Design, synthesis, and biological evaluation of novel piperidine-4- carboxamide derivatives as potent CCR5 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.11.013

Based on a putative 'Y shape' pharmacophore model of CCR5 inhibitors, a series of novel piperidine-4-carboxamide derivatives were designed and synthesized using a group-reverse strategy. Among synthesized target compounds, 16g (IC₅₀ = 25.73 nM) and 16i (IC₅₀ = 25.53 nM) showed equivalent inhibitory activity against CCR5 to that of the positive control maraviroc (IC₅₀ = 25.43 nM) in calcium mobilization assay. Selected compounds were further tested for their antiviral activity in HIV-1 single cycle assay. Two compounds, 16g and 16i, displayed antiviral activity with IC ₅₀ values of 73.01 nM and 94.10 nM, respectively. Additionally, the pharmacokinetic properties and inhibitory potency against hERG of 16g were evaluated, providing a foundation for ongoing optimization.

Full text of DOI:10.1016/j.ejmech.2013.11.013

European Journal of Medicinal Chemistry 71 (2014) 259e266
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, and biological evaluation of novel
piperidine-4-carboxamide derivatives as potent CCR5 inhibitors
,
Suwen Hu a, Quan Gu a, Zhilong Wang b, Zhiyong Weng a 1, Yunrui Cai a, Xiaowu Dong a,
,
b,
**
Yongzhou Hu a, Tao Liu a , Xin Xie
*
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
b State Key Laboratory of Drug Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on a putative ‘Y shape’ pharmacophore model of CCR5 inhibitors, a series of novel piperidine-4-
carboxamide derivatives were designed and synthesized using a group-reverse strategy. Among syn-
thesized target compounds, 16g (IC50 ¼ 25.73 nM) and 16i (IC50 ¼ 25.53 nM) showed equivalent
inhibitory activity against CCR5 to that of the positive control maraviroc (IC50 ¼ 25.43 nM) in calcium
mobilization assay. Selected compounds were further tested for their antiviral activity in HIV-1 single
cycle assay. Two compounds, 16g and 16i, displayed antiviral activity with IC50 values of 73.01 nM and
94.10 nM, respectively. Additionally, the pharmacokinetic properties and inhibitory potency against hERG
of 16g were evaluated, providing a foundation for ongoing optimization.
Received 5 May 2013
Received in revised form
28 October 2013
Accepted 9 November 2013
Available online 19 November 2013
Keywords:
CCR5 inhibitor
Anti-HIV-1 agent
Ó 2013 Elsevier Masson SAS. All rights reserved.
Piperidine-4-carboxamide derivatives
1. Introduction
anti-HIV activity with an IC50 value of 6.17 nM [8]. Although with
the emergence of these encouraging results, various challenges
The unfolding of the close connection between chemokine
receptor 5 (CCR5) and HIV-1 provides a better understanding
about how HIV-1 enters human cells [1]. CCR5, one of the seven-
transmembrane G-protein coupled receptors, plays an intimate
role in the R5-tropic HIV-1 entry process by serving as a critical
co-receptor for the viral envelope protein glycoprotein-120
(gp120) [2,3]. Homozygous individuals with the CCR5 delta 32
allele deletion do not express the functional receptor and are
ultimately resistant to R5-tropic HIV-1 infection, while hetero-
zygotes are prone to be infected but with a slowed progress to-
ward HIV-1 [4]. These facts have stimulated a great amount of
efforts in identifying new CCR5 inhibitors, among which include
several clinic candidates (TAK-220 in Phase I, 1, TBR-652 in Phase
II, 2, Fig. 1) and one FDA approved-CCR5 inhibitor, maraviroc (UK-
427,857, 3, Fig. 1) [5e7]. Recently, Dong and co-workers synthe-
sized a novel series of 1,4-disubstituted piperidine/piperazine
derivatives, among which compound B07 (4, Fig. 1) exhibited
associated with CCR5 inhibitors such as viral tropism, drug
resistance and possible long term adverse events still exist.
Therefore, current studies have been focused on the develop-
ment of second generation CCR5 inhibitors with improved
properties.
In our previous work, we have designed and synthesized a series
of novel piperazine derivatives based on a ‘Y shape’ pharmacophore
model, whichcontainsone basic center, three hydrophobic domains,
and an amide linker. The results obtained from CCR5 inhibitoryassay
were not very satisfactory with inhibitory activities (IC50 value) for
most compounds varied from several hundred nanomolar to low
micromolar [9]. During our continuing searching for more potent
compound, an interesting structure 5 disclosed by Long et al. [10],
which showed potent inhibitory effect on RANTES-stimulated [35S]-
GTPgS
binding to CCR5-expressing CHO cell membranes
(IC50 ¼ 7.00 nM), attracted our attention. A novel series of piperi-
dine-4-carboxamide derivatives were then designed through the
utilization of a group-reverse strategy by replacing the piperidin-4-
amine moiety of 5 with a piperidine-4-carboxamide scaffold. Eval-
uation of CCR5 inhibitory activity revealed that the obtained piper-
idine-4-carboxamide derivative 6 (IC50 ¼ 525.00 nM) and lead
compound 5 (IC50 ¼ 251.67 nM) shared equivalent potency against
CCR5 in a calcium mobilization assay (Fig. 2).
* Corresponding author. Tel.: þ86 571 88208458; fax: þ86 571 88981051.
** Corresponding author. Tel./fax: þ86 21 50801313.
E-mail addresses: Lt601@zju.edu.cn, lt601@163.com (T. Liu), xxie@mail.shcnc.ac.
cn (X. Xie).
1
Current address: Hangzhou No. 2 High School of Zhejiang Province, Hangzhou,
Zhejiang, China.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.11.013

260
S. Hu et al. / European Journal of Medicinal Chemistry 71 (2014) 259e266
N
O
O
N
N
NH2
N
BuO
NH
O
O
O
Cl
TAK-220, 1
TBR-652, 2
S
O
N
N
N
N
F
O
N
F
F
H
N
N
N
N
N
N
O
Maraviroc, 3
O
B07, 4
Cl
Fig. 1. Structures of TAK-220, TBR-652, maraviroc, and B07.
O
N
O
N
N
N
O
N
N
N
N
O
O
Cl
O
Cl
6
5
O
R2
R1
N
N
N
A
N
n
O
O
Cl
Target Compounds
Fig. 2. Design of the target compounds.
In order to explore the structureeactivity relationship of these
Besides, N-Boc-4-piperidinecarboxylic acid was added to a so-
lution of the different substituted amines 13aer at room temper-
ature to afford amides 14aer, which were de-protected with 6 N
HCl to yield the corresponding intermediates 15aer. 15aer were
then reacted with amide 12 in the presence of potassium iodide and
potassium carbonate to afford target compounds 6 and 16ber.
novel piperidine-4-carboxamide derivatives, we firstly modified
the phenyl ring A of the target compounds with a variety of other
functionalities. Then the nitrogen atom of the amide was
substituted with different alkyl groups (16bej). Thirdly, the length
of the linker between the phenyl ring and nitrogen atom of the
amide was changed from null to two-methylene units (16ker) to
determine an optimal linker. Herein, we report the design,
synthesis, and biological evaluation of a series of novel piperidine-
4-carboxamide derivatives, as well as the antiviral activity of
representative compounds, to develop novel compounds as potent
CCR5 inhibitors for HIV.
