Synthesis, Biological Evaluation, and Molecular Modeling Study of Substituted Benzyl Benzamides as CETP Inhibitors

Article abstract of DOI:10.1002/ardp.201700204

Cardiovascular disease is the most common cause for mortality and morbidity in the developed world; its risk is inversely related to the high-density lipoprotein (HDL) cholesterol levels. Therefore, there is a great interest in developing new cholesteryl ester transfer protein (CETP) inhibitors capable of raising HDL as a novel approach for the prevention of cardiovascular disease. Herein, the synthesis and characterization of ten benzyl benzamides 8a–j that aim at CETP inhibition was performed. The in vitro CETP inhibition bioassay revealed that benzamide 8j had the best activity, with a percent inhibition of 82.2% at 10 μM concentration and an IC₅₀ value of 1.3 μM. The docking study shows that the verified compounds accommodate the binding cleft of CETP and are enclosed by a hydrophobic lining. Furthermore, the scaffold of 8a–j matches the pharmacophoric points of CETP inhibitors, particularly in its hydrophobic and aromatic functionalities.

Full text of DOI:10.1002/ardp.201700204

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Arch. Pharm. Chem. Life Sci. 2017, 350, e1700204
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Full Paper
Synthesis, Biological Evaluation, and Molecular Modeling Study of
Substituted Benzyl Benzamides as CETP Inhibitors
Reema Abu Khalaf
, Dima Sabbah, Eveen Al-Shalabi, Samar Bishtawi, Ghadeer Albadawi,
and Ghassan Abu Sheikha
Faculty of Pharmacy, Department of Pharmacy, Al-Zaytoonah University of Jordan, Amman, Jordan
Cardiovascular disease is the most common cause for mortality and morbidity in the developed world;
its risk is inversely related to the high-density lipoprotein (HDL) cholesterol levels. Therefore, there is
a great interest in developing new cholesteryl ester transfer protein (CETP) inhibitors capable of
raising HDL as a novel approach for the prevention of cardiovascular disease. Herein, the synthesis
and characterization of ten benzyl benzamides 8a–j that aim at CETP inhibition was performed. The
in vitro CETP inhibition bioassay revealed that benzamide 8j had the best activity, with a percent
inhibition of 82.2% at 10 mM concentration and an IC50 value of 1.3 mM. The docking study shows
that the verified compounds accommodate the binding cleft of CETP and are enclosed by a
hydrophobic lining. Furthermore, the scaffold of 8a–j matches the pharmacophoric points of CETP
inhibitors, particularly in its hydrophobic and aromatic functionalities.
Keywords: Atherosclerosis / Benzyl benzamides / CETP inhibitors / Docking / Pharmacophore mapping
Received: June 22, 2017; Revised: October 8, 2017; Accepted: October 10, 2017
DOI 10.1002/ardp.201700204
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
cardiovascular events, which is independent of the LDL-C
levels and remains relevant even after the achievement of
optimal LDL-C levels [4, 5], and it is thus considered to be an
Introduction
Cardiovascular disease is the most common cause for
mortality and morbidity in the developed world [1]. It
encompasses coronary heart disease (CHD), as well as
congestive heart failure, stroke, peripheral artery disease,
carotid artery disease, and aortoiliac disease [2]. CHD is usually
caused by a build-up of fatty deposits on the walls of the
arteries around the heart in a process called atherosclerosis, in
which low-density lipoprotein cholesterol (LDL-C) plays a
major role in its pathophysiology [3].
anti-atherogenic lipoprotein [6].
Development of novel therapies is in process to further
enhance the atheroprotective properties of HDL [7]. It is
believed that reverse cholesterol transport (RCT) is a primary
atheroprotective property of HDL and its major protein,
apolipoprotein A-I (apoA-I) [8]. So, there is considerable
interest in the potential use of pharmacological inhibitors of
cholesteryl ester transfer protein (CETP) as a novel approach
for cardiovascular disease prevention [9].
On the other hand, epidemiological data have clearly
demonstrated a strong inverse relationship between high-
density lipoprotein cholesterol (HDL-C) levels and the risk of
Plasma CETP was first described as a high molecular
weight glycoprotein [10], consisting of 476 amino acid
residues and has a molecular mass of 74 kDa [10, 11]. It
belongs to a family of proteins involved in lipid binding [12],
stimulating the transfer of cholesteryl ester between
lipoproteins [8, 13, 14].
Correspondence: Dr. Reema Abu Khalaf, Faculty of Pharmacy,
Department of Pharmacy, Al-Zaytoonah University of Jordan,
Amman 11180, Jordan.
E-mail: reema.abukhalaf@zuj.edu.jo,
rima_abu_khalaf@yahoo.com
̊
Moreover, the structure of CETP revealed a 60-A long
tunnel structure that demonstrated binding of two neutral
lipids, hydrophobic cholesteryl esters in the tunnel, and two
phospholipids, phosphatidylcholines, that plug the two
distinct openings at each end [15, 16].
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Therefore, CETP may play a dual role in RCT. It may represent
an additional route for the delivery of cholesterol to the liver
or, if LDL receptors are not fully functional, CETP promotes the
accumulation of LDL-C in the plasma, thus favoring the
atheroma formation [17]. CETP seems to be anti-atherogenic.
However, when it is altered, either by receptor malfunction or
defective ligands, CETP becomes pro-atherogenic [18].
Early discovered CETP inhibitors (Fig. 1) are torcetrapib,
dalcetrapib, anacetrapib, and evacetrapib [19]. A randomized
evaluation of the effects of anacetrapib shows that anace-
trapib appears to be safe, well-tolerated, and delivers a
substantial increases in HDL-C and reductions in LDL-C as
monotherapy and when combined with a statin [20, 21].
Although CETP acts as an antiatherogenic protein in
certain human mutant studies, most clinical and experi-
mental evidences support that CETP is atherogenic in
nature [22, 23].The role of CETP-mediated lipid transfer in
the development of atherosclerosis and CETP inhibition as a
potential strategy for prevention of atherosclerosis have
been controversial [24].
Our research group identified different potential CETP
inhibitors including benzenesulfonamides and toluene-4-
sulfonic acid esters [5], benzylamino-methanones [11], fluori-
nated benzamides [16], benzylideneamino-methanones [23],
and chlorobenzyl benzamides [25].
