Discovery of SMP-304, a novel benzylpiperidine derivative with serotonin transporter inhibitory activity and 5-HT1Aweak partial agonistic activity showing the antidepressant-like effect

Article abstract of DOI:10.1016/j.bmc.2016.10.034

We report the discovery of a novel benzylpiperidine derivative with serotonin transporter (SERT) inhibitory activity and 5-HT_1Areceptor weak partial agonistic activity showing the antidepressant-like effect. The 3-methoxyphenyl group and the ph

Full text of DOI:10.1016/j.bmc.2016.10.034

Accepted Manuscript
Discovery of SMP-304, a Novel Benzylpiperidine Derivative with Serotonin
Transporter Inhibitory Activity and 5-HT1A Weak Partial Agonistic Activity
showing the Antidepressant-like Effect
Hidefumi Yoshinaga, Shuji Masumoto, Koji Koyama, Naoya Kinomura, Yuji
Matsumoto, Taro Kato, Satoko Baba, Kenji Matsumoto, Tomoko Horisawa,
Hitomi Oki, Kazuki Yabuuchi, Toru Kodo
PII:
S0968-0896(16)31080-X
http://dx.doi.org/10.1016/j.bmc.2016.10.034
BMC 13361
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 August 2016
25 October 2016
28 October 2016
Please cite this article as: Yoshinaga, H., Masumoto, S., Koyama, K., Kinomura, N., Matsumoto, Y., Kato, T., Baba,
S., Matsumoto, K., Horisawa, T., Oki, H., Yabuuchi, K., Kodo, T., Discovery of SMP-304, a Novel Benzylpiperidine
Derivative with Serotonin Transporter Inhibitory Activity and 5-HT1A Weak Partial Agonistic Activity showing the
Antidepressant-like Effect, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.10.034
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Bioorganic & Medicinal Chemistry
Discovery of SMP-304, a Novel Benzylpiperidine Derivative with Serotonin
Transporter Inhibitory Activity and 5-HT1A Weak Partial Agonistic Activity showing
the Antidepressant-like Effect
Hidefumi Yoshinaga *, Shuji Masumoto, Koji Koyama, Naoya Kinomura, Yuji Matsumoto, Taro Kato,
Satoko Baba, Kenji Matsumoto, Tomoko Horisawa, Hitomi Oki, Kazuki Yabuuchi and Toru Kodo
Drug Research Division, Sumitomo Dainippon Pharma Co., Ltd., 33-94 Enoki-cho, Suita, Osaka 564-0053, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We report the discovery of a novel benzylpiperidine derivative with serotonin transporter
(SERT) inhibitory activity and 5-HT1A receptor weak partial agonistic activity showing the
antidepressant-like effect. The 3-methoxyphenyl group and the phenethyl group of compound 1,
which has weak SERT binding activity, but potent 5-HT1A binding activity, were optimized,
leading to compound 35 with potent and balanced dual SERT and 5-HT1A binding activity, but
also potent CYP2D6 inhibitory activity. Replacement of the methoxy group in the left part of
compound 35 with a larger alkoxy group, such as ethoxy, isopropoxy or methoxy-ethoxy group
ameliorated CYP2D6 inhibition, giving SMP-304 as a candidate. SMP-304 with serotonin
uptake inhibitory activity and 5-HT1A weak partial agonistic activity, which could work as a 5-
HT1A antagonist, displayed faster onset of antidepressant-like effect than a representative SSRI
paroxetine in an animal model.
Available online
Keywords:
Serotonin transporter
5-HT1A receptor
Antidepressant
Fast onset
2009 Elsevier Ltd. All rights reserved.
Benzylpiperidine
1. Introduction
was observed in combination administration with SSRIs and
WAY-100635, a highly selective 5-HT1A receptor antagonist,
Major depressive disorder (MDD), a major worldwide health
issue, is commonly treated with selective serotonin reuptake
inhibitors (SSRIs), which are the first line drugs for depression.
However, the use of SSRIs has a number of drawbacks, including
slow onset and insufficient efficacy.
weeks of treatment for SSRIs to start producing beneficial
antidepressant effect. Furthermore, over one third of patients with
MDD do not response to SSRIs. This is because psychiatric
disorders, such as depression and schizophrenia have complex
etiology. Therefore, it is believed that drugs that can act on a
broad range of neuron network would be advantageous for the
suggesting the contribution of 5-HT1A antagonistic activity in
human. 12 Based on the evidences, it is believed that 5-HT1A auto-
receptor antagonists would presumably accelerate SSRIs onset.
Accordingly, we launched an exploratory research program
aimed at finding antidepressant drug candidates with serotonin
transporter (SERT) inhibitory activity and 5-HT1A receptor
antagonistic activity.
1-4
Indeed, it takes 2 to 3
Morphy and Rankovic propose three approaches to multiple
ligands (DMLs) lead generation, i.e. “designing in”, “designing
13
out” and “balancing” activity.
The “designing in” approach
5
focuses on incorporating a second activity into a highly selective
ligand, which originally has little or no activity for a second
target. The “designing out” approach emphasizes the removal or
weakening of an undesired activity in a non-selective ligand,
which has both the desired and undesired activities. The
“balancing” approach entails enhancing the second-activity of a
suboptimally selective ligand with weak activity for a second
target. In the lead optimization phase, the activity ratio of the
lead compound for multiple target proteins is appropriately
balanced to achieve maximum therapeutic efficacy and minimum
side effects. In the case of our study, it is important that a
compound with dual function shows comparable levels of SERT
and 5-HT1A receptor occupancy in vivo. This allowed us to set a
specific criterion for appropriate balance based on the ratio
treatment of psychiatric diseases. The slow onset of SSRIs is
due to increased serotonin levels, which in turn induces an initial
decrease in the firing activity of serotonin-producing neurons. As
this decrease is mediated via activation of somatodendritic 5-
HT1A auto-receptor, serotonin levels in the synaptic cleft can be
6,7
increased only after desensitization of 5-HT1A auto-receptor.
It has been reported that the use of pindolol, an adjuvant 5-HT1A
and β adrenergic receptor antagonist, accelerates the onset of
SSRIs therapy. 8,9 It is believed that this acceleration is mediated
by the blockage based on 5-HT1A antagonistic activity of negative
feedback inhibition via 5-HT1A auto-receptor in response to
increased serotonin.
10,11
There is another non-clinical evidence
to support the role of 5-HT1A receptors in this response, which
comes from the results that a rapid onset of antidepressant effect

between in vitro SERT and 5-HT1A receptor inhibitory activity
(within 10-fold of the concentration range). 14,15
Scheme 3. Demethylation of 8b with BBr3 followed by N-
protection with Boc2O gave the phenol 9. Alkylation with ethyl
iodide, isopropyl iodide or 2-methoxyethyl bromide transformed
the intermediate 9 to 10a – 10c. Finally, deprotection of 10a –
10c with hydrochloric acid furnished the benzyl piperidine 11a –
11c.
Based on the above, compound 1 with weak SERT binding
activity and potent 5-HT1A binding activity (SERT: Ki = 212 nM,
5-HT1A: Ki = 29.3 nM) was identified as a promising candidate.
It is reported that binding assay for SERT could be used as a
rapid method to find serotonin uptake inhibitors, because binding
activity for SERT would be well correlated with serotonin uptake
inhibitory activity. 16,17 The throughput of our radio binding assay
for SERT is higher than that of our functional assay system for
serotonin uptake inhibition. Moreover, 5-HT1A partial agonists
could be rather antagonistic for 5-HT1A receptor under an
environment where serotonin levels in the synaptic cleft are
increased by serotonin uptake inhibition, therefore we thought
that it is less reasonable to set a criterion of intrinsic activity for
Scheme 1. Reagents and Conditions: (i) PPh3, toluene, reflux, (ii) tert-butyl 4-oxopiperidine-1-
carboxylate, K2CO3, 2-PrOH, reflux, (iii) H2, 10% Pd-C, MeOH, r.t., (iv) 4N HCl/1,4-dioxane,
CHCl3, r.t.
18
5-HT1A receptor. Consequently, from the labor efficiency and
cost-effectiveness points of view, we selected an exploratory
strategy to find a compound with potent and balanced dual SERT
and 5-HT1A binding activity followed by characterizing the
functional activities for SERT and 5-HT1A receptor and in vivo
anti-depressant efficacy in the forced swim model. Optimization
of the left substituted phenyl group of compound 1 as well as the
right aryl group and finally the alkoxy group led to SMP-304
(Figure 1) with increased dual binding activity for SERT and 5-
HT1A receptor. SMP-304 with SERT inhibitory activity and 5-
HT1A weak partial agonistic activity, which could work as a 5-
HT1A antagonist, showed antidepressant-like effect in vivo with
faster onset than a known antidepressant. In this report we
describe our optimization of compound 1 as well as our
pharmacological evaluation of the obtained SMP-304.
Scheme 2. Reagents and Conditions: (v) Boc2O, THF, r.t., (vi) NCS, DMF, r.t., (vii) NBS, DMF,
r.t., (iv) 4N HCl/1,4-dioxane, CHCl3, r.t.
Scheme 3. Reagents and Conditions: (viii) BBr3, CH2Cl2, r.t., then Boc2O, THF, 20%aq.K2CO3,
r.t., (ix) R-X (X = Br or I), K2CO3, DMF, (iv) 4N HCl/1,4-dioxane, CHCl3, r.t.
Figure 1.
2. Result and Discussion
2.1. Chemistry
We started our study by optimizing the left methoxyphenyl
and the right phenyl groups of compound 1. The target
compounds were efficiently synthesized by coupling various
benzyl piperidine intermediates with 2-arylethyl intermediates.
The benzyl piperidine 5 was synthesized as shown in Scheme 1.
Treatment of the 2-(bromomethyl)-1-fluoro-4-methoxybenzene
with triphenylphosphine provided the phosphonium salt 2. Wittig
reaction of the phosphonium salt
2 with tert-butyl 4-
oxopiperidine-1-carboxylate afforded the intermediate 3, which
was hydrogenated over palladium on carbon and then deprotected
with hydrochloric acid to give the benzyl piperidine 5. As for the
preparation of 2-bromo and 2-chloro-5-methoxybenzyl
derivatives (such as 18, Figure 1), protection of the commercially
available 4-(3-methoxybenzyl)piperidine with Boc2O gave the
intermediate 6. Regiospecific chlorination or bromination of 6
with NCS or NBS followed by deprotection with hydrochloric
acid provided the benzyl piperidine 8a and 8b (Scheme 2).
Substitution of the methoxy group in the benzyl piperidine
intermediates was possible by the sequential protocol shown in
Scheme 4. Reagents and Conditions: (x) BH3-THF, THF, r.t. (xi) MeS+I-, KOH, DMSO,
40C, (xii) NaBH4, BF3-OEt2, THF, r.t., (xiii) TsCl, MeN3HCl, Et3N, CH2Cl2, 0C
To optimize the right part of compound 18 (Figure 1), various
2-arylethyl intermediates were synthesized according to the
procedure shown in Scheme 4. Reduction of 2-(2-chloro-4-
methoxyphenyl)acetic
acid
or
2-(2,3-
dihydrobenzo[b][1,4]dioxin-6-yl)acetic acid with borane-THF

