
12
These facts indicate that the P2Y12 antagonists distinct from
the thienopyridine and nucleotide chemical scaffolds have
received extensive attention and will hopefully minimize these
disadvantages of currently available drugs. Furthermore, two
novel P2Y12 receptor antagonists are currently in phase II
flow cytometric assay. Moreover, the probable binding models
of the target compounds 24w and 25w with P2Y and P2Y
receptors were explored by molecular modeling. Additionally,
metabolic stability, pharmacokinetic profile, and acute toxicity
studies were performed to support the pharmacological
1
12
16
C
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a
Scheme 1
a
Reagents and conditions: (a) NaNO , CH COOH, 0−5 °C; (b) Benzaldehyde derivatives, CH COOH/CH COONH , 50 °C; (c) Me SO ,
2
3
3
3
4
2
4
K CO , DMF, 0 °C.
2
3
a
Scheme 2
a
Reagents and conditions: (a) Me SiCl, NaI, CH CN, reflux; (b) Me SO , K CO , DMF, 0 °C; (c) RX, K CO , KI, N , DMF, 0−50 °C; (d)
3
3
2
4
2
3
2
3
2
LiOH, THF, H O, 50 °C.
2
characterization of the most potent compounds 24w and 25w.
Lastly, the compound 24w was further evaluated in a rat ferric
chloride model as well as a rat-tail-bleeding model to
investigate its antithrombotic effect and bleeding risk,
respectively. These results obtained from the investigations
supported that 24w and 25w could be promising drug
candidates for the treatment of arterial thrombosis and related
diseases.
Then, the 1-hydroxyl moiety of compound 21a was removed
with chlorotrimethylsilane and sodium iodide by refluxing in
53
acetonitrile to provide compound 23, which was further
alkylated with iodomethane or 1-bromo-2-methylpropane in
N,N-dimethylformamide in the presence of anhydrous
potassium carbonate and potassium iodide to provide
51
compounds 24a and 24b. Compounds 24c−y were prepared
through the alkylation reaction of the compound 21a with
appropriate alkyl halides in DMF in the presence of K CO
2
3
and KI, which were hydrolyzed using an aqueous solution of
RESULTS AND DISCUSSION
■
lithium hydroxide to afford the compounds 25a, 25d, 25k, 25s,
51
Chemistry. The key intermediate ethyl 2-hydroxyimino-3-
oxobutanoate 20 was obtained by nitrosation of the
commercially available ethyl 3-oxobutanoate 19 with sodium
and 25w (Scheme 2). The structures were elucidated by
high-resolution mass spectrometry (HRMS), infared (IR),
1
13
proton nuclear magnetic resonance ( H NMR), and C NMR
spectra (Supporting Information). All spectral data were in
accordance with the assumed structures.
51
nitrite in acetic acid. Commercially available benzaldehyde
derivatives were cyclized with the key intermediate 20 to give
compounds 21a−d, which were further alkylated with
iodomethane in N,N-dimethylformamide in the presence of
anhydrous potassium carbonate to provide target compounds
Discovery of Lead Compound 22a. Since the ethyl ester
of WSJ-557 (compound 18) displays an equivalent antiplatelet
potency (IC50 = 16.825 μM), the ethyl ester group of 5-
position was retained for further structural optimization for the
51
2
2a−d (Scheme 1).
D
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possibility of higher bioavailability. Initially, we found that
removing the 4′-isobutoxy group had no apparent effect on its
antiplatelet potency (21a vs 18, IC = 17.636 and 16.825 μM,
6.796, and 9.134 μM, respectively). For comparison, the more
polar compounds 24g, 24h, and 24i containing hydroxypro-
poxy, 2-amino-2-oxoethoxy, and 2-carboxyethyloxy groups at
the 1-position were synthesized, and the results showed that
compound 24g exhibited an IC50 value of 11.352 μM and the
antiplatelet potency for compounds 24h and 24i disappeared
completely. This indicated that enhancing the polarity of
substituents at the 1-position was not beneficial for antiplatelet
potency.
50
respectively, Table 1). In addition, the introduction of a small
Table 1. In Vitro Antiplatelet Aggregation Potency of Lead
Optimization on Imidazole Derivatives
In order to explore the antiplatelet effect of alkoxy groups at
the 1-position further, the pyridin-4-ylmethoxy and benzloxy
substitutions were introduced to provide compounds 24j and
2
4k, and they exhibited an apparent inhibitory potency in the
platelet aggregation assay than that of compound 22a (24j,
4k vs 22a, IC50 = 6.196, 5.934, and 9.134 μM, respectively).
2
The remarkable antiplatelet potency of compound 24k
encouraged us to tune the substituents on the phenyl moiety.
First, the electron withdrawing substituents, such as fluoro and
chloro substituents, were inserted at ortho, meta, and para
positions of the benzyloxy group. Among them, the para-
substituted derivatives showed a better antiplatelet aggregation
potency compared to ortho- and meta-substituted derivatives
(
2
24n > 24l > 24m; 24q > 24o > 24p, IC50 = 5.879, 8.987,
2.358, 6.825, 8.705, and 19.357 μM, respectively), These
results showed that the insertion of fluoro and chloro atoms at
the para position was beneficial for improving the inhibitory
potency. Consequently, the continued investigations of
substituents at the para position of the benzyloxy group
were carried out. The introduction of bromo, methyl, methoxy,
methoxycarbonyl, nitro, and cyano groups into the para
a
ADP-induced platelet aggregation ([ADP] = 2.27 μM, n = 3), rabbit
position led to compounds 24r−y with IC values of 24.590,
platelet-rich plasma (rPRP).
50
6.311, 6.826, 9.466, 8.754, and 4.237 μM, respectively. To our
surprise, compound 24w (IC50 = 4.237 μM) with a cyano
group at the para position displayed the most remarkable
inhibitory potency, and it was comparable to that of ticagrelor
(IC50 = 7.213 μM). Subsequently, the corresponding ortho and
meta counterparts were examined. The results showed that 24x
displayed a 2.2-fold decrease in inhibitory potency in
comparison to compound 24w, and the inhibitory potency
of compound 24y was lost (24w and 24y, IC50 = 9.376 and
>30 μM, respectively). This showed the same inhibitory
tendency as compounds with fluoro and chloro atoms
substituted at benzyloxy moiety. Presumably, the cyano
group substituted at the para position kept the 4-
cyanobenzyloxy moiety in a more favorable position so that
it could form better interactions with amino acid residues at
the active pocket.
hydrophobic methyl group into the 1-hydroxy position of
imidazole moiety was beneficial for remarkably increasing the
antiplatelet potency (21a < 22a, IC = 17.636 and 9.134 μM,
respectively). Consequently, 22a was further examined to
determine the antiplatelet influence by cyano group positions
on the phenyl moiety. The corresponding ortho-, para-, and no
cyano substituted derivatives were synthesized, and these
compounds showed a marked decrease in inhibitory potency
50
(
9
22b, 22c, and 22d vs 22a, IC50 = 14.857, 13.353, > 30, and
.134 μM, respectively), which implied that meta-cyano
substitution was necessary for the antiplatelet potency.
Moreover, removing the 1-hydroxy group led to compound
2
3, which displayed a loss in antiplatelet potency (23, IC50
>
3
0 μM). Therefore, on the basis of these data, the potent lead
compound 22a containing the 2-phenyl-1H-imidazole scaffold
was selected for further optimization.
Lastly, to explore the inhibitory potency of the correspond-
ing acids, compounds 22a, 24d, 24k, 24s, and 24w were
hydrolyzed to obtain compounds 25a, 25d, 25k, 25s, and 25w,
and the compounds showed equal potency in comparison to
their ester counterparts. (25a vs 24a; 25d vs 24d; 25k vs 24k;
25s vs 24s, IC50 = 7.987 vs 9.134 μM; IC50 = 6.350 vs 5.856
μM; IC50 = 6.238 vs 5.934 μM; IC50 = 6.563 vs 6.311 μM,
respectively). Among them, compound 25w (IC50 = 3.875
μM), the acid of compound 24w, displayed the same
remarkable inhibitory potency as that of compound 24w,
and it was also comparable to that of ticagrelor (IC50 = 7.213
μM).
Identification of Compounds 24w and 25w. The
observation that introducing a methyl at the hydroxyl group at
the 1-position could increase the antiplatelet potency greatly
inspired us to investigate the substitution pattern at the 1-
position further. First, the compounds containing methyl,
isopropyl, and isopropoxy groups were synthesized. The results
suggested that the antiplatelet potency of inserting alkoxy
groups into the 1-position was stronger than those of
corresponding alkyl groups (24a < 22a, 24b < 24c, IC50
=
2
2.136, 9.134, 9.259, and 6.736 μM, respectively, Table 2).
P2Y -Mediated Cytosolic Ca2+ Increases Assay. ADP
Then, introducing allyloxy, ethoxyethoxy, and 2-ethoxy-2-
1
oxoethoxy groups at the 1-position led to compounds 24d,
activates platelets by simultaneously acting on two platelet G
18
2
4e, and 24f, and their antiplatelet potency was further
enhanced (24d, 24e, and 24f vs 22a, IC50 = 5.856, 6.247,
protein−coupled receptors P2Y and P2Y . However, it is
1
12
not clear which of the two receptors was inhibited by the test
E
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Table 2. In Vitro Antiplatelet Aggregation Potency of Variations Around the Imidazole Moiety
a
ADP-induced platelet aggregation ([ADP] = 2.27 μM, n = 3), rabbit platelet-rich plasma (rPRP).
F
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compounds to exert their antiplatelet effect. To elucidate the
action mechanism of these test compounds in its antiplatelet
potency, the antagonist effects of the most potent compound
2
5w on platelet P2Y were determined by measuring the P2Y -
1
1
2+
mediated cytosolic Ca increase from intraplatelet stores after
stimulation by ADP as previously described with minor
modifications.
2
2,23,46,54,55
The results are listed in Table 3.
Table 3. Inhibitory Potency of ADP-Induced Ca2+ Increase
a
compounds
5w
BPTU
inhibition of Ca2+ rise in platelets (IC , μM)
50
2
2.59 ± 0.36
3.03 ± 0.63
Figure 3. Inhibition of ADP-induced, P2Y12-mediated decrease in
VASP phosphorylation by compound 25w or ticagrelor. PGE1-
stimulated VASP phosphorylation and its attenuation by ADP in the
presence and absence of the test compounds were measured by flow
cytometry. The data were reported as the mean ± SD (n = 3; *P >
a
In diluted rPRP, n = 3.
Compound 25w displayed a potent inhibitory effect against
P2Y on platelet with an IC value of 2.59 μM, which was
0.05, as compared to PGE alone).
1
50
1
comparable to that of BPTU (IC50 = 3.03 ± 0.63 μM),
suggesting that compound 25w exerted its antiplatelet potency
specifically binding to P2Y . Among them, the antagonist
1
2
mainly by inhibiting receptor P2Y on platelet. Besides, several
potency of compound 25w was higher than that of MRS2395,
1
research studies have already demonstrated that the P2Y1
antagonists could have an equivalent antithrombotic efficacy
but less bleeding compared with the P2Y antagonist
an antagonist for the P2Y purinoceptor reported in the
12
61
literature. Nevertheless, the antagonist potency of both
compound 25w and MRS2395 was lower than that of
ticagrelor (ticagrelor > 25w > MRS2395, IC50 = 5.14,
148.92, and 176−196 μM, respectively). In addition,
compound 25w and ticagrelor both could not cause a decrease
of VASP phosphorylation in the absence of ADP, which was
1
2
1
9,20
clopidogrel.
Therefore, this activity evaluation of P2Y1
rationalized that compound 24w (parent compound 25w
released from prodrug 24w in rats after oral administration)
exhibited a therapeutically equivalent antithrombotic effect as
that of ticagrelor at the same oral dose, with remarkably less
blood loss than ticagrelor.
comparable to that of PGE alone, suggesting that they did not
1
22,23
possess P2Y12 agonistic effects.
The results above proved
P2Y12 Binding Assay. To examine the effect of compound
5w on the platelet P2Y12 receptor in response to ADP (no
test of compound 24w due to poor solubility), P2Y12 binding
that compound 25w could bind to P2Y12 purinoceptor as an
antagonist. Therefore, we could confirm that compound 25w
exerted its antiplatelet effect through both P2Y and P2Y
2
1
12
assay was performed by measuring the P2Y -mediated
decrease in intraplatelet phosphorylated vasodilator-stimulated
receptors.
1
2
Xanthine Oxidase Inhibitory Activity. The evaluation of
the in vitro bovine XO inhibitory potency of compounds 24w
and 25w was performed with febuxostat as a reference
compound. The results showed that compounds 24w and
25w displayed no significant inhibition against XO (not active,
inhibition at 10 μM < 50%), and the IC50 value of febuxostat
was 0.0189 μM.
phosphoprotein (VASP) using a flow cytometric PLT VASP/
22,23,56−59
P2Y12 kit (Biocytex, Marseille, France).