In order to optimize the activity of our previously
discovered hit compounds [25], variable substituents at the
meta and para positions were utilized (p-OCH3, p-CH3, m-Br,
m-F, and p-F) followed by in vitro CETP inhibition bioassay.
Results and discussion
Chemistry
A series of benzyl benzamides 8a–j were synthesized as shown
in Scheme 1. The synthesis started with the activation of the
Figure 1. Chemical structures CETP inhibitors.
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Benzyl Benzamides as CETP Inhibitors
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Scheme 1. Synthesis of benzyl benzamide derivatives 8a–j. Reagents and conditions: (a) CH3OH/reflux (60–70°C), 24 h; (b) DCM, TEA,
RT, 5 days; (c) (1) 1 M NaOH (100°C), overnight, (2) 1 M HCl; (d) (COCl)2, TEA, DCM, RT, 5 days.
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 partially 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 inter-
mediates (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
carboxylic acid group of 3-aminobenzoic acid methyl ester
intermediates (5a,b) was carried out by alkaline hydrolysis
using 1 M NaOH under reflux followed by neutralization with
1 M 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 corresponding
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). Subse-
quently, amide formation was attained by the nucleophilic
attack of the amine moiety of benzylamine (7a–e) on the
partially positive carbonyl carbon of the previously produced
benzoyl chloride and acyl chloride to get the targeted benzyl
benzamide derivatives 8a–j.
The best yield was obtained upon reacting intermediate 6a
with 4-methoxy benzylamine (7a) to produce 8a in 53% yield.
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In vitro CETP inhibition bioassay
Docking studies
The results of CETP inhibition bioassay, presented in Table 1,
demonstrate that all the synthesized benzamides 8a–j have
appreciable CETP inhibitory activity with the compound 8j
exhibiting promising activity against CETP with a percent
inhibition of 82.1% at 10 mM concentration and an IC50 of
1.3 mM.
In order to identify the structural basis of binding of the co-
crystallized ligand (ORP: torcetrapib) and 8a–j in CETP (PDB ID:
4EWS), we carried out docking studies employing Glide
[26, 27] docking approach against 4EWS. The Glide docking
data for 8a–j and ORP show that these compounds bind to the
active site of 4EWS. Indeed, Fig. 2 demonstrates that the
docked pose of 8i superposed the conformation of ORP
(torcetrapib).
Structure–activity relationship study of the synthesized
compounds 8a–j reveals that (Scheme 1 and Table 1) the
presence of para trifluoromethoxy group (as in compounds 8a
and 8b) gives greater inhibitory activity than the two meta
trifluoromethyl groups (as in compounds 8f and 8g) when the
Of note is the dominance of hydrophobic interaction
between the bound ligands and the backbones of the key
binding residues of 4EWS (Table 2; Figs. 3 and 4; Supporting
Information Figs. S1 and S2).
structure is substituted with
a lipophilic and electron
donating R groups, i.e. OCH3 and CH3. Conversely, existence
of lipophilic and electron withdrawing R groups such as F and
Br enhances the inhibitory activity more in the presence of the
two meta trifluoromethyl groups (as in compounds 8h–j).
Furthermore, it looks that the position of the fluorine
substituent whether at the meta or para position has greater
influence on the CETP inhibitory activity of the synthesized
compound when the structure is substituted with 3,5-
ditrifluoromethyl moieties (as in compounds 8h and 8j) rather
than 4-trifluoromethoxy group (as in compounds 8c and 8e).
Moreover, comparing the activities of the synthesized
compounds having m-F groups (as in compounds 8c and 8h)
with those containing m-Br moieties (as in compounds 8d and
8i) shows that m-F derivatives are more active than m-Br ones.
The docking scores demonstrate the high binding affinity
of this series toward 4EWS. The more negative the docking
score, the higher is the binding affinity and consequently the
better is the binder. Distinctly, a 2 kcal/mol difference in the
docking score of the co-crystallized ligand (ORP: torcetrapib)
and 8g,h infers that this scaffold might be a promising core
structure for CETP inhibition. Figure 5 shows that there is a
positive correlation between the % inhibition and Glide
docking scores against CETP (R2 ¼ 0.81).
The in vitro biological data elucidates that compounds
(8a–e) tailored with p-OCF3 moiety exerted promising CETP
inhibition; particularly 8a–c and 8e. Results suggests that
hydrophobic and/or H-bond acceptor cleft accommodates the
p-OCH3 motifs in 8a. In addition, the inhibitory activity of 8b
Table 1. In vitro bioactivities of synthesized benzyl benzamides 8a–j.
Compound
Y
X
Z
R
% Inhibition
IC50 (mM)
8a
8b
8c
8d
8e
8f
8g
8h
OCF3
OCF3
OCF3
OCF3
OCF3
H
H
H
H
H
H
H
H
H
H
CF3
CF3
CF3
CF3
CF3
–
H
H
H
H
H
CF3
CF3
CF3
CF3
CF3
–
4-OCH3
4-CH3
3-F
3-Br
4-F
4-OCH3
4-CH3
3-F
3-Br
4-F
65.8a)
62.1a)
50.0a)
21.9a)
51.1a)
54.8a)
37.5a)
59.6a)
28.5a)
82.1a)
82.2b)
3.7 (R2 ¼ 0.98)
4.4 (R2 ¼ 0.96)
–
–
–
–
–
–
8i
8j
–
1.3 (R2 ¼ 0.99)
Torcetrapib
–
–
0.04
b)
a) Tested at 10 mM concentration. Tested at 0.08 mM concentration.
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confirms that a steric effect hinders the orientation of p-OCH3
motifs in the binding domain and therefore clarifies its
moderate activity. Additionally, incorporating a fluoro moiety
on m-position (8h) implies that small binding cleft encloses
the m-position and thus explains the weak inhibitory activity
of 8i. The weak activity of 8g confirms the steric hindrance
effect and recommends attaching a small motif on p-position.
