Pharmacophore-based design, synthesis, and biological evaluation of novel 3-((3,4-dichlorophenyl)(4-substituted benzyl)amino) propanamides as cholesteryl ester transfer protein (CETP) inhibitors

Article abstract of DOI:10.1016/j.cclet.2013.11.033

Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that plays an important role in decreasing high-density lipoprotein cholesterol (HDL-C) levels and increasing low-density lipoprotein cholesterol (LDL-C) levels. Inhibition of CETP may be a new therapy for treating atherosclerosis. Herein, we report the development of a ligand-based pharmacophore model and pharmacophore-based virtual screening of the ZINC/big-n-greasy database, leading to the identification of compound H-10 as a potential CETP inhibitor in vitro. Based on H-10, a series of 3-((3,4-dichlorophenyl)(4-substituted benzyl)amino) propanamides were designed, synthesized, and evaluated against CETP. Compound 4l was found to have the best activity, resulting in 85.0% inhibition of CETP at 10 μmol/L.

Full text of DOI:10.1016/j.cclet.2013.11.033

Chinese Chemical Letters 25 (2014) 299–304
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Pharmacophore-based design, synthesis, and biological evaluation of
novel 3-((3,4-dichlorophenyl)(4-substituted benzyl)amino)
propanamides as cholesteryl ester transfer protein (CETP) inhibitors
Dong-Mei Zhao a, Wen-Yan Li a, Yu-Fang Shi b, Xu-Qiong Xiong a, Shuai Song a,
b,
Chen-Zhou Hao a, Mao-Sheng Cheng a, , Jing-Kang Shen
*
*
a Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenyang 110016, China
b Central Research Institute, Shanghai Pharmaceuticals Holding Co., Ltd., Shanghai 201203, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that plays an important role in
decreasing high-density lipoprotein cholesterol (HDL-C) levels and increasing low-density lipoprotein
cholesterol (LDL-C) levels. Inhibition of CETP may be a new therapy for treating atherosclerosis. Herein,
we report the development of a ligand-based pharmacophore model and pharmacophore-based virtual
screening of the ZINC/big-n-greasy database, leading to the identification of compound H-10 as a
potential CETP inhibitor in vitro. Based on H-10, a series of 3-((3,4-dichlorophenyl)(4-substituted
benzyl)amino) propanamides were designed, synthesized, and evaluated against CETP. Compound 4l
Received 14 June 2013
Received in revised form 17 October 2013
Accepted 23 October 2013
Available online 27 November 2013
Keywords:
Cholesteryl ester transfer protein
CETP inhibitors
Pharmacophore
Virtual screening
Synthesis
was found to have the best activity, resulting in 85.0% inhibition of CETP at 10
mmol/L.
ß 2013 Mao-Sheng Cheng and Jing-Kang Shen. Published by Elsevier B.V. on behalf of Chinese Chemical
Society. All rights reserved.
1. Introduction
Although the active mechanisms of most CETP inhibitors are not
very clear, preliminary analysis of highly active CETP inhibitors
Despite continuing advances in its understanding and treat-
ment, atherosclerosis remains the leading cause of death
internationally [1]. High-density lipoprotein cholesterol (HDL-C),
which is protective against cardiovascular disease [2], plays an
important role in the transfer of excess cholesterol from peripheral
tissues to the liver (reverse cholesterol transport, RCT) [3,4].
Therefore, drug discovery efforts focused on raising HDL-C in
plasma may potentially provide a therapeutic benefit for coronary
heart disease (CHD) patients [5]. Cholesteryl ester transfer protein
(CETP), a 74-kDa plasma glycoprotein secreted principally by the
liver, facilitates the transfer of cholesteryl ester (CE) from HDL to
very low-density lipoprotein (VLDL) and low-density lipoprotein
(LDL) [6]. The net effect of CETP is to raise LDL-C levels and reduce
HDL-C levels; thus, inhibition of CETP is expected to be a new
therapy for atherosclerosis [7].
indicates that most of them are hydrophobic compounds featuring
anaromaticringanda nitrogenatom. Thequestionremainswhether
there are more specific pharmacophores in these inhibitors.
