CETP inhibitory activity of chlorobenzyl benzamides: QPLD docking, pharmacophore mapping and synthesis

Article abstract of DOI:10.2174/1570180814666170412122304

Background: Elevated levels of serum LDL and total cholesterol are considered important risk factors for the development of atherosclerosis. Cholesteryl ester transfer protein inhibition raises HDL levels and reduces atherosclerotic lesions. Objective: Consequently, there is a great interest in developing new CETP inhibitors. Methods: Herein, synthesis of four chlorobenzyl benzamides 8a-d that aim at CETP inhibition was performed. Results: Benzamide 8a showed the best CETP inhibitory activity with an IC₅₀ of 1.6 μM. In vitro biological data shows that the presence of p-trifluoromethoxy group enhances CETP inhibitory activity more than m-trifluoromethyl groups. QPLD docking shows that the verified compounds accommodate the binding cleft of CETP and are enclosed by hydrophobic lining. The scaffold of 8a-d matches the pharmacophoric points of CETP inhibitors; particularly hydrophobic and aromatic functionalities. Conclusion: Future structural modification is needed to improve CETP inhibitory activity and to enhance understanding of the structure-activity relationship.

Full text of DOI:10.2174/1570180814666170412122304

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1
391
Letters in Drug Design & Discovery, 2017, 14, 1391-1400
RESEARCH ARTICLE
eISSN: 1 58 7 05 - 16 82 08 8X
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1
CETP Inhibitory Activity of Chlorobenzyl Benzamides: QPLD Docking,
Pharmacophore Mapping and Synthesis
SB EC N
I
ET NH AC ME
*
Reema Abu Khalaf , Hamada Abd El-Aziz, Dima Sabbah, Ghadeer Albadawi and
Ghassan Abu Sheikha
Department of Pharmacy, Faculty of Pharmacy, Al-Zaytoonah University of Jordan, Amman, Jordan
Abstract: Background: Elevated levels of serum LDL and total cholesterol are considered impor-
tant risk factors for the development of atherosclerosis. Cholesteryl ester transfer protein inhibition
raises HDL levels and reduces atherosclerotic lesions.
Objective: Consequently, there is a great interest in developing new CETP inhibitors.
A R T I C L E H I S T O R Yꢀ
Methods: Herein, synthesis of four chlorobenzyl benzamides 8a-d that aim at CETP inhibition was
performed.
Received: January 07, 2017
Revised: March 21, 2017
Accepted: March 22, 2017
Results: Benzamide 8a showed the best CETP inhibitory activity with an IC50 of 1.6 µM. In vitro
biological data shows that the presence of p-trifluoromethoxy group enhances CETP inhibitory
activity more than m-trifluoromethyl groups. QPLD docking shows that the verified compounds
accommodate the binding cleft of CETP and are enclosed by hydrophobic lining. The scaffold of
DOI:
1
0.2174/1570180814666170412122304
8
a-d matches the pharmacophoric points of CETP inhibitors; particularly hydrophobic and aromatic
functionalities.
Conclusion: Future structural modification is needed to improve CETP inhibitory activity and to
enhance understanding of the structure-activity relationship.
Keywords: Atherosclerosis, CETP inhibitors, chlorobenzyl benzamides, docking, QPLD, pharmacophore mapping.
1
. INTRODUCTION
protective HDL to the proatherogenic LDL and VLDL. Epi-
demiologic studies support an inverse relation between HDL
cholesterol and coronary heart disease [9]. Therefore, there
has been a pronounced interest in developing new CETP
inhibitors [10].
Hyperlipidemia is characterized by abnormal lipid levels:
higher levels of blood total cholesterol (TC), triglycerides
(
TG), and low density lipoprotein (LDL) cholesterol along
with lower levels of high density lipoproteins (HDL) choles-
terol [1]. The increased levels of serum LDL and TC are
considered important risk factors for the development of
atherosclerosis [2]. The elevation in serum levels of TC, TG
and LDL, could be either alone or in combination [3, 4].
The crystal structure of CETP displays a long hydropho-
bic groove that comprises two molecules of cholesteryl ester
and two phospholipid molecules [4]. CETP, the main key
binding residues are hydrophobic and this concludes that
hydrophobic binding interactions are dominating [11].
Cholesteryl ester transfer protein (CETP) is considered to
be pro-atherogenic by lowering HDL and increasing LDL
levels [5]. CETP inhibition increases HDL levels and re-
duces atherosclerotic lesions [6]. HDL has numerous proper-
ties that protect against atherosclerosis progress [7].
CETP inhibitors prevent the transfer of cholesteryl ester
from HDL to VLDL and LDL, thus increasing HDL choles-
terol and decreasing LDL cholesterol [12-14]. Several re-
ported CETP inhibitors are undergoing clinical trials such as,
dalcetrapib [15], anacetrapib [16], and evacetrapib [17].