Moreover, the difference between the activity of 8f and 8g is
due to oxygen atom that might direct the methyl deeper in
the binding domain. Also, the O-atom provides a water
accessible surface area that facilitates the interaction
between the polar key binding residues and the backbone
of 8f. Altogether, p- and m-substituents are favored for small
motif provided the core structure is tailored with two m-CF3
groups. On the other hand, analogues bearing p-OCF3
favored hydrophobic and/or H-bond acceptor on p- and
m-positions provided small size attachment on m-position
whereas the p-position shows no restriction on the motif’s size
and favors p-OCH3 (8a), p-CH3 (8b), and p-F (8e). Comparing
the activity of analogues incorporating p-OCF3 with those
tailored with two m-CF3 recommends the two m-CF3 group
and this accords with the hydrophobic interaction that guides
ligand/protein complex formation.
Figure 2. Superposition of the Glide docked pose of 8i
(represented in pink color) and the cocrystallized ligand (gold
color). H atoms and some of key binding residues are hidden for
clarity purpose. Picture made by PYMOL.
confirms that hydrophobic interaction is favored on
p-position. Remarkably, the % inhibition of 8e endorses that
hydrophobic interaction is essential on p-position to elicit a
biological activity. It is worth noting that 8c exerted
comparable inhibitory activity to that of 8e; such finding
recommends small hydrophobic and/or H-bond acceptor
motifs on m-position. Actually, the activity of 8d implies that
a tight binding cleft surrounds the m-position and hinders the
proper accommodation of ꢀBr in 8d.
Pharmacophore mapping
In order to explore the backbones of 8a–j and their attach-
ments, we screened them against CETP inhibitors pharmaco-
phore model [16]. We found that 8a–j match the CETP
inhibitors fingerprint (Figs. 6 and 7; Supporting Information
Figs. S3 and S4) and this explains the affinity of 8a–j toward
CETP active domain. Moreover, 8a–j accommodate CETP
active binding domain thus explaining their inhibitory
activity.
Comparable inhibitory activity was noticed for analogues
bearing two m-CF3 groups instead of p-OCF3 (8f–j). Surpris-
ingly, the inhibitory activity of 8j matches that of the
reference inhibitor (torcetrapib). Result suggests that the
two m-CF3 groups occupy part of the binding site thus favors
small moiety such as p-F in 8j. Accordingly, the activity of 8f
Conclusion
Successful synthesis, characterization, biological evaluation,
and modeling studies of ten new benzamides were carried
out. Synthesized compound 8j was found to present a worthy
lead compound for CETP inhibitors with an IC50 of 1.3 mM.
Benzyl benzamides can serve as a promising new class of CETP
inhibitors that can be further structurally optimized to
improve CETP inhibitory activity and to enhance understand-
ing of the structure–activity relationship.
Table 2. The Glide docking scores (Kcal/mol) of the
verified compounds.
Glide docking
scores
(kcal/mol)
Compound
H-Bond
8a
8b
8c
8d
8e
8f
8g
8h
ꢀ11.26
ꢀ12.29
ꢀ11.13
ꢀ12.32
ꢀ11.24
ꢀ12.35
ꢀ12.52
ꢀ12.56
ꢀ12.26
ꢀ11.90
ꢀ10.57
H232
NA
NA
R201
NA
NA
NA
NA
NA
Experimental
Chemistry
General
8i
8j
All chemicals, reagents, and solvents were of analytical grade
and used directly without extra purification. Chemicals and
solvents were purchased from the corresponding companies
(Alfa Aesar, Acros Organics, Sigma–Aldrich, Fluka, SD Fine
Chem Limited, Tedia and Fisher Scientific).
NA
NA
Torcetrapib
(ORP)
NA: not available.
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Figure 3. The ligand/protein complex of (A) 8a and (B) 8b. The hydrophobic lining is represented in green color. Picture made by
MAESTRO [28].
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Figure 4. The ligand/protein complex of (A) 8f and (B) 8g. The hydrophobic lining is represented in green color. Picture made by
MAESTRO [28].
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Methyl 3-(4-(trifluoromethoxy)benzylamino)benzoate
(5a) [25]
Addition of 1-(bromomethyl)-4-(trifluoromethoxy) benzene
(4a, 4.2 mL, 26.4 mmol), and triethylamine (9.2 mL, 66 mmol)
to the solution of 3 was done. The mixture was left under
stirring 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; Rf ¼ 0.46 (cyclo-
hexane/ethyl acetate, 9:1); 1H NMR (300 MHz, CDCl3): d 3.84 (s,
2H, NHCH2), 4.63 (s, 3H, OCH3), 6.83 (dd, J ¼ 4, 15 Hz, 1H, Ar-H),
7.20 (d, J ¼ 12 Hz, 2H, Ar-H), 7.22–7.25 (m, 4H, Ar-H), 7.40 (d,
J ¼ 12 Hz, 1H, Ar-H), 7.44 ppm (s, 1H, NHCH2); 13C NMR
(75 MHz, CDCl3): d 53.6, 59.6, 113.4, 117.1, 118.7, 121.4,
128.1, 129.5, 136.5, 150.0, 168.0 ppm; IR (KBr): 3480, 3225,
Figure 5. The correlation between % inhibition of CETP and
Glide docking scores for the verified compounds. Outliers are
deleted for clarity purpose.
2986, 1713, 1620, 1458, 1404, 1303, 1250 cmꢀ1
.
Melting points were measured using Gallenkamp melting
point apparatus and are uncorrected. Infrared (IR) spectra
were recorded using Shimadzu IR Affinity1 FTIR spectro-
photometer. All samples were prepared with potassium
bromide and pressed into a disc. 1H NMR and 13C NMR
spectra were measured on Bruker Avance DPX 500, 400
and Bruker 300 MHz-Avance III spectrometers. Chemical
shifts are given in d (ppm) using TMS as internal reference;
the samples were dissolved in CDCl3 or DMSO-d6. High
resolution mass spectrometry (HR-MS) was performed
using LC Mass Bruker Apex-IV mass spectrometer utilizing
an electrospray interface. AFLX800TBI microplate fluorim-
eter was used in the in vitro bioassay (BioTek Instruments,
Winooski, VT, USA).
Thin-layer chromatography (TLC) was performed on
20 ꢁ 20 cm with layer thickness of 0.2 mm aluminum
cards pre-coated with fluorescent silica gel GF254 DC-
Alufolien-Kieselgel (Fluka Analytical, Germany), and visual-
ized by UV light indicator (at 254 and/or 360 nm). Commer-
cially available CETP inhibition drug screening kit was used
(BioVision, Linda Vista Avenue, USA).
