Discovery and biological evaluation of tetrahydrothieno[2,3-c]pyridine derivatives as selective metabotropic glutamate receptor 1 antagonists for the potential treatment of neuropathic pain

Article abstract of DOI:10.1016/j.ejmech.2015.04.060

Abstract Metabotropic glutamate receptor 1 (mGluR1) has been a prime target for drug discovery due to its heavy involvement in various brain disorders. Recent studies suggested that mGluR1 is associated with chronic pain and can serve as a promising targe

Full text of DOI:10.1016/j.ejmech.2015.04.060

European Journal of Medicinal Chemistry 97 (2015) 245e258
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery and biological evaluation of tetrahydrothieno[2,3-c]pyridine
derivatives as selective metabotropic glutamate receptor 1 antagonists
for the potential treatment of neuropathic pain
Mina Nam a , TaeHun Kim
,
b
,
1
a
,
c
,
1, Jinsook Kwak a , Seon Hee Seo , Min Kyung Ko ,
,
d
a
a
a
a
a
a
b
Eun Jeong Lim , Sun-Joon Min , Yong Seo Cho , Gyochang Keum , Du-Jong Baek ,
e
,
*
a, c, **
Jiyoun Lee , Ae Nim Pae
a
Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, Republic
of Korea
Department of Chemistry, College of Natural Sciences, Sangmyung University, Hong-ji moon 2-gil 20, Jongno-gu, Seoul 110-743, Republic of Korea
Biological Chemistry, Korea University of Science and Technology, Yuseong-Gu, Daejon 305-350, Republic of Korea
Department of Chemistry, Sogang University, 35 Baekbeom-ro, Mapo-gu, Seoul 121-742, Republic of Korea
b
c
d
e
Department of Global Medical Science, Sungshin University, Seoul 142-732, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 October 2014
Received in revised form
Metabotropic glutamate receptor 1 (mGluR1) has been a prime target for drug discovery due to its heavy
involvement in various brain disorders. Recent studies suggested that mGluR1 is associated with chronic
pain and can serve as a promising target for the treatment of neuropathic pain. In an effort to develop a
novel mGluR1 antagonist, we designed and synthesized a library of compounds with tetrahydrothieno
30 March 2015
Accepted 28 April 2015
Available online 30 April 2015
[
2,3-c]pyridine scaffold. Among these compounds, compound 9b and 10b showed excellent antagonistic
activity in vitro and demonstrated pain-suppressing activity in animal models of pain. Both compounds
were orally active, and compound 9b exhibited a favorable pharmacokinetic profile in rats. We believe
that these compounds can provide a promising lead compound that is suitable for the potential treat-
ment of neuropathic pain.
Keywords:
mGluR1 antagonist
Metabotropic glutamate receptor
Neuropathic pain
© 2015 Elsevier Masson SAS. All rights reserved.
Thiophene derivatives
1. Introduction
according to their sequence homology, pharmacological profile,
and signal transduction pathways [3,4]. Among these subtypes,
Glutamate is the major excitatory neurotransmitter in the
mammalian central nervous system and participates in a wide
range of physiological and pathophysiological functions [1].
Glutamate activates two types of receptors, ionotropic receptors
group I mGluRs, mGluR1 and mGluR5, are located in postsynaptic
regions predominantly, and induce increases in intracellular cal-
cium upon activation [1,5e8].
Group I mGluRs are one of the thoroughly studied mGluRs due
to their therapeutic implications in various brain disorders
including anxiety, schizophrenia, drug addiction, Parkinson's dis-
ease and chronic pain [2,4,9e11]. Group I mGluRs share a high
degree of sequence similarity, and are also distributed in the mostly
same region, however, mGluR1 and mGluR5 affect distinct
signaling pathways and are regulated by different mechanisms
(
iGluRs) and metabotropic glutamate receptors (mGluRs). The
iGluRs are ligand gated ion channels mediating fast excitatory
synaptic transmission, whereas the mGluRs are G-protein-coupled
receptors (GPCRs) modulating neuronal excitability in a much
slower manner [2]. The mGluRs comprise eight subtypes, mGluR1
through mGluR8, which can be further divided into three groups
[11e14]. Particularly, many studies suggested that mGluR1 is
located in the brain regions including dorsal root ganglia, spinal
cord, thalamus, and cerebral cortex, most of which are known to be
involved in pain sensing [15e18]. Additionally, suppressing the
activation of mGluR1 via genetic knockdown or small molecule
antagonists attenuated nociceptive responses in animal models of
pain [19e25] indicating that mGluR1 is associated with chronic
*
Corresponding author.
* Corresponding author. Center for Neuro-Medicine, Brain Science Institute, Ko-
rea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul
36-791, Republic of Korea.
*
1
E-mail addresses: jlee@sungshin.ac.kr (J. Lee), anpae@kist.re.kr (A.N. Pae).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2015.04.060
223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
0

246
M. Nam et al. / European Journal of Medicinal Chemistry 97 (2015) 245e258
pain and can serve as a promising target for the treatment of
neuropathic pain [26,27]. Since the first non-competitive antago-
nist of mGluR1, CPCCOEt, was identified [28,29], many small
molecule antagonists were developed and utilized to establish the
location and structure of the allosteric binding site [30]. Further-
more, with the help of the structural information, a number of
highly potent allosteric antagonists of mGluR1 have been reported
to date, and several examples are shown in Fig. 1. Unfortunately,
however, preclinical and clinical trials of these compounds failed to
deliver a successful drug candidate possibly due to side effects [31].
In an effort to develop selective mGluR1 antagonists that are
suitable for clinical use, we designed a new scaffold based on tet-
rahydrothieno pyridine core as shown in Fig. 2. To provide struc-
tural diversity and subtype selectivity, we adapted a previously
published pharmacophoric model for allosteric mGluR1 antago-
nists [32]. In this model, it was reported that the allosteric binding
site of mGluR1 requires three essential functionalities. We
perceived that the relative position of these groups in the binding
site would be important for selectivity, therefore, the tetrahy-
drothieno pyridine scaffold would provide appropriate spatial
distribution of these groups. With the tetrahydrothieno pyridine
core at the center, heterocyclic and aromatic rings with various
Fig. 2. Design strategy of novel mGluR1 antagonists.
sodium borohydride to afford 7 [35]. Subsequent removal of the Boc
protecting group, followed by coupling to a pyrazole carboxylic acid
with various substituents afforded compounds 9aei. Coupling to
various benzoyl chloride analogs instead of the pyrazole carboxylic
acids further afforded compounds 10aev.
3
. Results and discussion
1 2
hydrophobic substituents can be added on R and R region. In this
report, we present the synthesis and biological evaluation of novel
mGluR1 antagonists with tetrahydrothieno pyridine scaffold.
The in vitro antagonistic activity of the prepared compounds on
human mGluR1 and mGluR5 were determined as summarized in
Tables 1 and 2. First, we measured % inhibition of calcium release
induced by glutamate at the 10 mM of each compound, and then
2
. Chemistry
further determined IC50 values of selected compounds. Structur-
eeactivity relationships of the compounds with a pyrazole sub-
stituent on the tetrahydrothieno pyridine core were described in
Table 1. Most noticeable feature in this series is that the benzyl-
The synthesis of tetrahydrothieno[2,3-c]pyridine derivatives is
described in Schemes 1 and 2. The tetrahydrothieno pyridine core
was constructed at the first step from commercially available 1-
Boc-4-piperidone via Gewald reaction [33,34] as shown in
Scheme 1. Subsequently, the amine group on the thiophene ring
was modified with various substituted benzoyl chloride to generate
the intermediate 3. Removal of the Boc protecting group, followed
by coupling to a pyrazole carboxylic acid with various substituents
yielded compounds 5aeh.
amine derivatives (9aei; X ¼ CH
2
) appeared to be more potent
against mGluR1 compared to the benzamide derivatives (5aeh;
X ¼ C]O) indicating that hydrogen-bond interactions via eNH is
important, but the presence of a carbonyl group is not required. In
the benzamide derivatives (5aeh), the size of the substituent on
1
the pyrazole ring (R ) does not seem to affect antagonistic activity
significantly, since compounds with an isopropyl group and a
Next, we decided to replace the benzamide group on the thio-
phene ring with a benzylamine to generate compounds 9 and 10.
The synthesis of these derivatives started from the tetrahy-
drothieno pyridine intermediate 2. Refluxing compound 2 with
aromatic aldehydes in toluene yielded the benzylideneamine 6. The
benzylideneamine 6 was then reduced to an amine by using
methyl group showed similar % inhibition values. More impor-
tantly, the electron density of the substituents on the R position
appears to be crucial for selectivity. For example, compounds with a
4-Cl substituent (5e and 5f) moderately inhibited mGluR1, but did
not inhibit mGluR5, whereas compounds with a 4-OMe substituent
(5g and 5h) showed a reversed selectivity.
2
Fig. 1. Structures of representative allosteric mGluR1 antagonists (AeD). Compound A developed by Merck-Banyu [23]; compound B developed by Eli Lilly [25]; compound C and D
developed by Schering-Plough [22].

M. Nam et al. / European Journal of Medicinal Chemistry 97 (2015) 245e258
247
ꢀ
ꢀ
Scheme 1. Reagents and conditions. (a) Malononitrile, MeOH, sulfur, morpholine, 45 / 60 C, 75% (b) benzoyl chloride, pyridine, 80 C 80e92% (c) TFA, MC, MeOH, r,t, 95% (d)
pyrazole caboxylic acid, DMF, HATU, r.t. 45e70%.
Scheme 2. Reagents and conditions. (a) Benzaldehyde, toluene, reflux, 90% (b) sodium borohydride, MC, MeOH, 70e90% (c) TFA, MC, MeOH, r,t, 95% (d) pyrazole caboxylic acid for 6,
DMF, HATU, r.t. 45e70% or benzoyl chloride for 7, DMF, TEA, r.t, 50e75%.
The benzylamine derivatives (9aei) generally showed higher
that the R1 substituent is potentially useful to improve receptor
selectivity. In addition, compounds with a simple benzylamine
activity compared to the benzamide derivatives. More interestingly,
1
2
it was observed that the R site clearly favors a small group. Com-
group (R ¼ H; 10aeg) showed slightly higher activity compared to
1
2
pounds without any substituent (R ¼ H, 9c and 9f) demonstrated
the R -substituted analogs. Overall, we found that compounds with
1
higher % inhibition against mGluR1 compared to compounds with a
methyl or isopropyl substituent. It should be also noted that com-
pounds 9c and 9f appeared to show higher activity against mGluR1
than mGluR5, whereas the rest of the compounds showed similar
a fluorine atom on the benzamide (R ¼ F) and a simple benzyl-
2
amine group (R ¼ H) showed excellent in vitro activity and
selectivity.
Before continuing to evaluate biological activity of these com-
pounds in animal models, we assessed metabolic stability of several
compounds with potent in vitro activity. We measured the
remaining activity of cytochrome P450 (CYP) in the presence of the
selected compounds and also determined the remaining percent-
age of the selected compounds in human liver microsome after
30 min as described in Table 3. All the tested compounds inhibited
activity of CYP to some extent, however, compounds 9b and 10b
inhibited CYP slightly less than the rest of the compounds. Micro-
somal stability data showed that 77% of 9b and 49% of 10b
remained after 30 min, whereas the rest of the compounds
remained less than 40% of the initial concentrations. Based on these
results, we chose compounds 9b and 10b for further in vivo study.
In addition to metabolic profiling, we performed a panel of GPCR
selectivity tests of compound 9b against 17 different classes of
GPCR receptors to investigate off-target activity (See supplemen-
1
activity against both mGluR1 and mGluR5 upon changing the R
2
group. Additionally, the introduction of a halide atom (R ¼ 4-F and
4
-Cl) did not generate the same increasing selectivity as observed
with the benzamide derivatives, suggesting that the interaction
2
around the pyrazole binding region is more critical than the R site.
Based on these findings, we retained the benzylamine group
(
X ¼ CH
2
) and replaced the pyrazole moiety with a more hydro-
phobic group such as benzyl or pyridyl ring as shown in Table 2. As
we expected, most compounds in this series showed slightly better
antagonistic activity against mGluR1 compared to the pyrazole
derivatives with a benzamide group (compounds 5aeh), however,
they also exhibited some degree of antagonistic activity against
mGluR5. Notably, compounds with a fluorine atom on the 4-
1
position of the benzamide ring (R ¼ 4-F; 10b, 10c, and 10n)
showed excellent antagonistic activity against mGluR1, whereas
1
compounds with a methoxy group (R ¼ 4-OMe; 10l, 10p, and 10v)
tary information). When tested at fixed concentrations (12.5
activation and 10 M for inhibition), compound 9b showed no
agonistic activity towards all tested receptors. However, compound
mM for
demonstrated higher antagonistic activity against mGluR5 with
lower activity against mGluR1. This reversed selectivity indicates
m

