Discovery of novel N,N-3-phenyl-3-benzylaminopropionanilides as potent inhibitors of cholesteryl ester transfer protein in vivo

Article abstract of DOI:10.1016/j.bmc.2016.03.002

Epidemiological studies have identified that the risk of cardiovascular events increases due to the decreased levels of high density lipoprotein-cholesterol and the elevated levels of low density lipoprotein-cholesterol. Herein, we report a novel series of N,N-3-phenyl-3-benzylaminopropionanilide derivatives, which were identified as potent cholesteryl ester transfer protein (CETP) inhibitor. The initial lead compound L10 (IC₅₀ 8.06 μM) was found by pharmacophore-based virtual screening (Dong-Mei Zhao et al., Chin. Chem. Lett. 2014, 25, 299). After systematic structure variation and biological testing against CETP, two different series were identified as scaffolds for potent CETP inhibitors. One is N,N-3-phenyl-3-benzylaminopropanamide derivatives, which were investigated in our previous paper (Bioorg. Med. Chem. 2015, doi: http://dx.doi.org/10.1016/j.bmc.2015.12.010). The most potent compound HL16 in that series has the IC₅₀ of 0.69 μM. The other series is N,N-3-phenyl-3-benzylaminopropionanilide derivatives, which was investigated in current study. Further optimization of the structure-activity relationship (SAR) resulted in H16 (IC₅₀ 0.15 μM), which was discovered as a potent CETP inhibitor in vitro by BODIPY-CE fluorescence assay. In addition, the results of pharmacodynamics studies showed that H16 exhibited both favorable HDL-C enhancement and LDL-C reduction in vivo by hamster. It also has an excellent stability in rat liver microsomal.

Full text of DOI:10.1016/j.bmc.2016.03.002

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel N,N-3-phenyl-3-benzylaminopropionanilides
as potent inhibitors of cholesteryl ester transfer protein in vivo
Honglei Xie, Yiqun Li, Changlin Bai, Ruifeng Wang, Chunchi Liu, Chenzhou Hao, Bin Lin,
⇑
Maosheng Cheng, Dongmei Zhao
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016,
PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Epidemiological studies have identified that the risk of cardiovascular events increases due to the
decreased levels of high density lipoprotein-cholesterol and the elevated levels of low density lipopro-
tein-cholesterol. Herein, we report a novel series of N,N-3-phenyl-3-benzylaminopropionanilide deriva-
tives, which were identified as potent cholesteryl ester transfer protein (CETP) inhibitor. The initial lead
Received 3 February 2016
Revised 1 March 2016
Accepted 2 March 2016
Available online xxxx
compound L10 (IC50 8.06 lM) was found by pharmacophore-based virtual screening (Dong-Mei Zhao
et al., Chin. Chem. Lett. 2014, 25, 299). After systematic structure variation and biological testing against
CETP, two different series were identified as scaffolds for potent CETP inhibitors. One is N,N-3-phenyl-3-
benzylaminopropanamide derivatives, which were investigated in our previous paper (Bioorg. Med. Chem.
2015, doi: http://dx.doi.org/10.1016/j.bmc.2015.12.010). The most potent compound HL16 in that series
Keywords:
CETP inhibitor
N,N-3-Phenyl-3-benzylaminopropionanilide
derivatives
Microsomal stability
Dose-dependent
has the IC50 of 0.69
which was investigated in current study. Further optimization of the structure–activity relationship
(SAR) resulted in H16 (IC50 0.15 M), which was discovered as a potent CETP inhibitor in vitro by
lM. The other series is N,N-3-phenyl-3-benzylaminopropionanilide derivatives,
l
BODIPY-CE fluorescence assay. In addition, the results of pharmacodynamics studies showed that H16
exhibited both favorable HDL-C enhancement and LDL-C reduction in vivo by hamster. It also has an
excellent stability in rat liver microsomal.
Ó 2016 Published by Elsevier Ltd.
1. Introduction
plasma.10 CETP has been demonstrated to participate in the move-
ment of transferring cholesterol from peripheral tissue to the liver
Cardiovascular disease (CVD) is the leading cause of morbidity
and mortality in both developed and developing countries.1–3 Clin-
ical studies have established an inverse relationship between
serum levels of high density-cholesterol (HDL-C) and the incidence
of cardiovascular events. Epidemiological studies have identified
that for every 1.0 mg/dL increase in HDL-C, there is an approxi-
mately reduction of 2–3% in CVD risk.4–6 Niacin, can robustly ele-
vate HDL-C levels in current available treatments, but lacks of
compliance.7 The statins are used to reduce low density-lipoprotein
(LDL-C) levels in patients at risk for CVD in clinical treatment. How-
ever, many patients on statins treatment remain at high risk to
develop CVD events.8 Thus, there is a demand for specific HDL-C
raising therapies that confer better efficacy with less side effects.
Plasma cholesteryl ester transfer protein (CETP) is the key mod-
ulator facilitating the transfer cardioprotective HDL-C into very
low density-lipoprotein (VLDL-C) and LDL-C.9 CETP, secreted
mainly from the liver, is a carrier glycoprotein circulating in
for catabolism, a process known as reverse cholesterol transport
(CRT).10 There are several suggestions that CETP plays a key
proatherogenic role.10 Inhibition of CETP activities is considered
to be one of the most effective mechanisms to elevate the level
of HDL-C.11,12 Four small molecule inhibitors have entered into
phase III of clinical trials: (1) torcetrapib, (2) anacetrapib, (3)
evacetrapib and (4) dalcetrapib shown in Scheme 1. However,
torcetrapib was prematurely terminated due to more mortality in
the torcetrapib/atorvastatin group than the atorvastatin group.13–
15
Dalcetrapib showed modest potency, whose clinical trial III
was also terminated due to the failure to show a clinically mean-
ingful reduction in cardiovascular (CV) events.16,17 It was shown
that anacetrapib and evacetrapib led to elevated HDL-C and low-
ered LDL-C while avoiding torcetrapib’s side effects.18–21 TA-
8995, a new CETP inhibitor, has successfully completed phase IIb
trial in 2014. The clinical II 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.22,23 It remains to be seen whether these drug candidates
will prevail eventually.
