Synthesis and biological evaluation of 1-(isoxazol-5-ylmethylaminoethyl)-4- phenyl tetrahydropyridine and piperidine derivatives as potent T-type calcium channel blockers with antinociceptive effect in a neuropathic pain model

Article abstract of DOI:10.1016/j.ejmech.2013.12.056

New tetrahydropyridinyl and piperidinyl ethylamine derivatives were designed with hypothetical mapping on pharmacophore model generated from ligand-based virtual screening. The designed compounds were synthesized, and their inhibitory activities on T-type calcium channel were assayed using FDSS and patch-clamp assay. Among them, compounds 7b and 10b showed potent T-type calcium current blocking activity against Ca_v3.1 (α _1G) and Ca_v3.2 (α_1H) channel simultaneously. With hERG and pharmacokinetics studies, compounds 7b and 10b were evaluated for the antinociceptive effect on rat model of neuropathic pain. They were significantly effective in decreasing the pain responses to mechanical and cold allodynia induced by spinal nerve ligation. These results suggest that modulation of α_1G and α_1H subtype T-type calcium channels may provide a promising approach for the treatment of neuropathic pain.

Full text of DOI:10.1016/j.ejmech.2013.12.056

European Journal of Medicinal Chemistry 74 (2014) 246e257
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of 1-(isoxazol-5-
ylmethylaminoethyl)-4-phenyl tetrahydropyridine and piperidine
derivatives as potent T-type calcium channel blockers with
antinociceptive effect in a neuropathic pain model
,
,
Ju-Hyeon Lee a b, Seon Hee Seo a, Eun Jeong Lim a, Nam-Chul Cho a c, Ghilsoo Nam a,
Soon Bang Kang a, Ae Nim Pae a, Nakcheol Jeong b, Gyochang Keum a
,
*
a Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology (KIST), Hwarangro 14-gil 5, Seongbuk-gu, Seoul 136-791,
Republic of Korea
b Department of Chemistry, Korea University, Anam-ro 145, Seongbuk-gu, Seoul 136-701, Republic of Korea
c Department of Biotechnology, Yonsei University, 220, Seoul 120-749, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
New tetrahydropyridinyl and piperidinyl ethylamine derivatives were designed with hypothetical
mapping on pharmacophore model generated from ligand-based virtual screening. The designed com-
pounds were synthesized, and their inhibitory activities on T-type calcium channel were assayed using
FDSS and patch-clamp assay. Among them, compounds 7b and 10b showed potent T-type calcium
current blocking activity against Cav3.1 (a1G) and Cav3.2 (a1H) channel simultaneously. With hERG and
pharmacokinetics studies, compounds 7b and 10b were evaluated for the antinociceptive effect on rat
model of neuropathic pain. They were significantly effective in decreasing the pain responses to me-
chanical and cold allodynia induced by spinal nerve ligation. These results suggest that modulation of a1G
and a1H subtype T-type calcium channels may provide a promising approach for the treatment of
neuropathic pain.
Received 14 November 2013
Received in revised form
20 December 2013
Accepted 24 December 2013
Available online 8 January 2014
Keywords:
T-type calcium channel blocker
Neuropathic pain
Tetrahydropyridine
Piperidine
Ó 2014 Elsevier Masson SAS. All rights reserved.
Allodynia
1. Introduction
different biophysical functions, and inhibition of T-type calcium
channels have significant potential for the treatment of peripheral
Voltage-dependent calcium channels are transmembrane pro-
teins that regulate various intracellular signaling via rapid calcium
entry into cells in a voltage-dependent manner, and associated
with various physiological and pathological processes which
functions are neuronal excitability, neurotransmitter release, acti-
vation of intracellular signaling pathways, and regulation of gene
expression [1]. Voltage-dependent calcium channels are classified
into high voltage-activated (HVA) (L-, N-, P/Q- and R-types), and
low voltage-activated (LVA) channels (T-type) [1]. T-type calcium
channels are characterized by activation at low membrane poten-
tials, small conductance, and fast inactivation relative to HVA
channels. T-type calcium channels have been cloned to three iso-
forms Cav3.1 (a1G), Cav3.2 (a1H), and Cav3.3 (a1I) expressed in the
central and peripheral nerve tissues [2]. All the three isoforms have
and CNS disorders [3,4], such as absence epilepsy [5], schizophrenia
[6], tremor (Parkinson’s disease) [7], insomnia [8], cancer [9], and
neuropathic pain [10].
Neuropathic pain is a chronic pain results from a lesion or
dysfunction in the peripheral or central nervous system, and
characterized by enhanced responsiveness to noxious (hyper-
algesia) as well as nonnoxious stimuli (allodynia). Neuropathic pain
is estimated to afflict more than 6 million people worldwide, and
current treatments of neuropathic pain include antidepressants,
anticonvulsants, especially pregabalin and gabapentin, opioids and
topical anesthetics such as capsaicin and lidocaine. However
treatment failure is high in the patient group with existing thera-
peutics due to unpredictable effectiveness, complicated dosing,
delayed analgesic onset, and common side effects [11,12].
T-type calcium channels are involved in pain signaling with a
prominent role in primary afferent neurons in the dorsal root
ganglia (DRG). Several reports have revealed that T-type calcium
currents are linked to acute peripheral nociception and neuropathic
* Corresponding author. Tel.: þ82 2 958 5148; fax: þ82 2 958 5189.
E-mail address: gkeum@kist.re.kr (G. Keum).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.056

J.-H. Lee et al. / European Journal of Medicinal Chemistry 74 (2014) 246e257
247
pain in animal models [3]. Indeed, Cav3.2 T-type channel knockout
mice exhibited hyposensitivity to pain [13] as well as resistance to
chronic mechanical hyperalgesia [14]. However Cav3.2 knockout
mice did not show significantly attenuated neuropathic pain re-
sponses induced by spinal nerve ligation (SNL), which may reveal
that Cav3.2 channel is concerned in pain signals of the peripheral
processing [13]. On the other hand, antisense T-type channel
knockdown studies in rats DRG neurons provided evidence for the
involvement of Cav3.2 and Cav3.3 subtype T-type channels in
neuropathic pain [15,16]. Meanwhile, Cav3.1 KO mice experiment in
the SNL-induced neuropathic pain model showed that Cav3.1
channel is involved in the pathogenesis of neuropathic pain by
central sensitization in the spinal dorsal horn [17]. Moreover, a
study of Cav3.1 T-type channel gene knockout in mice thalamic
neurons demonstrated hyperalgesia to visceral pain [18].
Several T-type calcium channel inhibitors also exhibited efficacy
for the treatment of neuropathic pain [3,4,10]. Mibefradil, the first
selective T-type calcium channel blocker acting peripherally,
exhibited antihyperalgesic effects in the neuropathic pain rat
model with spinal nerve ligation [19]. Also mibefradil and antiep-
ileptic ethosuximide (Fig. 1), T-type channel blocker in sensory
neurons, suppressed nociception in rats [20,21] and mice [18].
Zonisamide, a newer antiepileptic drug that blocks T-type calcium
channels, is analgesic in the clinic [22] and displays anti-
hyperalgesic actions in rats with sciatic nerve injury [23]. Anti-
convulsants gabapentin and pregabalin, targeting a2d auxiliary
subunits of high-voltage-activated (HVA) calcium channels, are
current blockbuster drugs for the treatment of neuropathic pain
despite side effects, low effectiveness and late onset [24]. Recently,
TTA-P2 (Fig. 1), a potent and selective blocker of T-type calcium
channels in sensory neurons, showed potent antihyperalgesic ef-
fects in animal models of both inflammatory and neuropathic pain
[25]. These results emphasize the urgency and importance of
developing potent and selective T-type channel blocker as a
candidate drug for pain relief.
Fig. 2. Designed structures (7 and 10) of T-type calcium channel blocker and structural
mapping of ligand-based in silico 5-feature pharmacophore with 7a (dark blue) and 7b
(yellow). Cyan: hydrophobic, green: hydrogen bond acceptor, red: positive ionizable.
KST5468, among them, increased significantly the mechanical pain
threshold in neuropathic animal model [28].
Inspired by such compounds and other second generation T-
type calcium channel inhibitors [3,26,29], we designed new tet-
rahydropyridine 7 and piperidine 10 scaffolds substituted with
isoxazol-5-ylmethylaminoethyl group on the ring nitrogen to
discover new potent T-type calcium channel blockers for the
treatment of neuropathic pain (Fig. 2). In order to predict a
binding affinity of the designed compounds to T-type calcium
channel prior to synthesis them, we attempted to map them to the
3D ligand-based pharmacophore model generated by common
feature generation approach (HipHop) implemented in CATALYST
program [30].
Fig. 2 shows results of the mapping of designed 4-phenyl and
4-(3,4-dichloro)phenyl tetrahydropyridine compounds 7a and 7b
to the selected five feature hypothesis which was generated from
ligand-based virtual screening [30]. The compounds were ex-
pected to match well with three hydrophobic regions, single
hydrogen bonding acceptor and single positive ionizable region of
the pharmacophore model. Especially, 7a have one unoccupied
hydrophobic region, however 7b occupied all the three hydro-
phobic regions by positioning phenyl ring and its meta-substituent
at two hydrophobic regions respectively with an appropriate
conformational change. The corresponding piperidine analogs 10a
and 10b showed similar mapping pattern to their tetrahydropyr-
idine analogs 7.
Herein, we described the design and synthesis of novel series of
tetrahydropyridine and piperidine derivatives, and evaluation of
their Cav3.1 (a1G) and Cav3.2 (a1H) T-type calcium channel blocking
activities in both fluorescence-based HTS (high throughput
screening) FDSS6000 assay and whole-cell patch clamp recordings.
Selected compounds from in vitro T-type channel blocking assay
were evaluated their analgesic efficacies in a rat model of neuro-
pathic pain.
2.2. Chemistry
The synthesis of tetrahydropyridine derivatives 7aef is shown
in Scheme 1. The key intermediates 4aef, 4-phenyl tetrahy-
dropyridine derivatives, were synthesized from 1-Boc-4-
2. Result and discussion
2.1. Design
piperidone 1 and substituted bromobenzenes via different
methods depend on bromobenzene parts. Addition of lithiated
benzene to piperidone 1 and subsequent dehydration was used
generally starting from the corresponding substituted bromo-
benzenes 3. Especially, the reported two-step reaction was modi-
fied to a convenient one-pot process [31]. In case of 3,4-dichloro
and 4-chloro bromobenzene (3b and 3f), Suzuki and Stille coupling
As revealed from literature survey, some compounds having
piperidine moiety, such as haloperidol, penfluridol, pimozide, flu-
nazirine, and TTA-P2, were well known as T-type calcium channel
inhibitors [3,26,27]. Piperazinylalkylisoxzole derivatives were re-
ported to have good T-type calcium channel inhibitory activity and
Fig. 1. Representative T-type calcium channel blockers which have antinociceptive effect on animal pain models.

248
J.-H. Lee et al. / European Journal of Medicinal Chemistry 74 (2014) 246e257
dichlorophenyl derivatives of 7b and 10b showed good blocking
activity in 55e69% inhibition range. Most of the compounds in this
series showed similar activity against both a1G and a1H T-type
calcium channels in the FDSS assay. For examination of IC50 values
of the synthesized compounds, whole-cell patch-clamp assay was
performed [33]. It was observed that T-type calcium channel
blocking activities obtained by patch-clamp method are well
correlated with those obtained by FDSS assay. Moreover, IC50 values
obtained by patch-clamp methods are lower than those inferred
from % inhibition data by FDSS assay as shown Table 1.
