Synthesis and biological evaluation of novel piperidin-4-ol derivatives

Article abstract of DOI:10.1007/s00706-011-0645-9

A series of novel piperidin-4-ol derivatives were designed, synthesized, and evaluated for potential treatment of HIV. The compounds were obtained via an efficient synthetic route in excellent yields and have been characterized by ¹H NMR, ¹³C NMR, MS, and elemental analysis. The CCR5 antagonistic activities of the compounds have also been evaluated.

Full text of DOI:10.1007/s00706-011-0645-9

Monatsh Chem (2012) 143:303–308
DOI 10.1007/s00706-011-0645-9
ORIGINAL PAPER
Synthesis and biological evaluation of novel piperidin-4-ol
derivatives
•
Zhiyong Weng Wei Wei Xiaowu Dong
•
•
•
•
•
Yongzhou Hu Shufang Huang Tao Liu
Xin Xie
Received: 7 April 2010 / Accepted: 31 August 2011 / Published online: 24 September 2011
Ó Springer-Verlag 2011
Abstract A series of novel piperidin-4-ol derivatives
were designed, synthesized, and evaluated for potential
treatment of HIV. The compounds were obtained via an
efficient synthetic route in excellent yields and have been
slowly to AIDS and respond better to treatment [3].
Blockade of the CCR5 receptor seems not only to treat
HIV-1 infections but also, importantly, not to be associated
with any mechanism-related side effects [4]. These facts
have inspired much research to identify new CCR5
antagonists. Widespread research has resulted in an FDA-
approved drug, maraviroc, and several clinical candidates
[5].
1
characterized by H NMR, 13C NMR, MS, and elemental
analysis. The CCR5 antagonistic activities of the com-
pounds have also been evaluated.
Keywords Piperidin-4-ol ꢀ HIV ꢀ CCR5 ꢀ Heterocycles ꢀ
Ketones ꢀ Reductions
Although CCR5 antagonists are very different in struc-
ture, all contain one basic nitrogen atom which is believed
to anchor the ligands to the CCR5 receptor via a strong
salt-bridge interaction. Another common feature of most of
the antagonists is the presence of two or more lipophilic
groups in the ligand [6].
Introduction
The chemokine receptor CCR5, belonging to the seven
transmembrane G-protein coupled receptor family, has
been identified as the essential coreceptor in the process of
HIV-1 entry [1]. Zaitseva and coworkers reported that cells
could not become infected with macrophagetropic (R5)
HIV-1 strains in vitro if they do not express CCR5 [2].
Furthermore, CCR5D32 homozygous individuals are ulti-
mately resistant to R5-tropic HIV-1 infection whereas
heterozygotes do become infected, but progress more
Our CCR5 program was initiated from compound 1,
which was a potent CCR5 antagonist of ligand binding
(IC50: 45 nM) [7]. Researchers at AstraZeneca had earlier
reported a series of piperidin-4-ol CCR5 antagonists
(exemplified by compound 2, with an IC50 value of
1 lM in ligand-binding assay) [8, 9]. As illustrated in
Scheme 1, considering the common features of CCR5
antagonists, we designed a chemical scaffold that com-
bined the attractive characteristics of compounds 1 and 2
by fragment assembly. Herein, we report the synthesis and
biological evaluation with ligand-induced calcium mobili-
zation assay of a series of novel piperidin-4-ol derivatives
13a–13g, with our primary interest in finding CCR5
antagonists with a novel skeleton as potential treatments
for HIV-1.
Z. Weng ꢀ X. Dong ꢀ Y. Hu ꢀ S. Huang ꢀ T. Liu (&)
ZJU-ENS Joint Laboratory of Medicinal Chemistry,
College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou 310058, China
e-mail: lt601@zju.edu.cn
W. Wei ꢀ X. Xie (&)
State Key Laboratory of Drug Research,
National Center for Drug Screening, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China
Results and discussion
The synthetic route to compounds 13a–13g is shown in
Scheme 2. 6-Benzyl-1-oxa-6-azaspiro[2.5]octane (4),
e-mail: xxie@mail.shcnc.ac.cn
123

304
Z. Weng et al.
Scheme 1
prepared from N-benzyl-4-piperidone (3) with trimethyl-
sulfonium iodide in high yield according to the reported
method [10], was reacted with several corresponding het-
erocycles in methanol under reflux to provide compounds
5a–5f in excellent yields. Deprotection of 5a–5f by
hydrogenation afforded amines 6a–6f.
the three new heteroaromatic analogs, the 1,2,3-1H-ben-
zotriazole analog 13d had the most potent CCR5
antagonistic activity, with an IC50 value of 1.28 lM, and
the 1H-benzo[d]imidazole analog 13e had activity similar
to that of compound 13a, whereas the 1H-indole analog 13f
was one ninth as potent as compound 13d. The data indi-
cated that the number of nitrogen atoms in the fused
heteroaromatic systems affected the activity.