248
M. Nam et al. / European Journal of Medicinal Chemistry 97 (2015) 245e258
Table 1
In vitro antagonistic activity of the pyrazole derivatives 5 and 9 against mGluR1 and mGluR5.
a
Compd
X
R1
R2
% Inhibitionb
mGluR1
IC50 (m
M)c
mGluR5
10
mGluR1
mGluR5
10
m
M
mM
c
c
5
5
5
5
5
5
5
5
9
9
9
9
9
9
9
9
a
b
c
d
e
f
g
h
a
b
c
d
e
f
C]O
C]O
C]O
C]O
C]O
C]O
C]¼O
C]O
Isopropyl
Me
Isopropyl
Me
Isopropyl
Me
Isopropyl
Me
Isopropyl
Me
H
Isopropyl
Me
H
H
H
14.6 ± 0.12
8.51 ± 0.84
28.5 ± 2.1
31.0 ± 3.8
62.5 ± 1.5
42.2 ± 2.4
ꢁ6.42 ± 0.02
ꢁ9.58 ± 0.09
54.4 ± 1.5
71.2 ± 5.3
94.9 ± 3.3
40.7 ± 2.2
84.2 ± 5.1
91.4 ± 4.0
46.3 ± 1.8
40.9 ± 1.2
39.3 ± 1.5
27.8 ± 1.0
e
e
c
c
e
e
c
c
4-F
4-F
4-Cl
4-Cl
4-OMe
4-OMe
H
ꢁ2.30 ± 0.03
13.7 ± 0.72
ꢁ4.28 ± 0.07
ꢁ2.15 ± 0.06
63.1 ± 1.1
28.3 ± 0.68
8.89 ± 0.32
59.8 ± 3.2
4.48 ± 0.02
43.1 ± 0.95
37.6 ± 0.79
13.5 ± 0.21
24.8 ± 0.09
31.2 ± 0.45
e
e
c
c
e
e
c
12.2 ± 2.7
e
c
c
e
e
c
e
4.83 ± 1.4
c
c
e
e
c
CH
CH
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
2
2
43.2 ± 3.0
1.42 ± 0.38
1.09 ± 0.08
e
c
H
H
e
c
e
c
c
4-F
4-F
4-F
4-Cl
4-Cl
e
e
c
2.45 ± 0.37
2.03 ± 0.09
e
c
e
c
c
h
i
Isopropyl
Me
e
e
c
c
e
e
a
b
c
The mean ± SE values were obtained from three independent measurements.
Ca flux assay using glutamate as agonist.
Not determined.
2þ
Table 2
In vitro antagonistic activity of the benzamide derivatives 10 against mGluR1 and mGluR5.
a
Compd
X
R1
R2
% Inhibitionb
mGluR1
IC50 (mM)
mGluR5
mGluR1
mGluR5
10
mM
10 mM
c
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0f
0g
0i
0j
C
C
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
H
4-F
4-F
3-F
4-Me
3-Me
H
H
H
H
H
H
H
66.4 ± 2.3
91.5 ± 0.95
90.1 ± 2.3
79.0 ± 1.1
69.7 ± 2.6
73.5 ± 2.0
77.8 ± 1.1
73.1 ± 2.0
46.6 ± 0.9
47.7 ± 0.9
63.9 ± 2.6
82.0 ± 3.6
43.9 ± 2.3
42.8 ± 1.4
41.2 ± 1.0
35.2 ± 0.14
67.5 ± 0.33
15.5 ± 0.07
36.8 ± 0.5
10.9 ± 0.02
46.0 ± 1.7
26.1 ± 0.58
16.0 ± 0.53
29.8 ± 0.49
24.3 ± 0.61
8.80 ± 0.33
26.4 ± 0.07
22.3 ± 0.25
37.6 ± 0.40
68.0 ± 1.7
44.2 ± 1.7
36.5 ± 1.3
54.8 ± 2.8
69.0 ± 2.0
60.0 ± 1.9
25.3 ± 0.18
31.1 ± 0.18
33.2 ± 0.12
3.16 ± 0.03
41.5 ± 0.11
2.82 ± 0.10
0.60 ± 0.22
1.24 ± 0.17
2.9 ± 0.8
e
c
e
c
e
c
e
c
4.72 ± 1.1
3.14 ± 1.1
1.81 ± 0.3
e
c
e
c
4-F
4-F
4-F
4-F
3-F
3-F
3-F
3-F
3-F
4-Cl
3-OMe
3-OMe
4-CN
4-CN
e
c
c
4-F
e
e
c
c
0k
0l
4-Me
4-OMe
H
e
e
c
e
1.43 ± 0.4
c
0m
0n
0o
0p
0q
0r
0s
0t
5.31 ± 1.9
2.38 ± 0.6
e
c
4-F
e
c
c
4-Me
4-OMe
4-Cl
4-F
4-F
3-F
e
e
c
e
4.73 ± 1.6
c
c
e
e
c
c
e
e
c
3.09 ± 0.44
e
c
c
e
e
c
c
0u
0v
H
e
e
c
c
4-OMe
e
e
a
b
c
The mean ± SE values were obtained from three independent measurements.
Ca flux assay using glutamate as agonist.
Not determined.
2þ

M. Nam et al. / European Journal of Medicinal Chemistry 97 (2015) 245e258
249
Table 3
Cytochrome P450 inhibition and microsomal stability of the selected compounds.
a
CYP (10
CYP1A2
m
M, remaining activity (% of control))
Stability in human hepatic microsome
% remaining after 30 min)
(
CYP2D6
CYP2C9
CYP3A4
CYP2C19
9
1
1
1
1
b
72.7 ± 1.4
58.1 ± 0.7
42.6 ± 0.6
52.0 ± 1.1
78.5 ± 1.2
33.7 ± 0.3
42.0 ± 0.4
35.7 ± 1.1
36.4 ± 0.7
35.0 ± 1.5
6.77 ± 0.4
5.48 ± 0.3
4.33 ± 0.3
5.48 ± 0.1
6.46 ± 0.1
20.5 ± 0.3
20.0 ± 0.6
9.36 ± 0.8
24.2 ± 0.7
12.5 ± 0.6
14.2 ± 1.2
9.94 ± 0.8
4.57 ± 0.5
3.81 ± 0.2
7.99 ± 0.6
77.6 ± 2.5
49.4 ± 4.1
16.4 ± 1.8
33.4 ± 3.2
12.1 ± 0.7
0b
0g
0i
0s
a
The mean ± SE values were obtained from three independent measurements.
9
b inhibited activity of mGluR1 exclusively, without affecting ac-
other hand, 10b, the most potent compound in vitro, demonstrated
poor analgesic activity toward mechanical allodynia as described in
Fig. 4A and B. Surprisingly, however, 10b showed excellent pain-
suppressing activity against cold allodynia as shown in Fig. 4C
and D demonstrating similar efficacy compared to gabapentin.
Given the excellent in vivo activity of compound 9b, we decided
to evaluate its pharmacokinetic profile in rats as described in
Table 4. Compound 9b exhibited good oral bioavailability and a
half-life of 89 min when injected orally. When injected intrave-
nously, 9b demonstrated moderate clearance and a half-life of
110 min. We also measured brain penetration of 9b after two hours
and brain-to-plasma ratio was approximately 14% for both orally
and intravenously administered rats. Overall, compound 9b dis-
played an acceptable oral bioavailability and maintained moderate
plasma concentrations. Although the brain level of 9b was not ideal,
however, it appeared to be enough to suppress pain in SNL model
indicating that it might be possible to optimize in vivo efficacy by
improving brain penetration.
tivity of other GPCRs including mGluR2.
To evaluate pain-suppressing activity in vivo, we tested com-
pounds 9b and 10b in the spinal nerve ligation (SNL) model of
neuropathic pain [36] as described in Figs. 3 and 4. Compounds 9b
and 10b were administered orally in mice on the 14th day after
ligation surgery and their behavioral responses to mechanical and
cold stimuli were observed. In addition, gabapentin, an FDA
approved analgesic, was also tested for comparison. As shown in
Fig. 3, compound 9b showed pain-suppressing activity in both
mechanical allodynia and cold allodynia. Upon mechanical stimuli,
as depicted in Fig. 3A and B, the treatment of 9b recovered
approximately 60% of the threshold after 3 h, while gabapentin
recovered 80% of the threshold at the same time point. Further-
more, compound 9b demonstrated even greater analgesic effects
against cold stimuli as shown in Fig. 3C and D. The administration
of 9b exhibited approximately 90% recovery from cold allodynia
after 1 h, whereas gabapentin demonstrated 40% recovery. On the
Fig. 3. Effects on mechanical allodynia (A and B) and cold allodynia (C and D) after oral administration of gabapentin (B, 100 mg/kg, n ¼ 4) and 9b (C, 100 mg/kg, n ¼ 4) to
neuropathic pain-induced rats. Experimental time expressed as D for days after neuropathic injury (N) and h for hours after gabapentin or 9b administration, *P < 0.05 (gabapentin
and 9b) vs. pre-administration value (paired t-test), )P < 0.05 gabapentin vs. 9b (unpaired t-test).