Plasma CETP is a hydrophobic glycoprotein that trans-
fers lipids between HDL and apolipoprotein B-containing
lipoproteins, and/or between HDL subparticles [8]. It facili-
tates the transfer of cholesteryl ester from the athero-
Our research group identified different potential CETP
inhibitors including benzylideneamino-methanones [18],
benzylamino-methanones [19], N-(4-benzyloxyphenyl)-4-
methyl-benzenesulfonamides, N-(4-benzylamino- phenyl)-
toluene-4-sulfonic acid esters [20], and fluorinated ben-
zamides [21].
*
Address correspondence to this author at the Department of Pharmacy,
Faculty of Pharmacy, Al-Zaytoonah University of Jordan, Amman, Jordan;
Tel: +962-64291511; Ext 239; Fax: 00962-64291432;
In order to optimize the activity of our previously discov-
ered popular compounds [19], hydrophobicity was enhanced
E-mails: reema.abukhalaf@zuj.edu.jo and rima_abu_khalaf@yahoo.com
1
875-628X/17 $58.00+.00
©2017 Bentham Science Publishers

1
392 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 12
Abu Khalaf et al.
by an additional aromatic ring. Moreover, modifications of
the linker groups together with the aromatic substitutions
were carried out. These modifications led to the discovery of
more hydrophobic molecules with greater CETP inhibition.
This result matches with the hydrophobic nature of CETP
binding pocket, and with the pharmacophore mapping and
QPLD docking outcomes of the newly synthesized CETP
inhibitors.
ring at room temperature for 5 days then the mixture was
evaporated. Column chromatography was carried out using
cyclohexane: ethyl acetate (9:1) as eluent to afford 5a as off
white powder (2.18 g, 44%); m.p. 85-86°C; Rƒ = 0.46 (cy-
1
clohexane:ethyl acetate, 9:1); H-NMR (300MH
Z
, CDCl
3
): δ
3.84 (s, 2H, NHCH
, 1H, Ar-H), 7.20 (d, J=12 H
4H, Ar-H), 7.40 (d, J=12 H , 1H, Ar-H), 7.44 ppm (s, 1H,
, CDCl ): δ 53.6, 59.6, 113.4,
17.1, 118.7, 121.4, 128.1, 129.5, 136.5, 150.0, 168.0 ppm;
2
), 4.63 (s, 3H, OCH
3
), 6.83 (dd, J=4, 15
H
Z
Z
, 2H, Ar-H), 7.22-7.25 (m,
Z
1
3
NHCH
2
); C-NMR (75MH
Z
3
1
2
2
. EXPERIMENTAL
IR (KBr): 3480, 3225, 2986, 1713, 1620, 1458, 1404, 1303,
-
1
1
250 cm .
.1. Methods and Materials
2
.2.1.2. Methyl 3-(3,5-bis(trifluoromethyl)benzyl) benzy-
All chemicals, reagents and solvents were of analytical
lamino) benzoate (5b)
grade and used directly without extra purification. Chemicals
and solvents were purchased from the corresponding compa-
nies (Alfa Aesar, Acros Organics, Sigma-Aldrich, Fluka, SD
fine Chem Limited, Tedia and Fisher Scientific).
1
-(bromomethyl)-3, 5-bis(trifluoromethyl) benzene (4b,
4
.6 mL, 25 mmol), and triethylamine (8.76 mL, 62 mmol)
were added to the solution of 3 . The mixture was left under
stirring at room temperature for 7 days then the mixture was
evaporated. Column chromatography was carried out using
cyclohexane: ethyl acetate (8.5:1.5) as eluent to afford 5b as
yellow powder (1.34 g, 28%); m.p. 109-110 °C; Rƒ = 0.66
Melting points were measured using Gallenkamp melting
point apparatus and uncorrected. Infrared (IR) spectra were
recorded using Shimadzu IR Affinity1 FTIR spectropho-
tometer. All samples were prepared with potassium bromide
1
1
13
(chloroform, 10); H-NMR (300MH , CDCl ): δ 3.86 (s, 3H,
OCH
HNCH
Z
3
and pressed into a disc. H-NMR and C-NMR spectra were
measured on Bruker, Avance DPX-400 and Bruker 300
MHz-Avance III spectrometers, Jordan University of Science
and Technology. Chemical shifts are given in δ (ppm) using
TMS as the internal reference; the samples were dissolved in
3
), 4.33 (t, J=6 H
Z
, 1H, NHCH
2
Z
), 4.49 (d, J=6 H , 2H,
2
), 6.73 (dd, J=3, 9 H
Z
Z
, 1H, Ar-H), 7.19 (t, J=9 H ,
1
H, Ar-H), 7.29 (d, J=3 HZ, 1H, Ar-H), 7.40 (d, J=9 HZ, 1H,
13
Ar-H), 7.82 ppm (d, J=12 H
Z Z
, 3H, Ar-H); C-NMR (75MH ,
3
CDCl ): δ 47.5, 52.1, 113.7, 117.4, 119.7, 121.4, 121.5,
3 6
CDCl or DMSO-d . High resolution mass spectrometry
1
21.6, 127.4, 129.5, 131.3, 142.0, 147.3, 168.0 ppm; IR
(
HR-MS) was performed using LC Mass Bruker Apex-IV
-1
(
KBr): 3402, 2955, 1713, 1605, 1512, 1443, 1381, 1297 cm .
mass spectrometer utilizing an electrospray interface.
