Design, synthesis and biological evaluation of N,N-3-phenyl-3-benzylaminopropanamide derivatives as novel cholesteryl ester transfer protein inhibitor

Article abstract of DOI:10.1016/j.bmc.2015.12.010

A series of N,N-3-phenyl-3-benzylaminopropanamide derivatives were identified as novel CETP (cholesteryl ester transfer protein) inhibitors. In our previous study, lead compound L10 was discovered by pharmacophore-based virtual screening (Dong-Mei Zhao et al., 2014). Based on L10 (IC₅₀ 8.06 μM), compound HL6 (IC₅₀ 10.7 μM) was discovered following systematic structure variation and biological tests. Further optimization of the structure-activity relationship (SAR) resulted in N,N-3-phenyl-3-benzylaminopro panamides derivatives as novel CETP inhibitors. They were synthesized and evaluated against CETP by BODIPY-CE fluorescence assay. Among them, HL16 (IC₅₀ 0.69 μM) was a highly potent CETP inhibitor in vitro. In addition, HL16 exhibited favorable HDL-C enhancement and LDL-C reduction in vivo by hamster. The molecular docking of HL16 into the CETP was performed. The binding mode demonstrated that HL16 occupied the CETP binding site and formed interactions with the key amino acid residues.

Full text of DOI:10.1016/j.bmc.2015.12.010

Accepted Manuscript
Design, synthesis and biological evaluation of N, N-3-phenyl-3-benzylamino-
propanamide derivatives as novel Cholesteryl Ester Transfer Protein inhibitor
Dongmei Zhao, Honglei Xie, Changlin Bai, Chunchi Liu, Chenzhou Hao,
Shizhen Zhao, Hongli Yuan, Changqun Luo, Jian Wang, Bin Lin, Jiang Zheng,
Maosheng Cheng
PII:
S0968-0896(15)30180-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.12.010
BMC 12703
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 October 2015
6 December 2015
7 December 2015
Please cite this article as: Zhao, D., Xie, H., Bai, C., Liu, C., Hao, C., Zhao, S., Yuan, H., Luo, C., Wang, J., Lin,
B., Zheng, J., Cheng, M., Design, synthesis and biological evaluation of N, N-3-phenyl-3-benzylaminopropanamide
derivatives as novel Cholesteryl Ester Transfer Protein inhibitor, Bioorganic & Medicinal Chemistry (2015), doi:
http://dx.doi.org/10.1016/j.bmc.2015.12.010
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Design, synthesis and biological evaluation of N,
N-3-phenyl-3-benzylaminopropanamide derivatives as novel
Cholesteryl Ester Transfer Protein inhibitor
Dongmei Zhaoa*, Honglei Xiea, Changlin Baia, Chunchi Liua, Chenzhou Haoa, Shizhen Zhaoa,
Hongli Yuana, Changqun Luoa, Jian Wanga, Bin Lina, Jiang Zhenga,b, Maosheng Chenga
a Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University,
103 Wenhua Road, Shenhe District, Shenyang 110016, P. R. China.
b Center for Developmental Therapeutics, Seattle Children’s Research Institute, Division of Gastroenterology and Hepatology,
Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98101, USA.
ABSTRACT
A series of N,N-3-phenyl-3-benzylaminopropanamide derivatives were identified as novel CETP
(Cholesteryl ester transfer protein) inhibitors. In our previous study, lead compound L10 was
discovered by pharmacophore-based virtual screening (Dong-Mei Zhao et al, Chin. Chem. Lett, 25,
299). Based on L10 (IC50 8.06 µM), compound HL6 (IC50 10.7 µM) was discovered following
systematic structure variation and biological tests. Further optimization of the structure-activity
relationship (SAR) resulted in N,N-3-phenyl-3-benzylaminopro panamides derivatives as novel CETP
inhibitors. They were synthesized and evaluated against CETP by BODIPY-CE fluorescence assay.
Among them, HL16 (IC50 0.69 µM) was a highly potent CETP inhibitor in vitro. In addition, HL16
exhibited favorable HDL-C enhancement and LDL-C reduction in vivo by hamster. The molecular
docking of HL16 into the CETP was performed. The binding mode demonstrated that HL16 occupied
the CETP binding site and formed interactions with the key amino acid residues.
Keywords: CETP inhibitor; N,N-3-phenyl-3-benzylaminopropanamides derivatives; Molecular
docking
Corresponding author: Dong-mei Zhao. Tel.: +86 24 2398 6413; fax: +86 24 2399 5043;
E-mail address: medchemzhao@163.com (D-M, Z).
1

1. Introduction
Cardiovascular disease (CVD) is the leading cause of death and morbidity in developed nations.1-3
Elevated levels of low density lipoprotein-cholesterol (LDL-C) and decreased levels of high density
lipoprotein-cholesterol (HDL-C) are considered to be the major risk factors for cardiovascular events.4,5
Plasma cholesteryl ester transfer protein (CETP) is the key modulator of cholesterol ester inverse
transport, facilitating the transfer of cholesteryl esters from high density lipoprotein to low density
lipoprotein cholesterol and very low density lipoprotein-cholesterol (VLDL-C). CETP, secreted mainly
from the liver, has 476 amino acid residues. It is a 74-kDa hydrophobic glycoprotein which circulates
in plasma, primary bound to HDL-C.12,13 CETP acts to elevated LDL-C levels and reduced HDL-C
levels therefore it is considered to be a proatherogenic factor. Inhibition of CETP activities is
considered to be one of the most effective mechanisms to elevate the level of HDL-C.6-8
Epidemiological studies showed that for each 1.0 mmol/dL decrease in plasma LDL-C, there is
approximately 22% decrease in cardiovascular risk3. The statins, which are HMG-CoA reductase
inhibitors, have been used to reduce LDL-C levels in patients at risk for cardiovascular disease in
clinical treatment. However, many patients on statins and other lipid lowering treatments remain at an
unacceptably high risk of having a future CVD event.8-11 Development of novel cholesterol lowering
drugs is of utmost importance for treating cardiovascular diseases.
Torcetrapib is the first inhibitor into phase III clinical trial with potent CETP inhibition (IC50 50
nM). However, it was prematurely terminated due to more mortality in the torcetrapib/atorvastatin
group than the atorvastatin group.14-16 Dalcetrapib showed modest potency, whose phase III clinical
trial was also terminated due to the failure to show a clinically meaningful reduction in cardiovascular
(CV) events.17,18 In recent years, potential CETP inhibitors anacetrapib and evacetrapib have also
entered phase III clinical trials. The clinical trial data showed that both anacetrapib and evacetrapib led
to elevated HDL-C and lowered LDL-C while avoiding torcetrapib’s side effects.19-22 TA-8995, a
newly-developed CETP inhibitor, has successfully completed phase IIb trial in 2014. The phase II
clinical trial data showed that TA-8995 reduced LDL-C levels by 45.3%, whereas increased HDL-C
levels by up to 179.1%, and without serious adverse events or signs of liver toxic effects.24,25 It remains
to be seen whether these drug candidates will prevail eventually.
Despite the setbacks from the terminations of torcetrapib and dalcetrapib, great progress has been
made in illuminating the inhibition mechanism of CETP. A co-crystal structure of CETP with
2

