Novel thienopyrimidinones as mGluR1 antagonists

Article abstract of DOI:10.1016/j.ejmech.2014.08.027

There has been much attention to discover mGluR1 antagonists for treating various central nervous system diseases such as seizures and neuropathic pain. Thienopyrimidinone derivatives were designed, synthesized, and biologically evaluated against mGluR1. Among the synthesized compounds, 3-(4-methoxyphenyl)- 7-(o-tolyl)thienopyrimidin-4-one 30 exhibited the most potent inhibitory activity with an IC₅₀ value of 45 nM and good selectivity over mGluR5. Also, the selective mGluR1 antagonist 30 showed marginal hERG channel activity (IC₅₀ = 9.87 μM), good profiles to CYP isozymes, and a good pharmacokinetic profile. Overall, the compound 30 was identified as a selective mGluR1 antagonist with a good pharmacokinetic profile, which is probably devoid of cardiac side effect and drug-drug interactions. Therefore, the compound 30 can be expected to be broadly used as mGluR1 antagonistic chemical probe in in vitro and in vivo study for investigating CNS diseases.

Full text of DOI:10.1016/j.ejmech.2014.08.027

European Journal of Medicinal Chemistry 85 (2014) 629e637
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel thienopyrimidinones as mGluR1 antagonists
,
,
Youngjae Kim a b, Jeeyeon Kim a b, Sora Kim a, Yooran Ki a, Seon Hee Seo a, Jinsung Tae b,
Min Kyung Ko a, Hyun-Seo Jang a, Eun Jeong Lim a, Chiman Song c, YoonJeong Cho d,
Hae-Young Koh d, Youhoon Chong e, Il Han Choo f, Gyochang Keum a, Sun-Joon Min a
,
, g, *, 1
, g, *, 1
Hyunah Choo a
a Center for Neuro-Medicine, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, South Korea
b Department of Chemistry, Yonsei University, Seodaemun-gu, Seoul 120-749, South Korea
c Chemical Kinomics Center, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, South Korea
d Center for Neuroscience, Korea Institute of Science and Technology, Seongbuk-gu, Seoul 136-791, South Korea
e Department of Bioscience and Biotechnology, Konkuk University, Gwangjin-gu, Seoul 143-701, South Korea
f School of Medicine, Chosun University, Dong-gu, Kwangju 501-759, South Korea
g Department of Biological Chemistry, University of Science and Technology, Youseong-gu, Daejeon 305-350, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
There has been much attention to discover mGluR1 antagonists for treating various central nervous
system diseases such as seizures and neuropathic pain. Thienopyrimidinone derivatives were designed,
synthesized, and biologically evaluated against mGluR1. Among the synthesized compounds, 3-(4-
methoxyphenyl)-7-(o-tolyl)thienopyrimidin-4-one 30 exhibited the most potent inhibitory activity
with an IC50 value of 45 nM and good selectivity over mGluR5. Also, the selective mGluR1 antagonist 30
Received 8 April 2014
Received in revised form
6 August 2014
Accepted 7 August 2014
Available online 8 August 2014
showed marginal hERG channel activity (IC50 ¼ 9.87
mM), good profiles to CYP isozymes, and a good
pharmacokinetic profile. Overall, the compound 30 was identified as a selective mGluR1 antagonist with
a good pharmacokinetic profile, which is probably devoid of cardiac side effect and drugedrug in-
teractions. Therefore, the compound 30 can be expected to be broadly used as mGluR1 antagonistic
chemical probe in in vitro and in vivo study for investigating CNS diseases.
Keywords:
mGluR1 antagonist
Thienopyrimidinone
CNS disease
© 2014 Elsevier Masson SAS. All rights reserved.
Glutamate
Metabotropic
1. Introduction
antagonists exhibit neuroprotective effects in traumatic brain
injury models. Administration of mGluR1 antagonist YM-202074
Glutamate is
a
major excitatory neurotransmitter in the
within 2 h of the onset of ischemia significantly reduced infarct
volumes in the brain and improved neurological scores [4]. The
intrathecal injection of group I mGluR agonists induced sponta-
neous pain behavior that was reversed by administration of a se-
lective mGluR1 antagonist [5]. Moreover, the intrathecal
administration of mGluR1 antibodies reduced cold hyperalgesia
and mechanical allodynia associated with chronic nerve constric-
tion in rats. mGluR1 knockout mice exhibit reduced sensitivity to
pain and are also more receptive to morphine than the wild types,
thus identifying mGluR1 as a potential target for treating neuro-
pathic pain.
mammalian central nervous system, which exerts its effects via
activation of two receptor families: ionotropic glutamate (iGlu)
receptors and metabotropic glutamate (mGlu) receptors [1]. The
iGlu receptors are responsible for fast excitatory effects via ligand
gated ion channels, which are divided into NMDA receptors, AMPA
receptors and kainate receptors, while the mGlu receptors which
belong to class C GPCR regulate these fast effects by modulating the
ion channel activity and neurotransmitter release [2].
It has been reported that mGluR1 is essential for motor coor-
dination and perception of pain, and may play an important role in
seizures and related disorders [3]. It has been proved that mGluR1
There has been much attention to discover novel mGluR1 an-
tagonists, and some of the most recently reported mGluR1 antag-
onists
include
pyridopyrimidines,
tricyclic
pyridothienopyrimidinones,
b
-carbolines, pyrrolopyrimidinones
* Corresponding authors. Center for Neuro-Medicine, Korea Institute of Science
and Technology, Seongbuk-gu, Seoul 136-791, South Korea.
E-mail address: hchoo@kist.re.kr (H. Choo).
and their analogs (Fig. 1) [6]. Despite the structural diversity of
known mGluR1 antagonists, selective and orally-bioavailable
small-molecule mGluR1 antagonists are still needed to be
1
These two corresponding authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.08.027
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

630
Y. Kim et al. / European Journal of Medicinal Chemistry 85 (2014) 629e637
which is an imaging based plate reader for cellular assays [8].
Initially, the %-inhibitions against mGluR1 were measured at 10
and 1 M, and the IC50 values of the selected compounds with
mM
m
potent inhibitory activities were obtained. The FDSS6000 assay
results of the thienopyrimidinones 6e23 with R1 as hydrogen were
shown in Table 1. The unsubstituted compound 6 showed inhibi-
tory activity against mGluR1 with an IC50 value of 136 nM, which
was less potent compared with the reference compound
(IC50 ¼ 6.7 nM). The compound 7 with 2-F as R2 was less active than
the unsubstituted compound 6, while the compounds 8 and 9 with
3-F and 4-F were more active with IC50 values of 58 nM and 66 nM,
respectively. In the case of chlorine substituents, three compounds
10e12 showed moderate inhibitory activities with IC50 values of
407 nM, 374 nM, and 162 nM, respectively. The inhibitory activity of
the methyl-substituted compounds 13e15 were the same as the
chlorine-substituted analogs (IC50 ¼ 499 nM, 184 nM, and 112 nM,
respectively). The compounds 16 and 17 with 2-OMe and 3-OMe
substituents showed only marginal inhibitory activities against
mGluR1. However, the compound 18 with 4-OMe group was the
most active among the thienopyrimidinones 6e23 (R1 ¼ H) with an
IC50 value of 50 nM. The compounds 19e23 with vinyl, cyano and
nitro groups as R2 were also tested against mGluR1; the vinyl
compounds 19 (IC50 ¼ 74 nM) and 20 (IC50 ¼ 128 nM) showed good
potency while the other compounds 21e23 are barely active. Taken
together, among the thienopyrimidinones 6e23 with hydrogen as
R1, the compounds with meta and para substituents showed better
inhibitory activities than the corresponding ortho-substituted an-
alogs with the 4-methoxy compound 18 being most active
(IC50 ¼ 50 nM).
Fig. 1. Known mGluR1 antagonists.
developed for treating various central nervous system diseases. The
pyridothienopyrimidinone and the pyrrolopyrimidinone were re-
ported to have good in vitro potency against mGluR1, selectivity
over mGluR5 and a good pharmacokinetic profile [6e,6h]. The tri-
cyclic pyridothienopyrimidinone reduced mechanical allodynia in
spinal nerve ligation animal model with an ED50 of 6.4 mg/kg.
Based on these results, we designed thienopyrimidinone de-
rivatives which was synthesized and biologically evaluated against
mGluR1 (Fig. 2). The compounds with good in vitro potency were
further evaluated for selectivity over mGluR5, activities on hERG
channel and CYP isozymes, and pharmacokinetic profiles in order
to make sure that the thienopyrimidine derivatives could be
developed as a drug candidate for treating CNS diseases.
