Discovery of novel AHLs as potent antiproliferative agents

Article abstract of DOI:10.1016/j.ejmech.2015.02.026

Three series of novel AHL analogs were synthesized and evaluated for their in vitro cytotoxic activity against four human cancer cell lines. The SARs investigation indicated that AHLs with a terminal phenyl group, especially those with the chalcone scaffold had remarkably enhanced cytotoxicity than those with the hydrophobic side chains. Besides, some of these compounds were much more potent than 5-Fu and natural OdDHL. Through the detailed SARs discussions, we found that compounds 10a-k and 14 with the 4-amino chalcone scaffold showed excellent inhibition against all the tested cancer cell lines and were much more potent than 5-Fu and AHLs. Such scaffold may act as a template for further lead optimization. Compound 10i with a 3, 4, 5-trimethoxy group was the most potent one against all the tested cancer cell lines. Flow cytometry analysis indicated that analog 11e induced the cellular apoptosis and cell cycle arrest of MCF-7 cells at G2/M phase in a concentration-and time-dependent manner.

Full text of DOI:10.1016/j.ejmech.2015.02.026

European Journal of Medicinal Chemistry 93 (2015) 321e329
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of novel AHLs as potent antiproliferative agents
Jing-Li Ren 1, Xu-Yao Zhang 1, Bin Yu, Xi-Xin Wang, Kun-Peng Shao, Xiao-Ge Zhu,
Hong-Min Liu*
School of Pharmaceutical Sciences and New Drug Research & Development Center, Zhengzhou University, Zhengzhou 450001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three series of novel AHL analogs were synthesized and evaluated for their in vitro cytotoxic activity
against four human cancer cell lines. The SARs investigation indicated that AHLs with a terminal phenyl
group, especially those with the chalcone scaffold had remarkably enhanced cytotoxicity than those with
the hydrophobic side chains. Besides, some of these compounds were much more potent than 5-Fu and
natural OdDHL. Through the detailed SARs discussions, we found that compounds 10a-k and 14 with the
4-amino chalcone scaffold showed excellent inhibition against all the tested cancer cell lines and were
much more potent than 5-Fu and AHLs. Such scaffold may act as a template for further lead optimization.
Compound 10i with a 3, 4, 5-trimethoxy group was the most potent one against all the tested cancer cell
lines. Flow cytometry analysis indicated that analog 11e induced the cellular apoptosis and cell cycle
arrest of MCF-7 cells at G2/M phase in a concentration-and time-dependent manner.
Received 12 August 2014
Received in revised form
17 February 2015
Accepted 18 February 2015
Available online 19 February 2015
Keywords:
AHLs
Chalcones
Dithiocarbamates
Cytotoxicity
Apoptosis
© 2015 Elsevier Masson SAS. All rights reserved.
Cell cycle arrest
1. Introduction
acridine-based AHL analogs showed excellent antiproliferative ac-
tivity against human oral squamous carcinoma cell lines and even
N-Acyl homoserine lactones (AHLs) have been identified to be
responsible for the largest proportion of quorum sensing signals of
induced radiation-sensitizing effects on Ca9-22 cells and poly-
ploidy in SAS at the concentration of 21.2 M [8,9].
m
Gram negative bacteria [1]. N-(3-oxododecanoyl)-
L
-homoserine
Dithiocarbamates have received considerable attention for their
excellent biological activities and abundance in nature. The natural
Brassinin (Fig. 1) has been indentified as potent antifungal agent
and moderate inhibitor of cancer immunosuppression target
Indoleamine 2, 3-dioxygenase (IDO). The dithiocarbamate group
was found to be crucial for the cytotoxic activity [10]. Besides, the
dithiocarbamate motif has always been used as a linkage to
combine different biologically active scaffold to design new
chemical entities [11e14]. Very recently, our group have reported a
series of selective LSD1 inhibitors, which can inhibit gastric cancer
cell growth, invasion and migration (Fig. 1) [12].