complex gave the alcohol 13a or 13b, respectively. Corey-
Chaykovsky reaction of 3-chloro-4-methoxybenzaldehyde or 2,3-
dihydrobenzo[b][1,4]dioxine-5-carbaldehyde followed by ring-
opening reaction of the epoxides afforded the alcohol 13c or 13d,
respectively. The alcohols 13a – 13d were transformed to the
tosylates 14a – 14e. Direct chlorination of 4-(2-bromoethyl)-1,2-
dimethoxy-benzene or the tosylate 14b yielded the intermediate
15a or 15b, respectively (Scheme 5). The target compounds were
synthesized by coupling reaction of the benzyl piperidine
intermediates with the 2-arylethyl intermediates (Scheme 6).
group substituted with a chlorine atom showed dual binding
activity for SERT and 5-HT1A receptor in the desired range (< 2-
fold).
Although compound 35 showed dual binding activity for
SERT and 5-HT1A receptor in the desired range, it also exhibited
very potent inhibition of CYP2D6 enzyme, which is a concern
for drug-drug interaction (DDI). In fact, antidepressants with
minimal potential for DDI are preferable, because patients with
depression are often prescribed multiple drugs. 21 With the aim of
ameliorating CYP2D6 inhibition, we next turned our attention to
substitution of the methoxy group in the left hand part of
compound 35. As shown in Table 3, various alkoxy groups were
tolerated at the 3-position of the left phenyl group with no
significant influence on the desired potent dual binding activity
for SERT and 5-HT1A receptor. Also, introduction of an alkoxy
group larger than the methoxy group resulted in improvement in
CYP2D6 inhibitory activity. Finally, we selected compound 38
(SMP-304) with potent dual binding activity for SERT and 5-
HT1A receptor in the desired range and comparably weak
CYP2D6 inhibitory activity against CYP2D6 for further
evaluation, including onset of antidepressant-like effect.
Scheme 5. Reagents and Conditions: (xiv) NCS, DMF, r.t.
2.3. Effects of SMP-304 in the rat forced swimming test
We evaluated the antidepressant-like effect of SMP-304,
which has potent dual binding activity for SERT and 5-HT1A
receptor (SERT: Ki = 32.7 nM, 5-HT1A: Ki = 9.4 nM). SMP-304
Scheme 6. Reagents and Conditions: (xv) K2CO3, MeCN, reflux
2.2. Evaluation of SERT and 5-HT1A receptor binding
activity
22
inhibits serotonin reuptake with an IC50 value of 306 nM and
possesses intrinsic activity for the 5-HT1A receptor estimated at
19.9%, which means SMP-304 shows weak partial agonistic
Using the “balancing approach”, we started with Compound 1
(X = H), which has weak SERT binding activity, but potent 5-
HT1A binding activity. As previous studies have shown that 4-(3-
methoxybenzyl)piperidine derivatives with a halogen atom at 6-
position of the methoxy phenyl group possess potent SERT
affinity with moderate 5-HT1A affinity (Figure 2) 19,20, we
introduced a halogen atom (F, Cl, or Br) into the 6-position of
the left phenyl group of compound 1 to enhance SERT binding
activity while maintaining or improving 5-HT1A binding activity.
As a result, introduction of a halogen atom indeed enhanced
SERT affinity and, fortunately, improved 5-HT1A affinity.
Compound 18 with a bromine atom at the 6-position of the left
phenyl group showed the most potent SERT binding activity
(Table 1).
activity for 5-HT1A. Pindolol, a 5-HT1A and β adrenergic receptor
8,9
antagonist, accelerated the onset of SSRIs therapy.
It is
believed that the acceleration by pindolol is caused by the
mechanism of 5-HT1A autoreceptor antagonistic activity. 10,11 One
of the reasons is that propranolol, a widely used blocker for β
6
adrenergic receptor, has no antidepressant efficacy. Pindolol
also shows weak partial agonistic activity for 5-HT1A receptor
(intrinsic activity: 13.3%). 23 It is reported pindolol works as a 5-
HT1A autoreceptor antagonist to prevent the suppression effect of
paroxetine, a representative SSRI, on 5-HT firing in the dorsal
6
raphe nucleus.
Although further study is needed, it is
presumable that SMP-304 with potent binding activity and weak
partial agonistic activity for 5-HT1A as with pindolol could work
as a 5-HT1A antagonist under the environment where serotonin
levels in the synaptic cleft are increased by serotonin uptake
inhibition. The rat forced swimming test is one of the most
As compound 18 showed relatively moderate SERT affinity
with potent 5-HT1A affinity (ratio: > 20-fold based on Ki values),
it was necessary to further improve its SERT affinity to meet our
set criteria for appropriate SERT and 5-HT1A binding activity.
First we investigated replacement of the right hand part of
compound 18 (Table 2). Introduction of a substituent into the 4-
position of the right phenyl group enhanced SERT affinity, while
maintaining 5-HT1A affinity. Among the resulting compounds
(20, 19, 23 and 24), 20 with a methoxy group showed the most
potent SERT affinity. Next, introduction of a substituent, such as
a methoxy or chlorine group into the right 4-methoxy phenyl
group of compound 20, afforded compounds 27, 28 and 29 with
improved SERT affinity. Compound 31 and 32 with a naphtyl
group in the right hand part also showed more potent SERT
affinity than compound 18. In particular, the beta-naphtyl group
was more appropriate for SERT affinity than the alpha-naphtyl
group. As these results pointed to the possible suitability of
oxygen-containing bicyclic groups for the right hand part, we
introduced the 1,4-benzodioxane group into the right hand part to
obtain compound 34, which exhibited potent dual SERT and 5-
HT1A binding activity. Compound 35 with the 1,4-benzodioxane
commonly used behavioral tests as a model to evaluate
23
antidepressant-like effect of drug candidates.
In the 2-day
administration test (Figure 3), treatment with SMP-304 at 1 and
3 mg/kg p.o. clearly reduced rats immobility time without
increasing locomotor activity, whereas paroxetine had no such
effect at 3 or 10 mg/kg p.o.. The total brain concentrations of
SMP-304 (1 and 3 mg/kg, p.o.), as measured following repeated
administration of SMP-304 at 26 hours and 2 hours before
concentration measurement, were 6.74 nmol/kg for 1 mg/kg and
30.3 nmol/kg for 3 mg/kg in the wet brain tissue (Figure 4).
These findings indicate that sufficient amounts of SMP-304
migrate to the brain and induce antidepressant-like effect at lower
doses and faster onset than paroxetine. In fact, whereas
paroxetime requires 3 weeks treatment for any efficacy 23, SMP-
304 produces antidepressant-like effect after the 2-day treatment.
It is conceivable that the effect was mediated by SERT inhibitory
activity and 5-HT1A antagonistic activity.

Figure 2.
Table 1. SERT and 5-HT1A receptor binding activity in the optimization of X
a
Compound
X
H
SERTa
212 + 46
274 + 70
102 + 15
66.4 + 11.3
5-HT1A
1
29.3 + 4.1
6.04 + 0.0
2.27 + 0.38
2.72 + 0.81
16
17
18
F
Cl
Br
a Ki values (nM) are the means of two independent experiments.
Table 2. SERT and 5-HT1A receptor binding activity in the optimization of Ar group
a
a
Compound
Ar
SERTa
5-HT1A
Compound
Ar
SERTa
5-HT1A
18
66.4 + 11.3
2.72 + 0.81
27
30.5 + 2.3
1.12 + 0.48
1.04 + 0.18
1.56 + 0.22
1.96 + 0.45
19
20
21
65.3 + 7.7
37.5 + 4.1
37.6 + 3.0
1.40 + 0.66
0.81 + 0.19
3.03 + 0.60
28
29
30
18.9 + 0.2
26.9 + 1.0
28.0 + 2.9
22
23
24
37.5 + 4.1
51.2 + 6.5
46.2 + 1.5
0.82 + 0.17
1.04 + 0.34
1.34 + 0.59
31
32
33
38.9 + 6.8
25.6 + 5.2
25.6 + 0.3
4.81 + 0.49
0.89 + 0.14
2.12 + 0.09
25
26
62.4 + 3.1
42.6 + 1.2
4.08 + 0.75
2.93 + 0.78
34
35
14.0 + 1.4
12.4 + 1.5
0.36 + 0.05
6.46 + 0.57
a Ki values (nM) are the means of two independent experiments.

Table 3. SERT and 5-HT1A receptor binding activity and CYP2D6 inhibitory activity in the optimization of R
a
Compound
R
Me
Et
SERTa
5-HT1A
CYP2D6b
<0.4
35
36
37
12.4 + 1.5
18.7 + 2.1
13.9 + 0.3
6.46 + 0.57
23.4 + 6.1
15.9 + 3.6
1.4
2-Pr
1.5
38
32.7 + 3.4
9.4 + 0.2
3.0
(HCl salt: SMP-304)
a Ki values (nM) are the means of at least two independent experiments.
b IC50 value (uM).
Figure 3. Effects of 2-day of SMP-304 and paroxetine on parameters in the forced swimming test in rats. (A) Effect of SMP-304 on immobility time in
the forced swimming test in rats. (B) Effect of paroxetine on immobility time in the forced swimming test in rats. (C) Effect of SMP-304 on spontaneous
locomotor activity in rats. Each bar represents the mean ± S.E.M. n = 10 (spontaneous locomotor activity) or 12 (forced swimming test) per group. * P
< 0.05, significantly different from the vehicle-treated group (Dunnett’s multiple comparison test).
Figure 4. Total plasma and brain concentrations of
SMP-304 (1 and 3 mg/kg, p.o.) as measured following
repeated administration of SMP-304 at 26 hours and 2
hours before concentration measurement.
3. Conclusion
SSRI. Although further studies with more compounds are needed
to validate our hypothesis, these results suggest that a compound
with SERT inhibitory activity and 5-HT1A antagonistic activity
would likely show antidepressant activities earlier time point than
SSRI alone.
Starting from compound 1, focusing on the binding activity
for SERT and 5-HT1A receptor from the labor efficiency and
cost-effectiveness points of view and using the DMLs
“balancing” approach, we found in this study SMP-304 with
potent and balanced dual SERT and 5-HT1A binding activity.
SMP-304 with serotonin uptake inhibition and 5-HT1A weak
partial agonistic activity, which could work as a 5-HT1A
antagonist, induced antidepressant-like effect in the rat
swimming test with faster onset than paroxetine, a representative