Racemic 3-amino-3-phenylpropanoic acid (8) and
3-amino-3-phenylpropan-1-ol (9) were synthesized fol-
lowing the method described in the literature with minor
modifications [11]. Acylation of 9 with benzoyl chlorides
10a and 10b in the presence of Et3N gave amides 11a and
11b, which were then subjected to a Swern oxidation
to give aldehydes 12a and 12b. Treatment of aldehydes
12a and 12b with compounds 6a–6f in the presence of
NaBH(OAc)3 afforded the racemic target compounds
13a–13g. The structures of the synthesized target com-
Replacement of fluorine atom with a hydrogen atom
(13a vs. 13g) reduced potency by approximately a factor of
three. This result showed that a fluorine atom substituted at
R1 had a favorable effect on activity.
In summary, we have designed and synthesized a series
of novel piperidin-4-ol derivatives by use of an efficient
synthetic route and studied their CCR5 antagonistic activ-
ities. Several compounds had promising antagonistic
activity. Among these, compound 13d was the most potent
with an IC50 of 1.28 lM. This work provided novel com-
pounds with CCR5 antagonistic activity for further
optimization. Further studies of the mechanism of action of
these compounds are under way.
1
pounds were confirmed by H NMR, 13C NMR, ESI–MS,
and elemental analysis.
The synthesized compounds 13a–13g were screened for
their antagonistic activity against CCR5 by employing a
calcium mobilization assay. As shown in Table 1, most of
tested compounds had potent CCR5 antagonistic activity.
Comparison of piperidin-4-ol derivatives 13a, 13b, and 13c
revealed that the benzoimidazole motif 13a provided a
significant potency advantage versus triazole 13b and car-
Experimental
bazole 13c, indicating that
a
smaller and bulkier
Melting points were obtained on a Bu¨chi B-540 apparatus
(Bu¨chi Labortechnik, Flawil, Switzerland) and were cor-
rected using standard compounds of known melting points.
heteroaromatic ring at the R2 position of the target com-
pounds was not favorable for CCR5 antagonistic activity.
Encouraged by the results for 13a, fused heteroaromatic
rings attached to the piperidine-4-ol moiety were further
investigated to improve CCR5 antagonistic activity. Among
1
All H NMR and 13C NMR spectra were recorded on a
Bruker 400 MHz spectrometer (Bruker Bioscience, Bille-
rica, MA, USA) with SiMe4 as the internal standard.
123

Novel piperidin-4-ol derivatives
305
Scheme 2
H
N
(CH3)3SO+I- Bu4N+Br-, NaOH
H2, 5%Pd/C
R2-H
N
,
N
N
Toluene, 80 o
C
CH3OH, reflux
CH3OH, reflux
HO
R2
O
HO
O
R2
5a-5f
6a-6f
4
3
N
d: R2=
a: R2=
N
N
N
N
N
N
N
N
e: R2=
f: R2=
b: R2=
N
N
c: R2=
N
R1
NH2
NH2
O
O
Et3N, CH2Cl2, 0 o
C
LiAlH4
NH4Ac, CH2(COOH)2
C2H5OH, reflux
OH
OH
H
THF,
Cl
65 oC
R1
9
8
O
7
O
NH
10a 10b
-
OH
R1
1
R1
a
: R =H
1
b
: R =F
11a-11b
a: R1=H
b: R1=F
ClCOCOCl, DMSO
NaBH(OAc)3, CH2Cl2, rt
O
NH
O
O
NH
CH2Cl2, -78 o
C
6a 6f
-
H
N
R2
OH
13a-13g
12a-12b
N
g: R1=H R2=
1
2
a: R1=H
b: R1=F
a: R1=F R2=
N
N
d
: R =F R =
N
N
N
N
N
N
N
N
b: R1=F R2=
c: R1=F R2=
N
N
e: R1=F R2=
f: R1=F R2=
N
Column chromatography was performed on 200–300 mesh
silica gel.
