Design, synthesis and biological evaluation of novel asperphenamate derivatives

Article abstract of DOI:10.1016/j.ejmech.2016.01.020

A series of novel asperphenamate derivatives were designed and synthesized, including series I (the A-phenyl group replaced with various aromatic heterocycles) and series II (the acyl group substituted by sulfonyl group). All compounds have been screened for their antiproliferative activity in vitro against MCF-7, HeLa, and BEL-7402 cell lines by the standard MTT method. Structure-activity relationship studies displayed the heterocycle type played an important role in activity. Six-membered ring derivatives displayed more potency than five-membered ring and the sulfonyl group in A-ring region made an important contribution to activity. Among all derivatives, tosyl derivative 8c exhibited the greatest potency in three human cancer cell lines. Especially in MCF-7 cells, the cellular potency of 8c was approximately 3.0-fold more potent than that of cisplatin. Firstly, the mechanism of cell death induced by 8c in MCF-7 cells was investigated. The results showed that the cell death was induced by autophagy instead of apoptosis or cell cycle arrest. Further studies indicated that 8c might induce autophagic cell death in HeLa and BEL-7402 cell lines.

Full text of DOI:10.1016/j.ejmech.2016.01.020

European Journal of Medicinal Chemistry 110 (2016) 76e86
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological evaluation of novel asperphenamate
derivatives
,
,
Qingyin Liu a 1, Wei Li b 1, Lei Sheng a, Chunyang Zou c, Hongxin Sun a, Chunfeng Zhang a,
Yang Liu a, Jiyue Shi a, Enlong Ma d *, Lei Yuan a
,
, **
a Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry of Education, Shenyang 110016, PR China
b Department of Neurology, General Hospital of Shenyang Military Command, Shenyang 110840, PR China
c Department of Pharmacy, Liaoning Vocational College of Medicine, Shenyang 110101, PR China
d Department of Pharmacology, Shenyang Pharmaceutical University, 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:
A series of novel asperphenamate derivatives were designed and synthesized, including series I (the A-
phenyl group replaced with various aromatic heterocycles) and series II (the acyl group substituted by
sulfonyl group). All compounds have been screened for their antiproliferative activity in vitro against
MCF-7, HeLa, and BEL-7402 cell lines by the standard MTT method. Structureeactivity relationship
studies displayed the heterocycle type played an important role in activity. Six-membered ring de-
rivatives displayed more potency than five-membered ring and the sulfonyl group in A-ring region made
an important contribution to activity. Among all derivatives, tosyl derivative 8c exhibited the greatest
potency in three human cancer cell lines. Especially in MCF-7 cells, the cellular potency of 8c was
approximately 3.0-fold more potent than that of cisplatin. Firstly, the mechanism of cell death induced by
8c in MCF-7 cells was investigated. The results showed that the cell death was induced by autophagy
instead of apoptosis or cell cycle arrest. Further studies indicated that 8c might induce autophagic cell
death in HeLa and BEL-7402 cell lines.
Received 25 October 2014
Received in revised form
13 January 2016
Accepted 13 January 2016
Available online 18 January 2016
Keywords:
Asperphenamate
Dipeptide analog
Synthesis
Anticancer activity
Structureeactivity relationship
Inducing autophagy
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
persistent stress or prolonged starvation can result in autophagic
cell death, or type II programmed cell death, which is distinct from
Cancer is one of the most prevalent diseases diagnosed in
developed countries and one of the leading causes of mortality
[1,2]. The development of cancer therapies over the past few de-
cades has focused principally on chemotherapy combined with
other treatment approaches such as surgery, radiotherapy, and
targeted therapy. Although a large majority of current anti-cancer
drugs exhibit their effects through the induction of apoptosis in
cancer cells, many types of cancer are resistant to proapoptotic
stimuli. Therefore, new types of drugs that display antiproliferative
effects against such apoptosis-resistant cancers are urgently
needed.
apoptotic cell death (type I programmed cell death) [5,6]. More
recently, induction of autophagy was considered as a mechanism
for some anti-cancer compounds and provided a novel target for
the development of chemotherapeutic agents [7e11].
Asperphenamate, a dipeptide analog, has an N, N’-substituted
phenylalanine-phenylalaninol ester framework. It was isolated
from raw malt, a traditional medicine for the treatment of mam-
mary hyperplasia [12]. In previous work, we found both the type
and position of substituent on A-ring displayed a remarkable in-
fluence on bioactivity and ortho-benzyloxy substituted derivative
(OBA) showed the greatest potency. It inhibited the growth of MCF-
7 cells by inducing autophagy instead of apoptosis [13]. The results
suggested that A-ring region had a significant impact on anticancer
effect of asperhenamate.
Autophagy is an important mechanism to maintain cell ho-
meostasis and promote cell survival during starvation [3,4]. But
Heterocyclic compounds occur widely in nature and are essen-
tial for life. The widespread use of them as a scaffold in medicinal
chemistry establishes this moiety as a member of the privileged
structures class [14,15]. Recently, many sulfonyl derivatives have
also been reported to display substantial anticancer activity in vitro
* Corresponding author.
** Corresponding author.
E-mail addresses: enlongma@hotmail.com (E. Ma), yuanlei3094@163.com
(L. Yuan).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2016.01.020
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

Q. Liu et al. / European Journal of Medicinal Chemistry 110 (2016) 76e86
77
and/or in vivo [16e19]. In our effort to discover and develop auto-
the presence of Et3N afforded compounds 5 and 6.
phagy regulator as potential new anticancer agents, we further
investigated the effect of the phenyl and acyl moiety in A-ring re-
gion on activity, respectively. And two series of derivatives were
designed and synthesized, including series I: the A-phenyl group
was replaced by various aromatic heterocycles; series II: the acyl
group was substituted by sulfonyl group.
In this paper, we described the synthesis of two series of de-
rivatives as well as their anti-proliferative activities in MCF-7, HeLa,
and BEL-7402 cell lines in vitro. The cell death mechanisms were
further explored in three human cancer cell lines.
2.2. Biological activity and structure-activity relationships (SAR)
All of these analogs were screened in vitro for antitumor activ-
ities against MCF-7, HeLa and BEL-7402 cells using the standard
MTT method, and cisplatin and OBA, the most potent compound in
our previous work [13], were used as the positive control. The re-
sults were presented in Table 1.
In heterocycles derivatives, almost all (S, S) and (S, R) configu-
ration compounds displayed no growth inhibitory effect. 2-Furyl
and 2-thienyl analogs were inactive with an average IC50 value
2. Results and discussion
greater than 100 mM. 1c and 2c with (R, S) configuration, 4-pyridyl
and 3-pyridyl derivatives, showed slightly more potency than
cisplatin against MCF-7 and BEL-7402 cells. The cytotoxicity of 1c
and 2c was approximately equal. Imidazolyl and 1, 2, 4-triazolyl
derivatives showed similar growth inhibitory effect. And imida-
zolyl and 1, 2, 4-triazolyl substitution type led to a three to four fold
decreased activity compared with 1c.
In sulfonyl derivatives, with the exception of mesyl derivative
with (S, R) configuration, other derivatives displayed moderate to
excellent growth inhibitory effect. The growth inhibitory activity
against three cancer cell lines for tosyl derivatives was superior to
mesyl derivatives. Tosyl derivative 8c showed the greatest potency
among tested compounds against three cell lines. The cellular po-
tency of 8c against MCF-7 and BEL-7402 cell lines was 3.0 and 1.6-fold
more potent than that of cisplatin, respectively. 8c exhibited a similar
2.1. Chemistry
The synthesis of novel asperphenamte derivatives 1e8 has been
accomplished as outlined in Scheme 1. Starting from optically pure
phenylalanine, phenylalaninol IM1 was prepared according to the
method developed by McKennon [20]. N-benzoyl-phenylalaninol
IM2 was synthesized by the method of Lewanowicz [21]. Conden-
sation of optically pure N-Boc-phenylalanine with IM2 was pro-
moted by 1, 1’-carbonyldiimidazole (CDI) to afford the key
intermediate IM3. After the removal of the Boc group by use of
1.5 mol/L HCl (g)/ethyl acetate, 1e4, 7 and 8 were directly obtained
through acylation reaction. Furthermore, the reaction of depro-
tected product with triphosgene and imidazole or 1,2,4-triazole in
Scheme 1. Reagents and conditions: (i) H2SO4, NaBH4, I2, THF, reflux, 98%; (ii) benzoyl chloride, K2CO3, MeOH, rt, 95%; (iii) N-Boc-(L or D)-phenylalanine, CDI, CHCl3, rt, 65%; (iv) (a)
1.5 N HCl/AcOEt, rt, 76%; (b) Different acyl chlorides and sulfonyl chlorides, Et3N, CH2Cl2, rt, 92e94%; (v) (a) 1.5 N HCl/AcOEt, rt, 76%; (b) Triphosgene, imidazole or 1,2,4-triazole,
Et3N, CH2Cl2, rt, 67e72%.

78
Q. Liu et al. / European Journal of Medicinal Chemistry 110 (2016) 76e86
Table 1
results indicated that 8c did not induce apoptosis or influence the
distribution of cell cycle in MCF-7 cell lines.
IC50 values of different compounds against three human cancer cell lines.
Compound
IC50(m
M)a
2.4. 8c inducing autophagy in MCF-7
MCF-7
HeLa
BEL-7402
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
4a
>100
>100
>100
Autophagy, type II programmed cell death, is a cell death
pathway distinct from apoptosis. More recently, induction of
autophagy was considered as a mechanism for some anticancer
compounds and provided a novel target for the development of
chemotherapeutic agents. For example, rapamycin derivatives CCI-
779 and RAD001 (mTOR-specific inhibitors) induced autophagy,
and inhibition of autophagy by siRNA against Beclin 1 gene atten-
uated their anti-cancer effects [23]. The treatment of breast carci-
noma cell line MCF-7 with estrogen antagonist tamoxifen caused
autophagic cell death [24].