To further improve inhibitory activity of the compound 18
against mGluR1, we fixed R2 as 4-OMe group and modified the
substituent R1 on the other aryl ring attached to the thiophene. The
mGluR1-overexpressing cell-based assay results were shown in
Table 2. The compound 24 with 2-F as R1 showed potent inhibitory
activity against mGluR1 with an IC50 value of 57 nM, while meta-F
and para-F compounds 25 and 26 were only marginally active. The
ortho-Cl compound 27 was less active than the original compound
28 with an IC50 value of 221 nM. The other Cl-substituted com-
pounds 28 and 29 were almost as active as their fluoro congeners,
25 and 26. The compound 30 with 2-methyl substituent showed
the most potent activity among all the synthesized compounds
with an IC50 value of 45 nM, while other methyl-substituted iso-
mers, 31 and 32, were shown moderate to low inhibition of
mGluR1. The compounds 33 and 34 with ortho-OMe and meta-OMe
as R1 showed moderate inhibitory activities with IC50 values of
747 nM and 257 nM, respectively, while the para-methoxy com-
pound 35 was not active. Taken together, substituents such as F,
OMe and vinyl attached to the meta- and/or para-positions of the
thiophenylphenyl ring (R2) afforded the resulting thiophenylpyr-
imidones with potent activities against mGluR1.
2. Chemistry
The designed thienopyrimidinones were synthesized in 4 steps
from various arylacetonitriles 1 which include substituents such as
H, F, Cl, Me and OMe at ortho, meta and para positions [7]. The
arylacetonitriles 1 underwent formylation by treating methyl
formate and NaH in THF to give the desired products 2 as tauto-
merized enol forms (72%e100% yields). The enols 2 were methyl-
ated by methyl sulfate and NaH in THF to afford 3 (19%e100%
yields), which were converted to thiophenes 4 in 17%e70% yields
by treatment with methyl thioglycolate under basic conditions. The
anilines 5 with various substituents such as H, F, Cl, OMe, Me, vinyl,
CN and NO2 reacted with the thiophenes 4 in the presence of
triethyl orthoformate under acidic conditions to afford the target
compounds 6e35 in 7%e88% yields (Scheme 1). The reference
compound, pyridothienopyrimidinone (Fig. 1) was synthesized by a
known method [6e].
The most potent mGluR1 inhibitors thus obtained (18, 24, and
30) were further evaluated for their selectivity over mGluR5, ac-
tivities on hERG channel and CYP isozymes, and human liver
microsomal stability, and the results are summarized in Table 3.
hERG channel is a potassium ion channel coded by the human
3. Results and discussion
The synthesized compounds were biologically evaluated against
Chem-3 cells which stably express mGluR1, where intracellular
calcium ion levels in the cells were measured using FDSS6000
ꢀ
Ether-a-go-go-related gene, and its inhibition is known to result in
long QT syndrome which can create a concomitant risk of sudden
death [9]. In order to avoid drugedrug interactions, inhibitory ac-
tivities of the thiophenylpyrimidones against CYP isozymes such as
CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were also tested
[10]. Finally, through the human liver microsomal stability assay,
in vitro stabilities of the tested compounds to metabolism in human
liver microsomes were measured [11]. Like the reference com-
pound (Table 1), all three compounds 18, 24, and 30 were very
Fig. 2. Designed thienopyrimidinones.
selective over mGluR5 with 40e60% inhibition of mGluR5 at 10 mM

Y. Kim et al. / European Journal of Medicinal Chemistry 85 (2014) 629e637
631
Scheme 1. Synthesis of the thienopyrimidinone derivativesa.
concentrations (Table 3). While the compound 24 did not block
hERG channel up to at 100 M, the two compounds 18 and 30
showed moderate inhibition against hERG channel with IC50 values
of 9.89 M and 31.0 M. Nevertheless, it should be noted that the
mGluR5. We further evaluated activities on hERG channel and CYP
isozymes, and pharmacokinetic profiles for the selective mGluR1
antagonist, and the compound 30 showed excellent pharmaco-
logical properties in terms of hERG channel and CYP isozymes and a
good pharmacokinetic profile. Overall, the compound 30 was
identified as a selective mGluR1 antagonist with a good pharma-
cokinetic profile, which is probably devoid of cardiac side effect and
drugedrug interactions. Therefore, the compound 30 can be ex-
pected to be broadly used as mGluR1 antagonistic chemical probe
in in vitro and in vivo study for investigating CNS diseases.
m
m
m
therapeutic indices of these compounds are high enough (>200
times in IC50). The microsomal stability was shown in %-remaining
concentration 30 min after incubation with human liver micro-
somes and the compound 30 was the most stable with 50.1% of the
initial concentration remaining after incubation. In the CYP
isozyme assay, the %-remaining activities of the CYP isozymes after
treatment with the thiophenylpyrimidones were evaluated. Even
though the activity of CYP2C9 was shown to be inhibited by 18
(18.3%-remaining activity), no other CYP isozyme was significantly
affected by the thiophenylpyrimidones derivatives. Thus, all the
selected compounds displayed excellent pharmacological proper-
ties in terms of selectivity and activities on hERG channel and CYP
isozymes, and the most promising candidate turned out to be 30.
The pharmacokinetic parameters for the compound 30 were then
evaluated after intravenous and oral administration in rats, and the
results are presented in Table 4. In the intravenous injection, the
values of AUC, half-life, mean residence time (MRT), and clearance
were proper and Vdss (apparent volume of distribution at steady
state) was also good. In contrast, in the oral administration, half-life
was a little bit high and AUCs fell short of our expectation, resulting
in moderate oral bioavailability with an F value of 16.5% [12]. The
concentrations of 30 in plasma and brain were also measured and
the brain-to-plasma ratios were good in both intravenous and oral
administrations.
5. Experimental section
5.1. Chemistry
General: All reactions were carried out under dry nitrogen un-
less otherwise indicated. Commercially available reagents were
used without further purification. Solvents and gases were dried
according to standard procedures. Organic solvents were evapo-
rated with reduced pressure using a rotary evaporator. Analytical
thin layer chromatography (TLC) was performed using glass plates
precoated with silica gel (0.25 mm). TLC plates were visualized by
exposure to UV light (UV), and then were visualized with a p-ani-
saldehyde stain followed by brief heating on hot plate. Flash col-
umn chromatography was performed using silica gel 60
(230e400 mesh, Merck) with the indicated solvents. 1H and 13C
spectra were recorded on Bruker 300, Bruker 400 or Varian 300
NMR spectrometers. 1H NMR spectra are represented as follows:
chemical shift, multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet,
q ¼ quartet, m ¼ multiplet), integration, and coupling constant (J)
in Hertz (Hz). 1H NMR chemical shifts are reported relative to CDCl3
(7.26 ppm). 13C NMR was recorded relative to the central line of
CDCl3 (77.0 ppm). LC/MS analyses were performed on either
Micromass QUATTRO micro™ or Agilent 6410 Triple Quad system.
Overall, the compound 30 is a selective mGluR1 antagonist with
a good pharmacokinetic profile, which is probably devoid of cardiac
side effect and drugedrug interactions. Therefore, the compound
30 can be expected to be broadly used as mGluR1 antagonistic
chemical probe in in vitro and in vivo study for investigating CNS
diseases.
4. Conclusions
5.1.1. Synthesis of various 3-hydroxy-2-phenylacrylonitriles (2)
General: To a solution of phenyl acetonitrile 1 (1.0 eq) in methyl
formate (10 eq), THF was added. In anhydrous condition, solution of
NaH (1.25 eq) in THF was added dropwise to the reaction mixture at
Thienopyrimidinone derivatives were designed, synthesized,
and biologically evaluated against mGluR1. Among the synthesized
compounds, the compound 30 exhibited the most potent inhibitory
activity with an IC50 value of 45 nM and good selectivity over
0
ꢀC and the resulting mixture was stirred at room temperature
overnight. After the reaction, the mixture was quenched with H2O,

632
Y. Kim et al. / European Journal of Medicinal Chemistry 85 (2014) 629e637
Table 1
5.1.2. Synthesis of various 3-methoxy-2-phenylacrylonitriles (3)
General: To a solution of 3-hydroxy-2-phenylacrylonitrile (1 eq)
and THF, NaH (2 eq) was added and the reaction mixture was
stirred at room temperature for 2 h. Then dimethylsulfate (1.7 eq)
was added to the mixture and the resulting solution was stirred at
40 ꢀC. The progress of reaction was ensured using TLC. After the
reaction, the mixture was quenched with H2O and evaporated.
Then it was diluted with ether by extraction and washed with H2O.