Table 1 CCR5 antagonistic
activity of the piperidin-4-ol
derivatives 13a–13g
Compound
IC50/lM
13a
13b
13c
13d
13e
13f
3.95 1.25
[30
General method for synthesis of compounds 5a–5f
[30
A mixture of the corresponding heterocycle (3.0 mmol) and
0.61 g 6-benzyl-1-oxa-6-azaspiro[2.5]octane (4, 3.0 mmol)
in 5 cm3 CH3OH in a sealed tube was stirred at reflux over-
night. After cooling to room temperature, the mixture was
concentrated in vacuo. The residue was purified by column
chromatography (petroleum ether–ethyl acetate = 1:10) to
afford the products 5a–5f.
1.28 0.10
2.20 0.69
10.96 3.05
13.33 3.93
13g
Chemical shifts were reported in d values (ppm), relative to
TMS as internal standard, and J values are reported in
Hertz (Hz). Mass spectra (ESI, positive ion) were recorded
on an Esquire-LC-00075 spectrometer (Bruker Biosci-
ence). Elemental analysis (C, H, N) was conducted using
an EA-1110 elemental analyzer; the results were found to
be in good agreement ( 0.2%) with calculated values.
1-Benzyl-4-(2-methyl-1H-benzo[d]imidazol-1-ylmethyl)-
piperidin-4-ol (5a, C21H25N3O)
1
Yield: 55%; yellow solid; m.p.: 115–118 °C; Rf = 0.25; H
NMR (400 MHz, CD3OD): d = 8.03 (dd, 2H, J = 5.0,
8.0 Hz, Ar–H), 7.59 (d, 2H, J = 7.0 Hz, Ar–H), 7.45–7.23
(m, 5H, Ar–H), 4.08 (s, 2H), 3.51 (s, 2H), 2.93–2.76 (m, 4H,
123

306
Z. Weng et al.
piperidine-H), 2.73–2.65 (m, 4H, piperidine-H), 2.60 (s, 3H)
ppm; 13C NMR (100 MHz, CD3OD): d = 154.9, 142.8,
138.4, 137.6, 130.9, 129.5, 128.6, 123.3, 123.2, 118.8, 112.4,
71.7, 64.1, 55.7, 49.7, 35.7, 14.7 ppm; ESI–MS: m/z = 336
[M ? H]?.
7.65 (d, 1H, J = 7.5 Hz, Ar–H), 7.60–7.55 (m, 2H, Ar–H),
7.30–7.00 (m, 6H, Ar–H), 3.89 (s, 2H), 3.53 (s, 2H),
2.97–2.78 (m, 4H, piperidine-H), 2.73–2.56 (m, 4H,
piperidine-H) ppm; 13C NMR (100 MHz, CD3OD):
d = 138.8, 138.2, 130.9, 130.8, 129.9, 129.4, 128.6,
122.3, 121.6, 120.1, 111.4, 102.1, 71.4, 64.0, 57.5, 49.8,
35.2 ppm; ESI–MS: m/z = 321 [M ? H]?.
1-Benzyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-4-ol
(5b, C15H20N4O)
1
Yield: 72%; yellow solid; m.p.: 98–102 °C; Rf = 0.40; H
General method for synthesis of compounds
11a and 11b
NMR (400 MHz, CDCl3): d = 8.18 (d, 1H, J = 2.5 Hz,
triazol-H), 8.06 (d, 1H, J = 9.5 Hz, triazol-H), 7.27–7.14 (m,
5H, Ar–H), 4.25 (s, 2H), 3.57 (s, 2H), 2.97–2.73 (m, 6H,
piperidine-H), 2.71–2.64 (m, 2H, piperidine-H) ppm; 13C
NMR (100 MHz, CDCl3): d = 151.0, 144.4, 137.9, 129.4,
128.3, 127.2, 69.0, 62.9, 59.2, 48.8, 34.6 ppm; ESI–MS:
m/z = 273 [M ? H]?.
To an ice-cooled stirred suspension of 0.45 g 3-amino-3-
phenylpropan-1-ol 9 (3 mmol) in 3 cm3 CH2Cl2 was added
1.2 cm3 Et3N (9 mmol) followed by compound 10a or 10b
(3.6 mmol). After stirring at 0 °C for 4 h, the mixture was
diluted with 30 cm3 saturated aqueous NaHCO3 solution,
and the organic layer was separated. The aqueous layer was
extracted with 30 cm3 CH2Cl2, and the organic layers were
combined and washed with brine (2 9 20 cm3), dried
(Na2SO4), filtered, and concentrated in vacuo. The residue
was purified by column chromatography (petroleum ether–
ethyl acetate = 1:1) to afford the products 11a or 11b.