53.61 0.76
15.26 0.35
64.75 0.8
>100
>100
16.72 0.22
>100
>100
>100
>100
>100
89.67 1.03
33.79 0.46
>100
>100
>100
24.85 0.31
>100
>100
>100
>100
64.71 0.85
19.33 0.39
>100
>100
>100
16.62 0.21
>100
>100
>100
>100
>100
>100
>100
>100
>100
Because 8c did not induce apoptosis and cell cycle arrest in MCF-
7 cells, we continued to investigate whether 8c could induce
autophagy. The formation of acidic vesicular organelles (AVOs) is a
characteristic of autophagy. To assess the development of AVOs,
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
8a
>100
>100
>100
>100
87.22 0.94
66.25 0.67
>100
>100
>100
>100
>100
79.09 0.81
72.46 0.72
>100
>100
>100
>100
>100
91.11 0.95
78.38 0.75
>100
MCF-7 cells were treated with 8c (5 and 10 mM) for 72 h. After
stained with AO and MDC [25,26], the morphological changes were
observed through fluorescence microscopy. As shown in Fig. 3A and
B, 8c could apparently increase the acid vesicles accumulation
compared with control cells. MDC fluorescent intensity was also
measured to determine AVOs by flow cytometry. As shown in
Fig. 3C, after treatment 5 and 10 mM of 8c for 72 h, MCF-7 cells
showed higher MDC associated fluorescence than that of the con-
trol cells (from 2.54% to 40.31% and 56.08%).
To exhibit a far more explicit function of 8c on autophagy of
MCF-7 cells, the conversion of LC3-I to LC3-II was investigated. It
was known that LC3-I was involved in the formation of autopha-
gosome and was incorporated into the autophagic vacuolemem-
brane. LC3-I must be conjugated to phosphatidylethanolamine
(PE). The PE-conjugated LC3-I is designated as LC3-II. The LC3-II
devise is a potential marker of activation of the autophagic
pathway [27]. Cellular extracts were prepared from cells incubated
with or without 8c for a period of 48 h and the level of both forms of
LC3 was assessed by western blot analysis. As shown in Fig. 3D, the
conversion of LC3-I to LC3-II was observed. These results indicated
that 8c was capable of inducing autophagy in MCF-7 cell lines.
>100
>100
>100
89.02 0.89
67.54 0.65
>100
76.3 0.74
59.97 0.61
21.85 0.22
>100
61.1 0.55
36.74 0.36
5.87 0.11
28.59 0.25
6.03 0.97
18.9 0.23
91.23 0.90
78.93 0.77
>100
93.85 0.86
63.95 0.62
22.11 0.23
>100
37.59 0.38
31.57 0.30
16.75 0.20
41.89 0.43
20.99 1.14
8.7 0.11
95.66 0.93
82.09 0.79
>100
70.42 0.67
52.21 0.47
37.03 0.37
>100
49.97 0.46
22.19 0.21
12.46 0.16
54.32 0.56
15.65 1.01
20.1 0.34
8b
8c
8d
OBA
Cisplatinb
Bold values show the IC50 values of the target compounds lower than the values of
the positive control.
a
The growth inhibitory effects of all compounds in MCF-7 (human breast cancer),
HeLa (human cervical cancer), BEL-7402 (human liver cancer) cell lines were
measured by MTT assay after 3 days of incubation. IC50 values were calculated using
the software of Statistical Product and Service Solutions. Data were shown as
mean SD from three independent experiments.
b
Used as a positive control.
activity to OBA against three cell lines. And 8c against MCF-7 cells
showed obvious selectivity over HeLa and BEL-7402 cells (see Fig. 1).
Taken together, the biological activity results indicated that (a)
the molecular configuration had an impact on antitumor activity;
(b) the heterocycle type played an important role in activity. Six-
membered ring displayed more potency than five-membered ring
derivatives. In pyridyl analogs, the position of nitrogen atom had no
significant influence on activity; (c) the sulfonyl group in A-ring
region made an important contribution to activity.
2.5. 8c inducing autophagy in HeLa and BEL-7402 cells
Based on above results, it was important to determine the cell
death mechanisms involved in HeLa and BEL-7402 cells. To detect
whether 8c could result in apoptosis, flow cytometry analysis was
carried out in two cell lines treated with 20
mM compound 8c
during 48 and 72 h Fig. 4A displayed that 8c did not influence the
Among the tested compounds, 8c showed the most potent
inhibitory activity against MCF-7 cells, and hence, the mechanism
of cell death induced by 8c in human breast cancer MCF-7 cells was
firstly investigated.
2.3. 8c did not induce cell cycle arrest and apoptosis in MCF-7 cells
MCF-7 cells were treated with 10 mM 8c for 24, 48 and 72 h, and
then examined by flow cytometry analysis after PI staining. The
positive control, 0.1 mM H2O2, led to a significant increase in the
SubG1 cell population (subG1 populations: 38.13%) which repre-
sents cells undergoing apoptosis [22]. Compared with it, 8c did not
influence the cell cycle progression and induce the characteristic
hypodiploid DNA content peak (subG1) (subG1 populations during
24, 48 and 72 h: 3.38%, 3.22% and 3.76%, respectively, Fig. 2). These
Fig. 1. The structure of asperphenamate and 8c.

Q. Liu et al. / European Journal of Medicinal Chemistry 110 (2016) 76e86
79
Fig. 2. Flow cytometric analysis of cell cycle distribution in MCF-7 cells treated with 10
m
M 8c for 24, 48 and 72 h. The 100
m
M of H2O2 treating for 12 h was used as a positive
control.
cell cycle progression. Meanwhile, the subG1 cell population
remained nearly unchanged compared with control (subG1 pop-
ulations during 48 and 72 h: 2.12 and 2.43% in HeLa and 2.89% in
control; 3.01 and 3.78% in BEL-7402 and 2.87% in control), indi-
cating that 8c did not promote apoptotic death or influence the
distribution of cell cycle in two cells.
In order to confirm if the growth inhibitory effects of 8c were
due to autophagy, we examined whether AVOs accumulated in
HeLa and BEL-7402 cells by AO staining assay. After treatment with
540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and were
uncorrected. High resolution accurate mass determinations (HRMS)
for all final target compounds were obtained on a Bruker Micromass
Time of Flight mass spectrophotometer equipped with electrospray
ionisation (ESI). 1H NMR and 13C NMR spectra were recorded on a
Bruker ARX-600 (600 MHz) spectrophotometer or Bruker ARX-300
(300 MHz) spectrophotometer (Bruker Bioscience, Billerica, MA,
USA), using CDCl3 and DMSO-d6 as solvent and tetramethylsilane
(TMS) as an internal standard. Unless otherwise indicated, reagents
were purchased from commercial suppliers andused without further
purification. Organic solvents were dried by standard methods when
necessary. The column chromatography was performed using silica
gel (200e300 mesh) from Qingdao Ocean Chemicals (Qingdao,
Shandong, China) with chloroform/acetone mixture as eluent.
Compounds IM1 and IM2 were synthesized in accordance with
literature procedures [20,21].
8c (20 and 40 mM) for 72 h, acidic vesicles noticeably accumulated
in HeLa and BEL-7402 cells (Fig. 4B). Further flow cytometry anal-
ysis showed the percentage of autophagic cells displayed a signif-
icant increase from 1.92% to 26.11% and 49.37% for HeLa cells;
3.94%e23.66% and 48.25% for BEL-7402, respectively (Fig. 4C). To
gain further insight in 8c inducing autophagy in two cells, we
examined the protein level of LC3-II. As shown in Fig. 4D, the LC3-I
was almost converted into LC3-II compared with control. This
observation, together with the formation of AVOs, demonstrated
that autophagy occurred in HeLa and BEL-7402 cells evidently.
4.2. General procedure for the synthesis of 2-benzamido-3-
phenylpropyl 2-(tert-butoxycarbonylamino)-3-phenylpropanoate
(IM 3a-d)
3. Conclusion
Commercially available N-tert-butoxycarbonyl-L (or )-phenyl-
D
In summary, two series of asperphenamate derivatives were
synthesized including aromatic heterocycles and sulfonyl de-
rivatives. The growth inhibitory activity in vitro for all derivatives
was evaluated. Among all compounds, 8c with tosyl moiety showed
the strongest inhibitory activity. The mechanism of cell death
induced by 8c in MCF-7, HeLa and BEL-7402 cells was further
investigated. The results showed that inhibition of growth was
attributed to the autophagy induction.
alanine (4.24 g, 16 mmol) and CDI (3.24 g, 20 mmol) in 200 mL
ethanol-free CHCl3 were stirred for 20 min at room temperature.
Then a solution of IM2 (3.98 g, 15.5 mmol) in 80 mL ethanol-free
CHCl3 was added in 4 portions at 20 min intervals and stirred for
an additional 48 h. The crude product was purified by chroma-
tography on silica gel (chloroform/acetone, 40:1) to give the key
intermediate IM 3a-d as a white solid.
4.3. Spectroscopy data of compounds IM 3a-d
4. Experimental
4.3.1. (S)-(S)-2-Benzamido-3-phenylpropyl 2-(tert-
butoxycarbonylamino)-3-phenylpropanoate tert-
butoxycarbonylamino)-3-phenylpropanoate (IM 3a)
Yield: 65.3%; M.p.: 135e137 ꢀC; ½aꢁD20 -15.1 (c 0.5,
4.1. Reagents and apparatus
All melting points were determined on a Büchi Melting Point B-

80
Q. Liu et al. / European Journal of Medicinal Chemistry 110 (2016) 76e86
Fig. 3. 8c induced autophagy in MCF-7 cells. (A) Autophagosomes observation. MCF-7 cells treated with 8c (5 and 10
observed by fluorescence microscope. (B) Autophagosomes observation. MCF-7 cells treated with 8c (5 and 10 M) for 72 h, and then the cells were stained with MDC and observed
by fluorescence microscope. (C) Autophagic cells detection. MCF-7 cells treated with 8c (5 and 10 M) for 72 h, and then the cells were stained with MDC and subjected to flow
M) for 48 h, and then the total proteins were extracted and detected by
mM) for 72 h, and then the cells were stained with AO and
m
m
cytometric analysis. (D) Western blotting analysis for autophagy. MCF-7 cells were treated with 8c (5 and 10
western blot analysis.
m
CHCl3:MeOH ¼ 1:1); 1H NMR (600 MHz, CDCl3):
d
7.16 (m, 15H,
4.09 (dd,1H, J ¼ 11.4, 3.6 Hz, 4H-b), 3.09 (dd,1H, J ¼ 13.8, 6.0 Hz,1H-
a), 3.05 (dd, 1H, J ¼ 13.8, 6.6 Hz, 1H-b), 2.95 (dd, 1H, J ¼ 13.8, 8.4 Hz,
6H-a), 2.81 (dd, 1H, J ¼ 13.8, 6.0 Hz, 6H-b), 1.38 (s, 9H, t-Bu).