(ESI) m/z calcd for C21H27ClNO [M+H]+ 344.1776; found
344.1773.
4. Experimental section
4.1. Chemistry
4.1.4. 4-(2-Bromo-5-methoxybenzyl)-1-(2-
phenylethyl)piperidine (18)
Melting points were determined on Stanford Research
Systems OptiMelt MPA 100 without correction. NMR spectra
were recorded at ambient temperature on a JEOL JMN-LA300
spectrometer. Chemical shifts are expressed in δ values (ppm)
relative to a tetramethylsilane as an internal standard, and signals
are expressed as s (singlet), d (doublet), t (triplet), m (multiplet)
or br (broad). High-resolution mass spectra (HRMS) were
recorded on a Thermo Fisher Scientific LTQ orbitrap Discovery
MS equipment. Elemental analysis was performed on a CE
Instrument EA1110 and a Yokokawa analytical system IC7000.
In general, reagents and solvents were used as obtained from
commercial suppliers without further purification. Reaction
progress was determined by thin layer chromatography (TLC)
analysis on a Merck silica gel 60 F254 precoated glass plate.
Visualization was done with UV light (254 nm) or iodine.
Column chromatography was carried out using a Yamazen W-
prep system and performed using prepacked silica-gel columns.
All reactions were carried out under a nitrogen atmosphere unless
otherwise mentioned.
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
8b and (2-bromoethyl)benzene. (72%) white solid. 1H NMR (300
MHz,CDCl3) δ: 1.31-1.49 (2H, m), 1.61-1.74 (3H, m), 1.90-2.03
(2H, m), 2.52-2.61 (2H, m), 2.64 (2H, d, J = 6.6 Hz), 2.76-2.85
(2H, m), 2.93-3.04 (2H, m), 3.78 (3H, s), 6.63 (1H, dd, J = 8.8,
2.9 Hz), 6.72 (1H, d, J = 3.1 Hz), 7.14-7.23 (3H, m), 7.24-7.31
(2H, m), 7.41 (1H, d, J = 8.6 Hz); HRMS (ESI) m/z calcd for
C21H27BrNO [M+H]+ 388.1271; found 388.1270.
4.1.5. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(4-
methylphenyl)ethyl]piperidine (19)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
1
8b and 4-methylphenethyl bromide. (98%) white solid. H NMR
(300 MHz,CDCl3) δ: 1.33-1.50 (2H, m), 1.62-1.73 (3H, m), 1.89-
2.03 (2H, m), 2.31 (3H, s), 2.50-2.59 (2H, m), 2.64 (2H, d, J =
6.4 Hz), 2.72-2.82 (2H, m), 2.94-3.05 (2H, m), 3.78 (3H, s), 6.63
(1H, dd, J = 8.7, 3.0 Hz), 6.71 (1H, d, J = 3.1 Hz), 7.09 (4H, s),
7.41 (1H, d, J = 8.8 Hz); HRMS (ESI) m/z calcd for C22H29BrNO
[M+H]+ 402.1427; found 402.1426.
4.1.1. 4-(3-Methoxybenzyl)-1-(2-
phenylethyl)piperidine (1)
To a mixture of 4-(3-methoxy-benzyl)-piperidine (100 mg,
0.487 mmol) and potassium carbonate (101 mg, 0.731 mmol) in
MeCN (2.4 mL) was added (2-bromoethyl)benzene (86.5 uL,
0.633 mmol). After reflux for 24 h, EtOAc (7.2 mL) was added
to the reaction mixture, and the whole was filtered. The filtrate
was evaporated in vacuo and the residue was purified by silica
4.1.6. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(4-
methoxyphenyl)ethyl]piperidine (20)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
8b and 4-methoxyphenethyl bromide. (98%) pale yellow oil. 1H
NMR (300 MHz,CDCl3) δ: 1.37-1.59 (2H, m), 1.62-1.76 (3H, m),
1.93-2.13 (2H, m), 2.52-2.71 (4H, m), 2.74-2.88 (2H, m), 2.98-
3.13 (2H, m), 3.78 (6H, s), 6.64 (1H, dd, J = 8.7, 3.0 Hz), 6.71
(1H, d, J = 2.9 Hz), 6.82 (2H, d, J = 8.8 Hz), 7.12 (2H, d, J = 8.6
Hz), 7.41 (1H, d, J = 8.8 Hz); HRMS (ESI) m/z calcd for
C22H29BrNO2 [M+H]+ 418.1376; found 418.1377.
gel chromatography using
25% EtOAc/hexane and 10%
MeOH/CHCl3 as eluent to give 100 mg (66%) of the title
compound as a pale yellow oil. 1H NMR (300 MHz,CDCl3) δ:
1.26-1.41 (2H, m), 1.46-1.59 (1H, m), 1.63-1.72 (2H, m), 1.95
(2H, td, J = 11.9, 2.3 Hz), 2.50-2.60 (4H, m), 2.76-2.84 (2H, m),
2.95-3.03 (2H, m), 3.80 (3H, s), 6.69-6.77 (3H, m), 7.15-7.23
(4H, m), 7.24-7.31 (2H, m); HRMS (ESI) m/z calcd for
C21H28NO [M+H]+ 310.2165; found 310.2164.
4.1.7. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(2-
methoxyphenyl)ethyl]piperidine (21)
4.1.2. 4-(2-Fluoro-5-methoxybenzyl)-1-(2-
phenylethyl)piperidine (16)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
1
8b and 2-methoxyphenethyl bromide. (86%) pale yellow oil. H
To a mixture of the benzyl piperidine intermediate 5 (200 mg,
0.770 mmol) and potassium carbonate (266 mg, 1.93 mmol) in
MeCN (3.0 mL) was added (2-bromoethyl)benzene (137 uL, 1.00
mmol). After reflux for 24 h, EtOAc (9.0 mL) was added to the
reaction mixture and the whole was filtered. The filtrate was
evaporated in vacuo and the residue was purified by silica gel
chromatography using 0%-1% MeOH/CHCl3 as eluent to give
228 mg (90%) of the title compound as a pale yellow solid. 1H
NMR (300 MHz,CDCl3) δ: 1.28-1.46 (2H, m), 1.50-1.74 (3H,
m), 1.96 (2H, br t, J = 10.7 Hz), 2.51-2.60 (4H, m), 2.75-2.84
(2H, m), 2.99 (2H, br d, J = 11.4 Hz), 3.77 (3H, s), 6.64-6.71
(2H, m), 6.88-6.97 (1H, m), 7.16-7.23 (3H, m), 7.24-7.32 (2H,
m); HRMS (ESI) m/z calcd for C21H27FNO [M+H]+ 328.2071;
found 328.2074.
NMR (300 MHz,CDCl3) δ: 1.36-1.54 (2H, m), 1.58-1.74 (3H,
m), 1.93-2.07 (2H, m), 2.50-2.60 (2H, m), 2.65 (2H, d, J = 6.4
Hz), 2.79-2.89 (2H, m), 2.98-3.09 (2H, m), 3.78 (3H, s), 3.80
(3H, s), 6.63 (1H, dd, J = 8.7, 3.0 Hz), 6.72 (1H, d, J = 3.1 Hz),
6.80-6.91 (2H, m), 7.11-7.22 (2H, m), 7.41 (1H, d, J = 8.8 Hz);
HRMS (ESI) m/z calcd for C22H29BrNO2 [M+H]+ 418.1376;
found 418.1371.
4.1.8. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(3-
methoxyphenyl)ethyl]piperidine (22)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
1
8b and 3-methoxyphenethyl bromide. (89%) pale yellow oil. H
NMR (300 MHz,CDCl3) δ: 1.31-1.51 (2H, m), 1.63-1.75 (3H,
m), 1.89-2.03 (2H, m), 2.52-2.61 (2H, m), 2.64 (2H, d, J = 6.6
Hz), 2.74-2.83 (2H, m), 2.94-3.04 (2H, m), 3.78 (3H, s), 3.79
(3H, s), 6.63 (1H, dd, J = 8.8, 2.9 Hz), 6.70-6.82 (4H, m), 7.19
(1H, dd, J = 8.1, 8.1 Hz), 7.41 (1H, d, J = 8.6 Hz); HRMS (ESI)
m/z calcd for C22H29BrNO2 [M+H]+ 418.1376; found 418.1376.
4.1.3. 4-(2-Chloro-5-methoxybenzyl)-1-(2-
phenylethyl)piperidine (17)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
8a and (2-bromoethyl)benzene. (94%) white solid. 1H NMR (300
MHz,CDCl3) δ: 1.32-1.49 (2H, m), 1.55-1.65 (2H, m), 1.68-1.74
(1H, m), 1.89-2.04 (2H, m), 2.49-2.61 (2H, m), 2.64 (2H, d, J =
6.4 Hz), 2.76-2.86 (2H, m), 2.95-3.04 (2H, m), 3.78 (3H, s),
6.65-6.74 (2H, m), 7.16-7.24 (4H, m), 7.24-7.31 (2H, m); HRMS
4.1.9. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(4-
fluorophenyl)ethyl]piperidine (23)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate

1
8b and 4-fluorophenethyl bromide. (98%) white solid. H NMR
d, J = 8.4 Hz), 7.05 (1H, dd, J = 8.3, 2.1 Hz), 7.20 (1H, d, J = 2.0
Hz), 7.41 (1H, d, J = 8.8 Hz); HRMS (ESI) m/z calcd for
C22H28BrClNO2 [M+H]+ 452.0986; found 452.0982.
(300 MHz,CDCl3) δ: 1.30-1.49 (2H, m), 1.63-1.74 (3H, m), 1.88-
2.03 (2H, m), 2.48-2.57 (2H, m), 2.64 (2H, d, J = 6.4 Hz), 2.73-
2.82 (2H, m), 2.93-3.02 (2H, m), 3.78 (3H, s), 6.63 (1H, dd, J =
8.7, 3.0 Hz), 6.71 (1H, d, J = 3.1 Hz), 6.95 (2H, dd, J = 8.8, 8.8
Hz), 7.14 (2H, dd, J = 8.6, 5.5 Hz), 7.41 (1H, d, J = 8.8 Hz);
HRMS (ESI) m/z calcd for C21H26BrFNO [M+H]+ 406.1176;
found 406.1188.
4.1.15. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(3,4-
dimethoxyphenyl)ethyl]piperidine (29)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
8b and 3,4-dimethoxyphenethyl bromide. (98%) pale yellow oil.
1H NMR (300 MHz,CDCl3) δ: 1.45-1.76 (5H, m), 1.95-2.15 (2H,
m), 2.58-2.70 (4H, m), 2.77-2.87 (2H, m), 3.01-3.15 (2H, m),
3.78 (3H, s), 3.85 (3H, s), 3.87 (3H, s), 6.64 (1H, dd, J = 8.6, 3.1
Hz), 6.70-6.81 (4H, m), 7.42 (1H, d, J = 8.8 Hz); HRMS (ESI)
m/z calcd for C23H31BrNO3 [M+H]+ 448.1482; found 448.1478.
4.1.10. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(4-
chlorophenyl)ethyl]piperidine (24)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
8b and 4-chlorophenethyl bromide. (98%) white solid. 1H NMR
(300 MHz,CDCl3) δ: 1.30-1.48 (2H, m), 1.61-1.74 (3H, m), 1.88-
2.01 (2H, m), 2.49-2.56 (2H, m), 2.64 (2H, d, J = 6.4 Hz), 2.73-
2.80 (2H, m), 2.92-3.00 (2H, m), 3.78 (3H, s), 6.63 (1H, dd, J =
8.7, 3.0 Hz), 6.71 (1H, d, J = 3.1 Hz), 7.12 (2H, d, J = 8.4 Hz),
7.24 (2H, d, J = 8.4 Hz), 7.41 (1H, d, J = 8.8 Hz); HRMS (ESI)
m/z calcd for C21H26BrClNO [M+H]+ 422.0881; found 422.0881.
4.1.16. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(2-
chloro-4,5-dimethoxyphenyl)ethyl]piperidine (30)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
1
8b and the bromide intermediate 15b. (97%) pale yellow oil. H
NMR (300 MHz,CDCl3) δ: 1.41-1.75 (5H, m), 1.99-2.15 (2H,
m), 2.53-2.62 (2H, m), 2.65 (2H, d, J = 6.4 Hz), 2.85-2.96 (2H,
m), 3.00-3.11 (2H, m), 3.78 (3H, s), 3.84 (3H, s), 3.85 (3H, s),
6.64 (1H, dd, J = 8.6, 3.1 Hz), 6.72 (1H, d, J = 3.1 Hz), 6.76 (1H,
s), 6.83 (1H, s), 7.42 (1H, d, J = 8.8 Hz); HRMS (ESI) m/z calcd
for C23H30BrClNO3 [M+H]+ 482.1092; found 482.1091.
4.1.11. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(2-
chlorophenyl)ethyl]piperidine (25)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
1
8b and 2-chlorophenethyl bromide. (54%) white solid. H NMR
(300 MHz,CDCl3) δ: 1.33-1.51 (2H, m), 1.63-1.75 (3H, m), 1.95-
2.08 (2H, m), 2.52-2.61 (2H, m), 2.65 (2H, d, J = 6.4 Hz), 2.90-
2.97 (2H, m), 2.98-3.06 (2H, m), 3.78 (3H, s), 6.63 (1H, dd, J =
8.6, 3.1 Hz), 6.72 (1H, d, J = 3.1 Hz), 7.09-7.21 (2H, m), 7.24
(1H, dd, J = 7.2, 2.3 Hz), 7.33 (1H, dd, J = 7.4, 1.7 Hz), 7.41 (1H,
d, J = 8.6 Hz); HRMS (ESI) m/z calcd for C21H26BrClNO
[M+H]+ 422.0881; found 422.0882.
4.1.17. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(5,8-
dihydronaphthalen-1-yl)ethyl]piperidine (31)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
1
8b and 2-(1-naphthyl)ethyl bromide. (99%) pale yellow oil. H
NMR (300 MHz,CDCl3) δ: 1.39-1.55 (2H, m), 1.67-1.77 (3H,
m), 2.00-2.14 (2H, m), 2.64-2.76 (4H, m), 3.05-3.16 (2H, m),
3.26-3.36 (2H, m), 3.79 (3H, s), 6.64 (1H, dd, J = 8.8, 2.9 Hz),
6.73 (1H, d, J = 3.1 Hz), 7.32-7.54 (5H, m), 7.71 (1H, d, J = 7.5
Hz), 7.82-7.87 (1H, m), 8.06 (1H, d, J = 7.7 Hz); HRMS (ESI)
m/z calcd for C25H29BrNO [M+H]+ 438.1427; found 438.1427.
4.1.12. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(3-
chlorophenyl)ethyl]piperidine (26)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
1
8b and 3-chlorophenethyl bromide. (86%) pale yellow oil. H
4.1.18. 4-(2-Bromo-5-methoxybenzyl)-1-[2-
(naphthalen-2-yl)ethyl]piperidine (32)
NMR (300 MHz,CDCl3) δ: 1.31-1.47 (2H, m), 1.62-1.73 (3H,
m), 1.90-2.01 (2H, m), 2.50-2.58 (2H, m), 2.64 (2H, d, J = 6.4
Hz), 2.74-2.81 (2H, m), 2.92-3.00 (2H, m), 3.78 (3H, s), 6.63
(1H, dd, J = 8.6, 3.1 Hz), 6.71 (1H, d, J = 2.9 Hz), 7.05-7.10 (1H,
m), 7.14-7.23 (3H, m), 7.42 (1H, d, J = 8.8 Hz); HRMS (ESI)
m/z calcd for C21H26BrClNO [M+H]+ 422.0881; found 422.0882.
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
8b and the tosylate intermediate 14e. (86%) white solid. 1H NMR
(300 MHz,CDCl3) δ: 1.34-1.52 (2H, m), 1.63-1.75 (3H, m), 1.95-
2.06 (2H, m), 2.61-2.71 (4H, m), 2.93-3.08 (4H, m), 3.78 (3H, s),
6.64 (1H, dd, J = 8.8, 3.1 Hz), 6.72 (1H, d, J = 2.9 Hz), 7.34 (1H,
dd, J = 8.3, 1.7 Hz), 7.38-7.48 (3H, m), 7.63 (1H, br s), 7.74-7.82
(3H, m); HRMS (ESI) m/z calcd for C25H29BrNO [M+H]+
438.1427; found 438.1429.
4.1.13. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(2-
chloro-4-methoxyphenyl)ethyl]piperidine (27)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
8b and the tosylate intermediate 14a. (99%) white solid. 1H NMR
(300 MHz,CDCl3) δ: 1.36-1.53 (2H, m), 1.58-1.74 (3H, m), 1.96-
2.08 (2H, m), 2.49-2.59 (2H, m), 2.64 (2H, d, J = 6.4 Hz), 2.83-
2.93 (2H, m), 2.98-3.07 (2H, m), 3.77 (3H, s), 3.78 (3H, s), 6.63
(1H, dd, J = 8.7, 3.0 Hz), 6.70-6.77 (2H, m), 6.89 (1H, d, J = 2.6
Hz), 7.13 (1H, d, J = 8.4 Hz), 7.41 (1H, d, J = 8.6 Hz); HRMS
(ESI) m/z calcd for C22H28BrClNO2 [M+H]+ 452.0986; found
452.0986.
4.1.19. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(2,3-
dihydro-1,4-benzodioxin-5-yl)ethyl]piperidine
(33)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
1
8b and the tosylate intermediate 14d. (99%) pale yellow oil. H
NMR (300 MHz,CDCl3) δ: 1.35-1.52 (2H, m), 1.63-1.74 (3H,
m), 1.92-2.05 (2H, m), 2.50-2.60 (2H, m), 2.64 (2H, d, J = 6.4
Hz), 2.74-2.84 (2H, m), 2.96-3.07 (2H, m), 3.78 (3H, s), 4.24
(4H, s), 6.63 (1H, dd, J = 8.7, 3.0 Hz), 6.68-6.77 (4H, m), 7.41
(1H, d, J = 8.6 Hz); HRMS (ESI) m/z calcd for C23H29BrNO3
[M+H]+ 446.1325; found 446.1324.
4.1.14. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(3-
chloro-4-methoxyphenyl)ethyl]piperidine (28)
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
1
8b and the tosylate intermediate 14c. (98%) pale yellow oil. H
4.1.20. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(2,3-
dihydro-1,4-benzodioxin-6-yl)ethyl]piperidine
(34)
NMR (300 MHz,CDCl3) δ: 1.32-1.50 (2H, m), 1.55-1.74 (3H, m),
1.89-2.02 (2H, m), 2.48-2.57 (2H, m), 2.64 (2H, d, J = 6.6 Hz),
2.69-2.77 (2H, m), 2.93-3.01 (2H, m), 3.78 (3H, s), 3.87 (3H, s),
6.63 (1H, dd, J = 8.7, 3.0 Hz), 6.71 (1H, d, J = 3.1 Hz), 6.84 (1H,