After the organic layer was dried over MgSO4 and concentrated, the
crude product was purified by column chromatography (SiO2,
Hexane:EtOAc ¼ 5:1) to give product 3 as oil in 19% ~ quant. yields.
Inhibitory activities of the thienopyrimidinones with hydrogen as R1 against
mGluR1.
Entry
Compd
R1
R2
%Inhibitiona
IC50b in nM
At 10
m
M
At 1 mM
5.1.2.1. 3-Methoxy-2-phenylacrylonitrile (3a). 100% Yield (17.9 g); a
1
2
3
4
5
6
7
8
6
7
8
9
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
2-Me
3-Me
4-Me
2-OMe
3-OMe
4-OMe
3-vinyl
4-vinyl
3-CN
4-CN
4-NO2
90.48
92.91
91.33
95.64
93.32
94.81
77.69
87.12
95.22
97.26
22.84
76.39
93.98
92.27
93.02
15.91
24.18
11.11
92.03
84.04
64.97
90.57
82.61
72.27
84.67
75.31
55.46
86.74
84.24
16.11
29.30
87.02
92.43
82.78
8.17
136 21
677 74
58 17
yellow solid, mp 66e69 ꢀC; 1H NMR (400 MHz, CDCl3)
(m, 4H), 7.31e7.26 (m, 1H), 7.21 (s, 1H), 3.99 (s, 3H).
d 7.40e7.32
66 15
10
11
12
13
14
15
16
17
18
19
20
21
22
23
407 66
374 15
162 57
499 94
184 20
5.1.2.2. 2-(2-Fluorophenyl)-3-methoxyacrylonitrile (3b). 66% Yield
(1.0 g: inseparable 2:1 mixture as an oil); major product 1H NMR
(400 MHz, CDCl3)
d
7.48 (s, 1H), 7.46 (td, J ¼ 8.0, 2.0 Hz, 1H),
9
7.27e7.22 (m, 1H), 7.17 (td, J ¼ 7.8, 2.0 Hz, 1H), 7.09 (ddd, J ¼ 11.6,
8.0, 1.2 Hz, 1H), 4.01 (s, 3H); minor product 1H NMR (400 Hz, DCl3)
10
11
12
13
14
15
16
17
18
19
112
NDc
NDc
4
d
7.43 (td, J ¼ 8.0, 2.0 Hz, 1H), 7.35e7.28 (m, 1H), 7.17 (td, J ¼ 7.8,
2.0 Hz, 1H), 7.14e7.07 (m, 1H), 7.06 (s, 1H), 3.93 (s, 3H).
50 19
74
7
128 10
5.1.2.3. 3-Methoxy-2-(o-tolyl)acrylonitrile (3c). 32% Yield (416 mg:
NDc
inseparable 8:1 mixture as an oil); major product 1H NMR
18.75
14.65
93.37
NDc
NDc
(400 MHz, CDCl3) d 7.29e7.10 (m, 4H), 6.82 (s, 1H), 3.96 (s, 3H), 2.43
(s, 3H); minor product 1H NMR (400 MHz, CDCl3)
4H), 7.02 (s, 1H), 3.86 (s, 3H), 2.34 (s, 3H).
d
7.29e7.10 (m,
the reference compoundd
6.7 1.8
a
%Inhibition of increased intracellular calcium ion levels by treating
by each tested compound at each concentration.
L-glutamate
b
Values are the mean SEM of triplicate binding experiments.
5.1.3. Synthesis of various methyl 3-amino-4-phenylthiophene-2-
carboxylates (4)
c
Not determined.
Structure of the reference compound.
d
General: NaOMe (1.4 eq) in MeOH followed by methyl thio-
glycolate (1.6 eq) were added to 3-methoxy-2-phenylacrylonitrile
(1 eq). The reaction mixture was stirred and refluxed at 65 ꢀC
overnight. After the reaction, the mixture was filtered using celite
Table 2
Inhibitory activities of the thienopyrimidinones with 4-OMe as R2 against mGluR1.
and several drops of 1 N HCl were added until the pH of the solution
is lowered to 4 to 5. Then the mixture was extracted with methy-
lene chloride and dried with MgSO4. After filtration and evapora-
tion, the resulting solution was concentrated under vacuum to give
the product 2 as yellowish oil in 72%e100% yields.
5.1.1.1. 3-Hydroxy-2-phenylacrylonitrile (2a). 99% Yield (12.3 g:
inseparable 1:1 mixture); a yellow solid, mp 157e162 ꢀC; 1H NMR
b
Entry
1
Compd
R1
R2
%Inhibitiona
IC50 in nM
At 10
92.74
m
M
At 1 mM
(400 MHz, DMSO-d6)
d 8.03 (s, 1H), 7.71e7.66 (m, 2H), 7.67 (s, 1H),
24
2-F
4-OMe
90.86
57
8
7.44e7.34 (m, 6H), 7.29e7.22 (m, 2H).
2
3
4
5
6
7
8
9
10
11
12
19
25
26
27
28
29
30
31
32
33
34
35
3-F
4-F
2-Cl
3-Cl
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
60.29
36.07
91.80
60.81
29.88
92.48
89.05
81.04
90.40
89.19
35.75
92.03
53.01
44.40
84.24
37.59
49.07
85.27
78.05
46.44
53.23
71.75
25.41
93.37
NDc
NDc
221 40
5.1.1.2. 2-(2-Fluorophenyl)-3-hydroxyacrylonitrile (2b). 94% Yield
(6.1 g: inseparable 2:1 mixture); a yellow solid, mp 118e124 ꢀC;
NDc
4-Cl
NDc
major product 1H NMR (400 MHz, DMSO-d6)
d 7.85 (s, 1H), 7.41 (td,
2-Me
3-Me
4-Me
2-OMe
3-OMe
4-OMe
45
9
J ¼ 7.8, 2.0 Hz, 1H), 7.38e7.21 (m, 3H); minor product 1H NMR
221 23
NDc
(400 MHz, DMSO-d6)
7.38e7.21 (m, 3H).
d
7.72 (s, 1H), 7.50 (td, J ¼ 8.0, 1.6 Hz, 1H),
747 145
257 34
NDc
the reference compound
6.7 1.8
5.1.1.3. 3-Hydroxy-2-(o-tolyl)acrylonitrile (2c). 100% Yield (1.2 g:
inseparable 1:1 mixture as an oil); 1H NMR (400 MHz, DMSO-d6)
a
%Inhibition of increased intracellular calcium ion levels by treating
by each tested compound at each concentration.
L-glutamate
b
d
7.62 (s, 1H), 7.47 (s, 1H), 7.28e7.17 (m, 8H), 2.34 (s, 3H), 2.27 (s,
Values are the mean SEM of triplicate binding experiments.
Not determined.
c
3H).

Y. Kim et al. / European Journal of Medicinal Chemistry 85 (2014) 629e637
633
Table 3
Selectivity over mGluR5, activities on hERG channel and CYP isozymes, and microsomal stability of the selected compounds.
Compd
mGluR1 IC50 %Inhibitiona
against mGluR5
hERG channel
Microsomal stabilityc CYP isozymes (%remaining activityd at 10
mM)
At 10
m
M
At 1
mM
IC50
Maximum inhibitionb
CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4
18
24
30
50 nM
57 nM
45 nM
37.58
59.30
51.27
36.56
4.70
10.8
>100
9.87
31.0
m
m
m
m
M
M
M
M
0.7
NDe
1.0
1.0
24.4%
17.8%
50.1%
12.1%
91.0
148.4
103.8
114.1
18.3
93.8
98.8
93.0
103.4
98.6
64.8
98.3
90.4
101.7
84.9
200.5
44.8
47.2
46.8
14.94
28.34
30.94
the reference compound 6.7 nM
80.0
a
%Inhibition of increased intracellular calcium ion levels by treating
L
-glutamate by each tested compound at each concentration.
Maximum inhibition of ionic current; 1.0 means totally blocking the ionic current by the tested compound.
%Remaining concentration after 30 min incubation with human liver microsomes (HLM) at 1 M.
b
c
m
d
e
%Remaining activity of each CYP isozyme at a 10
Not determined.
mM of each tested compound.
and washed with methylene chloride. The crude compound was
then purified by column chromatography (SiO2,
124.7, 124.5, 117.1 (d, J ¼ 20 Hz); LC/MS (ESIþ): m/z: calcd for
C
18H11FN2OS: 322.06, [MþH]þ; found: 323.3.
Hexane:EtOAc ¼ 5:1) to give white solid product 4 in 17e70%
yields.