Recently, pharmacophore modelling and pharmacophore-based
virtual screening have been applied to drug discovery, including
such aspects as lead identification, lead optimization, and de novo
design. To quickly identify novel chemical classes of CETP ligand,
ligand-based pharmacophore modelling of CETP inhibitors was
attempted. Pharmacophore modelling assumes that all compounds
bind to the same site of a common receptor. The crystal structure of
CETP displays an unprecedentedly large cavity at the active site, so
the results based on docking were not plausible. Therefore, we tried
to classify some highly active CETP inhibitors by clustering analysis
and to construct a pharmacophore model based on these clusters.
For this purpose, a diverse dataset of 56 potent CETP inhibitors was
assembled from the literature (Fig. 1) and used to construct the
pharmacophore model [8–31]. Basedon this pharmacophore model,
compound H-10 as a potential CETP inhibitor was found by
pharmacophore-based virtual screening and a series of 3-((3,4-
dichlorophenyl)(4-substituted benzyl)amino) propanamides were
designed and synthesized as CETP inhibitors.
*
Corresponding authors.
E-mail addresses: mscheng@syphu.edu.cn (M.-S. Cheng),
jkshen@mail.shcnc.ac.cn (J.-K. Shen).
1001-8417/$ – see front matter ß 2013 Mao-Sheng Cheng and Jing-Kang Shen. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.11.033

300
D.-M. Zhao et al. / Chinese Chemical Letters 25 (2014) 299–304
Fig. 1. The structures of CETP inhibitors assembled from literature.
2. Experimental
uncorrected. Mass spectra (MS) were taken in ESI mode on Agilent
1100 LC–MS (Agilent, Palo Alto, CA, USA). Proton (1H) nuclear
magnetic resonance spectroscopy was performed using Bruker
ARX-300, 300 MHz spectrometers (Bruker Bioscience, Billerica,
All melting points were obtained on a Bu¨chi Melting Point B-
540 apparatus (Bu¨chi Labortechnik, Flawil, Switzerland) and were

D.-M. Zhao et al. / Chinese Chemical Letters 25 (2014) 299–304
301
MA, USA) with TMS as an internal standard. IR spectra (KBr disks)
were recorded with a Bruker IFS-55 instrument (Bruker). All the
materials were obtained from commercially available sources and
used without further purification, unless otherwise specified. CETP
inhibition assay was conducted according to the literature [32].
3-(3,4-Dichlorophenylamino) propanoic acid (2): To a 50 mL
three-necked bottle 3,4-dichloroaniline (4.6 g, 28.4 mmol), acrylic
acid (17 mL) and 10% hydrochloric acid was added. The solution
was heated to 50 8C for 3 h and cooled to room temperature. The
reaction mixture was poured into ice water. The precipitate was
filtered, washed with water, and then purified by column
chromatography (silica gel) to give compound 2 (3.5 g, 72.1%
yield) as a yellow solid. mp 49.5–51.2 8C.
3.83–3.75 (m, 2H), 3.40–3.31 (m, 2H), 3.28–3.18 (m, 2H), 2.63–2.55
(m, 2H), 1.26 (s, 1H), 1.15–1.06 (m, 6H). 13C NMR (150 MHz, DMSO-
d6):
d 13.0, 14.1, 29.7, 41.3, 47.2, 52.5, 112.4, 113.0, 115.2, 115.3,
116.8, 128.3, 130.5, 131.5, 131.7, 134.3, 147.7, 160.3, 161.9, 169.4.
IR (KBr, cmÀ1): 2965, 2930, 1727, 1640, 1592, 1493, 1137, 820, 793.
ESI-MS (m/z): 397.2 [M+H]+, 419.3 [M+Na]+.