Among the tested compounds, tetrahydropyridine 7b and
piperidine 10b, having the 3,4-dichloro group on the terminal
phenyl ring, displayed most potent activity against both a1G and a1H
T-type calcium channels with IC50 values of 0.80, 1.37
mM and 1.47,
1.36 M, respectively. The results are consistent with the postula-
m
tion from the design that meta-substitution on phenyl ring will
increase the activity by occupying the two hydrophobic pockets.
Furthermore, 7b showed similar IC50 to mibefradil against a1G
Scheme 1. Reagent and reaction conditions: i) 3, nBuLi, THF, ꢀ78 ꢁC; MsCl, TEA, ꢀ78 ꢁ
C
to rt, 71% (4a), 49% (4c), 45% (4d), 56% (4e); ii) nBuLi, diisopropylamine, PhN(SO2CF3)2,
THF, ꢀ78 ꢁC to 0 ꢁC, 82%; iii) Bis(pinacolato)diboron, KOAc, PdCl2(dppf)2, dppf, 1,4-
dioxane, 80 ꢁC, 50%; iv) 1-bromo-3,4-dichlorobenzene (3b), Pd(PPh3)4, K3PO4, 1,4-
subtype with the value of 0.80 and 0.86 mM, respectively. However,
dioxane, H2O, 85 ꢁC, 72% (4b); v) nBuLi, iPr2NH, Bu3SnH, MsCl, TEA, THF,
0
ꢁC
tetrahydropyridine 7a, 7d, and 7e and piperidine 10a derivatives
possessing unsubstituted or electron withdrawing nitrile group on
the phenyl ring exhibited lower activities against both T-type cal-
cium channels (entry 1, 4, 5, 7). Tetrahydropyridine and piperidine
derivatives showed comparable blocking activities with poor sub-
type selectivity against the both T-type calcium channels.
The most active compounds 7b and 10b were also measured
hERG channel inhibition activity by the same patch-clamp methods
to expect cardiac side effects, and showed high hERG inhibition
to ꢀ78 ꢁC to rt, 45%; vi) 1-bromo-4-chlorobenzene (3f), Pd(PPh3)4, toluene, reflux,
100% (4f); vii) TFA, DCM, 0 ꢁC to rt, 91e100%; viii) N-(2-bromoethyl)phthalimide,
K2CO3, DMF, rt to 80 ꢁC, 19e85%; ix) N2H4eH2O (80%), EtOH, 70 ꢁC, 61e96%; x) 6,
ꢀ
NaBH(OAc)3, 4 A MS, DCM, rt, 25e77%.
reactions gave good results respectively by improving yields and
purities significantly.
Boc-deprotection of 4 followed by Gabriel reaction gave the
corresponding primary ethylamine 5. Then, N-tetrahydropyridinyl
ethylamine derivatives 7aef were generated through reductive
amination of 5-isoxazolecarboxaldehyde 6 [28] with sodium tri-
activities with the IC50 values of 0.21, 1.18 mM respectively (Table 1).
For comparison, the inhibitory activity of mibefradil was also
examined against those channels and found that it inhibited the a1G
and a1H T-type calcium channels and hERG channel with IC50 values
ꢀ
acetoxyborohydride in the presence of 4 A molecular sieves.
Piperidine derivatives 10a and 10b were prepared from the
corresponding tetrahydropyridine intermediates 4a and 4b as
illustrated in Scheme 2. It was found that complete hydrogenation
of double bonds in both tetrahydropyridine 7a and 7b was not
successful in mild hydrogenation conditions with Pd/C, PtO2, or
Wilkinson’s catalyst. Hence, piperidine derivatives 10a and 10b
were synthesized by hydrogenation of 4a and 4b respectively to
provide 8a and 8b, and followed by the same procedure for the
synthesis of 7 from 4.
of 0.86, 0.28 and 1.40 mM, respectively.
2.3.2. Stability and PK profile
In order to predict in vivo pharmacokinetic properties, in vitro
human hepatic microsomal stability and rat plasma stability were
measured for the most active compounds 7b and 10b [34]. The
results indicated that compounds 7b and 10b showed quite low
microsomal stability with 11.6% and 25.5% remaining concentration
respectively, analyzed after 30 min exposure, while 56.7% of initial
mibefradil was remained. However, it was found that 86.3% and
100% concentration of each compound maintained in rat plasma at
120 min with sufficiently high stability. Anticipating low micro-
somal stability and high plasma stability, the compounds were
subjected to pharmacokinetic studies in male rats. The pharmaco-
kinetic profile of 7b and 10b after intravenous and oral adminis-
tration (10 mg/kg) is shown in Table 2. The AUC0eN values of 7b are
204.2 and 97.6 mg min/ml in intravenous and oral administration,
respectively. Also 7b exhibited slow clearance, and a good oral
bioavailability of 47.8%.
In general, tetrahydropyridine 7b showed good pharmacoki-
netic profile regarding the absorption, clearance and bioavailability.
It is inferred that both compounds are metabolically stable in rat
liver enzymes (T1/2 ¼ 80.3 and 78.7 min). Meanwhile, piperidine
10b showed inferior PK profile to 7b except terminal half-life, and
increasing dose would be considered for the in vivo study of 7b on
animal model.
2.3. Biological activity
2.3.1. In vitro T-type calcium channel and hERG channel activity
The in vitro T-type calcium channel blocking activity of the
synthesized compounds was evaluated against Cav3.1 (a1G) or
Cav3.2 (a1H) T-type calcium channels exogenously expressed in
HEK293 cells, and the results are summarized in Table 1. The ac-
tivities were measured as % inhibitions of calcium currents at 10 mM
concentration of the compounds through fluorescence-based HTS
FDSS6000 assay [32]. Among the tested compounds, 3,4-
2.3.3. In vivo antinociceptive effects in the rat neuropathic pain
model
In attempt to identify antinociceptive effect on neuropathic pain
and the relationship between antinociceptive effect and T-type
calcium channel blocking activity, compounds 7b and 10b were
further evaluated for the mechanical and cold allodynia in rats
Scheme 2. Reagent and reaction conditions: i) Pd/C, H2, MeOH, rt, 86% (a), 89% (b); ii)
TFA, DCM, 0 ꢁC to rt, 100% (8a), 91% (8b); iii) N-(2-bromoethyl)phthalimide, NaI,
Na2CO3, DMF, 80 ꢁC, 47% (a), 45% (b); iv) N2H4eH2O (80%), EtOH, 70 ꢁC, 99% (9a), 80%
ꢀ
(9b); v) 6, NaHB(OAc)3, 4 A MS, DCM, rt, 37% (10a), 77% (10b).

J.-H. Lee et al. / European Journal of Medicinal Chemistry 74 (2014) 246e257
249
Table 1
In vitro activities of the synthesized compounds 7 and 10 against a1G (Cav3.1) and a1H (Cav3.2) T-type calcium channel and hERG channel.
En.
Compd.
R1
FDSS % inhibitiona
Patch-clamp IC50 (m
M)b
a1G 10
m
M
a1H 10
mM
T-type calcium channel
hERG channel
a1G
a1H
1
2
3
4
5
6
7
8
9
7a
7b
7c
7d
7e
7f
10a
10b
H
31.25
69.27
37.81
26.46
28.46
44.00
38.11
54.76
79.19
25.62
68.89
41.02
23.05
19.18
34.63
41.89
54.72
76.00
12.51 ꢂ 2.29
0.80 ꢂ 0.05
3.68 ꢂ 0.51
8.07 ꢂ 0.45
10.89 ꢂ 0.58
2.17 ꢂ 0.15
13.40 ꢂ 1.44
1.47 ꢂ 14
9.54 ꢂ 2.84
1.37 ꢂ 0.17
5.88 ꢂ 0.14
11.8 ꢂ 1.52
11.15 ꢂ 2.55
3.44 ꢂ 0.17
ed
2.00 ꢂ 0.10
0.21 ꢂ 0.08
0.78 ꢂ 0.06
ec
3,4-Cl2
4-CH3O
2-CN
3-CN
4-Cl
ec
ec
H
1.08 ꢂ 0.40
1.18 ꢂ 0.17
1.40 ꢂ 0.29
3,4-Cl2
1.36 ꢂ 47
0.28 ꢂ 0.01
Mibefradil
0.86 ꢂ 0.14
a
% inhibition value was obtained using FDSS6000 fluorescence-based HTS assay on HEK293 cell.
IC50 value (ꢂSD) was obtained from a doseeresponse curve.
Not determined.
Maximum % inhibition was 85 ꢂ 1.65 at 100
b
c
d
mM.
spinal nerve ligation model (SNL, Chung’s model) [35] of peripheral
nerve injury after oral administration (100 mg/kg). Gabapentin,
used primarily to treat neuropathic pain clinically, and the known
T-type current inhibitor mibefradil (100 mg/kg) were tested for
comparison. The results were shown in Fig. 3, and those of other
compounds in Table 1 were described in Supplementary material.
Two weeks after the nerve surgery, the rats exhibited significant
decrease of paw withdrawal threshold to mechanical stimuli and
increase of paw withdrawal frequency in response to cold stimuli
confirming induction of neuropathic pain. Then, test compounds
were administrated orally, and the mechanical threshold was
assessed by von Frey paw withdrawal threshold. Compounds 7b
and 10b were found to significantly reduce mechanical hypersen-
sitivity in SNL model. Compound 7b showed less potent relieving
effect for the mechanical allodynia than gabapentin, however
exhibited more potent effect than mibefradil after 3 h of treatment.
Compound 10b also displayed some increasing the pain threshold
in mechanical allodynia test. However mechanical threshold was
decreased after 3 h for 7b (P ¼ 0.05 vs. pretreatment value, one-way
repeated measured ANOVA t-test) and 1 h for 10b, which may be
caused by pharmacokinetic differences.
These results are consistent with the previous reports that
Cav3.1 knockout mouse and antisense silencing of Cav3.2 showed
reduced pain response to mechanical allodynia respectively [14e
18]. However the results are controversial that Cav3.2 knockout
mouse showed decreased pain response in peripheral pain, and did
not showed neuropathic pain response in SNL model [13]. There-
fore the high antinociceptive effect of compounds 7b and 10b in
neuropathic model can be explained by their inhibition of both
Cav3.1 and Cav3.2 T-type calcium channels. With additional in vivo
evaluation of 7a, 7c, and 7f (see Supplementary material), the pain-
relieving effects of tetrahydropyridine and piperidine compounds
were found to be well correlated with T-type calcium channel
blocking activity.
3. Conclusion
A series of novel 1-(isoxazol-5-ylmethylaminoethyl)-4-phenyl
tetrahydropyridine and piperidine derivatives were designed by
mapping to ligand-based in silico 5-feature pharmacophore model,
synthesized and evaluated their T-type calcium channel blocking
activity utilizing FDSS and patch-clamp assay. Among the tested
compounds, compounds 7b and 10b showed good inhibitory ac-
tivity against Cav3.1 and Cav3.2 T-type calcium channel simulta-
neously. In addition, compounds 7b and 10b exhibited significant
antinociceptive effects in mechanical and cold allodynia induced by
spinal nerve ligation as a neuropathic pain rat model. These results
revealed the importance of a1G and a1H T-type calcium channels in
the process of neuropathic pain, and suggest that modulation of a1G
and a1H subtype channels may provide a promising approach for
the treatment of neuropathic pain.