5.1.4.3. 3-(3-Fluorophenyl)-7-phenylthieno[3,2-d]pyrimidin-4(3H)-
one (8). 12% Yield (8.1 mg); a white solid, mp 164e167 ꢀC; 1H NMR
(300 MHz, CDCl3)
d 8.17 (s, 1H), 7.91 (s, 1H), 7.85e7.82 (m, 2H),
5.1.3.1. Methyl 3-amino-4-phenylthiophene-2-carboxylate (4a).
26% Yield (5.1 g); a yellow solid, mp 62e67 ꢀC; 1H NMR (300 MHz,
7.60e7.38 (m, 5H), 7.42e7.21 (m, 2H); 13C NMR (100 MHz, CDCl3)
d
162.8 (d, J ¼ 245 Hz), 156.9, 154.0, 146.8, 138.4, 138.2, 133.5, 131.2,
CDCl3)
d 7.49e7.35 (m, 5H), 7.25 (s, 1H), 5.63 (br, 2H), 3.86 (s, 3H);
13C NMR (100 MHz, CDCl3)
128.1, 128.0, 101.3, 51.3.
d 165.1, 151.6, 134.5, 133.4, 129.2, 128.72,
131.0 (d, J ¼ 9 Hz), 128.7, 128.4, 128.2, 125, 122.8 (d, J ¼ 3 Hz), 116.3
(d, J ¼ 20 Hz), 115 (d, J ¼ 26 Hz); LC/MS (ESIþ): m/z: calcd for
C
18H11FN2OS: 322.06, [MþH]þ; found: 323.3.
5.1.3.2. Methyl 3-amino-4-(2-fluorophenyl)thiophene-2-carboxylate
(4b). 53% Yield (750 mg); a white solid, mp 122e125 ꢀC; 1H NMR
5.1.4.4. 3-(4-Fluorophenyl)-7-phenylthieno[3,2-d]pyrimidin-4(3H)-
one (9). 64% Yield (68.5 mg); a brownish solid, mp 235e239 ꢀC; 1H
(300 MHz, CDCl3)
7.27e7.15 (m, 2H), 5.56 (s, 2H), 3.86 (s, 3H); 13C NMR (100 MHz,
CDCl3)
d
7.43e7.33 (m, 2H), 7.32 (d, J ¼ 0.8 Hz, 1H),
NMR (300 MHz, CDCl3)
d 8.18 (s, 1H), 7.92 (s, 1H), 7.90e7.83 (m, 2H),
7.54e7.40 (m, 5H), 7.31e7.24 (m, 2H); 13C NMR (75 MHz, CDCl3)
d
165.0, 159.8 (J ¼ 246 Hz), 152.0, 131.0 (J ¼ 3 Hz), 130.7
d
162.8 (d, J ¼ 248 Hz), 157.2, 154.0, 147.1, 138.3, 133.6, 132.8 (d,
(J ¼ 2 Hz), 130.0 (J ¼ 8 Hz), 126.8, 124.7 (J ¼ 3 Hz), 121.7 (J ¼ 15 Hz),
J ¼ 3.2 Hz), 131.1, 129.1, 128.9, 128.6 (d, J ¼ 27 Hz), 128.2, 125.1, 116.7
(d, J ¼ 23 Hz); LC/MS (ESIþ): m/z: calcd for C18H11FN2OS: 322.06,
[MþH]þ; found: 323.3.
116.3 (J ¼ 22 Hz), 101.3, 51.3.
5.1.3.3. Methyl 3-amino-4-(o-tolyl)thiophene-2-carboxylate (4c).
35% Yield (210 mg as an oil); 1H NMR (300 MHz, CDCl3)
d
7.31e7.28
(m, 2H), 7.26e7.21 (m, 1H), 7.20e7.16 (m, 1H), 5.33 (s, 2H), 3.85 (s,
3H), 2.20 (s, 3H); 13C NMR (100 MHz, CDCl3)
165.1, 152.1, 137.5,
5.1.4.5. 3-(2-Chlorophenyl)-7-phenylthieno[3,2-d]pyrimidin-4(3H)-
one (10). 13% Yield (19.2 mg); a yellowish solid, mp 163e168 ꢀC; 1H
d
NMR (300 MHz, CDCl3)
7.64e7.62 (m, 1H), 7.53e7.41 (m, 6H); 13C NMR (75 MHz, CDCl3)
156.6, 154.1, 147.2, 138.4, 134.6, 133.6, 132.5, 131.2, 131.1, 130.8,
d 8.04 (s,1H), 7.91 (s,1H), 7.87e7.83 (m, 2H),
133.4, 133.0, 130.6, 130.3, 128.9, 128.6, 126.2, 100.5, 51.3, 19.8.
d
129.9, 128.7, 128.4, 128.1, 128.1, 125.1; LC/MS (ESIþ): m/z: calcd for
5.1.4. Synthesis of various thienopyrimidinone derivatives (6e35)
General: To a pressure bottle containing methyl 3-amino-4-
phenylthiophene-2-carboxylate (1 eq), CH(OEt)3 (14 eq) was
added, followed by aniline 5 (1.9 eq) and AcOH (4 eq). The reaction
mixture was stirred and refluxed at 160 ꢀC overnight. After the
reaction, the mixture was evaporated then solidified with ether.
The produced solid was filtered and dried in vacuo to give the
product thienopyrimidinone in 7e88% yields.
C
18H11ClN2OS: 338.03, [MþH]þ; found: 339.4.
5.1.4.6. 3-(3-Chlorophenyl)-7-phenylthieno[3,2-d]pyrimidin-4(3H)-
one (11). 23% Yield (16 mg); a white solid, mp 123e126 ꢀC; 1H NMR
(300 MHz, CDCl3)
7.54e7.45 (m, 5H), 7.42e7.30 (m, 2H); 13C NMR (100 MHz, CDCl3)
156.9, 154.0, 146.8, 138.3, 138.0, 135.3, 133.6, 131.2, 130.6, 129.6,
d 8.13 (s, 1H), 7.89 (s, 1H), 7.85e7.81 (m, 2H),
d
128.7, 128.4, 128.2, 127.6, 125.4, 125.1; LC/MS (ESIþ): m/z: calcd for
C
18H11ClN2OS: 338.03, [MþH]þ; found: 339.4.
5.1.4.1. 3,7-Diphenylthieno[3,2-d]pyrimidin-4(3H)-one
(6). 33%
Yield (42 mg); a yellowish solid, mp 179e183 ꢀC; 1H NMR
(400 MHz, DMSO)
d
8.53 (s, 1H), 8.51 (s, 1H), 8.01 (d, J ¼ 7.2 Hz, 2H),
5.1.4.7. 3-(4-Chlorophenyl)-7-phenylthieno[3,2-d]pyrimidin-4(3H)-
7.63e7.55 (m, 5H), 7.52 (t, J ¼ 7.6 Hz, 2H), 7.42 (t, J ¼ 7.4 Hz, 1H); 13
C
one (12). 36% Yield (32 mg); a yellow solid, mp 235e239 ꢀC; 1H
NMR (100 MHz, DMSO)
d 157.0, 154.3, 149.2, 137.5, 137.1, 133.9,
NMR (300 MHz, CDCl3)
2H), 7.58e7.50 (m, 4H), 7.46e7.40 (m, 3H); 13C NMR (100 MHz,
CDCl3) 157.0, 154.0, 146.9, 138.3, 135.4, 133.5, 131.2, 129.9, 128.8,
d
8.19 (s, 1H), 7.93 (s, 1H), 7.86 (d, J ¼ 1.4 Hz,
132.4, 129.7, 129.5, 129.0, 128.5, 128.3, 128.1, 124.6; LC/MS (ESIþ): m/
z: calcd for C18H12N2OS: 304.37, [MþH]þ; found: 305.3.
d
128.4, 128.4, 128.2, 125.1; LC/MS (ESIþ): m/z: calcd for
C
18H11ClN2OS: 338.03, [MþH]þ; found: 339.3.
5.1.4.2. 3-(2-Fluorophenyl)-7-phenylthieno[3,2-d]pyrimidin-4(3H)-
one (7). 26% Yield (36 mg); a yellowish solid, mp 202e207 ꢀC; 1H
NMR (400 MHz, CDCl3)
d
8.12 (s, 1H), 7.91 (s, 1H), 7.84 (d, J ¼ 1.4 Hz,
5.1.4.8. 7-Phenyl-3-(o-tolyl)thieno[3,2-d]pyrimidin-4(3H)-one (13).