lactone (OdDHL, Fig. 1), as the well-known quorum sensing signal
of Pseudomonas aeruginosa, was reported to play a critical role in
the infection caused by P. aeruginosa [2] and to be able to interfere
the eukaryotic system. Besides, numerous studies have showed
that OdDHL can induce apoptosis of human breast cancer cell lines
through ablating the activity of the signal transducer and activator
of transcription protein 3 (STAT3) with little effect on normal breast
cells and serve as rTSb mimics down-regulating thymidylate syn-
thase, inhibiting the growth of human colorectal cancer cells
(H630) and enhancing the anti-tumor activity of 5-fluorouracil,
taxol and tomudex [3,4]. In immunity, OdDHL has been proved to
be capable of stimulating the production of IL-8 and inducing
apoptosis in specific host immune cells [5,6]. OdDHL and its ana-
logs, as the anticancer agents, have been extensively studied. For
example, phenacylhomoserine lactones presented potent anti-
cancer activity and minimum quorum sensing activation [7], and
Chalcones and their derivatives have been reported to possess
various biological activities [15e22], the anticancer activity, in
particular, has received significant attention in last decades
[16e18,20,21,23]. In addition to the
a, b-unsaturated carbonyl
scaffold [20], the amino group of 40-amino chalcone was also found
as the promising site for further modifications to obtain more
biologically promising chalcone derivatives with high efficacy and
selectivity [18,21].
Inspired by the above-mentioned findings and in continuation
with our previously efforts toward finding novel anticancer agents
* Corresponding author.
E-mail address: liuhm@zzu.edu.cn (H.-M. Liu).
1
J.-L. Ren and X.-Y. Zhang contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2015.02.026
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

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J.-L. Ren et al. / European Journal of Medicinal Chemistry 93 (2015) 321e329
Fig. 1. Structures of OdDHL, Brassinin and LSD1 inhibitor previously reported.
[12e14], we herein report the identification of novel anti-
proliferative AHL analogs with the chalcone and homoserine
lactone scaffold linked by the dithiocarbamate group through
extensive SARs investigations. Besides, the promising 40-amino
chalcone scaffold with potent cytotoxicity was first discovered.
human cancer cell lines including MGC-803 (human gastric cancer
cell line), MCF-7 (human breast cancer cell line), EC-9706 (human
esophageal cancer cell line) and SMMC-7721 (human hepatocel-
lular carcinoma cell line) were determined using MTT assay [26],
and OdDHL and 5-Fluorouracil were both used as positive controls.
The SARs were fully explored by changing the substituents
attached to the dithiocarbamate group. The in vitro inhibitory ac-
tivity was summarized in Table 1. It is evident that compounds 6a-g
with hydrophobic carbon chains showed weak or no inhibition
against the tested cancer cells. Specifically, no cytotoxicity was
observed for compounds 6a-e (n < 8), compounds 6f-g with rela-
tively prolonged side chains (n ¼ 11 and 13) showed slightly
improved inhibitory activity. This indicated that the hydrophobic
chain may be not beneficial for the activity.
2. Results and discussion
2.1. Chemistry
L
-homoserine lactone hydrochloride salt 1 was prepared from -
L
methionine following the literature reported method [24]. Com-
pounds 3a-r were obtained from the corresponding anilines
through the well established acylation reaction. AHL analogs 4a-r
and 6a-g were then synthesized from compound 1 and corre-
sponding intermediates 3a-r and 2a-g via a three-component one-
pot reaction following our previously reported method [23]
(Scheme 1).
The condensation of 4-amino acetophenone and 8a-k in the
presence of NaOH efficiently gave 4'-amino chalcones 9a-k, which
then reacted with chloroacetyl chloride, generating acylated 4'-
amino chalcones 10a-k. With these compounds in hand, analogs
11a-g were efficiently synthesized via a three-component one-pot
reaction (Scheme 2). To further explore the SARs, compound 14
with an additional double bond was also synthesized from cinna-
maldehyde 12.