In case of cold allodynia, the analgesic effect of 7b was higher
than the effect of gabapentin up to 3 h after administration
(P ¼ 0.02 vs. pretreatment value, one-way repeated measured
ANOVA t-test), then decreased to exhibit similar activity to gaba-
pentin after 5 h. Compound 10b also displayed attenuated pain
response to the rat SNL neuropathic pain model, and showed
similar effect to gabapentin and higher effect than mibefradil.
Table 2
Pharmacokinetic parameters after intravenous (n ¼ 5) and oral (n ¼ 5) adminis-
tration (10 mg/kg) of 7b and 10b to male rats.
4. Experimental protocols
7b
10b
iv
Oral
iv
Oral
4.1. General
AUC0eN
g min/ml)
AUClast
g min/ml)
Terminal
half-life (min)
Cmax (ng/ml)
Tmax (min)
CL (ml/min/kg)
F (%)
204.2 ꢂ 1.7
97.6 ꢂ 52.8
64.7 ꢂ 22.1
7.7 ꢂ 3.8
1H and 13C NMR spectra were recorded on a Bruker Avance 300
or 400 MHz spectrometer, using CDCl3 as a NMR solvent. TMS was
used as the internal standard and chemical shifts (d) are reported in
parts per million (ppm) and s, d, t, and m are designated as singlet,
doublet, triplet and multiplet respectively. Coupling constants (J)
are reported in hertz (Hz). Mass spectra were recorded on Waters
Acquity UPLC/Synapt G2 QTOF MS mass spectrometer. The reaction
progress was monitored on TLC plate (Merck, silica gel 60 F254), and
column chromatography was performed on silica gel (Merck, 230e
400 mesh). All solvents and reagents were commercially available
and used without further purification.
(
m
199.1 ꢂ 0.4
80.3 ꢂ 1.4
81.8 ꢂ 40.1
63.9 ꢂ 22.6
78.7 ꢂ 15.9
6.9 ꢂ 2.8
(
m
164.2 ꢂ 21.1
98.1 ꢂ 39.2
e
420 ꢂ 270
37.5 ꢂ 31.8
e
e
70 ꢂ 50
21 (15e30)
e
e
e
49.0 ꢂ 0.4
168.8 ꢂ 55.1
47.8
11.9
Values are presented as mean ꢂ SD. SD, standard deviation; AUC (area under the
plasma concentration versus time curve) ¼ dose/clearance; Cmax, peak plasma
concentration; Tmax, time to reach Cmax; CL, time-averaged body clearance; F,
bioavailability.

250
J.-H. Lee et al. / European Journal of Medicinal Chemistry 74 (2014) 246e257
Fig. 3. Effect on mechanical allodynia (A and B) and cold allodynia (C and D) after oral administration of gabapentin (C, 100 mg/kg, n ¼ 4), mibefradil (100 mg/kg, n ¼ 4), 7b
(100 mg/kg, n ¼ 4) and 10b (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 7b or 10b administration, *P < 0.05 gabapentin, #P < 0.05 7b or 10b vs. pre-treatment value (N14D) (one-way repeated measure ANOVA t-test), ) P < 0.05
gabapentin vs. 10b (unpaired t-test).
4.2. Synthesis
4.2.3. 2-(2-(4-Phenyl-5,6-dihydropyridin-1(2H)-yl)ethyl)
isoindoline-1,3-dione
4.2.1. tert-Butyl 4-phenyl-5,6-dihydropyridine-1(2H)-carboxylate
(4a)
To a solution of above 4-phenyl-1,2,3,6-tetrahydropyridine
(2.1 g, 13.09 mmol) in DMF (15 mL) were added N-(2-bromoethyl)
phthalimide (3.3 g, 13.09 mmol) and potassium carbonate (5.4 g,
39.28 mmol). This reaction solution was stirred at 80 ꢁC for 4 h, and
then was washed with sodium bicarbonate solution, and extracted
with hexane/ethyl acetate (2/1, v/v). The organic layer was washed
with brine, dried (Na2SO4), and filtered. The solvent was evaporated
under reduced pressure, and the residue was purified by column
chromatography with hexane/ethyl acetate (5/1, v/v) to obtain the
title compound (1.8 g, 40% yield) as a white solid: 1H NMR
To a solution of substituted bromobenzene (1.1 mL, 10.0 mmol)
in THF (90 mL) was dropwised n-butylithium solution in hexane
(4.0 mL, 2.5 M in hexane,10.0 mmol) at ꢀ78 ꢁC. After 30 min,1-Boc-
4-piperidone (2.0 g, 10.0 mmol) in THF (8.6 mL) was slowly added
to the reaction mixture. After 1 h, the mixture was stirred at room
temperature for 1 h, and then methanesulfonyl chloride (3.2 mL,
40.2 mmol) and triethylamine (11.2 mL, 80.3 mmol) were added
at ꢀ78 ꢁC. The mixture was stirred at room temperature for 15 h,
and was extracted with ethyl acetate, washed with brine, dried
(MgSO4), and filtered. The solvent was evaporated under reduced
pressure, and the residue was purified by column chromatography
with hexane/ethyl acetate (20/1, v/v) to obtain the compound 4a
(400 MHz, CDCl3)
d
7.83 (dd, J ¼ 5.3, 3.0 Hz, 2H), 7.69 (dd, J ¼ 5.4,
3.0 Hz, 2H), 7.37 (d, J ¼ 7.5 Hz, 2H), 7.35e7.27 (m, 2H), 7.22 (t,
J ¼ 7.2 Hz, 1H), 6.05 (bs, 1H), 3.91 (t, J ¼ 6.7 Hz, 2H), 3.25 (d,
J ¼ 2.8 Hz, 2H), 2.83e2.77 (m, 2H þ 2H), 2.55 (bs, 2H); 13C NMR
(1.85 g, 71% yield): 1H NMR (300 MHz, CDCl3)
d
7.40e7.29 (m, 4H),
(100 MHz, CDCl3) d 168.4, 140.7, 134.9, 133.9, 132.2, 128.3, 127.0,
7.27 (d, J ¼ 3.0 Hz, 1H), 6.04 (bs, 1H), 4.09 (bs, 2H), 3.65 (t, J ¼ 5.5 Hz,
124.8, 123.2, 121.6, 55.4, 53.4, 49.9, 35.5, 27.9.
2H), 2.54 (bs, 2H), 1.52 (s, 9H).
4.2.2. 4-Phenyl-1,2,3,6-tetrahydropyridine
4.2.4. 2-(4-Phenyl-5,6-dihydropyridin-1(2H)-yl)ethanamine (5a)
To a solution of above phthalimide compound (1.8 g, 5.30 mmol)
in ethanol (20 mL) was added hydrazine hydrate 80% solution
(1.0 mL, 15.88 mmol). This reaction solution was stirred at 70 ꢁC for
2 h, and then was extracted with dichloromethane. The organic
layer was washed with brine, dried (Na2SO4), and filtered. The
solvent was evaporated under reduced pressure to give compound
To a solution of compound 4a (1.8 g, 7.12 mmol) in dichloro-
methane (36 mL) was slowly added trifluoroacetic acid (18.5 mL,
242.01 mmol) at 0 ꢁC. The reaction solution was stirred at room
temperature for 5 h, and then 1 N NaOH was added. The mixture
was extracted with dichloromethane, and the organic layer was
washed with brine, dried (Na2SO4), and filtered. The solvent was
evaporated under reduced pressure to give a crude product
5a (683 mg, 64% yield): 1H NMR (400 MHz, CDCl3)
d 7.38 (d,
(423 mg, 99% yield): 1H NMR (300 MHz, CDCl3)
d
7.41e7.31 (m, 4H),
J ¼ 7.1 Hz, 2H), 7.34e7.28 (m, 2H), 7.27e7.20 (m, 1H), 6.06 (bs, 1H),
7.25 (d, J ¼ 2.6 Hz, 1H), 6.1 (bs, 1H), 3.55 (d, J ¼ 2.2 Hz, 2H), 3.12 (t,
3.16 (d, J ¼ 2.6 Hz, 2H), 2.86 (t, J ¼ 6.0 Hz, 2H), 2.71 (t, J ¼ 5.6 Hz, 2H),
J ¼ 5.6 Hz, 2H), 2.68 (bs, 1H), 2.48 (bs, 2H); 13C NMR (75 MHz,
2.61e2.52 (m, 4H), 1.82 (bs, 2H); 13C NMR (100 MHz, CDCl3)
d 140.8,
CDCl3)
d
141.3, 135.3, 128.3, 127.1, 124.8, 123.1, 45.4, 43.2, 27.6.
135.0, 128.3, 127.0, 124.9, 121.8, 61.0, 53.4, 50.5, 39.2, 28.1.

J.-H. Lee et al. / European Journal of Medicinal Chemistry 74 (2014) 246e257
251
4.2.5. N-((3-Isopropylisoxazol-5-yl)methyl)-2-(4-phenyl-5,6-
dihydropyridin-1(2H)-yl)ethanamine (7a)
A solution of 3-isopropylisoxazole-5-carbaldehyde 6 (470 mg,
3.38 mmol) in dichloromethane (5 mL) was added to a solution of
72% yield): 1H NMR (300 MHz, CDCl3)
d
7.45 (d, J ¼ 1.9 Hz, 1H), 7.40
(d, J ¼ 8.4 Hz, 1H), 7.49 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.06 (bs, 1H), 4.08 (d,
J ¼ 2.4 Hz, 1H), 3.64 (t, J ¼ 5.6 Hz, 2H), 2.48 (bs, 2H), 1.50 (s, 9H); 13
C
NMR (75 Hz, CDCl3)
d 154.8, 140.7, 132.6, 131.0, 130.3, 129.5, 126.9,
ꢀ
amine 5a (683 mg, 3.38 mmol) in DCM (81 mL), and then 4 A
124.2, 123.6, 80.0, 28.5, 27.2.
molecular sieve (68 beads) was added. After 2 h, sodium tri-
acetoxyborohydride (1.1 g, 5.07 mmol) was added to the reaction
mixture. This mixture was stirred at room temperature for 2 h, and
then 1 N NaOH was added. The mixture was extracted with
dichloromethane, and the organic layer was washed with brine,
dried (Na2SO4), and filtered. The solvent was evaporated under
reduced pressure, and the residue was purified by column chro-
matography with dichloromethane/methanol (50/1, v/v) to obtain
4.2.9. 4-(3,4-Dichlorophenyl)-1,2,3,6-tetrahydropyridine
To a solution of compound 4b (2.05 g, 6.25 mmol) in DCM
(31 mL) was slowly added trifluoroacetic acid (16.27 mL,
212.48 mmol) at 0 ꢁC. The reaction solution was stirred at room
temperature for 5 h, and then 1 N NaOH was added. The reaction
mixture was extracted with dichloromethane, and the organic layer
was washed with brine, dried (Na2SO4), and filtered. The solvent
was evaporated under reduced pressure to give a crude title com-
compound 7a (425 mg, 39% yield): 1H NMR (300 MHz, CDCl3)
d 7.24
(d, J ¼ 6.9 Hz, 2H), 7.15 (t, J ¼ 7.2 Hz, 2H), 7.07 (t, J ¼ 6.7 Hz, 1H), 5.90
(bs, 2H), 3.73 (s, 2H), 2.99 (bs, 2H), 2.93e2.84 (m, 1H), 2.64 (t,
J ¼ 5.4 Hz, 2H), 2.60 (bs, 1H), 2.53 (t, J ¼ 5.2 Hz, 2H), 2.46 (t,
J ¼ 5.6 Hz, 2H), 2.40 (bs, 2H), 1.13 (d, J ¼ 6.9 Hz, 6H); 13C NMR
pound (1.30 g, 91% yield). 1H NMR (300 MHz, CDCl3)
d 7.32 (s, 1H),
7.25 (d, J ¼ 8.4 Hz, 1H), 7.08 (dd, J ¼ 6.6, 1.8 Hz, 1H), 6.04 (bs, 1H),
3.42 (s, 2H), 2.98 (t, J ¼ 4.6 Hz, 2H), 2.27 (s, 2H), 1.99 (bs, 1H); 13C
NMR (75 Hz, CDCl3)
d 141.2, 133.2, 132.3, 130.5, 130.0, 126.6, 125.2,
(75 MHz, CDCl3)
d
171.2, 169.0, 140.5, 134.7, 128.2, 126.9, 124.7, 121.5,
124.0, 45.4, 43.0, 28.9, 27.4.