250
M. Nam et al. / European Journal of Medicinal Chemistry 97 (2015) 245e258
Fig. 4. Effects on mechanical allodynia (A and B) and cold allodynia (C and D) after oral administration of gabapentin (B, 100 mg/kg, n ¼ 4) and 10b (C, 100 mg/kg, n ¼ 4) to
neuropathic pain-induced rats. Experimental time expressed as D for days after neuropathic injury (N) and h for hours after gabapentin or 10b administration, *P < 0.05 (gaba-
pentin, 10b) vs. pre-administration value (paired t-test), )P < 0.05 gabapentin vs. 10b (unpaired t-test).
4
. Conclusion
each side of the core are beneficial for overall in vitro activity. In
addition, the size of the substituents on these rings also affected
antagonistic activity. It should be also noted that the electronic
property of the substituents appeared to be crucial for selectivity,
which warrants further investigation of structureeactivity rela-
tionship analysis. Among several selected compounds, we found
that compound 9b and 10b exhibited favorable metabolic stability
with low CYP inhibition, therefore evaluated their pain suppressing
activity in rat SNL model of neuropathic pain. Both compounds
appeared to be effective in cold allodynia, demonstrating the ac-
tivity comparable to gabapentin, however, they showed much less
efficacy in mechanical allodynia. We believe that the newly
We have developed novel mGluR1 antagonists with tetrahy-
drothieno pyridine scaffold. We identified several mGluR1 antag-
onists with potent in vitro activity. Our SAR analysis suggested that
hydrophobic functional groups such as benzene and pyridine on
Table 4
Pharmacokinetic parameters after intravenous and oral administration of 9b (10 mg/
kg) to SD male rats.a
Plasma
Intravenous (n ¼ 4)
Oral (n ¼ 5)
AUC0e∞
(
m
m
g min/ml)
g min/ml)
Terminal half-life (min)
g/ml)
max (min)
556.09 ± 199.80
543.46 ± 191.61
109.91 ± 8.95
e
254.96 ± 56.63
245.52 ± 52.01
88.96 ± 9.1
1.41 ± 0.25
72 (60e120)b
e
developed tetrahydrothieno pyridine scaffold can provide a
promising lead for further development of mGluR1 antagonists for
the treatment of neuropathic pain.
AUClast
(
C
max
(m
T
e
CL (ml/min/kg)
MRT (min)
20.15 ± 8.36
78.49 ± 22.31
1656.19 ± 168.08
0.00
1.24 ± 0.64
0.08 ± 0.06
0.1381 ± 0.0554
0.14
5. Experimental section
e
V
ss (ml/kg)
e
Ae (%)
Plasma concentration (
Plasma concentration (
Brain concentration (mg/ml) at 2 h
Brain-to-plasma ratio (B/P) at 2 h
0.00
5.1. Chemistry
m
m
g/ml) at 2 h
g/ml) at 8 h
1.19 ± 0.13
0.07 ± 0.03
0.1555 ± 0.0582
0.13
5.1.1. General
All reactions were carried out under dry nitrogen unless
F (%)
45.8
otherwise indicated. Commercially available reagents were used
without further purification. Solvents and gases were dried ac-
cording to standard procedures. Organic solvents were evaporated
under reduced pressure using a rotary evaporator. Analytical thin
layer chromatography (TLC) was performed using glass plates pre-
coated 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
Abbreviation. 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 reach Cmax; CL, time-averaged total body clearance; MRT, mean residence
time; Vss, apparent volume of distribution at steady state; Ae, Excreted amount; F,
bioavailability.
a
All values are reported as mean ± standard deviation.
Median (range) for Tmax
b
.

M. Nam et al. / European Journal of Medicinal Chemistry 97 (2015) 245e258
251
column chromatography was performed using silica gel 60
stirred at room temperature for 2 h. After evaporation of the solvent
and recrystallization from Ether or EtOH, the desired product is
obtained as TFA salt (317 mg, 78%).
1
13
(
230e400 mesh, Merck) with the indicated solvents. H and
C
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, ppm 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
1
H NMR (300 MHz, CHLOROFORM-d) ppm 9.23 (br. s., 2H),
7.92e8.01 (m, 2H), 7.54e7.71 (m, 3H) 4.32 (s, 2H), 3.45 (t, J ¼ 5.7 Hz,
2H), 2.86 (t, J ¼ 5.7 Hz, 2H).
1
3
CHLOROFORM-
d
(7.26 ppm). C NMR was recorded relative to the
(77.0 ppm).
5.1.2.7. 4-Fluoro-N-(3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-
2-yl)benzamide (4b). The title compound was prepared by method
B. The title compound was obtained as a white TFA salt form
(403 mg, 88%).
central line of CHLOROFORM-
d
5
5
.1.2. Synthesis of compounds 5aeh
.1.2.1. tert-Butyl 2-amino-3-cyano-4,5-dihydrothieno[2,3-c]pyri-
stirred solution of (5.0 g,
.025 mol) of 1-Boc-4-piperidone, malononitrile (1.82 g, 0.028 mol)
1H NMR (400 MHz, DMSO-d
6
) ppm 9.28 (br. s., 2H), 8.06 (dd,
dine-6(7H)-carboxylate (2). To
0
a
J ¼ 5.3, 8.8 Hz, 2H), 7.43 (t, J ¼ 8.8 Hz, 2H) 4.31 (s, 2H), 3.45 (t,
J ¼ 5.9 Hz, 2H), 2.80e3.01 (m, 2H).
and sulfur (0.88 g, 0.028 mol) in methanol (50 mL), morpholine
ꢀ
(
2.31 mL, 0.025 mol) was added over period of 15 min at 45 C. The
5.1.2.8. 4-Chloro-N-(3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-
2-yl)benzamide (4c). The title compound was prepared by method
B. The title compound was obtained as a white TFA salt form
(285 mg, 98%).
ꢀ
reaction mixture was heated at 80 C with stirring for 2 h and
cooled at room temperature. A precipitate formed which was
filtered and washed with cold ethanol. The precipitate was
recrystallized from methanol, filtered and dried to give pale pink
crystals (5.3 g, 73%).
1H NMR (400 MHz, DMSO-d
J ¼ 8.41 Hz, 3H), 3.65 (s, 3H), 2.95 (t, J ¼ 5.40 Hz,1H), 2.42 (br. s., 2H).
) 8.12 (d, J ¼ 8.41 Hz, 2H), 7.40 (d,
6
1H NMR (400 MHz, CHLOROFORM-d) ppm 4.75 (s, 2H), 4.39 (br.
s., 2H), 3.69 (t, J ¼ 5.5 Hz, 2H), 2.62 (t, J ¼ 5.4 Hz, 1H), 1.51 (s, 9H).
5.1.2.9. 4-Methoxy-N-(3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyr-
idin-2-yl)benzamide (4d). The title compound was prepared by
method B. The title compound was obtained as a white TFA salt
5
.1.2.2. tert-Butyl
pyridine-6(7H)-carboxylate (3a) (method A). The benzoyl chloride
0.23 mL, 1.97 mmol) was added to a solution tert-butyl 2-amino-3-
cyano-4,5-dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate
2-benzamido-3-cyano-4,5-dihydrothieno[2,3-c]
form (270 mg, 82%).
1
(
6
H NMR (400 MHz, DMSO-d ) ppm 9.15 (s, 2H), 7.99 (d,
J ¼ 8.4 Hz, 2H), 7.13 (d, J ¼ 8.5 Hz, 2H), 4.30 (s, 2H), 3.87 (s, 3H), 3.45
(
500 mg, 1.80 mmol) in pyridine (3 mL) and the reaction mixture
(t, J ¼ 5.4 Hz, 2H), 2.86 (t, J ¼ 5.4 Hz, 2H).
ꢀ
allowed to stir at 80 C for 2 h. Upon completion, the crude mixture
crystallized from 10% HCl, filtered and dried to give desired com-
pound (686 mg, 80%).
5.1.2.10. N-(3-cyano-6-(3-isopropyl-1H-pyrazole-5-carbonyl)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)benzamide (5a) (method
C). To a solution of HATU (174.17 mg, 0.459 mmol) and 3-
isopropylpyrazole-5-carboxylic acid (59.85 mg, 0.388 mmol) in
1H NMR (300 MHz, CHLOROFORM-d) ppm 8.92 (s, 1H),
7.96e8.03 (m, 2H), 7.56e7.73 (m, 3H), 4.58 (s, 2H), 3.76 (t,
J ¼ 5.7 Hz, 2H), 2.77 (br. s., 2H), 1.53 (s, 9H).
DMF (2 mL) was then added diisopropylethylamine (92
mL,
0.529 mmol). The reaction solution was stirred at room tempera-
5
.1.2.3. tert-Butyl 2-(4-fluorobenzamido)-3-cyano-4,5-dihydrothieno
ture for 2 h. The N-(3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyr-
idin-2-yl)benzamide (100 mg, 0.353 mmol) was added in one
portion. The solution was then stirred at room temperature over-
night. The reaction was diluted with methylene chloride and
washed. The aqueous layer was extracted with methylene chloride
twice, the combined organic phases were dried over anhydrous
[2,3-c]pyridine-6(7H)-carboxylate (3b). The title compound was
prepared by method A. The title compound was obtained as a white
solid (440 mg, 61%).
1H NMR (300 MHz, CHLOROFORM-d) ppm 8.87 (s, 1H), 8.0 (dd,
J ¼ 8.6, 5.6 Hz, 2H), 7.41 (t, J ¼ 8.6 Hz, 2H), 4.48 (s, 2H), 3.62 (t,
J ¼ 5.7 Hz, 2H), 2.60 (br. s., 2H), 1.45 (s, 9H).
4
MgSO . After evaporation of the solvent and recrystallization from
ethyl acetate, filtered and dried to give desired compound (50 mg,
5
.1.2.4. tert-Butyl 2-(4-chlorobenzamido)-3-cyano-4,5-
34%).
dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate (3c). The title
1H NMR (300 MHz, DMSO-d
6
) ppm 13.00 (s, 1H), 11.93 (s, 1H),
compound was prepared by method A. The title compound was
obtained as a white solid (694 mg, 92%).
7.98 (d, J ¼ 7.40 Hz, 2H), 7.67 (t, J ¼ 7.15 Hz, 1H), 7.59 (t, J ¼ 7.47 Hz,
2H), 6.40 (s, 1H), 5.18 (br. s., 0.9H), 4.75 (br. s., 1.1H), 4.20 (br. s.,
1.2H), 3.93 (br. s., 0.8H), 2,91e3.10 (m, 1H), 2.78 (br. s., 2H), 1.25 (d,
J ¼ 5.7 Hz, 6H).
1
H NMR (300 MHz, CHLOROFORM-d) 8.85 (s, 1H), 7.80 (d,
J ¼ 8.67 Hz, 1H), 7.45 (d, J ¼ 8.67 Hz, 1H), 4.47 (br. s., 2H), 3.65 (t,
13
J ¼ 5.56 Hz, 2H), 2.65 (br. s., 2H), 1.42 (s, 9H).
6
C NMR (101 MHz, DMSO-d ) ppm 166.7, 164.5, 146.7, 147.0,
139.5, 139.2, 133.3, 132.7, 131.0, 128.8, 125.9, 114.3, 104.1, 95.6, 44.0,
5
.1.2.5. tert-Butyl 2-(4-methoxybenzamido)-3-cyano-4,5-
41.5, 25.6, 25.4 22.7.
þ
dihydrothieno[2,3-c]pyridine-6(-7H)-carboxylate (3d). The title
compound was prepared by method A. The title compound was
obtained as a white solid (319 mg, 71%).
LC/MS (ESI , MeCN/H
2
O): m/z: calcd for C22
H
21
N
5
O
2
S: 419.14
þ
[MþH] ; found: 420.1.
1
H NMR (300 MHz, CHLOROFORM-d) ppm 8.88 (s, 1H), 7.91 (d,
5.1.2.11. N-(3-cyano-6-(3-methyl-1H-pyrazole-5-carbonyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridin-2-yl)benzamide (5b). The title com-
pound was prepared by method C. The title compound was ob-
J ¼ 8.1 Hz, 2H), 7.05 (d, J ¼ 8.1 Hz, 1H), 4.56 (s, 2H), 3.93 (s, 3H), 3.74
(
t, J ¼ 5.7 Hz, 2H), 2.75 (br. s., 2H), 1.52 (s, 9H).
tained as a white solid (86.28 mg, 62%).
1
5
.1.2.6. N-(3-cyano-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)ben-
zamide (4a) (method B). The trifluoroacetic acid (TFA, 2.2 mL) was
added to solution of tert-Butyl 2-benzamido-3-cyano-4,5-
6
H NMR (300 MHz, DMSO-d ) ppm 12.98 (s, 1H), 11.93 (s, 1H),
7.98 (d, J ¼ 7.28 Hz, 2H), 7.67 (t, J ¼ 7.34 Hz, 1H), 7.59 (t, J ¼ 7.53 Hz,
2H), 6.37 (s,1H), 5.16 (br. s., 0.7H), 4.75 (br. s.,1.3H), 4.19 (br. s.,1.3H),
3.93 (br. s., 0.7H), 2,74 (br. s., 2H), 2.28 (s, 3H).
a
dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate in methylene
chloride (MC, 5.5 mL) by using a dropping funnel. The mixture was
13
6
C NMR (101 MHz, DMSO-d ) ppm 165.8, 163.6, 147.8, 147.1,