23% Yield (62 mg); a white solid, mp 152e154 ꢀC; 1H NMR
2H), 7.53e7.30 (m, 7H); 13C NMR (75 MHz, CDCl3)
d
160.8 (d,
J ¼ 231 Hz), 156.4 (d, J ¼ 51 Hz), 154.1, 147.3, 138.4, 133.6, 131.6 (d,
J ¼ 8.3 Hz), 131.2, 129.5, 128.8, 128.5, 128.2, 125.1 (d, J ¼ 3.8 Hz),
(400 MHz, CDCl3) d 8.11 (s, 1H), 7.94 (s, 1H), 7.91e7.89 (m, 2H),
7.55e7.39 (m, 6H), 7.30 (d, J ¼ 7.6 Hz, 1H), 2.26 (s, 3H); 13C NMR

634
Y. Kim et al. / European Journal of Medicinal Chemistry 85 (2014) 629e637
129.6, 128.7, 128.4, 128.1, 125.1, 114.9, 55.6; LC/MS (ESIþ): m/z: calcd
for C19H14N2O2S: 334.08, [MþH]þ; found: 335.3.
Table 4
Mean ( SDa) pharmacokinetic parameters after intravenous (n ¼ 4) and oral (n ¼ 4)
administration (10 mg/kg) of 30 to SD male rats.
Plasma
Intravenous
Oral
5.1.4.14. 7-Phenyl-3-(3-vinylphenyl)thieno[3,2-d]pyrimidin-4(3H)-
one (19). 51% Yield (108 mg); a white solid, mp 168e171 ꢀC; 1H
AUC0e∞
(m
g min/ml)
g min/ml)
Terminal half-life (min)
Cmax g/ml)
544.48 373.81
496.00 340.60
163.67 36.72
e
90.05 23.96
56.86 8.15
309.72 97.7
0.19 0.05
53 (30e60)b
e
AUClast (m
NMR (300 MHz, CDCl3) d 8.22 (s, 1H), 7.9 (s, 1H), 7.86e7.83 (m, 2H),
7.61e7.48 (m, 5H), 7.44e7.38 (m, 1H), 7.36e7.31 (m, 1H), 6.76 (dd,
J ¼ 23.6, 14.4 Hz, 1H), 5.82 (d, J ¼ 23.6 Hz, 1H), 5.37 (d, J ¼ 14.4 Hz,
(
m
Tmax (min)
CL (ml/min/kg)
MRT (min)
Vdss (ml/kg)
Plasma concentration (
Brain concentration (
Brain-to-plasma ratio (B/P) at 2 h
F (%)
e
1H); 13C NMR (75 MHz, CDCl3)
d 157.2, 154.1, 147.3, 139.4, 138.3,
23.43 10.06
90.12 19.68
3569.69 1936.40
0.59 0.17
0.6347 0.1460
1.05
137.3, 135.5, 133.7, 131.0, 129.9,128.8,128.4, 128.1, 127.1,126.3,124.8,
116.0; LC/MS (ESIþ): m/z: calcd for C20H14N2OS: 330.08, [MþH]þ;
found: 331.3.
e
e
m
g/ml) at 2 h
0.15 0.01
0.1365 0.0175
0.92
mg/ml) at 2 h
5.1.4.15. 7-Phenyl-3-(4-vinylphenyl)thieno[3,2-d]pyrimidin-4(3H)-
16.5
one (20). 7% Yield (7 mg); a yellow solid, mp 177e182 ꢀC; 1H NMR
AUC0e∞, total area under the plasma concentrationetime curve from time zero to
time infinity; AUClast, total area under the plasma concentrationetime curve from
time zero to last measured time; Cmax, peak plasma concentration; Tmax, time to
(400 MHz, DMSO)
d
8.53 (s, 1H), 8.52 (s, 1H), 8.01 (d, J ¼ 8.4 Hz, 2H),
7.69 (d, J ¼ 8.4 Hz, 2H), 7.58e7.40 (m, 5H), 6.89e6.82 (m, 1H), 5.98
(d, J ¼ 17.6 Hz, 1H), 5.40 (d, J ¼ 10.8 Hz, 1H); 13C NMR (100 MHz,
reach Cmax; CL, time-averaged total body clearance; MRT, mean residence time; Vdss
,
apparent volume of distribution at steady state; F, oral bioavailability.
DMSO)
d 157.2, 154.3, 148.8, 138.3, 137.2, 136.5, 136.0, 133.7, 132.5,
a
SD: Standard deviations.
Median (range) for Tmax
129.1,128.5,128.5,128.1,127.3,124.5,116.6; LC/MS (ESIþ): m/z: calcd
for C20H14N2OS: 330.08, [MþH]þ; found: 331.3.
b
.
5.1.4.16. 3-(4-Oxo-7-phenylthieno[3,2-d]pyrimidin-3(4H)-yl)benzo-
(100 MHz, CDCl3)
d 156.9, 154.3, 147.6, 138.3, 136.2, 136.0, 133.7,
nitrile (21). 14% yield (20 mg); a white solid, mp 194e198 ꢀC; 1H
131.4, 130.9,129.9,128.7, 128.4,128.1, 128.0,127.4, 125.2, 17.8; LC/MS
(ESIþ): m/z: calcd for C19H14N2OS: 318.08, [MþH]þ; found: 319.3.
NMR (400 MHz, DMSO)
d
8.58 (s, 1H), 8.54 (s, 1H), 8.19 (t, J ¼ 1.6 Hz,
1H), 8.05 (d, J ¼ 6.0 Hz, 1H), 8.00 (d, J ¼ 7.2 Hz, 3H), 7.82 (t,
J ¼ 8.0 Hz, 1H), 7.52 (t, J ¼ 7.6 Hz, 2H), 7.43 (t, J ¼ 7.4 Hz, 1H); 13C
5.1.4.9. 7-Phenyl-3-(m-tolyl)thieno[3,2-d]pyrimidin-4(3H)-one (14).
15% Yield (40 mg); a yellowish solid, mp 132e134 ꢀC; 1H NMR
NMR (75 MHz, DMSO) d 157.0,148.6,137.9,137.3, 133. 7, 133.3, 132.8,
131.8, 131.1, 129.1, 128.5, 124.3, 112.6; LC/MS (ESIþ): m/z: calcd for
(400 MHz, CDCl3)
d
8.21 (s, 1H), 7.91 (s, 1H), 7.88 (d, J ¼ 7.6 Hz, 2H),
19H11N3OS: 329.06, [MþH]þ; found: 330.3.
7.54e7.25 (m, 7H), 2.48 (s, 3H); 13C NMR (100 MHz, CDCl3)
d
157.2,
C
154.0, 147.4, 139.8, 138.2, 136.9, 133.7, 130.9, 130.1, 129.5, 128.7,
128.4, 128.1, 127.8, 125.2, 124.1, 21.4; LC/MS (ESIþ): m/z: calcd for
5.1.4.17. 4-(4-Oxo-7-phenylthieno[3,2-d]pyrimidin-3(4H)-yl)benzo-
nitrile (22). 27% Yield (38 mg); as yellowish solid, mp 278e282 ꢀC;
C
19H14N2OS: 318.08, [MþH]þ; found: 319.3.
1H NMR (400 MHz, DMSO)
d 8.56 (s, 1H), 8.53 (s, 1H), 8.11 (d,
J ¼ 7.6 Hz, 2H), 7.99 (d, J ¼ 8.0 Hz, 2H), 7.85 (d, J ¼ 7.6 Hz, 2H), 7.52 (t,
5.1.4.10. 7-Phenyl-3-(p-tolyl)thieno[3,2-d]pyrimidin-4(3H)-one (15).
68% Yield (74 mg); a brown solid, mp 210e214 ꢀC; 1H NMR
J ¼ 8.4 Hz, 2H), 7.45e7.41 (m, 1H); 13C NMR (75 MHz, DMSO)
d
156.7, 154.3, 148.5, 141.4, 137.2, 133.8, 132.7, 129.4, 129.0, 128.5,
(300 MHz, CDCl3)
d
8.21 (s, 1H), 7.91e7.88 (m, 3H), 7.55e7.34 (m,
157.3, 154.1, 147.5,
128.4, 124.4, 118.7, 112.3; LC/MS (ESIþ): m/z: calcd for C19H11N3OS:
7H), 2.48 (s, 3H); 13C NMR (100 MHz, DMSO)
d
329.06, [MþH]þ; found: 330.3.
139.4, 138.2, 134.4, 133.7, 130.9, 130.3, 128.7, 128.4, 128.1, 126.8,
125.2, 21.2; LC/MS (ESIþ): m/z: calcd for C19H14N2OS: 318.08,
[MþH]þ; found: 319.3.