Methyl 3-(3,5-bis(trifluoromethyl)benzyl)benzylamino)-
benzoate (5b) [25]
Addition of 1-(bromomethyl)-3,5-bis(trifluoromethyl)ben-
zene (4b, 4.6 mL, 25 mmol), and triethylamine (8.76 mL,
62 mmol) to the solution of 3 was done. 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;
Rf ¼ 0.66 (chloroform, 10); 1H NMR (300 MHz, CDCl3): d 3.86 (s,
3H, OCH3), 4.33 (t, J ¼ 6 Hz, 1H, NHCH2), 4.49 (d, J ¼ 6 Hz, 2H,
HNCH2), 6.73 (dd, J ¼ 3, 9 Hz, 1H, Ar-H), 7.19 (t, J ¼ 9 Hz, 1H, Ar-
H), 7.29 (d, J ¼ 3 Hz, 1H, Ar-H), 7.40 (d, J ¼ 9 Hz, 1H, Ar-H),
7.82 ppm (d, J ¼ 12 Hz, 3H, Ar-H); 13C NMR (75 MHz, CDCl3): d
47.5, 52.1, 113.7, 117.4, 119.7, 121.4, 121.5, 121.6, 127.4, 129.5,
131.3, 142.0, 147.3, 168.0 ppm; IR (KBr): 3402, 2955, 1713,
1605, 1512, 1443, 1381, 1297 cmꢀ1
.
General procedure for the synthesis of the targeted
compounds (8a–e)
The intermediate methyl 3-(4-(trifluoromethoxy)benzyla-
mino)benzoate (5a) was dissolved in 1 M sodium hydroxide
(5 mL) and refluxed overnight at 100°C, then the reaction
mixture was neutralized with 1 M hydrochloric acid and
extracted three times using chloroform (3 ꢁ 20 mL). The
organic layer was dried using anhydrous sodium sulfate
and evaporated. Subsequently, the intermediate 3-(4-(tri-
The InChI codes of the investigated compounds together
with some biological activity data are provided as Supporting
Information.
General procedure for the synthesis of methyl benzoates
intermediates (5a,b) [25]
3-Aminobenzoic acid (1, 2.0 g, 14.58 mmol) was dissolved in
methanol (20 mL) and cooled in the ice bath. The solution
was treated with oxalyl chloride (2, 2.5 mL, 29 mmol) stirred
at room temperature for 20–30 min, and refluxed for 24 h at
60–70°C. The reaction mixture was then evaporated and
neutralized by 3 M potassium carbonate. Four times
extraction by chloroform (4 ꢁ 20 mL) was applied. The
organic layer was dried with sodium sulfate anhydrous
followed by evaporation to get 2.07 g of pure 3-amino-
benzoic acid methyl ester (3). Next, 3-aminobenzoic acid
methyl ester (3, 2.0 g, 13.2 mmol) was dissolved in (20 mL)
dichloromethane.
fluoromethoxy)benzylamino)benzoic
acid (6a,
0.31 g,
1.0 mmol) was dissolved in 10 mL dichloromethane and oxalyl
chloride (2, 0.17 mL, 2.0 mmol) was added. The reaction was
left under stirring for 5 days at 50–60°C. Later the reaction
mixture was evaporated.
3-{(4-(Trifluoromethoxybenzyl)-[3-(4-
trifluoromethoxybenzylamino)benzoyl]amino}-N-(4-
methoxyobenzyl)benzamide (8a)
4-Methoxy benzylamine (7a, 0.39 mL, 3.0 mmol) was added
together with 5 mL of triethylamine and stirred at room
temperature for 5 days. Then the crude product was purified
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Figure 6. CETP inhibitor pharmacophore model with (A) 8a and (B) 8b. Aro stands for aromatic rings; Acc for H-bond acceptor; Don
for H-bond donor; Cat for cationic group, PiN for p-ring; and Hyd for hydrophobic groups. Picture made by MOE [29].
by column chromatography using chloroform/Methanol
(99:1) as eluent to afford 8a as white powder (53%); m.p.
130.5–131.5°C; Rf ¼ 0.90 (CHCl3/MeOH, 90:10); 1H NMR
(500 MHz, DMSO-d6): d 3.71 (s, 3H, OCH3), 3.72 (s, 3H,
OCH3), 4.07 (d, J ¼ 5.85 Hz, 2H, CH2), 4.41 (d, J ¼ 5.80 Hz, 2H,
CH2), 5.02 (s, 2H, CH2), 6.77 (d, J ¼ 8.55 Hz, 2H, Ar-H), 6.82 (d,
J ¼ 8.50 Hz, 2H, Ar-H), 6.88 (d, J ¼ 8.55 Hz, 4H, Ar-H), 7.24 (d,
J ¼ 8.55 Hz, 4H, Ar-H), 7.27–7.31 (m, 1H, Ar-H), 7.36–7.39 (m,
2H, Ar-H), 7.82–7.85 (m, 1H, Ar-H), 8.98 (t, J ¼ 5.85 Hz, 1H,
CONH), 9.14 (t, J ¼ 5.80 Hz, 1H, CONH) ppm; 13C NMR
(125 MHz, DMSO-d6): d 41.4 (1C), 42.6 (1C), 50.9 (1C), 55.2
(2C), 114.1 (2C), 114.2 (4C), 121.4 (2C), 126.6 (1C), 126.8 (1C),
128.8 (2C), 129.1 (4C), 130.3 (2C), 131.9 (1C), 135.9 (1C), 136.4
(1C), 140.9 (1C), 148.0 (1C), 158.7 (2C), 163.3 (1C), 165.4 (1C),
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Figure 7. CETP inhibitor pharmacophore model with (A) 8f and (B) 8g. Aro stands for aromatic rings; Acc for H-bond acceptor; Don
for H-bond donor; Cat for cationic group; PiN for p-ring; and Hyd for hydrophobic groups. Picture made by MOE [29].
165.5 (1C) ppm; IR (KBr): 3318, 3094, 2963, 1651, 1512, 1443,
1319, 1258 cmꢀ1; HR-MS (ESI, negative mode) m/z [Mꢀ1]þ
620.20074 (C33H29F3N3O6 requires 620.20867).
temperature for 5 days. Then the crude product was purified
by column chromatography using cyclohexane/ethyl acetate
(65:35) as eluent to afford 8b as white powder (46%); m.p.