ArH), 6.49 (d, 1H, J ¼ 7.8 Hz, NH), 4.99 (d, 1H, J ¼ 7.2 Hz, NH), 4.57
(m, 1H, 2-H), 4.53 (m, 1H, 5-H), 4.35 (dd, 1H, J ¼ 11.4, 9.6 Hz, 4H-a),
4.09 (dd,1H, J ¼ 11.4, 3.6 Hz, 4H-b), 3.09 (dd,1H, J ¼ 13.8, 6.0 Hz,1H-
a), 3.05 (dd, 1H, J ¼ 13.8, 6.6 Hz, 1H-b), 2.95 (dd, 1H, J ¼ 13.8, 8.4 Hz,
6H-a), 2.81 (dd, 1H, J ¼ 13.8, 6.0 Hz, 6H-b), 1.38 (s, 9H, t-Bu).
4.3.3. (R)-(S)-2-Benzamido-3-phenylpropyl 2-(tert-
butoxycarbonylamino)-3-phenylpropanoate tert-
butoxycarbonylamino)-3-phenylpropanoate (IM 3c)
Yield: 65.5%; M.p.: 133e134 ꢀC; ½aꢁD20 -8.1 (c 0.5,
4.3.2. (R)-(R)-2-Benzamido-3-phenylpropyl 2-(tert-
butoxycarbonylamino)-3-phenylpropanoate (IM 3b)
Yield: 66.1%; M.p.: 132e134 ꢀC; ½aꢁD20 14.8 (c 0.5,
CHCl3:MeOH ¼ 1:1); 1H NMR (600 MHz, CDCl3):
d 7.14 (m, 15H,
ArH), 6.49 (d, 1H, J ¼ 7.2 Hz, NH), 4.99 (d, 1H, J ¼ 6.6 Hz, NH), 4.57
(m, 1H, 2-H), 4.54 (m, 1H, 5-H), 4.22 (dd, 1H, J ¼ 11.4, 4.1 Hz, 4H-a),
4.09 (dd, 1H, J ¼ 11.4, 3.0 Hz, 4H-b), 3.07 (m, 2H, 1-H), 2.93 (dd, 1H,
J ¼ 13.8, 8.4 Hz, 6H-a), 2.77 (dd, 1H, J ¼ 13.8, 9.0 Hz, 6H-b), 1.38 (s,
CHCl3:MeOH ¼ 1:1); 1H NMR (600 MHz, CDCl3):
d 7.16 (m, 15H,
ArH), 6.49 (d, 1H, J ¼ 7.8 Hz, NH), 4.99 (d, 1H, J ¼ 7.2 Hz, NH), 4.57
(m, 1H, 2-H), 4.53 (m, 1H, 5-H), 4.35 (dd, 1H, J ¼ 11.4, 9.6 Hz, 4H-a),

Q. Liu et al. / European Journal of Medicinal Chemistry 110 (2016) 76e86
81
Fig. 4. 8c induced autophagy in HeLa and BEL-7402 cells. (A) Flow cytometric analysis of cell cycle distribution in HeLa and BEL-7402 cells treated with 20
mM 8c for 48 and 72 h,
respectively. (B) Autophagosomes observation. HeLa and BEL-7402 cells treated with 8c (20 and 40
mM) for 72 h, and then the cells were stained with AO and observed by
fluorescence microscope, respectively. (C) Autophagic cells detection. HeLa and BEL-7402 cells treated with 8c (20 and 40
m
M) for 72 h, and then the cells were stained with MDC
and subjected to flow cytometric analysis, respectively. (D) Western blotting analysis for autophagy. HeLa and BEL-7402 cells were treated with 8c (20 and 40 mM) for 48 h, and then
the total proteins were extracted and detected by western blot analysis, respectively.
9H, t-Bu).
4.5. Spectroscopy data of compounds 1e4, 7 and 8
4.5.1. (S)-(S)-2-Benzamido-3-phenylpropyl 2-isonicotinamido-3-
phenylpropanoate (1a)
4.3.4. (S)-(R)-2-Benzamido-3-phenylpropyl 2-(tert-
butoxycarbonylamino)-3-phenylpropanoate tert-
butoxycarbonylamino)-3-phenylpropanoate (IM 3d)
Yield: 64.2%; M.p.: 135e136 ꢀC; ½aꢁD20 7.8 (c 0.5,
Yield: 93.1%; M.p.: 171e175 ꢀC; ½aꢁD20 -20.3 (c 0.5
CHCl3:MeOH
C
¼
1:1); HR-MS: 530.2057 [MþNa]þ (Calcd. for
31H29N3O4Na, 530.2056); 1H NMR (300 MHz, CDCl3):
d 7.18 (m,
CHCl3:MeOH ¼ 1:1); 1H NMR (600 MHz, CDCl3):
d 7.13 (m, 15H,
19H, ArH), 6.79 (d, 1H, J ¼ 6.8 Hz, NH), 6.43 (d, 1H, J ¼ 8.3 Hz, NH),
4.94 (m, 1H, 2-H), 4.63 (m, 1H, 5-H), 4.47 (dd, 1H, J ¼ 11.2, 3.4 Hz,
4H-a), 4.05 (dd, 1H, J ¼ 11.2, 5.3 Hz, 4H-b), 3.27 (dd, 1H, J ¼ 13.8,
6.3 Hz, 1H-a), 3.17 (dd, 1H, J ¼ 13.8, 6.6 Hz, 1H-b), 2.95 (dd, 1H,
J ¼ 13.8, 6.4 Hz, 6H-a), 2.85 (dd, 1H, J ¼ 13.8, 7.8 Hz, 6H-b); 13C NMR
ArH), 6.49 (d, 1H, J ¼ 7.2 Hz, NH), 5.01 (d, 1H, J ¼ 6.6 Hz, NH), 4.57
(m, 1H, 2-H), 4.54 (m, 1H, 5-H), 4.21 (dd, 1H, J ¼ 11.4, 4.1 Hz, 4H-a),
4.12 (dd, 1H, J ¼ 11.4, 3.0 Hz, 4H-b), 3.06 (m, 2H, 1-H), 2.93 (dd, 1H,
J ¼ 13.8, 8.4 Hz, 6H-a), 2.76 (dd, 1H, J ¼ 13.8, 9.0 Hz, 6H-b), 1.38 (s,
9H, t-Bu).
(75 MHz, CDCl3) d 171.33,167.46, 166.40, 150.21 (2C), 136.78, 135.59,
131.51, 129.13 (2C), 129.06 (2C), 128.76 (2C), 128.69 (2C), 128.42
(2C), 127.33, 126.94 (2C), 126.86, 120.99 (2C), 66.81, 54.44, 50.00,
37.33, 37.17.
4.4. General procedure for the synthesis of compounds 1e4, 7 and 8
IM 3 (0.3 g, 0.6 mmol) was treated with a solution of dry hy-
drochloride gas in ethyl acetate (1.5 mol/L, 3.0 mL) for 4 h at room
temperature. The solvent was removed under reduced pressure to
give a white solid. To a solution of the deprotected product and
triethylamine (0.5 mL) in anhydrous CH2Cl2 (10 mL) was added
dropwise corresponding acyl chloride or sulfonyl chloride
(0.8 mmol). The reaction mixture was stirred overnight at room
temperature and concentrated under vacuum to give the crude
product, which was purified by chromatography on silica gel
(chloroform/acetone, 35:1) to yield 1e4, 7 and 8 as a white solid.
4.5.2. (R)-(R)-2-Benzamido-3-phenylpropyl 2-isonicotinamido-3-
phenylpropanoate (1b)
Yield: 93.4%; M.p.: 173e174 ꢀC; ½aꢁD20 21.1 (c 0.5
CHCl3:MeOH
C
¼
1:1); HR-MS: 530.2057 [MþNa]þ (Calcd. for
31H29N3O4Na, 530.2056); 1H NMR (300 MHz, CDCl3):
d 7.18 (m,
19H, ArH), 6.79 (d, 1H, J ¼ 6.8 Hz, NH), 6.43 (d, 1H, J ¼ 8.3 Hz, NH),
4.94 (m, 1H, 2-H), 4.63 (m, 1H, 5-H), 4.47 (dd, 1H, J ¼ 11.2, 3.4 Hz,
4H-a), 4.05 (dd, 1H, J ¼ 11.2, 5.3 Hz, 4H-b), 3.27 (dd, 1H, J ¼ 13.8,

82
Q. Liu et al. / European Journal of Medicinal Chemistry 110 (2016) 76e86
6.3 Hz, 1H-a), 3.17 (dd, 1H, J ¼ 13.8, 6.6 Hz, 1H-b), 2.95 (dd, 1H,
J ¼ 13.8, 6.4 Hz, 6H-a), 2.85 (dd, 1H, J ¼ 13.8, 7.8 Hz, 6H-b); 13C NMR
4.5.7. (R)-(S)-2-Benzamido-3-phenylpropyl 2-nicotinamido-3-
phenylpropanoate (2c)
(75 MHz, CDCl3)
d
171.33,167.46, 166.40, 150.21 (2C), 136.78, 135.59,
Yield: 92.8%; M.p.: 174e178 ꢀC; ½aꢁD20 -16.9 (c 0.5
131.51, 129.13 (2C), 129.06 (2C), 128.76 (2C), 128.69 (2C), 128.42
(2C), 127.33, 126.94 (2C), 126.86, 120.99 (2C), 66.81, 54.44, 50.00,
37.33, 37.17.
CHCl3:MeOH
C
¼
1:1); HR-MS: 530.2057 [MþNa]þ (Calcd. for
31H29N3O4Na, 530.2056); 1H NMR (300 MHz, CDCl3):
d 7.17 (m,
19H, ArH), 6.82 (d, 1H, J ¼ 6.8 Hz, NH), 6.30 (d, 1H, J ¼ 9.0 Hz, NH),
5.01 (m, 1H, 2-H), 4.71 (m, 1H, 5-H), 4.36 (dd, 1H, J ¼ 12.0, 6.0 Hz,
4H-a), 4.17 (dd, 1H, J ¼ 12.0, 3.0 Hz, 4H-b), 3.20 (m, 2H, 1-H), 2.90
(dd, 1H, J ¼ 13.8, 9.0 Hz, 6H-a), 2.82 (dd, 1H, J ¼ 13.8, 9.0 Hz, 6H-b);
4.5.3. (R)-(S)-2-Benzamido-3-phenylpropyl 2-isonicotinamido-3-
phenylpropanoate (1c)
13C NMR (75 MHz, DMSO-d6)
d 171.58, 166.64, 165.42, 152.18,
Yield: 93.2%; M.p.: 195e197 ꢀC; ½aꢁD20 -10.6 (c 0.5
1:1); HR-MS: 530.2057 [MþNa]þ (Calcd. for
148.52, 138.65, 137.99, 135.87, 134.80, 129.41 (2C), 129.38 (2C),
128.64 (2C), 128.58 (4C), 127.56 (2C), 126.90, 126.58, 124.01, 66.02,
54.76, 50.01, 36.88, 36.41.