⇑
Corresponding author. Tel.: +86 24 2398 6413; fax: +86 24 2399 5043.
E-mail address: medchemzhao@163.com (D. Zhao).
http://dx.doi.org/10.1016/j.bmc.2016.03.002
0968-0896/Ó 2016 Published by Elsevier Ltd.
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Scheme 1. Structure of CETP inhibitors.
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 torcetrapib was published in 2012.13 It was reported
that the holo-CETP structure revealed an exceptionally long
hydrophobic tunnel made of the N-terminal and the C-terminal
pocket, connected by
a narrow neck. Lipids can transfer
through this hydrophobic tunnel between HDL and LDL, but
small molecule inhibitors within the N-terminal pocket occupy
the narrow neck and successfully block lipids transfer.13 In our
previous study, a pharmacophore-based virtual screening cam-
paign was carried out and a lead compound L10 was identified
to show modest inhibitory activity against CETP.24 In this
study, using L10 as a hit compound, we aimed to identify bet-
ter lead compounds with more potency and novel structure as
small-molecule CETP inhibitor as potential candidates for
atherosclerosis.
Scheme 2. General structure of the target compounds L10 derivatives.
2. Results and discussion
2.1. Chemistry
Following the structure–activity relationship (SAR) of com-
pound L10, we focused on N,N-3-phenyl-3-benzylamino-
propanamides derivatives. In our earlier work, we have
demonstrated that N,N-3-phenyl-3-benzylaminopropanamides
derivatives exhibited CETP inhibition activities.24 This discovery
have been briefly reported in an earlier paper (Dong-mei Zhao
et al. doi: http://dx.doi.org/10.1016/j.bmc.2015.12.010).24 Differ-
ent from most of reported 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 flexible family exhibiting inhibitory activity against
CETP. We hypothesized that propionamide acted as a link to con-
nect potential pharmacophore. Thus the formation of modified
amide terminus, benzyl and aniline moiety (Scheme 2) are impor-
tant to improve CETP inhibition. Here we report our further efforts
to optimize the activity of propionamide series through the modi-
fication or replacement of the amide terminus (B moiety), benzyl
(C moiety) and aniline moiety (A moiety). As a result, the introduc-
tion of 3-phenoxyl into A moiety and 4-fluobenzenesulfonyl into C
Since the propionamide moiety gives an important contribution
to the potency of CETP inhibition, we tried to explore the chemical
space based on this core moiety. Chemical diversity around the
propionamide scaffold was generated from a highly versatile and
straightforward synthesized intermediate that in one final step
could be converted in parallel to many desired compounds. Com-
pounds H1–H18 were prepared according to Scheme 3. Overall,
they were prepared in 4 or 5 steps. The key intermediate L3 was
prepared by standard reductive amination or alkylation sequences
with commercially corresponding aniline and substituted ben-
zaldehyde. The Secondary amine L3 was treated with acrylic acid
through michael reaction at 50 °C for 6 h to generate the key link
L4. In this reaction, we found the yield of product from the solvent
of acrylic acid was higher than some literature reported acetoni-
trile or toluene as solvent. The next step L4 was treated with HOBT,
EDCI and DIEA in DMF at 0 °C for 2 h, followed by various sub-
stituent aliphatic amine or arylpiperazines by condensation reac-
tion to obtain the desired target compounds. In another synthetic
route the first step was synthesizing the linker L5 of the scaffold
by commercially corresponding aniline with acrylic acid through
michael reaction at 50 °C. The next step L5 was treated with HOBT,
EDCI and DIEA in DMF at 0 °C for 2 h, followed by ethyl
moiety leads to discovery of H16 (IC50 0.15 lM), which was man-
ifested as a potent CETP inhibitor in vitro by BODIPY-CE fluores-
cence assay.
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H. Xie et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Scheme 3. Synthetic routes. Reagents and conditions: (a) AcOH, Pd/C, H2; (b) 10% HCl, acrylic acid, 50 °C; (c) substituted amines, HOBt, EDCI, DIEA, RT; (d) DCM, reflux; (e) n-
BuOH, K2CO3, reflux; (f) 10% HCl, acrylic acid, 50 °C; (g) ethyl 4-aminobenzoate, HOBt, EDCI, DIEA, RT; (h) DCM, azabenzene, 4-fluobenzenesulfonyl chloride; (k) DMF, K2CO3,
substituent benzyl bromide, 90 °C.
Table 1
4-aminobenzoate by condensation reaction to obtain the key inter-
Structure–activity relationship (SAR) of N,N-3-phenyl-3-benzylaminopropionanilides
mediate L6. These intermediates were used to in the next step to
furnish the desired target compounds. The amide terminus was
synthesized by two steps. Thionyl chloride and 2,20-azanediylbis
(ethan-1-ol) were used as starting materials to give bis(2-chlor-
oethyl)amine hydrochloride by chlorination, and then the corre-
sponding aryl piperazines were obtained by cyclization.
derivatives H1–H18
Compd
IC50
(
lM)
Compd
IC50 (lM)
H1
H2
H3
H4
H5
H6
H7
H8
5.4
9.5
2.0
H10
H11
H12
H13
H14
H15
H16
H17
H18
2.9
1.3
3.4
2.3
>50
6.7
0.15
0.24
0.69
0.06
4.9
18.2
12.4
9.1
16.7
2.7
2.2. Results and discussion
2.2.1. In vitro activity and structure–activity relationships
In an attempt to evaluate the ability of various N,N-3-phenyl-3-
benzylaminopropionanilide 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 exhibited
H9
Anacetrapiba
a
The positive control.
moderate CETP inhibition activity, while only one compound
showed unexpected activity with IC50 > 50 M, indicating that
the introduction of propionamide scaffold was able to fit the
l
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Figure 1. In vivo dose-dependent analysis of hamster treated with H16 for 4 weeks. (A) HDL-C plasma parameters (mg/dl); (B) LDL-C plasma parameters (mg/dl) (ig). Control:
high-fat diet-fed group untreated with H16; H16: high-fat diet-fed hamster treated with H16 (20, 50, 100 mg/kg/d). n = 6.
moiety into the large pocket of CETP. As summarized below, sev-
eral key structural changes provided critical insights into the gen-
eral SAR of this series.