99.7, 57.3, 53.2, 50.2, 45.9, 44.9, 27.8, 26.4, 21.7; HRMS (ESI-TOF) m/z
calcd for C20H28N3Oþ [M þ Hþ] 326.2227, found 326.2237.
4.2.10. 2-(2-(4-(3,4-Dichlorophenyl)-5,6-dihydropyridin-1(2H)-yl)
ethyl)isoindoline-1,3-dione
4.2.6. tert-Butyl 4-(trifluoromethylsulfonyloxy)-5,6-
To
a
solution of above 4-(3,4-dichlorophenyl)-1,2,3,6-
dihydropyridine-1(2H)-carboxylate
tetrahydropyridine (1.30 g, 5.68 mmol) in DMF (17 mL) were
added N-(2-bromoethyl)phthalimide (1.44 g, 5.68 mmol), sodium
iodide (170 mg, 1.14 mmol), and sodium carbonate (602 mg,
5.68 mmol). This reaction solution was stirred at 90 ꢁC for 5 h, and
then was washed with sodium bicarbonate solution, extracted with
hexane/ethyl acetate (2/1, v/v). The organic layer was washed with
brine, dried (Na2SO4), and filtered. The solvent was evaporated
under reduced pressure, and the residue was purified by column
chromatography with hexane/ethyl acetate (5/1, v/v) to obtain the
title compound (768 mg, 34% yield) as a white solid: 1H NMR
To a solution of diisopropylamine (0.8 mL, 5.52 mmol) in THF
(7 mL) was dropwised n-butyllithium solution in hexane (3.5 mL,
2.5 M in hexane, 5.52 mmol) at ꢀ78 ꢁC. After 15 min, 1-Boc-4-
piperidone (1.0 g, 5.02 mmol) in THF (7 mL) was slowly added to
the reaction mixture at ꢀ78 ꢁC. After 20 min, a solution of 1,1,1-
trifluoro-N-phenyl-N-(trifluoromethylsulfonyl)meth-
anesulfonamide (2.0 g, 5.52 mmol) was slowly added to the
mixture. The reaction mixture was stirred at 0 ꢁC for 3 h. The sol-
vent was evaporated under reduced pressure, and the residue was
purified by column chromatography with hexane/ethyl acetate (20/
1, v/v) to obtain the title compound (1.4 g, 82% yield): 1H NMR
(300 MHz, CDCl3)
d 7.87e7.81 (m, 2H), 7.73e7.68 (m, 2H), 7.43 (d,
J ¼ 2.1 Hz, 1H), 7.35 (d, J ¼ 8.4 Hz, 1H), 7.19 (dd, J ¼ 8.4, 2.1 Hz, 1H),
(300 MHz, CDCl3)
J ¼ 5.7 Hz, 2H), 2.47e2.44 (m, 2H), 1.48 (s, 9H).
d
5.77 (bs, 1H), 4.05 (d, J ¼ 2.9 Hz, 2H), 3.64 (t,
6.07 (bs, 1H), 3.90 (t, J ¼ 6.6 Hz, 2H), 3.23 (d, J ¼ 2.6 Hz, 2H), 2.82e
2.75 (m, 4H), 2.47 (bs, 2H); 13C NMR (75 MHz, CDCl3)
d 168.4, 140.7,
133.9, 133.0, 132.3, 132.2, 130.6, 130.1, 126.8, 124.1, 123.4, 123.2, 55.3,
53.2, 49.6, 35.4, 27.8.
4.2.7. tert-Butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
5,6-dihydropyridine-1(2H)-carboxylate (2b)
The suspension of above triflate compound (500 mg,
1.51 mmol), bis(pinacolato)diboron (425 mg, 1.68 mmol), potas-
sium acetate (447 mg, 4.56 mmol), 1,10-bis(diphenylphosphino)
ferrocene (42 mg, 0.075 mmol), and [1,10-bis(diphenylphosphino)
ferrocene]dichloropalladium (II) complex in dichloromethane
(62 mg, 0.075 mmol) were stirred in 1,4-dioxane (10 mL) at 80 ꢁC
for 17 h. The reaction mixture was extracted with ethyl acetate, and
the organic layer was washed with brine, dried (Na2SO4), and
filtered. The solvent was evaporated under reduced pressure, and
the residue was purified by column chromatography with hexane/
ethyl acetate (9/1, v/v) to obtain the compound 2b (233 mg, 50%
4.2.11. 2-(4-(3,4-Dichlorophenyl)-5,6-dihydropyridin-1(2H)-yl)
ethanamine (5b)
To a solution of above phthalimide compound (768 mg,
1.91 mmol) in ethanol (7 mL) was added hydrazine hydrate 80%
solution (0.40 mL, 5.74 mmol). This reaction solution was stirred at
70 ꢁC for 2 h, and then was extracted with dichloromethane. The
organic layer was washed with brine, dried (Na2SO4), and filtered.
The solvent was evaporated under reduced pressure to give the
compound 5b (424 mg, 82% yield): 1H NMR (300 MHz, CDCl3)
d 7.38
(d, J ¼ 1.5 Hz,1H), 7.29 (d, J ¼ 8.5 Hz,1H), 7.14 (dd, J ¼ 8.4,1.5 Hz,1H),
6.02 (bs, 1H), 3.10 (d, J ¼ 1.7 Hz, 2H), 2.80 (t, J ¼ 6.1 Hz, 2H), 2.64 (t,
yield): 1H NMR (300 MHz, CDCl3)
d
6.37 (bs, 1H), 3.86 (bs, 2H), 3.34
J ¼ 5.5 Hz, 2H), 2.48 (t, J ¼ 6.3 Hz, 2H), 2.43 (bs, 2H) 1.73 (bs, 2H); 13
C
(t, J ¼ 5.2 Hz), 2.13 (bs, 2H), 1.37 (s, 9H), 1.17 (s, 10H).
NMR (75 MHz, CDCl3) d 140.7, 133.0, 132.3, 131.1, 130.6, 130.1, 126.7,
124.1, 123.6, 60.8, 53.2, 50.1, 39.1, 29.7, 27.8.
4.2.8. tert-Butyl 4-(3,4-dichlorophenyl)-5,6-dihydropyridine-
1(2H)-carboxylate (4b)
4.2.12. 2-(4-(3,4-Dichlorophenyl)-5,6-dihydropyridin-1(2H)-yl)-N-
The suspension of tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-
((3-isopropylisoxazol-5yl)methyl) ethanamine (7b)
dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate
2b
A solution of 3-isopropylisoxazole-5-carbaldehyde 6 (281 mg,
2.02 mmol) in dichloromethane (3.54 mL) was added to a solution
of amine 5b (549 mg, 2.02 mmol) in dichloromethane (49 mL), and
(2.68 g, 8.72 mmol), 4-bromoanisole (1.19 mL, 9.53 mmol), potas-
sium phosphate tribasic (2.76 g, 13.0 mmol), tetrakis(-
triphenylphosphine)palladium(0) (362 mg, 0.31 mmol), and H2O
(18 mL) were stirred in 1,4-dioxane (70 mL) at 85 ꢁC for 18 h. The
reaction mixture solvent was evaporated under reduced pressure,
and the residue was purified by column chromatography with
hexane/ethyl acetate (6/1, v/v) to obtain the compound 4b (2.05 g,
ꢀ
then 4 A molecular sieve (42 beads) was added. After 2 h, sodium
triacetoxyborohydride (643 mg, 3.03 mmol) was added to a reac-
tion mixture. This mixture was stirred at room temperature for 2 h,
and then 1 N NaOH was added. The reaction mixture was extracted
with dichloromethane. The organic layer was washed with brine,

252
J.-H. Lee et al. / European Journal of Medicinal Chemistry 74 (2014) 246e257
dried (Na2SO4), and filtered. The solvent was evaporated under
reduced pressure, and the residue was purified by column chro-
matography with dichloromethane/methanol (50/1, v/v) to obtain
d 158.7, 134.3, 133.4, 125.9, 120.1, 113.6, 61.0, 55.2, 53.4, 50.5, 39.2,
28.1.
compound 7b (572 mg, 77% yield): 1H NMR (300 MHz, CDCl3)
d
7.31
4.2.17. N-((3-Isopropylisoxazol-5-yl)methyl)-2-(4-(4-
(d, J ¼ 1.6 Hz, 1H), 7.22 (d, J ¼ 8.4 Hz, 1H), 7.07 (dd, J ¼ 8.5, 1.6 Hz,
1H), 5.94 (bs, 1H), 3.78 (s, 2H), 3.02 (bs, 2H), 2.96e2.87 (m, 1H), 2.68
(t, J ¼ 5.7 Hz, 2H), 2.57e2.48 (m, 4H), 2.35 (bs, 2H), 2.26 (bs,1H),1.15
methoxyphenyl)-5,6-dihydropyridin-1(2H)-yl)ethanamine (7c)
A solution of 3-isopropylisoxazole-5-carbaldehyde 6 (411 mg,
2.95 mmol) in dichloromethane (12 mL) was added to a solution of
(d, J ¼ 6.9 Hz, 6H); 13C NMR (75 MHz, CDCl3)
d
171.1, 169.1, 140.6,
5c (686 mg, 2.95 mmol) in dichloromethane (71 mL), and then 4 A
ꢀ
132.8, 132.2, 130.4, 130.0, 126.6, 124.1, 123.5, 99.7, 57.2, 53.0, 50.0,
molecular sieve powder (1.6 g) was added. After 2 h, sodium tri-
acetoxyborohydride (938 mg, 4.43 mmol) was added to a reaction
mixture. This mixture was stirred at room temperature for 2 h, and
then 1 N NaOH was added. The mixture was extracted with
dichloromethane, and the organic layer was washed with brine,
dried (Na2SO4), and filtered. The solvent was evaporated under
reduced pressure, and the residue was purified by column chro-
matography with dichloromethane/methanol (50/1, v/v) to give the
46.0, 45.0, 27.7, 26.4, 21.7; HRMS (ESI-TOF) m/z calcd for
C
20H26Cl2N3Oþ [M þ Hþ] 394.1447, found 394.1450.