CHCl3:MeOH
C
¼
31H29N3O4Na, 530.2056); 1H NMR (300 MHz, CDCl3):
d 7.15 (m,
19H, ArH), 6.82 (d, 1H, J ¼ 6.8 Hz, NH), 6.30 (d, 1H, J ¼ 9.0 Hz, NH),
5.00 (m, 1H, 2-H), 4.71 (m, 1H, 5-H), 4.36 (dd, 1H, J ¼ 12.0, 6.0 Hz,
4H-a), 4.17 (dd, 1H, J ¼ 12.0, 3.0 Hz, 4H-b), 3.20 (m, 2H, 1-H), 2.91
(dd, 1H, J ¼ 13.8, 9.0 Hz, 6H-a), 2.84 (dd, 1H, J ¼ 13.8, 9.0 Hz, 6H-b);
4.5.8. (S)-(R)-2-Benzamido-3-phenylpropyl 2-nicotinamido-3-
phenylpropanoate (2d)
Yield: 93.3%; M.p.: 174e178 ꢀC; ½aꢁD20 17.8 (c 0.5
13C NMR (75 MHz, DMSO-d6)
d 171.46, 166.62, 165.44, 150.63 (2C),
CHCl3:MeOH
C
¼
1:1); HR-MS: 530.2057 [MþNa]þ (Calcd. for
140.93, 138.64, 137.96, 134.82, 131.56, 129.41 (2C), 129.37 (2C),
128.65 (2C), 128.58 (4C), 127.56 (2C), 126.90, 126.59, 121.64 (2C),
66.04, 54.75, 50.09, 36.69, 36.32.
31H29N3O4Na, 530.2056); 1H NMR (300 MHz, CDCl3):
d 7.17 (m,
19H, ArH), 6.82 (d, 1H, J ¼ 6.8 Hz, NH), 6.30 (d, 1H, J ¼ 9.0 Hz, NH),
5.01 (m, 1H, 2-H), 4.71 (m, 1H, 5-H), 4.36 (dd, 1H, J ¼ 12.0, 6.0 Hz,
4H-a), 4.17 (dd, 1H, J ¼ 12.0, 3.0 Hz, 4H-b), 3.20 (m, 2H, 1-H), 2.90
(dd, 1H, J ¼ 13.8, 9.0 Hz, 6H-a), 2.82 (dd, 1H, J ¼ 13.8, 9.0 Hz, 6H-b);
4.5.4. (S)-(R)-2-Benzamido-3-phenylpropyl 2-isonicotinamido-3-
phenylpropanoate (1d)
13C NMR (75 MHz, DMSO-d6)
d 171.58, 166.64, 165.42, 152.18,
Yield: 93.5%; M.p.: 195e197 ꢀC; ½aꢁD20 12.0 (c 0.5
148.52, 138.65, 137.99, 135.87, 134.80, 129.41 (2C), 129.38 (2C),
128.64 (2C), 128.58 (4C), 127.56 (2C), 126.90, 126.58, 124.01, 66.02,
54.76, 50.01, 36.88, 36.41.
CHCl3:MeOH
C
¼
1:1); HR-MS: 530.2057 [MþNa]þ (Calcd. for
31H29N3O4Na, 530.2056); 1H NMR (300 MHz, CDCl3):
d 7.15 (m,
19H, ArH), 6.82 (d, 1H, J ¼ 6.8 Hz, NH), 6.30 (d, 1H, J ¼ 9.0 Hz, NH),
5.00 (m, 1H, 2-H), 4.71 (m, 1H, 5-H), 4.36 (dd, 1H, J ¼ 12.0, 6.0 Hz,
4H-a), 4.17 (dd, 1H, J ¼ 12.0, 3.0 Hz, 4H-b), 3.20 (m, 2H, 1-H), 2.91
(dd, 1H, J ¼ 13.8, 9.0 Hz, 6H-a), 2.84 (dd, 1H, J ¼ 13.8, 9.0 Hz, 6H-b);
4.5.9. (S)-(S)-2-Benzamido-3-phenylpropyl 2-(furan-2-
carboxamido)-3-phenylpropanoate (3a)
Yield: 87.2%; M.p.: 150e152 ꢀC; ½aꢁD20 -41.2 (c 0.5
13C NMR (75 MHz, DMSO-d6)
d 171.46, 166.62, 165.44, 150.63 (2C),
CHCl3:MeOH
C
¼
1:1); HR-MS: 519.1896 [MþNa]þ (Calcd. for
140.93, 138.64, 137.96, 134.82, 131.56, 129.41 (2C), 129.37 (2C),
128.65 (2C), 128.58 (4C), 127.56 (2C), 126.90, 126.59, 121.64 (2C),
66.04, 54.75, 50.09, 36.69, 36.32.
30H28N2O5Na, 519.1896); 1H NMR (600 MHz, CDCl3):
d 7.06 (m,
18H, ArH), 6.76 (d, 1H, J ¼ 6.6z, NH), 6.66 (d, 1H, J ¼ 8.4Hz, NH), 4.87
(m, 1H, 2-H), 4.59 (m, 1H, 5-H), 4.51 (dd, 1H, J ¼ 11.4, 3.0 Hz, 4H-a),
4.02 (dd,1H, J ¼ 11.4, 4.2 Hz, 4H-b), 3.25 (dd,1H, J ¼ 13.8, 6.6 Hz,1H-
a), 3.18 (dd, 1H, J ¼ 13.8, 6.6 Hz, 1H-b), 2.97 (dd, 1H, J ¼ 13.8, 6.6 Hz,
6H-a), 2.71 (dd, 1H, J ¼ 13.8, 9.0 Hz, 6H-b); 13C NMR (150 MHz,
4.5.5. (S)-(S)-2-Benzamido-3-phenylpropyl 2-nicotinamido-3-
phenylpropanoate (2a)
Yield: 93.1%; M.p.: 176e178 ꢀC; ½aꢁD20 -34.5 (c 0.5
CDCl3)
d 171.60, 167.09, 158.18, 144.41, 131.32, 129.22 (2C), 129.05
CHCl3:MeOH
C
¼
1:1); HR-MS: 530.2057 [MþNa]þ (Calcd. for
(2C), 128.78 (2C), 128.59 (2C), 128.35 (2C), 127.29, 127.09 (2C),
126.69, 115.07, 112.14, 65.26, 53.77, 50.23, 37.56, 37.15.
31H29N3O4Na, 530.2056); 1H NMR (300 MHz, CDCl3):
d 7.20 (m,
19H, ArH), 6.95 (s, 1H, NH), 6.59 (d, 1H, J ¼ 8.2 Hz, NH), 4.96 (m, 1H,
2-H), 4.64 (m,1H, 5-H), 4.48 (dd,1H, J ¼ 11.4, 3.2 Hz, 4H-a), 4.05 (dd,
1H, J ¼ 11.4, 4.9 Hz, 4H-b), 3.28 (dd, 1H, J ¼ 13.6, 6.3 Hz, 1H-a), 3.19
(dd, 1H, J ¼ 13.6, 6.8 Hz, 1H-b), 2.97 (dd, 1H, J ¼ 13.8, 6.8 Hz, 6H-a),
2.85 (dd, 1H, J ¼ 13.8, 7.8 Hz, 6H-b); 13C NMR (75 MHz, DMSO-d6)
4.5.10. (R)-(R)-2-Benzamido-3-phenylpropyl 2-(furan-2-
carboxamido)-3-phenylpropanoate (3b)
Yield: 86.5%; M.p.: 154e158 ꢀC; ½aꢁD20 40.5 (c 0.5
CHCl3:MeOH
C
¼
1:1); HR-MS: 519.1896 [MþNa]þ (Calcd. for
30H28N2O5Na, 519.1896); 1H NMR (600 MHz, CDCl3):
d 7.06 (m,
d
171.66, 166.62, 165.59, 152.52, 148.82, 138.67, 137.99, 135.46,
134.84, 131.52, 129.56, 129.43 (2C), 129.37 (2C), 128.66 (2C), 128.59
(2C), 128.56 (2C), 127.55 (2C), 126.91, 126.59, 123.83, 66.01, 54.76,
50.20, 36.67, 36.40.
18H, ArH), 6.76 (d, 1H, J ¼ 6.6 Hz, NH), 6.66 (d, 1H, J ¼ 8.4 Hz, NH),
4.87 (m, 1H, 2-H), 4.59 (m, 1H, 5-H), 4.51 (dd, 1H, J ¼ 11.4, 3.0 Hz,
4H-a), 4.02 (dd, 1H, J ¼ 11.4, 4.2 Hz, 4H-b), 3.25 (dd, 1H, J ¼ 13.8,
6.6 Hz, 1H-a), 3.18 (dd, 1H, J ¼ 13.8, 6.6 Hz, 1H-b), 2.97 (dd, 1H,
J ¼ 13.8, 6.6 Hz, 6H-a), 2.71 (dd, 1H, J ¼ 13.8, 9.0 Hz, 6H-b); 13C NMR
4.5.6. (R)-(R)-2-Benzamido-3-phenylpropyl 2-nicotinamido-3-
phenylpropanoate (2b)
(150 MHz, CDCl3)
d 171.60, 167.09, 158.18, 144.41, 131.32, 129.22
Yield: 93.2%; M.p.: 176e178 ꢀC; ½aꢁD20 33.9 (c 0.5
(2C), 129.05 (2C), 128.78 (2C), 128.59 (2C), 128.35 (2C), 127.29,
127.09 (2C), 126.69, 115.07, 112.14, 65.26, 53.77, 50.23, 37.56, 37.15.