The title compound was prepared in a manner similar to that
5.75; N, 2.50; Cl, 12.63; Br, 14.23. Found: C, 53.69; H, 5.76; N,
2.57; Cl, 12.69; Br, 14.22.
for the preparation of 16 using the benzyl piperidine intermediate
1
8b and the tosylate intermediate 14b. (72%) colorless oil. H
4.1.25. (2-Fluoro-5-
methoxybenzyl)(triphenyl)phosphonium bromide
(2)
NMR (300 MHz,CDCl3) δ: 1.31-1.47 (2H, m), 1.61-1.72 (3H, m),
1.88-2.00 (2H, m), 2.47-2.56 (2H, m), 2.60-2.74 (4H, m), 2.92-
3.01 (2H, m), 3.78 (3H, s), 4.23 (4H, s), 6.60-6.68 (2H, m), 6.70-
6.72 (2H, m), 6.77 (1H, d, J = 8.3 Hz), 7.41 (1H, d, J = 8.6 Hz);
HRMS (ESI) m/z calcd for C23H29BrNO3 [M+H]+ 446.1325;
found 446.1330.
To a solution of 2-fluoro-5-methoxybenzyl bromide (12.5 g,
57.1 mmol) in toluene (150 mL) was added triphenylphosphine
(16.5 g, 62.8 mmol). After reflux for 4 h, the resulting solid was
collected and washed with toluene (20 mL x 3) to give 19.8 g
1
4.1.21. 4-(2-Bromo-5-methoxybenzyl)-1-[2-(7-
chloro-2,3-dihydro-1,4-benzodioxin-6-yl)ethyl]-
piperidine (35)
(64%) of the title compound as a white solid. H NMR (300
MHz,DMSO-D6) δ: 3.48 (3H, s), 5.10 (2H, d, J = 15.4 Hz), 6.47-
6.54 (1H, m), 6.87-6.95 (1H, m), 7.06 (1H, dd, J = 9.2, 9.2 Hz),
7.66-7.80 (12H, m), 7.87-7.97 (3H, m); HRMS (ESI) m/z calcd
for C26H23FOP [M+H]+ 401.1465; found 401.1472.
The title compound was prepared in a manner similar to that
for the preparation of 16 using the benzyl piperidine intermediate
1
8b and the tosylate intermediate 15b. (83%) white solid. H
4.1.26. tert-Butyl 4-(2-fluoro-5-
methoxybenzylidene)piperidine-1-carboxylate (3)
NMR (300 MHz,CDCl3) δ: 1.33-1.50 (2H, m), 1.63-1.73 (3H,
m), 1.93-2.05 (2H, m), 2.46-2.55 (2H, m), 2.64 (2H, d, J = 6.6
Hz), 2.76-2.85 (2H, m), 2.94-3.04 (2H, m), 3.78 (3H, s), 4.22
(4H, s), 6.63 (1H, dd, J = 8.7, 3.0 Hz), 6.71 (1H, d, J = 3.1 Hz),
6.73 (1H, s), 6.85 (1H, s), 7.41 (1H, d, J = 8.6 Hz); HRMS (ESI)
m/z calcd for C23H28BrClNO3 [M+H]+ 480.0936; found
480.0927.
To a mixture of the phosphonium salt 2 (15 g, 27.7 mmol) and
potassium carbonate (5.74 g, 41.6 mmol) in 2-PrOH was added
1-(tert-Butoxycarbonyl)-4-oxopiperidine (6.06 g, 30.4 mmol).
After reflux for 5 h, the reaction mixture was filtered, and the
filtrate was evaporated in vacuo. Et2O (100 mL) was then added
to the residue and stirred for 30 min at room temperature. The
resulting solid was filtered out, and the filtrate was concentrated.
The residue was purified by silica gel chromatography using 0-
13% EtOAc/hexane as eluent to give 7.32 g (82%) of the title
4.1.22. 4-(2-Bromo-5-ethoxybenzyl)-1-[2-(7-
chloro-2,3-dihydro-1,4-benzodioxin-6-yl)ethyl]-
piperidine (36)
1
The title compound was prepared in a manner similar to that
compound as a colorless oil. H NMR (300 MHz,CDCl3) δ: 1.47
for the preparation of 16 using the benzyl piperidine intermediate
11a and the tosylate intermediate 15b. (84%) white solid. H
(9H, s), 2.30-2.39 (4H, m), 3.41 (2H, t, J = 5.7 Hz), 3.52 (2H, t, J
= 5.9 Hz), 3.77 (3H, s), 6.23 (1H, s), 6.65-6.76 (2H, m), 6.96
(1H, dd, J = 9.1, 9.1 Hz).
1
NMR (300 MHz,CDCl3) δ: 1.32-1.50 (5H, m), 1.63-1.73 (3H,
m), 1.92-2.06 (2H, m), 2.46-2.57 (2H, m), 2.63 (2H, d, J = 6.4
Hz), 2.76-2.85 (2H, m), 2.94-3.04 (2H, m), 3.99 (2H, q, J = 7.0
Hz), 4.22 (4H, s), 6.61 (1H, dd, J = 8.8, 2.9 Hz), 6.71 (1H, d, J =
2.9 Hz), 6.73 (1H, s), 6.85 (1H, s), 7.39 (1H, d, J = 8.8 Hz);
HRMS (ESI) m/z calcd for C24H30BrClNO3 [M+H]+ 494.1092;
found 494.1099.
4.1.27. tert-Butyl 4-(2-fluoro-5-
methoxybenzyl)piperidine-1-carboxylate (4)
The olefin intermediate 3 (5.30 g, 16.5 mmol) was dissolved
in MeOH (30 mL) and hydrogenated over 10% Pd on carbon
(water ~50%, 1.00 g) at room temperature for 5 h. The catalyst
was removed by filtration, and the filtrate was concentrated in
vacuo. The residue was purified by silica gel chromatography
using 3-23% EtOAc/hexane as eluent to give 4.95 g (93%) of the
4.1.23. 4-[2-Bromo-5-(propan-2-yloxy)benzyl]-1-
[2-(7-chloro-2,3-dihydro-1,4-benzodioxin-6-
yl)ethyl]piperidine (37)
1
title compound as a colorless oil. H NMR (300 MHz,CDCl3) δ:
The title compound was prepared in a manner similar to that
1.08-1.24 (2H, m), 1.45 (9H, s), 1.58-1.66 (2H, m), 1.66-1.78
(1H, m), 2.54 (2H, dd, J = 7.1, 1.0 Hz), 2.57-2.71 (2H, m), 3.77
(3H, s), 3.99-4.15 (2H, m), 6.62-6.71 (2H, m), 6.93 (1H, dd, J =
9.1, 9.1 Hz).
for the preparation of 16 using the benzyl piperidine intermediate
1
11b and the tosylate intermediate 15b. (81%) colorless oil. H
NMR (300 MHz,CDCl3) δ: 1.32 (6H, d, J = 6.1 Hz), 1.35-1.50
(2H, m), 1.63-1.73 (3H, m), 1.93-2.07 (2H, m), 2.47-2.56 (2H,
m), 2.62 (2H, d, J = 6.4 Hz), 2.76-2.86 (2H, m), 2.94-3.05 (2H,
m), 4.22 (4H, s), 4.42-4.56 (1H, m), 6.61 (1H, dd, J = 8.8, 2.9
Hz), 6.70 (1H, d, J = 2.9 Hz), 6.73 (1H, s), 6.85 (1H, s), 7.39
(1H, d, J = 8.8 Hz); HRMS (ESI) m/z calcd for C25H32BrClNO3
[M+H]+ 508.1248; found 508.1251.
4.1.28. 4-(2-Fluoro-5-methoxybenzyl)piperidine
hydrochloride (5)
To a solution of intermediate 4 (4.00 g, 12.4 mmol) in CHCl3
(15 mL) was added 4 N HCl/1,4-dioxane (30 mL). After stirring
at room temperature for 21 h, the reaction mixture was
evaporated in vacuo. Et2O (50 mL) was then added to the residue
and stirred at room temperature for 30 min. The resulting solid
was collected to obtain 3.16 g (98%) of the title compound as a
4.1.24. 4-[2-Bromo-5-(2-methoxyethoxy)benzyl]-
1-[2-(7-chloro-2,3-dihydro-1,4-benzodioxin-6-
yl)ethyl]piperidine (38)
1
white solid. H NMR (300 MHz,DMSO-D6) δ: 1.27-1.46 (2H,
The title compound was prepared in a manner similar to that
m), 1.62-1.75 (2H, m), 1.75-1.89 (1H, m), 2.53 (2H, d, J = 6.1
Hz), 2.71-2.85 (2H, m), 3.16-3.26 (2H, m), 3.72 (3H, s), 6.76-
6.86 (2H, m), 7.08 (1H, dd, J = 9.2, 9.2 Hz), 8.72 (2H, br s);
HRMS (ESI) m/z calcd for C13H19FNO [M+H]+ 224.1445; found
224.1451.
for the preparation of 16 using the benzyl piperidine intermediate
1
11c and the tosylate intermediate 15b. (81%) colorless oil. H
NMR (300 MHz,CDCl3) δ: 1.30-1.50 (2H, m), 1.62-1.73 (3H,
m), 1.92-2.07 (2H, m), 2.46-2.57 (2H, m), 2.63 (2H, d, J = 6.2
Hz), 2.76-2.86 (2H, m), 2.94-3.05 (2H, m), 3.45 (3H, s), 3.71-
3.77 (2H, m), 4.05-4.11 (2H, m), 4.22 (4H, s), 6.65 (1H, dd, J =
8.8, 3.1 Hz), 6.73 (1H, s), 6.76 (1H, d, J = 2.9 Hz), 6.85 (1H, s),
7.40 (1H, d, J = 8.8 Hz); HRMS (ESI) m/z calcd for
C25H32BrClNO4 [M+H]+ 524.1197; found 524.1199. The product
was converted into the corresponding hydrochloride salt. Mp:
155-158°C. Anal. Calcd for C25H31BrClNO4HCl: C, 53.49; H,
4.1.29. tert-Butyl 4-(3-methoxybenzyl)piperidine-
1-carboxylate (6)
To a solution of 4-(3-methoxybenzyl)piperidine (1.70 g,
8.28mmol) in THF (15 mL) was added a solution of di-tert-butyl
dicarbonate (1.90 g, 8.69 mmol) in THF (5 mL). After stirring at
room temperature for 3h, the reaction mixture was concentrated.
The residue was purified by silica gel chromatography using 1-