4.2.13. tert-Butyl 4-(4-methoxyphenyl)-5,6-dihydropyridine-
1(2H)-carboxylate (4c)
The compound 4c (658 mg, 49% yield) was obtained from 1-
bromo-4-methoxybenzene (1.3 mL, 10.04 mmol) according to the
same procedure for the compound 4a: 1H NMR (300 MHz, CDCl3)
compound 7c (369 mg, 35% yield): 1H NMR (300 MHz, CDCl3)
d 7.31
d
7.28 (d, J ¼ 8.7 Hz, 2H), 6.83 (d, J ¼ 8.8 Hz, 2H), 5.90 (bs, 1H), 4.04
(d, J ¼ 8.8 Hz, 2H), 6.84 (d, J ¼ 8.9 Hz, 2H), 6.03 (s, 1H), 5.95 (bs, 1H),
3.90 (s, 2H), 3.78 (s, 3H), 3.12 (d, J ¼ 3.1 Hz, 2H), 3.08e2.99 (m, 1H),
2.80 (t, J ¼ 6.0 Hz, 2H), 2.68 (t, J ¼ 5.6 Hz, 2H), 2.61 (t, J ¼ 6.0 Hz, 2H),
2.52 (bs, 2H), 2.00 (bs, 1H), 1.26 (d, J ¼ 7.0 Hz, 6H); 13C NMR
(bs, 2H), 3.76 (s, 3H), 3.60 (bs, 2H), 2.46 (bs, 2H), 1.48 (s, 9H).
4.2.14. 4-(4-Methoxyphenyl)-1,2,3,6-tetrahydropyridine
To a solution of 4c (1.4 g, 4.94 mmol) in dichloromethane
(25 mL) was slowly added trifluoroacetic acid (12.9 mL,
167.79 mmol) at 0 ꢁC. The reaction solution was stirred at room
temperature for 5 h, and then 1 N NaOH was added. The mixture
was extracted with dichloromethane, and the organic layer was
washed with brine, dried (Na2SO4), and filtered. The solvent was
evaporated under reduced pressure to give the title compound
(75 MHz, CDCl3) d 171.3,169.3,158.7,134.3,133.4,125.9,120.0,113.6,
99.7, 57.5, 55.2, 53.3, 50.5, 46.2, 45.2, 28.1, 26.5, 21.8; HRMS (ESI-
TOF) m/z calcd for C21H30N3O2þ [M þ Hþ] 356.2333, found 356.2336.
4.2.18. tert-Butyl 4-(2-cyanophenyl)-5,6-dihydropyridine-1(2H)-
carboxylate (4d)
The compound 4d (658 mg, 45% yield) was obtained from 2-
bromobenzonitrile (914 mg, 5.02 mmol) according to the same
procedure for the compound 4a: 1H NMR (300 MHz, DMSO-d6)
(1.1 g, 100% yield): 1H NMR (300 MHz, CDCl3)
d
7.14 (d, J ¼ 8.7 Hz,
2H), 6.68 (d, J ¼ 8.7 Hz, 2H), 5.85 (bs, 1H), 3.57 (s, 3H), 3.29 (d,
J ¼ 2.7 Hz, 2H), 2.86 (t, J ¼ 5.6 Hz, 2H), 2.20 (bs, 2H),1.65 (bs,1H); 13
C
d
7.51 (d, J ¼ 7.8 Hz, 1H), 7.43 (dt, J ¼ 10.6, 3.9 Hz, 1H), 7.22 (t,
NMR (75 MHz, CDCl3)
45.5, 43.3, 27.6.
d
158.6, 134.3, 133.8, 125.6, 121.7, 113.5, 54.9,
J ¼ 7.8 Hz, 2H), 5.83 (bs, 1H), 3.96 (d, J ¼ 2.9 Hz, 2H), 3.52 (t,
J ¼ 5.6 Hz, 2H), 2.39 (bs, 2H), 1.37 (s, 9H).
4.2.15. 2-(2-(4-(4-Methoxyphenyl)-5,6-dihydropyridin-1(2H)-yl)
ethyl)isoindoline-1,3-dione
4.2.19. 2-(1,2,3,6-Tetrahydropyridin-4-yl)benzonitrile
To a solution of 4d (658 mg, 2.31 mmol) in dichloromethane
(12 mL) was slowly added trifluoroacetic acid (6.0 mL, 78.68 mmol)
at 0 ꢁC. The reaction solution was stirred at room temperature for
5 h, and then 1 N NaOH was added. The mixture was extracted with
dichloromethane, and the organic layer was washed with brine,
dried (Na2SO4), and filtered. The solvent was evaporated under
reduced pressure to give the title compound (423 mg, 99% yield),
which was used for the next step without further purification: 1H
To
a solution of 4-(4-methoxyphenyl)-1,2,3,6-tetrahydrop
yridine (1.1 g, 5.66 mmol) in DMF (7 mL) were added N-(2-
bromoethyl)phthalimide (1.4 g, 5.66 mmol) and potassium car-
bonate (2.3 g, 16.99 mmol). This reaction solution was stirred at
80 ꢁC for 4 h, and then washed with aqueous sodium bicarbonate
solution, extracted with hexane/ethyl acetate (2/1, v/v). The organic
layer was washed with brine, dried (Na2SO4), and filtered. The
solvent was evaporated under reduced pressure, and the residue
was purified by column chromatography with hexane/ethyl acetate
(5/1, v/v) to obtain the title compound (1.8 g, 85% yield) as a white
NMR (400 MHz, CDCl3)
d
7.63 (d, J ¼ 7.5 Hz, 1H), 7.54 (t, J ¼ 7.7 Hz,
1H), 7.34 (d, J ¼ 8.2 Hz, 2H), 6.04 (bs, 1H), 3.54 (bs, 2H), 3.10 (t,
J ¼ 5.3 Hz, 2H), 2.44 (bs, 2H), 1.96 (bs, 1H); 13C NMR (100 MHz,
solid: 1H NMR (300 MHz, CDCl3)
d
7.85 (dd, J ¼ 3.5 Hz, 1.8 Hz, 2H),
CDCl3) d 146.5, 134.0, 133.3, 132.5, 129.1, 127.9, 127.0, 118.6, 110.1,
7.71 (dd, J ¼ 5.5, 3.0 Hz, 2H), 7.31 (t, J ¼ 9.0 Hz, 2H), 6.85 (d,
J ¼ 8.8 Hz, 2H), 5.96 (bs,1H), 3.91 (t, J ¼ 6.8 Hz, 2H), 3.81 (s, 3H), 3.23
(d, J ¼ 2.8 Hz, 2H), 2.83e2.76 (m, 4H), 2.52 (bs, 2H); 13C NMR
45.0, 42.9, 29.5.
4.2.20. 2-(1-(2-(1,3-Dioxoisoindolin-2-yl)ethyl)-1,2,3,6-
tetrahydropyridin-4-yl) benzonitrile
(75 MHz, CDCl3)
d
168.4, 158.7, 134.3, 133.8, 133.4, 132.2, 125.9,
123.2, 119.9, 113.6, 55.4, 55.3, 53.4, 49.9, 35.5, 28.0.
To a solution of 2-(1,2,3,6-tetrahydropyridin-4-yl)benzonitrile
(423 mg, 2.30 mmol) in DMF (3 mL) were added N-(2-bromoethyl)
phthalimide (583 mg, 2.30 mmol) and potassium carbonate
(953 mg, 6.89 mmol). This reaction solution was stirred at 80 ꢁC for
4 h, and then washed with sodium bicarbonate solution, and
extracted with hexane/ethyl acetate (2/1, v/v). The organic layer
was washed with brine, dried (Na2SO4), and filtered. The solvent
was evaporated under reduced pressure, and the residue was pu-
rified by column chromatography with hexane/ethyl acetate (5/1, v/
v) to obtain the title compound (301 mg, 33% yield) as a white solid:
4.2.16. 2-(4-(4-Methoxyphenyl)-5,6-dihydropyridin-1(2H)-yl)
ethanamine (5c)
To a solution of above phthalimide compound (1.8 g, 4.84 mmol)
in ethanol (18 mL) was added hydrazine hydrate 80% solution
(0.9 mL, 14.51 mmol). This reaction solution was stirred at 70 ꢁC for
2 h, and then was extracted with dichloromethane. The organic
layer was washed with brine, dried (Na2SO4), and filtered. The
solvent was evaporated under reduced pressure to give the com-
pound 5c (686 mg, 61% yield): 1H NMR (300 MHz, CDCl3)
d
7.28 (d,
1H NMR (300 MHz, CDCl3)
d 7.84e7.81 (m, 2H), 7.71e7.67 (m, 2H),
J ¼ 8.7 Hz, 2H), 6.81 (d, J ¼ 8.7 Hz, 2H), 5.93 (bs,1H), 3.74 (s, 3H), 3.11
(d, J ¼ 2.3 Hz, 2H), 2.82 (t, J ¼ 12.8 Hz, 2H), 2.66 (t, J ¼ 5.4 Hz, 2H),
2.49 (t, J ¼ 6.1 Hz, 4H), 1.75 (bs, 2H); 13C NMR (75 MHz, CDCl3)
7.61 (td, J ¼ 4.6 Hz, 2.6 Hz, 1H), 7.49 (t, J ¼ 7.7 Hz, 1H), 7.30 (d,
J ¼ 7.8 Hz, 2H), 5.97 (bs, 1H), 3.89 (t, J ¼ 6.7 Hz, 2H), 3.27 (d,
J ¼ 2.6 Hz, 2H), 2.85e2.76 (m, 4H), 2.51 (bs, 2H); 13C NMR (75 MHz,

J.-H. Lee et al. / European Journal of Medicinal Chemistry 74 (2014) 246e257
253
CDCl3)
d
168.3, 146.2, 133.9, 133.7, 133.6, 132.5, 132.2, 128.0, 127.4,
4.2.25. 3-(1-(2-(1,3-Dioxoisoindolin-2-yl)ethyl)-1,2,3,6-
tetrahydropyridin-4-yl)benzonitrile
127.1, 123.2, 118.8, 110.3, 55.2, 52.9, 49.4, 35.4, 29.4.