CHCl3:MeOH
C
¼
1:1); HR-MS: 530.2057 [MþNa]þ (Calcd. for
31H29N3O4Na, 530.2056); 1H NMR (300 MHz, CDCl3):
d 7.20 (m,
19H, ArH), 6.95 (s, 1H, NH), 6.59 (d, 1H, J ¼ 8.2 Hz, NH), 4.96 (m, 1H,
2-H), 4.64 (m,1H, 5-H), 4.48 (dd,1H, J ¼ 11.4, 3.2 Hz, 4H-a), 4.05 (dd,
1H, J ¼ 11.4, 4.9 Hz, 4H-b), 3.28 (dd, 1H, J ¼ 13.6, 6.3 Hz, 1H-a), 3.19
(dd, 1H, J ¼ 13.6, 6.8 Hz, 1H-b), 2.97 (dd, 1H, J ¼ 13.8, 6.8 Hz, 6H-a),
2.85 (dd, 1H, J ¼ 13.8, 7.8 Hz, 6H-b); 13C NMR (75 MHz, DMSO-d6)
4.5.11. (R)-(S)-2-Benzamido-3-phenylpropyl 2-(furan-2-
carboxamido)-3-phenylpropanoate (3c)
Yield: 87.5%; M.p.: 168e170 ꢀC; ½aꢁD20 -19.6 (c 0.5
CHCl3:MeOH
C
¼
1:1); HR-MS: 519.1896 [MþNa]þ (Calcd. for
30H28N2O5Na, 519.1896); 1H NMR (600 MHz, CDCl3):
d 7.06 (m,
d
171.66, 166.62, 165.59, 152.52, 148.82, 138.67, 137.99, 135.46,
18H, ArH), 6.77 (d, 1H, J ¼ 6.6 Hz, NH), 6.56 (d, 1H, J ¼ 8.4 Hz, NH),
4.94 (m, 1H, 2-H), 4.57 (m, 1H, 5-H), 4.45 (dd, 1H, J ¼ 11.4, 3.0 Hz,
4H-a), 4.05 (dd, 1H, J ¼ 11.4, 4.2 Hz, 4H-b), 3.23 (m, 2H, 1-H), 2.87
(dd, 1H, J ¼ 13.8, 6.6 Hz, 6H-a), 2.70 (dd, 1H, J ¼ 13.8, 8.4 Hz, 6H-b);
134.84, 131.52, 129.56, 129.43 (2C), 129.37 (2C), 128.66 (2C), 128.59
(2C), 128.56 (2C), 127.55 (2C), 126.91, 126.59, 123.83, 66.01, 54.76,
50.20, 36.67, 36.40.

Q. Liu et al. / European Journal of Medicinal Chemistry 110 (2016) 76e86
83
13C NMR (150 MHz, CDCl3)
d
171.10, 167.00, 158.31, 147.11, 144.47,
4.5.16. (S)-(R)-2-Benzamido-3-phenylpropyl 2-(thiophene-2-
135.53, 131.45, 129.13 (2C), 128.99 (2C), 128.82 (2C), 128.57 (2C),
128.43 (2C), 127.34, 127.06 (2C), 126.69, 115.08, 112.22, 65.40, 53.50,
50.01, 37.60, 37.20.
carboxamido)-3-phenyl propanoate (4d)
Yield: 91.1%; M.p.: 158e160 ꢀC; ½aꢁD20 23.5 (c 0.5
CHCl3:MeOH
¼
1:1); HR-MS: 535.1668 [MþNa]þ (Calcd. for
C
30H28N2O4SNa, 535.1667); 1H NMR (600 MHz, CDCl3):
d 7.06 (m,
18H, ArH), 6.54 (d, 1H, J ¼ 8.4 Hz, NH), 6.46 (d, 1H, J ¼ 6.6 Hz, NH),
4.93 (m, 1H, 2-H), 4.61 (m, 1H, 5-H), 4.46 (dd, 1H, J ¼ 11.4, 3.6 Hz,
4H-a), 4.05 (dd, 1H, J ¼ 11.4, 4.2 Hz, 4H-b), 3.22 (m, 2H, 1-H), 2.87
(dd, 1H, J ¼ 13.8, 6.6 Hz, 6H-a), 2.71 (dd, 1H, J ¼ 13.8, 7.8 Hz, 6H-b);
4.5.12. (S)-(R)-2-Benzamido-3-phenylpropyl 2-(furan-2-
carboxamido)-3-phenylpropanoate (3d)
Yield: 87.7%; M.p.: 165e166 ꢀC; ½aꢁD20 19.9 (c 0.5
1:1); HR-MS: 519.1896 [MþNa]þ (Calcd. for
13C NMR (150 MHz, CDCl3)
d 171.11, 167.12, 161.93, 137.76, 136.91,
CHCl3:MeOH
C
¼
30H28N2O5Na, 519.1896); 1H NMR (600 MHz, CDCl3):
d 7.06 (m,
135.58, 134.02, 131.51, 130.75, 129.13 (2C), 129.02 (2C), 128.83 (2C),
128.68, 128.60 (2C), 128.46 (2C), 127.80, 127.36, 127.07 (2C), 126.73,
65.60, 54.04, 49.96, 37.50, 37.24.
18H, ArH), 6.77 (d, 1H, J ¼ 6.6 Hz, NH), 6.56 (d, 1H, J ¼ 8.4 Hz, NH),
4.94 (m, 1H, 2-H), 4.57 (m, 1H, 5-H), 4.45 (dd, 1H, J ¼ 11.4, 3.0 Hz,
4H-a), 4.05 (dd, 1H, J ¼ 11.4, 4.2 Hz, 4H-b), 3.23 (m, 2H, 1-H), 2.87
(dd, 1H, J ¼ 13.8, 6.6 Hz, 6H-a), 2.70 (dd, 1H, J ¼ 13.8, 8.4 Hz, 6H-b);
4.5.17. (S)-(S)-2-Benzamido-3-phenylpropyl 2-methylsulfonamido-
3-phenyl propanoate (7a)
13C NMR (150 MHz, CDCl3)
d 171.10, 167.00, 158.31, 147.11, 144.47,
Yield: 78.6%; M.p.: 186e187 ꢀC; ½aꢁD20 -26.2 (c 0.5
135.53, 131.45, 129.13 (2C), 128.99 (2C), 128.82 (2C), 128.57 (2C),
128.43 (2C), 127.34, 127.06 (2C), 126.69, 115.08, 112.22, 65.40, 53.50,
50.01, 37.60, 37.20.
CHCl3:MeOH
C
¼
1:1); HR-MS: 503.1616 [MþNa]þ (Calcd. for
26H28N2O5SNa, 503.1617); 1H NMR (600 MHz, CDCl3):
d 7.17 (m,
15H, ArH), 6.40 (d, 1H, J ¼ 8.4 Hz, NH), 4.81 (d, 1H, J ¼ 9.0 Hz, NH),
4.66 (m, 1H, 2-H), 4.36 (m, 1H, 5-H), 4.40 (dd, 1H, J ¼ 11.4, 3.6 Hz,
4H-a), 4.18 (dd, 1H, J ¼ 11.4, 4.8 Hz, 4H-b), 3.15 (dd, 1H, J ¼ 13.8,
5.4 Hz, 1H-a), 2.97 (dd, 1H, J ¼ 13.8, 7.2 Hz, 1H-b), 2.95 (dd, 1H,
J ¼ 14.4, 6.0 Hz, 6H-a), 2.71 (dd, 1H, J ¼ 14.4, 7.8 Hz, 6H-b), 2.64 (s,
4.5.13. (S)-(S)-2-Benzamido-3-phenylpropyl 2-(thiophene-2-
carboxamido)-3-phenyl propanoate (4a)
Yield: 89.7%; M.p.: 144e145 ꢀC; ½aꢁD20 -48.1 (c 0.5
CHCl3:MeOH
C
¼
1:1); HR-MS: 535.1668 [MþNa]þ (Calcd. for
3H, -CH3); 13C NMR (150 MHz, CDCl3)
d 171.56, 167.16, 136.71,
30H28N2O4SNa, 535.1667); 1H NMR (600 MHz, CDCl3):
d 7.05 (m,
135.33, 133.92, 131.59, 129.31 (2C), 129.15 (2C), 128.79 (2C), 128.70
(2C), 128.50 (2C), 127.46, 126.98 (2C), 126.88, 66.12, 57.44, 49.90,
41.37, 38.86, 37.14.
18H, ArH), 6.62 (d, 1H, J ¼ 8.4 Hz, NH), 6.43 (d, 1H, J ¼ 6.6 Hz, NH),
4.87 (m, 1H, 2-H), 4.60 (m, 1H, 5-H), 4.52 (dd, 1H, J ¼ 11.4, 3.6 Hz,
4H-a), 4.01 (dd, 1H, J ¼ 11.4, 4.2 Hz, 4H-b), 3.26 (dd, 1H, J ¼ 13.8,
6.6 Hz, 1H-a), 3.20 (dd, 1H, J ¼ 11.4, 4.2 Hz, 1H-b), 2.98 (dd, 1H,
J ¼ 13.8, 8.4 Hz, 6H-a), 2.86 (dd, 1H, J ¼ 13.8, 6.6 Hz, 6H-b). 13C NMR
4.5.18. (R)-(R)-2-Benzamido-3-phenylpropyl 2-
methylsulfonamido-3-phenyl propanoate (7b)
(150 MHz, CDCl3)
d 171.60, 167.13, 161.83, 137.60, 137.08, 135.65,
Yield: 74.6%; M.p.: 185e187 ꢀC; ½aꢁD20 25.9 (c 0.5
134.06, 131.34, 130.66, 129.21 (2C), 129.11 (2C), 128.78 (2C), 128.73,
128.60 (2C), 128.39 (2C), 127.69, 127.29, 127.05 (2C), 126.71, 65.34,
54.38, 50.18, 37.47, 37.15.