H15 (IC50 6.7 lM) with 4-phenoxyaniline moiety. Therefore, it was
concluded that 3-phenoxyaniline provides an important contribu-
tion to the potency.
Initial modifications focused on substituted benzyl moiety. The
results showed that 4-fluorobenzyl was the most important than
3,5-bis(trifluoromethyl)benzyl and 4-(trifluoromethyl)benzyl to
improve CETP inhibition activity. However, while 4-fluorobenzene-
sulfonyl was introduced to further explore the requirements for
2.2.2. In vitro rat liver microsomal stability
Microsomal incubation is an effective enzyme system for meta-
bolism studies. In human P450 metabolism stability was important
for drug discovery. Based on its in vitro properties, in order to
examine compound H16 metabolism stability, rat liver microsomal
was employed. We chose LC–MS/MS approach to detect H16 meta-
bolism stability. The compound H16 was eluted out LC–MS/MS at
17.1 min after quantitative assessment 36% was oxidized or
reduced. The result showed that it has an excellent stability in
rat liver microsomal.
activity. Compounds H11 (IC50 1.3
played marked improvement in potency compared to H9 (IC50
2.7 M) and H17 (IC50 0.24 M) respectively. For the modifications
lM) and H16 (IC50 0.15 lM) dis-
l
l
of the substituted amide terminus, in order to investigate
hydrophilicity and adjust lipophilicity, ethyl 4-(piperazin-1-yl)
benzoate, ethyl 4-aminobenzoate and 1-(4-methoxyphenyl)-piper-
azine were employed. The activity of compounds H3, H4 and H6
showed that ethyl 4-aminobenzoate seems to be optimal for CETP
2.2.3. In vivo dose-dependent study in hamsters
inhibition. Compound H3 (IC50 2.0
zoate through hydrolysis reaction changed to 4-aminobenzoic acid
was also investigated. But the effectiveness of compound H7 (IC50
9.1 lM) was diminished. For that reason ethyl 4-aminobenzoate
was chosen for further developed structures. Results from the
modification of the aniline moiety indicated that increased aniline
space volume with 4-chloro-3-biphenyl, 5-chloro-3-biphenyl or 4-
biphenyl the potency showed nearly no improvement. However,
introduction of flexible 3-phenoxyaniline moiety resulted in 10-
fold improvement over 3,4-dichloroaniline (H17 exhibit IC50
l
M) with ethyl 4-aminoben-
Based on the result of in vitro CETP inhibitory assay, potent
inhibitor H16 was selected for the in vivo assay. Male Golden Syr-
ian hamsters (8 weeks old, weight 120–130 g) were randomly
divided into control and treated groups (n = 6). Hamsters were
placed on high cholesterol and lipid diet for 5 weeks, and dyslipi-
demia model was induced. After oral administration of H16 (for-
mulation ethanol/cremophor/saline = 5:5:90) at dose of 20, 50
and 100 mg/kg/day for four 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 and no
hamster died. The results indicated that LDL-C was reduced by
19.3%, 36.2%, 45.8% and HDL-C was increased by 16.2%, 34.7%,
0.24
lM while H3 IC50 2.0 lM). Compound H18 (IC50 0.69 lM)
with 3-phenoxyaniline moiety was 10-fold potent than compound
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H. Xie et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
48.2%. The results were presented in Figure 1, confirming that H16
had significant lipid regulating potency, and also exhibits dose-
dependency in vivo.
4.1.3. 4-Substituted-phenylpiperazine (L6)
In
solution of 2,20-azanediylbis(ethan-1-ol) (11.0 g,
a
104.7 mmol), dissolved in dry DCM (100.0 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 °C.
3. Conclusion
In the present study, we report a novel CETP inhibitor and bio-
logical evaluation in vitro by BODIPY-CE fluorescence assay. Most
of the synthetic compounds showed moderate inhibition activity,
fortunately, several representatives of N,N-3-phenyl-3-benzy-
laminopropionanilide derivatives were identified to exhibit
remarkable potent CETP inhibition. After limited further struc-
ture–activity relationship study, compounds substituted with 4-
fluorobenzenesulfonyl and 3-phenoxyaniline were found to have
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.
potent CETP inhibition. H16 exhibits an IC50 of 0.15 lM in vitro
and dose-dependency for lipid regulating activity in vivo by ham-
ster. In addition, H16 also has an excellent stability in rat liver
microsomal. 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 struc-
tural modifications, the novel scaffold will produce promising CETP
inhibition agents.
4.1.4. 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 °C for 2 h and then cooled to room temperature.23 The reac-
tion mixture was poured into ice water. The precipitate was fil-
tered, 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 °C.
4. Experimental
4.1. Chemistry
4.1.5. 3-(3,4-Dichlorophenylamino)propanamides
All solvents were purchased from Aladdin (Shanghai China)
were used without further purification, all the chemicals were
purchased from commercial sources with purity >98%, Melting
points (mp) were determined in open capillaries on a Buchi 353
melting point apparatus (Buchi Labortechnik, Flawil, Switzerland)
and were uncorrected. The solvents used for reactions that mois-
ture sensitive were distilled and performed under argon atmo-
sphere. The purity and homogeneity of the compounds by
chromatography either on a glass column using silica gel (100–
200 mesh) assessed by TLC and HPLC chromatography. Mass
spectra were taken in ESI mode on Agilent 1100 LC–MS (Agilent,
Palo Alto, CA, USA). NMR spectra were recorded at 400 MHz for
1H and 400 MHz for 13C on a Bruker spectrometer with TMS as
an internal standard, CDCl3, or DMSO-d6 as solvent, and coupling
constants (J) were in hertz (Hz), and the signals were designated
as follows: s, singlet; d, doublet; t, triplet; m, multiplet; br s,
broad singlet.