torcetrapib was published in 2012.14 It was reported that the mechanism of lipid transfer is through the
hydrophobic tunnel between HDL-C and LDL-C, demonstrating the interactions of small molecule
compounds with CETP at the atomic level. In our previous study, based on this structure (compounds
1-4 shown in Scheme 1), a pharmacophore-based virtual screening campaign was carried out several
years ago and a lead compound L10 was identified to show modest inhibitory activity against CETP. In
this study, using L10 as a starting point, we attempted to identify better lead compounds with more
potency and novel structure as small-molecule CETP inhibitors as a therapy for atherosclerosis.
(Scheme 1. should be listed here)
Following the structure-activity relationship (SAR) of compound L10, HL6 of N-cyclohexyl-3-
(3,4-dichlorophenyl-4-fluorobenzyl)amino propanamide was discovered. It was shown to have a mild
CETP inhibition (IC50 10.7 µM). Molecular docking showed that HL6 are located at the binding site
cavity of CETP but cannot match with sub-pockets very well. Based on the result, our subsequent
studies focused on exploring the propionamide scaffold. Different from most of disclosed inhibitors
which require a central core ring for activity, the propionamide is a flexible scaffold as an unusually
simple class of CETP inhibitors. This novel class thereby represents a flexibility families exhibiting
inhibitory activity against CETP. We hypothesized that propionamide acted as a link to connect
potential pharmacophore, thus the formation of modified amide terminus and aniline moiety (Scheme 2)
are important to improve CETP inhibition.
We chose preponderant CETP inhibition drug fragments reported in literature as substituents for
hybrids. As an initial strategy we studied the replacement of the amide terminus of the propionamide
by different heterocycles, naphthene base and alkyl with the aim to achieve CETP affinity and mitigate
lipophicility. After the biological evaluation, compound HL3 with 1-phenylpiperazine as amide
terminus scaffold showed the most promising results. Further optimization of HL3 led to the discovery
of compound HL16 (IC50 0.69 µM) in vitro by BODIPY-CE fluorescence assay, as shown in Scheme 2.
Herein, we described the design, synthesis and biological evaluation N,N-3-phenyl-3-benzylaminoprop
anamide as novel CETP inhibitors.
(Scheme 2. should be listed here)
2. Results and discussion
2.1. Chemistry
Compounds HL1-HL17 were prepared according to Scheme 3. Overall, they were prepared in 5
3

steps. The first step was synthesizing the linker of the scaffold by commercially corresponding aniline
with acrylic acid through michael reaction at 40 oC. In this reaction, we found the yield of product from
the solvent of acrylic acid was higher than some literature reported acetonitrile or toluene as solvent.
The next step L2 was treated with HOBT, EDCI and DIEA in DMF at 0 °C for 2 h, followed by various
substituent aliphatic amine or arylpiperazines by condensation reaction to obtain the key intermediate
L3. These intermediates were used to in the next step to furnish the desired target compounds
o
HL1-HL17 by substitution reaction with 4-fluorobenzyl bromide and K2CO3 in DMF at 90 C. The
amide terminus scaffold was synthesized by two steps. Thionyl chloride and 2,2'-azanediyl
bis(ethan-1-ol) were used as starting materials to give bis(2-chloroethyl)amine hydrochloride by
chlorination, and then the corresponding aryl piperazines were obtained by cyclization.
(Scheme 3. should be listed here)
2.2. In vitro activity and Structure-Activity Relationships
(Table 1. should be listed here)
Structure−Activity Relationship (SAR) of N,N-3-phenyl-3-benzylamino propanamides derivatives
HL1-HL17
In an attempt to evaluate the ability of various N,N-3-phenyl-3-benzylaminopropanamide
derivatives to inhibit CETP, all these new synthetic compounds and reference compound anacetrapib
were initially assayed for their inhibitory effects against CETP by BODIPY-CE fluorescence assay with
CETP RP Activity Assay Kit (Catalog # RB-RPAK; Roar). The results of compound assay are shown in
Table 1. Most of the target compounds exhibit good CETP inhibition activity, while two compounds
have especially unexpected activity with IC50 >50 µM, indicating that the introduction of propionamide
scaffold to fit into the large pocket of CETP increased the inhibition activity. As summarized below,
several key structural changes provided critical insights into the general SAR of this series. First, the
effect of the N-substituted amide terminus on the inhibition of CETP was examined. Compounds with
N-cycloproryl and N-butyl have no inhibition activity, but these with N-tert-butyl HL7 exhibit IC50 of
29.6 µM while IC50 of HL6 with N-hexamethylene is nearly 2.8 fold higher than that of HL7. Thus we
could conclude that the shape of the hexamethylene moiety seems to influence the potency. HL3 with
1-phenylpiperazine exhibits nearly two times higher than HL1 and HL6. These results indicate that the
improvement of potency is related with the increase in the N-substituted space volume. However, the
potency of the 2-(piperazin-1-yl) pyrimidine compound HL2 is weaker than HL3, indicating that
4