252
M. Nam et al. / European Journal of Medicinal Chemistry 97 (2015) 245e258
1
39.5, 139.4, 133.1, 132.7, 131.0, 128.8, 125.8, 114.2, 106.5, 95.6, 44.0,
7.97 (d, J ¼ 8.48 Hz, 2H), 7.10 (d, J ¼ 8.85 Hz, 2H), 6.38 (s, 1H), 5.16
41.5, 25.3, 10.7.
(br. s., 0.8H), 4.72 (br. s., 1.2H), 4.18 (br. s., 2H), 3.85 (s, 3H),
þ
LC/MS (ESI , MeCN/H
2
O): m/z: calcd for C20
H
17
N
5
O
2
S: 391.11
2.93e3.02 (m, 1H), 2.76 (br. s., 2H), 1.24 (d, 6H J ¼ 6.78 Hz).
þ
13
[MþH] ; found: 392.0.
6
C NMR (101 MHz, DMSO-d ) ppm 165.0, 163.2, 148.3, 146.7,
139.6, 135.8, 130.9, 130.8, 125.7, 125.5, 124.5, 114.4, 104.0, 95.3, 56.0,
5
.1.2.12. 4-Fluoro-N-(3-cyano-6-(3-isopropyl-1H-pyrazole-5-
43.9, 41.6, 25.6, 25.4, 22.7.
þ
carbonyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)benzamide
5c). The title compound was prepared by method C. The title
compound was obtained as a white solid (65 mg, 50%).
LC/MS (ESI , MeCN/H
[MþH] ; found: 450.0.
2
O): m/z: calcd for C23
H
23
N
5
O
3
S: 449.15
þ
(
1
H NMR (300 MHz, DMSO-d
6
) ppm 13.00 (s, 1H), 11.94 (s, 1H),
5.1.2.17. 4-Methoxy-N-(3-cyano-6-(3-methyl-1H-pyrazole-5-
carbonyl)-4,5,6,7-tetrahydrothino[2,3-c]pyridin-2-yl)benzamide
(5h). The title compound was prepared by method C. The title
8
4
1
.03e8.11 (m, 2H), 7.45e7.37 (m, 2H), 6.39 (s, 1H), 5.19 (br. s., 0.8H),
.75 (br. s., 1.2H), 4.21 (br. s., 1.1H), 3.93 (br. s., 0.9H), 2.91e3.09 (m,
H), 2.76 (br. s., 2H), 1.25 (d, J ¼ 5.7 Hz, 6H).
compound was obtained as a white solid (28 mg, 38%).
13
1
C NMR (101 MHz, DMSO-d
6
) ppm 166.3, 164.7, 163.9, 151.5,
6
H NMR (300 MHz, DMSO-d ) ppm 12.94 (s, 1H), 11.72 (s, 1H),
1
47.9, 140.1, 135.1, 131.8, 131.7, 131.0, 129.2, 125.7, 116.1, 115.9, 114.3,
7.98 (d, 2H J ¼ 8.7 Hz), 7.12 (d, 2H J ¼ 8.9 Hz), 6.36 (s, 1H), 5.15 (br. s.,
104.1, 95.7, 44.0, 41.5, 25.6, 25.4, 22.7.
0.8H), 4.74 (br. s., 1.2H), 4.18 (br. s., 1.2H), 3.87 (s, 3H), 3.92 (br. s.,
þ
LC/MS (ESI , MeCN/H
2
O): m/z: calcd for C22
H
20FN
5
O
2
S: 437.13
0.8H), 2.74 (br. s., 2H), 2.28 (s, 3H).
þ
13
[MþH] ; found: 438.1.
6
C NMR (101 MHz, DMSO-d ) ppm 165.0, 163.2, 148.3, 146.7,
139.7, 135.6, 130.9, 129.3, 125.7, 125.4, 124.6, 114.2, 106.6, 95.1, 56.0,
5
.1.2.13. 4-Fluoro-N-(3-cyano-6-(3-methyl-1H-pyrazole-5-
44.0, 41.5, 25.4, 10.9.
þ
carbonyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)benzamide
5d). The title compound was prepared by method C. The title
compound was obtained as a white solid (45 mg, 42%).
LC/MS (ESI , MeCN/H
2
O): m/z: calcd for C21
H
19
N
5
O
3
S: 421.12
þ
(
[MþH] ; found: 421.9.
1
H NMR (300 MHz, DMSO-d
6
) ppm 12.95 (s, 1H), 11.94 (s, 1H),
5.1.2.18. tert-Butyl 2-(benzylideneamino)-3-cyano-4,5-dihydrothieno
8
4
2
.02e8.11 (m, 2H), 7.47e7.38 (m, 2H), 6.37 (s, 1H), 5.16 (br. s., 0.8H),
.75 (br. s., 1.2H), 4.19 (br. s., 1.1H), 3.93 (br. s., 0.9H), 2.74 (br. s., 2H),
.28 (s, 3H).
[2,3-c]pyridine-6(7H)-carboxylate (6a) (method D). The mixture of
tert-butyl
2-amino-3-cyano-4,5-dihydrothieno[2,3-c]pyridine-
6(7H)-carboxylate (300 mg, 0.78 mmol) and benzaldehyde(0.12 mL,
0.87 mmol) in 2 mL of toluene was refluxed for 2 h. After the
mixture was cooled the crystalline precipitate formed was filtered
off and washed with methanol: MC (1:4) and dried to give desired
compound (340 mg, 86%).
13C NMR (101 MHz, DMSO-d
) ppm 166.3, 164.7, 163.9, 151.6,
48.0, 140.1, 135.1, 131.8, 131.7, 130.9, 129.3, 125.6, 116.0, 115.8, 114.2,
6
1
106.6, 95.5, 44.0, 41.5, 25.3, 10.8.
þ
LC/MS (ESI , MeCN/H
2
O): m/z: calcd for C20
H16FN
5
O
2
S: 409.10
þ
1H NMR (400 MHz, CHLOROFORM-d) ppm 8.48 (s, 1H),
[MþH] ; found: 410.1.
7.96e8.07 (m, 2H), 7.48e7.64 (m, 3H), 4.59 (s, 2H), 3.76 (t,
5
.1.2.14. 4-Chloro-N-(3-cyano-6-(3-isopropyl-1H-pyrazole-5-
J ¼ 5.7 Hz, 2H), 2.80 (t, J ¼ 5.2 Hz, 2H), 1.53 (s, 9H).
carbonyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)benzamide
5e). The title compound was prepared by method C. The title
compound was obtained as a white solid (60 mg, 42%).
(
5.1.2.19. tert-Butyl 2-((4-fluorobenzylidene)amino)-3-cyano-4,5-
dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate
(6b). The
title
1
H NMR (300 MHz, DMSO-d
H), 7.97 (d, 2H J ¼ 8.2 Hz), 7.66 (d, J ¼ 8.4 Hz, 2H), 6.39 (s, 1H), 5.19
br. s., 0.8H), 4.75 (br. s., 1.2H), 4.21 (br. s., 1.2H), 3.78 (br. s., 0.8H),
,97e3.01 (m, 1.2H), 2.76 (br. s., 2H), 1.24 (d, J ¼ 5.7 Hz, 6H).
6
) ppm 13.00 (br. s., 1H), 11.99 (s,
compound was prepared by method D. The title compound was
obtained as a yellow solid (1.43 g, 80%).
1
(
2
1
H NMR (300 MHz, CHLOROFORM-d) ppm 8.44 (s, 1H), 8.00 (dd,
J ¼ 5.46, 8.85 Hz, 2H), 7.21 (t, J ¼ 8.57 Hz, 2H), 4.59 (br. s., 2H), 3.77
13
C NMR (101 MHz, DMSO-d
6
) ppm 164.8, 163.3, 151.3, 147.8,
(t, J ¼ 5.65 Hz, 2H), 2.81 (t, J ¼ 5.56 Hz, 2H), 1.54 (s, 9H).
140.1, 137.9, 135.1, 131.4, 130.8, 129.0, 125.7, 114.3, 104.1, 95.6, 44.0,
4
1.6, 25.6, 25.4, 22.7.
5.1.2.20. tert-Butyl 2-((3-fluorobenzylidene)amino)-3-cyano-4,5-
þ
LC/MS (ESI , MeCN/H
2
O): m/z: calcd for C22
H20ClN
5
O
2
S: 453.10
dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate
(6c). The
title
þ
[MþH] ; found: 454.0.
compound was prepared by method D. The title compound was
obtained as a yellow solid (1.5 g, 84%).
1
5
.1.2.15. 4-Chloro-N-(3-cyano-6-(3-methyl-1H-pyrazole-5-
H NMR (400 MHz, CHLOROFORM-d) ppm 8.43 (s, 1H),
carbonyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)benzamide
5f). The title compound was prepared by method C. The title
compound was obtained as a white solid (78 mg, 58%).
7.68e7.75 (m, 2H), 7.48 (dt, J ¼ 5.52, 8.03 Hz,1H), 7.24e7.29 (m, 1H),
4.59 (s, 2H), 3.76 (t, J ¼ 5.7 Hz, 2H), 2.80 (t, J ¼ 5.5 Hz, 2H), 1.53 (s,
9H).
(
1
H NMR (300 MHz, DMSO-d
d, J ¼ 8.4 Hz, 2H), 6.36 (s, 1H), 5.16 (br. s., 0.7H), 4.78 (br. s., 1.3H),
.19 (br. s., 1.2H), 3.93 (br. s., 0.8H), 2,74 (br. s., 2H), 2.28 (s, 3H).
6
) ppm 7.99 (d, J ¼ 8.0 Hz, 2H), 7.66
(
4
5.1.2.21. tert-Butyl 3-cyano-2-((3-methoxybenzylidene)amino)-4,5-
dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate
(6d). The
title
13
C NMR (101 MHz, DMSO-d
6
) ppm 164.9, 163.3, 151.6, 147.9,
compound was prepared by method D. The title compound was
obtained as a yellow solid (1.2 g, 84%).
140.1, 137.9, 135.0, 131.5, 130.7, 129.1, 125.8, 114.4, 106.5, 95.8, 44.0,
1
4
1.6, 25.3, 10.8.
H NMR (400 MHz, CHLOROFORM-d) ppm 8.44 (s, 1H), 7.57 (s,
þ
LC/MS (ESI , MeCN/H
2
O): m/z: calcd for C20
H16ClN
5
O
2
S: 425.07
1H), 7.49 (d, J ¼ 7.65 Hz, 1H), 7.41 (t, J ¼ 7.84 Hz, 1H), 7.11 (ddd,
J ¼ 0.88, 2.54, 8.13 Hz, 1H), 4.59 (br. s., 2H), 3.92 (s, 3H), 3.76 (t,
J ¼ 5.46 Hz, 2H), 2.80 (br. s., 2H), 1.53 (s, 9H).
þ
[MþH] ; found: 426.0.
5
.1.2.16. 4-Methoxy-N-(3-cyano-6-(3-isopropyl-1H-pyrazole-5-
carbonyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)benzamide
5g). The title compound was prepared by method C. The title
compound was obtained as a white solid (28 mg, 35%).
5.1.2.22. tert-Butyl 3-cyano-2-((4-cyanobenzylidene)amino)-4,5-
dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate (6f). The title com-
pound was prepared by method D. The title compound was ob-
tained as a yellow solid (1.58 g, 86.5%).
(
1
6
H NMR (400 MHz, DMSO-d ) ppm 12.99 (s, 1H), 11.70 (s, 1H),