2.2. Cell cytotoxicity assays
A total of 18 novel piperidine-4-carboxamide derivatives (6 and
16ber) were tested in CCK-8 assay, showing no significant cyto-
toxicity against HEK293 cells at a concentration of 50
mM. The
purity of all compounds used for biological test was more than 95%.
2. Results and discussion
2.3. Calcium mobilization assay
2.1. Chemistry
All compounds have been evaluated by calcium mobilization
assay for their CCR5 inhibitory activity with maraviroc employed as
the positive control, the results of which were summarized in
Table 1.
Compound 6, with the IC50 value of 525.00 nM, was obtained via
inverting the amide functionality that tethered to the terminal
benzene ring of 5. Provided its moderate CCR5 inhibitory activity, 6
was employed as a starting point for further optimization to pursue
compounds with more attractive potency and probe SARs associ-
ated with its derivatives. As an initial step for our structural
Target compounds were synthesized as outlined in Scheme 1.
The readily available material piperidine-4-carboxylic acid 7 was
firstly converted to 1-acetylpiperidine-4-carboxylic acid 8 accord-
ing to a reported method with minor modification [11]. Then
reaction of 8 with thionyl chloride gave chloride 9 [12]. Subsequent
N-alkylation of 3-chloroaniline 10 with 1-bromo-3-chloropropane
in the presence of potassium iodide under microwave conditions
afforded substituted aniline 11. Acylation of 11 with chloride 9 gave
amide 12 [13].

S. Hu et al. / European Journal of Medicinal Chemistry 71 (2014) 259e266
261
O
O
O
O
O
b)
a)
O
HN
N
N
OH
OH
Cl
N
Cl
7
8
9
d)
N
NH2
O
HN
Cl
12
Cl
c)
Cl
Cl
11
10
H
n
N
O
O
R1
e)
f)
N
R1
n
N
R1
n
R2
R2
N
HN
Boc
R2
14a-r
15a-r
13a-r
O
R2
R1
N
N
N
g)
N
12
+
15a-r
n
O
O
Cl
6, 16b-r
Scheme 1. Synthesis of target compounds 6 and 16ber. Reagents and conditions: a) Ac2O, DCM, rt, 12 h; b) SOCl2, rt, 30 min; c) 1-bromo-3-chloropropane, KI, MeCN, microwave
reactor, 110 ꢁC, 15 min; d) Et3N, DCM, rt, 5 h; e) N-Boc-4-piperidinecarboxylic acid, EDC$HCl, DCM; f) 6 N HCl, EtOAc, rt, 2 h; g) K2CO3, KI, MeCN, reflux, 48 h.
derivatization, a variety of substituents were introduced to the
4-position of the phenyl ring A while maintaining the ethyl
substitution on the nitrogen of the inversed amide functionality
(16bee). As displayed in Table 1, replacement of the 4-position with
methyl group (16b, IC50 ¼ 202.43 nM) only resulted in a slight
improvement in activity compared to 6. Subsequently, halogen
atoms, including fluorine and chloride, were employed as the C-4
substituents of the terminal benzene ring A. Between the attained
Cl) afforded compound 16n (IC50
¼
196.90 nM) and 16o
(IC50 ¼ 86.70 nM), whose activities were at least 2-fold less potent
than compounds 16g and 16i, respectively. These experimental
results indicated that the longer linker (n ¼ 2) was not preferable
compared to the linker with one methylene group (n ¼ 1). In
addition, similar results, decrease of activity comparable to com-
pounds 16g and 16i, were observed when ethyl group was intro-
duced to the nitrogen atom of compounds 16meo to afford
compound 16p (IC50 ¼ 26.37 nM), 16q (IC50 ¼ 151.80 nM), and 16r
(IC50 ¼ 125.81 nM), respectively. To this point, it was clear that
compounds with the linker of one methylene displayed better
activities than that of compounds with linker of none or two
methylenes.
compounds, known as 16c (IC50
¼
320.33 nM) and 16d
(IC50 ¼ 93.53 nM), the latter one exhibited a 6-fold enhancement in
activity by contrast to 6, indicating that the chloro substitution was
more beneficial for CCR5 inhibitory activity. Afterward, structural
alterations were examined at the nitrogen of the inversed amide
functionality to generate the optimum R1 replacement. Upon
changing the ethyl substitution of 6 into methyl substitution, a 9-
fold improvement in activity was obtained by compound 16f
(IC50 ¼ 64.07 nM). Encouraged by this observation, we prepared
compound 16g via removal the R1 replacement, which exhibited
comparable activity (IC50 ¼ 25.73 nM) to the positive control
maraviroc. On the insight that the amide functionality without
substitution on the nitrogen atom was preferable for potency, we
subsequently investigated compounds containing distinct
replacement at the 4-position of the phenyl ring A on the basis of
16g. As a consequence, most of the target compounds were eval-
uated to be more favorable in activity than 6. Particularly com-
pounds 16g (IC50 ¼ 25.73 nM) and 16i (IC50 ¼ 25.53 nM) which
showed equivalent CCR5 inhibitory activity to maraviroc. To this
point, it became evident that the inversed amide group without
substitution on the nitrogen was optimum for activity.
2.4. Viral infectivity assays
In order to assess the activities of target compounds for inhi-
bition of virus infectivity, the most potent compounds (16g and 16i)
were selected for further anti-HIV-1 single cycle antiviral assay. The
results are summarized in Table 2. The tested compounds showed
nanomolar inhibitory activities (16g, IC50 ¼ 73.01 nM and 16i,
IC50 ¼ 94.10 nM, respectively).
2.5. Evaluation of pharmacokinetic property and cardiovascular
safety (hERG) of 16g
Given that compound 16g was easily synthesized and exhibited
good anti-HIV-1 activity and no cytotoxicity at the concentration of
50 mM, it was selected for further evaluation. The pharmacokinetic
Further modification was focused on exploring the length of the
alkyl linker in order to optimize the spacing between the nitrogen
atom of the amide group and the phenyl ring A of the target
compounds. As shown in Table 1, compounds with a CH2 (n ¼ 1)
linker (16g and 16i) demonstrated better activity while shortening
the linker by a carbon (n ¼ 0) caused a loss in potency (16k,
IC50 > 1000.00 nM; 16l, IC50 > 1000.00 nM). Similarly, lengthening
the linker by a carbon atom (n ¼ 2) also resulted in a loss of potency,
as testified by the comparison of 16g with 16m. Moreover, substi-
tution of the 4-position of the phenyl ring A with halogen atom (F,
properties of 16g in rat are shown in Table 3. This compound dis-
played a large volume of distribution after oral administration
(Vdssiv ¼ 1.23 L kgꢀ1 and Vdsspo ¼ 8.99 L kgꢀ1), moderatelyclearance
(Clpiv ¼ 1.25 L hꢀ1 kgꢀ1 and Clppo ¼ 8.86 L hꢀ1 kgꢀ1), a lowblood level
(AUC0e24iv ¼ 3.25 mg Lꢀ1 h and AUC0e24PO ¼ 3.13 mg Lꢀ1 h) and a
short half-life (tiv1/2 ¼ 0.74 h and tpo1/2 ¼ 0.71 h), and the oral
bioavailability in rats was 15.00%.
Meanwhile a patch clamp assay for 16g was also conducted.
Disappointingly, it exhibited 15.20% inhibition of the hERG potas-
sium current at a concentration of 3 mM.