162–163°C; Rf ¼ 0.93 (CHCl3/MeOH, 90:10); 1H NMR (500 MHz,
DMSO-d6): d 2.23 (s, 3H, CH3), 2.28 (s, 3H, CH3), 4.10 (d,
J ¼ 5.85 Hz, 2H, CH2), 4.44 (d, J ¼ 5.70 Hz, 2H, CH2), 5.02 (s, 2H,
CH2), 6.79 (d, J ¼ 7.80 Hz, 2H, Ar-H), 6.83 (d, J ¼ 7.75 Hz, 2H, Ar-
H), 6.88 (d, J ¼ 8.55 Hz, 4H, Ar-H), 7.14 (d, J ¼ 7.90 Hz, 2H, Ar-
H), 7.20 (d, J ¼ 7.85 Hz, 2H, Ar-H), 7.29–7.30 (m, 1H, Ar-H),
3-{(4-(Trifluoromethoxybenzyl)-[3-(4-
trifluoromethoxybenzylamino)benzoyl]amino}-N-(4-
methylbenzyl)benzamide (8b)
4-Methyl benzylamine (7b, 0.38 mL, 3.0 mmol) was added
together with 5 mL of triethylamine and stirred at room
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7.36–7.39 (m, 2H, Ar-H), 7.83–7.86 (m, 1H, Ar-H), 9.00 (t,
J ¼ 5.85 Hz, 1H, CONH), 9.17 (t, J ¼ 5.70 Hz, 1H, CONH) ppm;
13C NMR (125 MHz, DMSO-d6): d 21.1 (1C), 26.8 (1C), 41.7 (1C),
42.9 (1C), 51.0 (1C), 121.4 (1C), 121.5 (1C) 126.7 (1C), 126.8 (1C),
127.4 (2C), 127.7 (4C), 129.2 (2C), 129.3 (4C), 130.3 (2C), 135.5
(1C), 135.8 (1C), 136.3 (2C), 136.9 (1C), 140.9 (1C), 148.0 (1C),
163.4 (1C), 165.5 (2C) ppm; IR (KBr): 3356, 3071, 2924, 2862,
1659, 1543, 1435, 1397, 1258 cmꢀ1; HR-MS (ESI, negative
mode) m/z [Mꢀ1]þ 588.21019 (C33H29F3N3O4 requires
588.21884).
3-{(4-(Trifluoromethoxybenzyl)-[3-(4-
trifluoromethoxybenzylamino)benzoyl]amino}-N-(4-
fluorobenzyl)benzamide (8e)
4-Fluorobenzylamine (7e, 0.34 mL, 3.0 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
(60:40) as eluent to afford 8e as off-white powder
(46.3%); m.p. 140.5–141.5°C; Rf ¼ 0.62 (CHCl3/MeOH, 98:2);
1H NMR (400 MHz, DMSO-d6): d 4.01 (d, J ¼ 4.0 Hz, 2H, CH2),
4.45 (d, J ¼ 4.0 Hz, 2H, CH2), 5.00 (s, 2H, CH2), 6.87–6.90 (m, 4H,
Ar-H), 6.98 (m, 1H, Ar-H), 7.13 (m, 1H, Ar-H), 7.20–7.48 (m, 8H,
Ar-H), 7.82 (s, 2H, Ar-H), 9.25 (t, J ¼ 4.0 Hz, 1H, CONH), 9.54 (t,
J ¼ 4.0 Hz, 1H, CONH) ppm; 13C NMR (100 MHz, DMSO-d6): d
45.5 (1C), 46.7 (1C), 55.0 (1C), 119.5 (1C), 119.6 (2C), 119.7 (1C),
119.8 (2C), 125.7 (1C), 131.0 (1C), 133.6 (1C), 133.7 (1C), 133.9
(2C), 134.0 (2C), 134.6 (2C), 135.1 (1C), 139.0 (1C), 139.9 (1C),
140.5 (1C), 140.6 (1C), 145.1 (1C), 152.2 (1C), 164.8 (1C), 167.0
(1C), 167.9 (1C), 170.1 (1C), 170.2 (1C) ppm; IR (KBr): 3279,
3-{(4-(Trifluoromethoxybenzyl)-[3-(4-
trifluoromethoxybenzylamino)benzoyl]amino}-N-(3-
fluorobenzyl)benzamide (8c)
3-Fluorobenzylamine (7c, 0.34 mL, 3.0 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 chloroform/acetone (90:10)
as eluent to afford 8c as ivory viscous liquid (29%); Rf ¼ 0.60
1
(CHCl3/ethylacetate, 70:30); H NMR (500 MHz, CDCl3): d 4.27
3078, 2924, 2855, 1674, 1543, 1512, 1435, 1204 cmꢀ1
.
(d, J ¼ 5.9 Hz, 2H, CH2), 4.56 (d, J ¼ 6.0 Hz, 2H, CH2), 4.94 (s, 2H,
CH2), 6.40–6.53 (m, 1H, Ar-H), 6.81 (d, J ¼ 9.25 Hz, 1H, Ar-H),
6.92 (m, 2H, Ar-H), 6.95–7.00 (m, 2H, Ar-H), 7.01–7.07 (m, 1H,
Ar-H), 7.08 (d, J ¼ 8.3 Hz, 2H, Ar-H), 7.21 (d, J ¼ 8.1 Hz, 2H, Ar-
H), 7.24 (s, 1H, Ar-H), 7.26–7.28 (m, 1H, Ar-H), 7.33 (m, 1H, Ar-
H), 7.44–7.52 (m, 1H, Ar-H), 7.55 (s, 1H, Ar-H), 8.55 (t,
J ¼ 5.9 Hz, 1H, CONH), 8.75 (t, J ¼ 6.0 Hz, 1H, CONH) ppm;
13C NMR (125 MHz, CDCl3): d 43.6 (2C), 54.0 (1C), 114.4 (1C),
114.5 (1C), 114.7 (2C), 114.8 (2C), 121.0 (2C), 123.2 (1C), 123.4
(1C), 125.8 (1C), 126.2 (1C), 129.4 (1C), 130.2 (1C), 130.3 (1C),
130.4 (2C), 134.3 (1C), 135.5 (1C), 139.5 (1C), 140.5 (1C), 142.1
(1C), 150.0 (1C), 160.0 (1C), 161.9 (1C), 162.0 (1C), 164.0 (1C),
166.2 (1C) ppm; IR (KBr): 3364, 3071, 2963, 2855, 1667, 1589,
General procedure for the synthesis of the targeted
compounds (8f–j)
The intermediate methyl 3-(3,5-bis(trifluoromethyl)benzyl)-
benzylamino)benzoate (5b) was dissolved in 1 M sodium
hydroxide (5 mL) and refluxed overnight at 100°C, then the
reaction mixture was neutralized with 1 M hydrochloric acid
and extracted three times using chloroform (3 ꢁ 20 mL). The
organic layer was dried using anhydrous sodium sulfate and
evaporated. Subsequently, the intermediate 3-(3,5-bis-
(trifluoromethyl)benzylamino)benzoic acid (6b, 0.36 g,
1.0 mmol) was dissolved in 10 mL dichloromethane and oxalyl
chloride (2, 0.17 mL, 2.0 mmol) was added. The reaction was
left under stirring for 5 days at 50–60°C. Later the reaction
mixture was evaporated.