CHCl3:MeOH
C
¼
1:1); HR-MS: 503.1616 [MþNa]þ (Calcd. for
26H28N2O5SNa, 503.1617); 1H NMR (600 MHz, CDCl3):
d 7.17 (m,
15H, ArH), 6.40 (d, 1H, J ¼ 8.4 Hz, NH), 4.81 (d, 1H, J ¼ 9.0 Hz, NH),
4.66 (m, 1H, 2-H), 4.36 (m, 1H, 5-H), 4.40 (dd, 1H, J ¼ 11.4, 3.6 Hz,
4H-a), 4.18 (dd, 1H, J ¼ 11.4, 4.8 Hz, 4H-b), 3.15 (dd, 1H, J ¼ 13.8,
5.4 Hz, 1H-a), 2.97 (dd, 1H, J ¼ 13.8, 7.2 Hz, 1H-b), 2.95 (dd, 1H,
J ¼ 14.4, 6.0 Hz, 6H-a), 2.71 (dd, 1H, J ¼ 14.4, 7.8 Hz, 6H-b), 2.64 (s,
4.5.14. (R)-(R)-2-Benzamido-3-phenylpropyl 2-(thiophene-2-
carboxamido)-3-phenyl propanoate (4b)
Yield: 90.2%; M.p.: 144e147 ꢀC; ½aꢁD20 49.4 (c 0.5
3H, -CH3); 13C NMR (150 MHz, CDCl3)
d 171.56, 167.16, 136.71,
CHCl3:MeOH
¼
1:1); HR-MS: 535.1668 [MþNa]þ (Calcd. for
C30H28N2O4SNa, 535.1667); 1H NMR (600 MHz, CDCl3):
d 7.05 (m,
135.33, 133.92, 131.59, 129.31 (2C), 129.15 (2C), 128.79 (2C), 128.70
(2C), 128.50 (2C), 127.46, 126.98 (2C), 126.88, 66.12, 57.44, 49.90,
41.37, 38.86, 37.14.
18H, ArH), 6.62 (d, 1H, J ¼ 8.4 Hz, NH), 6.43 (d, 1H, J ¼ 6.6 Hz, NH),
4.87 (m, 1H, 2-H), 4.60 (m, 1H, 5-H), 4.52 (dd, 1H, J ¼ 11.4, 3.6 Hz,
4H-a), 4.01 (dd, 1H, J ¼ 11.4, 4.2 Hz, 4H-b), 3.26 (dd, 1H, J ¼ 13.8,
6.6 Hz, 1H-a), 3.20 (dd, 1H, J ¼ 11.4, 4.2 Hz, 1H-b), 2.98 (dd, 1H,
J ¼ 13.8, 8.4 Hz, 6H-a), 2.86 (dd, 1H, J ¼ 13.8, 6.6 Hz, 6H-b). 13C NMR
4.5.19. (R)-(S)-2-Benzamido-3-phenylpropyl 2-methylsulfonamido-
3-phenyl propanoate (7c)
Yield: 75.7%; M.p.: 192e195 ꢀC; ½aꢁD20 -13.1 (c 0.5
(150 MHz, CDCl3)
d 171.60, 167.13, 161.83, 137.60, 137.08, 135.65,
CHCl3:MeOH
C
¼
1:1); HR-MS: 503.1616 [MþNa]þ (Calcd. for
134.06, 131.34, 130.66, 129.21 (2C), 129.11 (2C), 128.78 (2C), 128.73,
128.60 (2C), 128.39 (2C), 127.69, 127.29, 127.05 (2C), 126.71, 65.34,
54.38, 50.18, 37.47, 37.15.
26H28N2O5SNa, 503.1617); 1H NMR (600 MHz, CDCl3):
d 7.16 (m,
15H, ArH), 6.42 (d, 1H, J ¼ 8.4 Hz, NH), 4.77 (d, 1H, J ¼ 9.6 Hz, NH),
4.39 (m, 1H, 2-H), 4.33 (m, 1H, 5-H), 4.35 (dd, 1H, J ¼ 11.4, 3.6 Hz,
4H-a), 4.17 (dd, 1H, J ¼ 11.4, 4.8 Hz, 4H-b), 3.00 (dd, 1H, J ¼ 12.0,
6.6 Hz, 1H-a), 2.96 (dd, 1H, J ¼ 12.0, 6.0 Hz, 1H-b), 3.14 (dd, 1H,
J ¼ 13.8, 5.4 Hz, 6H-a), 2.89 (dd, 1H, J ¼ 13.8, 7.8 Hz, 6H-b), 2.64 (s,
4.5.15. (R)-(S)-2-Benzamido-3-phenylpropyl 2-(thiophene-2-
carboxamido)-3-phenyl propanoate (4c)
Yield: 90.4%; M.p.: 153e154 ꢀC; ½aꢁD20 -22.7 (c 0.5
3H, -CH3); 13C NMR (150 MHz, CDCl3)
d 171.15, 167.12, 136.86,
CHCl3:MeOH
C
¼
1:1); HR-MS: 535.1668 [MþNa]þ (Calcd. for
135.44, 133.95, 131.56, 129.27 (2C), 129.18 (2C), 128.80 (2C), 128.68
(2C), 128.46 (2C), 127.47, 127.01 (2C), 126.82, 66.20, 57.59, 49.83,
41.23, 38.88, 37.28.
30H28N2O4SNa, 535.1667); 1H NMR (600 MHz, CDCl3):
d 7.06 (m,
18H, ArH), 6.54 (d, 1H, J ¼ 8.4 Hz, NH), 6.46 (d, 1H, J ¼ 6.6 Hz, NH),
4.93 (m, 1H, 2-H), 4.61 (m, 1H, 5-H), 4.46 (dd, 1H, J ¼ 11.4, 3.6 Hz,
4H-a), 4.05 (dd, 1H, J ¼ 11.4, 4.2 Hz, 4H-b), 3.22 (m, 2H, 1-H), 2.87
(dd, 1H, J ¼ 13.8, 6.6 Hz, 6H-a), 2.71 (dd, 1H, J ¼ 13.8, 7.8 Hz, 6H-b);
4.5.20. (S)-(R)-2-Benzamido-3-phenylpropyl 2-
methylsulfonamido-3-phenyl propanoate (7d)
13C NMR (150 MHz, CDCl3)
d
171.11, 167.12, 161.93, 137.76, 136.91,
Yield: 75.5%; M.p.: 195e197 ꢀC; ½aꢁD20 12.8 (c 0.5
135.58, 134.02, 131.51, 130.75, 129.13 (2C), 129.02 (2C), 128.83 (2C),
128.68, 128.60 (2C), 128.46 (2C), 127.80, 127.36, 127.07 (2C), 126.73,
65.60, 54.04, 49.96, 37.50, 37.24.
CHCl3:MeOH
C
¼
1:1); HR-MS: 503.1616 [MþNa]þ (Calcd. for
26H28N2O5SNa, 503.1617); 1H NMR (600 MHz, CDCl3):
d 7.16 (m,
15H, ArH), 6.42 (d, 1H, J ¼ 8.4 Hz, NH), 4.77 (d, 1H, J ¼ 9.6 Hz, NH),

84
Q. Liu et al. / European Journal of Medicinal Chemistry 110 (2016) 76e86
4.39 (m, 1H, 2-H), 4.33 (m, 1H, 5-H), 4.35 (dd, 1H, J ¼ 11.4, 3.6 Hz,
4H-a), 4.17 (dd, 1H, J ¼ 11.4, 4.8 Hz, 4H-b), 3.00 (dd, 1H, J ¼ 12.0,
6.6 Hz, 1H-a), 2.96 (dd, 1H, J ¼ 12.0, 6.0 Hz, 1H-b), 3.14 (dd, 1H,
J ¼ 13.8, 5.4 Hz, 6H-a), 2.89 (dd, 1H, J ¼ 13.8, 7.8 Hz, 6H-b), 2.64 (s,
137.05, 136.02, 134.87, 134.05, 131.47, 129.61 (2C), 129.20 (2C),
129.07 (2C), 128.70 (2C), 128.62 (2C), 128.40 (2C), 127.30, 127.09
(2C), 127.03 (2C), 126.73, 65.84, 57.05, 49.92, 38.83, 37.14, 21.45.
3H, -CH3); 13C NMR (150 MHz, CDCl3)
d
171.15, 167.12, 136.86,
4.6. General procedure for the synthesis of compounds 5e6
135.44, 133.95, 131.56, 129.27 (2C), 129.18 (2C), 128.80 (2C), 128.68
(2C), 128.46 (2C), 127.47, 127.01 (2C), 126.82, 66.20, 57.59, 49.83,
41.23, 38.88, 37.28.
IM 3 (0.3 g, 0.6 mmol) was treated with 1.5 mL 1.35 mol/L dry
hydrochloride gas in ethyl acetate for 4 h at room temperature. The
solvent was removed under reduced pressure to give white solid
and suspended in anhydrous CH2Cl2 (10 mL). Triethylamine
(0.4 mL) and 0.4 equivalents of triphosgene were added at 0 ꢀC.
After stirred for 15 min, various heterocycles were added. The re-
action mixture was stirred at room temperature for 24 h. Then it
was concentrated and the residue upon column chromatographic
purification using 200e300 mesh silica gel and 10% acetone-
chloroform (v/v) as eluent, yielding target compound as a white
solid.
4.5.21. (S)-(S)-2-Benzamido-3-phenylpropyl 2-(4-
methylphenylsulfonamido)-3-phenyl propanoate (8a)
Yield: 85.8%; M.p.: 143e145 ꢀC; ½aꢁD20 -45.8 (c 0.4
CHCl3:MeOH
C
¼
1:1); HR-MS: 579.1930 [MþNa]þ (Calcd. for
32H32N2O5SNa, 579.1930); 1H NMR (600 MHz, CDCl3):
d 7.04 (m,
19H, ArH), 6.50 (d, 1H, J ¼ 8.4 Hz, NH), 5.06 (d, 1H, J ¼ 9.6 Hz, NH),
4.51 (m,1H, 2-H), 4.20 (m,1H, 5-H), 4.17 (dd,1H, J ¼ 11.4, 3.6 Hz, 4H-
a), 4.05 (dd, 1H, J ¼ 11.4, 4.2 Hz, 4H-b), 3.04 (dd, 1H, J ¼ 13.8, 6.0 Hz,
1H-a), 2.95 (dd, 1H, J ¼ 13.8, 7.2 Hz, 1H-b), 2.90 (dd, 1H, J ¼ 13.8,
6.6 Hz, 6H-a), 2.75 (dd, 1H, J ¼ 13.8, 7.8 Hz, 6H-b), 2.35 (s, 3H, -CH3);
4.7. Spectroscopy data of compounds 5e6
13C NMR (150 MHz, CDCl3)
d 171.51, 166.97, 143.78, 136.95, 136.26,
134.88, 133.92, 131.56, 129.59 (2C), 129.15 (4C), 128.64 (2C), 128.62
(2C), 128.47 (2C), 127.25, 127.02 (2C), 126.99 (2C), 126.77, 65.89,
56.88, 50.19, 38.83, 36.99, 21.42.