further improvement in potency may increase N-substituted lipophilicity. Then, we were particularly
interested in extended linker of 1-phenylpiperazine with 4-methoxyl or 4-ethoxycarbonyl. The results
showed that HL12 or HL13 salient improved CETP inhibition activities, further indicating that the
structure of ethyl 4-(piperazin-1-yl)benzoate is more important to improve CETP inhibition than
1-(4-methoxyphenyl)piperazine.
Next, the modified aniline moiety was examined as shown in Table 1. The small-group substituted
aniline moiety caused loss of activity compared with compound HL3. When we increased the space
volume of aniline with 3-biphenyl or 3-phenoxyl molecules, the result exhibited that HL16 with
3-phenoxyaniline has especially outstanding activity with IC50 of 0.69 µM. Therefore we can conclude
that 3-phenoxyaniline provided an important contribution to the potency.
2.3. In vivo study in hamsters
Based on the result of in vitro CETP inhibitory assay, potent inhibitor HL16 was selected for the
in vivo assay. Male Golden Syrian hamster (8 weeks old, weight 120-130 g) were randomly divided
into control and treated groups (n = 6) by weight. Hamsters were placed on high cholesterol and lipid
diet for 5 weeks, and dyslipidemia model was induced. After oral administration of HL16 (formulation
ethanol/cremophor/salin=5:5:90) at dose of 30 mg/kg/day for two weeks, the blood samples were
drawn from orbit and the serum lipid levels were measured. The weights of hamsters were also
observed during the dosing process. The results indicated that LDL-C was reduced by 24.6% and
HDL-C was increased by 21.7%. The results were presented in Figure 1, confirming that HL16 had
significant lipid regulating potency.
(Figure 1. should be list here)
2.4. Molecular docking of compound HL16 in CETP
To further elucidate the binding pattern observed, molecular docking of the most potent inhibitor
HL16 were performed in the binding site cavity of CETP. Crystal structure of CETP (PDB ID: 4EWS)
and inhibitors were both pretreated by Discovery Studio 3.5. All docking runs were utilizing LigandFit
Dock protocol of Discovery Studio 3.5. The image files are generated by the Accelrys DS visualizer 4.0
systems. The binding modes of compound HL16 and CETP were depicted in Figure 2 A and B, which
revealed that the inhibitor is well occupied in the binding pocket. As showed in Figure 2 A, flexibility
of 3-phenoxyaniline occupied the important sub-pocket, ethyl 4-(piperazin-1-yl)benzoate located at the
deep of CETP. The amino acid residues which had interaction with inhibitor were labeled. As
5

illustrated in Figure 2 B, hydrogen atom on the piperazidine and the oxygen atom on the main chain of
Ser230 contribute to the hydrogen bonding interaction together, being a probable explanation for its
activity. One hydrophobic interaction is formed between the 3-phenoxyl and Ile15. Two hydrophobic
bonding are formed between the aniline ring of HL16 and Leu23 and Cys13. One π-π interaction is
formed between the benzene ring of the compound HL16 and phe441, and one hydrophobic reaction is
formed between Leu261 and benzene ring. Two hydrophobic interactions are formed between
ethoxycarbonyl and Leu29 and Val136, which may be helpful to improve its activity.14
(Figure 2 A. should be list here)
(Figure 2 B. should be list here)
3. Conclusion
In the present study, we report a novel CETP inhibitor and biological evaluation in vitro by
BODIPY-CE fluorescence assay. Most of the synthetic compounds showed mild inhibition activity,
fortunately, several representatives of N,N-3-phenyl-3-benzylaminopropanamides derivatives were
identified to exhibit remarkable potent CETP inhibition. After limited further structure-activity
relationship study, compounds HL16 with ethyl 4-(piperazin-1-yl)benzoate and 3-phenoxyaniline were
found to have potent CETP inhibition. HL16 exhibits an IC50 of 0.69 μM in vitro and significant lipid
regulating activity in vivo in hamster. In addition, Molecular docking of HL16 into the CETP was
performed, demonstrating that HL16 occupies the CETP binding site, and forms interaction with key
amino acid residues. In summary, these results demonstrate that the newly synthetic compounds have
the potential to be developed as leads and novel scaffold. It is highly expected that through further
structural modifications, the novel scaffold will produce promising CETP inhibition agents.
4. Experimental
4.1. Chemistry
4.1.1. Materials
All solvents were purchased from Aladdin (Shanghai China) and used without further purification.
All the chemicals were purchased from commercial sources with purity>98%, and melting points (mp.)
were determined in open capillaries on a Buchi 353 melting point apparatus (Buchi Labortechnik,
Flawil, Switzerland) without further correction. The solvents used for moisture sensitive reactions were
distilled and performed under argon atmosphere. The purity and homogeneity of the compounds were
assessed by TLC and HPLC chromatography either on a glass column using silica gel(100-200 mesh).
6