Compounds 4a-r with different terminal phenyl groups were
then synthesized and evaluated for their cytotoxicity. Interestingly,
most of these compounds showed improved inhibitory effect
against the tested cell lines compared to compounds 6a-g. Com-
pounds 4a-r had weak or no activity against EC-9706 and SMMC-
7721 cells. However, an increased inhibition against MGC-803
and MCF-7 cells was observed. It is worth noting that compounds
4l-n showed excellent inhibition against MCF-7 cells (IC50 ¼ 3.13,
5.46 and 5.83
OdDHL and 5-Fu (IC50 ¼ 37.31 and 12.24
m
M, respectively) and were much more potent than
M, respectively). For MCF-
m
7 cells, analogs with electron-withdrawing groups showed better
cytotoxicity than those with electron-donating groups. In partic-
ular, compounds with electron-withdrawing substituents at meta-
position of the phenyl ring contributed more to the cytotoxicity,
such as analogs 4l (3-CF3) and 4m (3-NO2). Analogs with the same
electron-withdrawing group at meta-position showed better cyto-
toxicity against MCF-7, while weaker cytotoxicity was observed
when the group was replaced at the ortho-position, like 4f (3-Cl)
>4b (4-Cl) >4h (2-Cl). Analogs substituted by halogen atoms F, Cl
and Br at the same position on the phenyl ring exerted an activity
sequence of 4n (4-F) >4b (4-Cl) >4c (4-Br). For MGC-803 cells,
compounds 4a-r showed moderated inhibition with the IC50 values
Natural OdDHL was synthesized according to the literature re-
ported method [25]. Meldrum's acid 17 was easily formed via the
reaction of malonic acid 15 with acetone 16, followed by the
coupling reaction with n-decanoic acid in the presence of DCC and
DMAP, giving compound 19, which reacted with compound 1 to
give OdDHL in the presence of Et3N (Scheme 3).
2.2. Biological evaluation
2.2.1. Cytotoxic activity
ranging from 17.88
mM to >128
mM. However, most of them were
The IC50 values (concentration required to inhibit the prolifer-
ation of cancer cells by 50%) for all obtained AHL analogs 4a-r, 6a-g,
11a-g, 14 and substituted amino chalcones 10a-k against four
much more potent than OdDHL (IC50 ¼ 101.83
m
M). From the bio-
logical data of compounds 6a-g and 4a-r against MGC-803 cells, we
can conclude that compounds with a terminal phenyl group have
better cytotoxicity than those with a hydrophobic side chain
regardless of the length.
This interesting finding promoted us to further explore the SARs
by incorporating the chalcone scaffold into our molecules (com-
pounds 11a-g). A significant enhanced cytotoxicity against the
tested cancer cell lines was observed, especially for EC-9706 cells,
compared to compounds 6a-g and 4a-r. Specifically, compounds
11e and 11g represented excellent inhibitory effect against MCF-7
cells (IC50 ¼ 5.57 and 2.67
mM, respectively) and were much more
potent than OdDHL and 5-Fu. Besides, compound 11a also had
excellent inhibition against MGC-803 cells with the IC50 value of
6.90 mM. This finding indicated that the chalcone scaffold played an
Scheme 1. Synthesis of AHL analogs 4a-r and 6a-g. Reagents and conditions: (a) CS2,
Na3PO4$12H2O, acetone, rt.

J.-L. Ren et al. / European Journal of Medicinal Chemistry 93 (2015) 321e329
323
Scheme 2. Synthesis of compounds 10a-k, 11a-g and 14. Reagents and conditions: (a) NaOH, EtOH/H2O; (b) ClCH2COCl, anhydrous K2CO3, acetone, rt; (c) 1, CS2, Na3PO4$12H2O,
acetone, rt.
Scheme 3. Synthesis of natural OdDHL. Reagents and conditions: (a) Ac2O, H2SO4, 0 ꢀC-rt. (b) DCC, DMAP, rt. (c) Et3N, CH3CN, reflux.
important role in the activity.
was monitored by flow cytometry. As shown in Fig. 2A, after
treatment for 12 h, compound 11e caused early apoptosis with the
percentage increased from 7.2% (DMSO control) to 19.0%. After 24 h,
the extent of early apoptosis increased significantly from 4.5%
(DMSO control) to 43.2% as shown in Fig. 2B. The results showed
that compound 11e effectively induced the cellular apoptosis in a
concentration-and time-dependent manner.
Based on the above findings, we further tested the cytotoxicity
of 40-amino chalcone intermediates 10a-k. To compare the activity,
compound 14 with an additional double bond was also synthesized.