ADP, as an
agonist, activated the P2Y12 receptor to trigger the reaction of
VASP phosphorylation. Meanwhile, it also antagonized the
P2Y12 receptor to induce the decrease of VASP phosphor-
ylation. Then, the test compounds were added to antagonize
the P2Y receptor to attenuate the antagonistic effect of ADP,
Docking Studies. To explore a probable interaction model
1
2
resulting in the increase of VASP phosphorylation, and this
of compounds 24w and 25w with P2Y1 and P2Y , the
molecular docking of compounds 24w and 25w in MRS2500
and AZD1283 binding pockets of proteins was performed
12
protocol was used to measure the antagonistic effect of
5
6−59
compounds.
Thus, the percentage inhibition of prosta-
glandin E (PGE )-stimulated VASP phosphorylation was set
as 100%, and no stimulation was set as 0% inhibition.
using the Glide XP docking protocol (2016, Schrodinger
1
1
59
62
Suite). The X-ray crystal structures of the P2Y /MRS2500
1
63
64
Specifically, the percentage inhibition was calculated relative to
vehicle (0% inhibition) and PGE1 (100% inhibition):
inhibition (%) = [(MFI(PGE1) − MFI(PGE1+ADP+compound))/
(PDB: 4XNW) and P2Y /AZD1283 (PDB: 4NTJ) used
12
in the docking studies were obtained from the RCSB Protein
Data Bank, and MRS2500 as well as AZD1283 were adopted
as references. The proteins were prepared by removing all
water molecules and adding all hydrogen atoms using Protein
6
0
(
MFI(PGE1) − MFI(negative))] × 100, where MFI is the mean
59
fluorescence intensity of VASP phosphorylation. In the
absence of ADP, P2Y −VASP phosphorylation was performed
to investigate whether compounds possessed an agonistic effect
6
2
Preparation Wizard (2016, Schrodinger Suite).
The
12
phosphonic acid and carboxyl groups of all compounds were
calculated in dissociated forms using the LIGPREP module
on the P2Y12 receptor.
62
As expected, PGE is added to induce the full phosphor-
(2016, Schrodinger Suite).
The binding models of compounds 24w and 25w were
1
ylation of VASP, which was set as 100%, and VASP
phosphorylation in the presence of the P2Y12 agonist ADP
65
illustrated by Pymol (Figure 4). For the binding mode of
(
3 μM) was significantly reduced in comparison with PGE
P2Y , we found that the carboxylic acid group of compound
1
1
2
2,23
alone (Figure 3).
Furthermore, compound 25w and
25w could engage in three hydrogen bonds with key residues
Thr205, Tyr206, and Arg310, which was almost equivalent to
the interactions formed by the phosphate group of MRS2500,
and it could also interact with key residue Asn283 via two
hydrogen bonds. In addition, an extra electrostatic interaction
ticagrelor both were able to dose dependently antagonize the
ADP-induced reduction of VASP phosphorylation compared
to PGE1 alone, indicating that the antagonist effects of
compound 25w and ticagrelor were mediated through
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Figure 4. Binding modes of (A) MRS2500, (B) compound 24w, and (C) compound 25w in the P2Y receptor (PDB: 4XNW). Binding modes of
1
(
D) AZD-1283, (E) compound 24w, and (F) 25w in the P2Y12 receptor (PDB: 4NTJ). Protein is shown as a cartoon, and small molecules are
shown as sticks. Hydrogen bonds, π−π stacking interactions, and electrostatic interaction are depicted by red, purple, and orange dashed lines,
respectively. Residues of P2Y and P2Y interacting with inhibitors are depicted by green sticks.
1
12
Figure 5. (A and B) Stability profiles of compounds 24w and 25w in simulated gastric and intestinal fluids. Each value is the mean ± SEM; n = 3.
was formed between 25w and Arg287 (Figure 4C), and a π−π
stacking interaction was observed between the imidazole
moiety of 25w and Tyr203. Moreover, the cyano groups on
phenyl and benzyl moieties of 25w formed new hydrogen
bonds with Tyr203, Gln291, and Asn299, respectively, which
was lacking in MRS2500. In terms of compound 24w, the
similar key hydrogen bonds with Thr205, Tyr206, Arg310, and
Asn283 were also observed at the cyano group on the phenyl
moiety and the N atom on the imidazole ring, and the cyano
and ester carbonyl groups were able to interact with Tyr203
and Gln291 via two hydrogen bonds. The imidazole moiety of
compound 24w could also interact with Tyr 303 through a
π−π stacking interaction. Nevertheless, compounds 24w and
of the cyano group of AZD1283 in the binding pocket of
P2Y12 (Figure 4A). Moreover, the negatively charged
carboxylic acid group of compound 25w formed an electro-
static interaction with the positively charged side chain of
Lys280, which is consistent with those observed in acidic
groups such as sulfonate or carboxylic acid groups presented in
anthraquinone and glutamic acid piperazine P2Y antago-
nists, whereas the ester carbonyl group of compound 24w
only engaged in a hydrogen bond with the side chain of
Lys280. Meanwhile, the phenyl ring of compounds 24w and
25w occupied the same region as the pyridine moiety of
AZD1283 through an aromatic π−π stacking interaction with
Tyr105. Moreover, compared to AZD1283, the imidazole ring
of compounds 24w and 25w formed an additional aromatic
π−π stacking interaction with Tyr105, and the 4-cyanobenzyl
group further inserted into the cavity occupied by the
piperidine moiety of AZD1283.
1
2
66
2
5w both lacked hydrogen bonds formed by the other
phosphate group of MRS2500 with Lys46, Arg195, Thr201,
Tyr203, and Tyr303.
The docking results of P2Y12 showed that the cyano groups
of compounds 24w and 25w were able to form two hydrogen
bonds with the side chains of key residues Tyr109 and Gln195
Simulated Gastric and Intestinal Fluid Stability. To
characterize the in vitro metabolic stability of compounds 24w
and 25w, they were incubated with simulated gastric and
(Figure 4B,C), which was in agreement with the binding mode
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Figure 6. Stability and metabolism profiles of compounds (A) 24w and (B) 25w in rabbit and rat plasma. Each value is the mean ± SEM; n = 3.
intestinal fluid, and the remaining percentages of compounds
4w and 25w after incubation are summarized in Figure 5.
confirmed the formation of compound 25w in later PK studies
in rats after the oral administration of 24w. Moreover, the liver
2
After a 12 h incubation period in simulated gastric fluid,
approximately 90% of the parent compounds 24w and 25w
remained intact, which suggested that they were considerably
stable in simulated gastric fluid. After a 12 h incubation period
in simulated intestinal fluid, compound 24w was also stable
with about 93% remaining while compound 25w was
moderately stable in simulated intestinal fluid, due to 73% of
its parent compound left. These results suggested that
compounds 24w and 25w should be stable in stomach and
intestine.
microsomal stability of compound 24w in HLMs (t1/2
120.65 min, Figure 7C) was better in that in RLMs (t1/2
=
=
23.13 min, Figure 7A), suggesting that there was a species
difference of compound 24w in both RLMs and HLMs, and
compound 24w could be converted to compound 25w rapidly
in rat liver. In addition, compound 25w (t1/2 > 120 min in both
RLMs and HLMs) was considerably stable in both RLMs and
HLMs (Figure 7B,D).
CYP450 Inhibition Assay. CYP450-mediated drug−drug
interaction is one of the important reasons for the dropout of
68
Stability Studies in Plasma. The in vitro stability profiles
of compounds 24w and 25w were further studied by
incubation with both rabbit and rat plasma to observe the
disappearance of the prototype and formation of the
metabolite. Figure 6 showed that 12% of compound 24w
was converted to compound 25w by 45 min in rabbit plasma.
Meanwhile, in rat plasma, 43% of the compound 24w was
hydrolyzed to compound 25w by 45 min. These results
suggested that compound 24w could be rapidly converted to
compound 25w in plasma, which is consistent with the later
PK studies in rats after the oral administration of 24w. The
hydrolysis rate of compound 24w in rat plasma was greater
than that in rabbit plasma. Moreover, no significant
degradation of compound 25w was observed in these two
media during the incubation period, which indicated that
compound 25w was rather stable in both rabbit and rat plasma,
and compound 24w could be used as a prodrug of 25w.
Metabolic Stability in Liver Microsomes. The hepatic
metabolism plays a vital role in biotransformation in the
majority of prodrugs, and liver microsomal metabolic stability
drug candidates during new drug development. Therefore,
testing for drug−drug interaction potential of new chemical
68−70
entities is essential for developing a novel drug.
As a
result, compounds 24w and 25w were selected to further
evaluate their in vitro inhibitory potential of major CYP450
enzymes including CYP1A2, CYP2A6, CYP2C9, CYP2C19,
CYP2D6, CYP2E1, and CYP3A4 in a CYP450 inhibition
cocktail assay, and the results are summarized in Table 5.
Indeed, compound 25w was found to show no significant
inhibition against these CYP450 isoforms at 10 μM, which
indicated that its IC50 value against these major CYP450
isoforms was greater than 10 μM. This result implied that
compound 25w showed no apparent drug−drug interaction
potential at 10 μM. However, compound 24w exhibited a weak
inhibitory potency against CYP2D6 with an inhibition of
31.69% at 10 μM, suggesting that there might be low liability
for drug−drug interactions between 24w and CYP2D6.
Pharmacokinetic Studies. To explore the pharmacoki-
netic assessment of compounds 24w and 25w, they were
further evaluated for their PK properties in Sprague-Dawley
rats, as shown in Figure 8, and their noncompartmental PK
parameters are listed in Table 6.
For a single oral administration of 24w (10 mg/kg), the
prototype of compound 24w (below analytical detection limit
of quantification: 10 ng/mL) was not observed in the
beginning of the oral administration, while compound 25w
was immediately detected at 10 min after the oral
administration of 24w (Figure 8). This suggested that the
compound 24w could be rapidly converted into the metabolite
67
is widely used in the research of drug metabolism. Therefore,
compounds 24w and 25w were further evaluated for their in
vitro liver microsomal metabolic stability, and the data are
listed in Table 4. In Figure 7A,C, compound 24w was rapidly
converted to compound 25w in both rat liver microsomes
(
RLMs) and human liver microsomes (HLMs), which
indicated that compound 24w could be rapidly converted
into compound 25w by hepatic metabolism and further
a
Table 4. Microsomal Stability of Compounds 24w and 25w
2
5w, and these results were consistent with in vitro plasma and
b
c
liver microsomal stability. Furthermore, the average time for
compound
species
t1/2 (min)
Clint (mL/min/mg protein)
compounds 24w and 25w to reach the maximum concen-
2
4w
rat
human
rat
23.13
120.65
>120
>120
0.060
0.011
36
tration (Tmax) was shorter than that of ticagrelor (ticagrelor >
4w > 25w, Tmax = 4 > 0.46 > 0.167 h, respectively), indicating
2
2
5w
that they both were absorbed quickly into circulation. This
human
might demonstrate a faster onset of action than current clinical
14,16,35,36
a
oral P2Y antagonists.
Meanwhile, the half-lives (t1/2
)
Microsomal protein (0.50 mg/mL), NADPH-regenerating system,
inhibitor], 0.2 μM; incubation at 37 °C. T1/2: elimination half-life.
Cl : intrinsic body clearance.
12
b
[
of compounds 24w and 25w were both 13 h, suggesting that
they both could achieve a longer duration of action. Among
c
int
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Figure 7. Stability profiles of compounds 24w and 25w obtained in both RLMs and HLMs: (A) incubation of 24w in RLMs; (B) incubation of
2
5w in RLMs; (C) incubation of 24w in HLMs; (D) incubation of 25w in HLMs. The data represent the mean ± SEM of three independent
experiments (n = 3).
Table 5. CYP450 Inhibition of Compounds 24w and 25w
inhibition at 10 μM (%)
a
b
c
d
e
f
g
compounds
CYP1A2
3.85
0.11
CYP2A6
CYP2C9
CYP2C19
CYP2D6
CYP2E1
CYP3A4
2
2
4w
5w
7.69
3.64
0.024
0.41
4.52
1.97
31.69
2.42
8.68
6.79
2.16
7.40
a
b
c
d
e
f
g
Phenacetin. Coumarin. Tolbutamide. S-Mephenytoin. Dextromethorphan, Chlorzoxazone. Nifedipine.
them, compound 24w displayed slightly better PK properties
compared to those of 25w, including higher plasma exposure
control. The thrombus weights in compound 24w and
ticagrelor groups were significantly reduced compared to that
of the model group (***P < 0.001 for 5, 10, and 20 mg/kg of
24w and 10 mg/kg of ticagrelor vs model; Figure 10),
indicating that this ferric chloride model in rats was
(
AUC0−∞ = 1135.08 ng·h/mL) and moderate oral bioavail-
ability (F = 32.4%). On the basis of its favorable
pharmacokinetic profiles, compound 24w was selected for
further evaluation of its acute oral toxicity study, in vivo
antithrombotic efficacy, and associated bleeding risk assess-
ment in rats.