262
S. Hu et al. / European Journal of Medicinal Chemistry 71 (2014) 259e266
Table 1
25.53 to 76.03 nM. Among them, two compounds (16g,
IC50 ¼ 25.73 nM and 16i, IC50 ¼ 25.53 nM) showed equivalent
similar activity in comparison with that of the positive control,
maraviroc (IC50 ¼ 25.43 nM). Potent compounds identified by
calcium mobilization assay were selected for further antiviral
evaluation. As a result, two compounds showed a nanomolar
activity (16g, IC50 ¼ 73.01 nM and 16i, IC50 ¼ 94.10 nM, respec-
tively). Moreover, compound 16g was chosen for further evaluation
of pharmacokinetic property, and it exhibited oral bioavailability of
15.00% in rats. Meanwhile, a patch clamp assay revealed that
compound 16g exhibited 15.20% inhibition of the hERG potassium
CCR5 inhibitory activity of compounds 6 and 16ber by calcium mobilization assay.
O
R2
R1
N
N
N
N
n
O
O
Cl
Compd.
R1
R2
n
IC50 (nM)a
6
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3
H
H
H
H
H
CH3
F
Cl
CN
H
H
F
Cl
CN
H
H
H
F
Cl
H
1
1
1
1
1
1
1
1
1
1
0
0
2
2
2
2
2
2
525.00 ꢃ 191.85
202.43 ꢃ 111.63
320.33 ꢃ 169.36
93.53 ꢃ 30.98
473.33 ꢃ 155.73
64.07 ꢃ 14.73
25.73 ꢃ 3.85
35.47 ꢃ 5.67
25.53 ꢃ 5.84
75.30 ꢃ 23.86
>1000
current at a concentration of 3
mM. Although we were able to
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
16m
16n
16o
16p
16q
16r
identify compound 16g with acceptable oral bioavailability, it had
hERG inhibition in rats, suggesting an ion channel effect on car-
diovascular safety. As a consequence, we will describe our efforts in
redesigning this chemotype in due course.
4. Experimental protocols
4.1. Chemistry
H
CH3CH2
H
H
>1000
76.03 ꢃ 12.60
196.90 ꢃ 32.85
86.70 ꢃ 6.95
26.37 ꢃ 8.43
151.80 ꢃ 25.72
125.81 ꢃ 21.87
25.43 ꢃ 1.35
4.1.1. General procedures
Melting points were obtained on a B-540 Buchi melting point
apparatus which were uncorrected. 1H NMR spectra was recorded
on a Bruker Avance III instrument at 500 MHz (chemical shifts were
H
CH3CH2
CH3CH2
CH3CH2
F
Cl
expressed as
d values relative to TMS as internal standard). Mass
Positive control
Maraviroc
spectra (MS) and ESI (positive) were recorded on an Esquire-LC-
00075 spectrometer. High-resolution mass (HRMS) were
measured by Agilent 6224 LC/MS.
a
Values are means of three experiments.
Table 2
4.1.2. Synthesis of 1-acetylpiperidine-4-carboxylic acid (8) and 1-
acetylpiperidine-4-carbonyl chloride (9)
Compound 8 and compound 9 were obtained according to the
known approach in Refs. [11,12].
Antiviral activity of two promising compounds.
O
R2
N
N
H
N
N
4.1.3. Synthesis of 3-chloro-N-(3-chloropropyl)aniline (11)
A mixture of 3-chloroaniline 10 (1.14 g, 9.00 mmol), 1-bromo-3-
chloropropane (0.49 g, 3.00 mmol), and KI (0.05 g, 0.3 mmol) in
MeCN (5 ml) was reacted in microwave reactor at 110 ꢁC for 15 min.
The solvent was evaporated in vacuo, diluted with water (30 ml),
and extracted with EtOAc (30 ml ꢂ 3). The combined organic layer
was dried (MgSO4) and filtered, then the solvent was evaporated in
vacuo. The residue was purified by column chromatography on
silica gel (EtOAc/PE 1/12) to yield yellow oil (0.34 g, 60%). 1H NMR
O
O
Cl
Compd.
R2
IC50, nM
16g
16i
H
Cl
73.01
94.10
1.10
Maraviroc
(CDCl3, 500 MHz) d: 7.34e7.20 (m, 2H, AreH), 7.18e7.00 (m, 2H,
AreH), 3.67e3.64 (m, 2H, CH2), 3.57e3.53 (m, 2H, CH2), 3.05e2.98
3. Conclusions
(m, 2H, CH2). ESI-MS m/z: 204 [M þ H]þ.
In conclusion, a series of novel piperidine-4-carboxyamide de-
rivatives were designed and synthesized by using a group-reverse
strategy. The CCR5 inhibitory activities of target compounds were
evaluated based on calcium mobilization assay. Several compounds
showed promising inhibitory activity, with IC50 values ranging from
4.1.4. Synthesis of 1-acetyl-N-(3-chlorophenyl)-N-(3-chloropropyl)
piperidine-4-carboxamide (12)
To an ice-cooled stirred suspension of 3-chloro-N-(3-
chloropropyl)aniline 11 (0.21 g, 1.00 mmol) in DCM (3.00 ml) was
added Et3N (0.40 ml, 3.00 mmol) followed by 1-acetylpiperidine-4-
carbonyl chloride 9 (0.23 g, 1.20 mmol), and the mixture was stirred
at room temperature for 5 h. Then it was poured to a saturated so-
lution of sodium bicarbonate (10.00 ml). The residue was extracted
by EtOAc (20 ml ꢂ 3), the organic layer was combined, then washed
with water (15 ml ꢂ 3) and brine (10.00 ml), dried (MgSO4), filtered
and concentrated in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc/PE 1/3) to afford the title
compound (0.30 g, 85%) as a brown solid; mp: 115e117 ꢁC (dec). 1H
Table 3
Pharmacokinetic property of 16g in rat.a
Parameter
IV (4 mg kgꢀ1
)
PO (25 mg kgꢀ1
)
Vdss (L kgꢀ1
)
1.23
1.25
3.25
0.74
15.00%
8.99
8.86
3.13
0.71
Clp (L hꢀ1 kgꢀ1
)
AUC0e24 (mg Lꢀ1 h)
NMR (CDCl3, 500 MHz) d: 7.31e6.98 (m, 4H, AreH), 4.55e4.50 (m,
t1/2 (h)
F%b
1H, piperidineeH), 3.83e3.80 (m, 1H, piperidineeH), 3.76 (t,
J ¼ 7.0 Hz, 2H, CH2), 3.54 (t, J ¼ 7.0 Hz, 2H, CH2), 2.94e2.81 (m, 1H,
piperidineeH), 2.44e2.34 (m, 2H, piperidineeH), 2.06 (s, 3H, CH3),
a
Drug was administered orally (PO) or intravenously (IV) to fasted SD rats (n ¼ 3).
F ¼ (AUCpo/25)/(AUCiv/4) ꢂ 100%.
b

S. Hu et al. / European Journal of Medicinal Chemistry 71 (2014) 259e266
263
2.10e2.06 (m, 2H, CH2), 1.87e1.62 (m, 4H, piperidineeH). ESI-MS m/
4.1.12. tert-Butyl-4-(benzylcarbamoyl)piperidine-1-carboxylate
(14g)
White solid (62%). Mp: 88e92 ꢁC. 1H NMR (CDCl3, 500 MHz)
z: 357 [M þ H]þ.
d
:
7.33e7.10 (m, 5H, AreH), 6.40 (s, 1H, NH), 4.52e4.48 (m, 2H, CH2),
4.16e4.10 (m, 2H, piperidineeH), 2.74e2.69 (m, 2H, piperidineeH),
2.22e2.18 (m, 1H, piperidineeH), 1.86e1.70 (m, 4H, piperidineeH),
1.40 (s, 9H, Boc-CH3). ESI-MS m/z: 319 [M þ H]þ.