To our surprise, almost all these compounds showed excellent
cytotoxic activity with the IC50 values less than 10 mM regardless of
the position and nature of the substituents on the phenyl group.
And these compounds were remarkably much more potent than 5-
Fu and OdDHL. This may explain why compounds 11a-g with a
chalcone scaffold had improved cytotoxicity. Compound 14 had a
comparable inhibition against the tested cell lines. Among them,
compound 10i with a 3, 4, 5-trimethoxy group showed the best
inhibitory effect toward the tested cancer cell lines with the IC50
values of 1.72, 1.32, 2.73 and 3.00 mM against MGC-803, MCF-7, EC-
9706 and SMMC-7721, respectively. This scaffold may serve as a
starting point for further lead optimization.
2.2.3. Cell cycle analysis
To better characterize the cytotoxic activity of analog 11e, a cell
cycle progression was performed by treating MCF-7 cells at
different concentrations of compound 11e (0, 2.0, 4.0, 8
mM).
Treatment of MCF-7 cells for 12 h, the percentage of cells at G2/M
phase increased to 33.6% for the high concentration group from
16.5% for the control group (Fig. 3A), whereas when treated for 24 h,
the proportion of cells at G2/M phase was added up to 63.1% from
16.27% (Fig. 3B). These data indicated that 11e caused an obvious
G2/M arrest in a concentration-and time-dependent manner.
OdDHL, the quorum sensing signal molecule of P. aeruginosa,
only inhibited the proliferation of MCF-7 cells moderately with an
IC50 value of 37.31 mM.
Next, we evaluated the cytotoxicity of compound 11e against
human normal cell lines GES-1 and HEK293. However, certain
cytotoxicity of compound 11e against these two normal cell lines
3. Conclusion
In conclusion, three series of novel AHL analogs were synthe-
sized and evaluated for their in vitro cytotoxic activity against four
human cancer cell lines. The SARs were fully explored by changing
the groups attached to the dithiocarbamate linker. Compounds 6a-
g with the hydrophobic side chains showed no or weak inhibition
against the tested cancer cell lines. By contrast, Compounds with
the terminal phenyl group (compounds 4a-r), especially those
(compounds 11a-g), with the 4’-amino-chalcone scaffold had
remarkably enhanced inhibition against the tested cancer cell lines,
an effect which was probably attributed to the introduction of the
biologically active chalcone scaffold. Compounds 10a-k and 14
showed excellent inhibition against all the tested cancer cell lines
and were much more potent than 5-Fu. Such scaffold may act as a
starting point for further lead optimization. Compound 10i with a 3,
was observed with IC50 values of 23.49 1.41 and 21.12 1.00
respectively.
mM,
2.2.2. Apoptosis assay
Analog 11e was chosen to further explore the mechanism of
action for its excellent cytotoxic activity against human breast
cancer cell line MCF-7. A biparametric cytofluorimetric analysis
using propidium iodide (PI) and annexin-V-FITC in MCF-7 cells was
performed to characterize the mode of cell death induced by
compound 11e. After treatment with compound 11e at different
concentrations (0, 2.0, 4.0, 8.0 mM) for 12 h and 24 h, respectively,
MCF-7 cells were labeled with the two dyes, and the resulting red
(in web version) (PI) and green (in web version) (FITC) fluorescence

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J.-L. Ren et al. / European Journal of Medicinal Chemistry 93 (2015) 321e329
Table 1
In vitro cytotoxic activity against four human cancer cell lines.