4.7.1. (S)-(S)-2-Benzamido-3-phenylpropyl 2-(1H-imidazole-1-
carboxamido)-3-phenylpropanoate (5a)
Yield: 45.2%; M.p.: 173e174 ꢀC; ½aꢁD20 -24.5 (c 0.5
CHCl3:MeOH
¼
1:1); HR-MS: 519.2007 [MþNa]þ (Calcd. for
4.5.22. (R)-(R)-2-Benzamido-3-phenylpropyl 2-(4-
C d 8.32 (d,
29H28N4O4Na, 519.2008); 1H NMR (600 MHz, DMSO-d6):
methylphenylsulfonamido)-3-phenyl propanoate (8b)
2H, J ¼ 9.0 Hz, ArH), 7.74 (d, 2H, J ¼ 7.2 Hz, ArH), 7.40 (m, 3H, ArH),
7.15 (m, 9H, ArH), 7.08 (d, 2H, J ¼ 7.2 Hz, ArH), 6.51 (d, 2H, J ¼ 7.8 Hz,
ArH), 4.37 (m, 2H, 2-H, 5-H), 4.10 (m, 2H, 4-H), 2.93 (dd,1H, J ¼ 13.8,
5.4 Hz, 1H-a), 2.77 (m, 3H, 1H-b, 6-H); 13C NMR (150 MHz, DMSO-
Yield: 85.9%; M.p.: 145e146 ꢀC; ½aꢁD20 45.5 (c 0.4
CHCl3:MeOH
¼
1:1); HR-MS: 579.1930 [MþNa]þ (Calcd. for
C
32H32N2O5SNa, 579.1930); 1H NMR (600 MHz, CDCl3):
d 7.04 (m,
19H, ArH), 6.50 (d, 1H, J ¼ 8.4 Hz, NH), 5.06 (d, 1H, J ¼ 9.6 Hz, NH),
4.51 (m,1H, 2-H), 4.20 (m,1H, 5-H), 4.17 (dd,1H, J ¼ 11.4, 3.6 Hz, 4H-
a), 4.05 (dd, 1H, J ¼ 11.4, 4.2 Hz, 4H-b), 3.04 (dd, 1H, J ¼ 13.8, 6.0 Hz,
1H-a), 2.95 (dd, 1H, J ¼ 13.8, 7.2 Hz, 1H-b), 2.90 (dd, 1H, J ¼ 13.8,
6.6 Hz, 6H-a), 2.75 (dd, 1H, J ¼ 13.8, 7.8 Hz, 6H-b), 2.35 (s, 3H, -CH3);
d6) d 172.54, 166.58, 157.25, 138.73, 137.29, 134.83, 131.54, 129.48
(3C), 129.46 (3C), 129.04 (3C), 128.59 (3C), 128.57 (3C), 127.56 (3C),
126.87, 126.56, 65.81, 54.39, 50.16, 37.55, 36.65.
4.7.2. (R)-(R)-2-Benzamido-3-phenylpropyl 2-(1H-imidazole-1-
carboxamido)-3-phenylpropanoate (5b)
13C NMR (150 MHz, CDCl3)
d 171.51, 166.97, 143.78, 136.95, 136.26,
134.88, 133.92, 131.56, 129.59 (2C), 129.15 (4C), 128.64 (2C), 128.62
(2C), 128.47 (2C), 127.25, 127.02 (2C), 126.99 (2C), 126.77, 65.89,
56.88, 50.19, 38.83, 36.99, 21.42.
Yield: 45.7%; M.p.: 177e179 ꢀC; ½aꢁD20 24.5 (c 0.5
CHCl3:MeOH
C
¼
1:1); HR-MS: 519.2007 [MþNa]þ (Calcd. for
29H28N4O4Na, 519.2008); 1H NMR (600 MHz, DMSO-d6):
d 8.32 (d,
2H, J ¼ 9.0 Hz, ArH), 7.74 (d, 2H, J ¼ 7.2 Hz, ArH), 7.40 (m, 3H, ArH),
7.15 (m, 9H, ArH), 7.08 (d, 2H, J ¼ 7.2 Hz, ArH), 6.51 (d, 2H, J ¼ 7.8 Hz,
ArH), 4.37 (m, 2H, 2-H, 5-H), 4.10 (m, 2H, 4-H), 2.93 (dd,1H, J ¼ 13.8,
5.4 Hz, 1H-a), 2.77 (m, 3H, 1H-b, 6-H); 13C NMR (150 MHz, DMSO-
d6) d 172.54, 166.58, 157.25, 138.73, 137.29, 134.83, 131.54, 129.48
(3C), 129.46 (3C), 129.04 (3C), 128.59 (3C), 128.57 (3C), 127.56 (3C),
126.87, 126.56, 65.81, 54.39, 50.16, 37.55, 36.65.
4.5.23. (R)-(S)-2-Benzamido-3-phenylpropyl 2-(4-
methylphenylsulfonamido)-3-phenyl propanoate (8c)
Yield: 83.6%; M.p.: 150e152 ꢀC; ½aꢁD20 -23.3 (c 0.4
CHCl3:MeOH
C
¼
1:1); HR-MS: 579.1930 [MþNa]þ (Calcd. for
32H32N2O5SNa, 579.1930); 1H NMR (600 MHz, CDCl3):
d
7.02 (m,
19H, ArH), 6.57 (d, 1H, J ¼ 8.4 Hz, NH), 4.99 (d, 1H, J ¼ 9.0 Hz, NH),
4.56 (m, 1H, 2-H), 4.10 (m, 1H, 5-H), 4.32 (dd, 1H, J ¼ 11.4, 3.6 Hz,
4H-a), 3.95 (dd, 1H, J ¼ 11.4, 4.2 Hz, 4H-b), 3.03 (dd, 1H, J ¼ 13.8,
6.0 Hz, 1H-a), 2.96 (dd, 1H, J ¼ 13.8, 7.8 Hz, 1H-b), 2.94 (dd, 1H,
J ¼ 13.8, 6.6 Hz, 6H-a), 2.82 (dd, 1H, J ¼ 13.8, 7.8 Hz, 6H-b), 2.38 (s,
4.7.3. (R)-(S)-2-Benzamido-3-phenylpropyl 2-(1H-imidazole-1-
carboxamido)-3-phenylpropanoate (5c)
Yield: 44.3%; M.p.: 167e168 ꢀC; ½aꢁD20 11.7 (c 0.5
3H, -CH3); 13C NMR (150 MHz, CDCl3)
d
170.89, 167.08, 143.80,
CHCl3:MeOH
¼
1:1); HR-MS: 519.2007 [MþNa]þ (Calcd. for
137.05, 136.02, 134.87, 134.05, 131.47, 129.61 (2C), 129.20 (2C),
129.07 (2C), 128.70 (2C), 128.62 (2C), 128.40 (2C), 127.30, 127.09
(2C), 127.03 (2C), 126.73, 65.84, 57.05, 49.92, 38.83, 37.14, 21.45.
C d 8.33 (d,
29H28N4O4Na, 519.2008); 1H NMR (600 MHz, DMSO-d6):
2H, J ¼ 8.4 Hz, ArH), 7.77 (d, 2H, J ¼ 8.4 Hz, ArH), 7.41 (m, 3H, ArH),
7.09 (m, 9H, ArH), 6.56 (d, 2H, J ¼ 7.8 Hz, ArH), 4.36 (m, 2H, 2-H, 5-
H), 4.12 (dd, 1H, J ¼ 10.8, 5.4 Hz, 4H-a), 4.01 (dd, 1H, J ¼ 10.8, 6.0 Hz,
4.5.24. (S)-(R)-2-Benzamido-3-phenylpropyl 2-(4-
4H-a), 2.78 (m, 4H,1H, 6-H); 13C NMR (150 MHz, DMSO-d6)
d 171.91,
methylphenylsulfonamido)-3-phenyl propanoate (8d)
169.90, 166.58, 138.70, 137.76, 134.81, 131.57, 129.44 (3C), 129.31
(3C), 128.60 (6C), 127.57 (3C), 126.87, 126.58, 65.80, 54.12, 50.06,
36.85, 36.68.
Yield: 84.3%; M.p.: 150e151 ꢀC; ½aꢁD20 23.8 (c 0.4
CHCl3:MeOH
¼
1:1); HR-MS: 579.1930 [MþNa]þ (Calcd. for
C
32H32N2O5SNa, 579.1930); 1H NMR (600 MHz, CDCl3):
d 7.02 (m,
19H, ArH), 6.57 (d, 1H, J ¼ 8.4 Hz, NH), 4.99 (d, 1H, J ¼ 9.0 Hz, NH),
4.56 (m, 1H, 2-H), 4.10 (m, 1H, 5-H), 4.32 (dd, 1H, J ¼ 11.4, 3.6 Hz,
4H-a), 3.95 (dd, 1H, J ¼ 11.4, 4.2 Hz, 4H-b), 3.03 (dd, 1H, J ¼ 13.8,
6.0 Hz, 1H-a), 2.96 (dd, 1H, J ¼ 13.8, 7.8 Hz, 1H-b), 2.94 (dd, 1H,
J ¼ 13.8, 6.6 Hz, 6H-a), 2.82 (dd, 1H, J ¼ 13.8, 7.8 Hz, 6H-b), 2.38 (s,
4.7.4. (S)-(R)-2-Benzamido-3-phenylpropyl 2-(1H-imidazole-1-
carboxamido)-3-phenylpropanoate (5d)
Yield: 41.3%; M.p.: 165e167 ꢀC; ½aꢁD20 -12.7 (c 0.5
CHCl3:MeOH
C
¼
1:1); HR-MS: 519.2007 [MþNa]þ (Calcd. for
29H28N4O4Na, 519.2008); 1H NMR (600 MHz, DMSO-d6):
d 8.33 (d,
3H, -CH3); 13C NMR (150 MHz, CDCl3)
d
170.89, 167.08, 143.80,
2H, J ¼ 8.4 Hz, ArH), 7.77 (d, 2H, J ¼ 8.4 Hz, ArH), 7.41 (m, 3H, ArH),

Q. Liu et al. / European Journal of Medicinal Chemistry 110 (2016) 76e86
85
7.09 (m, 9H, ArH), 6.56 (d, 2H, J ¼ 7.8 Hz, ArH), 4.36 (m, 2H, 2-H, 5-
H), 4.12 (dd, 1H, J ¼ 10.8, 5.4 Hz, 4H-a), 4.01 (dd, 1H, J ¼ 10.8, 6.0 Hz,
4H-a), 2.78 (m, 4H,1H, 6-H); 13C NMR (150 MHz, DMSO-d6)
171.91,
169.90, 166.58, 138.70, 137.76, 134.81, 131.57, 129.44 (3C), 129.31
(3C), 128.60 (6C), 127.57 (3C), 126.87, 126.58, 65.80, 54.12, 50.06,
36.85, 36.68.