M. Nam et al. / European Journal of Medicinal Chemistry 97 (2015) 245e258
253
1H NMR (400 MHz, CHLOROFORM-d) ppm 8.48 (s, 1H), 8.08 (d,
J ¼ 8.28 Hz, 1H), 7.80 (d, J ¼ 8.41 Hz, 1H), 4.61 (br. s., 2H), 3.77 (t,
J ¼ 5.65 Hz, 2H), 2.82 (t, J ¼ 5.58 Hz, 2H), 1.53 (s, 9H).
The title compound was obtained as a white TFA salt form (121 mg,
50%).
1H NMR (300 MHz, DMSO-d
6
) ppm 9.06 (br. s., 2H), 8.51 (t,
J ¼ 5.96 Hz,1H), 7.24e7.41 (m, 5H), 4.41 (d, J ¼ 5.90 Hz, 2H), 4.05 (br.
s., 2H), 3.36 (br. s., 2H), 2.68 (t, J ¼ 5.90 Hz, 2H).
5.1.2.23. tert-Butyl 2-(benzylamino)-3-cyano-4,5-dihydrothieno[2,3-
c]pyridine-6(7H)-carboxylate (7a) (method E). tert-Butyl 2-(benzy-
lideneamino)-3-cyano-4,5-dihydrothieno[2,3-c]pyridine-6(7H)-
carboxylate(340 mg, 0.92 mmol) was dispersed in 3.5 mL of
methanol and 5 mL methylene chloride under stirring at r.t. Sodium
tetrahydridoborate (70 mg, 1.85 mmol) was added. The mixture
turned white and the mixture was stirred for 30 min, diluted with
5.1.2.30. 2-((4-Fluorobenzyl)amino)-4,5,6,7-tetrahydrothieno[2,3-c]
pyridine-3-carbonitrile (8b). The title compound was prepared by
method B. The title compound was obtained as a white TFA salt
form (1.37 g, 97%).
1H NMR (300 MHz, DMSO-d6) ppm 9.28(br. s., 2H), 8.51 (t,
J ¼ 5.96 Hz, 1H), 7.39 (dd, J ¼ 5.58, 8.60 Hz, 2H), 7.20 (t, J ¼ 8.85 Hz,
2H), 4.39 (d, J ¼ 5.90 Hz, 2H), 4.04 (br. s., 2H), 3.34 (br. s., 2H), 2.68 (t,
J ¼ 5.83 Hz, 2H).
5
mL of methylene chloride, and acidified to pH 7.5. The collected
organic extracts were concentrated under reduced pressure. The
product was dried to give white solid (256 mg, 75%).
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.33e7.46 (m, 5H),
5
.29 (br. s., 1H), 4.42 (d, J ¼ 5.65 Hz, 2H), 4.38 (br. s., 2H), 3.68 (t,
5.1.2.31. 2-((3-Fluorobenzyl)amino)-4,5,6,7-tetrahydrothieno[2,3-c]
pyridine-3-carbonitrile (8c). The title compound was prepared by
method B. The title compound was obtained as a white TFA salt
form (899 mg, 62%).
J ¼ 5.08 Hz, 1H), 2.62 (br. s., 2H), 1.50 (s, 9H).
5
.1.2.24. tert-Butyl 2-((4-fluorobenzyl)amino)-3-cyano-4,5-
dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate (7b). The title
compound was prepared by method E. The title compound was
obtained as a white solid (1.33 g, 92%).
1H NMR (300 MHz, DMSO-d6) ppm 9.19 (br. s., 2H), 8.51 (t,
J ¼ 5.93 Hz, 1H), 7.29e7.52 (m, 1H), 7.02e7.27 (m, 3H), 4.42 (d,
J ¼ 6.03 Hz, 2H), 4.05 (s, 2H), 3.35 (t, J ¼ 6.03 Hz, 2H), 2.67 (t,
J ¼ 5.56 Hz, 2H).
1
H NMR (400 MHz, DMSO-d
6
) ppm 8.27 (t, J ¼ 6.02 Hz, 1H), 7.39
(
dd, J ¼ 5.52, 8.66 Hz, 2H), 7.19 (t, J ¼ 8.91 Hz, 2H), 4.36 (d,
J ¼ 5.90 Hz, 2H), 4.25 (s, 2H), 3.55 (t, J ¼ 5.71 Hz, 2H), 2.44 (t,
5.1.2.32. 2-((4-Chlorobenzyl)amino)-4,5,6,7-tetrahydrothieno[2,3-c]
pyridine-3-carbonitrile (8d). The title compound was prepared by
method B. The title compound was obtained as a white TFA salt
J ¼ 5.46 Hz, 2H), 1.41 (s, 9H).
5
.1.2.25. tert-Butyl 2-((3-fluorobenzyl)amino)-3-cyano-4,5-
form (667 mg, 107%).
1
dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate (7c). The title
6
H NMR (400 MHz, DMSO-d ) ppm 9.21 (br. s., 2H), 8.52 (t,
compound was prepared by method E. The title compound was
obtained as a white solid (1.43 mg, 95%).
J ¼ 5.96 Hz, 1H), 7.44 (d, J ¼ 8.41 Hz, 2H), 7.37 (d, J ¼ 8.53 Hz, 2H),
4.40 (d, J ¼ 5.77 Hz, 2H), 4.05 (br. s., 2H), 3.36 (br. s., 2H), 2.68 (t,
J ¼ 5.52 Hz, 2H).
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.37 (dt, J ¼ 5.90,
7
1
.91 Hz, 1H), 7.15 (d, J ¼ 7.65 Hz, 1H), 6.99e7.12 (m, 2H), 5.53 (br. s,
H), 4.43 (d, J ¼ 5.77 Hz, 2H), 4.37 (br. s., 2H), 3.68 (t, J ¼ 5.33 Hz,
5.1.2.33. 2-((3-Methoxybenzyl)amino)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carbonitrile (8e). The title compound was prepared by
method B. The title compound was obtained as a white TFA salt
2
H), 2.62 (br. s., 2H), 1.53 (s, 9H).
5
.1.2.26. tert-Butyl 2-((4-chlorobenzyl)amino)-3-cyano-4,5-
form (0.8 g, 78%).
1
dihydrothieno[2,3-c]pyridine-6-(7H)-carboxylate (7d). The title
compound was prepared by method E. The title compound was
obtained as a white solid (644 mg, 99%).
6
H NMR (300 MHz, DMSO-d ) ppm 9.28 (br. s., 2H), 8.50 (t,
J ¼ 5.93 Hz, 1H), 7.27 (t, J ¼ 8.01 Hz, 1H), 6.91 (br. s., 2H), 6.84 (d,
J ¼ 8.10 Hz, 1H), 4.36 (d, J ¼ 5.84 Hz, 2H), 4.04 (s, 2H), 3.73 (s, 3H),
3.34 (t, J ¼ 5.47 Hz, 2H), 2.67 (br. s., 2H).
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.38 (d, J ¼ 8.53 Hz,
2
2
9
H), 7.31 (d, J ¼ 8.41 Hz, 2H), 5.36 (br. s., 1H), 4.40 (d, J ¼ 5.77 Hz,
H), 4.38 (br. s., 2H), 3.68 (t, J ¼ 5.08 Hz, 2H), 2.62 (br. s., 2H), 1.49 (s,
5.1.2.34. 2-((4-Cyanobenzyl)amino)-4,5,6,7-tetrahydrothieno[2,3-c]
pyridine-3-carbonitrile (8f). The title compound was prepared by
method B. The title compound was obtained as a white TFA salt
H).
5
.1.2.27. tert-Butyl 3-cyano-2-((3-methoxybenzyl)amino)-4,5-
form (1.2 mg, 98%).
1
dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate
(7e). The
title
H NMR (400 MHz,DMSO-d
6
) ppm 8.59 (t, J ¼ 6.09 Hz, 2H), 7.85
compound was prepared by method E. The title compound was
obtained as a white solid (1.03 g, 85%).
(d, J ¼ 8.41 Hz, 1H), 7.85 (d, J ¼ 8.41 Hz, 1H), 4.51 (d, J ¼ 6.02 Hz, 2H),
4.05 (br. s., 2H), 3.36 (br. s., 2H), 2.68 (t, J ¼ 5.77 Hz, 2H).
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.24e7.35 (m, 1H),
6
.95 (d, J ¼ 7.65 Hz, 1H), 6.85e6.92 (m, 2H), 5.43 (br. s., 1H), 4.39 (d,
5.1.2.35. 2-(Benzylamino)-6-(3-isopropyl-1H-pyrazole-5-carbonyl)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (9a) (method
J ¼ 5.65 Hz, 4H), 3.84 (s, 3H), 3.67 (t, J ¼ 5.70 Hz, 2H), 2.61 (t,
J ¼ 5.30 Hz, 2H), 1.50 (s, 9H).
F). To
a solution of HATU (104 mg, 0.27 mmol) and 3-
isopropylpyrazole-5-carboxylic acid (36 mg, 0.22 mmol) in DMF
(14 mL) was then added diisopropylethylamine (0.055 mL,
0.31 mmol). The reaction solution was stirred at room temperature
for 2 h. 2-(Benzylamino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-
3-carbonitrile (75 mg, 0.21 mmol) was added in one portion. The
solution was then stirred at room temperature overnight. The re-
action was diluted with methylene chloride and washed. The
aqueous layer was extracted with methylene chloride twice, the
5
.1.2.28. tert-Butyl 3-cyano-2-((4-cyanobenzyl)amino)-4,5-
dihydrothieno[2,3-c]pyridine-6(7H)-carboxylate (7f). The title com-
pound was prepared by method E. The title compound was ob-
tained as a white solid (1.22 g, 77%).
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.69 (d, J ¼ 8.53 Hz,
2
2
9
H), 7.49 (d, J ¼ 8.41 Hz, 2H), 5.66 (br. s., 1H), 4.51 (d, J ¼ 6.02 Hz,
H), 4.36 (br. s., 2H), 3.67 (t, J ¼ 5.52 Hz, 2H), 2.61 (br. s., 2H), 1.49 (s,
H).
4
combined organic phases were dried over anhydrous MgSO . The
resulting residue was purified by column chromatography on silica
gel (EtOAc/hexane ¼ 2:1) to give title compound as a white solid
(33 mg, 37%).
5
.1.2.29. 2-(Benzylamino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carbonitrile (8a). The title compound was prepared by method B.