1-Benzyl-4-(9H-carbazol-9-ylmethyl)piperidin-4-ol
(5c, C25H26N2O)
1
Yield: 42%; white solid; m.p.: 65–67 °C; Rf = 0.35; H
NMR (400 MHz, CDCl3): d = 8.10 (d, 2H, J = 7.5 Hz, Ar–
H), 7.94 (dd, 2H, J = 5.5, 8.5 Hz, Ar–H), 7.85–7.73 (m, 3H,
Ar–H), 7.27–7.12 (m, 6H, Ar–H), 4.10 (s, 2H), 3.50 (s, 2H),
2.95–2.77 (m, 4H, piperidine-H), 2.71–2.57 (m, 4H, piper-
idine-H) ppm; 13C NMR (100 MHz, CDCl3): d = 141.7,
138.3, 129.2, 128.3, 127.1, 125.8, 123.2, 120.2, 119.4, 110.0,
72.3, 63.1, 54.6, 48.8, 35.6 ppm; ESI–MS: m/z = 371
[M ? H]?.
N-(3-Hydroxy-1-phenylpropyl)benzamide (11a)
1
Yield: 90%; yellow solid; m.p.: 85–87 °C; H NMR was
identical with that in Ref. [12].
4-Fluoro-N-(3-hydroxy-1-phenylpropyl)benzamide
(11b, C16H16FNO2)
1
4-(1H-Benzo[d][1,2,3]triazol-1-ylmethyl)-1-benzylpiperi-
din-4-ol (5d, C19H22N4O)
Yield: 88%; white solid; m.p.: 90–92 °C; Rf = 0.50; H
NMR (400 MHz, CDCl3): d = 7.95–7.87 (m, 2H, Ar–H),
7.83–7.78 (m, 2H, Ar–H), 7.49–7.36 (m, 4H, Ar–H),
7.32–7.29 (m, 1H, Ar–H), 7.05 (d, 1H, J = 7.0 Hz, NH),
5.40–5.36 (m, 1H), 3.72–3.65 (m, 2H), 3.65 (s, 1H, OH),
2.24–2.19 (m, 1H), 1.95–1.90 (m, 1H) ppm; 13C NMR
(100 MHz, CDCl3): d = 166.5, 166.1, 163.6, 141.4, 130.3,
130.2, 129.4, 129.3, 128.9, 127.7, 126.5, 115.8, 115.5,
59.3, 51.8, 38.2 ppm; ESI–MS: m/z = 274 [M ? H]?.
1
Yield: 70%; white solid; m.p.: 147–149 °C; Rf = 0.20; H
NMR (400 MHz, CDCl3): d = 8.03 (d, 1H, J = 8.0 Hz, Ar–
H), 7.86 (dd, 2H, J = 6.0, 8.0 Hz, Ar–H), 7.53–7.50 (m, 1H,
Ar–H), 7.39–7.11 (m, 5H, Ar–H), 4.55 (s, 2H), 3.55 (s, 2H),
2.93–2.73 (m, 6H, piperidine-H), 2.65–2.57 (m, 2H, piperidine-
H) ppm; 13C NMR (100 MHz, CDCl3): d = 145.5, 138.3,
134.2, 129.1, 128.3, 127.6, 127.1, 124.0, 119.8, 110.2, 70.5,
63.0, 58.0, 48.8, 35.1 ppm; ESI–MS: m/z = 323 [M ? H]?.
General method for synthesis of compounds
12a and 12b
4-(1H-Benzo[d]imidazol-1-ylmethyl)-1-benzylpiperi-
din-4-ol (5e, C20H23N3O)
Yield: 68%; yellow solid; m.p.: 155–158 °C; Rf = 0.25; 1H
NMR (400 MHz, CD3OD): d = 7.93–7.87 (m, 3H, Ar–H),
7.55–7.49 (m, 2H, Ar–H), 7.31–7.15 (m, 5H, Ar–H), 4.09 (s,
2H), 3.55(s, 2H), 2.95–2.73(m, 6H, piperidine-H), 2.63–2.49
(m, 2H, piperidine-H) ppm; 13C NMR (100 MHz, CD3OD):
d = 146.0, 143.6, 138.5, 136.3, 130.9, 129.4, 128.6, 124.3,
123.4, 120.0, 112.3, 70.6, 64.1, 56.3, 49.9, 35.2 ppm;
ESI–MS: m/z = 322 [M ? H]?.