4.1.5. General procedure for the synthesis of 14aer
To a solution of 1-(tert-butoxycarbonyl) piperidine-4-carboxylic
acid (0.23 g, 1.00 mmol) in DCM (10.00 ml), EDC$HCl (1.00 mmol),
13aer (1.00 mmol) and triethylamine (15.00 mL, 0.10 mmol) were
added successively at room temperature, and the resulting reaction
mixture was stirred for 18 h at the same temperature. Then the
reaction mixture was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (EtOAc/
PE 1/1) to furnish the title compound.
4.1.13. tert-Butyl-4-(4-fluorobenzylcarbamoyl)piperidine-1-
carboxylate (14h)
White solid (59%). Mp: 94e97 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
7.39e7.22 (m, 4H, AreH), 6.45 (s, 1H, NH), 4.53e4.49 (m, 2H, CH2),
4.17e4.10 (m, 2H, piperidineeH), 2.70e2.65 (m, 2H, piperidineeH),
2.32e2.23 (m, 1H, piperidineeH), 1.89e1.71 (m, 4H, piperidineeH),
1.41 (s, 9H, Boc-CH3). ESI-MS m/z: 337 [M þ H]þ.
4.1.6. tert-Butyl-4-(benzyl(ethyl)carbamoyl)piperidine-1-
carboxylate (14a)
Yellow oil (65%). 1H NMR (CDCl3, 500 MHz)
d: 7.55e7.45 (m, 5H,
4.1.14. tert-Butyl-4-(4-chlorobenzylcarbamoyl)piperidine-1-
AreH), 4.35e4.20 (m, 2H, CH2), 4.19e4.15 (m, 2H, piperidineeH),
2.79e2.74 (m, 2H, piperidineeH), 2.49e2.43(m, 2H, CH2), 2.35e2.29
(m, 1H, piperidineeH), 1.92e1.71 (m, 4H, piperidineeH), 1.43 (s, 9H,
Boc-CH3), 1.03e0.96 (m, 3H, CH3). ESI-MS m/z: 347 [M þ H]þ.
carboxylate (14i)
White solid (72%). Mp: 121e123 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
7.41e7.21 (m, 4H, AreH), 6.50 (s, 1H, NH), 4.55e4.51 (m, 2H, CH2),
4.20e4.11 (m, 2H, piperidineeH), 2.70e2.62 (m, 2H, piperidineeH),
2.34e2.28 (m, 1H, piperidineeH), 1.90e1.71 (m, 4H, piperidineeH),
1.43 (s, 9H, Boc-CH3). ESI-MS m/z: 353 [M þ H]þ.
4.1.7. tert-Butyl 4-(ethyl(4-methylbenzyl)carbamoyl)piperidine-1-
carboxylate (14b)
Yellow oil (72%). 1H NMR (CDCl3, 500 MHz)
d: 7.17e7.04 (m, 4H,
4.1.15. tert-Butyl-4-(4-cyanobenzylcarbamoyl)piperidine-1-
AreH), 4.54e4.51 (m, 2H, CH2), 4.26e4.00 (m, 2H, piperidineeH),
2.87e2.76(m, 2H, piperidineeH), 2.66e2.44 (m, 2H, CH2), 2.34e2.31
(m, 1H, piperidineeH), 2.21 (s, 3H, AreCH3), 1.95e1.72 (m, 4H,
piperidineeH),1.45 (s, 9H, Boc-CH3),1.03e0.97 (m, 3H, CH3). ESI-MS
m/z: 361 [M þ H]þ.
carboxylate (14j)
Yellow solid (67%). Mp: 89e94 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
7.57e7.45 (m, 4H, AreH), 6.43 (s, 1H, NH), 4.35e4.17 (m, 2H, CH2),
4.19e4.13 (m, 2H, piperidineeH), 2.78e2.73 (m, 2H, piperidineeH),
2.33e2.23 (m, 1H, piperidineeH), 1.90e1.71 (m, 4H, piperidineeH),
1.42 (s, 9H, Boc-CH3). ESI-MS m/z: 344 [M þ H]þ.
4.1.8. tert-Butyl-4-(ethyl(4-fluorobenzyl)carbamoyl)piperidine-1-
carboxylate (14c)
4.1.16. tert-Butyl-4-(phenylcarbamoyl)piperidine-1-carboxylate
(14k)
Yellow oil (43%). 1H NMR (CDCl3, 500 MHz)
d: 7.38e7.19 (m, 4H,
Yellow solid (65%). Mp: 155e158 ꢁC. 1H NMR (CDCl3, 500 MHz)
AreH), 4.52e4.48 (m, 2H, CH2), 4.17e4.10 (m, 2H, piperidineeH),
3.40e3.30 (m, 2H, CH2), 2.75e2.68 (m, 2H, piperidineeH), 2.37e2.26
(m, 1H, piperidineeH), 1.89e1.72 (m, 4H, piperidineeH), 1.45 (s, 9H,
Boc-CH3), 1.10e0.95 (m, 3H, CH3). ESI-MS m/z: 365 [M þ H]þ.
d: 7.35e7.06 (m, 5H, AreH), 6.63 (s, 1H, NH), 4.18e4.13 (m, 2H,
piperidineeH), 2.79e2.74 (m, 2H, piperidineeH), 2.36e2.33 (m, 1H,
piperidineeH), 1.91e1.65 (m, 4H, piperidineeH), 1.43 (s, 9H, Boc-
CH3). ESI-MS m/z: 305 [M þ H]þ.
4.1.9. tert-Butyl-4-((4-chlorobenzyl)(ethyl)carbamoyl)piperidine-
4.1.17. tert-Butyl-4-(ethyl(phenyl)carbamoyl)piperidine-1-
1-carboxylate (14d)
carboxylate (14l)
Yellow oil (74%). 1H NMR (CDCl3, 500 MHz)
d: 7.40e7.29 (m, 4H,
White solid (54%). Mp: 77e79 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
AreH), 4.45e4.19 (m, 2H, CH2), 4.13e4.13 (m, 2H, piperidineeH),
2.78e2.73 (m, 2H, piperidineeH), 2.59e2.52 (m, 2H, CH2), 2.39e2.30
(m, 1H, piperidineeH), 1.93e1.70 (m, 4H, piperidineeH), 1.44 (s, 9H,
Boc-CH3), 1.01e0.94 (m, 3H, CH3). ESI-MS m/z: 381 [M þ H]þ.
7.39e7.16 (m, 5H, AreH), 4.29e4.20(m, 2H, piperidineeH), 3.77e3.75
(m, 2H, CH2), 2.79e2.71 (m, 2H, piperidineeH), 2.45e2.42 (m, 1H,
piperidineeH), 1.98e1.72 (m, 4H, piperidineeH), 1.45 (s, 9H, Boc-
CH3), 1.04 (t, 3H, J ¼ 7 Hz, CH3). ESI-MS m/z: 333 [M þ H]þ.