In a solution of 3-((3,4-dichlorophenyl)(4-fluorobenzyl)amino)
propanoic acid (0.7 g, 2.0 mmol) dissolved in dry DMF (10.0 mL)
1-hydroxybenzotrizole (HOBt, 0.3 g, 2.0 mmol) and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 0.4 g,
2.0 mmol) was added. The mixture was stirred at room tempera-
ture for 1.5 h, and then the DIEA (0.6 g, 4.0 mmol) and correspond-
ing amine (2.0 mmol) were added. The reaction mixture was
stirred at room temperature for 2 h and poured into ice water.
The precipitate was filtered, washed with water, dried in infrared
lamp and obtained as a white solid, and the resulting residue
was purified by column chromatography (10% EtOAc/petroleum
ether, silica) or recrystallization (petroleum ether/ethyl acetate)
to provide the title compounds as a white to yellow solid.
4.1.6. Ethyl 4-(3-((3,5-bis(trifluoromethyl)benzyl)(3,4-dichloro-
phenyl)amino)propanamido)benzoate (H1)
Light white solid. Yield: 78%; mp: 108.4–109.2 °C. 1H NMR
(600 MHz, CDCl3) d 8.19 (s, 1H), 7.86 (d, J = 8.7 Hz, 2H), 7.64 (s,
1H), 7.48 (s, 2H), 7.46 (d, J = 8.6 Hz, 2H), 7.06 (d, J = 9.0 Hz, 1H),
6.64 (d, J = 2.9 Hz, 1H), 6.34 (dd, J = 9.0, 3.0 Hz, 1H), 4.57 (s, 2H),
4.24 (d, J = 7.1 Hz, 2H), 3.78 (s, 2H), 2.65 (s, 2H), 1.28 (t,
J = 7.1 Hz, 3H). 13C NMR (151 MHz, CDCl3) d 169.29 (s), 166.30
(s), 146.44 (s), 141.83 (s), 140.79 (s), 133.21 (s), 132.12 (s),
131.90 (s), 130.86 (s), 130.64 (s), 126.35 (s), 125.88 (s), 123.97
(s), 122.16 (s), 121.31 (s), 120.65 (s), 118.79 (s), 113.92 (s),
112.26 (s), 77.20 (s), 76.99 (s), 76.78 (s), 61.00 (s), 54.54 (s),
47.44 (s), 34.93 (s), 26.82 (s), 14.17 (s). HRMS calcd for
4.1.1. 3,4-Dichloro-N-(4-fluorobenzyl)aniline
To a 250 mL three-necked round bottom flask 3,4-dichloroani-
line (5.2 g, 32.1 mmol), 4-fluorobenzaldehyde (4.8 g, 38.5 mmol)
and acetic acid (0.5 mL) were dissolved in ethanol 30.0 mL. The
solution was heated to 40 °C for 2 h and then Pd/C (0.3 g) was
added, with hydrogen reaction 5 h. The reaction mixture was fil-
tered, washed with ethanol, the filtrate was then concentrated in
vacuum to give target product (7.5 g, 84.7% yield) as a yellow solid.
ESI-MS (m/z): 270.1[M+H]+.
C
27H22Cl2F6N2O3Na, [M+Na]+, 629.0809; found 629.0809.
4.1.2. 3-((3,4-Dichlorophenyl)(4-fluorobenzyl)amino)propanoic
acid
4.1.7. Ethyl 4-(3-((3,4-dichlorophenyl)(4-(trifluoromethyl)
benzyl)amino)propanamido)benzoate (H2)
In a solution of 3,4-dichloro-N-(4-fluorobenzyl)aniline (7.5 g,
27.8 mmol), dissolved acrylic acid (20.0 mL) and 10% hydrochloric
acid (0.5 mL) and then the mixture was heated at 50 °C for 6 h, and
then cooled to room temperature. The reaction mixture was
poured into ice water. The precipitate was filtered, washed with
water, and then purified by column chromatography (silica gel)
to give target product (7.9 g, 83.1% yield) as a white solid. ESI-MS
(m/z): 242.1[M+H]+.
White solid. Yield: 76%; mp: 121.2–122.4 °C. 1H NMR (400 MHz,
CDCl3) d 8.02 (d, J = 8.7 Hz, 2H), 7.55 (dd, J = 8.2, 3.2 Hz, 4H), 7.40 (s,
1H), 7.22 (s, 2H), 6.79 (d, J = 2.9 Hz, 1H), 6.54–6.51 (m, 1H), 4.65 (s,
2H), 4.40–4.35 (m, 2H), 3.89 (t, J = 6.4 Hz, 2H), 2.72 (d, J = 6.4 Hz,
2H), 1.41 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) d
169.59 (s), 164.54 (s), 161.41 (s), 142.15 (s), 136.97 (s), 128.46
(s), 126.04 (t, J = 5.3 Hz), 121.90 (s), 120.99 (d, J = 3.8 Hz), 115.46
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H. Xie et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
(s), 114.10 (s), 109.11 (s), 108.32 (s), 107.55 (s), 72.62 (s), 72.31 (s),
71.99 (s), 50.05 (s), 42.80 (s), 34.00 (s), 29.76 (s), 25.19–24.74 (m),
24.18 (s), 9.58 (s). HRMS calcd for C26H23Cl2F3N2O3Na, [M+Na]+,
561.0935; found 561.0936.