11,72−74
successfully established,
and compound 24w as well
as ticagrelor both resulted in a remarkable antithrombotic
effect. Among them, the oral administration of compound 24w
demonstrated dose-dependent antithrombotic efficacy, achiev-
ing thrombus weight reductions of 22.6%, 40.7%, and 46.7% at
doses of 5, 10, and 20 mg/kg, respectively. These results
showed that its antithrombotic ED50 was 20.8 mg/kg.
Specifically, compound 24w (10 mg/kg) resulted in a
thrombus weight reduction of 40.7%, which was comparable
Acute Oral Toxicity Study. Prior to the in vivo
pharmacodynamics evaluation, the acute oral toxicity study
of compound 24w was carried out to investigate its preliminary
toxicity profile in healthy mice according to Organization for
Economic Cooperation and Development (OECD) guide-
7
1
lines. The mice were carefully observed for any behavioral
change and mortality after the oral administration of
compound 24w at a single dose of 2000 mg/kg. No death
occurred after oral administration, and all mice in the drug
administration group (male and female) grew normally
compared to the mice in the control group. The body weights
of these mice gradually increased during the subsequent 14
days (Figure 9), and no significant behavioral abnormalities
were observed.
#
to that of ticagrelor (39.2%) at the same 10 mg/kg dose ( P >
0.05 for 10 mg/kg 24w vs ticagrelor). Consequently, the
results of in vivo antithrombotic efficacy evaluation suggested
that compound 24w could be a potentially efficacious
antithrombotic agent in the treatment of thrombotic disorders.
Bleeding Risk Assessment. Since bleeding complications
had largely been considered an expected complication of
antiplatelet therapy for all currently approved oral P2Y
1
2
1
1,28,43
In Vivo Antithrombotic Efficacy. On the basis of the
better in vitro antiplatelet potency and excellent PK properties,
in vivo antithrombotic efficacy of compound 24w was further
evaluated and ticagrelor was tested for comparison as a positive
antagonists,
the bleeding effect of compound 24w was
11,44,72
further evaluated in a rat-tail-bleeding model.
In this
study, the bleeding effect of compound 24w was compared to
those of ticagrelor by measuring the tail-vein-bleeding time and
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Figure 8. Plasma concentration−time profiles of compounds 24w and 25w after administration in rats (n = 6). (A) Single oral administration of
compounds 24w (parent compound 25w released from prodrug 24w; 10 mg/kg) and 25w (10 mg/kg). (B) Intravenous injection administration of
compound 25w (10 mg/kg). Data are presented as the mean ± SD.
Table 6. Main Pharmacokinetic Parameters of Compounds 24w and 25w in Sprague-Dawley Rats after Administration (n =
a
6
)
parameters
oral administration (mean ± SD; 24w)
oral administration (mean ± SD; 25w)
intravenous administration (mean ± SD; 25w)
Cmax (ng/mL)
Tmax (h)
67.99 ± 10.72
0.46 ± 0.29
214.05 ± 148.88
0.167
11225.0 ± 8214.12
0.14 ± 0.04
AUC0−∞ (ng·h/mL)
1135.08 ± 764.28
13.83 ± 10.82
12.40 ± 6.38
808.86 ± 382.05
13.13 ± 15.06
15.64 ± 8.64
239.60 ± 183.57
3500.34 ± 1003.87
4.89 ± 3.24
3.03 ± 0.73
t1/2 (h)
CLz (L·h 1·kg−1
−
)
Vz (L/kg)
190.56 ± 86.64
21.29 ± 15.29
a
Cmax, peak plasma concentration; Tmax, time to reach Cmax; AUC0−∞, area under the concentration−time curve from time zero to infinity; t1/2
,
elimination half-life; CLz, clearance; Vz, volume of distribution.
Figure 9. Body weight evolution of (A) male and (B) female mice. Each value is the mean ± SEM; n = 8.
weights in rats after oral administration. As shown in Figure 11,
the blood loss of the vehicle-treated rats within 2 h after the tail
snip amounted to 0.08 g with a bleeding time of 17.69 min,
significantly increased by 6.78-fold and 48.63-fold in
comparison to the vehicle, respectively (120 vs 17.69 min,
3.89 vs 0.08 g, ***P < 0.001 for ticagrelor group vs vehicle
group). Compound 24w only caused 1.92- and 5.00-times
more than those of the vehicle group in bleeding time and
44,72
indicating that this assay was highly reproducible.
In
ticagrelor-treated rats, the total bleeding time and weight were
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antiplatelet agents 24w and 25w (IC50 = 4.237 and 3.875 μM,
respectively). We completed the optimization process and SAR
of the target compounds. Moreover, P2Y -mediated cytosolic
1
2
+
Ca increase indicated that compound 25w exhibited a potent
inhibitory effect for P2Y with a IC value of 2.59 μM,
1
50
comparable with that of BPTU (IC50 = 3.03 μM), and P2Y12-
mediated vasodilator-stimulated phosphoprotein phosphoryla-
tion assay revealed that compound 25w also could dose
dependently antagonize P2Y12 receptor, which could confirm
that compound 25w exerted its antiplatelet effect through both
P2Y and P2Y . Molecular modeling studies revealed the
1
12
binding modes of 24w and 25w with two receptors, P2Y and
1
P2Y , which suggested that they could form hydrogen-bond
1
2
interactions with key residues in active pockets. The simulated
gastric and intestinal fluid stabilities indicated that they were
considerably stable within 10 h, and the in vitro plasma and
liver microsomal stability studies also showed that compound
24w could be rapidly converted to compound 25w. In
addition, the CYP450 inhibition assay suggested that
compound 25w showed no apparent drug−drug interaction
at 10 μM and there might be low liability for drug−drug
interactions between 24w and CYP2D6. Furthermore, the PK
studies showed the Tmax values of compounds 24w and 25w
were 0.46 and 0.167 h, respectively, suggesting that they were
both absorbed quickly into circulation, which could hopefully
overcome the slow onset of action of currently approved oral
P2Y12 antagonists. The acute oral toxicity study of compound
Figure 10. Effects of ticagrelor and compound 24w on thrombus
weight after FeCl -induced arterial injury in anesthetized Sprague-
3
Dawley rats at 1.5 h postdosing following the oral administration of
24w (5, 10, and 20 mg/kg) and ticagrelor (10 mg/kg). Data are
presented as the mean ± SD (n = 10, ***P < 0.001 and ***P < 0.001
#
vs model; P > 0.05 vs ticagrelor).
2
4w was evaluated in mice, and the results indicated that 24w
was nontoxic and tolerated at a dose up to 2000 mg/kg in
mice. In addition, the rat ferric chloride model study suggested
that compound 24w demonstrated the dose-dependent
antithrombotic efficacy and was comparable to what was
observed for ticagrelor at the same oral dose (10 mg/kg).
Importantly, compound 24w showed significantly lower
bleeding weight and time compared to ticagrelor at therapeuti-
cally equivalent antithrombotic dose (10 mg/kg) in a rat-tail-
bleeding model, indicating that it had a clearly wider
therapeutic window than ticagrelor in rats. This could
potentially address the concern of high bleeding risk in
clinically approved oral P2Y12 antagonists. Therefore, com-
pounds 24w and 25w were promising drug candidates for the
treatment of arterial thrombosis and related diseases. The
investigations performed in this research perfectly achieved the
transition from a nonpurine imidazole XO inhibitor to dual-
Figure 11. Effects of ticagrelor and compound 24w on (A) tail-vein-
bleeding weight and (B) time in anesthetized Sprague-Dawley rats at
2
h postdosing following the oral administration of 24w (10 mg/kg)
and ticagrelor (10 mg/kg). The data are presented as mean ± SD (n =
8
group).
#
##
, *P < 0.05, ***P < 0.001 vs model group; P < 0.001 vs ticagrelor
weight, respectively (33.94 vs 17.69 min, 0.40 vs 0.08 g, *P <
0
.05 for 24w group vs vehicle group), and were 3.54- and 9.73-
times less than those of ticagrelor on the bleeding time and
weight, respectively (33.94 vs 120 min, 0.40 vs 3.89 g, # P <
##
target P2Y and P2Y antagonists, which implied that other
0
.001 for 24w group vs ticagrelor group). These results
1
12
nonpurine XO inhibitors with a similar chemical structure to
WSJ-557 could also be adopted to design novel effective dual-
indicated that 24w resulted in much less blood loss than
ticagrelor in rats at a therapeutically equivalent antithrombotic
dose of 10 mg/kg. It was observed that, at this oral dose,
compound 24w could lead to a thrombosis inhibition of 40.7%,
which was comparable to that of ticagrelor (39% inhibition of
thrombosis). This reduction in bleeding time and weight when
compared to ticagrelor indicated that compound 24w clearly
had a wider therapeutic window than ticagrelor in a rat
thrombosis model and could potentially resolve the serious
concern of high bleeding risk associated with clinical oral P2Y
target P2Y and P2Y antagonists.
1
12
EXPERIMENTAL SECTION
■
Chemistry. Compounds WSJ-557 and 18 were prepared from our
51,52
previous report,
and the purity of each was more than 95%. In
addition, reagents and solvents were purchased from commercial
sources and used without further purification. All reactions were
monitored by TLC using silica gel aluminum cards (0.2 mm
thickness) with 254 and 365 nm fluorescent indicators. Melting
points were obtained using a YRT-3 melting apparatus and were
1
2
1
4,16,35,36
antagonists.
1
CONCLUSIONS
Starting from our previously reported nonpurine XO inhibitor
WSJ-557 with an apparent antiplatelet aggregation potency
uncorrected. H NMR spectra were recorded on a Bruker 400 or 600
■
13
MHz spectrometer, and C NMR spectra were recorded on a Bruker
4
00 or 600 MHz spectrometer. Chemical shifts were expressed in
parts per million using tetramethylsilane as an internal reference and
(
IC50 = 15.727 μM), the 2-phenyl-1H-imidazole moiety was
DMSO-d as the solvent. Electrospray ionization-mass spectrometry
6
adopted as a new chemical scaffold for the structural
optimization to lead to the identification of the most potent
(ESI-MS) data were gathered using an Agilent 1100 instrument and
ESI-HRMS data were recorded in an Agilent 6540 Series quadrupole
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time-of-flight mass spectrometer (Q-TOF-MS) system (Supporting
Information).
same manner as that described for 22a to yield a white solid, yield:
92.4%. Purity (HPLC): 98.1%. Mp 147.3−148.5 °C. ESI-HRMS
+
1
Ethyl 2-Hydroxyimino-3-oxobutanoate (20). A solution of
sodium nitrite (51.3 g, 0.74 mol) in water (102.6 mL) was added
dropwise at 0−5 °C to a stirred solution of ethyl acetoacetate (80 g,
calcd. for C H N O [M + H] 286.1186, found: 286.1208. H
15 15 3 3
NMR (400 MHz, DMSO-d ): δ 8.21 (dd, 2H), 7.99 (dd, 2H), 4.33
6
(q, J = 7.1 Hz, 2H), 3.98 (s, 3H), 2.42 (s, 3H), 1.34 (t, J = 7.1 Hz,
1
3
0
.62 mol) in acetic acid (240 mL). Upon completion of the reaction, a
3H). C NMR (100 MHz, DMSO-d ): δ 158.67, 143.97, 140.37,
6
mixture of DCM (500 mL) and water (250 mL) was added. The
organic phase was washed with water (2 × 150 mL) and brine (150
mL), dried (Na SO ), and concentrated to afford a crude product 20
as a yellow oil, which was used for the next reaction without further
purification.
133.37, 131.94, 128.22, 118.88, 117.29, 112.53, 67.72, 60.90, 16.28,
14.55.
Ethyl 1-Methoxy-4-methyl-2-phenyl-1H-imidazole-5-car-
boxylate (22d). Compound 22d was prepared in the same manner
as that described for 22a to yield a white solid, yield: 88.5%. Purity
2
4
(
HPLC): 99.8%. Mp 98.8−100.4 °C. ESI-HRMS calcd. for
Ethyl 2-(3-Cyanophenyl)-1-hydroxy-4-methyl-1H-imida-
zole-5-carboxylate (21a). A mixture of 3-formylbenzonitrile (30g,
+
1
C
H N O
[M + H] 261.1234, found: 261.1300. H NMR (600
): δ 8.10−7.99 (m, 2H), 7.57−7.45 (m, 3H), 4.30 (q,
J = 7.1 Hz, 2H), 3.93 (s, 3H), 2.40 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H).