4.1.10. tert-Butyl-4-((4-cyanobenzyl)(ethyl)carbamoyl)piperidine-
4.1.18. tert-Butyl-4-(phenethylcarbamoyl)piperidine-1-carboxylate
1-carboxylate (14e)
(14m)
Yellow oil (73%). 1H NMR (CDCl3, 500 MHz)
d: 7.59e7.57 (d, 2H,
White solid (73%). Mp: 139e143 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
AreH), 7.59e7.57 (d, 2H, AreH) 4.16e4.09 (m, 2H, piperidineeH),
3.66 (s, 2H, CH2), 2.66e2.62 (m, 2H, piperidineeH), 2.56e2.51 (m,
2H, CH2), 2.34e2.28 (m, 1H, piperidineeH) 1.73e1.64 (m, 4H,
piperidineeH), 1.44 (s, 9H, Boc-CH3), 1.02e0.95 (m, 3H, CH3). ESI-
MS m/z: 372 [M þ H]þ.
7.38e7.19 (m, 5H, AreH), 6.90 (s, 1H, NH), 4.24e4.18 (m, 2H,
piperidineeH), 3.41e3.35 (m, 2H, CH2), 2.84e2.79 (m, 2H, piperi-
dine), 2.75e2.70 (m, 2H, CH2), 2.32e2.27 (m, 1H, piperidineeH),
1.87e1.70 (m, 4H, piperidineeH), 1.39 (s, 9H, Boc-CH3). ESI-MS m/z:
333 [M þ H]þ.
4.1.11. tert-Butyl-4-(benzyl(methyl)carbamoyl)piperidine-1-
4.1.19. tert-Butyl 4-(4-fluorophenethylcarbamoyl)piperidine-1-
carboxylate (14n)
carboxylate (14f)
White solid (67%). Mp: 57e62 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
White solid (78%). Mp: 153e155 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
7.45e7.21 (m, 5H, AreH), 4.53e4.49 (m, 2H, CH2), 4.16e4.11 (m, 2H,
piperidineeH), 3.27 (s, 3H, CH3), 2.78e2.71 (m, 2H, piperidineeH),
2.22e2.19 (m, 1H, piperidineeH), 1.88e1.71 (m, 4H, piperidineeH),
1.41 (s, 9H, Boc-CH3). ESI-MS m/z: 333 [M þ H]þ.
7.14e7.11 (m, 2H, AreH), 7.01e6.97 (m, 2H, AreH), 5.43 (brs, 1H),
4.12e4.10 (m, 2H), 3.51e3.47 (m, 2H), 2.80e2.67 (m, 4H), 2.18e2.11
(m, 1H), 1.74e1.72 (m, 2H), 1.61e1.53 (m, 2H), 1.44 (s, 9H, 3ꢂ CH3).
ESI-MS m/z: 351 [M þ H]þ.

264
S. Hu et al. / European Journal of Medicinal Chemistry 71 (2014) 259e266
4.1.20. tert-Butyl 4-(4-chlorophenethylcarbamoyl)piperidine-1-
carboxylate (14o)
4.1.26. 1-Acetyl-N-(3-chlorophenyl)-N-(3-(4-(ethyl(4-
methylbenzyl)carbamoyl)piperidin-1-yl)propyl)piperidine-4-
carboxamide (16b)
Yellow solid (75%). Mp: 143e145 ꢁC. 1H NMR (CDCl3, 500 MHz)
d: 7.27e7.25 (m, 2H, AreH), 7.10e7.08 (m, 2H, AreH), 5.53 (brs, 1H),
Yellow solid (44%); mp: 50e56 ꢁC. 1H NMR (CDCl3, 500 MHz)
4.11e4.09 (m, 2H), 3.49e3.45 (m, 2H), 2.79e2.69 (m, 4H), 2.16e2.11
(m, 1H), 1.73e1.70 (m, 2H), 1.60e1.52 (m, 2H), 1.44 (s, 9H, 3ꢂ CH3).
ESI-MS m/z: 367.8 [M þ H]þ.
d:7.45e7.41 (m, 2H, AreH), 7.25e7.23 (m, 1H, AreH), 7.17e7.02 (m,
5H, AreH), 4.56e4.48 (m, 4H), 4.24e4.11 (m, 2H), 3.78e3.67 (m,
4H), 3.43e3.39 (m, 1H), 3.30e3.16 (m, 3H), 2.86e2.50 (m, 6H),
2.37e2.34 (m, 3H), 2.32 (s, 3H, AreCH3), 2.05 (s, 3H, CH3), 1.99e1.70
(m, 5H), 1.13e1.02 (m, 3H, CH3). HRMS (ESIþ) m/z calculated for
4.1.21. tert-Butyl-4-(ethyl(phenethyl)carbamoyl)piperidine-1-
C
33H46ClN4O3 [M þ H]þ, 581.3258; found, 581.3271.
carboxylate (14p)
Yellow oil (65%). 1H NMR (CDCl3, 500 MHz)
d: 7.39e7.20 (m, 5H,
4.1.27. 1-Acetyl-N-(3-chlorophenyl)-N-(3-(4-(ethyl(4-fluorobenzyl)
carbamoyl)piperidin-1-yl)propyl)piperidine-4-carboxamide (16c)
Brown solid (41%); mp: 48e52 ꢁC. 1H NMR (CDCl3, 500 MHz)
AreH), 4.25e4.18 (m, 2H, piperidineeH), 3.43e3.36 (m, 2H, CH2),
2.85e2.79 (m, 2H, piperidineeH), 2.75e2.52 (m, 4H, CH2), 2.32e2.27
(m, 1H, piperidineeH), 1.89e1.70 (m, 4H, piperidineeH), 1.37 (s, 9H,
Boc-CH3), 1.03e0.98 (m, 3H, CH3). ESI-MS m/z: 361 [M þ H]þ.
d
:
7.47e7.42 (m, 2H, AreH), 7.18e7.13 (m, 3H, AreH), 7.08e6.99 (m,
3H, AreH), 4.58e4.48 (m, 4H), 4.04e3.82 (m, 2H), 3.78e3.64 (m,
4H), 3.59e3.50 (m, 1H), 3.43e3.18 (m, 6H), 3.10e2.75 (m, 6H),
2.38e2.17 (m, 5H), 2.06 (s, 3H, CH3), 1.10e0.96 (m, 3H, CH3). HRMS
(ESIþ) m/z calculated for C32H43ClFN4O3 [M þ H]þ, 585.3008; found,
585.3015.
4.1.22. tert-Butyl 4-(ethyl(4-fluorophenethyl)carbamoyl)
piperidine-1-carboxylate (14q)
White solid (78%). Mp: 93e95 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
7.12e7.06 (m, 2H, AreH), 6.98e6.90 (m, 2H, AreH), 4.12e4.05 (m,
2H), 3.46e3.29 (m, 3H), 3.17e3.13 (m, 1H), 2.80e2.71 (m, 3H),
2.53e2.47 (m, 1H), 2.16e2.00 (m, 1H), 1.75e1.66 (m, 1H), 1.61e1.53
(m, 2H), 1.42 (s, 9H, 3ꢂ CH3), 1.36e1.24 (m, 1H), 1.11e1.06 (m, 3H,
CH3). ESI-MS m/z: 379 [M þ H]þ.