To a solution of 0.26 cm3 oxalyl chloride (3.0 mmol) in
20 cm3 CH2Cl2 at -78 °C was added a solution of 0.5 cm3
dimethyl sulfoxide (6.0 mmol) in 8 cm3 CH2Cl2. After the
addition was complete, the mixture was stirred for 3 min
before addition of a solution of compound 11a or 11b
(2.8 mmol) in 8 cm3 CH2Cl2. After 40 min 2.0 cm3 tri-
ethylamine (14.0 mmol) was added. The mixture was
stirred for 10 min and then left to warm to room temper-
ature. After 1 h, the reaction mixture was partitioned
between 10 cm3 H2O and 20 cm3 CH2Cl2. The organic
layer was washed with brine (2 9 10 cm3), dried
(Na2SO4), filtered, and concentrated in vacuo. The residue
1-Benzyl-4-(1H-indol-1-ylmethyl)piperidin-4-ol
(5f, C21H24N2O)
Yield: 65%; yellow solid; m.p.: 165–167 °C; Rf = 0.55; 1H
NMR (400 MHz, CD3OD): d = 7.97–7.93 (m, 2H, Ar–H),
123

Novel piperidin-4-ol derivatives
307
4-Fluoro-N-[3-[4-hydroxy-4-(1H-1,2,4-triazol-1-
ylmethyl)piperidin-1-yl]-1-phenylpropyl]benzamide
(13b, C24H28FN5O2)
was purified by column chromatography (petroleum ether–
ethyl acetate = 2:1) to afford the products 12a or 12b.
N-(3-Oxo-1-phenylpropyl)benzamide (12a)
Yield: 92%; yellow solid; m.p.: 112–114 °C; 1H NMR was
identical with that in Ref. [13].
1
Yield: 85%; yellow solid; m.p.: 73–75 °C; Rf = 0.25; H
NMR (400 MHz, CDCl3): d = 8.66 (d, 1H, J = 6.5 Hz,
NH), 8.15 (d, 1H, J = 2.5 Hz, triazol-H), 8.00 (d, 1H,
J = 9.5 Hz, triazol-H), 7.98–7.94 (m, 1H, Ar–H), 7.38–7.34
(m, 5H, Ar–H), 7.31–7.29 (m, 1H, Ar–H), 7.19–7.15 (m, 2H,
Ar–H), 5.31–5.27 (m, 1H), 4.20 (s, 2H), 2.89–2.57 (m, 8H,
piperidine-H), 2.52–2.43 (m, 3H), 2.25–2.20 (m, 1H) ppm;
13C NMR (100 MHz, CDCl3): d = 176.4, 166.0, 163.5,
151.9, 144.4, 142.0, 130.9, 129.7, 129.6, 128.7, 127.4, 126.1,
115.5, 115.3, 68.5, 58.7, 54.5, 53.2, 48.7, 48.5, 34.3, 33.9,
31.6 ppm; ESI–MS: m/z = 438 [M ? H]?.
4-Fluoro-N-(3-oxo-1-phenylpropyl)benzamide
(12b, C16H14FNO2)
1
Yield: 90%; yellow solid; m.p.: 85–88 °C; Rf = 0.25; H
NMR (400 MHz, CDCl3): d = 9.53 (s, 1H, CHO),
7.91–7.88 (m, 2H, Ar–H), 7.84–7.81 (m, 2H, Ar–H),
7.43–7.35 (m, 4H, Ar–H), 7.30–7.24 (m, 1H, Ar–H), 6.82
(d, 1H, J = 7.0 Hz, NH), 5.68–5.64 (m, 1H), 3.25–3.21 (m,
1H), 3.09–3.06 (m, 1H) ppm; 13C NMR (100 MHz,
CDCl3): d = 200.6, 165.9, 165.8, 163.9, 140.3, 130.1,
130.0, 129.5, 129.4, 129.0, 128.0, 126.5, 115.7, 115.6,
49.2, 48.8 ppm; ESI–MS: m/z = 272 [M ? H]?.