254
M. Nam et al. / European Journal of Medicinal Chemistry 97 (2015) 245e258
1H NMR (400 MHz, CHLOROFORM-d) ppm 7.37e7.40 (m, 5H),
.49 (s, 1H), 5.34 (br. s., 1H), 4.95 (br. s., 0.8H), 4.69 (br. s., 1.2H), 4.42
d, J ¼ 4.8 Hz,2H), 4.16 (br. s., 1.2H), 4.02 (br. s., 0.8H), 3.03e3.06 (m,
H), 2.76 (br. s., 2H), 1.33 (d, J ¼ 5.7 Hz, 6H).
obtained as a white solid (30 mg, 42%).
1
6
(
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.64 (br. s., 1H),
7.32e7.38 (m, 2H), 7.09 (t, J ¼ 8.41 Hz, 1H), 6.75 (d, J ¼ 2.38 Hz, 1H),
4.94 (br. s., 0.8H), 4.71 (br. s., 1.2H), 4.39 (br. s., 2H), 4.15 (br. s., 1.2H),
4.04 (br. s., 0.8H), 2.78 (br. s., 2H).
13
C NMR (101 MHz, CHLOROFORM-d) ppm 164.0, 152.4, 144.9,
40.1, 136.5, 131.4, 128.9, 128.1, 127.6, 115.8, 114.6, 103.8, 84.0, 51.5,
13
1
C NMR (101 MHz, CHLOROFORM-d) ppm 163.8, 163.7, 163.4,
161.2,145.0,144.3,132.3,131.4,130.9,129.4,129.3,115.9,115.7,114.5,
4
4.2, 41.7, 26.1, 25.6, 22.3.
1
07.2, 84.0, 50.6, 44.2, 41.6, 25.5.
þ
5
4
.1.2.36. 2-(Benzylamino)-6-(3-methyl-1H-pyrazole-5-carbonyl)-
,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (9b). The title
LC/MS (ESI , MeCN/H
2
O): m/z: calcd for C19
H16FN
5
OS: 381.10
þ
[MþH] ; found: 382.0.
compound was prepared by method F. The title compound was
obtained as a white solid (25 mg, 31%).
5.1.2.41. 2-((4-Chlorobenzyl)amino)-6-(3-isopropyl-1H-pyrazole-5-
carbonyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carbonitrile
(9h). The title compound was prepared by method F. The title
compound was obtained as a white solid (46 mg, 58%).
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.34e7.47 (m, 5H),
6
(
3
.46 (s, 1H), 5.35 (br. s., 1H), 4.92 (br. s., 0.9H), 4.68 (br. s., 1.1H), 4.41
s, 2H), 4.14 (br. s., 1.3H), 4.01 (br. s., 0.7H), 2.75 (br. s., 2H), 2.37 (s,
H).
13C NMR (101 MHz, CHLOROFORM-d) ppm 164.0, 150.6, 144.9,
41.6, 136.5, 131.4, 128.9, 128.1, 127.6, 115.7, 114.5, 106.3, 83.8, 51.5,
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.45 (d, J ¼ 8.4 Hz,
2H), 7.37 (d, J ¼ 8.4 Hz, 2H), 6.42 (s, 1H), 4.61 (br. s., 2H), 4.44 (d,
J ¼ 4.6 Hz, 2H), 4.11 (br. s., 1.1H), 3.99 (br. s., 0.9H), 3.02e3.05 (m,
1H), 2.68 (br. s., 2H), 1.32 (d, 6H J ¼ 5.7 Hz).
1
4
4.3, 41.7, 25.6, 11.2.
þ
13
LC/MS (ESI , MeCN/H
2
O): m/z: calcd for C20
H
19
N
5
OS: 377.13
C NMR (101 MHz, CHLOROFORM-d) ppm 164.3, 151.6, 145.1,
þ
[MþH] ; found: 378.0.
135.6, 133.3, 131.7, 131.4, 128.7, 115.9, 113.8, 103.7, 103.2, 82.8, 50.1,
4
4.3, 41.4, 25.7, 25.4, 22.0.
5
.1.2.37. 2-(Benzylamino)-6-(1H-pyrazole-5-carbonyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (9c). The title com-
pound was prepared by method F. The title compound was ob-
tained as a white solid (40 mg, 56%).
5.1.2.42. 2-((4-Chlorobenzyl)amino)-6-(3-methyl-1H-pyrazole-5-
carbonyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (9i).
The title compound was prepared by method F. The title compound
was obtained as a white solid (32 mg, 43%).
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.59 (br. s., 1H),
1
7
.31e7.46 (m, 5H), 6.70 (d, J ¼ 2.38 Hz, 1H), 4.88 (br. s., 0.8H), 4.67
H NMR (400 MHz, CHLOROFORM-d) ppm 7.43 (d, J ¼ 8.4 Hz,
(
(
br. s., 1.2H), 4.40 (br. s., 2H), 4.12 (br. s., 1.2H), 4.01 (br. s., 0.8H), 2.74
br. s., 2H).
2H), 7.36 (d, J ¼ 8.4 Hz, 2H), 6.46 (s, 1H), 4.54 (br. s., 2H), 4.46 (d,
J ¼ 4.6 Hz, 2H), 4.01 (br. s., 1.1H), 3.89 (br. s., 0.9H), 2.68 (br. s., 2H),
2.37 (s, 3H).
13C NMR (75 MHz, CHLOROFORM-d) ppm 164.2, 163.4, 144.4,
136.5, 132.0, 131.4, 130.8, 128.8, 128.1, 127.6, 114.3, 107.2, 83.6, 51.3,
13C NMR (101 MHz, CHLOROFORM-d) ppm 164.1, 145.2, 141.0,
135.3, 133.7, 131.9, 131.4, 128.9, 115.9, 114.4, 106.5, 106.0, 83.5, 50.4,
4
4.3, 41.7, 25.6.
þ
LC/MS (ESI , MeCN/H
2
O): m/z: calcd for C19
H
17
N
5
OS: 363.11
44.3, 41.5, 25.5, 10.8.
þ
þ
[MþH] ; found: 364.0.
LC/MS (ESI , MeCN/H
2
O): m/z: calcd for C20
H
18ClN
5
OS: 411.09
þ
[
MþH] ; found: 411.9.
5
.1.2.38. 2-((4-Fluorobenzy)lamino)-6-(3-isopropyl-1H-pyrazole-5-
carbonyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carbonitrile
9d). The title compound was prepared by method F. The title
5.1.2.43. 6-Benzoyl-2-(benzylamino)-4,5,6,7-tetrahydrothieno[2,3-c]
pyridine-3-carbonitrile (10a) (method G). To a solution of 2-(ben-
zylamino)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carbonitrile
(75 mg, 0.21 mmol) and benzoyl chloride (0.027 mL, 0.23 mmol) in
DMF (14 mL) was then added diisopropylethylamine (0.055 mL,
0.31 mmol). The reaction solution was stirred at room temperature
for 2 h and was diluted with methylene chloride and washed. The
aqueous layer was extracted with methylene chloride twice, the
(
compound was obtained as a white solid (52 mg, 66%).
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.30e7.41 (m, 2H),
7
4
0
.09 (t, J ¼ 7.9 Hz, 2H), 6.50 (s, 1H), 5.34 (br. s., 1H), 4.95 (br. s., 0.8H),
.69 (br. s., 1.2H), 4.39 (d, 2H J ¼ 4.6 Hz), 4.17 (br. s., 1.1H), 4.01(br. s.,
.9H), 3.02e3.09 (m, 1H), 2.76 (br. s., 2H), 1.33 (d, 6H J ¼ 5.7 Hz).
1
3
C NMR (101 MHz, CHLOROFORM-d) ppm 164.0, 163.6, 161.2,
152.0, 145.0, 132.4, 131.4, 129.3, 129.3, 116.0, 115.8, 115.6, 114.5,
4
combined organic phases were dried over anhydrous mgSO . The
1
03.6, 83.6, 50.4, 44.3, 41.6, 25.9, 25.6, 22.3.
resulting residue was purified by column chromatography on silica
gel (EtOAc/hexane ¼ 2:1) to give title compound as a white solid
(55 mg, 69%).
þ
LC/MS (ESI , MeCN/H
2
O): m/z: calcd for C22
H22FN
5
OS: 423.15
þ
[MþH] ; found: 424.0.
1H NMR (400 MHz, CHLOROFORM-d) ppm 7.44e7.56 (m, 5H),
7.31e7.44 (m, 5H), 5.40 (br. s., 1H), 4.70 (br. s., 1.5H), 4.42 (br. s.,
2.5H), 4.01 (br. s., 0.7H), 3.67 (br. s., 1.3H), 2.68 (br. s., 2H).
5
.1.2.39. 2-((4-Fluorobenzyl)amino)-6-(3-methyl-1H-pyrazole-5-
carbonyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carbonitrile
9e). The title compound was prepared by method F. The title
1
3
(
C NMR (101 MHz, CHLOROFORM-d) ppm 170.7, 164.3, 136.5,
compound was obtained as a white solid (28 mg, 38%).
135.8, 135.4, 133.5, 130.1, 129.7, 128.9, 128.4, 128.1, 127.7, 115.7, 83.5,
51.4, 44.7, 41.4, 25.4.
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.33e7.36 (m, 2H),
7
.09 (t, J ¼ 8.5 Hz, 2H), 6.46 (s, 1H), 5.35 (br. s., 1H), 4.93 (br. s., 0.8H),
.69 (br. s., 1.2H), 4.40 (d, J ¼ 4.89 Hz, 2H), 4.15 (br. s., 1.2H), 4.02(br.
s., 0.8H), 2.76 (br. s., 2H), 2.37 (s, 3H).
4
5.1.2.44. 2-(Benzylamino)-6-(4-fluorobenzoyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10b). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (31 mg, 37%).
13
C NMR (101 MHz, CHLOROFORM-d) ppm 163.7, 163.6, 161.3,
53.7, 144.8, 141.7, 132.3, 131.4, 129.4, 129.3, 116.3, 115.9, 115.7, 114.8,
06.4, 84.2, 50.7, 44.2, 41.6, 25.6, 11.3.
1
1
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.45e7.52 (m, 2H),
7
.33e7.44 (m, 5H), 7.11e7.19 (m, 2H), 5.36 (t, J ¼ 5.46 Hz, 1H), 4.66
5
4
.1.2.40. 2-((4-Fluorobenzyl)amino)-6-(1H-pyrazole-5-carbonyl)-
,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (9f). The title
(br. s., 1.5H), 4.42 (br. s., 2.5H), 4.01 (br. s., 0.7H), 3.68 (br. s., 1.3H),
2.70 (br. s., 2H).
compound was prepared by method F. The title compound was
13C NMR (101 MHz, CHLOROFORM-d) ppm 169.9, 164.8, 164.3,