4.1.28. 1-Acetyl-N-(3-(4-((4-chlorobenzyl)(ethyl)carbamoyl)
piperidin-1-yl)propyl)-N-(3-chlorophenyl)piperidine-4-
carboxamide (16d)
Brown solid (39%); mp: 43e47 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
7.45e7.28 (m, 6H, AreH), 7.14e7.09 (m, 2H, AreH), 4.56e4.49 (m,
4H), 4.14e4.08 (m, 2H), 3.79e3.67 (m, 4H), 3.44e3.27 (m, 3H),
2.98e2.79 (m, 6H), 2.39e2.29 (m, 3H), 2.06 (s, 3H, CH3), 1.98e1.65
(m, 6H), 1.15e1.04 (m, 3H, CH3). HRMS (ESIþ) m/z calculated for
4.1.23. tert-Butyl 4-((4-chlorophenethyl)(ethyl)carbamoyl)
piperidine-1-carboxylate (14r)
C
32H43Cl2N4O3 [M þ H]þ, 601.2712; found, 601.2721.
Yellow solid (71%). Mp: 87e89 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
7.28e7.22 (m, 2H, AreH), 7.11e7.06 (m, 2H, AreH), 4.15e4.05 (m,
2H), 3.48e3.36 (m, 3H), 3.18e3.14 (m, 1H), 2.81e2.70 (m, 3H),
2.56e2.49 (m, 1H), 2.14e2.10 (m, 1H), 1.77e1.69 (m, 1H), 1.61e1.58
(m, 2H), 1.44 (s, 9H, 3ꢂ CH3), 1.35e1.29 (m, 1H), 1.13e1.08 (m, 3H,
CH3). ESI-MS m/z: 396 [M þ H]þ.
4.1.29. 1-Acetyl-N-(3-chlorophenyl)-N-(3-(4-((4-
cyanobenzyl)(ethyl)carbamoyl)piperidin-1-yl)propyl)piperidine-4-
carboxamide (16e)
Yellow solid (38%); mp: 42e46 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
7.68e7.28 (m, 6H, AreH), 7.22e7.12 (m, 2H, AreH), 4.61e4.52 (m,
3H), 4.23e4.12 (m, 2H), 3.81e3.64 (m, 3H), 3.42e3.29 (m, 2H),
3.08e2.97 (m, 2H), 2.87e2.81 (m, 1H), 2.60e2.30 (m, 5H), 2.06 (s,
3H, CH3), 1.99e1.65 (m, 10H), 1.19e1.10 (m, 3H, CH3). HRMS (ESIþ)
m/z calculated for C33H43ClN5O3 [M þ H]þ, 592.3054; found,
592.3059.
4.1.24. General procedure for the synthesis of 6 and 16ber
To a solution of compound 14aer (1.00 mmol) in EtOAc (1.00 ml)
was added 6 N HCl (4.00 ml). The mixture was stirred at room
temperature for 2 h. The solution was concentrated at reduced
pressure, and the residue was poured into water and basified with
10% NaOH aqueous solution (pH ¼ 10). The mixture was extracted
with EtOAc (15 ml ꢂ 3). The organic phase was dried with Na2SO4
and concentrated. The obtained products 15aer were used for next
step without further purification.
A mixture of 1-acetyl-N-(3-chlorophenyl)-N-(3-chloropropyl)
piperidine-4-carboxamide 11 (0.10 mmol), compound 15aer
(0.10 mmol), KI (0.10 mmol) and K2CO3 (0.15 mmol) in MeCN (8 ml)
was stirred at reflux for 48 h. The mixture was concentrated in
vacuo, diluted with water (10.00 ml) and extracted with EtOAc
(15 ml ꢂ 3). The organic layer was dried (MgSO4), filtered and
concentrated in vacuo. The residue was purified by column chro-
matography on silica gel (EtOAc/MeOH 1/0 to 9/1) to afford product.
4.1.30. 1-Acetyl-N-(3-(4-(benzyl(methyl)carbamoyl)piperidin-1-yl)
propyl)-N-(3-chlorophenyl)piperidine-4-carboxamide (16f)
Yellow solid (46%); mp: 50e54 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
7.47e7.29 (m, 6H, AreH), 7.23e7.12 (m, 3H, AreH), 4.59e4.51 (m,
4H), 4.17e4.03 (m, 2H), 3.82e3.63 (m, 5H), 3.47e3.32 (m, 4H), 3.15
(s, 3H, CH3), 2.62 (s, 2H, CH2), 2.36e2.29 (m, 4H), 2.05 (s, 3H, CH3),
1.95e1.73 (m, 5H). HRMS (ESIþ) m/z calculated for C31H42ClN4O3
[M þ H]þ, 553.2945; found, 553.2953.
4.1.31. 1-Acetyl-N-(3-(4-(benzylcarbamoyl)piperidin-1-yl)propyl)-
N-(3-chlorophenyl)piperidine-4-carboxamide (16g)
Brown solid (25%); mp: 89e92 ꢁC. 1H NMR (500 MHz, CDCl3)
d
: 7.37e7.36 (m, 2H, AreH), 7.31e7.28 (m, 2H, AreH), 7.24e7.18
(m, 4H, AreH), 7.07e7.05 (m, 1H, AreH), 5.92 (brs, 1H), 4.49 (d,
J ¼ 13.5 Hz, 1H), 4.41e4.39 (m, 2H), 3.75 (d, J ¼ 13.5 Hz,
1H), 3.66e3.63 (m, 2H), 2.90e2.78 (m, 3H), 2.34e2.29 (m, 4H),
2.14e2.08 (m, 1H), 2.01 (s, 3H, CH3), 1.94e1.93 (m, 2H), 1.85e1.59
4.1.25. 1-Acetyl-N-(3-(4-(benzyl(ethyl)carbamoyl)piperidin-1-yl)
propyl)-N-(3-chlorophenyl)piperidine-4-carboxamide (6)
Yellow oil (35%); 1H NMR (CDCl3, 500 MHz)
d: 7.46e7.28 (m, 7H,
AreH), 7.18e7.13 (m, 2H, AreH), 4.57e4.53 (m, 4H), 4.07e4.01 (m,
2H), 3.82e3.66 (m, 5H), 3.52e3.43 (m, 4H), 3.32e3.27 (m, 2H),
2.86e2.81 (m, 2H), 2.37e2.29 (m, 4H), 2.05 (s, 3H, CH3), 1.98e1.75
(m, 5H), 1.09e0.99 (m, 3H, CH3). HRMS (ESIþ) m/z calculated for
(m, 2H). 13C NMR (100 MHz, CDCl3)
d: 174.57, 173.72, 168.68,
143.50, 138.30, 135.31, 130.83, 128.63, 128.51, 128.25, 127.63,
127.41, 126.39, 55.66, 53.02, 47.96, 45.44, 43.35, 40.59, 39.40,
28.76, 28.24, 25.10, 21.29. HRMS (ESIþ) m/z calculated for
C
32H44ClN4O3 [M þ H]þ, 567.3102; found, 567.3080.
C
30H40ClN4O3 [M þ H]þ, 539.2789; found, 539.2765.