To a solution of above 3-(1,2,3,6-tetrahydropyridin-4-yl)benzo-
nitrile (493 mg, 2.68 mmol) in DMF (3 mL) were added N-(2-
bromoethyl)phthalimide (680 mg, 2.68 mmol) and potassium car-
bonate (1.11 g, 8.03 mmol). This reaction solution was stirred at
80 ꢁC for 4 h, and then was washed with sodium bicarbonate so-
lution, extracted with hexane/ethyl acetate (2/1, v/v). The organic
layer was washed with brine, dried (Na2SO4), and filtered. The
solvent was evaporated under reduced pressure, and the residue
was purified by column chromatography with hexane/ethyl acetate
(5/1, v/v) to obtain the title compound (301 mg, 31% yield) as a
4.2.21. 2-(1-(2-Aminoethyl)-1,2,3,6-tetrahydropyridin-4-yl)
benzonitrile (5d)
To a solution of above phthalimide (272 mg, 0.76 mmol) in
ethanol (3 mL) was added hydrazine hydrate 80% solution (0.1 mL,
2.28 mmol). This reaction solution was stirred at 70 ꢁC for 2 h, and
then was extracted with dichloromethane. The organic layer was
washed with brine, dried (Na2SO4), and filtered. The solvent was
evaporated under reduced pressure to give the compound 5d
(184 mg, 94% yield), which was used for the next step without
white solid: 1H NMR (300 MHz, CDCl3)
d
7.78 (dd, J ¼ 5.1 Hz, 3.0 Hz,
further purification: 1H NMR (300 MHz, CDCl3)
d
7.51 (d, J ¼ 7.5 Hz,
2H), 7.67 (dd, J ¼ 5.9 Hz, 2.9 Hz, 2H), 7.57 (s, 1.6H), 7.54 (d, J ¼ 8.2 Hz,
1H), 7.44 (d, J ¼ 7.5 Hz, 1H), 7.35 (t, J ¼ 7.7 Hz, 1H), 6.08 (bs, 1H), 3.86
(t, J ¼ 6.5 Hz, 2H), 3.22 (d, J ¼ 2.8 Hz, 2H), 2.79e2.72 (m, 4H), 2.45
1H), 7.40 (t, J ¼ 7.7 Hz, 1H), 7.23e7.17 (m, 2H), 5.88 (bs, 1H), 3.08 (d,
J ¼ 2.9 Hz, 2H), 2.74 (t, J ¼ 5.7 Hz, 2H), 2.62 (t, J ¼ 5.5 Hz, 2H), 2.44 (t,
J ¼ 5.9 Hz, 4H), 1.49 (bs, 2H); 13C NMR (75 MHz, CDCl3)
d 146.2,
(bs, 2H); 13C NMR (75 MHz, CDCl3)
d 168.3, 141.7, 133.9, 133.1, 132.1,
133.7, 133.4, 132.5, 128.0, 127.5, 127.1, 118.7, 110.2, 60.7, 52.8, 50.0,
39.0, 29.4.
130.3, 129.1, 128.4, 124.1, 123.2, 119.0, 112.3, 55.3, 53.2, 49.5, 35.4,
27.7.
4.2.22. 2-(1-(2-((3-Isopropylisoxazol-5-yl)methylamino)ethyl)-
1,2,3,6-tetrahydropyridin-4-yl)benzonitrile (7d)
4.2.26. 3-(1-(2-Aminoethyl)-1,2,3,6-tetrahydropyridin-4-yl)
benzonitrile (5e)
A solution of 3-isopropylisoxazole-5-carbaldehyde 6 (99 mg,
0.71 mmol) in dichloromethane (6 mL) was added to a solution of
5d (162 mg, 0.71 mmol) in dichloromethane (17 mL), and then
To a solution of above phthalimide (301 mg, 0.84 mmol) in
ethanol (3 mL) was added hydrazine hydrate 80% solution (0.2 mL,
2.53 mmol). This reaction solution was stirred at 70 ꢁC for 2 h, and
then was extracted with dichloromethane. The organic layer was
washed with brine, dried (Na2SO4), and filtered. The solvent was
evaporated under reduced pressure to give the compound 5e
ꢀ
4 A molecular sieve powder (423 mg) was added. After 2 h, so-
dium triacetoxyborohydride (227 mg, 1.07 mmol) was added to
the reaction mixture. This mixture was stirred at room temper-
ature for 2 h, and then 1 N NaOH was added. The reaction
mixture was extracted with dichloromethane, and the organic
layer was washed with brine, dried (Na2SO4), and filtered. The
solvent was evaporated under reduced pressure, and the residue
was purified by column chromatography with dichloromethane/
methanol (50/1, v/v) to obtain compound 7d (141 mg, 57% yield):
(184 mg, 96% yield): 1H NMR (300 MHz, CDCl3)
d 7.53 (s, 1H), 7.50
(d, J ¼ 8.9 Hz, 1H), 7.39 (d, J ¼ 7.7 Hz, 1H), 7.30 (t, J ¼ 7.6 Hz, 1H), 6.04
(bs, 1H), 3.08 (d, J ¼ 2.9 Hz, 2H), 2.76 (t, J ¼ 6.1 Hz, 2H), 2.62 (t,
J ¼ 5.6 Hz, 2H), 2.45 (t, J ¼ 6.3 Hz, 4H), 1.42 (bs, 2H); 13C NMR
(75 MHz, CDCl3) d 141.8, 133.1, 130.2, 129.2, 129.1, 128.4, 124.3, 118.9,
112.3, 60.8, 53.2, 50.1, 41.3, 39.1, 27.7.
1H NMR (300 MHz, CDCl3)
d
7.64 (td, J ¼ 4.1, 2.5 Hz, 1H), 7.53 (dt,
J ¼ 10.5, 3.9 Hz, 1H), 7.35e7.27 (m, 2H), 6.06 (s, 1H), 5.99 (bs, 1H),
3.92 (s, 2H), 3.19 (q, J ¼ 2.7 Hz, 2H), 3.09e3.0 (m, 1H), 2.81 (t,
J ¼ 5.9 Hz, 2H), 2.73 (t, J ¼ 5.5 Hz, 2H), 2.65 (t, J ¼ 5.9 Hz, 2H),
2.55 (bs, 2H), 2.08 (bs 1H), 1.27 (d, J ¼ 7.0 Hz, 6H); 13C NMR
4.2.27. 3-(1-(2-((3-Isopropylisoxazol-5-yl)methylamino)ethyl)-
1,2,3,6-tetrahydropyridin-4-yl)benzonitrile (7e)
A solution of 3-isopropylisoxazole-5-carbaldehyde 6 (113 mg,
0.81 mmol) in dichloromethane (6 mL) was added to a solution of
(75 MHz, CDCl3)
d 171.2, 169.3, 146.3, 133.8, 133.6, 132.6, 128.1,
ꢀ
5e (184 mg, 0.81 mmol) in dichloromethane (19 mL), and then 4 A
127.4, 127.2, 118.8, 110.4, 99.8, 57.3, 52.8, 50.0, 46.1, 45.1, 29.5,
26.5, 21.8; HRMS (ESI-TOF) m/z calcd for C21H27N4Oþ [M þ Hþ]
351.2179, found 351.2194.
molecular sieve powder (423 mg) was added. After 2 h, sodium
triacetoxyborohydride (257 mg,1.21 mmol) was added to a reaction
mixture. This mixture was stirred at room temperature for 2 h, and
then 1 N NaOH was added. The reaction mixture was extracted with
dichloromethane, and the organic layer was washed with brine,
dried (Na2SO4), and filtered. The solvent was evaporated under
reduced pressure, and the residue was purified by column chro-
matography with dichloromethane/methanol (50/1, v/v) to obtain
the compound 7e (151 mg, 53% yield): 1H NMR (300 MHz, CDCl3)
4.2.23. tert-Butyl 4-(3-cyanophenyl)-5,6-dihydropyridine-1(2H)-
carboxylate (4e)
The compound 4e (796 mg, 56% yield) was obtained from 3-
bromobenzonitrile (900 mg, 5.02 mmol) according to the same
procedure for the compound 4a: 1H NMR (300 MHz, DMSO-d6)
d
7.45 (d, J ¼ 7.4 Hz, 2H), 7.38e7.25 (m, 2H), 5.96 (bs, 1H), 3.94 (d,
d
7.63 (s, 1H), 7.59 (d, J ¼ 7.9 Hz, 1H), 7.50 (d, J ¼ 7.7 Hz, 1H), 7.40 (t,
J ¼ 2.7 Hz, 2H), 3.49 (t, J ¼ 5.6 Hz, 2H), 2.32 (bs, 2H), 1.34 (s, 9H).
J ¼ 7.7 Hz, 1H), 6.12 (bs, 1H), 6.03 (s, 1H), 3.90 (s, 2H), 3.16 (q,
J ¼ 2.8 Hz, 2H), 3.08e2.99 (m, 1H), 2.81 (t, J ¼ 5.9 Hz, 2H), 2.70 (t,
4.2.24. 3-(1,2,3,6-Tetrahydropyridin-4-yl)benzonitrile
J ¼ 5.6 Hz, 2H), 2.63 (t, J ¼ 5.9 Hz, 2H), 2.52 (bs, 2H),1.97 (bs,1H); 13
C
To a solution of 4e (796 mg, 2.80 mmol) in dichloromethane
(14 mL) was slowly added trifluoroacetic acid (7.3 mL, 95.20 mmol)
at 0 ꢁC. The reaction solution was stirred at room temperature for
5 h, and then 1 N NaOH was added. The reaction mixture was
extracted with dichloromethane, and the organic layer was washed
with brine, dried (Na2SO4), and filtered. The solvent was evaporated
under reduced pressure to give the crude title compound (493 mg,
NMR (75 MHz, CDCl3) d 171.1, 169.3, 141.8, 133.3, 130.4, 129.1, 128.5,
124.2, 119.0, 112.5, 99.8, 57.4, 53.2, 50.1, 46.1, 45.1, 27.8, 26.5, 21.8;
HRMS (ESI-TOF) m/z calcd for C21H27N4Oþ [M þ Hþ] 351.2179,
found 351.2192.
4.2.28. tert-Butyl 4-(tributylstannyl)-5,6-dihydropyridine-1(2H)-
carboxylate (2f)
96% yield): 1H NMR (400 MHz, CDCl3)
d
7.60 (d, J ¼ 9.4 Hz, 2H), 7.49
To a solution of diisopropylamine (4.2 mL, 30.11 mmol) in THF
(90 mL) was dropwised n-butyllithium (15.1 mL, 2.5 M in hexane,
45.17 mmol) at 0 ꢁC. After 30 min, tributyltinhydride (7.5 mL,
25.09 mmol) was slowly added at 0 ꢁC. After 30 min, 1-Boc-4-
piperidone (5.0 g, 25.09 mmol) in THF (10 mL) was slowly added
(d, J ¼ 6.3 Hz, 1H), 7.42 (d, J ¼ 7.3 Hz,1H), 6.20 (bs, 1H), 3.53 (bs, 2H),
3.09 (bs, 2H), 2.40 (bs, 2H), 1.78 (bs, 1H); 13C NMR (100 MHz, CDCl3)
d
142.2,133.2,130.1, 129.0,128.9,128.1,126.0,118.8, 112.2, 44.9, 42.4,
27.3.

254
J.-H. Lee et al. / European Journal of Medicinal Chemistry 74 (2014) 246e257
at ꢀ78 ꢁC. After
2
h, methanesulfonyl chloride (7.8 mL,
NMR (75 MHz, CDCl3)
53.4, 50.3, 39.2, 28.0.
d 139.2, 135.0, 128.4, 126.1, 124.9, 122.5, 61.1,
100.38 mmol) and triethylamine (28.0 mL, 200.75 mmol) were
added at ꢀ78 ꢁC. The reaction mixture was stirred at room tem-
perature for 15 h, and was extracted with hexane and acetonitrile.