AFLX800TBI Microplate Fluorimeter was used in the in vitro
bioassay (BioTek Instruments, Winooski, VT, USA).
2
.2.2. General Procedure for the Synthesis of the Targeted
Compounds (8a, b)
Thin Layer Chromatography (TLC) was performed on 20
x 20 cm layer with the thickness of 0.2 mm aluminum cards
pre-coated with fluorescent silica gel GF254 DC- alufolien-
kieselgel (Fluka analytical, Germany), and visualized by UV
light indicator (at 254 and/ or 360 nm).
The intermediate methyl 3-(4-(trifluoromethoxy) benzy-
lamino) benzoate (5a) was dissolved in 1M sodium hydrox-
ide (5 mL) and refluxed overnight at 100 ºC, then the reac-
tion mixture was neutralized with 1M hydrochloric acid and
extracted three times using chloroform (3 x 20 mL). The
organic layer was dried using anhydrous sodium sulfate and
evaporated.
2
.2. Synthesis of the Compounds
2
.2.1. General Procedure for the Synthesis of Methyl Ben-
Subsequently, the intermediate 3-(4-(trifluoromethoxy)
benzylamino) benzoic acid (6a, 0.2 g, 0.64 mmol) was dis-
solved in 10 mL dichloromethane and oxalyl chloride (2,
zoates Intermediates (5a, b)
3
-Aminobenzoic acid (1, 2.0 g, 14.58 mmol) was dis-
0
.11 mL, 1.28 mmol) was added. The reaction was left under
solved in methanol (20 mL) and cooled down in the ice bath.
The solution was treated with oxalyl chloride (2, 2.5 mL, 29
mmol) stirred at room temperature for 20-30 minutes, and
refluxed for 24 hours at 60-70 ºC. The reaction mixture was
then evaporated and neutralized by 3M potassium carbonate.
Extraction by chloroform (4 x 20 mL) was carried out four
times . The organic layer was dried with sodium sulfate an-
hydrous followed by evaporation to get 2.07 g of pure 3-
aminobenzoic acid methyl ester (3). Afterward, 3-
aminobenzoic acid methyl ester (3, 2.0 g, 13.2 mmol) was
dissolved in (20 mL) dichloromethane.
stirring for 5 days at 50-60 ºC. Later the reaction mixture
was evaporated.
2
.2.2.1.
3-{(4-(Trifluoromethoxy-benzyl)-[3-(4-trifluoro-
methoxy-benzylamino)-benzoyl]-amino}-N-(4-chlorobenzyl)-
benzamide (8a)
4
-Chloro benzylamine (7a, 0.125 mL, 1.03 mmol) was
added together with 5 mL of triethylamine and stirred at
room temperature for 5 days. Then the crude product was
purified by column chromatography using cyclohexane:
ethyl acetate (5.9:4.1) as eluent to afford 8a as off white
2
.2.1.1. Methyl 3-(4-(trifluoromethoxy) benzylamino) ben-
powder (138.9 mg, 41%); m.p. 112-114°C; Rƒ = 0.36 (cy-
1
zoate (5a)
clohexane:ethyl acetate, 45:55); H-NMR (400MH
Z
, DMSO-
d
6
): δ 4.13 (d, J=4 H
NHCH ), 5.00 (s, 2H, NCH
.21 (d, J=8 H , 2H, Ar-H), 7.31-7.37 (m, 10H, Ar-H), 7.83-
Z
, 2H, NHCH
2
), 4,46 (d, J=4 HZ, 2H,
1
-(bromomethyl)-4-(trifluoromethoxy) benzene (4a, 4.2
2
2
), 6.86 (d, J=8 H
Z
, 2H, Ar-H),
mL, 26.4 mmol), and triethylamine (9.2 mL, 66 mmol) were
added to the solution of 3. The mixture was left under stir-
7
Z

CETP Inhibitory Activity of Chlorobenzyl Benzamides
Letters in Drug Design & Discovery, 2017, Vol. 14, No. 12 1393
7
.90 (m, 2H, Ar-H), 9.13 (t, J=4 H
Z
, 1H, CONH), 9.27 ppm
7.22 (d, J=4 H
7.72 (m, 3H, Ar-H), 7.83 (d, J=8 H
2H, Ar-H), 8.01 (s, 1H, Ar-H), 9.12 (t, J=8 HZ, 1H, CONH),
Z
, 2H, Ar-H), 7.31-7.38 (m, 3H, Ar-H), 7.66-
1
3
(
t, J=4 HZ, 1H, CONH); C-NMR (100MH , DMSO-d ): δ
Z
6
Z
, 2H, Ar-H), 7.91 (s,
4
1
1
2
2.0, 50.6, 62.5, 121.0, 126.2, 126.3, 128.1, 128.2, 129.1,
1
3
29.2, 130.2, 131.3, 135.1, 135.9, 137.1, 138.5, 140.4, 147.5,
63.0, 164.8, 165.1 ppm; IR (KBr): 3364, 3248, 3094, 2963,
Z
9.36 ppm (t, J=8 HZ, 1H, CONH); C-NMR (100MH ,
6
DMSO-d ): δ 40.1, 50.1, 67.4, 123.6, 126.5, 127.4, 128.1,
-
1
917, 1689, 1636, 1589, 1481, 1404, 1157, 1011 cm ; HR-
128.2, 128.3, 128.7, 128.9, 129.1, 131.6, 131.7, 135.3, 136.9,
141.0, 164.6, 166.8, 167.0 ppm; IR (KBr): 3341, 3240, 3086,
MS (ESI, positive mode) m/z [M+Na]+ 652.10723
-
1
(
C
31
H
24Cl
2
F
3
N
3
O
4
Na requires 652.10960).