Compound 4e: Yield 33.2%, mp 135.4–136.7 8C. 1H NMR
(600 MHz, CDCl3):
d 7.19–7.15 (m, 1H), 7.13–7.08 (m, 2H), 6.78
(t, 2H, J = 4.26 Hz), 6.75–6.70 (m, 1H), 6.50 (dd, 1H, J1 = 9.0 Hz,
J2 = 2.88 Hz), 4.49 (s, 2H), 3.71 (t, 2H, J = 6.73 Hz), 2.37 (t, 2H,
J = 6.77 Hz), 1.32 (s, 9H). 13C NMR (150 MHz, DMSO-d6):
d 28.4,
34.2, 47.7, 49.9, 52.5, 112.5, 113.1, 114.7, 115.2, 116.9, 128.2, 130.4,
131.4, 134.1, 147.6, 160.3, 161.9, 169.9. IR (KBr, cmÀ1): 3280, 3079,
2966, 2930, 1724, 1637, 1595, 1492, 1223, 818, 796. ESI-MS (m/z):
431.0 [M+Cl]À, 395.0 [MÀH]À
3-(3,4-Dichlorophenylamino)propanamides (3): To a solution
of 3-(3,4-dichlorophenylamino) propanoic acid (3.0 mmol) in
dry DMF (10 mL) 1-hydroxybenzotrizole (HOBt) (3.0 mmol) and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDCÁHCl, 3.0 mmol) was added. The mixture was stirred at room
temperature for 1 h, and then the corresponding amine (3.0 mmol)
and DIEA (6.0 mmol) were added. The reaction mixture was stirred
at room temperature for 2 h and poured into ice water. The
precipitate 3 was filtered, washed with water, and obtained as a
white solid used directly in the next step without any purification.
3-((3,4-Dichlorophenyl)(4-substituted benzyl)amino) propana-
mides (4a–4l): To a solution of 3-(3,4-dichlorophenylamino)pro-
panamides (1.0 mmol) in dry DMF (10 mL) was added potassium
carbonate (1.5 mmol) and 1-(bromomethyl)-4-fluorobenzene or 1-
(bromomethyl)-3,5-bis(trifluoromethyl) benzene (2.0 mmol). The
solution was heated to 90 8C for 10 h and cooled to room
temperature. The reaction mixture was poured into ice water. The
mixture was extracted with ethyl acetate. The organic layer was
washed with water and brine, and then dried over Na2SO4. Solvent
was removed under reduced pressure and the resulting residue was
purified by column chromatography (10% EtOAc/petroleum ether,
silica) orrecrystallization (petroleum ether/ethylacetate) to provide
the title 4a–4l as a white to yellow solid.
Compound 4f: Yield 31.5%, mp 142.5–143.7 8C. 1H NMR
(600 MHz, CDCl3):
d 7.76 (s, 1H), 7.58 (s, 2H), 7.21 (d, 1H,
J = 8.91 Hz), 6.73 (d, 1H, J = 3.0 Hz), 6.45 (dd, 1H, J1 = 3.0 Hz,
J2 = 9.0 Hz), 4.67 (s, 1H), 3.81 (t, 2H, J = 6.42 Hz), 3.78–3.67 (m, 1H),
2.47 (t, 2H, J = 6.36 Hz), 2.04 (s, 1H), 1.94–1.82 (m, 2H), 1.74–1.66
(m, 2H), 1.45–1.0 (m, 6H). 13C NMR (150 MHz, DMSO-d6):
d 24.5,
25.2, 32.3, 33.3, 47.5, 52.7, 59.7, 112.8, 113.6, 117.7, 120.7, 122.4,
124.2, 126.0, 127.2, 130.0, 130.2, 130.4, 130.6, 131.6, 142.4, 147.4,
169.2, 170.3. IR (KBr, cmÀ1): 3282, 3089, 2934, 2857, 1741, 1637,
1594, 1494, 1287, 980, 900, 704, 682. ESI-MS (m/z): 541.3 [M+H]+,
563.3 [M+Na]+, 575.0 [M+Cl]À, 585.1 [M+HCOO]À.