Mass spectra were taken in ESI mode on Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA). High
resolution accurate mass determinations (HRMS) for all final target compounds were obtained on a
Bruker Micromass Time of Flight mass spectrometer equipped with electrospray ionisation (ESI).
1
NMR spectra were recorded at 600 MHz for H and 600 MHz for 13C on a Bruker spectrometer with
TMS as an internal standard, CDCl3 or DMSO-d6 as solvent. The chemical shifts (d) were reported in
ppm relative to internal tetramethylsilane. Peak multiplicities were expressed as follows: s, singlet; d,
doublet; t, triplet; q, quartet; dd, doublet of doublet; m, multiplet.
4.1.2. 3-(3,4-Dichlorophenylamino) propanoic acid
A mixture of 3,4-dichloroaniline (4.6 g, 28.4 mmol), acrylic acid (15.0 mL) and 10% hydrochloric
acid(0.5 mL) was added to a 250 mL three-necked round bottom flask. The solution was heated to 40
oC for 2 h and then cooled to room temperature.23 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 target product (3.5 g, 72.1% yield) as a yellow solid. mp: 49.5-51.2 oC.
4.1.3. 4- substituted -phenylpiperazine (L7)
In a solution of 2,2'-azanediylbis(ethan-1-ol) (11.0 g, 104.7 mmol), dissolved in dry DCM (100.0
o
mL), thionyl chloride (37.4 g, 314.0 mmol) was added in a slow stream at below 5 C with ice bath,
and then the mixture was heated at reflux for 4.5 h, and then cooled to room temperature. The
precipitate was filtered, washed with ethanol, dried in infrared lamp and obtained as a white solid
bis(2-chloroethyl)aminehydrochloride, mp: 212-216 oC.
A mixture of bis(2-chloroethyl)aminehydrochloride (7.3 g, 41.0 mmol), K2CO3 (5.7 g, 41.3 mmol),
dissolved in 1-butanol, the substituted aniline (41.0 mmol) was added. The solution was heated at
reflux for 8 h and then cooled to room temperature. The precipitate was filtered, washed with water,
dried in infrared lamp and obtained as a white solid used directly in the next step without further
purification.
4.1.4. 3-(3,4-Dichlorophenylamino)propanamides
In a solution of 3-(3,4-dichlorophenylamino) propanoic acid (0.7 g, 3.0 mmol) dissolved in dry
DMF (10.0 mL)1-hydroxybenzotrizole (HOBt, 0.4 g, 3.0mmol) and 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDCI, 0.6 g, 3.0 mmol) was added. The mixture was stirred at room
temperature for 1.5 h, and then the DIEA (0.8 g, 6.0 mmol) and the corresponding amine (3.0 mmol)
were added. The reaction mixture was stirred at room temperature for 2 h and poured into ice water.
7

The precipitate was filtered, washed with water, dried in infrared lamp and obtained as a white solid
used directly in the next step without further purification.
4.1.4. General procedure for the synthesis of HL1-HL17
To a solution of 3-(3,4-dichlorophenylamino) propanamides (1.0 mmol) in dry DMF (10.0 mL)
was added potassium carbonate (2.0 mmol) and 1-(bromomethyl)-4-fluorobenzene (2.0 mmol). The
o
solution was heated to 90 C for 10 h and then 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 then dried over Na2SO4. Solvent was removed under reduced pressure and the resulting
residue was purified by column chromatography (10% EtOAc/petroleum ether, silica) or
recrystallization (petroleum ether/ethyl acetate) to provide the title HL1 to HL17 as a white to yellow
solid.
4.1.4.1. 3-(3,4-dichlorophenyl-4-fluorobenzyl)amino-1-morpholinopropan-1-one (HL1)
o
1
Compound HL1 was obtained as a light white solid. Yield: 35.4%; mp: 133.4-134.2 C. H
NMR(600 MHz, CDCl3): δ 7.25-7.19 (m, 3H), 7.16-7.01 (m, 2H), 6.98(d, 2H, J = 2.97 Hz), 6.73 (dd,
1H, J1 = 2.97 Hz, J2 = 8.97 Hz),6.51 (m, 1H), 4.51 (s, 2H), 3.77 (t, 2H, J = 7.08 Hz), 3.64-3.58 (m, 6H),
3.37-3.36 (m, 2H), 2.60-2.55 (d, 2H, J = 6.93 Hz). 13C NMR (150 MHz, DMSO-d6): δ 30.18, 39.99,
47.47, 52.91, 66.44, 66.48, 112.42, 112.90, 113.55, 115.72, 115.86, 117.39, 128.73, 128.78, 131.00,
132.01, 148.11, 160.32, 161.94, 169.90. ESI-MS (m/z): 411.1[M+H]+. HRMS calcd for
C20H21Cl2FN2O2Na, [M+Na]+, 433.0862; found 433.0861.
4.1.4.2. 3-(3,4-dichlorophenyl-4-fluorobenzyl)amino-1-(4-(pyrimidin-2-yl)piperazin-1-yl)propan
-1-one (HL2)
Compound HL2 was obtained as a light white solid. Yield: 34.69%; mp: 151.1-151.7 oC. 1H NMR
(600MHz, CDCl3) δ 8.33(d, J = 4.74Hz, 2H), 7.19(d, J = 8.94Hz, 1H), 7.16-7.11(m, 2H), 7.03-6.96(m,
2H), 6.75(d, J = 2.94Hz, 1H), 6.57-6.49(m, 2H), 4.54(s, 2H), 3.83-3.79(m, 6H), 3.71-3.67(m, 2H),
3.47-3.44(m, 2H), 2.66(t, J = 7.05Hz, 2H). 13C NMR (600 MHz, DMSO-d6): δ 169.78, 159.06, 147.99,
137.99, 134.61, 131.94, 130.93, 128.68, 117.30, 115.78, 115.64, 113.63, 113.47, 112.82, 107.59, 52.84,
47.44, 44.96, 44.75, 41.04, 40.25, 40.05, 39.91, 39.77, 39.63, 39.49, 30.30. ESI-MS (m/z):
488.1[M+H]+. HRMS calcd for C24H24Cl2FN5ONa, [M+Na]+, 510.1240; found 510.1240.
4.1.4.3. 3-(3,4-dichlorophenyl-4-fluorobenzyl)amino-1-(4-phenylpiperazin-1-yl)propan-1-one
(HL3)
8