2963, 2932, 1674, 1628, 1551, 1497, 1381 cm ; HR-MS
+
(
ESI, positive mode) m/z [M+Na] 704.10144 (C32
Na requires 704.10206).
2 6
H23Cl F
2
.2.2.2.
3-{(4-(Trifluoromethoxy-benzyl)-[3-(4-trifluoro-
3 3
N O
methoxy-benzylamino)-benzoyl]-amino}-N-(2-chlorobenzyl)-
benzamide (8b)
2.2.3.2.
3-{(3,5-Bis(trifluoromethyl-benzyl)-[3-(3,5-bis-
trifluoromethyl-benzylamino)-benzoyl]-amino}-N-(2-
chlorobenzyl)benzamide (8d)
2
-Chloro benzylamine (7b, 0.125 mL, 1.03 mmol) was
added together with 5 mL of triethylamine and stirred at
room temperature for 5 days. Then the crude product was
purified by column chromatography using cyclohexane:
ethyl acetate (6.8:3.2) as eluent to afford 8b as off white
2-Chloro benzylamine (7b, 0.1 mL, 0.89 mmol) was
added together with 5 mL of triethylamine and stirred at
room temperature for 5 days. Then the crude product was
purified by column chromatography using cyclohexane:
ethyl acetate (7.6:2.4) as eluent to afford 8d as white crystal-
powder (42.5 mg, 21.3%); m.p. 91-93 °C; Rƒ = 0.66 (cyclo-
1
hexane:ethyl acetate, 45:55); H-NMR (400MH
Z
, DMSO-
, 2H,
), 6.72 (d, J=8 HZ, 2H, Ar-H),
, 2H, Ar-H), 7.21 (t, J=8 H , 2H, Ar-H), 7.29-
.47 (m, 8H, Ar-H), 7.87 (d, J=8 H , 2H, Ar-H), 9.07 (t, J=4
, 1H, CONH), 9.30 ppm (t, J=4 HZ, 1H, CONH); C-
, DMSO-d ): δ 40.5, 50.6, 61.8, 121.0, 126.5,
27.1, 127.3, 128.6, 128.7, 129.1, 129.8, 131.9, 132.0, 135.0,
35.8, 136.2, 140.5, 147.8, 163.7, 164.7, 165.4 ppm; IR
d
6
): δ 4.21 (d, J=4 H
), 5.03 (s, 2H, NCH
.10 (t, J=8 H
Z
, 2H, NHCH
2
), 4.54 (d, J=4 H
Z
line powder (103 mg, 51.5%); m.p. 158-160 °C; Rƒ =0.71
1
NHCH
2
2
(cyclohexane:ethyl acetate, 55:45); H-NMR (400MH
Z
,
7
7
H
Z
Z
DMSO-d
2H, NHCH
H), 7.11 (t, J=8 H
7.30-7.46 (m, 7H, Ar-H), 7.86 (d, J=8 H
(s, 2H, Ar-H), 8.02 (s, 1H, Ar-H), 9.07 (t, J=4 H
6
): δ 4.24 (d, J=4 H
), 5.24 (s, 2H, NCH
, 1H, Ar-H), 7.19 (t, J=8 H
, 2H, Ar-H), 7.92
, 1H,
CONH), 9.38 ppm (t, J=8 HZ, 1H, CONH); C-NMR
Z
, 2H, NHCH
), 6.77 (d, J=8 H
, 1H, Ar-H),
2
), 4.53 (d, J=8 HZ,
Z
2
2
Z
, 1H, Ar-
1
3
Z
Z
Z
NMR (100MH
Z
6
Z
1
1
Z
1
3
(
KBr): 3364, 3256, 3094, 2963, 2932, 1674, 1643, 1582,
Z 6
(100MH , DMSO-d ): δ 40.6, 50.4, 66.6, 124.0, 126.6,
-
1
1
543, 1481, 1435, 1108, 1018 cm ; HR-MS (ESI, positive
126.9, 127.1, 128.3, 128.5, 128.6, 128.7, 128.8, 129.0, 129.1,
132.0, 135.0, 136.2, 140.3, 165.0, 165.4, 167.2 ppm; IR
(KBr): 3364, 3217, 3086, 2924, 1667, 1635, 1559, 1505,
+
+
mode) m/z [M+Na] 652.10801 (C31
H
24Cl
2
F
3
N
3
O
4
Na re-
quires 652.10960).