Compound 4g: Yield 38.4%, mp 117.4–118.5 8C. 1H NMR
(600 MHz, CDCl3):
d 7.28 (s, 1H), 7.24–7.18 (m, 2H), 7.15–7.07
(m, 2H), 7.02–6.91 (m, 3H), 6.78 (d, 1H, J = 2.88 Hz), 6.55 (dd, 1H,
J1 = 2.85 Hz, J2 = 8.97 Hz), 4.53 (s, 2H), 3.83 (t, 2H, J = 6.57 Hz), 2.63
(t, 2H, J = 6.51 Hz), 2.33 (s, 3H). 13C NMR (150 MHz, DMSO-d6):
d
21.2, 34.4, 47.3, 52.7, 112.6, 113.2, 115.2, 115.4, 116.3, 117.1, 119.7,
123.9, 128.3, 128.5, 130.5, 131.5, 134.1, 137.8, 138.9, 147.5, 160.3,
161.9, 169.3. IR (KBr, cmÀ1): 3326, 1653, 1592, 1492, 813. ESI-MS
(m/z): 431.0 [MÀH]À, 453.4 [M+Na]+, 466.9 [MÀCl]À.
Compound 4h: Yield 35.4%, mp 125.5–127.0 8C. 1H NMR
Compound 4a: Yield 30.5%, mp 159.3–159.9 8C. 1H NMR
(600 MHz, CDCl3): d 7.23–7.16 (m, 1H), 7.15–7.06 (m, 2H), 7.04–
(600 MHz, CDCl3):
d
7.78 (s, 1H), 7.60 (s, 1H), 7.22 (d, 1H,
6.95 (m, 2H), 6.73 (s, 1H), 6.50 (dd, 1H, J1 = 2.97 Hz, J2 = 8.91 Hz),
4.50 (s, 2H), 3.74 (t, 2H, J = 6.66 Hz), 2.72–2.61 (m, 1H), 2.41 (t, 2H,
J = 6.66 Hz), 0.80–0.73 (m, 2H), 0.49–0.41 (m, 2H). 13C NMR
J = 8.88 Hz), 6.74 (d, 2H, J = 2.94 Hz), 6.40 (dd, 1H, J1 = 9.0 Hz,
J2 = 3.0 Hz), 4.70 (s, 2H), 3.68–3.65 (m, 2H), 3.65–3.62 (m, 2H),
3.61–3.58 (m, 2H), 3.44–3.37 (m, 2H), 2.63 (t, 2H, J = 3.0 Hz). 13C
(150 MHz, DMSO-d6):
d 5.5, 22.2, 33.2, 47.5, 52.6, 112.5, 113.1,
NMR (150 MHz, DMSO-d6):
d
29.7, 41.4, 45.2, 47.1, 52.6, 65.9, 66.0,
115.2, 115.3, 116.9, 128.2, 130.5, 131.5, 134.1, 147.5, 160.3, 161.9,
171.4. IR (KBr, cmÀ1): 3301, 3067, 2967, 1640, 1596, 1492, 819,
792. ESI-MS (m/z): 381.2 [M+H]+, 383.2 [M+Na]+, 417.0 [M+Cl]À,
425.0 [M+HCOO]À.
112.6, 113.4, 117.6, 122.4, 124.2, 127.2, 130.2, 130.4, 130.6, 131.7,
142.5, 147.3, 169.3. IR (KBr, cmÀ1): 3071, 2973, 2864, 1646, 1597,
1493, 1279, 1130. ESI-MS (m/z): 529.08 [M+H]+, 551.08 [M+Na]+.