4.1.12. 4-(3-((3,4-Dichlorophenyl)(4-fluorobenzyl)amino)
propanamido)benzoic acid (H7)
White solid. Yield: 68%; mp: 109.6–110.2 °C. 1H NMR (400 MHz,
DMSO-d6) d 10.31 (s, 1H), 7.87 (d, J = 8.7 Hz, 2H), 7.67 (d, J = 8.6 Hz,
2H), 7.31 (d, J = 9.0 Hz, 1H), 7.21 (d, J = 5.3 Hz, 2H), 7.14 (d,
J = 8.8 Hz, 3H), 6.89 (d, J = 2.9 Hz, 1H), 6.68 (dd, J = 9.1, 3.0 Hz,
1H), 4.59 (s, 2H), 3.77 (t, J = 6.7 Hz, 2H), 2.69 (t, J = 6.8 Hz, 2H).
13C NMR (101 MHz, DMSO-d6) d 170.52 (s), 167.38 (s), 162.80 (s),
147.99 (s), 143.51 (s), 132.02 (d, J = 7.4 Hz), 130.93 (d,
J = 17.9 Hz), 129.13 (s), 128.73 (d, J = 8.1 Hz), 118.81 (s), 117.62
(s), 115.78 (d, J = 21.4 Hz), 113.74 (s), 113.09 (s), 65.49 (s), 53.27
(s), 47.63 (s), 40.60 (s), 40.39 (s), 40.18 (s), 39.97 (s), 39.76 (s),
39.59 (s), 39.45 (d, J = 21.0 Hz), 34.88 (s), 30.47 (s), 19.12 (s),
14.01 (s). HRMS calcd for C23H19Cl2FN2O3Na, [M+Na]+, 483.0654;
found 483.0653.
4.1.8. Ethyl 4-(3-((3,4-dichlorophenyl)(4-fluorobenzyl)amino)
propanamido)benzoate (H3)
White solid. Yield: 78%; mp: 104.2–104.8 °C. 1H NMR (400 MHz,
DMSO-d6) d 10.34 (s, 1H), 7.90 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.8 Hz,
2H), 7.31 (d, J = 9.0 Hz, 1H), 7.22 (dd, J = 8.6, 5.6 Hz, 2H), 7.12 (t,
J = 8.8 Hz, 2H), 6.89 (d, J = 2.9 Hz, 1H), 6.68 (dd, J = 9.1, 3.0 Hz,
1H), 4.59 (s, 2H), 4.28 (q, J = 7.1 Hz, 2H), 3.76 (d, J = 6.8 Hz, 2H),
2.70 (t, J = 6.8 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz,
CDCl3) d 166.02 (s), 141.51 (s), 133.24 (s), 130.83 (s), 128.15 (d,
J = 7.9 Hz), 118.81 (s), 115.80 (s), 115.59 (s), 114.37 (s), 112.74
(s), 77.27 (d, J = 11.6 Hz), 77.01 (s), 76.69 (s), 60.94 (s), 54.63 (s),
47.35 (s), 35.30 (s), 14.33 (s). HRMS calcd for C25H23Cl2FN2O3Na,
[M+Na]+, 511.0967; found 511.0968.
4.1.13. Ethyl 4-(3-([1,10-biphenyl]-4-yl(4-fluorobenzyl)amino)
propanamido)benzoate (H8)
White solid. Yield: 73%; mp: 143.2–143.8 °C. 1H NMR (600 MHz,
CDCl3) d 8.02 (d, J = 8.4 Hz, 1H), 7.59 (s, 1H), 7.58–7.50 (m, 6H),
7.41 (d, J = 7.7 Hz, 2H), 7.30 (s, 1H), 7.21 (s, 2H), 6.99 (t,
J = 8.6 Hz, 2H), 6.83 (d, J = 49.2 Hz, 2H), 4.59 (s, 2H), 4.38 (q,
J = 7.1 Hz, 2H), 3.90 (s, 2H), 2.74 (s, 2H), 1.40 (t, J = 7.1 Hz, 3H).
13C NMR (101 MHz, CDCl3) d 166.08 (s), 141.75 (s), 130.81 (s),
129.03–127.46 (m), 127.88–127.46 (m), 126.35 (s), 118.79 (s),
115.56 (d, J = 21.5 Hz), 115.39–115.22 (m), 77.29 (d, J = 11.5 Hz),
77.03 (s), 76.72 (s), 60.91 (s), 35.41 (s), 31.94 (s), 29.72 (s), 22.71
(s), 21.27 (s), 20.50 (s), 14.36 (s). HRMS calcd for C31H28FN2O3Na,
[M+Na]+, 519.2059; found 519.2060.
4.1.9. 3-((3,4-Dichlorophenyl)(4-fluorobenzyl)amino)-1-(4-(4-
methoxyphenyl)piperazin-1-yl)propan-1-one (H4)
White solid. Yield: 80%; mp: 115.5–116.4 °C. 1H NMR (400 MHz,
CDCl3) d 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 (101 MHz, CDCl3) d 169.33 (s), 147.27
(s), 133.21 (d, J = 10.0 Hz), 130.76 (s), 128.00 (d, J = 8.0 Hz),
119.59 (s), 119.13 (s), 115.77 (s), 115.56 (s), 114.62 (s), 113.66
(s), 112.04 (s), 77.28 (d, J = 11.5 Hz), 77.02 (s), 76.70 (s), 55.57 (s),
54.05 (s), 47.32 (s), 30.24 (s). HRMS calcd for C27H28Cl2FN3O2Na,
[M+Na]+, 538.1440; found 538.1440.