22% EtOAc/hexane as eluent to give 2.57 g (quant.) of the title
compound as a colorless oil. 1H NMR (300 MHz,CDCl3) δ: 1.05-
1.22 (2H, m), 1.45 (9H, s), 1.52-1.74 (3H, m), 2.51 (2H, d, J =
6.8 Hz), 2.63 (2H, t, J = 12.4 Hz), 3.80 (3H, s), 3.97-4.16 (2H,
m), 6.66-6.71 (1H, m), 6.71-6.78 (2H, m), 7.20 (1H, dd, J = 7.8,
7.8 Hz).
EtOAc (50 mL x 2). The combined organic layer was dried over
MgSO4, filtered and concentrated. The residue was crystallized
using EtOAc (4 mL) and stirred at room temperature for 10min.
Hexane (8 mL) was then added to the mixture, and the whole was
stirred at room temperature for 20min. The resulting solid was
filtered, rinsed with EtOAc/hexane = 1/2 (1 mL) and collected to
1
give 1.38 g (80%) of the title compound as a white solid. H
4.1.30. tert-Butyl 4-(2-chloro-5-
methoxybenzyl)piperidine-1-carboxylate (7a)
NMR (300 MHz,CDCl3) δ: 1.11-1.27 (2H, m), 1.46 (9H, s), 1.60-
1.69 (2H, m), 1.70-1.87 (1H, m), 2.49-2.75 (4H, m), 3.99-4.15
(2H, m), 5.93 (1H, s), 6.58 (1H, dd, J = 8.6, 2.9 Hz), 6.68 (1H, d,
J = 3.1 Hz), 7.35 (1H, d, J = 8.6 Hz).
To a solution of intermediate 6 (300 mg, 0.982 mmol) in DMF
(3 mL) was added N-chlorosuccinimide (138 mg, 1.03 mmol).
After stirring at 60°C for 12h, H2O (30 mL) was added to the
reaction mixture and the whole was extracted with EtOAc (30
mL x 2). The combined organic layer was dried over MgSO4,
filtered and concentrated. The residue was purified by silica gel
chromatography using 1-22% EtOAc/hexane as eluent to give
4.1.35. tert-butyl 4-(2-bromo-5-
ethoxybenzyl)piperidine-1-carboxylate (10a)
To a mixture of intermediate 9 (1.00 g, 2.70 mmol) and
potassium carbonate (1.16 g, 8.37 mmol) in DMF (5 mL) was
added iodoethane (0.648 mL, 8.10 mmol). After stirring at 70°C
for 8 h, the reaction mixture was treated with H2O (40 mL) and
extracted with EtOAc (40 mL). To the organic layer was added
toluene (40 mL), and the mixture was washed with H2O (40 mL).
The organic layer was dried over MgSO4, filtered and
concentrated in vacuo. The residue was purified by silica gel
chromatography using 0~16% EtOAc/hexane as eluent to give
1.12 g (quant.) of the title compound as a colorless oil. 1H NMR
(300 MHz,CDCl3) δ: 1.12-1.28 (2H, m), 1.40 (3H, t, J = 7.0 Hz),
1.45 (9H, s), 1.59-1.67 (2H, m), 1.69-1.86 (1H, m), 2.56-2.71
(4H, m), 3.94-4.15 (4H, m), 6.62 (1H, dd, J = 8.7, 3.0 Hz), 6.69
(1H, d, J = 3.1 Hz), 7.40 (1H, d, J = 8.8 Hz).
1
290 mg (87%) of the title compound as a colorless oil. H NMR
(300 MHz,CDCl3) δ: 1.11-1.28 (2H, m), 1.45 (9H, s), 1.58-1.67
(2H, m), 1.69-1.85 (1H, m), 2.56-2.72 (4H, m), 3.78 (3H, s),
3.99-4.17 (2H, m), 6.67-6.72 (2H, m), 7.21-7.26 (1H, m).
4.1.31. 4-(2-Chloro-5-methoxybenzyl)piperidine
hydrochloride (8a)
The title compound was prepared in a manner similar to that
for the preparation of 5 using intermediate 7a. (89%) white solid.
1H NMR (300 MHz,DMSO-D6) δ: 1.33-1.50 (2H, m), 1.64-1.75
(2H, m), 1.77-1.95 (1H, m), 2.62 (2H, d, J = 7.0 Hz), 2.73-2.86
(2H, m), 3.17-3.26 (2H, m), 3.75 (3H, s), 6.84 (1H, dd, J = 8.8,
3.0 Hz), 6.91 (1H, d, J = 3.0 Hz), 7.33 (1H, d, J = 8.8 Hz), 8.69
(2H, br s).
4.1.36. 4-(2-bromo-5-ethoxybenzyl)piperidine
hydrochloride (11a)
4.1.32. tert-butyl 4-(2-bromo-5-
methoxybenzyl)piperidine-1-carboxylate (7b)
The title compound was prepared in a manner similar to that
for the preparation of 5 using intermediate 10a. (94%) white
solid. 1H NMR (300 MHz,DMSO-D6) δ: 1.31 (3H, t, J = 7.0 Hz),
1.34-1.51 (2H, m), 1.63-1.74 (2H, m), 1.78-1.94 (1H, m), 2.61
(2H, d, J = 7.0 Hz), 2.71-2.85 (2H, m), 3.17-3.26 (2H, m), 4.00
(2H, q, J = 7.0 Hz), 6.76 (1H, dd, J = 8.6, 2.9 Hz), 6.90 (1H, d, J
= 3.1 Hz), 7.46 (1H, d, J = 8.8 Hz), 8.73 (2H, br s); HRMS (ESI)
m/z calcd for C14H21BrClNO [M+H]+ 298.0801; found 298.0814.
To a solution of intermediate 6 (150 mg, 0.491 mmol) in DMF
(2.0 mL) was added N-bromosuccinimide (96.0 mg, 0.540 mmol)
at 0oC. After stirring at room temperature for 12h, H2O (20 mL)
was added to the reaction mixture, and the whole was extracted
with EtOAc (20 mL x 2). The combined organic layers were
dried over MgSO4, filtered and concentrated. The residue was
purified by silica gel chromatography using 1-22%
EtOAc/hexane as eluent to give 169 mg (90%) of the title
compound as a colorless oil. 1H NMR (300 MHz,CDCl3) δ: 1.12-
1.29 (2H, m), 1.46 (9H, s), 1.52-1.68 (2H, m), 1.70-1.86 (1H, m),
2.56-2.71 (4H, m), 3.78 (3H, s), 3.98-4.16 (2H, m), 6.64 (1H, dd,
J = 8.7, 3.1 Hz), 6.70 (1H, d, J = 3.1 Hz), 7.42 (1H, d, J = 8.7
Hz).
4.1.37. tert-butyl 4-[2-bromo-5-(propan-2-
yloxy)benzyl]piperidine-1-carboxylate (10b)
The title compound was prepared in a manner similar to that
for the preparation of 10a using intermediate 9. (quant.) white
1
solid. H-NMR (300 MHz,CDCl3) δ: 1.11-1.28 (2H, m), 1.32
(6H, d, J = 6.1 Hz), 1.46 (9H, s), 1.61-1.65 (2H, m), 1.69-1.87
(1H, m), 2.55-2.73 (4H, m), 3.95-4.19 (2H, m), 4.42-4.56 (1H,
m), 6.62 (1H, dd, J = 8.6, 3.0 Hz), 6.68 (1H, d, J = 3.0 Hz), 7.39
(1H, d, J = 8.6 Hz).
4.1.33. 4-(2-bromo-5-methoxybenzyl)piperidine
hydrochloride (8b)
The title compound was prepared in a manner similar to that
for the preparation of 5 using intermediate 7b. (91%) white solid.
1H NMR (300 MHz,DMSO-D6) δ: 1.33-1.52 (2H, m), 1.63-1.75
(2H, m), 1.78-1.95 (1H, m), 2.62 (2H, d, J = 7.2 Hz), 2.72-2.86
(2H, m), 3.16-3.28 (2H, m), 3.74 (3H, s), 6.78 (1H, dd, J = 8.6,
3.0 Hz), 6.91 (1H, d, J = 3.0 Hz), 7.48 (1H, d, J = 8.6 Hz), 8.66
(2H, br s).
4.1.38. 4-[2-bromo-5-(propan-2-
yloxy)benzyl]piperidine hydrochloride (11b)
The title compound was prepared in a manner similar to that
for the preparation of 5 using intermediate 10b. (99%) white
solid. 1H-NMR (300 MHz,DMSO-D6) δ: 1.24 (6H, d, J = 6.1 Hz),
1.30-1.48 (2H, m), 1.61-1.74 (2H, m), 1.75-1.92 (1H, m), 2.59
(2H, d, J = 7.0 Hz), 2.70-2.84 (2H, m), 3.14-3.25 (2H, m), 4.52-
4.64 (1H, m), 6.74 (1H, dd, J = 8.8, 3.1 Hz), 6.87 (1H, d, J = 3.1
Hz), 7.43 (1H, d, J = 8.8 Hz), 8.50 (1H, br s) ; HRMS (ESI) m/z
calcd for C15H23BrClNO [M+H]+ 312.0957; found 312.0971.
4.1.34. tert-butyl 4-(2-bromo-5-
hydroxybenzyl)piperidine-1-carboxylate (9)
To a suspension of intermediate 8b (1.50 g, 4.68 mmol) in
CH2Cl2 (10 mL) was added dropwise 1N BBr3 in CH2Cl2 solution
(5.61 mL, 5.61 mmol) at 0°C for 10min. After stirring at room
temperature for 12h, MeOH (10 mL) was added to the reaction
mixture, and the whole was stirred at room temperature for
30min and concentrated. To a mixture of the residue, THF (10
mL) and 20% aqueous K2CO3 (50 g) was added di-tert-butyl
dicarbonate (1.12 g, 5.15 mmol). After stirring at room
temperature for 12h, the reaction mixture was extracted with
4.1.39. tert-butyl 4-[2-bromo-5-(2-
methoxyethoxy)benzyl]piperidine-1-carboxylate
(10c)
To a mixture of intermediate 9 (1.5 g, 4.05 mmol) and
potassium carbonate (1.12 g, 8.10 mmol) in DMF (10 mL) was
added 2-bromoethylmethyl ether (0.571 mL, 6.08 mmol). After
stirring at 100°C for 3h, the reaction mixture was treated with