Compound 4b: Yield 38.1%, mp 126.8–127.9 8C. 1H NMR
Compound 4i: Yield 36.7%, mp 121.6–122.5 8C. 1H NMR
(600 MHz, CDCl3):
d
7.37–7.27 (m, 3H), 7.24–7.03 (m, 5H), 7.04–
(600 MHz, CDCl3): d 7.33 (d, 2H, J = 8.94 Hz), 7.20 (d, 1H,
6.90 (m, 2H), 6.72 (dd, 1H, J1 = 2.94 Hz, J2 = 2.12 Hz), 6.57–6.36 (m,
1H), 4.60–4.40 (m, 4H), 3.89–3.74 (m, 2H), 3.03–2.97 (m, 3H), 2.72–
2.58 (m, 2H). 13C NMR (150 MHz, DMSO-d6):
d 30.1, 34.6, 47.0, 49.8,
J = 8.94 Hz), 7.15–7.05 (m, 3H), 6.97 (t, 2H, J = 8.64 Hz), 6.85 (d,
2H, J = 8.97 Hz), 6.79 (d, 1H, J = 2.94 Hz), 6.54 (dd, 1H, J1 = 2.97 Hz,
J2 = 9.0 Hz), 4.53 (s, 2H), 3.87–3.81 (m, 2H), 3.79 (s, 3H), 2.62 (t, 2H,
52.5, 112.4, 113.1, 115.2, 115.4, 116.9, 126.4, 127.0, 127.5, 128.3,
128.4, 128.6, 130.5, 131.5, 134.2, 137.7, 147.7, 160.3, 161.9, 170.8. IR
(KBr, cmÀ1): 3029, 2912, 1630, 1592, 1494, 1220, 802, 734. ESI-MS
(m/z): 445.2 [M+H]+, 467.2 [M+Na]+, 483.2 [M+K]+.
J = 6.57 Hz), 1.56 (s, 2H). 13C NMR (150 MHz, DMSO-d6):
d 35.7,
47.6, 52.9, 55.1, 112.5, 112.8,113.5, 113.7, 116.1, 117.8, 120.6,
127.2, 130.4, 130.7, 131.3, 131.6, 132.1, 142.4, 147.3, 148.7, 155.0,
168.9. IR (KBr, cmÀ1): 3307, 3063, 2929, 2835, 1649, 1599, 1511,
823. ESI-MS (m/z): 336.9 [MÀH]À, 361.3 [M+Na]+, 339.2 [M+K]+.
Compound 4j: Yield 34.5%, mp 160.3–161.0 8C. 1H NMR
Compound 4c: Yield 35.4%, mp 133.4–134.2 8C. 1H NMR
(600 MHz, CDCl3):
d 7.21–7.07 (m, 3H), 7.04–6.93 (m, 2H), 6.73
(d, 2H, J = 2.97 Hz), 6.49 (dd, 1H, J1 = 2.97 Hz, J2 = 8.97 Hz), 4.51 (s,
1H), 3.77 (t, 2H, J = 7.08 Hz), 3.69–3.55 (m, 6H), 3.40–3.33 (m, 2H),
(600 MHz, CDCl3): d 7.33 (d, 2H, J = 8.91 Hz), 7.20 (d, 1H,
J = 8.97 Hz), 7.16–7.07 (m, 3H), 6.97 (t, 2H, J = 8.61 Hz), 6.85 (d,
2H, J = 8.94 Hz), 6.79 (d, 1H, J = 2.91 Hz), 6.55 (dd, 1H, J1 = 2.94 Hz,
J2 = 8.97 Hz), 4.52 (s, 2H), 3.87–3.81 (m, 2H), 3.79 (s, 3H), 2.62 (d,
2.58 (d, 2H, J = 6.93 Hz). 13C NMR (150 MHz, DMSO-d6):
d 29.7,
41.4, 45.3, 46.9, 52.4, 65.9, 66.0, 112.4, 113.1, 115.2, 115.4, 116.9,
128.2, 130.5, 131.5, 134.2, 147.6, 160.3, 161.9, 169.4. IR (KBr,
cmÀ1): 2991, 2858, 1640, 1590, 1500, 803. ESI-MS (m/z): 411.1
[M+H]+, 433.1 [M+Na]+.