4.8. Cell culture
d
MCF-7 human breast cancer cells, HeLa human cervical cancer
cells and BEL-7402 human hepatic cancer cells were cultured in
RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated
fetal bovine serum (FBS), 1 mM-glutamine, 100 U/ml penicillin and
100 m
g/ml streptomycin at 37 ꢀC in 100% humidified and 5% CO2.
4.7.5. (S)-(S)-2-Benzamido-3-phenylpropyl 3-phenyl-2-(1H-
1,2,4-triazole-1-carboxamido)propanoate (6a)
4.9. Cell growth inhibition assay
Yield: 56.2%; M.p.: 154e155 ꢀC; ½aꢁD20 -21.7 (c 0.5
CHCl3:MeOH
C
¼
1:1); HR-MS: 520.1961 [MþNa]þ (Calcd. for
The growth inhibitory effects of asperphenamate analogs on
various tumor cells in vitro were measured by MTT assay. The cells
seeded in 96-well plates were treated with different concentra-
tions. DMSO (end-concentration of 0.1%) was used as a control
group. After 48 and 72 h incubation, 5.0 mg/mL MTT solution was
added, and the plates were incubated for another 4 h at 37 ꢀC. The
29H28N4O4Na, 520.1961); 1H NMR (600 MHz, CDCl3):
d 8.71 (s, 1H,
ArH), 7.91 (s, 1H, ArH), 7.66 (d, 2H, J ¼ 7.2 Hz, ArH), 7.19 (m, 13H,
ArH), 6.31 (d, 1H, J ¼ 8.4 Hz, NH), 4.85 (m, 1H, 2-H), 4.63 (m, 1H, 5-
H), 4.40 (dd, 1H, J ¼ 10.2, 4.8 Hz, 4H-a), 4.11 (dd, 1H, J ¼ 11.4, 5.4 Hz,
4H-b), 3.28 (dd, 1H, J ¼ 14.4, 6.0 Hz, 1H-a), 3.19 (dd, 1H, J ¼ 13.8,
7.2 Hz, 1H-b), 2.94 (dd, 1H, J ¼ 13.8, 6.0 Hz, 6H-a), 2.82 (dd, 1H,
purple formazan crystals were dissolved in 100 ml DMSO and the
J ¼ 13.8, 7.8 Hz, 6H-b); 13C NMR (150 MHz, CDCl3)
d
170.52, 167.12,
plates were read on an ELISA reader at 570 nm. The cell viability
was calculated as the ratio of the absorbance of the treated cells to
the absorbance of the control groups. The IC50 values were calcu-
lated using the software of Statistical Product and Service Solutions.
All experiments were performed in triplicate in three independent
experiments.
152.49, 147.25, 143.40, 136.60, 134.84, 134.11, 131.60, 129.14 (2C),
128.99 (2C), 128.90 (2C), 128.71 (2C), 128.47 (2C), 127.54, 126.90
(3C), 65.99, 54.60, 49.85, 37.68, 37.23.
4.7.6. (R)-(R)-2-Benzamido-3-phenylpropyl 3-phenyl-2-(1H-
1,2,4-triazole-1-carboxamido)propanoate (6b)
Yield: 55.6%; M.p.: 156e157 ꢀC; ½aꢁD20 21.9 (c 0.5
4.10. AO and MDC staining assay for autophagy detection
CHCl3:MeOH
C
¼
1:1); HR-MS: 520.1961 [MþNa]þ (Calcd. for
29H28N4O4Na, 520.1961); 1H NMR (600 MHz, CDCl3):
d
8.71 (s, 1H,
As a marker of autophagy, the volume of the cellular acidic
compartment can be visualized after AO staining. Three cell lines
seeded in 24-well plate were exposed to various concentrations of
8c or 0.1% DMSO for indicated times. Cells were then incubated
with medium containing 1 mg/mL AO for 15 min. After washing
with PBS, the fluorescent micrographs were taken using an inver-
ted fluorescent microscope. MDC staining was used to confirm the
abundance of autophagic vacuoles in cells. Following 8c treatment,
ArH), 7.91 (s, 1H, ArH), 7.66 (d, 2H, J ¼ 7.2 Hz, ArH), 7.19 (m, 13H,
ArH), 6.31 (d, 1H, J ¼ 8.4 Hz, NH), 4.85 (m, 1H, 2-H), 4.63 (m, 1H, 5-
H), 4.40 (dd, 1H, J ¼ 10.2, 4.8 Hz, 4H-a), 4.11 (dd, 1H, J ¼ 11.4, 5.4 Hz,
4H-b), 3.28 (dd, 1H, J ¼ 14.4, 6.0 Hz, 1H-a), 3.19 (dd, 1H, J ¼ 13.8,
7.2 Hz, 1H-b), 2.94 (dd, 1H, J ¼ 13.8, 6.0 Hz, 6H-a), 2.82 (dd, 1H,
J ¼ 13.8, 7.8 Hz, 6H-b); 13C NMR (150 MHz, CDCl3)
d 170.52, 167.12,
152.49, 147.25, 143.40, 136.60, 134.84, 134.11, 131.60, 129.14 (2C),
128.99 (2C), 128.90 (2C), 128.71 (2C), 128.47 (2C), 127.54, 126.90
(3C), 65.99, 54.60, 49.85, 37.68, 37.23.
cells were stained with 50
m
M MDC for 1 h at 37 ꢀC, and then
examined by fluorescence microscopy or analyzed on a flow
cytometry using CellQuest software.
4.7.7. (R)-(S)-2-Benzamido-3-phenylpropyl 3-phenyl-2-(1H-
1,2,4-triazole-1-carboxamido)propanoate (6c)
4.11. Western blot analysis
Yield: 55.6%; M.p.: 147e149 ꢀC; ½aꢁD20 -11.3 (c 0.5
CHCl3:MeOH
C
¼
1:1); HR-MS: 520.1961 [MþNa]þ (Calcd. for
Three cell lines (1 ꢂ 106 cells) were incubated with various
concentrations of 8c or 0.1% DMSO for indicated times. After har-
vesting by trypsinisation, the cells were washed with PBS, and
harvested by centrifugation. The pellets were resuspended in RIPA
lysis buffer [50 mM Tris-HCl, 150 mM NaCl, 0.1% sodium dodecyl
sulfate (SDS), 1% nonidet P-40, 0.5% sodium deoxycholate, 1 mM
29H28N4O4Na, 520.1961); 1H NMR (600 MHz, DMSO-d6):
d 9.07 (s,
1H, ArH), 9.03 (d, 1H, J ¼ 8.4 Hz, NH), 8.35 (d, 1H, J ¼ 8.4 Hz, NH),
8.25 (s, 1H, ArH), 7.72 (d, 2H, J ¼ 7.8 Hz, ArH), 7.16 (m, 13H, ArH),
4.68 (m, 1H, 2-H), 4.40 (m, 1H, 5-H), 4.22 (dd, 1H, J ¼ 10.8, 5.4 Hz,
4H-a), 4.11 (dd,1H, J ¼ 10.8, 5.4 Hz, 4H-b), 3.18 (m, 2H,1-H), 2.82 (m,
2H, 6-H). 13C NMR (150 MHz, DMSO-d6)
d
170.64, 166.67, 153.06,
phenylmethylsulfonyl fluoride (PMSF), 100 mM leupeptin, and 2 mg/
148.06, 144.85, 138.65, 137.73, 134.84, 131.54, 129.41 (2C), 129.40
(2C), 128.68 (2C), 128.60 (2C), 128.57 (2C), 127.56 (2C), 126.96,
126.60, 66.26, 55.10, 50.13, 36.63, 35.83.
mL aprotinin, pH 8.0] on ice for 30 min and centrifuged. After mixed
with 2 ꢂ SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
sample buffer [20% glycerol, 4% SDS, 0.04% bromphenol blue,
200 nM L-mercaptoethanol, and 100 mM Tris-HCl, (pH 6.8)]. Equal
amounts of protein extracts (10 mg) were separated by 8e15% SDS-
4.7.8. (S)-(R)-2-Benzamido-3-phenylpropyl 3-phenyl-2-(1H-
1,2,4-triazole-1-carboxamido)propanoate (6d)
PAGE, and transferred onto polyvinylidene difluoride membranes.
The membranes were treated with specified antibodies, and then
incubated with horseradish peroxidaseconjugated secondary anti-
body. Blots were detected using enhanced chemiluminescence
western blotting detection reagents. All experiments were repeated
at least three times.
Yield: 55.6%; M.p.: 147e149 ꢀC; ½aꢁD20 11.8 (c 0.5
CHCl3:MeOH
C
¼
1:1); HR-MS: 520.1961 [MþNa]þ (Calcd. for
29H28N4O4Na, 520.1961); 1H NMR (600 MHz, DMSO-d6):
d 9.07 (s,
1H, ArH), 9.03 (d, 1H, J ¼ 8.4 Hz, NH), 8.35 (d, 1H, J ¼ 8.4 Hz, NH),
8.25 (s, 1H, ArH), 7.72 (d, 2H, J ¼ 7.8 Hz, ArH), 7.16 (m, 13H, ArH),
4.68 (m, 1H, 2-H), 4.40 (m, 1H, 5-H), 4.22 (dd, 1H, J ¼ 10.8, 5.4 Hz,
4H-a), 4.11 (dd,1H, J ¼ 10.8, 5.4 Hz, 4H-b), 3.18 (m, 2H,1-H), 2.82 (m,
Acknowledgements
2H, 6-H). 13C NMR (150 MHz, DMSO-d6)
d 170.64, 166.67, 153.06,
148.06, 144.85, 138.65, 137.73, 134.84, 131.54, 129.41 (2C), 129.40
(2C), 128.68 (2C), 128.60 (2C), 128.57 (2C), 127.56 (2C), 126.96,
126.60, 66.26, 55.10, 50.13, 36.63, 35.83.
The work was supported by the National Nature Science Foun-
dation of China (81302638) and Specialized Research Fund for the
Doctoral Program of Higher Education (20112134120007).

86
Q. Liu et al. / European Journal of Medicinal Chemistry 110 (2016) 76e86
inducer, Bioorg. Med. Chem. Lett. 22 (2012) 2216e2220.
Appendix A. Supplementary data
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