4.1.14. Ethyl 4-(3-((6-chloro-[1,10-biphenyl]-3-yl)(4-fluoroben-
zyl)amino)propanamido)benzoate (H9)
White solid. Yield: 82%; mp: 104.6–105.4 °C. 1H NMR (400 MHz,
DMSO-d6) d 10.34 (s, 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.68 (d, J = 8.8 Hz,
2H), 7.40 (d, J = 6.7 Hz, 2H), 7.35 (dd, J = 7.4, 5.8 Hz, 3H), 7.29–7.21
(m, 3H), 7.12 (t, J = 8.8 Hz, 2H), 6.73–6.68 (m, 1H), 6.66 (d,
J = 3.0 Hz, 1H), 4.60 (s, 2H), 4.27 (q, J = 7.1 Hz, 2H), 3.80 (t,
J = 6.7 Hz, 2H), 2.71 (t, J = 6.7 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H). 13C
NMR (101 MHz, DMSO-d6) d 170.74 (s), 165.78 (s), 162.76 (s),
160.35 (s), 147.10 (s), 143.83 (s), 140.51 (s), 139.98 (s), 130.67
(s), 129.62 (s), 128.98–128.51 (m), 128.51–128.39 (m), 127.95
(s), 124.60 (s), 118.89 (s), 118.53 (s), 115.81 (s), 115.60 (s),
115.23 (s), 113.45 (s), 60.87 (s), 53.43 (s), 47.68 (s), 40.61 (s),
40.40 (s), 40.19 (s), 39.98 (s), 39.77 (s), 39.57 (s), 39.36 (s), 35.11
4.1.10. Ethyl 4-(4-(3-((3,4-dichlorophenyl)(4-(trifluoromethyl)
benzyl)amino)propanoyl)piperazin-1-yl)benzoate (H5)
White solid. Yield: 73%; mp: 121.2–122.2 °C. 1H NMR
(400 MHz, DMSO-d6) d 7.45 (s, 1H), 7.38 (dd, J = 13.9, 7.7 Hz,
2H), 7.34–7.28 (m, 5H), 7.23 (d, J = 7.8 Hz, 1H), 6.86 (d,
J = 2.6 Hz, 1H), 6.64 (dd, J = 9.1, 2.8 Hz, 1H), 4.65 (s, 2H), 4.29
(d, J = 7.1 Hz, 2H), 3.62–3.54 (m, 4H), 3.14 (d, J = 4.3 Hz, 4H),
2.71 (t, J = 6.9 Hz, 2H), 1.31 (d, J = 7.1 Hz, 3H). 13C NMR
(151 MHz, CDCl3) d 169.27 (s), 166.40 (s), 153.41 (s), 146.91
(s), 141.95 (s), 131.16 (s), 130.78 (s), 126.58 (s), 125.68 (d,
J = 3.6 Hz), 121.10 (s), 114.04 (s), 113.47 (s), 111.87 (s), 77.17
(s), 76.96 (s), 76.75 (s), 60.43 (s), 54.39 (s), 47.57 (d,
J = 26.5 Hz), 47.32 (s), 47.28–47.17 (m), 44.91 (s), 41.12 (s),
(s), 14.68 (s). HRMS calcd for
553.1670; found 553.1670.
C
31H28ClFN2O3Na, [M+Na]+,
4.1.15. Ethyl 4-(4-(3-((6-chloro-[1,10-biphenyl]-3-yl)(4-fluoro-
benzyl)amino)propanoyl)piperazin-1-yl)benzoate (H10)
White solid. Yield: 67%; mp: 98.5–99.4 °C. 1H NMR (400 MHz,
DMSO-d6) d 7.79 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 15.8 Hz, 4H), 7.24
(s, 3H), 7.18–7.06 (m, 3H), 6.95 (d, J = 8.4 Hz, 2H), 6.68 (d,
J = 8.6 Hz, 1H), 6.62 (s, 1H), 4.60 (s, 2H), 4.24 (d, J = 6.9 Hz, 2H),
3.72 (s, 2H), 3.56 (s, 4H), 3.28 (s, 4H), 2.71 (s, 2H), 1.28 (d,
J = 7.0 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) d 169.87 (s), 166.01
(s), 153.87 (s), 147.06 (s), 140.43 (s), 139.94 (s), 135.09 (s),
131.06 (s), 129.51 (s), 128.71 (d, J = 8.0 Hz), 128.42 (s), 119.07 (s),
115.71 (s), 115.57 (s), 114.94 (s), 113.80 (s), 113.13 (s), 60.28 (s),
52.99 (s), 47.42 (s), 44.66 (s), 40.18 (s), 40.04 (s), 39.90 (s), 39.77
(s), 39.63 (s), 30.34 (s), 14.68 (s). HRMS calcd for C35H35ClFN3O3Na,
[M+Na]+, 622.2248; found 622.2247.
30.27 (s), 14.34 (s). HRMS calcd for
C30H30Cl2F3N3O3Na, [M
+Na]+, 630.1514; found 630.1514.
4.1.11. Ethyl 4-(4-(3-((3,4-dichlorophenyl)(4-fluorobenzyl)
amino)propanoyl)piperazin-1-yl)benzoate (H6)
White solid. Yield: 76%; mp: 99.7–100.2 °C. 1H NMR
(400 MHz, CDCl3) d 7.97 (d, J = 8.9 Hz, 2H), 7.58 (d, J = 8.2 Hz,
2H), 7.30 (s, 2H), 7.22 (d, J = 9.0 Hz, 1H), 6.87 (d, J = 9.0 Hz,
2H), 6.76 (d, J = 3.0 Hz, 1H), 6.50 (dd, J = 9.0, 3.0 Hz, 1H), 4.66
(s, 2H), 4.35 (q, J = 7.1 Hz, 2H), 3.85 (d, J = 6.9 Hz, 2H), 3.78 (d,
J = 5.3 Hz, 2H), 3.58 (d, J = 5.3 Hz, 2H), 3.32 (d, J = 4.6 Hz, 4H),
2.70 (t, J = 6.8 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H). 13C NMR
(101 MHz, CDCl3) d 169.47 (s), 166.48 (s), 153.50 (s), 147.19
(s), 133.18 (s), 131.25 (s), 130.78 (s), 128.01 (d, J = 8.0 Hz),
121.20 (s), 119.68 (s), 115.78 (s), 115.57 (s), 114.12 (s), 113.68
(s), 112.07 (s), 77.34 (s), 77.03 (s), 76.71 (s), 60.50 (s), 54.12
(s), 47.68 (d, J = 17.6 Hz), 47.25 (s), 45.01 (s), 41.19 (s), 30.26
4.1.16. Ethyl 4-(3-((N-(6-chloro-[1,10-biphenyl]-3-yl)-4-fluoro-
phenyl)sulfonamido)propanamido)benzoate (H11)
White solid. Yield: 78%; mp: 138.6–139.4 °C. 1H NMR (400 MHz,
CDCl3) d 8.01 (d, J = 8.7 Hz, 2H), 7.74 (s, 1H), 7.70–7.66 (m, 2H),
(s), 29.70 (s), 14.42 (s). HRMS calcd for
C29H30Cl2FN3O3Na,
[M+Na]+, 580.1546; found 580.1546.