2H, J = 6.54 Hz). 13C NMR (150 MHz, DMSO-d6):
d 34.1, 47.4, 52.7,
55.1, 112.6, 113.2, 113.7, 115.2, 115.4, 117.1, 120.6, 120.7, 128.2,
128.3, 130.5, 131.5, 132.2, 134.1, 147.5, 155.1, 160.3, 161.9, 168.4.
IR (KBr, cmÀ1): 3274, 3143, 3131, 3011, 2973, 2938, 2841, 1651,
1604, 1592, 1510, 1491, 1253, 832. ESI-MS (m/z): 447.3 [M+H]À,
469.3 [M+Na]+, 481.0 [M+Cl]À, 485.2 [M+K]+.
Compound 4d: Yield 39.2%, colourless oil. 1H NMR (600 MHz,
CDCl3):
d 7.22–7.09 (m, 3H), 7.04–6.96 (m, 2H), 6.77–6.73 (d, 2H,
J = 2.97 Hz), 6.51 (dd, 1H, J1 = 9.0 Hz, J2 = 2.97 Hz), 4.52 (s, 2H),

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D.-M. Zhao et al. / Chinese Chemical Letters 25 (2014) 299–304
Compound 4k: Yield 33.3%, mp 132.5–134.0 8C. 1H NMR
(600 MHz, CDCl3): 7.29 (d, 1H, J = 9.0 Hz), 7.14–7.06 (m, 2H),
d
6.98 (t, 2H, J = 8.61 Hz), 6.73 (d, 1H, J = 2.76 Hz), 6.51 (dd, 1H,
J1 = 2.85 Hz, J2 = 8.82 Hz), 5.32–5.23 (m, 1H), 4.50 (s, 2H), 3.81–
3.67 (m, 3H), 2.42 (t, 2H, J = 6.66 Hz), 1.91–1.81 (m, 2H), 1.75–1.61
(m, 3H), 1.15–1.26 (m, 2H), 1.21–0.99(m, 3H). 13C NMR (150 MHz,
DMSO-d6):
d 24.5, 25.2, 32.3, 33.5, 47.4, 47.6, 52.6, 112.5, 113.2,
115.2, 115.3, 116.9, 128.3, 130.4, 131.5, 134.1, 147.6, 160.3, 161.9,
169.2. IR (KBr, cmÀ1): 3314, 3071, 2930, 2856, 1637, 1595, 1548,
1490, 817, 795. ESI-MS (m/z): 423.3 [M+H]+, 445.3 [M+Na]+.
Compound 4l: Yield 37.1%, mp 135.0–135.8 8C. 1H NMR
(600 MHz, CDCl3):
d 7.31–7.26 (d, 1H, J = 7.95 Hz), 7.20 (d, 1H,
J = 9.0 Hz), 7.16–7.09 (m, 2H), 7.01 (t, 2H, J = 8.61 Hz), 6.95–6.87
(m, 3H), 6.75 (d, 1H, J = 2.97 Hz), 6.50 (dd, 1H, J1 = 3.0 Hz,
J2 = 9.0 Hz), 4.52 (s, 2H), 3.83–3.74 (m, 4H), 3.53 (t, 2H,
J = 4.89 Hz), 3.17–3.08 (m, 4H), 2.64 (t, 2H, J = 6.75 Hz). 13C NMR
(150 MHz, DMSO-d6):
d 29.8, 40.8, 44.7, 47.0, 48.2, 48.6, 52.4,
112.4, 113.1, 115.2, 115.4, 115.8, 116.9, 119.3, 128.2, 128.3, 128.9,
130.5, 131.5, 134.2, 134.3, 147.7, 150.8, 160.3, 161.9, 169.2. IR (KBr,
cmÀ1): 2820, 1640, 1629, 1591, 1507, 1447, 1231, 806. ESI-MS (m/
z): 486.4 [M+H]+, 508.4 [M+Na]+.