5.1.4.18. 3-(4-Nitrophenyl)-7-phenylthieno[3,2-d]pyrimidin-4(3H)-
one (23). 10% Yield (31 mg); as yellowish solid, mp 219e221 ꢀC; 1H
NMR (400 MHz, DMSO)
1H), 7.86 (d, J ¼ 7.6 Hz, 2H), 7.72 (d, J ¼ 8.4 Hz, 2H), 7.54 (t, J ¼ 7.4 Hz,
2H), 7.47e7.45 (m, 1H); 13C NMR (100 MHz, DMSO)
156.5, 123.9,
147.9, 145.9, 142.1, 138.5, 133.3, 131.6, 128.8, 128.4, 128.3, 128.1,
125.0; LC/MS (ESIþ): m/z: calcd for C18H11N3O3S: 349.05, [MþH]þ;
found: 350.3.
d
8.46 (d, J ¼ 8.8 Hz,1H), 8.22 (s, 1H), 7.97 (s,
5.1.4.11. 3-(2-Methoxyphenyl)-7-phenylthieno[3,2-d]pyrimidin-
4(3H)-one (16). 71% Yield (204 mg); a white solid, mp 210e212 ꢀC;
d
1H NMR (400 MHz, CDCl3)
d 8.09 (s, 1H), 7.91e7.88 (m, 3H),
7.54e7.38 (m, 5H), 7.14 (t, J ¼ 8.2 Hz, 2H), 3.85 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 157.1,154.7,154.1,148.5,138.2,133.8,131.1,130.6,
129.2, 128.7, 128.4, 128.0, 125.5, 125.3, 121.0, 112.4, 55.9; LC/MS
(ESIþ): m/z: calcd for C19H14N2O2S: 334.08, [MþH]þ; found: 335.3.
5.1.4.19. 7-(2-Fluorophenyl)-3-(4-methoxyphenyl)thieno[3,2-d]pyr-
imidin-4(3H)-one (24). 16% Yield (22 mg); a yellowish solid, mp
139e142 ꢀC; 1H NMR (400 MHz, CDCl3)
d 8.17 (s, 1H), 8.01 (d,
5.1.4.12. 3-(3-Methoxyphenyl)-7-phenylthieno[3,2-d]pyrimidin-
4(3H)-one (17). 83% Yield (83 mg); a brown solid, mp 126e130 ꢀC;
J ¼ 1.2 Hz, 1H), 7.85 (td, J ¼ 7.6, 2.0 Hz, 1H), 7.43e7.32 (m, 3H), 7.29
1H NMR (300 MHz, CDCl3)
d
8.22 (s, 1H), 7.93e7.81 (m, 3H),
(dd, J ¼ 7.2, 1.4 Hz, 1H), 7.22 (ddd, J ¼ 10.8, 8.4, 1.2 Hz,1H), 7.08e7.03
7.55e7.41 (m, 4H), 7.12e7.04 (m, 3H), 3.88 (s, 3H); 13C NMR
(m, 2H), 3.88 (s, 3H); 13C NMR (75 MHz, CDCl3)
d 160.1, 160.1 (d,
(100 MHz, CDCl3) d 16.4, 157.1, 154.0, 147.3, 138.2, 138.0, 133.7, 130.9,
J ¼ 248 Hz), 157.4, 154.6, 147.8, 133.7 (d, J ¼ 6 Hz), 131.6, 131.4 (d,
J ¼ 3 Hz), 129.8 (d, J ¼ 8 Hz), 129.6, 128.2, 124.5, 124.2 (d, J ¼ 4 Hz),
121.3 (d, J ¼ 14 Hz), 116.1 (d, J ¼ 23 Hz), 114.9, 55.6; LC/MS (ESIþ): m/
z: calcd for C19H13FN2O2S: 352.07, [MþH]þ; found: 353.3.
130.4, 128.4, 128, 119.2, 115.3, 113.0, 55.6; LC/MS (ESIþ): m/z: calcd
for C19H14N2O2S: 334.08, [MþH]þ; found: 335.3.
5.1.4.13. 3-(4-Methoxyphenyl)-7-phenylthieno[3,2-d]pyrimidin-
4(3H)-one (18). 31% Yield (26 mg); as brownish solid, mp
5.1.4.20. 7-(3-Fluorophenyl)-3-(4-methoxyphenyl)thieno[3,2-d]pyr-
144e148 ꢀC; 1H NMR (300 MHz, CDCl3)
d
8.20 (s, 1H), 7.90 (s, 1H),
imidin-4(3H)-one (25). 39% Yield (30 mg); a yellow solid, mp
7.89e7.85 (m, 2H), 7.51 (t, J ¼ 7.4 Hz, 2H), 7.43 (d, J ¼ 7.3 Hz, 1H),
207e210 ꢀC; 1H NMR (300 MHz, CDCl3)
d
8.22 (s, 1H), 7.95 (s, 1H),
7.68e7.10 (m, 8H), 3.84 (s, 3H); 13C NMR (75 MHz, CDCl3)
162.7 (d,
J ¼ 242 Hz), 160.0, 157.2, 154.1, 149.6, 136.0 (d, J ¼ 8 Hz), 135.3,133.2,
7.42e7.33 (m, 2H), 7.10e7.02 (m, 2H), 3.89 (s, 3H); 13C NMR
d
(75 MHz, CDCl3) d 162.3, 160.1, 157.4, 154.1, 147.7, 138.2, 133.7, 130.8,

Y. Kim et al. / European Journal of Medicinal Chemistry 85 (2014) 629e637
635
131.0 (d, J ¼ 9 Hz), 130.0, 129.2, 124.7, 124.4 (d, J ¼ 3 Hz), 115.1 (d,
5.1.4.28. 7-(2-Methoxyphenyl)-3-(4-methoxyphenyl)thieno[3,2-d]
J
¼
7
Hz), 114.9, 114.9, 56.0; LC/MS (ESIþ): m/z: calcd for
pyrimidin-4(3H)-one (33). 57% Yield (31 mg); a white solid, mp
C
19H13FN2O2S: 352.07, [MþH]þ; found: 353.3.
168e171 ꢀC; 1H NMR (400 MHz, CDCl3)
d
8.14 (s, 1H), 7.99 (s, 1H),
7.65 (dd, J ¼ 7.6, 1.6 Hz, 1H) 7.41e7.33 (m, 3H), 7.11e7.03 (m, 4H),
3.87 (s, 3H), 3.85 (s, 3H); 13C NMR (100 MHz, CDCl3)
160.1, 157.5,
5.1.4.21. 7-(4-Fluorophenyl)-3-(4-methoxyphenyl)thieno[3,2-d]pyr-
d
imidin-4(3H)-one (26). 51% Yield (57 mg); a white solid, mp
157.1,155.1,147.5, 134.3,133.4, 131.4,129.8,129.5,128.3,124.0,122.5,
235e239 ꢀC; 1H NMR (300 MHz, CDCl3)
d
8.50 (s, 1H), 8.48 (s, 1H),
8.08e8.04 (m, 2H), 7.53e7.45 (m, 2H), 7.39e7.26 (m, 2H), 7.15e7.03
(m, 2H), 3.84 (s, 3H); 13C NMR (75 MHz, CDCl3)
162.3 (d,
120.7, 114.9, 111.5, 55.7, 55.6; LC/MS (ESIþ): m/z: calcd for
C
20H16N2O3S: 364.09, [MþH]þ; found: 365.4.
d
J ¼ 244 Hz), 160.0, 157.2, 154.1, 149.5, 135.9, 132.2, 130.5 (d, J ¼ 8 Hz),
130.4 (d, J ¼ 3 Hz),130.1,129.2,124.5,115.9 (d, J ¼ 21 Hz),114.9, 56.0;
LC/MS (ESIþ): m/z: calcd for C19H13FN2O2S: 352.07, [MþH]þ; found:
353.3.