14
16 2 3
0
.229 mol), ethyl 2-hydroxyimino-3-oxobutanoate (43.7 g, 0.285
MHz, DMSO-d
6
mol), ammonium acetate (176.3 g, 2.29 mol), and acetic acid (600
mL) was stirred at 50 °C under a nitrogen atmosphere for 24 h. The
reaction mixture was cooled to room temperature and then slowly
poured into cold water (2000 mL). The resulting precipitate was
filtered, dried, and washed with ethyl acetate to obtain the compound
1
3
C NMR (100 MHz, DMSO-d ): δ 158.84, 143.68, 142.27, 130.39,
6
129.36, 127.95, 127.72, 116.35, 67.21, 60.62, 16.28, 14.57.
Ethyl 2-(3-Cyanophenyl)-4-methyl-1H-imidazole-5-carboxy-
late (23). A suspension of compound 21a (2.6 g, 10 mmol),
potassium iodide (1.16g, 10 mmol), chlorotrimethylsilane (1.63g, 15
mmol), and acetonitrile (30 mL) was stirred at 60 °C for 6 h. The
reaction mixture was poured into a solution of 1 M sodium hydroxide
aqueous and was stirred for 1 h. The precipitate was filtered and
washed with ethyl acetate to obtain compound 23 as a white solid,
2
1a as a white solid, yield: 75.5%. Purity (HPLC): 98.4%. Mp 167.7−
+
1
70.2 °C. ESI-HRMS calcd. for C H N O [M + H] 272.1030,
1
4
13
3
3
1
found: 272.1057. H NMR (400 MHz, DMSO-d ): δ 12.28 (s, 1H),
6
8
7
2
.39 (t, J = 1.7 Hz, 1H), 8.34 (dt, J = 8.1, 1.4 Hz, 1H), 7.91 (dt, J =
.8, 1.4 Hz, 1H), 7.71 (t, J = 7.9 Hz, 1H), 4.30 (q, J = 7.1 Hz, 2H),
1
3
.39 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSO-
yield: 77.3%. Purity (HPLC): 97.7%. Mp 209.6−210.7 °C. ESI-
−
HRMS calcd. for C H N O [M − H] 254.0935, found: 254.0959.
d6): δ 159.33, 142.72, 141.14, 133.16, 132.35, 131.03, 130.49, 129.77,
15 15
3
2
1
1
18.94, 118.83, 112.29, 60.53, 16.14, 14.67.
Ethyl 2-(2-Cyanophenyl)-1-hydroxy-4-methyl-1H-imida-
H NMR (400 MHz, DMSO-d
): δ 8.35 (s, 1H), 8.28 (d, J = 8.0 Hz,
6
1H), 7.80 (d, J = 7.7 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 4.80 (s, 1H),
1
3
zole-5-carboxylate (21b). Compound 21b was prepared in the
same manner as that described for 21a and yielded a brown oil, which
was used for the next reaction without further purification.
4.25 (q, J = 7.1 Hz, 2H), 2.47 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H). C
NMR (100 MHz, DMSO-d ): δ 162.99, 144.34, 140.85, 132.04,
6
130.50, 130.13, 128.92, 126.99, 118.97, 112.31, 59.83, 14.86, 12.89.
Ethyl 2-(3-Cyanophenyl)-1,4-dimethyl-1H-imidazole-5-car-
boxylate (24a). A solution of compound 23 (0.51 g, 2.0 mmol),
anhydrous potassium carbonate (0.33g, 2.40 mmol), and iodo-
methane (0.33 g, 2.4 mmol) in DMF (5.4 mL) was stirred at 35 °C
under a nitrogen atmosphere for 1 h. After the completion of the
reaction, the reaction mixture was poured into 11 mL of water and
stirred for 10 min. The precipitate was filtered and washed with water,
and then, ethyl acetate (5.0 mL) was added to wash the residue to
yield compound 24a as a white solid, yield: 76.2%. Purity (HPLC):
97.9%. Mp 137.3−138.1 °C. ESI-HRMS calcd. for C H N O [M +
Ethyl 2-(4-Cyanophenyl)-1-hydroxy-4-methyl-1H-imida-
zole-5-carboxylate (21c). Compound 21c was prepared in the
same manner as that described for 21a to yield a white solid, yield:
1
7
1
2.8%. Mp 170.5−171.8 °C. H NMR (400 MHz, DMSO-d ): δ
6
2.36 (s, 1H), 8.22 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H), 4.29
(
q, J = 7.1 Hz, 2H), 2.38 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H).
Ethyl 1-Hydroxy-4-methyl-2-phenyl-1H-imidazole-5-car-
boxylate (21d). Compound 21d was prepared in the same manner
as that described for 21a to yield a white solid, yield: 83.7%. Mp
+
1
1
31.5−133.1 °C. ESI-MS m/z: 247.1 [M + H] . H NMR (400
1
5
15
3
2
+
1
MHz, DMSO-d ): δ 8.22−8.13 (m, 2H), 7.46−7.33 (m, 3H), 4.23 (q,
J = 7.1 Hz, 2H), 2.34 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H).
H] 270.1237, found: 270.1250. H NMR (400 MHz, DMSO-d ): δ
6
6
8.13 (s, 1H), 8.07−7.91 (m, 2H), 7.73 (t, J = 7.8 Hz, 1H), 4.30 (q, J =
1
3
Ethyl 2-(3-Cyanophenyl)-1-methoxy-4-methyl-1H-imida-
zole-5-carboxylate (22a). A solution of compound 21a (0.51 g,
7.3 Hz, 2H), 3.84 (s, 3H), 2.42 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H). C
NMR (100 MHz, DMSO-d ): δ 160.95, 148.48, 146.87, 134.35,
6
2
.0 mmol), anhydrous potassium carbonate (0.33g, 2.40 mmol), and
iodomethane (0.33 g, 2.4 mmol) in DMF (5.4 mL) was stirred at 35
C under a nitrogen atmosphere for 1 h. After the completion of the
133.45, 132.92, 131.23, 130.38, 120.94, 118.74, 112.32, 60.54, 35.03,
16.05, 14.65.
°
Ethyl 2-(3-Cyanophenyl)-1-isopropyl-4-methyl-1H-imida-
zole-5-carboxylate (24b). Compound 23 (0.51 g, 2.0 mmol),
anhydrous potassium carbonate (0.33 g, 2.4 mmol), potassium iodide
(39.84 mg, 0.24 mmol), and 2-bromopropane (295 mg, 2.4 mmol)
were dissolved in DMF (5.4 mL), and the reaction mixture was stirred
at 35 °C for 1 h. Then, the mixture was poured into water (11 mL)
and stirred for 10 min, which was filtered and washed with ethyl
acetate to provide the compound 24b as a white solid, yield: 68.3%.
Purity (HPLC): 99.2%. Mp 105.3−106.7 °C. ESI-MS m/z: 298.2 [M
reaction, the reaction mixture was poured into 11 mL of water and
stirred for 10 min. The precipitate was filtered and washed with water,
and then, ethyl acetate (5.0 mL) was added to wash residue to yield
compound 22a as a white solid, yield: 79.3%. Purity (HPLC): 99.5%.
+
Mp 122.4−123.5 °C. ESI-HRMS calcd. for C H N O [M + H]
1
5
15
3
3
1
2
8
1
7
1
1
85.1186, found: 286.1207. H NMR (400 MHz, DMSO-d ): δ
6
.37−8.27 (m, 2H), 7.97 (dt, J = 7.6, 1.4 Hz, 1H), 7.75 (t, J = 7.8 Hz,
H), 4.32 (q, J = 7.1 Hz, 2H), 3.98 (s, 3H), 2.40 (s, 3H), 1.33 (t, J =
.1 Hz, 3H). 13C NMR (100 MHz, DMSO-d ): δ 158.68, 143.82,
+ 1
+ H] . H NMR (400 MHz, DMSO-d ): δ 8.33−8.24 (m, 2H), 7.94
6
6
40.26, 133.78, 132.14, 130.90, 130.82, 129.02, 118.70, 116.97,
(d, J = 7.7 Hz, 1H), 7.73 (t, J = 7.8 Hz, 1H), 4.44 (hept, J = 6.2 Hz,
1H), 4.30 (q, J = 7.1 Hz, 2H), 2.41 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H),
1.03 (d, J = 6.0 Hz, 6H). C NMR (100 MHz, DMSO-d ): δ 158.90,
12.70, 67.65, 60.84, 16.23, 14.54.
1
3
Ethyl 2-(2-Cyanophenyl)-1-methoxy-4-methyl-1H-imida-
zole-5-carboxylate (22b). Compound 22b was prepared in the
same manner as that described for 22a to yield a yellow solid, yield:
6
144.08, 142.05, 133.54, 132.72, 131.59, 130.54, 129.89, 118.62,
117.68, 112.39, 83.52, 60.76, 20.24, 16.32, 14.55.
5
2
3
3
1.4%. Purity (HPLC): 99.3%. Mp 144.2−145.6 °C. ESI-MS m/z:
Ethyl 2-(3-Cyanophenyl)-1-isopropoxy-4-methyl-1H-imida-
zole-5-carboxylate (24c). A mixture of compound 21a (0.54 g, 2.0
mmol), 2-bromopropane (295 mg, 2.4 mmol), anhydrous potassium
carbonate (0.33 g, 2.4 mmol), potassium iodide (39.84 mg, 0.24
mmol), and DMF (5.4 mL) was reacted at 35 °C for 1 h under a
nitrogen atmosphere. After the reaction was completed, the mixture
was poured into water (11.0 mL) and stirred for 10 min. The
precipitate was filtered, washed with water, and stirred for 30 min in a
+
1
86.17 [M + H] . H NMR (400 MHz, DMSO-d ): δ 7.68−7.57 (m,
6
H), 7.52 (dd, J = 7.4, 1.4 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 3.79 (s,
1
3
H), 2.42 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H). C NMR (100 MHz,
DMSO-d ): δ 158.69, 143.83, 140.26, 133.76, 132.12, 130.90, 130.81,
6
1
29.04, 118.69, 116.98, 112.71, 67.64, 60.83, 16.21, 14.53.
Ethyl 2-(4-Cyanophenyl)-1-methoxy-4-methyl-1H-imida-
zole-5-carboxylate (22c). Compound 22c was prepared in the
M
https://dx.doi.org/10.1021/acs.jmedchem.0c01524
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
solution of ethyl acetate (5.0 mL), which was filtered to yield
compound 24c as a white solid, yield: 79.3%. Purity (HPLC): 99.4%.
3-{[2-(3-Cyanophenyl)-5-(ethoxycarbonyl)-4-methyl-1H-
imidazol-1-yl] oxy} Propanoic acid (24i). Compound 24i was
prepared in the same manner as that described for 24c to yield a white
solid, yield: 75.4%. Purity (HPLC): 99.0%. Mp 142.1−143.9 °C. ESI-
+
Mp 99.6−101.5 °C. ESI-HRMS calcd. for C H N O [M + H]
1
7
19
3
3
1
3
14.1499, found: 314.1513. H NMR (600 MHz, DMSO-d ): δ 8.31
6
+
1
(
t, J = 1.7 Hz, 1H), 8.29 (dt, J = 8.0, 1.4 Hz, 1H), 7.95 (dt, J = 7.8, 1.4
MS m/z: 344.2 [M + H] . H NMR (400 MHz, DMSO-d ): δ 12.52
6
Hz, 1H), 7.74 (t, J = 7.9 Hz, 1H), 4.44 (h, J = 6.2 Hz, 1H), 4.31 (q, J
(s, 1H), 8.43−8.30 (m, 2H), 7.95 (d, J = 7.7 Hz, 1H), 7.71 (t, J = 7.9
=
7.1 Hz, 2H), 2.42 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H), 1.14−0.93 (m,
Hz, 1H), 4.47−4.18 (m, 4H), 2.67 (t, J = 5.9 Hz, 2H), 2.41 (s, 3H),
H). 13C NMR (100 MHz, DMSO-d ): δ 158.93, 144.09, 142.11,
13
6
1
6
1.33 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSO-d
6
): δ 171.77,
6
33.62, 132.78, 131.64, 130.60, 129.87, 118.66, 117.67, 112.39, 83.57,
0.81, 20.27, 16.36, 14.59.
158.80, 143.95, 140.78, 133.80, 132.41, 131.23, 130.59, 128.94,
118.67, 117.07, 112.68, 76.00, 60.87, 33.14, 16.26, 14.58.