Compound
R/n
IC50 (m
M)a
MGC-803
MCF-7
EC-9706
SMMC-7721
6a
6b
6c
6d
6e
6f
6g
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
11a
11b
11c
11d
11e
11f
11g
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
14
n ¼ 0
n ¼ 1
n ¼ 3
n ¼ 5
n ¼ 7
n ¼ 11
n ¼ 13
2-F
4-Cl
4-Br
2,3-dimethyl
3-CH3
3-Cl
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
65.05 6.28
50.28 3.96
56.79 2.87
>128
>128
>128
>128
33.28 4.01
36.60 3.15
122.19 4.88
76.92 4.13
42.78 7.73
ndb
61.09 2.42
30.46 2.03
28.61 3.02
50.34 3.63
66.48 5.83
>128
63.23 4.04
46.55 3.49
>128
90.80 4.33
33.52 2.59
29.23 2.80
>128
3.13 0.20
5.46 0.81
5.83 0.86
>128
58.30 6.06
66.18 3.55
>128
>128
67.43 2.13
>128
>128
106.63 4.51
>128
>128
93.87 7.42
>128
>128
79.67 3.91
>128
67.11 4.09
>128
>128
112.28 7.39
44.24 4.34
38.91 2.67
44.90 2.94
91.20 3.25
46.11 3.45
34.06 2.54
32.54 2.50
>128
8.34 1.33
6.37 0.94
10.11 1.54
8.60 1.38
6.98 1.10
5.56 0.80
3.67 0.30
5.97 0.90
2.73 0.07
7.15 1.12
4.63 0.57
10.73 1.67
>128
>128
28.31 2.25
42.15 2.59
>128
>128
>128
49.90 2.83
32.72 3.86
>128
>128
>128
>128
>128
>128
>128
64.87 2.61
27.34 2.73
>128
>128
18.04 1.94
>128
36.60 3.31
21.77 2.54
39.69 3.51
31.95 2.90
44.34 2.43
48.52 3.30
62.06 3.89
17.88 2.19
58.86 3.72
37.61 3.32
80.02 2.69
99.44 2.36
21.72 1.73
20.52 2.20
6.90 1.08
24.76 2.36
19.64 1.94
45.69 2.88
12.35 1.62
15.63 1.83
29.35 2.38
4.96 0.66
6.94 1.06
6.05 0.95
4.68 0.54
2.75 0.85
2.01 1.27
2.35 0.27
2.91 0.68
1.72 0.44
3.60 0.64
3.19 0.08
5.97 0.59
101.83 4.98
10.72 1.51
H
2-Cl
4-CH3
2,4-dichloro
4-OCH3
3-CF3
3-NO2
4-F
4-OH
4-COCH3
4-CO2C2H5
4-C4H9
4-Cl
H
4-Br
4-F
2-Cl
3-Cl
4-NO2
4-Cl
H
4-Br
>128
60.25 2.04
40.17 4.35
20.25 2.07
44.31 2.93
18.71 1.91
83.69 4.21
5.57 0.89
11.62 1.56
2.67 0.25
4.10 0.41
2.48 0.43
4.64 0.65
5.47 0.76
5.26 0.73
5.34 0.75
3.91 0.38
3.02 0.003
1.32 0.74
9.18 1.46
2.29 0.34
4.59 0.53
37.31 3.51
12.24 0.42
20.44 2.08
39.09 0.91
>128
8.16 1.30
12.56 1.92
13.34 1.86
15.07 2.14
11.12 1.71
7.76 1.24
9.06 1.72
10.33 1.62
3.00 0.17
22.92 2.73
5.96 0.90
36.73 3.31
>128
4-F
2-Cl
3-Cl
4-NO2
3-OCH3
3, 4, 5-trimethoxyl
4-CH3
3, 4-difluoro
e
OdDHL
5-Fu
e
e
15.67 0.85
20.40 1.31
a
Inhibitory activity was assayed by exposure for 72 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). Data are
presented as the means SDs of three independent experiments,
m
M ¼
mmol/L.
b
Not detected.
4, 5-trimethoxy group was the most potent one against all the
tested cancer cell lines. Further exploration indicated that analog
11e induced the cellular apoptosis and cell cycle arrest of MCF-7
cells at G2/M phase in a concentration-and time-dependent
manner.
Micromass spectrometer. Melting points were determined on an
electro thermal melting point apparatus and reported uncorrected.
4.2. Procedure for the synthesis of intermediates 3n-r
4. Experimental section
Compounds 3a-m were synthesized previously in our group
[23], so only the spectra data of compounds 3n-r were given below.