o
1
Compound HL3 was obtained as a light white solid. Yield: 32.43%; mp: 135.0-135.8 C. H
NMR(600MHz, CDCl3) δ 7.28-7.26(d, 1H, J = 7.95Hz), 7.22-7.19(d, 1H, J = 9.0Hz), 7.14-7.09(m, 2H),
7.00-6.95(t, 2H, J = 8.61Hz), 6.79-6.78(m, 3H), 6.56-6.55(d, 1H, J = 2.97Hz), 6.54-6.53(dd, 1H, J =
3.0, 9.0Hz), 4.52(s, 2H), 3.83-3.80(m, 4H), 2.65-2.60(t, 2H, J = 4.89Hz), 3.17-3.08(m, 4H), 2.68-2.61(t,
2H, J = 6.75Hz). 13C NMR (150 MHz, DMSO-d6): δ 169.62, 162.32, 160.72, 151.16, 148.05, 134.62,
131.94, 130.92, 129.36, 128.68, 119.70, 117.31, 116.24, 115.78, 115.64, 113.49, 112.83, 52.84, 48.99,
48.61, 47.45, 45.06, 41.26, 40.33, 40.19, 40.05, 39.91, 39.77, 39.63, 39.49, 30.24. ESI-MS(m/z):
486.4[M+H]+. HRMS calcd for C26H26Cl2FN3ONa, [M+Na]+, 508.1331; found 508.1331.
4.1.4.4. N-(cyclopropylmethyl)-3-(3,4-dichlorophenyl-4-fluorobenzyl)amino-N-butylpropanamide
(HL4)
Compound HL4 was obtained as a light white solid. Yield: 32.61%; mp: 115.7-116.3 oC. 1H NMR
(600MHz, CDCl3): δ 7.41(d, J = 8.61Hz, 2H), 7.16-7.11(m, 2H), 7.01-6.95(m, 2H), 6.71(d, J = 8.55Hz,
2H), 4.60(s, 2H), 3.90-3.84(m, 2H), 3.38(t, J = 7.23Hz, 1H), 3.24-3.19(m, 2H), 3.07(d, J = 6.45Hz, 1H),
2.68-2.62(m, 2H). 13C NMR (150 MHz, DMSO-d6): δ 25.22, 28.43, 34.19, 47.72, 49.88, 52.52, 112.49,
113.11, 114.72, 115.21, 116.93, 128.18, 130.38, 131.42, 134.09, 147.62, 160.31, 161.94, 169.91.
ESI-MS(m/z): 451.1[M+H]+. HRMS calcd for C24H29Cl2FN2ONa, [M+Na]+, 473.1641; found
473.1639.
4.1.4.5. N-cyclopropyl-3-(3,4-dichlorophenyl-4-fluorobenzyl)aminopropanamide (HL5)
Compound HL5 was obtained as a light white solid. Yield: 31.59%; mp: 125.5-127.0 oC. 1H NMR
(600MHz, CDCl3): δ 7.19-7.16(m, 1H), 7.13-7.08(m, 2H), 7.01-6.95(m, 2H), 6.73(d, 1H), 6.50(dd, J =
2.97,8.91Hz, 1H), 4.50(s, 2H), 3.74(t, J = 6.66Hz, 2H), 2.72-2.61(m, 1H), 2.41(t, J = 6.66Hz, 2H),
0.80-0.73(m, 2H), 0.49-0.41(m, 2H). 13C NMR: (150 MHz, DMSO-d6): δ 5.51, 22.24, 33.22, 47.49,
52.61, 112.54, 113.09, 115.18, 115.32, 116.92, 128.24, 130.48, 131.51, 134.09, 147.52, 160.33, 161.91.
ESI-MS(m/z): 381.2 [M+H]+. HRMS calcd for C19H19Cl2FN2ONa, [M+Na]+, 403.0756; found
403.0755.
4.1.4.6. N-cyclohexyl-3-(3,4-dichlorophenyl-4-fluorobenzyl)aminopropanamide (HL6)
o
1
Compound HL6 was obtained as a white solid. Yield: 33.3%; mp: 132.5-134.0 C. H NMR(600
MHz, CDCl3): δ 7.20 (d, 1H, J = 9.0 Hz), 7.13-7.01 (m, 2H), 6.98 (t, 2H, J = 8.61 Hz), 6.74 (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), 3.77-3.72 (m, 3H),
2.42 (t, 2H, J = 6.66 Hz), 1.89-1.86 (m, 2H), 1.71-1.57(m, 3H), 1.15-1.26 (m, 2H), 1.21-0.99(m, 3H).
9

13C NMR (150 MHz, DMSO-d6): δ 24.52, 25.23, 32.31, 33.49, 47.42, 47.58, 52.60, 112.52, 113.09,
115.21, 115.32, 116.89, 128.29, 130.42, 131.49, 134.12, 147.63, 160.34, 161.91, 169.23. ESI-MS (m/z):
423.3 [M+H]+. HRMS calcd for C22H25Cl2FN2ONa, [M+Na]+, 445.1226; found 445.1226.
4.1.4.7. N-tert-butyl-3-(3,4-dichlorophenyl-4-fluorobenzyl)amino-propanamide (HL7)
o
Compound HL7 was obtained as a white solid. Yield: 33.2%; mp: 135.4-136.7 C. 1H NMR(600
MHz, CDCl3): δ 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): δ 28.42, 34.24, 47.67, 49.92, 52.51, 112.54, 113.09,
114.67, 115.22, 116.91, 128.23, 130.42, 131.39, 134.12, 147.61, 160.32, 161.89, 169.92. ESI-MS (m/z):
397.1 [M+H]+. HRMS calcd for C20H23Cl2FN2ONa, [M+Na]+, 419.1069; found 419.1067.
4.1.4.8. 3-((4-chloro-3-(trifluoromethyl)phenyl)(4-fluorobenzyl)amino)-1-(4-phenylpiperazin-1-
yl)propan-1-one (HL8)
o
Compound HL8 was obtained as a white solid. Yield: 42.79; mp: 137.1-138.4 C. 1H NMR (600
MHz, CDCl3) δ 7.31-7.27 (m, 2H), 7.26 (dd, J = 8.6, 5.3 Hz, 1H), 7.19-7.16 (m, 4H), 6.93 (dd, J = 8.9,
3.1 Hz, 1H), 4.62 (s, 2H), 3.87 (dd, J = 17.6, 10.4 Hz, 2H), 3.79 (dd, J = 15.2, 9.3 Hz, 2H), 3.76 (s, 2H),
3.24 – 3.11 (m, 4H), 2.69 (t, J = 7.0 Hz, 2H). 13C NMR(150 MHz, DMSO-d6): δ 29.81, 40.82, 44.78,
47.03, 48.19, 48.56, 52.40, 112.43, 113.12, 115.24, 115.39, 115.79, 116.92, 119.31, 128.23, 128.28,
128.89, 130.51, 131.53, 134.24, 134.29, 147.71, 150.82, 160.31, 161.89, 169.22. ESI-MS (m/z):
519.2[M+H]+. HRMS calcd for C27H26ClF4N3ONa, [M+Na]+, 542.9584; found 542.9584.
4.1.4.9. 3-([1,1'-biphenyl]-3-yl(4-fluorobenzyl)amino)-1-(4-phenylpiperazin-1-yl)propan-1-one
(HL9)
Compound HL9 was obtained as a white solid. Yield: 37.42%; mp: 140.7-141.5 oC. 1H NMR (600
MHz, CDCl3) δ 7.52- 7.50 (t, 2H), 7.43 (m, J = 7.5 Hz, 8H), 7.36-7.28 (dd, J = 8.4, 3.7 Hz, 2H), 7.20 –
7.17 (m, 5H), 6.94 (t, J = 8.7 Hz, 1H), 4.66 (s, 2H), 3.95-3.91 (t, J = 7.1 Hz, 2H), 3.79 (d, J = 5.0 Hz,
2H), 3.57 (d, J = 5.0 Hz, 2H), 3.16-3.08 (m, 4H), 2.75 (s, 2H). 13C NMR (150 MHz, CDCl3) δ 169.84,
163.18, 160.74, 150.77, 148.06, 142.68, 134.29, 129.87, 129.28, 128.74, 127.24, 120.74, 116.74,
115.62, 115.41, 111.38, 77.33, 77.07, 76.75, 54.24, 49.58, 47.33, 45.48, 41.52, 30.44, 29.72. ESI-MS
(m/z): 494.2[M+H]+. HRMS calcd for C32H32FN3ONa, [M+Na]+, 516.2427; found 516.2423.
4.1.4.10. 3-((4-chloro-3-ethylphenyl)(4-fluorobenzyl)amino)-1-(4-phenylpiperazin-1-yl)propan-1
-one (HL10)
10