-
1
1
420, 1381 cm ; HR-MS (ESI, positive mode) m/z [M+Na]
2
.2.3. General Procedure for the Synthesis of the Targeted
32 23 2 6 3 3
704.10087 (C H Cl F N O Na requires 704.10206).
Compounds (8c, d)
2
.3. Computational Methods
The intermediate methyl 3-(3,5-bis(trifluoromethyl)
benzyl) benzylamino) benzoate (5b) was dissolved in 1M
sodium hydroxide (5 mL) and refluxed overnight at 100 ºC,
then the reaction mixture was neutralized with 1M hydro-
chloric acid and extracted three times using chloroform (3 x
2
.3.1. Preparation of Protein Structure
The X-ray crystal structure of cholesteryl ester transfer
protein (CETP) (PDB ID: 4EWS) [11] was retrieved from
the RCSB Protein Data Bank. The coordinates were prepared
and energetically minimized using the Protein Preparation
2
0 mL). The organic layer was dried using anhydrous so-
dium sulfate and evaporated.
[
22] wizard in the Schrödinger software suite to maximize
Subsequently, the intermediate 3-(3,5-bis (trifluoro-
methyl) benzylamino) benzoic acid (6b, 0.2 g, 0.64 mmol)
was dissolved in 10 mL dichloromethane and oxalyl chloride
H-bond interactions.
2.3.2. Preparation of Ligand Structure
(
2, 0.1 mL, 1.1 mmol) was added. The reaction was left un-
The verified compounds (ligands) were built based on the
coordinates of (ORP) in 4EWS [11]. The ligands were built
using MAESTRO build panel and energy minimized by
MacroModel [22] program using the OPLS2005 force field.
der stirring for 5 days at 50-60 ºC. Later the reaction mixture
was evaporated.
2
.2.3.1.
3-{(3,5-Bis(trifluoromethyl-benzyl)-[3-(3,5-bis-
trifluoromethyl-benzylamino)-benzoyl]-amino}-N-(4-
chlorobenzyl)benzamide (8c)
2
.4. Quantum-Polarized Ligand Docking (QPLD)
4
-Chloro benzylamine (7a, 0.1 mL, 0.89 mmol) was
QPLD [22] is a docking approach that recruits the
added together with 5 mL of triethylamine and stirred at
room temperature for 5 days. Then the crude product was
purified by column chromatography using cyclohexane:
ethyl acetate (6:4) as eluent to afford 8c as white powder
combined quantum mechanical/ molecular mechanical
(
QM/MM) approach to determine ligand/protein interactions.
The QPLD procedure starts with Glide [22] docking to gen-
erate a list of docked poses of a ligand that fit the protein
binding cleft. The interaction energy of the protein/newly
generated ligand pose is calculated while treating the protein
with the MM method and the ligand pose with QM method
employing the QSite program in Schrödinger [22]. The Qsite
(
124 mg, 62%); m.p. 184-186 °C; Rƒ = 0.46 (cyclohex-
1
ane:ethyl acetate, 65:45); H-NMR (400MH
.15 (d, J=8 H , 2H, NHCH ), 4.45 (d, J=8 HZ, 2H,
NHCH ), 5.21 (s, 2H, NCH ), 6.89 (d, J=8 H , 2H, Ar-H),
Z 6
, DMSO-d ): δ
4
Z
2
2
2
Z

1
394 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 12
Abu Khalaf et al.
program produces a new set of atomic partial charges for the
ligand pose within the protein environment. The ligand poses
with QM-generated partial charges are redocked to the bind-
ing site using Glide [22] program with XP-scoring function.
QPLD takes into account the polarization effect of protein
binding pocket during the docking process. And, the ligand
pose with the lowest root mean square deviation is extracted.
The binding site of the protein domain is determined using
the ligand as a centroid. The Vander Waals scaling of the
non-polar receptor atoms is set to 1.0.
535 nm) was read using FLX800TBI Microplate Fluorimeter
(BioTek Instruments, Winooski, VT, USA).