Please cite this article in press as: Xie, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.002

H. Xie et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
7.57 (d, J = 8.6 Hz, 2H), 7.43 (s, 1H), 7.42–7.39 (m, 3H), 7.30 (s, 1H),
7.21 (s, 2H), 7.03 (d, J = 2.6 Hz, 1H), 6.96 (dd, J = 8.5, 2.6 Hz, 1H),
4.38 (d, J = 7.1 Hz, 2H), 3.94 (t, J = 6.8 Hz, 2H), 2.73 (t, J = 6.8 Hz,
2H), 1.40 (d, J = 7.1 Hz, 3H). 13C NMR (151 MHz, CDCl3) d 168.44
(s), 166.06 (s), 141.49 (s), 137.87 (s), 137.57 (s), 132.66 (s),
131.42 (s), 130.74 (d, J = 12.6 Hz), 130.41 (d, J = 9.4 Hz), 129.10
(s), 128.13 (dd, J = 17.1, 13.9 Hz), 118.80 (s), 116.69–116.55 (m),
116.37 (d, J = 22.5 Hz), 60.86 (s), 47.58 (s), 37.33 (s), 29.62 (s),
14.27 (s). HRMS calcd for C30H25ClFN2O5SNa, [M+Na]+, 603.1132;
found 603.1133.
(d, J = 8.0 Hz), 123.34 (s), 118.66 (s), 115.66 (s), 115.52 (s), 113.79
(s), 107.83 (s), 106.27 (s), 103.12 (s), 60.28 (s), 53.13 (s), 47.52
(s), 44.65 (s), 40.04 (s), 39.90 (s), 39.76 (s), 30.39 (s), 14.68 (s).
HRMS calcd for
604.2587.
C
35H36FN3O4Na, [M+Na]+, 604.2588; found
4.1.21. Ethyl 4-(3-((4-fluoro-N-(3-phenoxyphenyl)phenyl)sul-
fonamido)propanamido)benzoate (H16)
White solid. Yield: 72%; mp: 143.7–144.2 °C. 1H NMR
(400 MHz, CDCl3)
d 8.01 (d, J = 8.6 Hz, 2H), 7.78 (s, 1H),
7.71–7.38 (m, 4H), 7.34 (t, J = 7.9 Hz, 2H), 7.27 (d, J = 8.4 Hz,
2H), 7.16 (dd, J = 11.8, 5.1 Hz, 3H), 6.94 (d, J = 7.9 Hz, 2H), 6.81
(d, J = 7.5 Hz, 1H), 6.63 (s, 1H), 4.38 (q, J = 7.1 Hz, 2H), 3.91 (t,
J = 6.8 Hz, 2H), 2.69 (t, J = 6.8 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). 13C
NMR (101 MHz, CDCl3) d 166.15 (s), 158.13 (s), 156.05 (s),
141.72 (s), 140.09 (s), 130.78 (s), 129.92 (s), 124.06 (s), 123.24
(s), 119.28 (s), 118.60 (s), 118.34 (s), 116.48 (s), 116.25 (s),
77.37 (s), 77.05 (s), 76.73 (s), 47.47 (s), 37.15 (s), 29.72 (s),
14.36 (s). HRMS calcd for C30H27FN2O6SNa, [M+Na]+, 585.1472;
found 585.1471.
4.1.17. Ethyl 4-(3-((5-chloro-[1,10-biphenyl]-3-yl)(4-fluoroben-
zyl)amino)propanamido)benzoate (H12)
White solid. Yield: 75%; mp: 146.8–147.8 °C. 1H NMR (400 MHz,
CDCl3) d 8.01 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.49–7.35
(m, 6H), 7.21–7.16 (m, 2H), 6.99 (dd, J = 14.8, 6.1 Hz, 3H), 6.76 (d,
J = 29.4 Hz, 2H), 4.60 (s, 2H), 4.37 (d, J = 7.1 Hz, 2H), 3.91 (t,
J = 6.5 Hz, 2H), 2.74 (t, J = 6.4 Hz, 2H), 1.40 (t, J = 7.1 Hz, 3H). 13C
NMR (101 MHz, CDCl3) d 169.70 (s), 166.21 (s), 163.18 (s), 160.74
(s), 148.95 (s), 143.89 (s), 140.45 (s), 135.68 (s), 130.77 (s),
128.84 (s), 128.15 (d, J = 8.0 Hz), 127.87 (s), 127.09 (s), 118.87 (s),
115.63 (d, J = 21.5 Hz), 115.47–115.07 (m), 111.44 (s), 110.03 (s),
77.41 (s), 77.09 (s), 76.77 (s), 60.98 (s), 54.44 (s), 47.24 (s), 35.35
4.1.22. Ethyl 4-(3-((4-fluorobenzyl)(3-phenoxyphenyl)amino)
propanamido)benzoate (H17)
(s), 14.36 (s). HRMS calcd for
553.1670; found 553.1670.