o
1
Compound HL10 was obtained as a white solid. Yield: 37.42%; mp: 138.3-138.9 C. H NMR
(600 MHz, CDCl3) δ 7.37 (s, 2H), 7.30 – 7.14 (m, 3H), 7.01 (t, J = 8.1 Hz, 2H), 6.93 (d, J = 6.9 Hz,
3H), 6.57 (s, 1H), 6.49 (d, J = 8.0 Hz, 1H), 4.53 (s, 2H), 3.81 (d, J = 15.0 Hz, 4H), 3.54 (s, 2H), 3.13 (d,
J = 19.0 Hz, 4H), 2.67 (d, J = 7.1 Hz, 4H), 1.18 (t, 3H). 13C NMR (150 MHz, CDCl3) δ 169.58, 162.37,
159.80, 150.98, 146.76, 133.53, 131.23, 129.21, 128.10, 125.8, 125.55, 120.58, 116.62, 115.91, 115.50,
115.36, 111.78, 77.18, 76.97, 76.76, 54.04, 49.58, 49.32, 47.15, 45.40, 41.46, 38.41, 30.22, 29.63,
21.45. ESI-MS (m/z): 480.2[M+H]+. HRMS calcd for C28H31ClFN3ONa, [M+Na]+, 502.2037; found
502.2038.
4.1.4.11. 3-((4-bromo-3-methylphenyl)(4-fluorobenzyl)amino)-1-(4-phenylpiperazin-1-yl)propan
-1-one (HL11)
1
Compound HL11 was obtained as a white solid. Yield: 37.42%; mp: 143.8-144.6 oC. H NMR
(600 MHz, CDCl3) δ 7.21 (d, J = 9.2 Hz, 2H), 7.08 (s, 2H), 6.95-6.91 (d, J = 9.4 Hz, 3H), 6.83 (s, 4H),
6.47 (d, J = 8.2 Hz, 1H), 4.42 (s, 2H), 3.69 (s, 4H), 3.45 (s, 2H), 3.04 (d, J = 15.4 Hz, 4H), 2.57 (s, 2H),
2.19 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 169.62, 162.64, 161.02, 150.77, 146.89, 133.83, 131.16,
129.19, 128.05, 125.83, 125.55, 120.58, 116.62, 115.91, 115.50, 115.36, 111.78, 77.18, 76.97, 76.76,
54.04, 49.58, 49.32, 47.15, 45.40, 41.46, 30.22, 29.63, 21.45. ESI-MS (m/z): 510.1[M+H]+. HRMS
calcd for C27H29BrFN3ONa, [M+Na]+, 532.1376; found 532.1376.
4.1.4.12. 3-((3,4-dichlorophenyl)(4-fluorobenzyl)amino)-1-(4-(4-methoxyphenyl)piperazin-1-yl)
propan-1-one (HL12)
o
1
Compound HL12 was obtained as a white solid. Yield: 39.87%; mp: 142.5-143.2 C. H NMR
(600 MHz, CDCl3) δ 7.21 (d, J = 9.0 Hz, 1H), 7.15 (dd, J = 8.5, 5.4 Hz, 2H), 7.01 (t, J = 8.6 Hz, 2H),
6.87 (t, J = 8.8 Hz, 4H), 6.77 (d, J = 3.0 Hz, 1H), 6.53 (dd, J = 9.0, 3.0 Hz, 1H), 4.54 (s, 2H), 3.82 (d, J
= 6.7 Hz, 2H), 3.79 (s, 5H), 3.55 (s, 2H), 3.02 (dd, J = 11.2, 4.9 Hz, 4H), 2.66 (t, J = 7.0 Hz, 2H). 13C
NMR(150 MHz, CDCl3) δ 169.33, 163.23, 160.79, 147.27, 133.16, 130.76, 128.00, 119.59, 119.13,
115.77, 115.56, 114.62, 113.66, 112.04, 77.28, 77.02, 76.70, 55.57, 54.05, 47.32, 30.24. ESI-MS (m/z):
516.1[M+H]+. HRMS calcd for C27H28Cl2FN3O2Na, [M+Na]+, 538.1440; found 538.1438.
4.1.4.13. Ethyl 4-(4-(3-((3,4-dichlorophenyl)(4-fluorobenzyl)amino)propanoyl)piperazin-1-yl)
benoate (HL13)
o
Compound HL13 was obtained as a white solid. Yield: 41.52%; mp: 150.2-150.9 C. 1H NMR
(600 MHz, CDCl3) δ 7.19 (d, J = 8.9 Hz, 1H), 7.12 (d, J = 9.0 Hz, 1H), 7.07 (dd, J = 8.5, 5.4 Hz, 2H),
11