S. Hu et al. / European Journal of Medicinal Chemistry 71 (2014) 259e266
265
4.1.32. 1-Acetyl-N-(3-chlorophenyl)-N-(3-(4-(4-
4.1.38. 1-Acetyl-N-(3-chlorophenyl)-N-(3-(4-(4-
fluorophenethylcarbamoyl)piperidin-1-yl)propyl)piperidine-4-
carboxamide (16n)
fluorobenzylcarbamoyl)piperidin-1-yl)propyl)piperidine-4-
carboxamide (16h)
Yellow solid (43%); mp: 75e78 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
White solid (44%); mp: 66e68 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
7.44e7.41 (m, 2H, AreH), 7.25e7.19 (m, 4H, AreH), 7.03e6.99 (m, 2H,
AreH), 4.51 (d, J ¼ 13.5 Hz,1H), 4.38 (d, J ¼ 6.0 Hz, 2H), 3.78e3.66 (m,
4H), 3.22e3.10 (m, 2H), 2.86e2.81 (m, 1H), 2.67e2.63 (m, 1H), 2.62
(s, 2H, CH2), 2.45e2.30 (m, 5H), 2.18e2.10 (m, 2H), 2.05 (s, 3H, CH3),
1.92e1.60 (m, 6H). HRMS (ESIþ) m/z calculated for C30H39ClFN4O3
[M þ H]þ, 557.2695; found, 557.2705.
7.36e7.35 (m, 2H, AreH), 7.16 (s,1H, AreH), 7.10e7.03 (m, 3H, AreH),
6.96e6.92 (m, 2H, AreH), 5.65 (brs, 1H), 4.48 (d, J ¼ 13.5 Hz, 1H),
3.74e3.71 (d, J ¼ 13.5 Hz,1H), 3.64e3.61 (m, 2H), 3.45e3.41 (m, 2H),
2.83e2.72 (m, 5H), 2.31e2.24 (m, 4H), 2.00 (s, 3H, CH3), 1.98e1.94
(m, 1H), 1.87e1.82 (m, 2H), 1.76e1.56 (m, 10H). 13C NMR (CDCl3,
100 MHz) d: 174.77, 173.65, 168.64, 162.47, 160.53, 143.47, 135.25,
134.48, 130.78, 130.07, 128.46, 128.21, 126.33, 55.67, 53.08, 47.97,
45.40, 43.14, 40.55, 39.34, 34.75, 28.75, 28.20, 25.14, 21.25. HRMS
(ESIþ) m/z calculated for C31H40ClFN4O3 [M þ H]þ571.2851; found,
571.2860.
4.1.33. 1-Acetyl-N-(3-(4-(4-chlorobenzylcarbamoyl)piperidin-1-yl)
propyl)-N-(3-chlorophenyl)piperidine-4-carboxamide (16i)
Brown solid (45%); mp: 46e49 ꢁC. 1H NMR (500 MHz, CDCl3)
d
: 7.38e7.37 (m, 2H, AreH), 7.27e7.26 (m, 2H, AreH), 7.18e7.15
4.1.39. 1-Acetyl-N-(3-(4-(4-chlorophenethylcarbamoyl)piperidin-
(m, 3H, AreH), 7.07e7.06 (m, 1H, AreH), 5.99 (brs, 1H), 4.99 (d,
J ¼ 13.5 Hz, 1H), 4.37 (d, J ¼ 6 Hz, 2H), 3.75 (d, J ¼ 13.5 Hz, 1H),
3.67 (t, J ¼ 7.5 Hz, 2H), 2.91e2.79 (m, 3H), 2.32 (brs, 4H), 2.14e
2.09 (m, 1H), 2.01 (s, 3H, CH3), 1.95e1.94 (m, 2H), 1.85e1.83 (m,
1-yl)propyl)-N-(3-chlorophenyl)piperidine-4-carboxamide (16o)
Yellow solid (49%); mp: 58e60 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
7.36e7.35 (m, 2H, AreH), 7.23e7.21 (d, J ¼ 8 Hz, 2H, AreH), 7.16 (s,
1H, AreH), 7.07e7.03 (m, 3H, AreH), 5.71 (brs, 1H), 4.45 (d,
J ¼ 10 Hz, 1H), 3.74e3.61 (m, 3H), 3.45e3.41 (m, 2H), 2.84e2.72
(m, 5H), 2.33e2.24 (m, 4H), 2.00 (s, 3H, CH3), 1.98e1.94 (m, 1H),
2H), 1.76e1.55 (m, 8H). 13C NMR (100 MHz, CDCl3)
d 174.71,
173.82, 168.75, 143.55, 137.00, 135.40, 133.24, 130.91, 129.03,
128.81, 128.60, 128.32, 126.45, 55.70, 53.04, 48.02, 45.51, 42.69,
40.66, 39.46, 28.83, 28.32, 25.15, 21.36. HRMS (ESIþ) m/z
calculated for
573.2406.
1.87e1.83 (m, 2H), 1.72e1.54 (m, 10H). 13C NMR (CDCl3, 100 MHz)
d:
174.83, 173.66, 168.68, 143.43, 137.30, 135.26, 132.24, 130.79, 130.00,
128.57, 128.48, 128.20, 126.32, 55.65, 53.06, 47.97, 45.41, 43.13,
40.56, 39.34, 34.91, 28.71, 28.20, 25.11, 21.25. HRMS (ESIþ) m/z
calculated for C31H40Cl2N4O3 [M þ H]þ587.2555; found, 587.2561.
C
30H39Cl2N4O3 [M
þ
H]þ, 573.2399; found,
4.1.34. 1-Acetyl-N-(3-chlorophenyl)-N-(3-(4-(4-
cyanobenzylcarbamoyl)piperidin-1-yl)propyl)piperidine-4-
carboxamide (16j)
4.1.40. 1-Acetyl-N-(3-chlorophenyl)-N-(3-(4-(ethyl(phenethyl)
carbamoyl)piperidin-1-yl)propyl)piperidine-4-carboxamide (16p)
Yellow oil (35%); 1H NMR (CDCl3, 500 MHz)
d: 7.63e7.30 (m,
Yellow oil (32%). 1H NMR (CDCl3, 500 MHz)
d: 7.44e7.28 (m, 4H,
8H, AreH), 4.53e4.45 (m, 3H), 3.80e3.48 (m, 5H), 3.33e3.29 (m,
1H), 3.12e3.08 (m, 1H), 2.97e2.82 (m, 2H), 2.40e2.07 (m, 7H),
2.05 (s, 3H, CH3), 1.98e1.63 (m, 7H). HRMS (ESIþ) m/z calculated
for C31H39ClN5O3 [M þ H]þ, 564.2741; found, 564.2752.
AreH), 7.25e7.14 (m, 5H, AreH), 4.56e4.53 (m, 1H), 3.80e3.62 (m,
4H), 3.56e3.41 (m, 3H), 3.20e3.11 (m, 3H), 2.86e2.80 (m, 3H),
2.78e2.61 (m, 2H), 2.40e2.25 (m, 4H), 2.20e2.06 (m, 4H), 2.07 (s,
3H, CH3),1.96e1.62 (m, 6H),1.16e1.08 (m, 3H, CH3). HRMS (ESIþ) m/
z calculated for C33H46ClN4O3 [M þ H]þ581.3258; found, 581.3266.