Ethyl 1-(Allyloxy)-2-(3-cyanophenyl)-4-methyl-1H-imida-
Ethyl 2-(3-Cyanophenyl)-4-methyl-1-(pyridin-4-ylmethoxy)-
1H-imidazole-5-carboxylate (24j). Compound 24j was prepared
in the same manner as that described for 24c to yield a white solid,
zole-5-carboxylate (24d). Compound 24d was prepared in the
same manner as that described for 24c to yield a white solid, yield:
7
3.1%. Purity (HPLC): 99.6%. Mp 94.7−96.2 °C. ESI-HRMS calcd.
yield: 76.2%. Purity (HPLC): 99.2%. Mp 133.5−135.1 °C. ESI-
+
1
+
for C H N O [M + H] 312.1343, found: 312.1361. H NMR (600
HRMS calcd. for C20
H
N
18
O
4
3
[M + H] 363.1452, found: 363.1471.
17
17
3
3
1
MHz, DMSO-d ): δ 8.34−8.32 (m, 1H), 8.31−8.27 (m, 1H), 7.97
H NMR (600 MHz, DMSO-d ): δ 8.58−8.42 (m, 2H), 8.14 (t, J =
6
6
(
5
2
dt, J = 7.8, 1.4 Hz, 1H), 7.79−7.72 (m, 1H), 5.95−5.76 (m, 1H),
1.7 Hz, 1H), 8.11 (dt, J = 8.1, 1.4 Hz, 1H), 7.92 (dt, J = 7.8, 1.4 Hz,
1H), 7.66 (t, J = 7.9 Hz, 1H), 7.31−7.23 (m, 2H), 5.19 (s, 2H), 4.34
.41−5.25 (m, 2H), 4.66 (d, J = 6.4 Hz, 2H), 4.32 (q, J = 7.1 Hz,
13
13
H), 2.41 (s, 3H), 1.33 (t, J = 7.1 Hz, 3H). C NMR (100 MHz,
(q, J = 7.1 Hz, 2H), 2.44 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H). C NMR
DMSO-d ): δ 158.83, 143.83, 140.95, 133.69, 132.35, 131.11, 130.71,
(150 MHz, DMSO-d ): δ 158.87, 150.26, 144.02, 141.67, 133.67,
6
6
1
29.33, 123.02, 118.66, 112.56, 80.69, 60.85, 16.24, 14.56.
Ethyl 2-(3-Cyanophenyl)-1-(2-ethoxyethoxy)-4-methyl-1H-
132.49, 131.32, 130.55, 128.99, 124.24, 118.59, 116.95, 112.37, 79.87,
60.97, 16.36, 14.57.
imidazole-5-carboxylate (24e). Compound 24e was prepared in
the same manner as that described for 24c to yield a white solid, yield:
8
for C H N O [M + H] 344.1605, found: 344.1619. H NMR (600
MHz, DMSO-d ): δ 8.46 (t, J = 1.7 Hz, 1H), 8.39 (dt, J = 8.1, 1.4 Hz,
Ethyl 1-(Benzyloxy)-2-(3-cyanophenyl)-4-methyl-1H-imida-
zole-5-carboxylate (24k). Compound 24k was prepared in the
same manner as that described for 24c to yield a white solid, yield:
94.3%. Purity (HPLC): 98.5%. Mp 124.8−126.3 °C. ESI-HRMS
3.2%. Purity (HPLC): 97.5%. Mp 88.6−89.4 °C. ESI-HRMS calcd.
+
1
18
21
3
4
+
1
calcd. for C21
NMR (600 MHz, DMSO-d
6
H
N
O
[M + H] 362.1499, found: 362.1539. H
6
20
3
3
1
H), 7.95 (dt, J = 7.7, 1.3 Hz, 1H), 7.70 (t, J = 7.9 Hz, 1H), 4.37−
): δ 8.13−8.09 (m, 2H), 7.90 (dt, J = 7.7,
4
.25 (m, 4H), 3.59 (dd, J = 4.9, 3.2 Hz, 2H), 3.35−3.33 (m, 2H), 2.40
1.4 Hz, 1H), 7.65 (td, J = 7.8, 0.6 Hz, 1H), 7.36−7.31 (m, 1H), 7.30−
1
3
(
s, 3H), 1.32 (t, J = 7.1 Hz, 3H), 1.09 (t, J = 7.0 Hz, 3H). C NMR
7.25 (m, 2H), 7.23−7.18 (m, 2H), 5.12 (s, 2H), 4.36 (q, J = 7.1 Hz,
1
3
(
100 MHz, DMSO-d ): δ 158.76, 143.93, 133.67, 132.57, 131.07,
2H), 2.43 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H). C NMR (100 MHz,
6
1
1
30.45, 128.97, 118.76, 117.18, 112.48, 79.70, 67.18, 66.16, 60.84,
6.25, 15.30, 14.52.
Ethyl 2-(3-Cyanophenyl)-1-(2-ethoxy-2-oxoethoxy)-4-meth-
DMSO-d ): δ 158.93, 143.95, 141.46, 133.43, 132.95, 132.51, 131.21,
6
130.57, 130.35, 129.86, 129.17, 128.85, 118.62, 117.06, 112.25, 81.89,
60.90, 16.36, 14.63.
yl-1H-imidazole-5-carboxylate (24f). Compound 24f was pre-
pared in the same manner as that described for 24c to yield a white
solid, yield: 79.8%. Purity (HPLC): 99.0%. Mp 126.3−127.7 °C. ESI-
Ethyl 2-(3-Cyanophenyl)-1-[(2-fluorobenzyl)oxy]-4-methyl-
1H-imidazole-5-carboxylate (24l). Compound 24l was prepared
in the same manner as that described for 24c to yield a white solid,
+
HRMS calcd. for C H N O [M + H] 358.1397, found: 358.1431.
yield: 83.2%. Purity (HPLC): 95.9%. Mp 135.5−136.4 °C. ESI-
1
8
19
3
5
1
+
H NMR (600 MHz, DMSO-d ): δ 8.41 (s, 1H), 8.33 (dt, J = 8.1, 1.4
HRMS calcd. for C21
H
18FN
3
O
3
[M + H] 380.1405, found: 380.1453.
6
1
H NMR (600 MHz, DMSO-d ): δ 8.02 (s, 1H), 7.98 (d, J = 8.0 Hz,
Hz, 1H), 7.96 (dt, J = 7.8, 1.4 Hz, 1H), 7.73 (t, J = 7.9 Hz, 1H), 4.95
6
(
3
s, 2H), 4.31 (q, J = 7.1 Hz, 2H), 4.13 (q, J = 7.1 Hz, 2H), 2.41 (s,
H), 1.31 (t, J = 7.1 Hz, 3H), 1.18 (t, J = 7.1 Hz, 3H). C NMR (100
1H), 7.86 (d, J = 7.7 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.32 (d, J = 6.9
Hz, 1H), 7.15 (t, J = 7.1 Hz, 1H), 7.05 (t, J = 7.4 Hz, 1H), 6.96 (t, J =
9.2 Hz, 1H), 5.20 (s, 2H), 4.35 (q, J = 7.0 Hz, 2H), 2.43 (s, 3H), 1.35
1
3
MHz, DMSO-d ): δ 166.39, 158.74, 143.85, 140.75, 133.76, 132.54,
1
1
6
1
3
31.19, 130.58, 129.05, 118.72, 117.20, 112.51, 75.54, 61.60, 61.00,
6.26, 14.44, 14.32.
Ethyl 2-(3-Cyanophenyl)-1-(3-hydroxypropoxy)-4-methyl-
H-imidazole-5-carboxylate (24g). Compound 24g was prepared
(t, J = 7.0 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ 158.92,
6
144.05, 141.86, 133.33, 132.73, 132.65, 132.58, 131.25, 130.16,
129.03, 124.81, 124.78, 118.61, 116.96, 115.69, 115.48, 112.10, 75.22,
60.90, 16.34, 14.59.
1
in the same manner as that described for 24c to yield a white solid,
yield: 66.4%. Purity (HPLC): 95.8%. Mp 78.6−80.4 °C. ESI-HRMS
Ethyl 2-(3-Cyanophenyl)-1-[(3-fluorobenzyl)oxy]-4-methyl-
1H-imidazole-5-carboxylate (24m). Compound 24m was pre-
pared in the same manner as that described for 24c to yield a white
+
1
calcd. for C H N O [M + H] 330.1448, found: 330.1483. H
17
19
3
4
NMR (600 MHz, DMSO-d ): δ 8.33 (t, J = 1.7 Hz, 1H), 8.30 (dt, J =
solid, yield: 76.9%. Purity (HPLC): 99.7%. Mp 136.3−138.0 °C. ESI-
6
+
8
1
.1, 1.4 Hz, 1H), 7.96 (dt, J = 7.7, 1.4 Hz, 1H), 7.74 (t, J = 7.9 Hz,
H), 4.54 (t, J = 5.1 Hz, 1H), 4.31 (q, J = 7.1 Hz, 2H), 4.15 (t, J = 6.5
HRMS calcd. for C21
H
20FN
3
O
3
[M + H] 380.1405, found: 380.1464.
1
H NMR (600 MHz, DMSO-d ): δ 8.12−8.03 (m, 2H), 7.90 (dt, J =
6
Hz, 2H), 3.48 (q, J = 6.0 Hz, 2H), 2.40 (s, 3H), 1.80 (p, J = 6.5 Hz,
7.7, 1.4 Hz, 1H), 7.64 (t, J = 7.8 Hz, 1H), 7.36−7.26 (m, 1H), 7.19−
1
3
2
1
1
H), 1.33 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ
7.12 (m, 1H), 7.10−6.98 (m, 2H), 5.15 (s, 2H), 4.35 (q, J = 7.1 Hz,
6
1
3
58.78, 143.93, 140.66, 133.71, 132.24, 131.09, 130.72, 129.10,
18.64, 117.08, 112.62, 78.14, 60.83, 57.62, 31.26, 16.22, 14.57.
Ethyl 1-(2-Amino-2-oxoethoxy)-2-(3-cyanophenyl)-4-meth-
yl-1H-imidazole-5-carboxylate (24h). Compound 24h was
prepared in the same manner as that described for 24c to yield a
2H), 2.43 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H). C NMR (100 MHz,
DMSO-d ): δ 158.93, 144.04, 141.75, 135.30, 133.50, 133.48, 132.60,
6
131.30, 130.67, 130.52, 130.28, 129.75, 129.08, 129.06, 118.59,
116.94, 112.22, 80.84, 60.93, 16.36, 14.60.
Ethyl 2-(3-Cyanophenyl)-1-[(4-fluorobenzyl)oxy]-4-methyl-
1H-imidazole-5-carboxylate (24n). Compound 24n was prepared
in the same manner as that described for 24c to yield a white solid,
white solid, yield: 86.7%. Purity (HPLC): 96.7%. Mp 179.6−181.2
+
°
3
C. ESI-HRMS calcd. for C H N O [M + H] 329.1244, found:
1
6
16
4
4
1
29.1364. H NMR (600 MHz, DMSO-d ): δ 8.51 (t, J = 1.7 Hz,
yield: 82.7%. Purity (HPLC): 98.0%. Mp 140.7−141.8 °C. ESI-
6
+
1
H), 8.38 (dt, J = 8.1, 1.5 Hz, 1H), 7.95 (dt, J = 7.8, 1.4 Hz, 1H), 7.72
HRMS calcd. for C21
H
18FN
3
O
3
[M + H] 380.1405, found: 380.1472.
1
(
=
t, J = 7.9 Hz, 1H), 7.66 (s, 1H), 7.54 (s, 1H), 4.62 (s, 2H), 4.31 (q, J
7.1 Hz, 2H), 2.41 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H). C NMR (100
H NMR (600 MHz, DMSO-d ): δ 8.12−8.02 (m, 2H), 7.89 (d, J =
6
1
3
7.7 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.22 (dd, J = 8.3, 5.5 Hz, 2H),
7.06 (t, J = 8.6 Hz, 2H), 5.11 (s, 2H), 4.35 (q, J = 7.1 Hz, 2H), 2.42
MHz, DMSO-d ): δ 167.20, 158.79, 143.89, 140.67, 133.84, 132.48,
6
1
3
1
1
31.19, 130.59, 128.94, 118.71, 117.15, 112.63, 77.05, 61.00, 16.23,
4.47.
(s, 3H), 1.35 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ
6
158.93, 143.99, 141.64, 133.40, 133.06, 132.97, 132.51, 131.25,
N
https://dx.doi.org/10.1021/acs.jmedchem.0c01524
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1
1
30.28, 129.19, 118.60, 117.00, 115.80, 115.58, 112.18, 80.93, 60.90,
6.38, 14.62.
Ethyl 1-[(2-Chlorobenzyl)oxy]-2-(3-cyanophenyl)-4-methyl-
H-imidazole-5-carboxylate (24o). Compound 24o was prepared
160.64, 158.96, 143.99, 141.78, 133.26, 132.53, 132.35, 131.21,
130.15, 129.27, 124.83, 118.65, 116.96, 114.11, 112.07, 81.62, 60.85,
55.59, 16.39, 14.64.