N-[3-[4-(9H-Carbazol-9-ylmethyl)-4-hydroxypiperidin-1-
yl]-1-phenylpropyl]-4-fluorobenzamide
(13c, C34H34FN3O2)
1
Yield: 56%; yellow solid; m.p.: 84–86 °C; Rf = 0.35; H
General method for synthesis of compounds 13a–13g
NMR (400 MHz, CDCl3): d = 8.77 (d, 1H, J = 6.5 Hz,
NH), 8.08 (d, 2H, J = 7.5 Hz, Ar–H), 7.92 (dd, 2H,
J = 5.5, 8.5 Hz, Ar–H), 7.46–7.43 (m, 5H, Ar–H),
7.34–7.30 (m, 5H, Ar–H), 7.25–7.22 (m, 1H, Ar–H), 7.07
(t, 2H, J = 8.5 Hz, Ar–H), 5.22–5.19 (m, 1H), 4.29 (s,
2H), 2.85–2.35 (m, 8H, piperidine-H), 2.34–2.28 (m, 2H),
2.20–2.16 (m, 2H) ppm; 13C NMR (100 MHz, CDCl3):
d = 174.9, 166.1, 163.5, 142.0, 141.6, 130.8, 129.7, 129.6,
128.7, 127.3, 126.2, 126.0, 123.3, 120.4, 119.7, 115.5,
115.3, 109.6, 71.6, 54.7, 54.1, 53.2, 48.8, 48.6, 34.7, 34.5,
31.4 ppm; ESI–MS: m/z = 536 [M ? H]?.
A mixture of 5a–5f (1 mmol) and 100 mg Pd/C (5% on
carbon) in 8 cm3 CH3OH was heated under reflux under H2
for 4 h. The mixture was cooled to room temperature and
the resulting precipitate was isolated by filtration. The fil-
trate was evaporated to give 6a–6f, which was used directly
without purification. Compounds 6a–6f (1 mmol) and
compounds 12a and 12b (1 mmol) were dissolved in
10 cm3 anhydrous CH2Cl2. NaBH(OAc)3 (0.64 g, 3 mmol)
was added, and the mixture was stirred for 8 h at room
temperature. The reaction was quenched by addition of
10 cm3 saturated NaHCO3 solution and extracted with
CH2Cl2 (3 9 10 cm3). The organic layer was washed with
brine (2 9 10 cm3), dried (Na2SO4), filtered, and concen-
trated in vacuo. The residue was purified by column
chromatography (petroleum ether–ethyl acetate = 1:2) to
afford the products 13a–13g.
N-[3-[4-(1H-Benzo[d][1,2,3]triazol-1-ylmethyl)-4-hydro-
xypiperidin-1-yl]-1-phenylpropyl]-4-fluorobenzamide
(13d, C28H30FN5O2)
1
Yield: 78%; white solid; m.p.: 90–93 °C; Rf = 0.15; H
NMR (400 MHz, CDCl3): d = 8.64 (d, 1H, J = 6.5 Hz,
NH), 8.01 (d, 1H, J = 8.0 Hz, Ar–H), 7.88 (dd, 2H,
J = 6.5, 8.0 Hz, Ar–H), 7.55–7.47 (m, 2H, Ar–H), 7.35 (t,
1H, J = 7.0 Hz, Ar–H), 7.30–7.27 (m, 4H, Ar–H),
7.24–7.20 (m, 1H, Ar–H), 7.09 (t, 2H, J = 8.0 Hz, Ar–
H), 5.24–5.20 (m, 1H), 4.58 (s, 2H), 2.88–2.52 (m, 8H,
piperidine-H), 2.50–2.45 (m, 3H), 2.18–2.14 (m, 1H) ppm;
13C NMR (100 MHz, CDCl3): d = 176.8, 166.1, 163.5,
145.4, 142.0, 134.2, 130.8, 129.7, 129.6, 128.7, 127.8,
127.3, 126.2, 124.2, 119.8, 115.5, 115.3, 110.0, 69.5, 57.6,
54.6, 53.2, 48.7, 48.6, 34.0, 33.9, 31.5 ppm; ESI–MS:
m/z = 488 [M ? H]?.
4-Fluoro-N-[3-[4-hydroxy-4-(2-methyl-1H-benzo[d]imida-
zol-1-ylmethyl)-piperidin-1-yl]-1-phenylpropyl]benzamide
(13a, C30H33FN4O2)
1
Yield: 83%; white solid; m.p.: 78–80 °C; Rf = 0.20; H
NMR (400 MHz, CDCl3): d = 8.68 (d, 1H, J = 6.5 Hz,
NH), 7.96 (dd, 2H, J = 5.0, 8.0 Hz, Ar–H), 7.53 (d, 1H,
J = 7.0 Hz, Ar–H), 7.38–7.34 (m, 5H, Ar–H), 7.31–7.28
(m, 1H, Ar–H), 7.21–7.16 (m, 4H, Ar–H), 5.28–5.23 (m,
1H), 4.08 (s, 2H), 2.95–2.65 (m, 8H, piperidine-H), 2.62 (s,
3H, CH3), 2.53–2.42 (m, 3H), 2.24–2.20 (m, 1H) ppm; 13
C
N-[3-[4-(1H-Benzo[d]imidazol-1-ylmethyl)-4-hydroxypi-
peridin-1-yl]-1-phenylpropyl]-4-fluorobenzamide
(13e, C29H31FN4O2)
NMR (100 MHz, CDCl3): d = 175.9, 166.0, 163.5, 153.0,
142.7, 142.1, 135.9, 131.1, 129.6, 129.5, 128.7, 127.3,
126.2, 122.2, 122.1, 118.7, 115.5, 115.3, 110.2, 70.6, 54.7,
54.4, 53.3, 48.6, 48.4, 35.0, 34.8, 31.7, 14.5 ppm; ESI–MS:
m/z = 501 [M ? H]?.