6.95 (t, J = 8.6 Hz, 3H), 6.77 (d, J = 9.0 Hz, 2H), 6.69 (d, J = 3.0 Hz, 2H), 6.46 – 6.42 (m, 1H), 4.46 (s,
2H), 4.28 – 4.26 (m, 2H), 3.74 (dd, J = 12.7, 6.0 Hz, 4H), 3.47 (d, J = 5.4 Hz, 2H), 3.27 – 3.17 (m, 4H),
2.58 (t, J = 7.0 Hz, 2H), 1.30 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 169.47, 166.48, 163.23, 160.79,
153.50, 147.19, 133.18, 131.25, 130.78, 128.05, 127.97, 115.78, 118.69, 115.57, 114.12, 113.82,
112.07, 77.34, 77.03, 76.71, 60.50, 54.12, 47.76, 47.59, 47.25, 45.01, 41.19, 30.26, 19.19, 14.42.
ESI-MS (m/z): 558.1[M+H]+. HRMS calcd for C29H30Cl2FN3O3Na, [M+Na]+, 580.1546; found
580.1546.
4.1.4.14. 3-([1,1'-biphenyl]-3-yl(4-fluorobenzyl)amino)-1-(4-(4-methoxyphenyl)piperazin-1-yl)
propan-1-one (HL14)
o
1
Compound HL14 was obtained as a white solid. Yield: 41.52%; mp: 148.9-149.5 C. H NMR
(400 MHz, CDCl3) δ 7.73 (s, 1H), 7.68 (d, J = 7.7 Hz, 1H), 7.58 (s, 1H), 7.53 (d, J = 7.7 Hz, 1H), 7.29
(s, 3H), 7.24 (dd, J = 8.2, 5.6 Hz, 2H), 7.03 (s, 2H), 6.92 (d, J = 8.0 Hz, 5H), 6.79 (s, 1H), 4.63 (s, 2H),
3.93 (d, J = 7.1 Hz, 2H), 3.79 (s, 2H), 3.74 (d, J = 40.7 Hz, 3H), 3.57 (s, 2H), 3.18 – 3.03 (m, 4H), 2.74
(s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 169.84, 158.87, 156.57, 142.68, 132.08, 129.87, 129.28,
128.70, 127.24, 120.74, 116.74, 115.62, 115.41, 111.38, 77.39, 77.07, 76.75, 49.69, 49.47, 45.48, 41.52,
30.44, 29.72. ESI-MS (m/z): 524.2[M+H]+. HRMS calcd for C33H34FN3O2Na, [M+Na]+, 546.2533;
found 546.2532.
4.1.4.15. Ethyl 4-(4-(3-([1,1’-biphenyl]-3-yl(4-fluorobenzyl)amino)propanoyl)piperazin-1-yl)ben
zoate (HL15)
o
1
Compound HL15 was obtained as a white solid. Yield: 43.61%; mp: 156.2-156.9 C. H NMR
(600 MHz, DMSO-d6) δ 7.54 – 7.51 (m, 2H), 7.40 (t, J = 7.7 Hz, 2H), 7.31 (s, 1H), 7.29 – 7.25 (m, 2H),
7.20 (s, 1H), 7.14 (t, J = 8.9 Hz, 2H), 6.89 – 6.83 (m, 4H), 6.80 (d, J = 9.1 Hz, 2H), 6.67 (dd, J = 8.3,
2.4 Hz, 1H), 4.63 (s, 2H), 3.76 (t, J = 7.0 Hz, 2H), 3.67 (s, 3H), 3.61 – 3.49 (m, 4H), 2.95 – 2.86 (m,
4H), 2.72 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 169.85, 166.01, 157.94, 157.10, 153.87, 149.67,
131.07, 130.68, 130.15, 128.67, 128.61, 123.34, 119.06, 118.66, 115.66, 115.52, 113.79, 107.83,
106.27, 103.12, 60.28, 40.32, 40.18, 40.04, 39.90, 39.76, 39.62, 39.48. ESI-MS (m/z): 566.6 [M+H]+.
HRMS calcd for C35H36FN3O3Na, [M+Na]+, 588.2638; found 588.2638.
4.1.4.16. Ethyl 4-(4-(3-((4-fluorobenzyl)(3-phenoxyphenyl)amino)propanoyl)piperazin-1-yl)ben
zoate (HL16)
o
Compound HL16 was obtained as a yellow solid. Yield: 42.59%; mp: 162.7-163.9 C. 1H NMR
12