1
Ethyl 2-(3-Cyanophenyl)-1-{[4-(methoxycarbonyl)benzyl]-
oxy}-4-methyl-1H-imidazole-5-carboxylate (24u). Compound
24u was prepared in the same manner as as that described for 24c
to yield a white solid, yield: 85.2%. Purity (HPLC): 97.4%. Mp
167.5−169.4 °C. ESI-HRMS calcd. for C H N O [M + H]+
in the same manner as that described for 24c to yield a white solid,
yield: 76.5%. Purity (HPLC): 98.4%. Mp 124.6−125.7 °C. ESI-
−
HRMS calcd. for C H ClN O [M − H] 394.1037, found:
2
1
18
3
3
1
3
94.0975. H NMR (400 MHz, DMSO-d ): δ 7.96 (s, 1H), 7.90 (d, J
6
23 21
3
5
1
=
8.0 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.54 (t, J = 7.9 Hz, 1H),
420.1554, found: 420.1676. H NMR (400 MHz, DMSO-d ): δ
6
7
.28−7.21 (m, 1H), 7.21−7.14 (m, 3H), 5.24 (s, 2H), 4.35 (q, J = 7.1
8.05 (dt, J = 8.1, 1.4 Hz, 1H), 8.01 (t, J = 1.6 Hz, 1H), 7.88 (dt, J =
7.8, 1.3 Hz, 1H), 7.81 (d, J = 8.3 Hz, 2H), 7.62 (t, J = 7.9 Hz, 1H),
7.33 (d, J = 8.2 Hz, 2H), 5.21 (s, 2H), 4.36 (q, J = 7.1 Hz, 2H), 1.34
1
3
Hz, 2H), 2.43 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H). C NMR (100 MHz,
DMSO-d ): δ 158.94, 144.16, 142.20, 134.86, 133.37, 133.22, 132.69,
1
1
6
1
3
31.89, 131.46, 130.81, 130.05, 129.62, 128.97, 127.55, 118.61,
16.86, 112.01, 78.84, 60.89, 16.38, 14.58.
Ethyl 1-[(3-Chlorobenzyl)oxy]-2-(3-cyanophenyl)-4-methyl-
H-imidazole-5-carboxylate (24p). Compound 24p was prepared
(t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ 166.14,
6
158.91, 144.04, 141.62, 137.99, 133.42, 132.58, 131.28, 130.79,
130.64, 130.32, 129.54, 129.09, 118.51, 116.96, 112.23, 80.98, 60.94,
52.71, 16.36, 14.60.
1
in the same manner as that described for 24c to yield a white solid,
yield: 83.8%. Purity (HPLC): 97.0%. Mp 118.3−119.7 °C. ESI-
HRMS calcd. for C H ClN O [M + H]+ 396.1109, found:
Ethyl 2-(3-Cyanophenyl)-4-methyl-1-[(4-nitrobenzyl)oxy]-
1H-imidazole-5-carboxylate (24v). Compound 24v was prepared
in the same manner as that described for 24c to yield a white solid:
78.9%. Purity (HPLC): 98.1%. Mp 159.8−160.6 °C. ESI-HRMS
2
1
18
3
3
1
3
7
1
7
1
1
1
96.1156. H NMR (600 MHz, DMSO-d ): δ 8.07−8.00 (m, 2H),
6
+
1
.89 (dt, J = 7.8, 1.4 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.37−7.31 (m,
H), 7.27 (t, J = 7.8 Hz, 1H), 7.24 (t, J = 1.8 Hz, 1H), 7.11 (dt, J =
.5, 1.3 Hz, 1H), 5.14 (s, 2H), 4.35 (q, J = 7.1 Hz, 2H), 2.43 (s, 3H),
calcd. for C H N O [M + H] 407.1350, found: 407.1398. H
21 18 4 5
NMR (600 MHz, DMSO-d ): δ 8.09 (d, J = 8.6 Hz, 2H), 8.07−8.00
6
(m, 2H), 7.89 (dt, J = 7.7, 1.4 Hz, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.49
(d, J = 8.6 Hz, 2H), 5.29 (s, 2H), 4.35 (q, J = 7.1 Hz, 2H), 2.44 (s,
1
3
.35 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ 158.91,
6
1
3
44.03, 141.69, 135.29, 133.48, 132.57, 131.27, 130.66, 130.50,
30.27, 129.74, 129.05, 116.92, 112.22, 80.82, 60.92, 16.36, 14.59.
Ethyl 1-[(4-Chlorobenzyl)oxy]-2-(3-cyanophenyl)-4-methyl-
3H), 1.34 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ
6
158.90, 148.31, 144.05, 141.55, 140.35, 133.51, 132.55, 131.48,
131.36, 130.37, 129.05, 123.80, 116.93, 112.25, 80.26, 60.97, 16.37,
14.59.
1H-imidazole-5-carboxylate (24q). Compound 24q was prepared
Ethyl 1-[(4-Cyanobenzyl)oxy]-2-(3-cyanophenyl)-4-methyl-
1H-imidazole-5-carboxylate (24w). Compound 24w was prepared
in the same manner as that described for 24c to yield a white solid:
81.6%. Purity (HPLC): 99.6%. Mp 174.4−176.3 °C. ESI-HRMS
2
1
18
3
3
1
+
1
calcd. for C22
H N O [M + H] 387.1452, found: 387.1540. H
18 4 3
NMR (400 MHz, DMSO-d
1.4 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.63 (t, J = 7.8 Hz, 1H), 7.41
(d, J = 8.2 Hz, 2H), 5.23 (s, 2H), 4.35 (q, J = 7.1 Hz, 2H), 2.44 (s,
): δ 8.10−7.98 (m, 2H), 7.90 (dt, J = 7.8,
6
1
3
3H), 1.33 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ
6
Ethyl 1-[(4-Bromobenzyl)oxy]-2-(3-cyanophenyl)-4-methyl-
158.89, 144.02, 141.55, 138.34, 133.52, 132.69, 132.53, 131.33,
131.11, 130.37, 129.06, 118.76, 118.54, 116.95, 112.49, 112.26, 80.69,
60.95, 16.37, 14.59.
Ethyl 1-[(2-Cyanobenzyl)oxy]-2-(3-cyanophenyl)-4-methyl-
1H-imidazole-5-carboxylate (24x). Compound 24x was prepared
in the same manner as that described for 24c to yield a white solid,
1
H-imidazole-5-carboxylate (24r). Compound 24r was prepared
in the same manner as that described for 24c to yield a white solid,
yield: 79.4%. Purity (HPLC): 99.7%. Mp 137.6−139.3 °C. ESI-
HRMS calcd. for C H BrN O [M + H]+ 440.0604, found:
2
1
18
3
3
1
4
7
2
2
40.0653. H NMR (600 MHz, DMSO-d ): δ 8.08−8.00 (m, 2H),
.90 (d, J = 7.7 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.42 (d, J = 7.9 Hz,
H), 7.11 (d, J = 7.9 Hz, 2H), 5.12 (s, 2H), 4.35 (q, J = 7.1 Hz, 2H),
6
yield: 73.9%. Purity (HPLC): 99.7%. Mp 186.8−188.4 °C. ESI-
+
HRMS calcd. for C22
H
N
18
O
4
[M + H] 387.1452, found: 387.1481.
3
1
3
1
.43 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSO-
H NMR (400 MHz, DMSO-d ): δ 7.91−7.79 (m, 3H), 7.62−7.52
6
d6): δ 158.88, 143.99, 141.57, 133.34, 132.63, 132.44, 132.28, 131.75,
31.20, 130.22, 129.12, 123.54, 118.58, 112.19, 80.91, 60.90, 16.37,
4.60.
(m, 2H), 7.56−7.46 (m, 1H), 7.42 (td, J = 7.6, 1.3 Hz, 1H), 7.32 (d, J
= 8.0 Hz, 1H), 5.32 (s, 2H), 4.35 (q, J = 7.1 Hz, 2H), 2.43 (s, 3H),
1
1
1
3
1.35 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ 158.94,
6
Ethyl 2-(3-Cyanophenyl)-4-methyl-1-[(4-methylbenzyl)-
144.29, 142.20,135.74, 133.45, 133.35, 133.21, 132.74, 132.50,
131.51, 130.77, 130.21, 128.94, 118.54, 116.98, 116.85, 113.50,
112.20, 79.22, 60.94, 16.38, 14.59.
oxy]-1H-imidazole-5-carboxylate (24s). Compound 24s was
prepared in the same manner as that described for 24c to yield a
white solid, yield: 81.3%. Purity (HPLC): 99.2%. Mp 119.4−120.7
Ethyl 1-[(3-Cyanobenzyl)oxy]-2-(3-cyanophenyl)-4-methyl-
1H-imidazole-5-carboxylate (24y). Compound 24y was prepared
in the same manner as that described for 24c to yield a white solid,
yield: 76.8%. Purity (HPLC): 99.1%. Mp 172.3−173.7 °C. ESI-
+
°
C. ESI-HRMS calcd. for C H N O [M + H] 376.1656, found:
2
2
21
3
3
1
3
7
4
3
1
1
2
76.1745. H NMR (600 MHz, DMSO-d ): δ 8.07−8.01 (m, 2H),
6
.89 (dt, J = 7.8, 1.3 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.05−6.98 (m,
+
H), 5.06 (s, 2H), 4.36 (q, J = 7.1 Hz, 2H), 2.43 (s, 3H), 2.26 (s,
HRMS calcd. for C H N O [M + H] 387.1452, found: 387.1466.
2
2
18
4
3
1
3
1
H), 1.35 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ
H NMR (400 MHz, DMSO-d ): δ 8.07−7.98 (m, 2H), 7.89 (dt, J =
6
6
58.93, 143.97, 141.66, 139.54, 133.27, 132.56, 131.16, 130.64,
30.14, 129.88, 129.33, 129.21, 118.64, 116.97, 112.08, 81.75, 60.86,
1.24, 16.36, 14.62.
7.7, 1.4 Hz, 1H), 7.75 (dt, J = 7.1, 1.7 Hz, 1H), 7.67−7.59 (m, 2H),
7.53−7.41 (m, 2H), 5.21 (s, 2H), 4.36 (q, J = 7.1 Hz, 2H), 2.44 (s,
1
3
3H), 1.35 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ
6
Ethyl 2-(3-Cyanophenyl)-1-[(4-methoxybenzyl)oxy]-4-
158.92, 144.08, 141.72, 135.23, 134.59, 134.28, 133.53, 133.40,
methyl-1H-imidazole-5-carboxylate (24t). Compound 24t was
prepared in the same manner as that described for 24c to yield a white
solid, yield: 74.2%. Purity (HPLC): 99.3%. Mp 99.2−101.3 °C. ESI-
1
1
32.61, 131.35, 130.34, 130.05, 129.05, 118.55, 116.93, 112.22,
11.81, 80.40, 60.95, 16.38, 14.59.
2
-(3-Cyanophenyl)-1-methoxy-4-methyl-1H-imidazole-5-
+
HRMS calcd. for C H N O [M + H] 392.1605, found: 392.1635.
carboxylic acid (25a). A mixture of ethyl 2-(3-cyanophenyl)-1-
methoxy-4-methyl-1H-imidazole-5-carboxylate 22a (2.9 mmol), 1 M
LiOH aqueous (11 mL), THF (5 mL), and ethanol (5 mL) was
stirred at 50 °C for 6 h. The solvent was concentrated in a vacuum,
and the residue was acidified with dilute hydrochloric acid to pH 1.
22
21
3
4
1
H NMR (400 MHz, DMSO-d ): δ 8.09−8.00 (m, 2H), 7.87 (dt, J =
6
7
(
3
.8, 1.5 Hz, 1H), 7.69−7.57 (m, 1H), 7.10−7.00 (m, 2H), 6.80−6.67
m, 2H), 5.04 (s, 2H), 4.36 (q, J = 7.1 Hz, 2H), 3.72 (s, 3H), 2.42 (s,
1
3
H), 1.36 (t, J = 7.1 Hz, 3H). C NMR (100 MHz, DMSO-d ): δ
6
O
https://dx.doi.org/10.1021/acs.jmedchem.0c01524
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
The resulting precipitate was filtered, dried, and recrystallized with a
mixture of methanol and ethyl acetate (2:1) to yield the
corresponding 2-(3-cyanophenyl)-1-methoxy-4-methyl-1H-imidazole-
μL of fresh PRP and incubated for 5 min. Following that incubation
period, 15 μL of ADP (25 μM) was added to the reaction mix, leading
to a final concentration of 2.27 μM ADP. The plates were then
transferred to the microplate reader, and their absorbance was
monitored every 1 min at 655 nm up to 6 min. The IC50 was further
calculated on the basis of the aggregation of platelets (n = 3).