H2O (50 mL) and extracted with EtOAc (50 mL x 2). The
combined organic layer was dried over MgSO4, filtered and
concentrated in vacuo. The residue was purified by silica gel
chromatography using 8~29% EtOAc/hexane as eluent to give
2.2 Hz), 6.61 (1H, d, J = 2.0 Hz), 6.74 (1H, d, J = 8.1 Hz), 7.30
(2H, d, J = 8.4 Hz), 7.72 (2H, d, J = 8.3 Hz).
4.1.45. 2-(3-chloro-4-methoxyphenyl)oxirane
(12a)
1
1.70 g (98%) of the title compound as a colorless oil. H NMR
To a mixture of 3-chloro-4-methoxybenzaldehyde (2.00 g,
11.7 mmol) and trimethylsulfonium iodide (3.35 g, 16.4 mmol)
in DMSO (12 mL) was added potassium hydroxide (0.920 g,
16.4 mmol). After stirring at 40°C for 7 h, the reaction mixture
was diluted with EtOAc (50 mL) and washed with H2O (100
mL). The aqueous layer was extracted with EtOAc (50 mL). To
the combined organic layer was added toluene (100 mL), and the
whole was washed with H2O (100 mL), dried over Na2SO4,
filtered and concentrated in vacuo to give 2.25 g (quant.) of the
title compound as a pale yellow oil. The obtained compound 12a
(300 MHz,CDCl3) δ: 1.11-1.28 (2H, m), 1.46 (9H, s), 1.57-1.68
(2H, m), 1.69-1.85 (1H, m), 2.55-2.71 (4H, m), 3.45 (3H, s), 3.74
(2H, t, J = 4.6 Hz), 4.00-4.16 (4H, m), 6.66 (1H, dd, J = 8.7, 3.0
Hz), 6.74 (1H, d, J = 3.0 Hz), 7.41 (1H, d, J = 8.7 Hz).
4.1.40. 4-[2-bromo-5-(2-
methoxyethoxy)benzyl]piperidine hydrochloride
(11c)
The title compound was prepared in a manner similar to that
for the preparation of 5 using intermediate 10c. (97%) white
solid. 1H NMR (300 MHz,DMSO-D6) δ: 1.33-1.51 (2H, m), 1.64-
1.75 (2H, m), 1.79-1.95 (1H, m), 2.62 (2H, d, J = 7.2 Hz), 2.72-
2.86 (2H, m), 3.16-3.27 (2H, m), 3.30 (3H, s), 3.64 (2H, t, J = 4.6
Hz), 4.07 (2H, t, J = 4.6 Hz), 6.78 (1H, dd, J = 8.8, 3.1 Hz), 6.93
(1H, d, J = 3.1 Hz), 7.47 (1H, d, J = 8.8 Hz), 8.68 (2H, br s).
1
was used in the next reaction without further purification. H
NMR (300 MHz,CDCl3) δ: 2.77 (1H, dd, J = 5.3, 2.6 Hz), 3.13
(1H, dd, J = 5.3, 4.0 Hz), 3.80 (1H, dd, J = 4.0, 2.6 Hz), 3.90 (3H,
s), 6.90 (1H, d, J = 8.4 Hz), 7.16 (1H, dd, J = 8.5, 2.1 Hz), 7.28
(1H, d, J = 2.2 Hz).
4.1.41. 2-(2-chloro-4-methoxyphenyl)ethanol
(13a)
4.1.46. 2-(3-chloro-4-methoxyphenyl)ethanol
(13c)
To a solution of (2-chloro-4-methoxyphenyl)acetic acid (1.50
g, 7.48 mmol) in THF (20 mL) was added 0.9 M BH3-THF
complex in THF solution (10.8 mL, 9.72 mmol). After stirring at
room temperature for 3 h, MeOH (10 mL) was added, and the
reaction mixture was stirred at room temperature for 30 min and
concentrated in vacuo. The residue was purified by silica gel
chromatography using 26~47% EtOAc/hexane as eluent to give
To a suspension of sodium borohydride (168 mg, 4.44 mmol)
in THF (12 mL) was added BF3-Et2O complex (0.903 mL, 7.13
mmol). After stirring at room temperature for 15 min, a solution
of intermediate 12a (2.00 g, 10.8 mmol) in THF (12 mL) was
added dropwise for 10 min to the reaction mixture at 0°C. After
stirring at room temperature for 2 h, H2O (50 mL) was added to
the reaction mixture, and the whole was extracted with EtOAc
(40 mL). The organic layer was washed with brine (20 mL),
dried over MgSO4, filtered and concentrated in vacuo. The
residue was purified by silica gel chromatography using 30~51%
EtOAc/hexane as eluent to give 1.65 g (81%) of the title
1
1.44 g (quant.) of the title compound as a colorless oil. H NMR
(300 MHz,CDCl3) δ: 1.36 (1H, t, J = 5.9 Hz), 2.95 (2H, t, J = 6.7
Hz), 3.79 (3H, s), 3.85 (2H, td, J = 6.4, 6.4 Hz), 6.77 (1H, dd, J =
8.4, 2.6 Hz), 6.93 (1H, d, J = 2.8 Hz), 7.17 (1H, d, J = 8.4 Hz).
1
compound as a colorless oil. H NMR (300 MHz,CDCl3) δ: 1.37
4.1.42. 2-(2-chloro-4-methoxyphenyl)ethyl 4-
methylbenzenesulfonate (14a)
(1H, t, J = 5.9 Hz), 2.79 (2H, t, J = 6.5 Hz), 3.83 (2H, td, J = 6.2,
6.2 Hz), 3.89 (3H, s), 6.88 (1H, d, J = 8.3 Hz), 7.09 (1H, dd, J =
8.4, 2.2 Hz), 7.25 (1H, d, J = 2.2 Hz).
To a mixture of intermediate 13a (1.20 g, 6.43 mmol),
triethylamine (1.08 mL, 7.72 mmol) and trimethylamine
hydrochloride (61.5 mg, 0.643 mmol) in CH2Cl2 (12 mL) was
added p-toluenesulfonyl chloride (1.35 g, 7.07 mmol) at 0°C.
After stirring at 0oC for 1 h, the reaction mixture was treated with
H2O (50 mL) and extracted with CHCl3 (30 mL x 2). The organic
layer was dried over MgSO4, filtered and concentrated in vacuo.
The residue was purified by silica gel chromatography using
4~25% EtOAc/hexane as eluent to give 2.08 g (95%) of the title
4.1.47. 2-(3-chloro-4-methoxyphenyl)ethyl 4-
methylbenzenesulfonate (14c)
The title compound was prepared in a manner similar to that
for the preparation of 14a using intermediate 13c. (89%)
1
colorless oil. H NMR (300 MHz,CDCl3) δ: 2.44 (3H, s), 2.86
(2H, t, J = 6.8 Hz), 3.88 (3H, s), 4.17 (2H, t, J = 6.8 Hz), 6.80
(1H, d, J = 8.4 Hz), 6.99 (1H, dd, J = 8.3, 2.2 Hz), 7.04 (1H, d, J
= 2.0 Hz), 7.28 (2H, d, J = 8.4 Hz), 7.66 (2H, d, J = 8.4 Hz).
1
compound as a colorless oil. H NMR (300 MHz,CDCl3) δ: 2.43
(3H, s), 3.01 (2H, t, J = 6.9 Hz), 3.77 (3H, s), 4.20 (2H, t, J = 6.9
Hz), 6.71 (1H, dd, J = 8.4, 2.6 Hz), 6.82 (1H, d, J = 2.6 Hz), 7.08
(1H, d, J = 8.4 Hz), 7.27 (2H, d, J = 7.9 Hz), 7.68 (2H, d, J = 8.3
Hz).
4.1.48. 5-(oxiran-2-yl)-2,3-dihydro-1,4-
benzodioxine (12b)
The title compound was prepared in a manner similar to that
for the preparation of 12a using 2,3-dihydro-1,4-benzodioxine-5-
1
4.1.43. 2-(2,3-dihydro-1,4-benzodioxin-6-
yl)ethanol (13b)
carbaldehyde. (quant.) colorless oil. H NMR (300 MHz,CDCl3)
δ: 2.75 (1H, dd, J = 5.7, 2.6 Hz), 3.14 (1H, dd, J = 5.7, 4.0 Hz),
4.14 (1H, dd, J = 4.0, 2.6 Hz), 4.26-4.30 (2H, m), 4.30-4.34 (2H,
m), 6.69-6.73 (1H, m), 6.79-6.82 (2H, m).
The title compound was prepared in a manner similar to that
for the preparation of 13a using (2,3-dihydro-benzo[1,4]dioxin-6-
yl)-acetic acid. (88%) 1.59 g. 1H NMR (300 MHz,CDCl3) δ: 1.39
(1H, t, J = 5.9 Hz), 2.76 (2H, t, J = 6.5 Hz), 3.82 (2H, td, J = 6.1,
6.1 Hz), 4.25 (4H, s), 6.70 (1H, dd, J = 8.3, 2.0 Hz), 6.74 (1H, d,
J = 2.0 Hz), 6.81 (1H, d, J = 8.3 Hz).
4.1.49. 2-(2,3-dihydro-1,4-benzodioxin-5-
yl)ethanol (13d)
The title compound was prepared in a manner similar to that
for the preparation of 13c using intermediate 12b. (72%)
1
4.1.44. 2-(2,3-dihydro-1,4-benzodioxin-6-yl)ethyl
4-methylbenzenesulfonate (14b)
The title compound was prepared in a manner similar to that
for the preparation of 14a using intermediate 13b. (quant.) 1.94
g. 1H NMR (300 MHz,CDCl3) δ: 2.44 (3H, s), 2.84 (2H, t, J = 7.2
Hz), 4.15 (2H, t, J = 7.2 Hz), 4.23 (4H, s), 6.57 (1H, dd, J = 8.1,
colorless oil. H NMR (300 MHz,CDCl3) δ: 2.88 (2H, t, J = 6.5
Hz), 3.84 (2H, td, J = 6.2, 6.2 Hz), 4.23-4.31 (4H, m), 6.72-6.80
(3H, m).
4.1.50. 2-(2,3-dihydro-1,4-benzodioxin-5-yl)ethyl
4-methylbenzenesulfonate (14d)