1
Yield: 70%; white solid; m.p.: 98–100 °C; Rf = 0.20; H
NMR (400 MHz, CDCl3): d = 8.59 (d, 1H, J = 6.5 Hz,
123

308
Z. Weng et al.
NH), 7.92–7.88 (m, 3H, Ar–H), 7.60 (d, 1H, J = 8.0 Hz,
Ar–H), 7.33–7.28 (m, 5H, Ar–H), 7.27–7.17 (m, 3H,
Ar–H), 7.09 (t, 2H, J = 8.0 Hz, Ar–H), 5.23–5.19 (m, 1H),
4.10 (s, 2H), 3.05–2.57 (m, 8H, piperidine-H), 2.54–2.50
(m, 1H), 2.47–2.36 (m, 2H), 2.16–2.13 (m, 1H) ppm; 13C
NMR (100 MHz, CDCl3): d = 176.0, 166.1, 163.5, 144.2,
142.7, 142.1, 134.6, 131.1, 129.6, 129.5, 128.7, 127.3,
126.2, 123.2, 122.3, 119.8, 115.5, 115.3, 110.1, 69.5, 55.2,
54.7, 53.3, 48.7, 48.6, 34.6, 34.2, 31.8 ppm; ESI–MS:
m/z = 487 [M ? H]?.
antagonist or 1% DMSO (negative control) was added. After
incubation at room temperature for 10 min, 25 mm3 RAN-
TES was dispensed into the well to achieve a final
concentrationof3 nmol/dm3 usingaFlexStation3microplate
reader (Molecular Devices), and intracellular calcium change
was recorded at an excitation wavelength of 485 nm and an
emission wavelength of 525 nm.
Data analysis
The calcium response towards CCR5 ligand RANTES after
compound treatment was calculated by use of the equation:
4-Fluoro-N-[3-[4-hydroxy-4-(1H-indol-1-ylmethyl)piperi-
din-1-yl]-1-phenylpropyl]benzamide
(13f, C30H32FN3O2)
Response % ¼ ðD ꢁ BÞ=ðS ꢁ BÞ ꢂ 100%
ð1Þ
1
Yield: 73%; yellow solid; m.p.: 69–72 °C; Rf = 0.35; H
where D stands for RANTES stimulated calcium peak
value after compound incubation; B stands for RANTES
stimulated calcium peak value after 10 lmol/dm3 maravi-
roc (positive control) incubation; and S stands for RANTES
stimulated calcium peak value after 1% DMSO (negative
control) incubation.
NMR (400 MHz, CDCl3): d = 8.73 (d, 1H, J = 6.5 Hz,
NH), 7.95–7.91 (m, 2H, Ar–H), 7.62 (d, 1H, J = 7.5 Hz, Ar–
H), 7.36–7.31 (m, 6H, Ar–H), 7.25–7.21 (m, 2H, Ar–H),
7.13–7.09 (m, 4H, Ar–H), 5.24–5.21 (m, 1H), 4.11 (s, 2H),
2.89–2.45 (m, 8H, piperidine-H), 2.43–2.30 (m, 3H),
2.19–2.16 (m, 1H) ppm; 13C NMR (100 MHz, CDCl3):
d = 176.0, 166.0, 163.5, 142.6, 138.5, 131.1, 130.7, 129.9,
129.7, 129.6, 128.7, 127.3, 126.2, 122.4, 121.6, 120.0, 115.5,
115.3, 110.4, 101.8, 70.4, 56.7, 54.4, 53.7, 48.8, 48.7, 33.9,
33.8, 31.6 ppm; ESI–MS: m/z = 486 [M ? H]?.
The response values were analyzed with GraphPad Prism
software. Non-linear regression analyses were performed to
generate dose–response curves and calculate IC50 values.