The solvent was evaporated under reduced pressure to give a crude,
which was purified by column chromatography with hexane/ethyl
acetate (50/1, v/v) to obtain the compound 2f (5.4 g, 45% yield): 1H
4.2.33. 2-(4-(4-Chlorophenyl)-5,6-dihydropyridin-1(2H)-yl)-N-((3-
isopropylisoxazol-5-yl)methyl)ethanamine (7f)
A solution of 3-isopropylisoxazole-5-carbaldehyde 6 (503 mg,
3.62 mmol) in dichloromethane (5 mL) was added to a solution of
NMR (300 MHz, CDCl3)
(t, J ¼ 5.5 Hz, 2H), 2.26 (bs, 2H),1.51e1.43 (m, 6H), 1.46 (s, 9H), 1.36e
1.27 (m, 6H), 0.89 (t, J ¼ 7.2 Hz, 15H).
d
5.73 (bs, 1H), 3.90 (d, J ¼ 2.6 Hz, 2H), 3.45
ꢀ
5f (856 mg, 3.62 mmol) in DCM (87 mL), and then 4 A molecular
sieve (86 beads) was added. After 2 h, sodium triacetoxyborohy-
dride (1.1 mg, 5.42 mmol) was added to a reaction mixture. This
mixture was stirred at room temperature for 2 h, and then 1 N
NaOH was added. The reaction mixture was extracted with
dichloromethane, and the organic layer was washed with brine,
dried (Na2SO4), and filtered. The solvent was evaporated under
reduced pressure, and the residue was purified by column chro-
matography with dichloromethane/methanol (50/1, v/v) to obtain
the compound 7f (329 mg, 25% yield): 1H NMR (300 MHz, CDCl3)
4.2.29. tert-Butyl 4-(4-chlorophenyl)-5,6-dihydropyridine-1(2H)-
carboxylate (4f)
The suspension of 2f (5.4 g, 11.36 mmol), 1-bromo-4-
chlorobenzene (2.6 g, 13.64 mmol), and tetrakis(triphenyl
phosphine)palladium(0) (1.3 g, 1.14 mmol) was stirred in toluene
(81 mL) at 120 ꢁC for 17 h. The reaction mixture was concentrated
under reduced pressure, and the residue was purified by column
chromatography with hexane/ethyl acetate (15/1, v/v) to obtain the
d
7.27e7.20 (m, 4H), 6.00 (bs, 2H), 3.86 (s, 2H), 3.09 (d, J ¼ 2.9 Hz,
2H), 3.04e2.95 (m, 1H), 2.76 (t, J ¼ 5.9 Hz, 2H), 2.64 (t, J ¼ 4.7 Hz,
2H), 2.58 (t, J ¼ 5.9 Hz, 2H), 2.46 (bs, 2H), 2.21 (bs, 1H), 1.23 (d,
compound 4f (3.5 g, 100% yield): 1H NMR (400 MHz, CDCl3)
d 7.34e
7.27 (m, 4H), 6.01 (bs, 1H), 4.06 (bs, 2H), 3.63 (bs, 2H), 2.47 (bs, 2H),
1.48 (s, 9H).
J ¼ 7.0 Hz, 6H); 13C NMR (75 MHz, CDCl3)
d 171.2, 169.3, 139.1, 134.0,
132.7, 128.4, 126.1, 122.3, 99.8, 57.5, 53.3, 50.3, 46.1, 45.1, 28.0, 26.5,
21.8; HRMS (ESI-TOF) m/z calcd for C20H27ClN3Oþ [M þ Hþ]
360.1837, found 360.1831.
4.2.30. 4-(4-Chlorophenyl)-1,2,3,6-tetrahydropyridine
To a solution of 4f (3.5 g, 11.84 mmol) in dichloromethane
(59 mL) was slowly added trifluoroacetic acid (30.8 mL,
402.51 mmol) at 0 ꢁC. The reaction solution was stirred at room
temperature for 5 h, and then 1 N NaOH was added. The reaction
mixture was extracted with dichloromethane, and the organic layer
was washed with brine, dried (Na2SO4), and filtered. The solvent
was evaporated under reduced pressure to give the title compound
4.2.34. tert-Butyl 4-phenylpiperidine-1-carboxylate (8a)
Pd/C (268 mg, 2.52 mmol) was added to the solution of 4a (3.3 g,
12.58 mmol) in methanol (105 mL). This reaction mixture was
stirred under hydrogen gas at room temperature for 15 h, and then
the suspension was filtered on celite and silica gel. The solvent was
evaporated under reduced pressure to give compound 8a (2.8 g,
(3.3 g, 100% yield): 1H NMR (300 MHz, CDCl3)
d 7.42e7.22 (m, 4H),
86% yield): 1H NMR (300 MHz, CDCl3)
d 7.35e7.30 (m, 2H), 7.25e
6.04 (bs, 1H), 3.43 (bs, 2H), 3.00 (t, J ¼ 4.7 Hz, 2H), 2.33 (bs, 2H), 1.20
7.21 (m, 3H), 4.29e4.26 (m, 2H), 2.82 (br. t, J ¼ 12.4 Hz, 2H), 2.66
(tt, J ¼ 12.1, 3.6 Hz, 1H), 1.86e1.82 (m, 2H), 1.73e1.58 (m, 2H), 1.52
(s, 9H).
(bs, 1H); 13C NMR (75 MHz, CDCl3)
123.9, 45.4, 43.1, 27.5.
d 139.6, 134.1, 128.3, 126.6, 124.7,
4.2.31. 2-(2-(4-(4-Chlorophenyl)-5,6-dihydropyridin-1(2H)-yl)
ethyl)iso-indoline-1,3-dione
4.2.35. 4-Phenylpiperidine
To a solution of 8a (2.8 g,10.86 mmol) above in dichloromethane
(54 mL) was slowly added trifluoroacetic acid (28.3 mL,
369.31 mmol) at 0 ꢁC. The reaction solution was stirred at room
temperature for 5 h, and then 1 N NaOH was added. The reaction
mixture was extracted with dichloromethane, and the organic layer
was washed with brine, dried (Na2SO4), and filtered. The solvent
was evaporated under reduced pressure to give the title compound
To a solution of 4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridine
(3.3 g, 17.13 mmol) in DMF (20 mL) were added N-(2-bromoethyl)
phthalimide (4.4 g, 17.13 mmol) and potassium carbonate (7.1 g,
51.38 mmol). This reaction solution was stirred at 80 ꢁC for 4 h, and
then was washed with sodium bicarbonate solution, extracted with
hexane/ethyl acetate (2/1, v/v). The organic layer was washed with
brine, dried (Na2SO4), and filtered. The organic layer was concen-
trated under reduced pressure, and the residue was purified by
column chromatography with hexane/ethyl acetate (5/1, v/v) to
obtain the title compound (1.2 g, 19% yield) as a white solid: 1H
(1.8 g, 100% yield): 1H NMR (300 MHz, CDCl3)
d 7.33e7.28 (m, 2H),
7.24e7.17 (m, 3H), 3.18e3.14 (m, 2H), 2.71 (bt, J ¼ 12.1 Hz, 2H), 2.60
(tt, J ¼ 12.0, 3.3 Hz, 1H), 1.84e1.80 (m, 2H), 1.70e1.57 (m, 1H þ 2H);
13C NMR (75 MHz, CDCl3)
d 146.8, 128.4, 126.8, 126.1, 47.2, 43.1, 34.6.
NMR (300 MHz, CDCl3)
d 7.86e7.80 (m, 2H), 7.72e7.68 (m, 2H),
7.32e7.19 (m, 4H), 6.03 (bs, 1H), 3.92e3.88 (m, 2H), 3.24 (d,
J ¼ 3.1 Hz, 2H), 2.83e2.75 (m, 4H), 2.49 (bs, 2H); 13C NMR (75 MHz,
4.2.36. 2-(2-(4-Phenylpiperidin-1-yl)ethyl)isoindoline-1,3-dione
To a solution of 4-phenylpiperidine (1.8 g, 11.46 mmol) in DMF
(35 mL) were added N-(2-bromoethyl)phthalimide (2.9 g,
11.46 mmol), sodium iodide (344 mg, 0.34 mmol), and sodium
carbonate (1.2 g, 11.46 mmol). This reaction solution was stirred at
80 ꢁC for 4 h, and then was washed with sodium bicarbonate so-
lution, extracted with hexane/ethyl acetate (2/1, v/v). The organic
layer was washed with brine, dried (Na2SO4), and filtered. The
solvent was evaporated under reduced pressure, and the residue
was purified by column chromatography with hexane/ethyl acetate
(5/1, v/v) to obtain the title compound (1.8 g, 47% yield) as a white
CDCl3)
d 168.4, 139.1, 133.9, 132.2, 128.3, 126.9, 126.1, 124.8, 123.2,
122.2, 55.3, 53.3, 49.7, 35.5, 27.9.
4.2.32. 2-(4-(4-Chlorophenyl)-5,6-dihydropyridin-1(2H)-yl)
ethanamine (5f)
To a solution of above phthalimide compound (1.2 g, 3.32 mmol)
in ethanol (12 mL) was added hydrazine hydrate 80% solution
(0.62 mL, 9.97 mmol). This reaction solution was stirred at 70 ꢁC for
2 h, and then was extracted with dichloromethane. The organic
layer was washed with brine, dried (Na2SO4), and filtered. The
solvent was evaporated under reduced pressure to give the com-
solid: 1H NMR (300 MHz, CDCl3)
d
7.86 (dd, J ¼ 5.3 Hz, 3.1 Hz, 2H),
pound 5f (787 mg, 92% yield): 1H NMR (300 MHz, CDCl3)
d
77.32e
7.72 (dd, J ¼ 5.5 Hz, 3.0 Hz, 2H), 7.32e7.27 (m, 2H), 7.22e7.16 (m,
2H), 3.88 (t, J ¼ 6.8 Hz, 2H), 3.15e3.11 (m, 2H), 2.69 (t, J ¼ 6.8 Hz,
2H), 2.49 (tt, J ¼ 11.9, 4.0 Hz, 1H), 2.21e2.13 (m, 2H), 1.85e1.81 (m,
7.19 (m, 4H), 6.05 (bs, 1H), 3.15 (d, J ¼ 2.7 Hz, 2H), 2.85 (t, J ¼ 6.0 Hz,
2H), 2.70 (t, J ¼ 5.4 Hz, 2H), 2.53 (t, J ¼ 6.1 Hz, 4H), 1.25 (bs, 2H); 13
C

J.-H. Lee et al. / European Journal of Medicinal Chemistry 74 (2014) 246e257
255
2H), 1.79e1.65 (m, 2H); 13C NMR (75 MHz, CDCl3)
133.8, 132.3, 128.4, 126.9, 126.1, 123.2, 56.1, 54.3, 42.6, 35.6, 33.5.
d
168.3, 146.4,
4.2.41. 2-(2-(4-(3,4-Dichlorophenyl)piperidin-1-yl)ethyl)
isoindoline-1,3-dione
To
a solution of 4-(3,4-dichlorophenyl)piperidine (1.3 g,
5.65 mmol) in DMF (17 mL) were added N-(2-bromoethyl)phtha-
limide (1.4 g, 5.65 mmol), sodium iodide (169 mg, 1.13 mmol), and
sodium carbonate (599 g, 5.65 mmol). This reaction solution was
stirred at 90 ꢁC for 4 h, and then washed with aqueous sodium
bicarbonate solution, extracted with hexane/ethyl acetate (2/1, v/v).