C
31H27ClFN2O3Na, [M+Na]+,
White solid. Yield: 76%; mp: 133.2–133.8 °C. 1H NMR
(400 MHz, CDCl3) d 8.01 (d, J = 8.7 Hz, 2H), 7.54 (d, J = 8.4 Hz,
3H), 7.34–7.29 (m, 2H), 7.21–7.07 (m, 4H), 7.02–6.92 (m, 4H),
6.55 (s, 1H), 6.43 (s, 2H), 4.51 (s, 2H), 4.41–4.35 (m, 2H), 3.83
(t, J = 6.5 Hz, 2H), 2.70 (s, 2H), 1.41 (d, J = 7.1 Hz, 3H). 13C NMR
(101 MHz, CDCl3) d 166.16 (s), 163.14 (s), 160.70 (s), 158.57 (s),
156.91 (s), 141.85 (s), 130.79 (s), 129.68 (s), 123.24 (s), 118.87
(d, J = 9.2 Hz), 115.60 (s), 115.39 (s), 77.38 (s), 77.06 (s), 76.74
(s), 65.64 (s), 60.94 (s), 54.83 (s), 47.50 (s), 29.73 (s), 14.36 (s).
4.1.18. Ethyl 4-(3-((N-(5-chloro-[1,10-biphenyl]-3-yl)-4-fluoro-
phenyl)sulfonamido)propanamido)benzoate (H13)
White solid. Yield: 72%; mp: 143.5–144.2 °C. 1H NMR (400 MHz,
CDCl3) d 8.00 (d, J = 8.7 Hz, 2H), 7.70 (dd, J = 8.8, 4.9 Hz, 3H), 7.57
(d, J = 8.6 Hz, 2H), 7.53 (t, J = 1.6 Hz, 1H), 7.45–7.36 (m, 5H), 7.22
(t, J = 8.5 Hz, 2H), 7.16 (t, J = 1.6 Hz, 1H), 6.99 (t, J = 1.8 Hz, 1H),
4.38 (q, J = 7.1 Hz, 2H), 3.97 (t, J = 6.9 Hz, 2H), 2.74 (t, J = 6.9 Hz,
2H), 1.41 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) d 168.63
(s), 166.77 (s), 166.20 (s), 164.22 (s), 143.80 (s), 141.82 (s),
140.52 (s), 138.28 (s), 135.04 (s), 133.15 (s), 130.90–130.29 (m),
129.03 (s), 128.49 (s), 127.52–127.37 (m), 127.37–126.83 (m),
125.93 (d, J = 12.8 Hz), 118.89 (s), 116.60 (s), 116.38 (s), 77.42 (s),
77.10 (s), 76.79 (s), 60.94 (s), 53.50 (s), 47.67 (s), 37.29 (s), 29.72
HRMS calcd for
535.2001.
C
31H29FN2O4Na, [M+Na]+, 535.2001; found
4.1.23. Ethyl 4-(4-(3-((4-fluorobenzyl)(3-phenoxyphenyl)
amino)propanoyl)piperazin-1-yl)benzoate (H18)
White solid. Yield: 75%; mp: 152.7–153.9 °C. 1H NMR (600 MHz,
DMSO-d6) d 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
(s), 14.36 (s). HRMS calcd for
603.1132; found 603.1133.
C
30H25ClFN2O5SNa, [M+Na]+,
4.1.19. 3-((4-Fluorobenzyl)(4-phenoxyphenyl)amino)-1-(4-(4-
methoxyphenyl)piperazin-1-yl)propan-1-one (H14)
White solid. Yield: 78%; mp: 155.4–156.2 °C. 1H NMR (600 MHz,
CDCl3) d 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) d 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.
(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.
4.2. Biological evaluation
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 trans-
fer 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 recom-
mended to vibrate the solution on oscillator for more than 30 s
then store them at nitrogen cabinet. Dilute the stocking
4.1.20. Ethyl 4-(4-(3-((4-fluorobenzyl)(4-phenoxyphenyl)amino)-
propanoyl)piperazin-1-yl)benzoate (H15)
White solid. Yield: 74%; mp: 162.2–162.9 °C. 1H NMR (400 MHz,
DMSO-d6) d 7.79 (d, J = 9.0 Hz, 2H), 7.27 (dt, J = 8.2, 6.6 Hz, 4H),
7.14 (d, J = 8.8 Hz, 2H), 6.98 (dd, J = 11.2, 8.3 Hz, 3H), 6.86 (t,
J = 9.3 Hz, 4H), 6.71 (d, J = 9.1 Hz, 2H), 4.55 (s, 2H), 4.23 (d,
J = 7.1 Hz, 2H), 3.68 (s, 2H), 3.57 (s, 4H), 3.30 (d, J = 4.4 Hz, 4H),
2.70 (t, J = 7.0 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H). 13C NMR (151 MHz,
DMSO-d6) d 157.94 (s), 157.10 (s), 131.07 (s), 130.15 (s), 128.64
Please cite this article in press as: Xie, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.002

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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 kit
and recombinant CETP.
4.2.2. In vitro rat liver microsomal stability
Rat liver microsomes were prepared from KM rats (200–220 g)
by differential centrifugation according to the method reported by
our laboratory.25 Microsomes (final concentration: 2.0 mg protein/
mL) were mixed with compound H16 at concentrations of 100 lM
in 0.1 M potassium phosphate buffer (pH 7.4) to a total volume of
5.0 mL. The reactions were initiated by adding NADPH (1.0 mM). In
order to investigate P450 metabolism the control groups excluded
NADPH. Each incubation were performed in thrice. The mixtures
were incubated at 37 °C for 60 min with gentle shaking, 10 point
times were detected and then the reactions were quenched by
mixing with two volumes of ice-cold acetonitrile. The resulting
mixtures were vortex-mixed and centrifuged at 16,000 rpm for
10 min. The precipitated protein was washed with cold 0.05 M
Tris–HCl (pH 7.4) for three times then reconstituted with 4.0 mL
of water for LC–MS/MS analysis.
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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.
23. John, F.; Matt, L.; David, F.; Nobuko, M.; Seiji, M.; Koichi, T.; Shuji, K.; Chisa, S.;
Atsuhiro, K.; Patrick, R.; Malcdm, B.; Steve, W.; Werner, W.; Sander, V. D.; John,
J. P. K. Br. J. Clin. Pharmacol. 2014, 78, 498.
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Please cite this article in press as: Xie, H.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.03.002