The synthesized molecules were dissolved in DMSO
yielding 10 mM stock solutions. Next, dilution to the re-
quired concentration was attained using distilled deionized
water. DMSO concentration was adjusted to 0.1%. CETP
activity is not affected by DMSO. The percentage of residual
CETP activity was identified in the presence and absence of
the tested molecules.
Negative control samples missing rabbit serum were used
as a contrast background. Torcetrapib was used as a standard
CETP inhibitor. The experimental protocol and measure-
ments were carried out in duplicates. The % inhibition of
CETP by the synthesized compounds was calculated using
the following equation [18]:
2
.5. Pharmacophore Mapping
Utilizing a previously generated pharmacophore model
by our group [21], and in order to get further details about
the functionalities of the synthesized compounds responsible
for activity, 8a-d were mapped against the adopted pharma-
cophore model of CETP inhibitors [21].
% Inhibition = [1 –
Inhibitor read– Blank read ] *100%
Positive control– Negative control
2
.6. In Vitro CETP Inhibition Bioassay
An aliquot of rabbit serum (1.5 μL) was mixed with 160
3
. RESULTS AND DISCUSSION
μL of the tested sample. The donor and acceptor molecules
in the assay buffer were added, mixed well, and the volume
was adjusted to 203 μL using the assay buffer.
3.1. Chemistry
A series of chlorobenzyl benzamides 8a-d were synthe-
sized as shown in Scheme 1.
Then, the mixture was incubated at 37°C for 1 hour.
Fluorescence intensity (Excitation λ: 465 nm; Emission λ:
Y
O
X
Z
O
H N
H N
O
2
2
a
b,
+
Cl
HN
Br
Cl
O
C
4a: Y=OCF , X=Z=H
C
3
O
O
O
OH
4b: X=Z=CF , Y=H
3
X
Z
1
2
3
Y
5
5
a: Y=OCF , X=Z=H
3
b: X=Z=CF , Y=H
3
c
Z
H N
2
Y
X
O
HO
Cl
N
O
d,
NH
7
7
a: 4-Cl
b: 2-Cl
O
NH
N
O
H
Cl
X
Z
Cl
Y
8
8
8
8
a: Y=OCF
3
, X=Z=H, 4-Cl
, X=Z=H, 2-Cl
, Y=H, 4-Cl
, Y=H, 2-Cl
b: Y=OCF
3
6
a: Y=OCF , X=Z=H
3
c: X=Z=CF
d: X=Z=CF
3
3
6
3
b: X=Z=CF , Y=H
Scheme (1). Synthesis of chlorobenzyl benzamide derivatives 8a-d. Reagents and conditions: (a) CH
3
OH/reflux (60-70°C), 24 hours, (b)
DCM, TEA, RT, 5 days, (c) (1) 1M NaOH (100°C), overnight, (2) 1M HCl, (d) (COCl) , TEA, DCM, RT, 5 days.
2

CETP Inhibitory Activity of Chlorobenzyl Benzamides
Letters in Drug Design & Discovery, 2017, Vol. 14, No. 12 1395
Table 1. In vitro bioactivities of synthesized chlorobenzyl benzamides 8a-d.
Compound
% Inhibition
IC50 (µM)
3
F CO
N
O
O
a
8
8.7
1.6
NH
N
H
O
Cl
Cl
8
a
3
F CO
N
O
O
a
3
3.9
-----
Cl
NH
N
H
O
Cl
8
b
CF
3
3
F C
N
O
O
a
1
9.2
-----
NH
N
H
O
Cl
Cl
8
c
CF
3
F
3
C
N
O
O
a
2
6.6
-----
0.04
NH
N
H
O
Cl
Cl
8
d
b
Torcetrapib
88.2
a
Tested at 10 µM concentration.
b
Tested at 0.08 µM concentration.
The synthesis started with the activation of the carboxylic
acid moiety of 3-aminobenzoic acid (1) using oxalyl chloride
2) in the presence of methanol to produce the corresponding
methyl ester protecting group (3). Next, the amine nitrogen
of 3-amino benzoic acid methyl ester (3) attacked the par-
tially positive methylene group of the benzyl bromide (4a, b)
in the presence of DCM as a solvent to produce substituted
3-benzylaminobenzoic acid methyl ester intermediates (5a,
b). Triethylamine was used as an acid scavenger.
(
It was found that 5a (44% yield) was produced in higher
yield than 5b (28%). Afterward, deprotection of the carbox-
ylic acid group of 3-aminobenzoic acid methyl ester inter-
mediates (5a, b) was carried out by alkaline hydrolysis using

1
396 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 12
Abu Khalaf et al.
1
M NaOH under reflux followed by neutralization with 1M
On the other hand, it looks that the position of the chlo-
rine substituent whether at the ortho or para position has
greater influence on the CETP inhibitory activity of the syn-
thesized compound when the structure is substituted with 4-
trifluoromethoxy group rather than 3,5- ditrifluoromethyl
moieties.