4
2
5
9
-carboxylic acid 25a as a white solid, yield: 91.2%. Purity (HPLC):
5.5%. Mp 203.7−205.3 °C. ESI-HRMS calcd. for C H N O [M −
1
3
11
3
3
−
1
H] 256.0801, found: 256.0725. H NMR (400 MHz, DMSO-d ): δ
6
2+
1
7
3.12 (s, 1H), 8.36−8.29 (m, 2H), 7.99−7.93 (m, 1H), 7.75 (t, J =
P2Y -Mediated Cytosolic Ca Increase Assay on PRP. The
1
1
3
2+
.8 Hz, 1H), 3.97 (s, 3H), 2.40 (s, 3H). C NMR (100 MHz,
ADP-dependent, P2Y -mediated increase in platelet cytosolic Ca
1
DMSO-d ): δ 160.19, 143.51, 140.01, 133.65, 132.12, 130.86, 130.81,
was measured by detecting changes in FLUO-4 fluorescence using a
6
22,23,46,54,55
1
29.23, 118.75, 117.59, 112.67, 67.59, 16.21.
-(Allyloxy)-2-(3-cyanophenyl)-4-methyl-1H-imidazole-5-
previously described method with minor modifications.
1
PRP was prepared as stated above, and it was added to HEPES−saline
buffer (1:8 dilutions; 10 mM HEPES, 0.15 M NaCl, pH 7.4). Diluted
PRP (70 μL, 8-fold) was incubated with 5 μM FLUO-4 AM (10 μL)
in the presence of 0.02 U/mL apyrase (10 μL) and 10 μM
indomethacin (10 μL) at 30 °C for 30 min. Then, 10 μL of the
vehicle or the tested compounds at various concentrations was added
into the mixture, which was incubated at 30 °C for 5 min. This
mixture (110 μL) was added to 430 μL of HEPES−saline buffer, and
the mixture was analyzed by a flow cytometer (BD FACSCalibur).
After obtaining a 30 s baseline recording, the acquisition was paused,
and then, 60 μL of ADP (5 μM final concentration) was quickly
added, the mixture was mixed, and the acquisition resumed (total
pause time less than 10 s). FLUO-4 fluorescence was monitored for 3
min. Fluo-4 fluorescence was plotted vs time, and the mean fluo-4
fluorescence of the baseline 30 s interval and of the 10 s poststimulant
carboxylic acid (25d). Compound 25d was prepared in the same
manner as that described for 25a to yield a white solid, yield: 91.5%.
Purity (HPLC): 97.9%. Mp 176.6−178.2 °C. ESI-HRMS calcd. for
−
1
C H N O [M − H] 282.0957, found: 282.0882. H NMR (400
15
13
3
3
MHz, DMSO-d ): δ 13.15 (s, 1H), 8.33 (t, J = 1.7 Hz, 1H), 8.30 (dt, J
6
=
7.9, 1.4 Hz, 1H), 7.95 (dt, J = 7.8, 1.4 Hz, 1H), 7.74 (t, J = 7.9 Hz,
1
H), 5.83 (ddt, J = 16.9, 10.3, 6.5 Hz, 1H), 5.40−5.26 (m, 2H), 4.67
1
3
(
d, J = 6.5 Hz, 2H), 2.40 (s, 3H). C NMR (100 MHz, DMSO-d ): δ
6
1
1
60.30, 143.44, 140.76, 133.55, 132.35, 131.08, 130.88, 130.69,
29.55, 122.93, 118.72, 117.96, 112.52, 80.61, 16.25.
1
-(Benzyloxy)-2-(3-cyanophenyl)-4-methyl-1H-imidazole-5-
carboxylic acid (25k). Compound 25k was prepared in the same
manneras that described for 25a to yield a white solid, yield: 90.2%.
Purity (HPLC): 95.7%. Mp 195.3−197.4 °C. ESI-HRMS calcd. for
−
1
C H N O [M − H] 332.1041, found: 332.1003. H NMR (400
19
15
3
3
2+
intervals were calculated. The cytosolic Ca increase was calculated
MHz, DMSO-d ): δ 13.22 (s, 1H), 8.15−8.08 (m, 2H), 7.89 (dt, J =
6
as the raise of the maximal poststimulant fluo-4 fluorescence over the
baseline fluo-4 fluorescence. The percentage inhibition was calculated
7
(
.7, 1.4 Hz, 1H), 7.69−7.60 (m, 1H), 7.37−7.30 (m, 1H), 7.31−7.24
13
m, 2H), 7.24−7.18 (m, 2H), 5.13 (s, 2H), 2.43 (s, 3H). C NMR
relative to ADP + vehicle (0% inhibition) and vehicle alone (100%
(
100 MHz, DMSO-d ): δ 160.43, 143.65, 141.15, 133.27, 133.06,
6
2+
inhibition): inhibition (%) = [(cytosolic Ca increase
−
(ADP)
(
ADP)
1
1
32.45, 131.13, 130.61, 130.32, 129.82, 129.37, 128.84, 118.67,
17.65, 112.21, 81.72, 16.31.
2
+
2+
cytosolic Ca increase (ADP+compound))/(cytosolic Ca increase
2
+
−
cytosolic Ca increase (Black))] × 100.
2
-(3-Cyanophenyl)-4-methyl-1-((4-methyl benzyl) oxy)-1H-
P2Y -Mediated Vasodilator-Stimulated Phosphoprotein
VASP) Phosphorylation Assay. This assay was measured by
imidazole-5-carboxylic acid (25s). Compound 25s was prepared
in the same manner as that described for 25a to yield a white solid,
yield: 81.2%. Purity (HPLC): 95.1%. Mp 168.2−169.8 °C. ESI-
12
(
flow cytometry using a kit (BioCytex, Marseilles, France) according to
the manufacturer’s recommendations. First, 2 μL of the test
compound solution or model (HEPES−saline) was added to each
−
HRMS calcd. for C H N O [M − H] 346.1264, found: 346.1191.
20
17
3
3
1
H NMR (400 MHz, DMSO-d ): δ 8.10−8.01 (m, 2H), 7.88 (dt, J =
.7, 1.4 Hz, 1H), 7.63 (t, J = 7.9 Hz, 1H), 7.03 (d, 4H), 5.08 (s, 2H),
.43 (s, 3H), 2.26 (s, 3H). C NMR (100 MHz, DMSO-d ): δ
67.82, 160.39, 143.68, 141.45, 139.51, 133.17, 132.56, 131.14,
30.67, 130.14, 130.00, 129.32, 118.69, 117.51, 112.04, 81.62, 21.25,
6.28.
6
23
set of assay tubes, followed by 9 μL of PGE or 9 μL of PGE /ADP.
1
1
7
2
1
1
1
1
3
Next, the citrated human whole blood was added to each tube and
6
23
incubated for 10 min at room temperature. Lastly, the samples were
fixed, permeabilized, and labeled with a fluorescently conjugated
monoclonal antibody (clone 16C2) directed against the serine 239
phosphorylated form of VASP, and CD61 was used as a platelet
1
-[(4-Cyanobenzyl)oxy]-2-(3-cyanophenyl)-4-methyl-1H-
23,56
identifier.
The analysis was performed in a flow cytometer (BD
imidazole-5-carboxylic acid (25w). Compound 25w was prepared
in the same manner as that described for 25a to yield a white solid,
yield: 92.4%. Purity (HPLC): 99.6%. Mp 201.5−202.3 °C. ESI-
FACSCalibur). The PGE -stimulated condition showed maximum
1
VASP phosphorylation, and HEPES−saline buffer and ticagrelor were
added as a control (no inhibition) and positive control, respectively.
Furthermore, the agonist effects of the test compounds were checked
−
HRMS calcd. for C H N O [M − H] 357.1066, found: 357.0983.
20
14
4
3
1
H NMR (400 MHz, DMSO-d ): δ 13.17 (s, 1H), 8.09−8.00 (m,
H), 7.88 (dt, J = 7.7, 1.4 Hz, 1H), 7.71 (d, J = 8.1 Hz, 2H), 7.62 (t, J
6
2
2,23
using PGE alone without ADP on samples.
The MFI was the
2
1
49
mean fluorescence intensity of VASP phosphorylation, and
=
7.8 Hz, 1H), 7.40 (d, J = 8.2 Hz, 2H), 5.23 (s, 2H), 2.42 (s, 3H).
1
3
C NMR (100 MHz, DMSO-d ): δ 160.35, 143.76, 141.22, 138.42,
inhibition (%) = [(MFI(PGE1) − MFI(PGE1+ADP+compound))/(MFI(PGE1)
6
−
MFI(Negative))] × 100.
Xanthine Oxidase Inhibitory Activity. The XO inhibitory
1
1
33.34, 132.66, 132.44, 131.22, 131.10, 130.31, 129.23, 118.77,
18.57, 117.47, 112.46, 112.22, 80.56, 16.31.
ADP-Induced Rabbit PRP Aggregation Assay. All target
potency with xanthine as the substrate was assayed spectrophoto-
metrically by measuring uric acid formation at 295 nm at 25 °C
compounds were evaluated for their antiplatelet potency in ADP-
5
2
11,22,23,44,56,75
according to the procedure previously reported by us with
modifications. Febuxostat was used as a reference. XO (Sigma,
X4875) was suspended in a buffer (0.1 M sodium pyrophosphate and
induced rabbit platelet-rich plasma (rPRP) aggregation.
Preparation of Platelet-Rich Plasma (PRP). Blood was drawn from
a rabbit carotid into a new tube containing 3.8% sodium citrate. The
platelet-rich plasma (PRP) was obtained by centrifugation of the
blood at 800 rpm for 10 min at room temperature, and the
supernatant was carefully transferred to a fresh tube. Then, the
remaining blood was again centrifuged at 3000 rpm for 10 min to
prepare platelet-poor plasma (PPP), which was carefully transferred
into a new tube.
0.3 mM Na EDTA buffer, pH 8.3). The buffer (67 mL), enzyme
2
solution (50/L, 40 μL), and sample (53 μL) or blank solution (the
buffer) were added to 96-well plates (COSTAR 3599) and incubated
at 25 °C for 15 min. Then, the mixture was added with substrate
xanthine (40 μL, 500 μM) to the plates to a total volume of 200 μL,
which was further scanned to measure the absorbance change
immediately at 295 nm and at 30 s intervals for 2 min. All the tests
were performed in triplicate. Compounds presenting inhibitory effects
over 50% at a concentration of 10 μM were further tested at a wide
range of concentrations to calculate their IC50 values using SPSS 20.0
software (SPSS Inc., Chicago, IL).
Rabbit Platelet Aggregation 96-Well Assay. The rabbit platelet
aggregation assay was performed in 96-well plates (COSTAR 3599)
75
using a microplate reader. In brief, PRP was added into a
prewarmed 96-well microplate at 37 °C. Then, 15 μL of the test
compounds at a 10× final concentration in NaCl was mixed with 135
P
https://dx.doi.org/10.1021/acs.jmedchem.0c01524
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Docking Studies. Molecular docking studies were performed
SYPU-IACUC-2019-9-23-201) were purchased from the Animal
Center of Shenyang Pharmaceutical University (Shenyang, China).
Animal maintenance and treatment met the protocols approved by
the Ethics Review Committee for Animal Experimentation of
Shenyang Pharmaceutical University. The rats had free access to
food and water and were maintained on a 12 h light/dark cycle in a
62
using GLIDE (2016, Schrodinger Suite). The crystal structures of
̈
63
64
P2Y (PDB: 4XNW) and P2Y (PDB: 4NTJ) were retrieved
1
12
from the RCSB Protein Data Bank, which was further prepared using
the Protein Preparation Wizard tool implemented in the Schrodinger
̈
suite by adding all hydrogen atoms as well as the missed side chains of
residues and deleting all bound water. The ligands were built within
8
5,86
temperature- and humidity-controlled room for 1 week.
Maestro BUILD (2016, Schro
LIGPREP module (2016, Schrodinger Suite). The tautomeric forms
of ligands, which include the keto and enol forms of ligands, were
̈
dinger Suite) and prepared by the
A total of 18 Sprague-Dawley rats were randomly distributed into
three experimental groups (n = 6 in each group). The oral groups
were given compounds (24w or 25w) suspended in 0.5% CMC-Na at
an oral dose of 10 mg/kg, and the other group received a single
intravenous injection of compound 25w dissolved in a solution
(saline/PBS/NaOH aqueous) at dose of 10 mg/kg. Whole blood
samples (0.3 mL) were gathered into heparinized tubes via the
suborbital vein after oral administration at 0.17, 0.33, 0.67, 1, 1.5, 2, 3,
4, 8, 16, and 24 h and after the intravenous administration at 0.083,
0.17, 0.33, 0.67, 1, 1.5, 2, 3, 4, 8, and 12 h. All blood samples were
centrifuged (8000g, 10 min, 4 °C), and the resulting plasma samples
were immediately stored at −80 °C until LC−MS analysis.