(600 MHz, DMSO-d6) δ 7.29 (d, J = 9.0 Hz, 2H), 7.28 – 7.26 (m, 2H), 7.13 (dd, J = 8.5, 5.6 Hz, 2H),
7.11 (dd, J = 16.0, 8.5 Hz, 3H), 7.06 (t, J = 7.4 Hz, 1H), 6.95 (d, J = 9.1 Hz, 2H), 6.91 – 6.86 (m, 2H),
6.47 (dd, J = 8.4, 2.3 Hz, 1H), 6.27 (t, J = 2.3 Hz, 1H), 6.17 (dd, J = 7.9, 2.0 Hz, 1H), 4.53 (s, 2H), 4.23
(d, J = 7.1 Hz, 2H), 3.67 (t, J = 7.0 Hz, 2H), 3.60 – 3.49 (m, 4H), 3.32 (s, 4H), 2.68 (d, J = 7.0 Hz, 2H),
1.28 (t, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) d 169.85, 166.01, 162.24, 160.64, 157.94,
157.10, 153.87, 149.67, 135.24, 131.07, 130.68, 130.15, 128.67, 128.61, 123.34, 119.06, 118.66,
115.66, 115.52, 113.79, 107.83, 106.27, 103.12, 60.28, 40.32, 40.18, 40.04, 39.90, 39.76, 39.62, 39.48,
30.39, 14.68.ESI-MS (m/z): 582.2[M+H]+. HRMS calcd for C35H36FN3O4Na, [M+Na]+, 604.2588;
found 604.2687.
4.1.4.17. 3-((4-fluorobenzyl)(3-phenoxyphenyl)amino)-1-(4-(4-methoxyphenyl)piperazin-1-yl)
propan-1-one (HL17)
o
1
Compound HL17 was obtained as a yellow solid. Yield: 46.15%; mp: 155.4-156.2 C. H NMR
(600 MHz, CDCl3) δ 7.79 (d, J = 9.0 Hz, 2H), 7.29 (m, 2H), 7.12 (dd, J = 8.5, 5.6 Hz, 2H), 7.10(dd, J =
16.0, 8.5 Hz, 3H), 6.96 (t, J = 7.4 Hz, 2H), 6.95 (d, J = 9.1 Hz, 2H), 6.91 – 6.86 (m, 2H), 6.48 (dd, J =
8.4, 2.3 Hz, 1H), 6.16 (dd, J = 7.9, 2.0 Hz, 1H), 4.53 (s, 2H), 3.83 (d, J = 7.1 Hz, 2H), 3.79 (s, 3H),
3.55 (m, 2H), 3.03 (m, 2H), 2.68 (d, J = 7.0 Hz, 4H), 2.64 (m, 2H). 13C NMR (150 MHz, CDCl3) δ
169.87, 166.01, 160.66, 153.87, 147.07, 140.43, 139.94, 131.06, 129.51, 128.43, 119.06, 118.22,
115.66, 115.57, 114.94, 113.80, 113.15, 60.28, 52.99, 47.44, 46.69, 44.66, 30.35. ESI-MS (m/z):
540.2[M+H]+. HRMS calcd for C33H34FN3O3Na, [M+Na]+, 562.2482; found 562.2481.
4.2. Biology
4.2.1. In Vitro CETP inhibitory assay
The CETP RP Activity Assay Kit (Catalog # RB-RPAK;Roar) uses a donor molecule containing a
fluorescent self-quenched neutral lipid that is transferred to an acceptor by CETP(Catalog #
R8899;Roar). CETP-mediated transfer of the fluorescence neutral lipid to the acceptor molecule results
in an increase in fluorescence (ExEm = 465/535 nm). Inhibitor of CETP will inhibit the lipid transfer
and therefore decrease the fluorescence intensity. The testing compounds are dissolved using 100%
DMSO. It is important to make sure that each compound is dissolved totally. It is recommended to
vibrate the solution on oscillator for more than 30 seconds then store them at nitrogen cabinet. Dilute
the stocking compounds (10 mM) with DMSO for 8 points titration (1:5 serial dilutions) in 96-well
dilution plate. The assay was performed according to the instruction for the CETP inhibitor screening
13

kit and recombinant CETP.
Acknowledgements
The work was supported by 81373324 and grants (J1103606 and J1210029) of the National
Natural Science Foundation of China and the program for Innovative Research Team of the Ministry of
Education and Program for Liaoning Innovative Research Team in University.
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15

Scheme and figure legends
Scheme 1. Structure of CETP inhibitors
Scheme 2. Optimization of L10 led to discovery HL16
o
Scheme 3. Synthetic routes. Reagents and conditions: (a) 10% HCl, acrylic acid, 40 C; (b) substituted
amines, HOBt, EDCI, DIEA, RT; (d) DCM, reflux; (e) n-BuOH, K2CO3, reflux; (c) 4-fluorobenzyl
bromide, K2CO3, DMF, 90 oC.
Table 1. Structure−Activity Relationship (SAR) of N,N-3-phenyl-3-benzylamino propanamides
derivatives HL1-HL17
Figure 1. The in vivo analysis of hamster treated with HL16 for 2 weeks. (ig). Control: high-fat
diet-fed group untreated with HL16; HL16: high-fat diet-fed hamster treated with HL16 (30 mg/kg/d).
n=6.
Figure 2. The proposed binding mode of compound HL16 and CETP. (A) HL16 occupy the pocket of
CETP-inhibitor binding site. (B) HL16 interactions with amino acid. HL16 with carbon atoms colored
green light magenta, oxygen atoms colored red, nitrogen atoms colored blue, fluorine colored light blue.
Amino acid carbon atoms are colored gray, oxygen colored red, hydrogen atoms colored light gray,
nitrogen atom colored blue, the name of amino acid colored geen. Hydrogen bond colored pistachio
green.
16

Table(s)
Table 1.
Structure−Activity Relationship (SAR) of N,N-3-phenyl-3-benzylamino propanamides derivatives
HL1-HL17
Compd
R1
R2
IC50 µM
14.2
HL1
HL2
HL3
11.8
5.2
HL4
HL5
>50c
>50c
HL6
10.7
HL7
HL8
29.6
37.8
HL9
11.3
HL10
HL11
19.5
13.9

HL12
HL13
HL14
3.5
2.8
7.3
HL15
3.1
HL16
HL17
0.69
2.8
anacetrapibb
0.06
b The positive control; c considered with no CETP inhibition activity

Figure(s)
Scheme 1. Structure of CETP inhibitors
Scheme 2. Optimization of L10 led to discovery HL16

Scheme 3. Synthetic routes. Reagents and conditions: (a) 10% HCl, acrylic acid, 40 oC; (b)
substituted amines, HOBt, EDCI, DIEA, RT; (d) DCM, reflux; (e) n-BuOH, K2CO3, reflux; (c)
4-fluorobenzyl bromide, K2CO3, DMF, 90 oC.

Figure(s)
Figure 1. The in vivo analysis of hamster treated with HL16 for 2 weeks. (ig). Control: high-fat
diet-fed group untreated with HL16; HL16: high-fat diet-fed hamster treated with HL16 (30 mg/kg/d).
n=6.

Figure 2. The proposed binding mode of compound HL16 and CETP. (A) HL16 occupy the pocket of
CETP-inhibitor binding site. (B) HL16 interactions with amino acid. HL16 with carbon atoms colored
green light magenta, oxygen atoms colored red, nitrogen atoms colored blue, fluorine colored light blue.
Amino acid carbon atoms are colored gray, oxygen colored red, hydrogen atoms colored light gray,
nitrogen atom colored blue, the name of amino acid colored geen. Hydrogen bond colored pistachio
green.

Graphical Abstract