Fig. 2. Five-feature pharmacophore model for cluster 1. Orange: ring aromatic;
Blue: hydrophobic; Green: hydrogen bond acceptor.
3. Results and discussion
3.1. Pharmacophore modelling
database, a conformational library of the resulting hits was
generated, with a maximum of 100 conformations per molecule,
and a new multi-conformational database was obtained. Then, the
derived 3D pharmacophore hypotheses were used to screen this
database, and 296 hits were returned. The hepatotoxicity of these
hits was predicted (DS2.5.1/ADME hepatotoxicity probability >
0.7), and 49 compounds that might have potent hepatotoxicity
were deleted. The remaining 247 hits were then clustered by Sybyl
Jarvis Patrick analysis for further downsizing. The fit value is a
measure of how well the ligand fits the pharmacophore; thus, the
higher the fit score, the better the match. Ligands with high fit
values were retained in each cluster, at which point there were 99
total remaining candidates. Using ADME/Tox Boxes and PreADME
predictions, 29 ‘‘problem’’ compounds were removed. Finally, 28 of
these compounds were purchased from Specs Company and
ChemBridge Corporation, and their CETP inhibitory activity was
tested.
Conformations (3–8 kcal/mol) were generated for each com-
pound assembled from the literature using OMEGA 2.5.1 [33] and
Corina 3.2/Rotate 1.20 [34]. Using FTrees, ROCS, FlexS, SYBYL/
Hierarchical clustering and SYBYL/Jarvis-Patrick Clustering meth-
ods, compounds that may share pharmacophore features were
assembled and 3 big clusters were obtained (Table 1). Rigid
templates for each cluster were determined by the FlexS rigid fit
method, and then the corresponding alignments were generated
using the ROCS rigid fit method, FlexS rigid/flexible fit methods,
and ‘‘Diverse Conformation Generation’’ in Accelrys Discovery
Studio 2.5.1 (DS 2.5.1). Ultimately, 28 alignments were obtained
and imported into DS 2.5.1. Pharmacophore hypotheses were
generated using ‘Common Feature Pharmacophore Generation’
technology, based on the HipHop algorithm. All 56 compounds
were used as a training set to validate these pharmacophore
hypotheses, and finally, the best 3 pharmacophore models in each
cluster were selected (Fig. 2).
Eleven compounds exhibited activity against CETP; compound
H-10 had the highest CETP inhibitory activity (IC50: 8.06
mmol/L).
3.2. Virtual screening
Furthermore, the pharmacophore of compound H-10, which
includes one H-bond acceptor, three hydrophobic interactions
and one aromatic ring, matched Anacetrapib very well (Fig. 2). In
consideration of the therapeutic prospects of Anacetrapib,
compound H-10 was used as a lead compound. A series of novel
3-(3,4-dichlorophenyl-4-substituted benzyl)amino-N-propana-
mides were designed by incorporating key pharmacophore
perssads that appeared in previously reported highly active CETP
inhibitors. These compounds were then evaluated by ligand
pharmacophore mapping technology to identify ligands mapping
to these pharmacophore models (Fig. 3).
These 3D pharmacophore hypotheses were then used to screen
the ZINC/big-n-greasy database (2 < xlogp < 6; 150 ꢀ p.mwt <
600; 577, 555 molecules) using the DS 2.5.1/Search 3D database.
Given this huge number of molecules, it would take a prohibitively
long amount of time to generate and screen the conformational
library directly. In addition, many false positives would be
generated. The aforementioned 56 compounds represent most
structural types of CETP inhibitor, so these structures were used for
similarity analysis (similarity level x > 0.8) in the ZINC/big-n-
greasy database using FTrees 1.50.1. Using the DS 2.5.1/Build 3D
Table 1
Cl
Cl
O
O
Clustering analysis result for 56 CETP inhibitors.