HCl. Again, activation of the carboxylic acid moiety of 3-
benzylamino benzoic acid intermediates (6a, b) was per-
formed using oxalyl chloride (2) to produce the correspond-
ing acyl chloride derivatives in the presence of TEA and
DCM. Additionally, oxalyl chloride reacted with the amine
moiety of 3-benzylamino benzoic acid intermediates (6a, b).
Subsequently, amide formation was attained by the nucleo-
philic attack of the amine moiety of chloro-benzylamine (7a,
b) on the partially positive carbonyl carbon of the previously
produced benzoyl chloride and acyl chloride to get the tar-
geted chlorobenzyl benzamide derivatives 8a-d.
3
.3. Computational Results
3
.3.1. Quantum-Polarized Ligand Docking (QPLD)
In order to determine the structural basis of binding of
the synthesized compounds in 4EWS, QPLD was applied
against 4EWS [23]. Cho et al. illustrated that incorporating
the derived charges of quantum mechanical/molecular me-
chanical (QM/MM) approach into molecular docking sig-
nificantly improved the predictive outcome of a docking
program [24]. Our QPLD docking data for 8a-d (Scheme 1)
and ORP demonstrate that these compounds bind to the ac-
tive domain of 4EWS. Interestingly, the hydrophobic inter-
actions between the backbone of the binding site and the
compounds' scaffold drive the activity (Fig. 1).
The best yield was obtained upon reacting intermediate
6
b with 4-chloro benzylamine (7a) to produce 8c in 62%
yield. It is obvious that lower yield was achieved when using
-chloro benzylamine (7b) which can be attributed to steric
effect.
2
3
.2. In Vitro CETP Inhibition Bioassay
The results of CETP inhibition bioassay, presented in
Table 1, demonstrate that compound 8a exhibit good activity
against CETP with a percent inhibition of 88.7% at 10 µM
concentration and an IC50 of 1.6 µM.
The fact that our verified molecules 8a-d harbor four
aromatic rings clarifies the dominance of hydrophobic (van
der Waals) interactions (Fig. 2). Table 2 shows the QPLD
docking scores of the synthesized compounds 8a-d and ORP
against 4EWS. The more the negative the docking score the
better the binder. The fact that our compounds harbor four
aromatic rings and other functionalities explains their higher
binding scores compared to that of ORP. The absence of
By comparing the structure of the synthesized com-
pounds 8a-d with their activities (Scheme 1, Table 1), it ap-
pears that the presence of the trifluoromethoxy group at the
para position (as in compounds 8a and 8b) gives greater in-
hibitory activity than the two trifluoromethyl groups at the
meta positions (as in compounds 8c and 8d).
Fig. (1). The ORP/protein interaction. The hydrophobic lining depicted in green dominates the binding interaction. (The color version of the
figure is available in the electronic copy of the article).

CETP Inhibitory Activity of Chlorobenzyl Benzamides
Letters in Drug Design & Discovery, 2017, Vol. 14, No. 12 1397
VAL
MET
459
A
B
1
98
MET
33
4
PHE
263
ILE
ALA
95
1
5
1
F
PHE
65
2
LEU
457
VAL
36
O
1
F
ILE
15
2
F
ILE
GLN
199
1
1
O
ALA
02
2
LEU
06
HIS
32
2
ILE
05
2
LEU
17
2
2
O
VAL
N
H
4
38
ILE
15
CYS
13
NH+
2
CI
PHE
65
VAL
438
2
H
N
HIS
HN
232
ILE
443
SER
30
2
O
O
NH+
ILE
ALA
02
1
1
2
O
CI
PHE
41
PHE
441
4
O
LEU
261
ARG
201
PHE
HN
CYS
13
2
63
LEU
61
MET
459
2
ARG
01
ILE
SER
230
2
205
ILE
5
PHE
461
1
VAL
98
1
ILE
43
4
GLN
CI
O
F
PHE
461
LEU
206
199
MET
33
4
ALA
95
1
LEU
29
1
F
F
VAL
1
36
LEU
28
2
LEU
57
4
LEU
129
ALA
95
1
THR
1
38
C
D
CI
GLN
VAL
136
1
99
LEU
28
2
ILE
11
ILE
HIS
15
2
32
PHE
65
2
LEU
206
LEU
457
ALA
95
O
1
F
F
VAL
198
VAL
136
PHE
263
SER
230
THR
138
N
H
F
F
VAL
98
1
LEU
129
ALA
02
ILE
205
2
O
VAL
438
F
ARG
01
2
F
PHE
63
NH+
2
ILE
82
ILE
05
O
NH
2
PHE
41
ILE
4
443
NH+
O
ILE
CI
H
N
1
5
PHE
441
F
F
HN
O
ILE
PRO
221
F
443
SER
230
LEU
57
F
CI
4
O
CI
F
F
MET
59
LEU
228
ALA
202
4
CYS
1
3
PHE
461
PHE
461
CYS
13
GLN
199
ILE
215
ILE
11
LEU
61
LEU
261
PHE
463
2
LEU
2
ILE
215
06
HIS
32
2
PHE
265
Fig. (2). The ligand/protein complex of (A) 8a, (B) 8b, (C) 8c and (D) 8d.