1543, 1451, 1250 cmꢀ1
.
3-{(4-(Trifluoromethoxybenzyl)-[3-(4-
trifluoromethoxybenzylamino)benzoyl]amino}-N-(3-
bromobenzyl)benzamide (8d)
3-{(3,5-Bis(trifluoromethylbenzyl)-[3-(3,5-bis-
trifluoromethylbenzylamino)benzoyl]amino}-N-(4-
methoxybenzyl)benzamide (8f)
3-Bromobenzylamine (7d, 0.38 mL, 3.0 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 chloroform/methanol
(95:5) as eluent to afford 8d as yellow viscous liquid (20%);
Rf ¼ 0.70 (CHCl3/MeOH, 98:2); 1H NMR (300 MHz, CDCl3): d 4.27
(d, J ¼ 6.0 Hz, 2H, CH2), 4.57 (d, J ¼ 6.0 Hz, 2H, CH2), 4.91 (s, 2H,
CH2), 6.24–6.51 (m, 1H, Ar-H), 7.13 (d, J ¼ 6.0 Hz, 2H, Ar-H),
7.20 (s, 2H, Ar-H), 7.24 (s, 1H, Ar-H), 7.29–7.43 (m, 6H, Ar-H),
7.45–7.51 (m, 2H, Ar-H), 7.55 (s, 1H, Ar-H), 7.65 (d, J ¼ 6.0 Hz,
1H, Ar-H), 8.55 (t, J ¼ 6.0 Hz, 1H, CONH), 8.63 (t, J ¼ 6.0 Hz, 1H,
CONH) ppm; 13C NMR (75 MHz, CDCl3): d 42.8 (1C), 43.6 (1C),
54.2 (1C), 121.1 (2C), 125.8 (1C), 126.2 (1C), 126.4 (1C), 126.6
(1C), 129.5 (1C), 130.3 (1C), 130.4 (2C), 130.5 (2C), 130.8 (2C),
130.9 (2C), 131.0 (2C), 134.2 (1C), 139.1 (1C), 140.0 (1C), 142.1
(1C), 144.5 (1C), 149.3 (1C), 152.5 (1C), 160.0 (1C), 162.1 (1C),
166.5 (1C) ppm; IR (KBr): 3325, 3071, 2963, 2855, 1651, 1543,
4-Methoxy benzylamine (7a, 0.39 mL, 3.0 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 chloroform/methanol
(98:2) as eluent to afford 8f as transparent semisolid (22%);
Rf ¼ 0.94 (CHCl3/MeOH, 95:5); 1H NMR (300 MHz, CDCl3): d 3.79
(s, 6H, 2 ꢁ OCH3), 4.21 (d, J ¼ 6.0 Hz, 2H, CH2), 4.55 (d,
J ¼ 6.0 Hz, 2H, CH2), 4.99 (s, 2H, CH2), 6.28–6.49 (m, 1H,
Ar-H), 6.82 (d, J ¼ 9.0 Hz, 2H, Ar-H), 6.85 (d, J ¼ 9.0 Hz, 2H, Ar-
H), 7.02 (d, J ¼ 6.0 Hz, 1H, Ar-H), 7.10 (d, J ¼ 9.0 Hz, 2H, Ar-H),
7.18–7.26 (m, 2H, Ar-H), 7.33–7.42 (m, 1H, Ar-H), 7.57 (s, 1H,
Ar-H), 7.62–7.67 (m, 2H, Ar-H), 7.77 (m, 1H, Ar-H), 8.56 (t,
J ¼ 6.0 Hz, 1H, CONH), 8.87 (t, J ¼ 6.0 Hz, 1H, CONH) ppm;
13C NMR (75 MHz, CDCl3): d 43.0 (1C), 43.8 (2C), 55.4 (2C), 114.2
(2C), 114.3 (2C), 123.0 (1C), 125.0 (1C), 125.3 (1C), 125.7 (1C),
126.3 (1C), 126.8 (1C), 128.9 (1C), 129.2 (1C), 129.3 (2C), 129.5
(2C), 129.8 (1C), 129.9 (1C), 130.2 (1C), 131.8 (1C), 132.2 (1C),
1427, 1265 cmꢀ1
.
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R. Abu Khalaf et al.
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136.1 (1C), 138.1 (1C), 142.0 (1C), 159.3 (2C), 162.0 (1C), 162.1
(1C), 165.9 (1C) ppm; IR (KBr): 3511, 3071, 2924, 2845, 1660,
3.67 (s, 2H, CH2), 4.30 (d, J ¼ 6.3 Hz, 2H, CH2), 4.51 (d, J ¼ 6.4 Hz,
2H, CH2), 6.44–6.58 (m, 1H, Ar-H), 6.59–6.73 (m, 1H, Ar-H),
6.85–7.19 (m, 3H, Ar-H), 7.20–7.38 (m, 3H, Ar-H), 7.39–7.60 (m,
3H, Ar-H), 7.89–8.02 (m, 1H, Ar-H), 8.10–8.21 (m, 1H, Ar-H),
8.32 (s, 2H, Ar-H), 8.45 (t, J ¼ 6.3 Hz, 1H, CONH), 8.55 (t,
J ¼ 6.4 Hz, 1H, CONH) ppm; 13C NMR (125 MHz, DMSO-d6): d
42.6 (2C), 48.3 (1C), 121.1 (1C), 122.5 (2C), 123.6 (1C), 124.9
(2C), 126.1 (1C), 126.2 (1C), 126.5 (1C), 129.1 (1C), 129.2 (1C),
129.5 (2C), 130.2 (2C), 130.9 (2C), 131.0 (1C), 134.6 (2C), 138.2
(1C), 139.3 (2C), 141.2 (1C), 144.5 (1C), 148.6 (1C), 160.8 (1C),
161.5 (1C), 165.4 (1C) ppm; IR (KBr): 3418, 3086, 2994, 2878,
1582, 1512, 1443, 1381, 1250 cmꢀ1
.