M. Nam et al. / European Journal of Medicinal Chemistry 97 (2015) 245e258
255
1
62.3, 136.8, 131.6, 131.6, 130.9, 129.4, 129.3, 128.8, 127.9, 127.6,
4.70 (br. s., 1.5H), 4.40 (br. s., 2.5H), 4.01 (br. s., 0.6H), 3.66 (br. s.,
1.4H), 2.68 (br. s., 2H).
115.8, 115.6, 114.2, 83.5, 51.2, 44.9, 41.7, 25.4.
LC/MS (ESIþ, MeCN/H
2
O): m/z: calcd for C22
H19FN
3
OS: 391.11
13C NMR (101 MHz, CHLOROFORM-d) ppm 170.9, 163.7, 163.6,
161.3, 135.6, 133.3, 132.3, 130.1, 129.4, 129.3, 128.6, 128.3, 126.9,
þ
[MþH] ; found: 392.1.
115.9, 115.7, 115.5, 84.2, 50.7, 44.6, 41.3, 25.4.
5
.1.2.45. 2-(Benzylamino)-6-(6-fluoronicotinoyl)-4,5,6,7-
LC/MS (ESIþ, MeCN/H
2 3
O): m/z: calcd for C22H19FN OS: 391.11
þ
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10c). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (49 mg, 68%).
[MþH] ; found: 392.1.
5.1.2.50. 6-(4-Fluorobenzoyl)-2-((4-fluorobenzyl)amino)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10j). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (53 mg, 69%).
1
H NMR (400 MHz, CHLOROFORM-d) ppm 8.51e8.76 (m, 2H),
7
.72 (d, J ¼ 7.65 Hz, 1H), 7.48 (dd, J ¼ 5.33, 8.72 Hz, 2H), 7.36 (dd,
J ¼ 4.83, 7.84 Hz, 1H), 7.15 (t, J ¼ 8.60 Hz, 2H), 5.40 (t, J ¼ 5.52 Hz,
1
1
3
H), 4.66 (br. s., 1.4H), 4.47 (d, J ¼ 5.02 Hz, 2.6H), 4.00 (br. s., 0.6H),
H NMR (400 MHz, CHLOROFORM-d) ppm 7.48 (dd, J ¼ 5.33,
.70 (br. s., 1.4H), 2.72 (br. s., 2H).
8.72 Hz, 2H), 7.31e7.39 (m, 2H), 7.15 (t, J ¼ 8.60 Hz, 2H), 7.09 (t,
J ¼ 8.60 Hz, 2H), 5.36 (t, 1H J ¼ 5.7 Hz), 4.66 (br. s., 1.5H), 4.39 (m,
2.5H), 3.97 (br. s., 0.6H), 3.77 (br. s., 1.4H), 2.70 (br. s., 2H).
13
C NMR (101 MHz, CHLOROFORM-d) ppm 170.1, 164.9, 163.6,
62.4, 149.2, 149.0, 135.5, 132.5, 131.4, 131.4, 131.1, 129.3, 129.3,
29.2, 123.8, 115.9, 115.7, 115.5, 84.3, 48.7, 44.8, 41.4, 25.3.
1
1
13
C NMR (101 MHz, CHLOROFORM-d) ppm 170.0, 164.9, 163.9,
LC/MS (ESIþ, MeCN/H
2
O): m/z: calcd for C21
H18FN
4
OS: 392.11
163.7,162.4,161.2,136.0,132.3,132.3,131.5,131.5,129.4,129.3,115.9,
115.6, 115.5, 114.6, 84.0, 50.7, 44.7, 41.5, 25.4.
þ
[
MþH] ; found: 393.0.
5
.1.2.46. 2-(Benzylamino)-6-(3-fluorobenzoyl)-4,5,6,7-
5.1.2.51. 2-((4-Fluorobenzyl)amino)-6-(4-methylbenzoyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyidine-3-carbonitrile (10k). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (37.51 mg, 56%).
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10d). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (23 mg, 50%).
1
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.38e7.44 (m, 6H),
H NMR (400 MHz, CHLOROFORM-d) ppm 7.32e7.39 (m, 4H),
7
2
.16e7.23 (m, 3H), 5.43 (br. s., 1H), 4.68 (br. s., 1.4H), 4.42 (br. s.,
7.26 (d, J ¼ 7.78 Hz, 2H), 7.10 (t, J ¼ 8.53 Hz, 2H), 5.29 (t, J ¼ 5.52 Hz,
1H), 4.67 (br. s.,1.5H), 4.40 (br. s., 2.5H), 3.98 (br. s., 0.6H), 3.69 (br. s.,
1.3H), 2.69 (br. s., 2H), 2.42 (s, 3H).
C NMR (101 MHz, CHLOROFORM-d) ppm 171.2, 163.8, 163.7,
161.2,140.4,132.5,132.3,132.3,130.1,129.4,129.3,129.2,129.1,127.1,
.6H), 4.01 (br. s., 0.6H), 3.50 (br. s., 1.4H), 2.71 (br. s., 2H).
13
C NMR (75 MHz, CHLOROFORM-d) ppm 169.4, 164.3, 163.9,
61.0,137.6, 137.5,136.3,130.6,130.5,129.3,129.1,129.0,128.3,127.7,
22.5, 117.3, 117.1, 115.4, 114.4, 114.1, 83.5, 51.6, 44.7, 41.3, 25.4.
13
1
1
LC/MS (ESIþ, MeCN/H
2
O): m/z: calcd for C22
H
19FN
3
OS: 391.11
115.9, 115.7, 115.6, 84.1, 50.7, 44.7, 41.4, 25.3, 21.4.
þ
þ
[
MþH] ; found: 392.1.
LC/MS (ESI , MeCN/H
MþH] ; found: 406.1.
2
O): m/z: calcd for C23
H
21FN
3
OS: 405.13
þ
[
5
.1.2.47. 2-(Benzylamino)-6-(4-methylbenzoyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10f). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (40 mg, 49%).
5.1.2.52. 2-((4-Methoxybenzyl)amino)-6-(4-fluorobenzoyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10l). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (49 mg, 56%).
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.35e7.41 (m, 6H),
1
7
.25e7.31 (m, 3H), 5.33 (t, 1H J ¼ 5.3 Hz), 4.66 (br. s., 1.4H), 4.43 (br.
s., 2.4H), 4.01 (br. s., 0.6H), 3.69 (br. s., 1.4H), 2.69 (br. s., 2H), 2.42 (s,
H).
13C NMR (101 MHz, CHLOROFORM-d) ppm 170.5, 164.1, 144.2,
40.4, 136.5, 132.4, 130.2, 129.2, 129.1, 128.9, 128.1, 127.7, 115.7, 83.8,
1.5, 44.7, 41.5, 25.4, 21.4.
H NMR (400 MHz, CHLOROFORM-d) ppm 7.45 (d, J ¼ 8.78 Hz,
2H), 7.35 (dd, J ¼ 5.40, 8.28 Hz, 2H), 7.10 (t, J ¼ 8.60 Hz, 2H), 6.96 (d,
J ¼ 8.66 Hz, 2H), 5.27 (t, J ¼ 5.7 Hz, 1H), 4.60 (br. s., 2H), 4.39 (d, 2H
J ¼ 5.7 Hz), 3.87 (s, 3H), 3.79 (br. s., 2H), 2.72 (br. s., 2H).
3
13
1
5
C NMR (101 MHz, CHLOROFORM-d) ppm 170.9, 163.8, 163.7,
161.2, 161.1, 132.4, 131.1, 129.4, 129.3, 129.0, 127.5, 115.8, 115.6, 114.7,
13.9, 83.9, 55.4, 50.6, 44.8, 41.6, 25.1.
1
5
.1.2.48. 2-(Benzylamino)-6-(3-methylbenzoyl)-4,5,6,7-
LC/MS (ESIþ, MeCN/H
2 3 2
O): m/z: calcd for C23H21FN O S: 421.13
þ
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10g). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (44 mg, 54%).
[MþH] ; found: 422.1.
5.1.2.53. 6-Benzoyl-2-((3-fluorobenzyl)amino)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10m). The title
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.30e7.47 (m, 7H),
7
2
3
.20e7.29 (m, 2H), 5.35 (br. s., 1H), 4.69 (br. s., 1.6H), 4.43 (br. s.,
.4H), 4.01 (br. s., 0.6H), 3.72 (br. s., 1.4H), 2.70 (br. s., 2H), 2.44 (s,
compound was prepared by method G. The title compound was
obtained as a white solid (61 mg, 81%).
1
H.)
H NMR (400 MHz, CHLOROFORM-d) ppm 7.38e7.54 (m, 6H),
13C NMR (101 MHz, CHLOROFORM-d) ppm 171.2, 163.9, 138.6,
7.04e7.18 (m, 3H), 7.09 (t, 2H J ¼ 7.6 Hz), 5.31 (t, 1H J ¼ 5.7 Hz), 4.70
(br. s., 1.5H), 4.40 (br. s., 2.5H), 4.01 (br. s., 0.6H), 3.66 (br. s., 1.4H),
2.68 (br. s., 2H).
137.5, 136.4, 135.5, 131.4, 130.8, 128.9, 128.5, 128.2, 127.7, 127.0,
123.8, 115.5, 84.0, 51.6, 44.6, 41.3, 25.4, 21.3.
LC/MS (ESIþ, MeCN/H
2
O): m/z: calcd for C23
22
H N
3
OS: 387.14
13C NMR (101 MHz, CHLOROFORM-d) ppm 170.7, 164.3, 164.0,
161.8,139.3,139.2,135.3,133.5,130.5,130.4,130.1,128.7,128.4,127.1,
þ
[MþH] ; found: 388.1.
123.1, 115.6, 115.1, 114.9, 114.5, 114.3, 83.9, 50.7, 44.6, 41.4, 25.3.
5
.1.2.49. 6-Benzoyl-2-((4-fluorobenzyl)amino)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10i). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (35 mg, 48%).
5.1.2.54. 6-(4-Fluorobenzoyl)-2-((3-fluorobenzyl)amino)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10n). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (41 mg, 53%).
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.41e7.54 (m, 5H),
1
7.31e7.41 (m, 2H), 7.10 (t, J ¼ 8.28 Hz, 2H), 5.34 (t, J ¼ 5.46 Hz, 1H),
H NMR (300 MHz, CHLOROFORM-d) ppm 7.48 (dd, J ¼ 5.27,