62
̈
62
generated at a physiological pH (7.0 ± 2.0). The Glide Grid was
built using an inner box of dimensions 14 × 14 × 14 Å around the
centroid of the ligand, assuming that the ligands to be docked were of
a size similar to that of the cocrystallized ligand. This docking
methodology has been validated by extracting the crystallographic
bound ligand and redocking it with the Glide module using extra
precision (SP). Different docking poses of ligands were generated and
3
65
analyzed for interpretation of the final results. Pymol was used for
graphic display.
7
6,77
Simulated Gastric and Intestinal Fluid Stability.
Acute Oral Toxicity Study. Healthy Kunming mice of both sexes
(18−22 g; n = 8; Number of Approval from Ethics Committee:
SYPU-IACUC-2019-5-29-202) were purchased from the Animal
Center of Shenyang Pharmaceutical University (Shenyang, China).
Animal maintenance and treatment met the protocols approved by
the Ethics Review Committee for Animal Experimentation of
Shenyang Pharmaceutical University. The mice had free access to
food and water and were maintained on a 12 h light/dark cycle in a
Incubations of tested compounds were performed at a concentration
of 10 μM in simulated gastric and intestinal fluids. Then, these
reaction solutions were kept at 37 °C and sampled hourly for 12 h. An
aliquot (50 μL) of the mixtures was terminated hourly by the addition
of 200 μL of ice-cold acetonitrile containing an internal standard
compound 24c). After immediate mixing, extracts were centrifuged
at 12 000 rpm for 10 min at 4 °C, and the supernatants were analyzed
(
8
5−87
by HPLC.
temperature- and humidity-controlled room for 1 week.
7
8,79
Stability Studies in Plasma.
The plasma stability and
After fasting for 12 h with free access to water prior to the
experiment, four groups of animals (male control group, female
control group, male test group, and female test group; n = 8) were
used for acute oral toxicity study. The control groups were treated
with the 0.5% CMC-Na, and the test groups were treated with a single
dose (2000 mg/kg) of the test compound 24w, which was suspended
in a 0.5% CMC-Na solution. All treatments were intragastrically
administered immediately after 12 h of fasting. The mice were
observed continuously for any signs and symptoms of toxicity, and
body weights of these mice were monitored every day over the 14 day
metabolism of compounds 24w and 25w (initial concentration: 10
μM) were incubated in rabbit and rat plasma, respectively, and were
placed in a water bath shaker at 37 °C. Reactions were terminated
following 0, 5, 15, 30, and 45 min by ice-cold acetonitrile containing
an internal standard (compound 24c). After immediate mixing, the
extracts were centrifuged at 12 000 rpm for 10 min at 4 °C, and the
supernatants were analyzed by HPLC.
8
0−83
Stability Studies in Liver Microsomes.
The assay was
performed using RLMs and HLMs, which were purchased from the
Research Institute for Liver Diseases (Shanghai, China). The
compounds (final concentration of 0.2 μM in 0.1% DMSO) were
incubated with liver microsomes (0.50 mg/mL in 0.1 M PBS buffer
88
period after treatment.
Rat FeCl3 Thrombosis Model. The FeCl -induced arterial
3
thrombosis was instigated according to the previously described
11,72,73
(
pH 7.4), 3.2 mM MgCl , and reduced nicotinamide adenine
method with minor modifications.
Male Sprague-Dawley rats
2
dinucleotide phosphate (NADPH, 1 mM)) at 37 °C. The reactions
were stopped by transferring 50 μL of the reaction solutions into 200
μL of ice-cooled methanol containing 0.03 μM internal standard
(300−320 g, n = 10; Number of Approval from Ethics Committee:
SYPU-IACUC-2019-9-23-201) were purchased from the Animal
Center of Shenyang Pharmaceutical University (Shenyang, China).
Animal maintenance and treatment met the protocols approved by
the Ethics Review Committee for Animal Experimentation of
Shenyang Pharmaceutical University. The rats had free access to
food and water and were maintained on a 12 h light/dark cycle in a
(
compound 24c) at 0, 10, 20, 30, 60, and 90 min. Then, the mixtures
were vortex-mixed for 1 min and centrifuged at 12 000 rpm for 10
min, and supernatants were analyzed by LC−MS/MS.
6
8−70
CYP450 Inhibition Cocktail Assay.
The incubation
8
5−87
mixture containing HLMs (final concentration: 0.5 mg/mL), MgCl2
final concentration: 3.2 mM), compounds 24w or 25w (final
temperature- and humidity-controlled room for 1 week.
(
Solutions of compound 24w, ticagrelor, or model suspended in
0.5% CMC-Na were administered once a day for 7 consecutive days
(including the last day) as a single oral dose of 10 mg/kg. Rats were
anesthetized with urethane (1.25 g/kg, i.p.) and then placed on a
heating pad to maintain body temperature. Through a median
incision of the ventral side in the neck, a 1.5 cm long portion of the
left carotid artery of rats was exposed via blunt dissection and carefully
dissected clear of the vagus nerve and surrounding tissue. Then, this
left carotid artery was put on a piece of plastic membrane (1.0 × 1.5
concentration: 10 μM), and specific CYP substrates in 0.1 M PBS
buffer (pH 7.4) was preincubated for 5 min at 37 °C. Then, this
reaction was initiated by the addition of NADPH solution (1 mM).
The total content of organic solvent was maintained at <3%. The
specific CYP substrates used in this cocktail assay included
phenacetin, coumarin, tolbutamide, S-mephenytoin, dextromethor-
phan, chlorzoxazone, and testosterone (as probe substrates for
enzymes of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6,
CYP2E1, and CYP3A4, respectively). After a 120 min incubation at
2
11,72
cm ) to protect surrounding tissues.
A total of 1.5 h after drug
2
3
7 °C, the reactions were quenched by the addition of cooled
administration, a strip of filter paper (0.8 × 1.0 cm ) saturated with
20% FeCl in water was placed on the anterior of the carotid artery to
methanol with a mixture of internal standard (2-chloro-5-methoxyani-
line hydrochloride, 0.5 μM). The mixture was vortex-mixed for 1 min
and centrifuged at 12 000 rpm for 10 min, and the supernatants were
used for a simultaneous analysis of the probe substrate metabolites
3
11,72
induce thrombosis formation.
Subsequently, the plastic mem-
brane and filter paper were removed 2 h after drug administration and
the right artery was cut immediately, gently blotted dry, and
11,72
(
acetaminophen, 7-hydroxycoumarin, 4-hydroxytolbutamide, 4-hy-
weighed.
The entire thrombosis was then scraped from artery,
droxymephenytoin, dextrorphan, 6-hydroxychlorzoxazone, and 6β-
hydroxytestosterone) and internal standard by LC−MS/MS.
and the vessel wall was reweighed; the wet weight of thrombosis was
11,72
obtained by subtraction.
Rat Blood Loss Model. The rat blood loss model was performed
8
4
Rat Pharmacokinetic Studies. Eighteen male Sprague-Dawley
rats (300−320 g; Number of Approval from Ethics Committee:
11,44,48,72
as described previously with minor modifications.
Male
Q
https://dx.doi.org/10.1021/acs.jmedchem.0c01524
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Sprague-Dawley rats (300−320 g, n = 8; Number of Approval from
Ethics Committee: SYPU-IACUC-2019-9-23-201) were purchased
from the Animal Center of Shenyang Pharmaceutical University
Qing Mao − Key Laboratory of Structure-Based Drugs Design
Discovery of Ministry of Education, School of
&
Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenhe District, Shenyang 110016, China
Yao Feng − Key Laboratory of Structure-Based Drugs Design
(
Shenyang, China). Animal maintenance and treatment met the
protocols approved by the Ethics Review Committee for Animal
Experimentation of Shenyang Pharmaceutical University. The rats had
free access to food and water and were maintained on a 12 h light/
dark cycle in a temperature- and humidity-controlled room for 1
week. Compound 24w and ticagrelor in 0.5% CMC-Na solution were
administered by oral gavage at the 10 mg/kg dose. At 0.5 h after
administration, the rats were anesthetized with urethane (1.25 kg, i.p.)
and placed on a 37 °C heating pad to maintain body temperature with
&
Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenhe District, Shenyang 110016, China
Jiaxing Zhao − Key Laboratory of Structure-Based Drugs
Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenhe District, Shenyang 110016, China
Fengwei Lin − Key Laboratory of Structure-Based Drugs
Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenhe District, Shenyang 110016, China
Yulin Duan − Key Laboratory of Structure-Based Drugs
Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenhe District, Shenyang 110016, China
Yan Zhang − Department of Pharmacology, Shenyang
Pharmaceutical University, Shenhe District, Shenyang
11,44,72
their tails straightened.
Then, 1.5 h after administration, rat tails
were transected 4 mm from the tip with a scalpel blade and
immediately immersed in a 20 mL graduated cylinder filled with 12
11,44,72
mL of saline held at 37 °C.
The observation period was stopped
when no additional bloodstains were observed in a period of 30
seconds, and the bleeding time (including those of rebleeding) was
11,44,72
recorded within 2 h.
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01524.
■
*
110016, China
Worksheet of molecular formula strings (XLSX)
Ziyang Bao − Key Laboratory of Structure-Based Drugs
Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenhe District, Shenyang 110016, China
Yuwei Yang − Key Laboratory of Structure-Based Drugs
Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenhe District, Shenyang 110016, China
Figures of HPLC spectra, H and 13C NMR spectra,
1
HRMS spectra, and MS spectra (PDF)
AUTHOR INFORMATION
Corresponding Authors
Yanhua Mou − Department of Pharmacology, Shenyang
Pharmaceutical University, Shenhe District, Shenyang
■
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01524
1
10016, China; Phone: 024-23986339;
Email: mu_hua_jj@sina.com; Fax: 024-23986339
Shaojie Wang − Key Laboratory of Structure-Based Drugs
Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenhe District, Shenyang 110016, China;
Phone: (+86)24-23986421; Email: wangshaojie@
syphu.edu.cn; Fax: (+86)24-23986421
Author Contributions
Y.L. and B.Z. contributed equally to this work.
∥
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors would like to thank Wei Zhang, Yanli Diao, and
Jingyi Sun for their technical support in animal experiments.
■
Authors
Yu Lei − Key Laboratory of Structure-Based Drugs Design &
Discovery of Ministry of Education, School of Pharmaceutical
Engineering, Shenyang Pharmaceutical University, Shenhe
District, Shenyang 110016, China
Bing Zhang − Key Laboratory of Structure-Based Drugs
Design & Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenhe District, Shenyang 110016, China;
orcid.org/0000-0002-4427-8771
Dan Liu − Shenyang Hinewy Pharmaceutical Technology Co.,
Ltd., Shenhe District, Shenyang 110016, China
Jian Zhao − Department of Pharmacology, Shenyang
Pharmaceutical University, Shenhe District, Shenyang
ABBREVIATIONS USED
■
ACS, acute coronary syndromes; AMI, acute myocardial
infarction; ADP, adenosine diphosphate; PCI, percutaneous
coronary intervention; XO, xanthine oxidase; SAR, structure−
activity relationships; rPRP, rabbit platelet-rich plasma; PK,
pharmacokinetic; VASP, vasodilator-stimulated phosphopro-
tein; PGE , prostaglandin E ; MFI, mean fluorescence
1
1
intensity; PDB, protein data bank; PBS, phosphate buffer
saline; NADPH, nicotinamide adenine dinucleotide phos-
phate; HLMs, human liver microsomes; RLMs, rat liver
1
10016, China
Xiwen Dai − Key Laboratory of Structure-Based Drugs Design
Discovery of Ministry of Education, School of
microsomes; Cl , intrinsic body clearance; Cmax, peak plasma
int
concentration; Tmax, time to reach Cmax; t1/2, elimination half-
life; AUC0−∞, area under the concentration−time curve; CLz,
clearance; Vz, volume of distribution; F, absolute oral
&
Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenhe District, Shenyang 110016, China
Jun Gao − Key Laboratory of Structure-Based Drugs Design &
Discovery of Ministry of Education, School of Pharmaceutical
Engineering, Shenyang Pharmaceutical University, Shenhe
District, Shenyang 110016, China
bioavailability; CYP450, cytochrome P450; NaNO , sodium
2
nitrite; Me SiCl, chlorotrimethylsilane; NaI, sodium iodide;
3
DMF, N,N-dimethylformamide; K CO , potassium carbonate;
2
3
KI, potassium iodide; LiOH, lithuium hydroxide; Me SO ,
2
4
dimethyl sulfate
R
https://dx.doi.org/10.1021/acs.jmedchem.0c01524
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1
-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea, a potent, selective,
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