The title compound was prepared in a manner similar to that
for the preparation of as 14a using intermediate 13d. (89%) white
solid. 1H NMR (300 MHz,CDCl3) δ: 2.43 (3H, s), 2.92 (2H, t, J =
7.0 Hz), 4.11-4.18 (4H, m), 4.21 (2H, t, J = 7.0 Hz), 6.64 (1H, dd,
J = 6.6, 2.6 Hz), 6.68-6.78 (2H, m), 7.26 (2H, d, J = 8.1 Hz), 7.65
(2H, d, J = 8.3 Hz).
(SMP-304, paroxetine, milnaciplan or imipramine) was diluted
with [3H]5-HT solution in another 96-well plate. After cells pre-
incubation, 50 μL of the prepared [3H]5-HT solution containing
dimethyl sulfoxide or test substance was added to the wells, and
the mixture was incubated at 37°C for 20 min. After the
incubation, the liquid layer was discarded, and the cells were
rinsed twice with 200 μL reaction buffer before being lysed with
100 μL of the Solvable solution. Radioactivity in each lysate
sample was measured as described in the previous section.
4.1.51. 2-(naphthalen-2-yl)ethyl 4-
methylbenzenesulfonate (14e)
The title compound was prepared in a manner similar to that
4.2.3. Guanosine 5’-(γ-thio) Triphosphate, [3 5 S]-
(GTPγS) assay for 5-HT1A receptor
for the preparation of 14a using 2-naphthalene ethanol. (91%)
1
white solid. H NMR (300 MHz,CDCl3) δ: 2.34 (3H, s), 3.11
(2H, t, J = 6.9 Hz), 4.31 (2H, t, J = 6.9 Hz), 7.08-7.14 (2H, m),
7.21 (1H, dd, J = 8.4, 1.7 Hz), 7.41-7.50 (2H, m), 7.52 (1H, br s),
7.60 (2H, d, J = 8.5 Hz), 7.71 (2H, d, J = 8.5 Hz), 7.76-7.83 (1H,
m).
To make up a total volume of 500 μL, 2.5 μL of test
compound, GTPγS (2 mM, to measure nonspecific binding),
DMSO (to measure basal [35S]GTPγS binding) or serotonin (20
mM, to measure maximal [35S]GTPγS binding); 50 μL of
reaction buffer [HEPES-NaOH buffer (20 mM, pH 7.4)
containing 100 mM NaCl, 10 mM MgCl2, 0.1 mM DTT, and 1
μM GDP] containing 0.5 nM [35S]GTPγS; and 447.5 μL of cell
membranes expressing human 5-HT1A receptors were mixed. The
following manipulation was carried out as described in the above
5-HT transporter binding assay. Intrinsic activity was expressed
as relative value of the activity of 100 μM serotonin, which was
considered to be 100%.
4.1.52. 1-(2-bromoethyl)-2-chloro-4,5-
dimethoxybenzene (15a)
To a solution of 3,4-dimethoxyphenethyl bromide (300 mg,
1.22 mmol) in DMF (6.0 mL) was added N-chlorosuccinimide
(179 mg, 1.34 mmol). After stirring at room temperature for 20h,
saturated aqueous NaHCO3 (4 mL) and H2O (20 mL) were added
to the reaction mixture, and the whole was extracted with EtOAc
(30 mL x 2). The combined organic layer was dried over MgSO4,
filtered and concentrated. The residue was purified by silica gel
chromatography using 0-15% EtOAc/hexane as eluent to give
The 5-HT1A agonistic activity of SMP-304 was evaluated
three times. The means of the results at each concentration are as
follows:
1
324 mg (95%) of the title compound as a white solid. H NMR
(300 MHz,CDCl3) δ: 3.21 (2H, t, J = 7.6 Hz), 3.56 (2H, t, J = 7.5
Hz), 3.86 (3H, s), 3.88 (3H, s), 6.74 (1H, s), 6.86 (1H, s).
1 nM: -0.87 ± 5.70%, 10 nM: 0.54 ± 4.28%, 100 nM: 6.66 ±
4.03%, 1000 nM: 16.2 ± 4.37%, 10000 nM: 19.6 ± 5.41%
4.1.53. 2-(7-chloro-2,3-dihydro-1,4-benzodioxin-
6-yl)ethyl 4-methylbenzenesulfonate (15b)
4.2.3.1. Data analyses
The following formulae were used:
1) Basal [35S]GTPγS binding
To a solution of 14b (500 mg, 1.50 mmol) in DMF (7.0 mL)
was added N-chlorosuccinimide (179 mg, 1.34 mmol). After
stirring at 50°C for 4h, H2O (40 mL) was added to the reaction
mixture, and the whole was extracted with EtOAc (40 mL x 2).
The combined organic layer was dried over MgSO4, filtered and
concentrated. The residue was purified by silica gel
chromatography using 10-31% EtOAc/hexane as eluent to give
Basal [35S]GTPγS binding (dpm) = Binding activity of the
DMSO group (dpm) − Binding activity of the GTPγS group
(dpm)
2) Maximal [35S]GTPγS binding
1
481 mg (87%) of the title compound as a white solid. H-NMR
(300 MHz,CDCl3) δ: 2.44 (3H, s), 2.94 (2H, t, J = 7.0 Hz), 4.18
(2H, t, J = 7.0 Hz), 4.22 (4H, s), 6.67 (1H, s), 6.79 (1H, s), 7.29
(2H, d, J = 8.3 Hz), 7.71 (2H, d, J = 8.3 Hz).
Maximal [35S]GTPγS binding (dpm) = Binding activity of the
serotonin group (dpm) − Binding activity of the GTPγS group
(dpm)
4.2. Biology
3) Specific binding of the test substance
Specific binding of the test substance (dpm) = Binding activity of
test substance group (dpm) − Binding activity of GTPγS group
(dpm)
4.2.1. Radio assay
Radio assays were conducted according to the methods of
Owens et al. for serotonin transporter 24 and Yabuuchi et al. for 5-
HT1A 25. Briefly, the following were used: for serotonin
transporter assays, cell membranes expressing human serotonin
transporter and [3H]citalopram; for 5-HT1A assays, cell
membranes expressing human 5-HT1A and [3H]8-OH-DPAT.
The inhibition constant (Ki) was calculated in Microsoft Office
Excel 2003 (Microsoft Corporation) using the Cheng–Prusoff
equation [Ki = IC50/(1 + ([L]/Kd)], where L is the concentration
of radioligand in the assay and Kd is the dissociation constant of
the radioligand for the receptor.
4) Maximal specific binding
The maximal specific binding of the test substance was
calculated using the Dx calculation (logistic curve fitting) with
the “measurement value input” function in Stat Preclinica Client
Version 1.0. The direct estimation method was used. The
maximal specific binding was calculated using the logistic curve
of the concentrations of the test substance and the specific
binding values.
4.2.2. [3 H]5-HT uptake assay
5) Intrinsic activity of the test substance
Phosphate-buffered saline containing 0.1 mmol/L CaCl2 and 1
mmol/L MgCl2 was used as reaction buffer. One microliter of
dimethyl sulfoxide or test substance and 199 μL of the reaction
buffer were mixed, and 50 μL of the mixed solution was added to
human serotonin transporter-expressing CHO cells cultured in
96-well assay plates. The plates were pre-incubated at 37°C for
10 min. During that time, dimethyl sulfoxide or test substance
When the increment in maximal [35S]GTPγS binding
(Maximal [35S]GTPγS binding − Basal [35S]GTPγS binding)
was considered as 100%, intrinsic activity of the test substance,
which is the percentage of the increment in maximal specific
binding of the test substance (Maximal specific binding of test
substance − Basal [35S]GTPγS binding), was calculated using
the following formula:

Intrinsic activity of the test substance (%) = 100 ×{ [Maximal
specific binding of test substance (dpm) − Basal [35S]GTPγS
binding (dpm)] ∕ [Maximal [35S]GTPγS binding (dpm) − Basal
[35S]GTPγS binding (dpm)]}
the remaining activity (50%). A larger IC50 value for CYP2D6
inhibition indicates weak CYP2D6 inhibition.
4.2.5. Rat forced swimming test
On the first day of the experiment, male Wistar rats weighing
121.3 – 178.1 g were placed in plastic cylinders (40 cm in height,
19 cm in diameter) containing water (25 ± 1°C) to a depth of 19
cm. After spending 15 min in the water, the rats were removed
from the cylinders and wiped with paper towels (training
session). SMP-304 (1, 3 mg/kg), paroxetine (3, 10 mg/kg), or
vehicle (0.5% methylcellulose) was orally administered to each
rats 30 min after the beginning of the training session. The test
session was carried out the following day with each rat treated
with one of the test-drugs or vehicle 2 h before the start of the
test session. During the test session each animal behavior was
videotaped for 6 min, and a rat was judged to be immobile
whenever it remained floating in water without movement,
except for slight movement to keep posture. An observer blind to
test-drugs measured twice the immobility time for each animal.
When the difference between the first and second measured
immobility time was within 30 sec, the first recorded immobility
time was used for data analysis. When the difference between the
two measured immobility times was more than 30 sec, another
measurement was conducted, and the obtained immobility time
was used for data analysis. After all measurements were
completed, animals’ assignment to test drugs was disclosed.
4.2.4. CYP2D6 inhibition assay
4.2.4.1. Materials
Bufuralol was purchased from Sigma-Aldrich Corp., and
Pooled Human Liver Microsomes were purchased from
Xenotech, LLC.
4.2.4.1.1. Preparation of 0.5 M Potassium
Phosphate Buffer (pH 7.4)
Monopotassium phosphate solution (150 mL, 0.5 M) and
dipotassium phosphate solution (700 mL, 0.5 M) were mixed,
giving a solution with pH 7.4.
4.2.4.1.2. Preparation of Magnesium Chloride
Solution (165 mM)
Magnesium chloride hexahydrate (3.35 g) was dissolved in
distilled water (100 ml) to a final concentration of 165 mM
(MgCl2.6H2O).
4.2.4.1.3. Preparation of Human Liver Microsome
Solution
Human Liver Microsome Solution was prepared by mixing
Pooled Human Liver Microsomes (150 μL, 20 mg/mL),
potassium phosphate buffer (12 mL, 0.5 M), magnesium chloride
solution (1.2 mL, 165 mM), and distilled water (34.65 mL).
4.2.6. Spontaneous locomotion
This experiment was performed using male Wistar rats
weighing 121.3 – 178.1 g. SMP-304 (1, 3 mg/kg), or vehicle
(0.5% methylcellulose) was orally administered to the rats 26hr
and 2hr before the test. The animals were placed in plastic cages,
and their spontaneous locomotion was measured for 30 min using
an automatic behavior analyzing system (Supermex; Muromachi
Kikai Co. Ltd., Tokyo, Japan).
4.2.4.1.4. Preparation of β-NADPH Solution (13
mM)
β-NADPH was dissolved in distilled water to a final
concentration of 11.75 mg/mL.
4.2.7. Pharmacokinetics of SMP-304
4.2.4.1.5. Preparation of Substrate Solution
Pharmacokinetic study of SMP-304 was carried out in male
Wistar rats weighing 121.3 – 178.1 g. SMP-304 (1, 3 mg/kg) was
orally administered to the rats 26hr and 2hr before blood and
brain sampling (3 rats). SMP-304 plasma and brain
concentrations were determined using high-performance liquid
chromatography (HPLC)/tandem mass spectrometry.
Bufuralol was dissolved in DMSO to a final concentration of
2.0 mM and then diluted 200-fold with distilled water.
4.2.4.2. Experimental Procedures
Step 1: The test drug in DMSO solution (10 mM) was serially
diluted 5-fold with DMSO to prepare 10, 2, 0.4 and 0.08 mM test
drug solutions.
Acknowledgments
We would lie to thank Tomohiro Toyoda, Tomoaki Nishida
and Daisuke Tanaka for their valuable suggestions on the
preparation of this manuscript. We also would like to thank
Hisayo Emoto for conduction receptor binding assays; Yoshie
Nakatsuji for the high resolution MS spectra; and Keiko Bando
for her elemental analysis; Kenji Miyazaki for conducting the
locomotor activity test; and Itaru Natsutani for the
pharmacokinetics study.
Step 2: Each test drug solution and DMSO were separately
diluted 96-fold with the human liver microsome solution, and 80
uL of each dilution was dispensed into 96-well microplates.
Step 3: The substrate solution (10 uL) and β-NADPH solution
(10 uL) were added to each well, and the plate was incubated at
37 °C for 10 min.
Step 4: The reaction was terminated by addition of 300 uL of
methanol.
References and notes
Step 5: The reaction mixture was filtered, and the filtrate was
loaded onto an LC-MSMS system.
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 Corresponding author. Tel.: +81-6-6337-5885; fax: +81-6-6337-6010; e-mail: hidefumi-yoshinaga@ds-pharma.co.jp

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Discovery of SMP-304, a Novel
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Benzylpiperidine Derivative with Serotonin
Transporter Inhibitory Activity and 5-HT1A
Weak Partial Agonistic Activity showing the
Antidepressant-like Effect
Hidefumi Yoshinaga, Shuji Masumoto, Koji Koyama, Naoya Kinomura, Yuji Matsumoto, Taro Kato, Satoko
Baba, Kenji Matsumoto, Tomoko Horisawa, Hitomi Oki, Kazuki Yabuuchi and Toru Kodo
Drug Research Division, Sumitomo Dainippon Pharma Co., Ltd., 33-94 Enoki-cho, Suita, Osaka 564-0053,
Japan