4.1.35. 1-Aacetyl-N-(3-chlorophenyl)-N-(3-(4-(phenylcarbamoyl)
piperidin-1-yl)propyl)piperidine-4-carboxamide (16k)
4.1.41. 1-Acetyl-N-(3-chlorophenyl)-N-(3-(4-(ethyl(4-
fluorophenethyl)carbamoyl)piperidin-1-yl)propyl)piperidine-4-
carboxamide (16q)
Yellow solid (34%); mp: 92e95 ꢁC. 1H NMR (CDCl3, 500 MHz)
d
: 7.54e7.40 (m, 5H, AreH), 7.34e7.31 (m, 2H, AreH), 7.12e7.09
White oil (42%). 1H NMR (CDCl3, 500 MHz)
d: 7.34e7.33 (m, 2H,
(m, 2H, AreH), 4.52 (d, J ¼ 13.5 Hz, 1H), 3.76 (d, J ¼ 13.5 Hz, 1H),
3.71e3.68 (m, 2H), 3.04 (d, J ¼ 10.5 Hz, 2H), 2.87e2.82 (m, 1H),
2.45e2.10 (m, 13H), 2.06 (s, 3H, CH3), 2.00e1.94 (m, 4H). HRMS
(ESIþ) m/z calculated for C29H38ClN4O3 [M þ H]þ525.2632; found,
525.2637.
AreH), 7.16e7.15 (m,1H, AreH), 7.11e7.04 (m, 3H, AreH), 6.96e6.88
(m, 2H, AreH), 4.47 (d, J ¼ 12 Hz, 1H), 3.72e3.70 (d, J ¼ 12 Hz, 1H),
3.63e3.62 (m, 2H), 3.43e3.40 (m, 2H), 3.34e3.30 (m, 1H), 3.14e3.11
(m, 1H), 2.86e2.74 (m, 5H), 2.29e2.21 (m, 5H), 1.99 (s, 3H, CH3),
1.89e1.85 (m, 1H), 1.81e1.55 (m, 10H), 1.34e1.32 (m, 1H), 1.08e1.04
(m, 3H). 13C NMR (CDCl3, 100 MHz)
d: 174.62, 173.72, 168.69, 143.57,
4.1.36. 1-Acetyl-N-(3-chlorophenyl)-N-(3-(4-(ethyl(phenyl)
carbamoyl)piperidin-1-yl)propyl)piperidine-4-carboxamide (16l)
Yellow solid (33%); mp: 70e72 ꢁC. 1H NMR (CDCl3, 500 MHz)
135.28, 134.96, 130.82, 130.27, 130.16, 128.48, 128.26, 127.77, 126.40,
55.82, 53.21, 48.08, 45.46, 42.75, 40.61, 39.41, 34.96, 33.27, 28.78,
28.26, 25.17, 21.30, 14.73, 12.87. HRMS (ESIþ) m/z calculated for
d
: 7.40e7.26 (m, 5H, AreH), 7.11e7.00 (m, 4H, AreH), 4.43 (d,
C
33H44ClFN4O3 [M þ H]þ599.3164; found, 599.3156.
J ¼ 12.5 Hz, 1H), 4.16e4.03 (m, 1H), 3.70e3.50 (m, 5H), 2.95e
2.66 (m, 4H), 2.50e2.05 (m, 10H), 1.97 (s, 3H, CH3), 1.92e1.73 (m,
5H), 1.00 (t, J ¼ 7.0 Hz, 3H, CH3). HRMS (ESIþ) m/z calculated for
4.1.42. 1-Acetyl-N-(3-(4-((4-chlorophenethyl)(ethyl)carbamoyl)
piperidin-1-yl)propyl)-N-(3-chlorophenyl)piperidine-4-
carboxamide (16r)
C
31H42ClN4O3 [M þ H]þ553.2945; found, 553.2952.
Yellow oil (48%). 1H NMR (CDCl3, 500 MHz)
d: 7.36 (brs, 2H, Are
4.1.37. 1-Acetyl-N-(3-chlorophenyl)-N-(3-(4-(phenethylcarbamoyl)
piperidin-1-yl)propyl)piperidine-4-carboxamide (16m)
H), 7.26e7.25 (m, 1H, AreH), 7.22e7.17 (m, 2H, AreH), 7.11e7.05 (m,
3H, AreH), 4.50 (d, J ¼ 13.5 Hz, 1H), 3.75e3.73 (d, J ¼ 13.5 Hz, 1H),
3.67e3.62 (m, 2H), 3.46e3.41 (m, 2H), 3.36e3.32 (m, 1H), 3.16e3.11
(m, 1H), 2.89e2.76 (m, 5H), 2.35e2.24 (m, 5H), 2.02 (s, 3H, CH3),
1.92e1.88 (m, 1H), 1.84e1.58 (m, 10H), 1.37e1.35 (m, 1H), 1.10e1.06
Yellow solid (46%); mp: 82e85 ꢁC. 1H NMR (CDCl3, 500 MHz)
d:
7.44e7.29 (m, 4H, AreH), 7.25e7.14 (m, 5H, AreH), 4.51 (d,
J ¼ 13.0 Hz, 1H), 3.83e3.65 (m, 5H), 3.56e3.47 (m, 2H), 3.19e3.05
(m, 2H), 2.90e2.77 (m, 4H), 2.72e2.60 (m, 2H), 2.40e2.28 (m, 4H),
2.05 (s, 3H, CH3), 1.95e1.65 (m, 8H). HRMS (ESIþ) m/z calculated for
(m, 3H). 13C NMR (CDCl3, 100 MHz)
d: 174.61, 173.67, 168.64, 143.43,
137.74, 135.24, 131.91, 130.78, 130.14, 128.78, 128.39, 128.21, 126.36,
55.77, 53.14, 48.03, 45.42, 42.75, 40.57, 39.36, 35.12, 33.41, 28.74,
C
31H42ClN4O3 [M þ H]þ553.2945; found, 553.2950.

266
S. Hu et al. / European Journal of Medicinal Chemistry 71 (2014) 259e266
28.21, 25.13, 21.26, 14.70, 12.83. HRMS (ESIþ) m/z calculated for
C
Acknowledgments
33H44ClFN4O3 [M þ H]þ615.2868; found, 615.2870.
The authors are grateful to the support of the National Natural
Science Foundation of China (81072515 and 81202341), the Min-
istry of Science and Technology of China (2012ZX09301001-005),
the Ministry of Health-medical Science Critical Technological Pro-
gram of Zhejiang Province (WKJ20122024)and the Shanghai Com-
mission of Science and Technology (12XD1402100). The authors
also thank Dr. Shan Cen (Institute of Medicinal Biotechnology,
Chinese Academy of Medical Sciences, Beijing, China) for evaluation
of antiviral activity of representative compounds and Jianyang Pan
(Institute of Pharmaceutical Informatics, Zhejiang University,
China) for performing NMR spectrometry and Mass spectrometry
for structure elucidation.
4.2. Biological assay methods
4.2.1. Cell cytotoxicity assays
Cytotoxicities of target compounds were tested using CCK-8
(SigmaeAldrich) assay. Briefly, 100
(5000 cells/well) were dispensed in a 96-well plate, and then
preincubated in the plate for 24 h at 37 ꢁC in 5% CO2.10
L of various
concentrations of substances to be tested were added to the plate.
mL of HEK293 cell suspension
m
After 7 h incubation, 10 mL of CCK-8 solution were added to each
well of the plate. The plate was incubated at 37 ꢁC for another 2 h in
the incubator and the optical absorbance was measured at 430 nm
using a microplate reader.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.11.013.
4.2.2. Calcium mobilization assay
CHO cells stably expressing CCR5 and Ga16 were loaded with
2.00
mmol/L Fluo-4 AM in Hanks balanced salt solution (HBSS,
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0.50 mmol/L, Mg2SO4 0.60 mmol/L, NaCl 137.00 mmol/L, BSA
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