O
No.
Compd.
N
A
NH
O
Cluster 1
16, 17, 18, 19, 20, 21, 22, 24, 26, 27, 28, 29, 30, 31, 33, 34,
36, 37, 3, 46, 47
R
1
Cluster 2
Cluster 3
3, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 18, 21, 24, 26, 28,
29, 31, 37, 38, 45, 46, 47
Cl
H-10
Fig. 3. Designof3-((3,4-dichlorophenyl)(4-substitutedbenzyl)amino)propanamides.

D.-M. Zhao et al. / Chinese Chemical Letters 25 (2014) 299–304
303
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
b
c
O
a
O
O
N
A
N
H
A
NH
N
H
OH
2
R
2
3
4
Scheme 1. The synthesis of 3-((3,4-dichlorophenyl)(4-substituted benzyl)amino) propanamides. Reagents and conditions: (a) Conc. HCl, acrylic acid, 50 8C, 72.1%; (b) amines,
HOBt, EDCÁHCl, EIEA, r.t.; (c) 4-fluorobenzyl bromine or 3,5-di-triflourobenzyl bromine, K2CO3, DMF, 90 8C, 30–40% (two steps).
Table 2
3. Twelve 3-((3,4-dichlorophenyl)(4-substituted benzyl)amino)-
propanamides were obtained via alkylation of compound 3
CETP inhibitory rate of the target compounds (4a–4l).
Cl
with 3,5-bis(trifluoromethyl) benzyl bromide or 4-fluorobenzyl
bromide.
O
Cl
N
A
3.4. Biological activity
R
The in vitro inhibitory activity of all twelve target compounds
against CETP was evaluated by a radioisotope-based assay. The
inhibition results (%) are presented in Table 2. Most newly
synthesized derivatives exhibited considerable CETP-inhibitory
activity (inhibition: 1.9–85.0%). The N-phenylpiperazinyl substitut-
edcompound4lexhibitedthebestactivitytowardsCETP, with85.0%
inhibition, which is comparable to the positive control Dalcetrapib.
Compd.
R
A
Inhibition (%)a
Dalcetrapibb
82.5 Æ 0.4
2.6 Æ 0.8
4a
3,5-bistrifluoromethyl
4-fluoro
N
O
O
4b
5.1 Æ 1.0
N
4. Conclusion
4c
4-fluoro
4-fluoro
31.2 Æ 1.2
1.9 Æ 0.8
N
N
In summary, we have constructed 9 pharmacophore hypothe-
ses for 56 diverse CETP inhibitors via clustering analysis and
common-feature pharmacophore generation. Then, the pharma-
cophores were validated for their rationality by all compounds. The
positive rates were above 95% and the highest rank value was
236.789. These pharmacophore models were then used to screen
the ZINC/big-n-greasy database, and compound H-10 was found
to exhibit good CETP-inhibitory activity. A series of 3-((3,4-
dichlorophenyl)(4-substituted benzyl)amino) propanamides were
designed, synthesized and evaluated against CETP, using com-
pound H-10 as a lead. Compound 4l exhibited the best activity,
4d
4e
4f
4-fluoro
4.7 Æ 0.9
N
H
3,5-bistrifluoromethyl
56.8 Æ 2.1
N
H
4g
4-fluoro
29.9 Æ 1.8
with 85.0% inhibition at 10
mmol/L.
N
H
4h
4i
4-fluoro
17.4 Æ 0.5
36.6 Æ 1.9
Acknowledgments
N
H
The authors thank Yi-Ping Wang of the Shanghai Institute of
Materia Medica, Chinese Academy of Science for the activity
assays.
3,5-bistrifluoromethyl
O
O
N
H
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27.9 Æ 2.0
47.7 Æ 1.4
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