256
M. Nam et al. / European Journal of Medicinal Chemistry 97 (2015) 245e258
8
4
2
.67 Hz, 2H), 7.34e7.43 (m, 1H), 7.02e7.24 (m, 5H) 5.46 (br. s., 1H),
obtained as a white solid (55 mg, 73%).
1
.63 (br. s., 2H), 4.44 (d, 2H J ¼ 5.1 Hz), 3.72 (br. s., 2H), 2.20 (br. s.,
H NMR (400 MHz, CHLOROFORM-d) ppm 7.48 (dd, J ¼ 5.27,
H).
8.66 Hz, 2H), 7.30e7.35 (m, 1H), 7.15 (t, J ¼ 8.47 Hz, 2H), 6.95 (d,
J ¼ 7.28 Hz, 1H), 6.87e6.92 (m, 2H), 5.36 (t, J ¼ 5.5 Hz, 1H), 4.67 (br.
s., 1.4H), 4.40 (d, 2.6H, J ¼ 4.2 Hz), 4.00 (br. s., 0.6H), 3.85 (s, 3H),
3.68 (br. s., 1.4H), 2.71 (br. s., 2H).
13C NMR (101 MHz, CHLOROFORM-d) ppm 170.0, 164.9, 164.3,
1
64.0, 162.4, 161.8, 139.2, 139.2, 131.4, 131.4, 130.9, 130.5, 130.4,
29.5, 129.3, 129.2, 123.1, 123.1, 115.9, 115.7, 115.5, 115.1, 114.9, 114.5,
14.3, 84.1, 50.7, 44.8, 41.6, 25.3.
1
1
13
C NMR (101 MHz, CHLOROFORM-d) ppm 170.0, 164.9, 164.1,
62.4, 160.0, 138.1, 131.5, 131.5, 130.9, 130.0, 129.9, 129.8, 129.3,
1
5
.1.2.55. 2-((3-Fluorobenzyl)amino)-6-(4-methylbenzoyl)-4,5,6,7-
119.8, 115.9, 115.7, 115.6, 113.4, 113.3, 83.8, 55.3, 51.3, 44.8, 41.5, 25.4.
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10o). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (48 mg, 63%).
5.1.2.60. 6-(3-Fluorobenzoyl)-2-((3-methoxybenzyl)amino)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10t). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (42 mg, 55%).
1
H NMR (300 MHz, CHLOROFORM-d) ppm 7.40 7.33e7.42 (m,
3
2
0
H), 7.26 (d, J ¼ 8.00 Hz, 2H), 7.16 (d, J ¼ 7.35 Hz, 1H), 7.01e7.12 (m,
H), 5.34 (br. s., 1H), 4.67 (br. s., 1.5H), 4.40 (br. s., 2.5H), 3.99 (br. s.,
.6H), 3.67 (br. s., 1.4H), 2.64 (br. s., 2H), 2.41 (s, 3H).
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.40e7.52 (m, 1H),
7.30e7.37 (m, 1H), 7.15e7.27 (m, 3H), 6.87e6.98 (m, 3H), 5.38 (br. s.,
1H), 4.69 (br. s., 1.4H), 4.41 (br. s., 2.6H), 4.02 (br. s., 0.7H), 3.85 (s,
3H), 3.66 (br. s., 1.3H), 2.73 (br. s., 2H).
13
C NMR (101 MHz, CHLOROFORM-d) ppm 171.3, 164.3, 163.9,
61.8, 140.4, 139.4, 139.3, 132.5, 130.4, 130.3, 130.0, 129.2, 129.1,
27.0, 127.0, 123.1, 115.7, 115.0, 114.8, 114.5, 114.3, 84.0, 50.7, 44.8,
1.5, 25.4, 21.4.
1
1
4
13
C NMR (101 MHz, CHLOROFORM-d) ppm 168.8, 163.8, 163.3,
160.9, 159.6, 137.6, 137.0, 136.9, 131.5, 129.7, 129.7, 125.4, 122.5,
20.0, 120.0, 119.4, 116.6, 116.4, 114.1, 113.9, 112.9, 83.4, 76.7, 54.9,
1
5
.1.2.56. 2-(4-Methoxybenzyl)amino)-6-(3-fluorobenzoyl)-4,5,6,7-
51.0, 44.2, 41.0, 24.9.
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10p). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (43 mg, 54%).
5.1.2.61. 6-Benzoyl-2-((4-cyanobenzyl)amino)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10u). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (62 mg, 81%).
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.44 (d, J ¼ 8.66 Hz,
2
6
(
H), 7.34e7.41 (m, 1H), 7.15 (d, J ¼ 7.65 Hz, 1H), 7.01e7.11 (m, 2H),
.96 (d, J ¼ 8.66 Hz, 2H), 5.37 (t, 1H J ¼ 5.7 Hz), 4.59 (br. s., 2H), 4.43
1
H NMR (400 MHz, CHLOROFORM-d) ppm 7.70 (d, J ¼ 7.78 Hz,
d, J ¼ 5.52 Hz, 2H), 3.87 (s, 3H), 3.85 (br. s., 2H), 2.72(br. s., 2H).
2H), 7.38e7.58 (m, 7H), 5.42 (t, 1H J ¼ 5.7 Hz), 4.65 (br. s., 1.5H), 4.52
(br. s., 2.3H), 4.03 (br. s., 0.6H), 3.69 (br. s.,1.4H), 2.70 (br. s., 2H), 2.42
(s, 3H).
1
3
C NMR (101 MHz, CHLOROFORM-d) ppm 171.3, 164.4, 163.9,
61.3, 139.3, 139.2, 131.1, 130.5, 130.4, 129.0, 128.2, 127.5, 127.1, 118.4,
15.6, 115.1, 114.9, 114.5, 114.3, 113.9, 83.9, 55.4, 50.6, 44.8, 41.6, 25.1.
1
1
13C NMR (101 MHz, CHLOROFORM-d) ppm 171.2, 163.9, 142.7,
135.3, 132.5, 131.1, 130.2, 128.7, 128.0, 126.7, 118.6, 115.6, 114.6, 111.5,
83.9, 50.4, 44.6, 41.3, 25.4.
LC/MS (ESIþ, MeCN/H
2 3 2
O): m/z: calcd for C23H20FN O S: 421.13
þ
[
MþH] ; found: 422.0.
5
.1.2.57. 6-(4-chlorobenzoyl)-2-((3-fluorobenzyl)amino)-4,5,6,7-
5.1.2.62. 2-((4-Cyanobenzyl)amino)-6-(4-methoxybenzoyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10v). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (42 mg, 52%).
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10q). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (41 mg, 52%).
1
1
H NMR (300 MHz, CHLOROFORM-d) ppm 7.33e7.49 (m, 5H),
H NMR (300 MHz, CHLOROFORM-d) ppm 7.71 (d, J ¼ 7.91 Hz,
7
4
1
.16 (d, J ¼ 7.54 Hz, 1H), 7.02e7.12 (m, 2H), 5.39 (t, J ¼ 5.65 Hz, 1H),
.67 (br. s., 1.4H), 4.39 (br. s., 2.6H), 3.99 (br. s., 0.6H), 3.68 (br. s.,
.4H), 2.70 (br. s., 2H).
13C NMR (101 MHz, CHLOROFORM-d) ppm 169.8, 164.3, 163.9,
61.8, 139.2, 139.1, 139.0, 136.3, 133.8, 132.2, 130.5, 130.4, 129.0,
28.4, 123.1, 115.5, 115.1, 114.9, 114.5, 114.3, 84.3, 50.8, 44.7, 41.4,
5.4.
2H), 7.50 (d, J ¼ 8.10 Hz, 2H), 7.45 (d, J ¼ 8.10 Hz, 2H), 6.96 (d,
J ¼ 8.48 Hz, 2H), 5.51 (t, 1H J ¼ 5.7 Hz), 4.59 (br. s., 2H), 4.52 (d, 2H
J ¼ 5.4 Hz), 3.88 (s, 3H), 3.77 (br. s., 2H), 2.73 (br. s., 2H).
13
C NMR (101 MHz, CHLOROFORM-d) ppm 170.9, 163.6, 161.2,
1
1
2
142.4, 132.6, 132.3, 129.0, 128.0, 127.4, 118.6, 115.5, 114.9, 113.9, 111.7,
84.5, 55.4, 50.6, 44.6, 41.5, 25.3.
þ
LC/MS (ESI , MeCN/H
2
O): m/z: calcd for C24
H
21
N
4
O
2
S: 428.13
þ
LC/MS (ESIþ, MeCN/H
2
O): m/z: calcd for C22
H18ClFN
3
OS: 425.08
[MþH] ; found: 429.1.
þ
[MþH] ; found: 425.2.
5.2. Biological evaluation
5
.1.2.58. 2-((4-Chlorobenzyl)amino)-6-(4-fluorobenzoyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10r). The title com-
pound was prepared by method G. The title compound was ob-
tained as a white solid (34 mg, 44%).
5.2.1. In vitro assay for mGluR1
Chem-3 cells stably expressing mGluR1 were purchased from
Millipore Corporation (Bedford, MA, USA). Cells were grown in
RPMI medium supplemented with 10% (v/v) fetal bovine serum,
penicillin (100 U/ml), streptomycin (100
Chem3 growth supplement at 37 C in a humid atmosphere of 5%
1
H NMR (400 MHz, CHLOROFORM-d) ppm7.47 (dd, J ¼ 5.33,
8
(
.72 Hz, 2H), 7.37 (d, J ¼ 8.00 Hz, 2H), 7.30 (d, J ¼ 8.00 Hz, 2H), 5.54
mg/mL), and 1% (v/v)
ꢀ
t, 1H J ¼ 5.6 Hz), 4.65 (br. s., 1.4H), 4.40 (d, J ¼ 4.27 Hz, 2H), 3.97 (br.
s., 0.6H), 3.67 (br. s., 1.4H), 2.69 (br. s., 2H).
2
CO and 95% air. For calcium assay, cells were harvested and seeded
1
3
C NMR (101 MHz, CHLOROFORM-d) ppm 170.0, 164.9, 163.6,
62.4, 134.9, 134.0, 133.0, 131.5, 131.5, 129.5, 129.3, 129.2, 129.1,
28.9, 126.7, 119.3, 115.9, 115.7, 115.3, 84.5, 50.8, 44.8, 41.5, 24.6.
into 96-well black wall clear bottom plates at a density of 100,000
cells per a well. After 1 h of stabilization, cells were treated with
Calcium-5 assay reagent (Molecular Devices Corporation, CA, USA).
Subsequently, cells were treated with compounds at various con-
1
1
5
.1.2.59. 6-(4-Fluorobenzoyl)-2-((3-methoxybenzyl)amino)-4,5,6,7-
centrations, followed by addition of a high concentration of L-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10s). The title com-
glutamate (30 mM) to activate mGluR1. All data were collected and
pound was prepared by method G. The title compound was
analyzed using an FDSS6000 automated fluorescent plate reader

M. Nam et al. / European Journal of Medicinal Chemistry 97 (2015) 245e258
257
and related software (Hamamatsu, Japan).
responses modified from the same literature, and d is the mean
difference between stimuli in log units (0.17).
5
.2.2. In vitro assay for mGluR5
Human embryonic kidney cells stably expressing mGluR5 were
Cold allodynia: To quantify cold sensitivity of the paw, brisk paw
withdrawal in response to acetone application was measured as
reported previously [39]. The rat was placed under a transparent
plastic box on a metal mesh floor and acetone was applied to the
plantar surface of the hind paw. To do this, an acetone bubble was
formed at the end of a small piece of polyethylene tubing which
was connected to a syringe. The bubble was then gently touched to
the heel. The acetone quickly spread over the proximal half of the
plantar surface of the hind paw. The acetone was applied 5 times to
hind paw at 2 min interval. The frequency of paw withdrawal was
expressed as a percentage [(no. of trials accompanied by brisk foot
withdrawal/total no. of trials) ꢂ l00].
obtained from Yonsei University. Cells were grown in DMEM me-
dium supplemented with 10% (v/v) fetal bovine serum, penicillin
(
100 U/mL), streptomycin (100 lg/mL), and puromycin (10 lg/mL) at
ꢀ
3
7
C in a humid atmosphere of 5% CO
2
and 95% air. For calcium
assay, cells were harvested and seeded 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 (Mo-
lecular Devices Corporation, California). Subsequently, cells were
treated with compounds at various concentrations, followed by
addition of a high concentration of L-glutamate (10 mM) to activate
mGluR5. All data were collected and analyzed using an FDSS6000
automated fluorescent plate reader and related software (Hama-
matsu, Japan).
Data analysis: The results of behavioral tests are expressed as a %
MPE. For example, paw withdrawal thresholds were converted to %
MPE by the following formula, by using a cutoff of 15 g (the
threshold for normal rats) to define maximum possible effect: (post
5
.2.3. Human liver microsomal stability assay
Human liver microsomes (0.5 mg/mL, BD Biosciences, Bedford,
drug
threshold-baseline
threshold)/(cutoff
ꢁ
baseline
threshold) ꢂ 100.%MPE values near 100 indicate normal mechan-
ical thresholds (i.e., at or near 15 g), whereas values near 0 indicate
allodynia. The result of cold allodynia was also expressed as %MPE.
MA) in phosphate buffer (100 mM, pH 7.4) were incubated with a
M solution of the test compound at 37C in the presence of
NADPH (1.2 mM). After 30 min of incubation, the reaction was
terminated by addition of cold acetonitrile containing 0.1 g/mL of
1
m
m
5.2.6. Pharmacokinetic study
internal standard. After spinning down each sample, the superna-
tants were analyzed by LC-MS/MS.
Each compound at a dose of 10 mg/kg was administered intra-
venously or orally to male SD rats. Blood samples were collected at
various time points after drug administration and were stored
5.2.4. CYP inhibition assay
ꢀ
at ꢁ70 C until LC-MS/MS analysis. To determine the drug con-
Inhibitions of CYP (CYP1A2, 2C9, 2D6 and 3A4) activities were
centration in the plasma, a 0.1 mg/mL of internal standard in 100 mL
®
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. Positive controls
of acetonitrile was added to 50
mL of plasma samples. After vortex-
mixing and centrifugation at 10,000 rpm for 10 min, 5
mL of the
supernatant was analyzed by LC-MS/MS. The LC-MS/MS system
®
including the
a-naphthoflavone (CYP1A2), sulfaphenazole
consisted of a HP1100 HPLC system (Agilent, Santa Clara, CA) and
®
(
CYP2C9), quinidine (CYP2D6) and ketoconazole (CYP3A4) were
API3200
triple-quadrupole mass spectrometer (Applied
prepared in acetonitrile to give a final concentration of 10 mM.
Briefly, each sample (test compounds, positive inhibition control
and solvent control) and the Master Pre-Mix [CYP450 BACULO-
SOMES Reagent (recombinant human CYP450 isozyme and rabbit
NADPHP450 reductase) and Regeneration System (3.3 mM glucose-
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 (Waters Corporation, Milford,
MA) using gradient elution at a flow rate of 0.3 mL/min.
6
-phosphate and 0.3 U/ml glucose-6-phosphate dehydrogenase in
1
00 mM potassium phosphate, pH 8.0)] were added to each well.
Acknowledgments
ꢀ
After incubating the mixtures for 5 min at 37 C, the enzyme re-
action was started by the addition of the Vivid CYP substrates and
®
This work was supported by Korea Institute of Science and
Technology (2E25240). J.L. was supported by the Sungshin
Women's University Research Grant of 2015-1-21-007.
0
.1 mM NADP þ buffer. The remaining enzyme activity was
measured after 20 min by using a fluorescent plate reader.
5
.2.5. In vivo efficacy test in spinal nerve (L5/L6) ligation model of
Appendix A. Supplementary data
neuropathic pain
Two behavioral tests (mechanical allodynia and cold allodynia)
were conducted for rats at 1 day prior to surgery and 14 days after
surgery. After the postoperative behavioral test, the animals were
treated orally with 100 mg/kg compound 9b, 10b or gabapentin.
The tests were re-evaluated at 1 h, 3 h, and 5 h after administration.
Mechanical allodynia: Testing was performed according to
methods described in the literature [37]. Rats were acclimated in a
transparent plastic boxes permitting freedom of movement with a
wire mesh floor to allow access to the planter surface of the hind
paws.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.04.060.
References
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