3-{(3,5-Bis(trifluoromethylbenzyl)-[3-(3,5-bis-
trifluoromethylbenzylamino)benzoyl]amino}-N-(4-
methylbenzyl)benzamide (8g)
4-Methyl benzylamine (7b, 0.38 mL, 3.0 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 chloroform/methanol
(98:2) as eluent to afford 8g as transparent semisolid
(39%); Rf ¼ 0.63 (cyclohexane/ethyl acetate, 60:40); 1H NMR
(500 MHz, DMSO-d6): d 2.28 (s, 6H, 2 ꢁ CH3), 4.12 (d, J ¼ 5.25
Hz, 2H, CH2), 4.43 (d, J ¼ 5.50 Hz, 2H, CH2), 5.03 (s, 2H, CH2),
6.78 (d, J ¼ 7.35 Hz, 2H, Ar-H), 6.97 (d, J ¼ 7.35 Hz, 2H, Ar-H),
7.14 (d, J ¼ 7.35 Hz, 2H, Ar-H), 7.21–7.25 (m, 3H, Ar-H), 7.29–
7.31 (m, 2H, Ar-H), 7.33–7.38 (m, 2H, Ar-H), 7.90 (d, J ¼ 7.30 Hz,
2H, Ar-H), 9.03 (t, J ¼ 5.3 Hz, 1H, CONH), 9.19 (t, J ¼ 5.5 Hz, 1H,
CONH) ppm; 13C-NMR (125 MHz, DMSO-d6): d 21.1 (2C), 41.8
(1C), 42.9 (1C), 50.7 (1C), 119.5 (1C), 121.5 (2C), 121.9 (1C),
126.9 (1C), 127.2 (1C), 127.7 (3C), 128.6 (1C), 129.1 (1C), 129.3
(3C), 129.6 (1C), 130.3 (2C), 133.8 (1C), 135.5 (1C), 136.2 (2C),
136.4 (1C), 137.0 (2C), 143.1 (1C), 148.0 (1C), 163.4 (1C), 165.5
(1C), 165.6 (1C) ppm; IR (KBr): 3279, 3032, 2932, 2862, 1651,
1674, 1597, 1566, 1458, 1427, 1281 cmꢀ1
.
3-{(3,5-Bis(trifluoromethylbenzyl)-[3-(3,5-bis-
trifluoromethylbenzylamino)benzoyl]amino}-N-(4-
fluorobenzyl)benzamide (8j)
4-Fluorobenzylamine (7e, 0.34 mL, 3.0 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
(60:40) as eluent to afford 8j as off-white powder
(23.2%); m.p. 126–127°C; Rf ¼ 0.64 (CHCl3/MeOH, 98:2);
1H NMR (500 MHz, DMSO-d6): d 4.13 (d, 2H, CH2), 4.47 (d,
2H, CH2), 5.02 (s, 2H, CH2), 6.90–7.00 (m, 4H, Ar-H), 7.09–7.20
(m, 3H, Ar-H), 7.31–7.37 (m, 6H, Ar-H), 7.85 (s, 2H, Ar-H), 9.09
(t, J ¼ 4.1 Hz, 1H, CONH), 9.25 (t, J ¼ 4.2 Hz, 1H, CONH) ppm;
13C NMR (125 MHz, DMSO-d6): d 43.1 (1C), 43.3 (1C), 45.2 (1C),
115.2 (1C), 115.3 (1C), 115.4 (1C), 115.5 (2C), 119.5 (1C), 121.5
(2C), 126.7 (1C), 127.8 (1C), 129.3 (1C), 129.4 (1C), 129.5 (1C),
129.6 (1C), 129.7 (1C), 130.3 (2C), 130.7 (1C), 134.7 (1C), 135.7
(1C), 136.1 (1C), 136.4 (1C), 140.9 (1C), 148.0 (1C), 160.7 (1C),
162.6 (1C), 163.4 (1C), 165.4 (1C), 165.5 (1C) ppm; IR (KBr):
1512, 1505, 1451, 1381, 1281 cmꢀ1
.
3-{(3,5-Bis(trifluoromethylbenzyl)-[3-(3,5-bis-
trifluoromethylbenzylamino)benzoyl]amino}-N-(3-
fluorobenzyl)benzamide (8h)
3-Fluorobenzylamine (7c, 0.34 mL, 3.0 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 chloroform/acetone (99:1)
as eluent to afford 8h as off-white powder (20.4%); m.p. 166–
167°C; Rf ¼ 0.40 (CHCl3/acetone, 95:5); 1H NMR (500 MHz,
CDCl3): d 3.56 (s, 2H, CH2), 4.48 (d, J ¼ 6.0 Hz, 4H, 2 ꢁ CH2), 6.97
(d, J ¼ 8.7 Hz, 4H, Ar-H), 7.05 (d, J ¼ 7.4 Hz, 2H, Ar-H), 7.24 (s,
2H, Ar-H), 7.28–7.30 (m, 4H, Ar-H), 7.79–7.86 (m, 3H, Ar-H),
8.89 (t, J ¼ 6.0 Hz, 1H, CONH), 9.15 (t, J ¼ 6.0 Hz, 1H, CONH)
ppm; 13C NMR (125 MHz, CDCl3): d 29.7 (1C), 43.2 (2C), 114.6
(1C), 114.7 (2C), 114.8 (1C), 114.9 (2C), 123.2 (2C), 123.3 (2C),
126.6 (1C), 127.4 (1C), 128.1 (2C), 129.3 (1C), 130.3 (2C), 130.4
(2C), 139.2 (1C), 139.3 (1C), 142.3 (1C), 143.4 (1C), 148.2 (1C),
159.6 (2C), 162.0 (1C), 163.9 (1C), 165.6 (1C) ppm; IR (KBr):
3441, 3081, 2940, 1651, 1506, 1474, 1397 cmꢀ1
.