5.1.4.29. 7-(3-Methoxyphenyl)-3-(4-methoxyphenyl)thieno[3,2-d]
pyrimidin-4(3H)-one (34). 51% Yield (56 mg); a white solid, mp
123e125 ꢀC; 1H NMR (400 MHz, CDCl3)
d
8.18 (s, 1H), 7.89 (s, 1H),
7.44e7.40 (m, 3H), 7.36e7.34 (m, 2H), 7.06e7.04 (m, 2H), 6.98e6.93
(m, 1H), 3.88 (s, 6H); 13C NMR (100 MHz, CDCl3)
160.1, 159.8,157.4,
d
5.1.4.22. 7-(2-Chlorophenyl)-3-(4-methoxyphenyl)thieno[3,2-d]pyr-
154.0, 147.6, 137.9, 135.0, 131.0, 129.7, 129.6, 128.2, 125.1, 120.7, 114.9,
114.2, 113.4, 55.6, 55.3; LC/MS (ESIþ): m/z: calcd for C20H16N2O3S:
364.09, [MþH]þ; found: 365.4.
imidin-4(3H)-one (27). 35% yield (49 mg); a white solid, mp
207e209 ꢀC; 1H NMR (300 MHz, CDCl3)
d
8.18 (s, 1H), 7.96 (s, 1H),
7.65e7.54 (m, 2H), 7.45e7.36 (m, 4H), 7.11e7.07 (m, 2H), 3.91 (s,
3H); 13C NMR (75 MHz, CDCl3)
160.1, 157.4, 154.8, 148.0, 135.6,
d
5.1.4.30. 7-(4-Methoxyphenyl)-3-(4-methoxyphenyl)thieno[3,2-d]
133.7, 133.7,132.5,132.1,130.1,129.7, 129.6,128.2,126.8,124.2,114.9,
55.6; LC/MS (ESIþ): m/z: calcd for C19H13ClN2O2S: 368.04, [MþH]þ;
found: 369.3.
pyrimidin-4(3H)-one (35). 88% Yield (96 mg); a white solid, mp
229e233 ꢀC; 1H NMR (400 MHz, CDCl3)
d 8.47 (s, 1H), 8.39 (s, 1H),
7.97 (d, J ¼ 8.8 Hz, 2H), 7.5 (d, J ¼ 8.8 Hz, 2H), 7.13 (d, J ¼ 8.8 Hz, 2H),
7.07 (d, J ¼ 8.8 Hz, 2H), 3.85 (s, 3H), 3.83 (s, 3H); 13C NMR (100 MHz,
5.1.4.23. 7-(3-Chlorophenyl)-3-(4-methoxyphenyl)thieno[3,2-d]pyr-
DMSO)
d 159.9, 159.5, 157.3, 154.2, 149.2, 136.8, 130.7, 130.1, 129.7,
imidin-4(3H)-one (28). 76% Yield (84 mg); a white solid, mp
129.2, 126.5, 124.4, 114.8, 114.4, 56.0, 55.6; LC/MS (ESIþ): m/z: calcd
for C20H16N2O3S: 364.09, [MþH]þ; found: 365.4.
190e193 ꢀC; 1H NMR (400 MHz, CDCl3)
d
8.19 (s, 1H), 7.92 (s, 1H),
7.90e7.89 (m, 1H), 7.76e7.36 (m, 1H), 7.44e7.33 (m, 4H), 7.08e7.04
(m, 2H), 3.88 (s, 3H); 13C NMR (100 MHz, CDCl3)
160.1,157.3,153.8,
d
5.2. Biological assays
147.8, 136.4, 135.3, 131.4, 129.9, 129.5, 128.3, 128.2, 128.0, 126.4,
125.2, 114.9, 55.6; LC/MS (ESIþ): m/z: calcd for C19H13ClN2O2S:
368.04, [MþH]þ; found: 369.3.
5.2.1. mGluR1 and mGluR5 assay using FDSS6000
Chem-3 cells which stably express mGluR1 were purchased
from Milipore Co. and human embryonic kidney cells which stably
express mGluR5 were obtained from Yonsei University. Cells were
grown in DMEM medium supplemented with 10% (v/v) fetal bovine
serum, penicillin (100 U/mL), streptomycin (100 lg/mL), and pu-
romycin (10 lg/mL) at 37 ꢀC in a humid atmosphere of 5% CO2 and
95% air. For calcium assay, cells were harvested and dispensed into
96-well black wall clear bottom plates at a density of 40,000 cells
per a well. After 18 h of incubation, cells were treated with Calcium-
5 assay reagent, which is prepared by manufacture's instruction
(Molecular Devices Corporation, California). During fluorescence-
based FDSS6000 assay, mGluR1 or mGluR5 was activated using a
5.1.4.24. 7-(4-Chlorophenyl)-3-(4-methoxyphenyl)thieno[3,2-d]pyr-
imidin-4(3H)-one (29). 70% Yield (97 mg); a white solid, mp
236e237 ꢀC; 1H NMR (300 MHz, CDCl3)
d
8.22 (s, 1H), 7.93 (s, 1H),
7.87e7.83 (m, 2H), 7.52e7.48 (m, 2H), 7.41e7.36 (m, 2H), 7.12e7.07
(m, 2H), 3.92 (s, 3H); 13C NMR (75 MHz, CDCl3)
160.0, 157.5, 154.2,
d
149.5, 135.5, 133.0, 132.7, 130.1, 130.0, 129.2, 129.1, 124.7, 114.8, 56.0;
LC/MS (ESIþ): m/z: calcd for C19H13ClN2O2S: 368.04, [MþH]þ;
found: 369.3.
5.1.4.25. 3-(4-Methoxyphenyl)-7-(o-tolyl)thieno[3,2-d]pyrimidin-
4(3H)-one (30). 60% Yield (83 mg);
a
brownish solid, mp
147e149 ꢀC; 1H NMR (400 MHz, CDCl3)
d
8.11 (s, 1H), 7.70 (s, 1H),
high concentration of L-glutamate (10 mM) in HBSS, and various
7.36e7.30 (m, 6H), 7.06e7.02 (m, 2H), 3.87 (s, 3H), 2.28 (s, 3H); 13
C
concentrations of synthesized compounds were treated to cells 75 s
before mGluR activation. All data were collected and analyzed using
FDSS6000 and related software (Hamamatsu, Japan).
NMR (100 MHz, CDCl3) d 160.1, 157.5, 155.0,147.9,138.9,137.0, 133.5,
132.1, 130.6, 130.4, 129.7, 128.3, 125.8, 124.2, 114.9, 55.6, 20.5; LC/MS
(ESIþ): m/z: calcd for C20H16N2O2S: 348.09, [MþH]þ; found: 349.3.
5.2.2. hERG channel assay
5.1.4.26. 3-(4-Methoxyphenyl)-7-(m-tolyl)thieno[3,2-d]pyrimidin-
CHO-K1 cells expressing hERG channels via the inducible Tet-On
gene expression system (CHO-K1 Tet-On hERG cells) were pur-
chased from IonGate Biosciences GmbH (Frankfurt, Germany).
CHO-K1 Tet-On hERG cells were cultured in Dulbecco's modified
Eagle's medium containing 10% (v/v) fetal bovine serum, penicillin
4(3H)-one (31). 33% Yield (36 mg);
a
brownish solid, mp
138e142 ꢀC; 1H NMR (300 MHz, CDCl3)
d
8.17 (s, 1H), 7.86 (s, 1H),
7.63e7.62 (m, 2H), 7.37e7.33 (m, 3H), 7.22e7.20 (m, 1H), 7.06e7.03
(m, 2H), 3.87 (s, 3H), 2.44 (s, 3H); 13C NMR (75 MHz, CDCl3)
d
160.1,
157.5, 154.1, 147.6, 138.4,138.3,133.7, 130.8, 129.7, 129.1, 128.9,128.6,
(100 U/ml), streptomycin (100 mg/ml), fungizone (2.5 Pg/ml) as well
128.2, 125.5, 125.1, 114.9, 55.6, 21.6; LC/MS (ESIþ): m/z: calcd for
as selection antibiotics, geneticin G418 (200 Pg/ml), hygromycin
(200 Pg/ml) and puromycin (2 Pg/ml) in humidified 5% CO2 at 37 ꢀC.
Cells were passaged every three days by trypsin-EDTA treatment.
For hERG channel expression, the Tet-On gene expression was
induced by adding 5 Pg/ml of doxycyclin (Sigma, St. Louis, MO, USA)
into the growth medium. For automated patch-clamp recordings,
CHO-K1 Tet-On hERG cells were plated into the 100-mm culture
dishes. Cells at 50e80% confluency were harvested by treatment
with trypsin-EDTA, washed twice and resuspended in the extra-
cellular solution at a final cell density of 1e5 ꢁ 106 cells/ml. Cells
C
20H16N2O2S: 348.09, [MþH]þ; found: 349.3.
5.1.4.27. 3-(4-Methoxyphenyl)-7-(p-tolyl)thieno[3,2-d]pyrimidin-
4(3H)-one (32). 70% Yield (98 mg); a brown solid, mp 175e178 ꢀC;
1H NMR (300 MHz, CDCl3)
d
8.21 (s, 1H), 7.88 (s, 1H), 7.78e7.75 (m,
2H), 7.41e7.33 (m, 4H), 7.11e7.08 (m, 2H), 3.92 (s, 3H), 2.45 (s, 3H);
13C NMR (75 MHz, CDCl3)
160.1, 157.5, 154.2, 147.6, 138.3, 137.9,
d
130.9, 130.3, 129.7, 129.4, 128.3, 128.2, 125.1, 114.9, 55.6, 21.3; LC/MS
(ESIþ): m/z: calcd for C20H16N2O2S: 348.09, [MþH]þ; found: 349.3.