Table 2. The QPLD docking scores (Kcal/mol) of the verified compounds.
Compound
QPLD Docking Score (Kcal/mol)
H-bond
RMSD (Å)
ORP
-7.52
-11.44
-10.30
-9.44
NA
H232
NA
1.950
0.049
0.002
0.027
0.033
8
8
a
b
8
c
NA
8
d
-11.16
NA
NA: Not available
RMSD: Root mean square deviation of the re-docked position and the original position for ORP into 4EWS.

1
398 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 12
Abu Khalaf et al.
3
.4. Validation of the QPLD
In order to assess the QPLD performance, the docked
pose of ORP in 4EWS was compared to its native conforma-
tion in the crystal structure. As can be seen in Fig. (3), there
is a superposition of the QPLD-generated ORP pose and the
native conformation in 4EWS. The RMSD for heavy atoms of
ORP between the native poses and QPLD-generated docked
poses was 1.950 Å, which designates the ability of QPLD
docking to recognize the native poses in crystal structures
and to effectively predict the ligand binding conformation.
3
.5. Pharmacophore Mapping
Fig. (3). The superposition of the QPLD-docked pose of ORP and
its native conformation in 4EWS. The native coordinates are yellow
colored and the docked pose is green colored. Picture made by
PYMOL. (The color version of the figure is available in the elec-
tronic copy of the article).
In order to gain insight about the functionalities of the
synthesized compounds, 8a-d were screened against an
adopted pharmacophore model [21] of CETP inhibitors (Fig.
4
). The pharmacophore model demonstrates that the CETP
inhibitor should harbor two aromatic (π-ring) or hydrophobic
moieties (F1 and F2); three hydrophobic functionalities (F4,
F5, and F6); two H-bond acceptors (F2 and F6); H-bond
donor or cationic center (F6) to incite an activity [21].
H-bond interaction and the dominance of hydrophobic inter-
action is observed.
The docking scores for 8a-d compared to ORP confirms
that the more the attaching moieties the more the interaction.
The in vitro biological data illustrates that 8a is the most
active derivative of this series followed by 8b. Compounds
The verified compounds matched the CETP inhibitors
pharmacophoric features (Fig. 5). This describes the affinity
of the targeted compounds toward CETP active domain. Fur-
thermore, the accommodation of 8a-d in the active binding
site could explain their inhibitory activity.
8
c and 8d having aromatic rings tailored with two -CF
3
moieties showed less CETP inhibition. This confirms that
the bulkiness of these compounds hinders their proper orien-
tation in the binding domain and thus in turn decreases their
activity.
The physicochemical properties of the synthesized mole-
cules such as high molecular weight, number of aromatic
rings, and log P violate Lipinski's rule of five [25, 26].
Fig. (4). Pharmacophore model of CETP inhibitor [21] with ORP. Aro stands for aromatic rings; Acc for H-bond acceptor; Don for H-bond
donor; Cat for cationic group; PiN for π-ring; and Hyd for hydrophobic groups. Picture made by MOE [24].

CETP Inhibitory Activity of Chlorobenzyl Benzamides
Letters in Drug Design & Discovery, 2017, Vol. 14, No. 12 1399
Fig. (5). Pharmacophore model of CETP inhibitor with (A) 8a, (B) 8b, (C) 8c, and (D) 8d.
Therefore, further optimization of this core nucleus is highly
required to improve their physicochemical properties and
better understand their structure-activity relationship.
The QPLD docking shows that compounds 8a-d accom-
modate the binding cleft of CETP and the hydrophobic inter-
action mediates ligand/complex formation. The scaffold of
8
a-d matches the pharmacophoric points of CETP inhibitors;
particularly hydrophobic and aromatic functionalities. The in
vitro biological data shows that 8a is the most potent deriva-
tive of this series followed by 8b. The bulkiness of 8c and 8d
CONCLUSION
This work identified chlorobenzyl benzamides as a new
scaffold targeting CETP activity. Benzamide 8a showed the
highest inhibitory activity with an IC50 of 1.6 µM. The study
found that the presence of p-trifluoromethoxy (as in 8a, 8b)
enhances CETP inhibitory activity more than m-trifluoromethyl
groups (as in 8c, 8d).
having aromatic rings tailored with two -CF moieties de-
3
creases their activity. This confirms that the bulkiness of
these compounds impedes their proper orientation in the
binding domain. Consequently, optimization of their struc-

1
400 Letters in Drug Design & Discovery, 2017, Vol. 14, No. 12
Abu Khalaf et al.
ture is recommended to enhance their physicochemical prop-
erties and clarify their structure-activity relationship.
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CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
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