Computational methods
Preparation of protein structures
The X-ray crystal structure of CETP (PDB ID: 4EWS) [30] was
adopted from the RCSB Protein Data Bank. CETP structure was
prepared and energetically minimized using the Protein
€
Preparation [28] panel in the Schrodinger software enterprise
to maximize H-bond interactions.
Preparation of ligand structures
The synthesized compounds (ligands) were built based on the
co-crystallized ligand’s (ORP) coordinates in 4EWS [30]. The
ligands were built using MAESTRO build wizard and
energetically minimized by MacroModel [28] program
recruiting the OPLS2005 force field.
3325, 3094, 2924, 2855, 1659, 1551, 1466, 1258 cmꢀ1
.
3-{(3,5-Bis(trifluoromethylbenzyl)-[3-(3,5-bis-
trifluoromethylbenzylamino)benzoyl]amino}-N-(3-
bromobenzyl)benzamide (8i)
Glide docking
3-Bromobenzylamine (7d, 0.38 mL, 3.0 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 chloroform/methanol
(95:5) as eluent to afford 8i as light brown liquid (17.1%);
Rf ¼ 0.70 (CHCl3/MeOH, 95:5); 1H NMR (500 MHz, DMSO-d6): d
The Grid file for CETP was generated employing the Glide Grid
Generation [28] procedure with the bound ligand as centroid.
During the docking process, the scaling factor for van der
Waals for the nonpolar atoms was set to 0.8 to facilitate some
flexibility for the protein side chains. All other parameters
were set as defaults. The binding affinity was represented in
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Arch. Pharm. Chem. Life Sci. 2017, 350, e1700204
Benzyl Benzamides as CETP Inhibitors
Archiv der Pharmazie
terms of docking scores (kcal/mol). The more negative the
docking score, the better the binder.
[7] C. G. Santos-Gallego, J. J. Badimon, R. S. Rosenson,
Endocrinol. Metab. Clin. North Am. 2014, 43(4),
913–947.
Pharmacophore mapping
[8] T. Aghebati, A. Badiee, A. H. Mohammadpour,
M. Afshar, M. R. Jaafari, K. Abnous, S. Issazadeh,
S. Hashemzadeh, M. Zareh, H. Hashemizadeh,
S. Nazemi, Biomed. Pharmacother. 2016, 81, 468–473.
[9] H. L. Xie, C. C. Liu, Y. Q. Li, C. L. Bai, C. Z. Hao, J. Guo,
C. Q. Luo, D. M. Zhao, M. S. Cheng, jcclet. 2017, 28(2),
260–263.
Utilizing a previously generated pharmacophore model by
our group [16], and in order to get further details about the
functionalities of the synthesized compounds responsible for
activity, 8a–j were mapped against the adopted pharmaco-
phore model of CETP inhibitors [16].
In vitro CETP inhibition bioassay
[10] D. Zhao, H. Xie, C. Bai, C. Liu, C. Hao, S. Zhao, H. Yuan,
C. Luo, J. Wang, B. Lin, J. Zheng, M. Cheng, Bioorg. Med.
Chem. 2016, 24(8), 1589–1597.
[11] G. Abu Sheikha, R. Abu Khalaf, A. Melhem, G. Albadawi,
Molecules 2010, 15, 5721–5733.
An aliquot of rabbit serum (1.5 mL) was mixed with 160 mL of
the tested sample. The donor and acceptor molecules in the
assay buffer were added, mixed well, and the volume was
adjusted to 203 mL using the assay buffer.
Then, the mixture was incubated at 37°C for 1 h. Fluores-
cence intensity (excitation l: 465 nm; emission l: 535 nm) was
read using FLX800TBI microplate fluorimeter (BioTek Instru-
ments, Winooski, VT, USA).
[12] H. Xie, Y. Li, C. Bai, R. Wang, C. Liu, C. Hao, B. Lin,
M. Cheng, D. Zhao, Bioorg. Med. Chem. 2016, 24(8),
1811–1818.
[13] B. Simic, P. Mocharla, M. Crucet, E. Osto, A. Kratzer,
€
The synthesized molecules were dissolved in DMSO yielding
10 mM stock solutions. Next, dilution to the required
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 measurements
were carried out in duplicates [23].
S. Stivala, S. Kuhnast, T. Speer, P. Doycheva,
H. M. Princen, J. W. van der Hoorn, J. W. Jukema,
H. Giral, A. Tailleux, U. Landmesser, B. Staels,
T. F. Luscher, Atherosclerosis 2017, 257, 186–194.
€
[14] D. Pirim, X. Wang, V. Niemsiri, Z. H. Radwan,
C. H. Bunker, J. E. Hokanson, R. F. Hamman,
M. M. Barmada, F. Y. Demirci, M. I. Kamboh, Metabolism
2016, 65(1), 36–47.
[15] Y. Chang, S. Zhou, E. Li, W. Zhao, Y. Ji, X. Wen, H. Sun,
H. Yuan, Eur. J. Med. Chem. 2017, 126, 143–153.
[16] R. Abu Khalaf, S. Al-Rawashdeh, D. Sabbah, G. Abu
Sheikha, Med. Chem. 2017, 13(3), 239–253.
[17] V. Garcıa-Gonzalez, B. Delgado-Coello, A. Perez-Torres,
J. Mas-Oliva, J. Arch. Med. Res. 2015, 46(5), 427–437.
[18] M. Hoekstra, Atherosclerosis 2017, 01, 034.
[19] P. J. Barter, K. A. Rye, R. M. Touyz, Pharmacol. Rev. 2016,
68(2), 458–475.
%Inhibition ¼ ½1 ꢀ ðInhibitorread ꢀ BlankreadÞ=
ðPositive control ꢀ Negative controlÞꢂ ꢁ 100%
ꢀ
ꢀ
ꢀ
The authors acknowledge the Scientific Research and
Postgraduate Deanship at Al-Zaytoonah University of Jordan
for sponsoring this project.
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