The organic layer was washed with brine, dried (Na2SO4), and
filtered. The solvent was evaporated under reduced pressure, and
the residue was purified by column chromatography with hexane/
ethyl acetate (5/1, v/v) to obtain the title compound (1.0 g, 45%
4.2.37. 2-(4-Phenylpiperidin-1-yl)ethanamine (9a)
To a solution of 2-(2-(4-phenylpiperidin-1-yl)ethyl)isoindo-
line-1,3-dione (1.8 g, 5.41 mmol) in ethanol (20 mL) was added
hydrazine hydrate 80% solution (1.0 mL, 16.24 mmol). This reaction
solution was stirred at 70 ꢁC for 2 h, and then was extracted with
dichloromethane. The organic layer was washed with brine, dried
(Na2SO4), and filtered. The solvent was evaporated under reduced
pressure to give the compound 9a (1.1 g, 99% yield): 1H NMR
(300 MHz, CDCl3)
d 7.27e7.22 (m, 2H), 7.19e7.11 (m, 3H), 2.98e
yield) as a white solid: 1H NMR (300 MHz, CDCl3)
d
7.82 (dd, J ¼ 5.3,
2.94 (m, 2H), 2.78e2.74 (m, 2H), 2.50e2.37 (m, 1H þ 2H), 2.07e
1.98 (m, 2H), 1.79e1.66 (m, 2H þ 2H), 1.42 (bs, 2H); 13C NMR
3.1 Hz, 2H), 7.68 (dd, J ¼ 5.4, 3.0 Hz, 2H), 7.28 (d, J ¼ 8.2 Hz, 1H), 7.23
(d, J ¼ 1.7 Hz, 1H), 6.99 (dd, J ¼ 8.2, 1.8 Hz, 1H), 3.82 (t, J ¼ 6.6 Hz,
2H), 3.10e3.06 (m, 2H), 2.64 (t, J ¼ 6.6 Hz, 2H), 2.40 (tt, J ¼ 12.0, 3.6
Hz, 1H), 2.13e2.06 (m, 2H), 1.76e1.73 (m, 2H), 1.66e1.52 (m, 2H);
(75 MHz, CDCl3)
33.6.
d 146.4, 128.4, 126.7, 126.1, 61.6, 54.5, 42.7, 39.1,
13C NMR (75 MHz, CDCl3)
d 168.3, 146.7, 133.8, 132.21, 132.15, 130.2,
4.2.38. N-((3-Isopropylisoxazol-5-yl)methyl)-2-(4-
phenylpiperidin-1-yl)ethanamine (10a)
129.7, 128.9, 126.3, 123.1, 56.0, 54.0, 41.7, 35.5, 33.2.
A solution of 3-isopropylisoxazole-5-carbaldehyde 6 (1.7 g,
8.06 mmol) in dichloromethane (17 mL) was added to a solution of
9a (1.1 g, 5.38 mmol) in dichloromethane (129 mL), and then 4 A
4.2.42. 2-(4-(3,4-Dichlorophenyl)piperidin-1-yl)ethanamine (9b)
To a solution of above phthalimide compound (1.0 g, 2.48 mmol)
in ethanol (9 mL) was added hydrazine hydrate 80% solution
(0.47 mL, 7.44 mmol). This reaction solution was stirred at 70 ꢁC for
2 h, and then extracted with dichloromethane. The organic layer
was washed with brine, dried (Na2SO4), and filtered. The solvent
was evaporated under reduced pressure to give compound 9b
ꢀ
molecular sieve powder (2.6 g) was added. After 2 h, sodium tri-
acetoxyborohydride (1.7 g, 8.06 mmol) was added to a reaction
mixture. This mixture was stirred at room temperature for 2 h, and
then 1 N NaOH was added. The reaction mixture was extracted
with dichloromethane, and the organic layer was washed with
brine, dried (Na2SO4), and filtered. The solvent was evaporated
under reduced pressure, and the residue was purified by column
chromatography with dichloromethane/methanol (50/1, v/v) to
obtain the compound 10a (647 mg, 37% yield): 1H NMR (300 MHz,
(542 mg, 80% yield): 1H NMR (300 MHz, CDCl3)
d
7.36 (d, J ¼ 8.3 Hz,
2H), 7.31 (d, J ¼ 2.0 Hz, 1H), 7.06 (dd, J ¼ 8.3, 2.0 Hz, 1H), 3.04e3.00
(m, 2H), 2.85 (t, J ¼ 9.0 Hz, 2H), 2.52e2.42 (m, 1H þ 2H), 2.12e2.03
(m, 2H), 1.83e1.65 (m, 2H þ 2H), 1.50 (bs, 2H); 13C NMR (75 MHz,
CDCl3)
33.3.
d 146.7, 132.3, 130.3, 129.9, 129.0, 126.3, 61.4, 54.2, 42.0, 39.1,
CDCl3)
d 7.29e7.24 (m, 2H), 7.21e7.13 (m, 3H), 6.02 (s, 1H), 3.87 (s,
2H), 3.06e2.94 (m, 1H þ 2H), 2.74 (t, J ¼ 5.8 Hz, 2H), 2.52e2.41 (m,
2H þ 1H), 2.07e1.99 (m, 2H þ 1H), 1.78e1.72 (m, 2H þ 2H), 1.25 (d,
4.2.43. 2-(4-(3,4-Dichlorophenyl)piperidin-1-yl)-N-((3-
isopropylisoxazol-5-yl)methyl)ethanamine (10b)
J ¼ 7.0 Hz, 6H); 13C NMR (75 MHz, CDCl3)
d 171.3, 169.2, 146.3,
128.4, 126.8, 126.1, 99.7, 58.0, 54.5, 46.1, 45.2, 42.7, 33.5, 26.5, 21.8;
HRMS (ESI-TOF) m/z calcd for C20H30N3Oþ [M þ Hþ] 328.2383,
found 328.2392.
A solution of 3-isopropylisoxazole-5-carbaldehyde 6 (276 mg,
1.98 mmol) in dichloromethane (2.8 mL) was added to a solution of
ꢀ
9b (542 mg, 1.98 mmol) in dichloromethane (35 mL), and then 4 A
molecular sieve powder (1.27 g) was added. After 2 h, sodium tri-
acetoxyborohydride (631 mg, 2.98 mmol) was added to a reaction
mixture. This mixture was stirred at room temperature for 2 h, and
then 1 N NaOH was added. The reaction mixture was extracted with
dichloromethane, and the organic layer was washed with brine,
dried (Na2SO4), and filtered. The solvent was evaporated under
reduced pressure, and the residue was purified by column chro-
matography with dichloromethane/methanol (50/1, v/v) to obtain
compound 10b (605 mg, 77% yield): 1H NMR (400 MHz, CDCl3)
4.2.39. tert-Butyl 4-(3,4-dichlorophenyl)piperidine-1-carboxylate
(8b)
Pd/C (147 mg, 1.38 mmol) was added to the solution of 4b
(2.30 g, 6.91 mmol) in methanol (58 mL). This reaction mixture was
stirred under hydrogen gas at room temperature for 18 h, and then
the suspension was filtered on celite and silica gel. The solvent was
evaporated under reduced pressure to give compound 8b (2.03 g,
89% yield): 1H NMR (300 MHz, CDCl3)
d
7.34 (d, J ¼ 8.2 Hz, 1H), 7.27
(d, J ¼ 1.7 Hz, 3H), 7.02 (dd, J ¼ 8.2, 1.9 Hz, 1H), 4.26e4.22 (m, 2H),
2.77 (t, J ¼ 12.3 Hz, 2H), 2.60 (tt, J ¼ 12.1, 3.4 Hz, 1H), 1.80e1.76 (m,
2H), 1.61e1.41 (m, 2H þ 9H).
d
7.35 (d, J ¼ 8.2 Hz, 1H), 7.31 (s, 1H), 7.0 (d, J ¼ 8.2 Hz, 1H), 6.04 (bs,
1H), 3.91 (s, 2H), 3.08e2.97 (m, 2H þ 1H), 2.76 (t, J ¼ 5.9 Hz, 2H), 2.52
(t, J ¼ 5.9 Hz, 2H), 2.50e2.43 (m, 1H), 2.08e2.02 (m, 2H), 1.95 (bs,
1H), 1.81e1.66 (m, 4H), 1.28 (d, J ¼ 6.9 Hz, 6H); 13C NMR (100 MHz,
4.2.40. 4-(3,4-Dichlorophenyl)piperidine
CDCl3) d 171.2, 169.3, 146.6, 132.3, 130.3, 129.9, 129.0, 126.3, 99.8,
To a solution of 8b (2.03 g, 6.15 mmol) in dichloromethane
(31 mL) was slowly added trifluoroacetic acid (16.0 mL,
209.0 mmol) at 0 ꢁC. The reaction solution was stirred at room
temperature for 5 h, and then 1 N NaOH was added. The reaction
mixture was extracted with dichloromethane, and the organic layer
was washed with brine, dried (Na2SO4), and filtered. The solvent
was evaporated under reduced pressure to give the title compound
57.9, 54.2, 46.0, 45.2, 41.9, 33.3, 26.5, 21.8; HRMS (ESI-TOF) m/z calcd
for C20H28Cl2N3Oþ [M þ Hþ] 396.1604, found 396.1606.
4.3. Biological assay
4.3.1. FDSS6000 assay (calcium imaging plate reader)
HEK293 cells which stably express both a1G and a1H subunits
[32] were grown in Dulbecco’s modified Eagle’s medium supple-
mented with 10% (v/v) fetal bovine serum, penicillin (100 U/mL),
(1.3 g, 91% yield): 1H NMR (300 MHz, CDCl3)
d
7.33 (d, J ¼ 8.3 Hz,
1H), 7.28 (d, J ¼ 1.9 Hz, 1H), 7.03 (dd, J ¼ 8.3, 2.0 Hz, 1H), 3.18e3.15
(m, 2H), 2.74e2.66 (m, 2H), 2.48 (tt, J ¼ 12.0, 3.5 Hz, 1H), 1.80e1.76
streptomycin (100
(1
g/mL) at 37 ꢁC in a humid atmosphere of 5% CO2 and 95% air.
Cells were seeded into 96-well black wall clear bottom plates at a
mg/mL), geneticin (500 mg/mL), and puromycin
(m, 1H), 1.62e1.48 (m, 2H þ 1H); 13C NMR (75 Hz, CDCl3)
d
147.0,
m
132.2, 130.3, 129.8, 128.9, 126.3, 47.0, 42.3, 34.3.

256
J.-H. Lee et al. / European Journal of Medicinal Chemistry 74 (2014) 246e257
density of 40,000 cells/well and were used on the next day for high-
throughput screening (HTS) FDSS6000 assay. For FDSS6000 assay,
(i.e., at or near 15 g), whereas values near 0 indicate allodynia. The
result of cold allodynia was also expressed as %MPE.
cells were incubated for 60 min at room temperature with 5 mM
fluo3/AM and 0.001% Pluronic F-127 in a HEPES-buffered solution
composed of (in mM): 115 NaCl, 5.4 KCl, 0.8 MgCl2, 1.8 CaCl2, 20
HEPES, and 13.8 glucose (pH 7.4). During fluorescence-based
FDSS6000 assay, a1G T-type Ca2þ channels were activated using a
high concentration of KCl (70 mM) in 10 mM CaCl2 containing
HEPES-buffered solution and the increase in [Ca2þ]i by KCl-induced
depolarization was detected. All data were collected and analyzed
using FDSS6000 and related software (Hamamatsu, Japan).
Acknowledgments
This research was supported by the KIST Institutional Program
(2E23870). We thank Prof. Perez-Reyes (Department of Pharma-
cology, University of Virginia) for providing HEK293/Cav3.1 and
Cav3.2 cells.
Appendix A. Supplementary data
4.3.2. Whole-cell patch-clamp assay (electro-physiological
recordings)
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.12.056.
For the recordings of a1G and a1H T-type Ca2þ currents, the
standard whole-cell patch-clamp method was utilized as previ-
ously described [33]. Briefly, borosilicate glass electrodes with a
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