Data are generatedfrom three independent experiments (each
in triplicate) and presented as mean SEM.
N-[3-[4-Hydroxy-4-(2-methyl-1H-benzo[d]imidazol-1-
ylmethyl)piperidin-1-yl]-1-phenylpropyl]benzamide
(13g, C30H34N4O2)
Acknowledgments The authors are grateful for support from the
National Natural Science Foundation of China (no. 81072515 and no.
90713047), the National Key Tech Project for Major Creation of New
drugs (no. 2009ZX09501-003), the Ministry of Science and Tech-
nology (no. 2009ZX09302-001), and the Shanghai Commission of
Science and Technology (no. 08DZ2291300).
1
Yield: 68%; yellow solid; m.p.: 79–82 °C; Rf = 0.30; H
NMR (400 MHz, CDCl3): d = 8.56 (d, 1H, J = 6.5 Hz,
NH), 7.92 (d, 2H, J = 7.5 Hz, Ar–H), 7.52–7.42 (m, 4H,
Ar–H), 7.38–7.31 (m, 5H, Ar–H), 7.25–7.21 (m, 1H, Ar–
H), 7.20–7.16 (m, 2H, Ar–H), 5.25–5.21 (m, 1H), 4.08 (s,
2H), 2.98–2.64 (m, 8H, piperidine-H), 2.60 (s, 3H, CH3),
2.51–2.43 (m, 2H), 2.31–2.18 (m, 2H) ppm; 13C NMR
(100 MHz, CDCl3): d = 173.8, 153.2, 142.7, 140.9, 136.2,
135.7, 133.4, 129.7, 128.2, 127.4, 126.9, 126.0, 122.5,
122.3, 118.4, 110.6, 69.5, 55.7, 54.8, 53.1, 48.5, 48.4, 34.9,
34.8, 31.9, 14.4 ppm; ESI–MS: m/z = 483 [M ? H]?.
References
1. Berger EA, Murphy PM, Farber JM (1999) Annu Rev Immunol
17:657
2. Zaitseva M, Blauvelt A, Lee S, Lapham CK, Klaus-Kovtun V,
Mostowski H, Manischewitz J, Golding H (1997) Nat Med 3:369
3. Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber
CM, Saragosti S, Lapoumeroulie C, Cognaux J, Forceille C,
Muyldermans G, Verhofstede C, Burtonboy G, Georges M, Imai
T, Rana S, Yi Y, Smyth RJ, Collman RG, Doms RW, Vassart G,
Parmentier M (1996) Nature 382:722
Calcium mobilization assay
4. Perros M (2007) Adv Antivir Drug Des 5:185
HEK 293 cells stably expressing CCR5 and Ga16 were see-
ded on to a 96-well plate 24 h before the assay. After removal
of the culture medium, cells were incubated in 2 lmol/dm3
fluo-4 AM in Hanks’ balanced salt solution (HBSS;
containing 5.4 mmol/dm3 KCl, 0.3 mmol/dm3 Na2HPO4,
0.4 mmol/dm3 KH2PO4, 4.2 mmol/dm3 NaHCO3, 1.3 mmol/
dm3 CaCl2, 0.5 mmol/dm3 MgCl2, 0.6 mmol/dm3 MgSO4,
137 mmol/dm3 NaCl, 5.6 mmol/dm3 D-glucose, and
250 lmol/dm3 sulfinpyrazone, pH 7.4) at 37 °C for 45 min.
After removal of the fluo-4 solution, cells were washed with
HBSS buffer once, and 50 mm3 HBSS containing either
5. Dorr P, Perros M (2008) Expert Opin Drug Discov 3:1345
6. Kondru R, Zhang J, Ji CH, Mirzadegan T, Rotstein D, Sankuratri
S, Dioszegi M (2008) Mol Pharmacol 73:789
7. Armour D, de Groot MJ, Edwards M, Perros M, Price DA,
Stammen BL, Wood A (2006) ChemMedChem 1:706
8. Faull A, Tucker H (2006) PCT Int Appl WO 2006001752
9. Yang HB, Rotstein DM (2009) Expert Opin Ther Pat 20:325
10. Sheng R, Hu YZ (2004) Synth Commun 34:3529
11. Torre O, Gotor-Fernandez V, Gotor V (2006) Tetrahedron
Asymmetr 17:860
12. Hayashi Y, Okano T, Itoh T, Urushima T, Ishikawa H, Uchimaru
T (2008) Angew Chem Int Ed 47:9053
13. Gizecki P, Dhal R, Toupet L, Dujardin G (2000) Org Lett 2:585
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