636
Y. Kim et al. / European Journal of Medicinal Chemistry 85 (2014) 629e637
were used for whole-cell recordings approximately 20e32 h after
doxycyclin addition.
5.2.4. Human CYP isozyme assay
Inhibitions of CYP (CYP1A2, 2C9, 2D6 and 3A4) activities were
measured using the Vivid® CYP450 screening kit (Invitrogen,
Madison, WI, USA) in a 96-well plate according to the manufac-
turer's instructions with some modifications. Test compounds
The automated patch-clamp device, NPC-16 Patchliner (Nanion
Technologies, Munchen, Germany) was used for whole-cell re-
cordings. Whole-cell currents were recorded with the intracellular
solution containing: 50 mM KCl, 60 mM KF, 10 mM NaCl, 2 mM
MgCl2, 20 mM EGTA and 10 mM HEPES (pH 7.2), and with the
extracellular solution containing: 140 mM NaCl, 4 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 5 mM Glucose and 10 mM HEPES (pH 7.4). To
assist stable seal formations, the seal enhancer containing: 80 mM
NaCl, 3 mM KCl, 35 mM CaCl2, 10 mM MgCl2 and 10 mM HEPES (pH
7.4) was used only at the seal formation step. Prior to the whole-cell
recordings, the external seal enhancing solution was exchanged to
the extracellular solution described above. hERG channel currents
were recorded using the parallel EPC-10 patch-clamp amplifiers
(HEKA Elektronik, Lambrecht/Pfalz, Germany), and low-pass
filtered (10 kHz) with a 4-pole Bessel filter. Cell suspension and
patch solutions were automatically added onto the four recording
wells in the microfabricated disposable chip (NPC-16 Chip, Nanion
Technologies, Munchen, Germany). To obtain the inhibitory con-
stants, hERG tail currents were evoked by repolarizing steps
to ꢂ50 mV for 500 ms preceded by a 500-ms depolarization po-
tential of þ20 mV at a holding potential of ꢂ80 mV with a 20-s
sweep interval. Whole-cell currents were acquired and digitized
at 5 kHz using the Patchmaster (HEKA Elektronik, Lambrecht/Pfalz,
Germany). The extracellular solution was exchanged to the extra-
cellular solution containing each blocker via four pipette tips of
including the
a-naphthoflavone, sulfaphenazole, quinidine and
ketoconazole known as CYP1A2, 2C9, 2D6 and 3A4 inhibitors,
respectively, were prepared in acetonitrile to give a final concen-
tration of 10 mM. Briefly, the compound solution (test compound,
positive inhibition control and solvent control) and the Master Pre-
Mix [CYP450 BACULOSOMES® Reagent (recombinant human
CYP450 isozyme and rabbit NADPHP450 reductase) and Regener-
ation System (3.3 mM glucose-6-phosphate and 0.3 U/ml glucose-
6-phosphate dehydrogenase in 100 mM potassium phosphate, pH
8.0)] were added to each well and then the mixtures were incu-
bated for 5 min at 37 ꢀC to allow the compounds to interact with the
CYP. The enzyme reaction was started by the addition of the Vivid®
substrate (CYP1A2 Blue, CYP2C9 Green, CYP2D6 Blue and CYP3A4
Green) and 0.1 mM NADPþ mixture. The plate was transferred into
the fluorescent plate reader for the kinetic analysis. The fluores-
cence of Vivid® substrate metabolites (blue-fluorescent 3-cyano-7-
hydroxycoumarin and green-fluorescent fluorescein) was
measured for 20 min using a fluorescence plate reader with an
excitation wavelength of 409-, 485 nm and an emission wavelength
of 460-, 530 nm, respectively. Percent inhibition for test com-
pounds on CYP450 was calculated by using the following equation.
% Inhibition ¼ ð1 ꢂ ðrX ꢂ rPCÞ=ðrSC ꢂ rPCÞÞ ꢁ 100
NPC-16 Patchliner using a 4-fold volume of solution (40
ml) with a
speed of 4 l/s, and the exchanged blocker solution was applied for
m
where rx is the rate in the presence of test compound, rPC is the rate
in the presence of the positive inhibition control, and rSC is the rate
in the solvent control.
100e200 s to the patch-clamped cells until blocker binding had
reached equilibrium by monitoring hERG tail currents.
Whole-cell recordings were analyzed using the Patchmaster/
Fitmaster (HEKA Elektronik, Lambrecht/Pfalz, Germany), IGOR Pro
(WaveMetrics Inc., Portland, OR, USA), and the GraphPad Prism 4
(GraphPad Software, Inc., La Jolla, CA, USA) software.
5.2.5. Pharmacokinetic studies
To yield concentrationeresponse curves, the concen-
trationeresponse data were fitted with a Hill equation (sigmoidal
concentrationeresponse equation).
Each compound at a dose of 10 mg/kg was administered intra-
venously or orally to male SpragueeDawley rats. Blood samples
were collected via carotid artery at 0 (to serve as a control), 10 (IV
only), 50, 150, 0.5, 1, 2, 4, 6, and 8 h after administration of each
.ꢀ
ꢁ
compound. After centrifugation at 3000 rpm for 10 min, a 50-mL
I ¼ Imin þ ðImax ꢂ IminÞ 1 þ 10ððLog IC50ꢂCÞꢁhÞ
aliquot of plasma samples were stored at ꢂ70 ꢀC until analysis.
Pharmacokinetic parameters were determined by
a
non-
where Imin and Imax are the normalized minimum and maximum
hERG tail currents, respectively. IC50 is the halfmaximum inhibition
concentration. C is the logarithm of molar concentration and h is
the Hill coefficient.
compartmental analysis using WinNonlin® (Pharsight Corpora-
tion, Mountain View, CA) program. The total area under the plasma
concentrationetime curve from time zero to last measured time
(AUClast) was calculated by the trapezoidal ruleeextrapolation
method. Standard methods in the literature[23] were used to
calculate the following pharmacokinetic parameters; the time-
averaged total body clearance (CL), total area under the first
moment of plasma concentrationetime curve from time zero to
time infinity (AUC0e∞), terminal half-life, mean residence time
(MRT), apparent volume of distribution at steady state (Vss).
Concentrations of each compound in the above samples were
5.2.3. Human liver microsomal stability assay
To determine the microsomal stability of the selected com-
pounds, pooled human liver microsomes (HLM's) containing
20 mg/mL of total protein were used and purchased from BD Bio-
sciences (Bedford, MA). A compound was incubated with HLM at
37 ꢀC, in the presence of an NADPH, in a final incubation volume of
analyzed using LC-MS/MS. To a 50
100 L aliquot of acetonitrile containing 0.1
standard was added. After vortex mixing and centrifugation at
10,000 rpm for 10 min, a 5 L of supernatant was injected onto LC-
mL aliquot of plasma sample, a
m
mg/mL of internal
200 mL. The final incubation solutions contain 1 mM of the com-
pound, 1.2 mM NADPH, 0.5 mg/mL (total protein) microsomes,
100 mM phosphate buffer (pH 7.4). The incubation tubes were then
incubated in a water-bath shaker at 37 ꢀC for 30 min. At the time
point, two incubation tubes were removed from the water-bath for
m
MS/MS system. The LC-MS/MS system consisted of a HP1100® HPLC
system (Agilent, Santa Clara, CA) and API3200® triple-quadrupole
mass spectrometer (Applied Biosystems-SCIEX, Concord, Canada).
The HPLC mobile phases consisted of 0.1% formic acid (A) and 0.1%
formic acid in 90% acetonitrile (B). Chromatographic separation was
achieved on a reversed-phase Xterra® C18 column (50 ꢁ 2.1 mm,
duplicate and the reaction was terminated immediately. The 400
mL
of ice-cold acetonitrile containing 0.1 g/mL of internal standard
m
was added to microsomal incubate to terminate the reaction and
precipitate the proteins. The incubation solutions were then
centrifuged and the supernatants were analyzed by LC-MS/MS.
3.5
mm, Waters Corporation, Milford, MA) using gradient elution at
a flow rate of 0.3 mL/min. The lower limit of quantitation of each

Y. Kim et al. / European Journal of Medicinal Chemistry 85 (2014) 629e637
637
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Acknowledgments
This research was supported by the Basic Science Research
Program (NRF-2013-R1A1A2A10009907) and the Priority Research
Centers Program (2009-0093824) funded by the National Research
Foundation of Korea (NRF) and a grant of the Korean Health Tech-
nology R&D Project, Ministry of Health & Welfare, Republic of
Korea (HI11C0998). Additional funding was provided by the Korea
Institute of Science and Technology (KIST) Institutional Program
(2E24510 and 2E24670).
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