To the mixture of corresponding amine (4.2 mmol) and K2CO3
(0.691 g, 5.0 mmol) in acetone was added chloroacetyl chloride
(0.565 g, 5.0 mmol) dropwise. The reaction mixture was stirred at
room temperature for 1e1.5 h. Upon completion of the reaction, the
mixture was poured into 20 mL of ice water. The precipitate formed
was filtrated and washed with water. The product was dried under
vacuum to obtain compounds 3n-r, which were pure enough
without further purification.
4.1. General
All commercial reagents were used without further purification.
The reaction process was monitored by TLC with silica gel plates.
The target analogs were purified by column chromatography with
silica gel (200-300 meshes). The structures reported were identi-
fied by 1H NMR and 13C NMR (400 MHz and 100 MHz) in DMSO-d6
or CDCl3 with TMS as internal standard, high resolution mass
spectra (HRMS) were recorded on a Waters Micromass Q-T of

J.-L. Ren et al. / European Journal of Medicinal Chemistry 93 (2015) 321e329
325
Fig. 2. Apoptotic effect on human MCF-7 cell line induced by compound 11e. Apoptotic cells were detected with Annexin V-FITC/PI double staining after incubation with compound
11e (0, 2, 4, 8 M) for 12 h or 24 h. (A) Incubated for 12 h; (B) incubated for 24 h. The lower left quadrants represent live cells, the lower right quadrants are for early/primary
m
apoptotic cells, upper right quadrants are for late/secondary apoptotic cells, while the upper left quadrants represent cells damaged during the procedure. The experiments were
performed three times, and a representative experiment is shown.
4.2.1. 2-Chloro-N-(4-fluorophenyl) acetamide (3n)
ppm): 10.06 (s, 1H), 9.30 (s, 1H), 7.37 (d, J ¼ 8.9 Hz, 2H), 6.72 (d,
Yield 67%, gray solid; 1H NMR (400 MHz, CDCl3,
d
, ppm): 8.23 (s,
J ¼ 8.9 Hz, 2H), 4.19 (s, 2H); 13C NMR (100 MHz, DMSO-d6,
d, ppm):
1H), 7.71e7.36 (m, 2H), 7.16e6.83 (t, J ¼ 8.6 Hz, 2H), 4.19 (s, 2H); 13
C
164.41, 154.26, 130.51, 121.65, 115.65, 43.97.
NMR (100 MHz, CDCl3,
d, ppm): 163.84, 161.14, 158.71, 132.67,
132.64, 122.11, 122.03, 115.99, 115.76, 42.81.
4.2.3. N-(4-acetylphenyl)-2-chloroacetamide (3p)
Yield 73%, yellow solid; 1H NMR (400 MHz, CDCl3,
d
, ppm): 8.49
(s, 1H), 7.99 (d, J ¼ 8.7 Hz, 2H), 7.70 (d, J ¼ 8.7 Hz, 2H), 4.23 (s, 2H),
2.61 (s, 3H); 13C NMR (100 MHz, CDCl3,
, ppm): 196.91, 164.11,
4.2.2. 2-Chloro-N-(4-hydroxyphenyl) acetamide (3o)
Yield 71%, adobe brown solid; 1H NMR (400 MHz, DMSO-d6,
d
,
d
Fig. 3. Effect of compound 11e on the cell cycle distribution of MCF-7 cells. Cells were treated with different concentrations (0, 2, 4, 8 mM) for 12 h or 24 h. Then the cells were fixed
and stained with PI to analyze DNA content by flow cytometry. (A) Incubated for 12 h; (B) incubated for 24 h. The experiments were performed three times, and a representative
experiment is shown.

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J.-L. Ren et al. / European Journal of Medicinal Chemistry 93 (2015) 321e329
140.97, 133.74, 129.76, 119.31, 42.89, 26.49.
4.3.4. (S)-ethyl 4-(2-(((2-oxotetrahydrofuran-3-yl) carbamothioyl)
thio) acetamido) benzoate (4q)
Yield 60%, dark yellow solid, m.p.: 141e142 ꢀC; 1H NMR
4.2.4. Ethyl 4-(2-chloroacetamido) benzoate (3q)
Yield 68%, white solid; 1H NMR (400 MHz, CDCl3,
d
, ppm): 8.44
(400 MHz, DMSO-d6,
d
, ppm): 10.62 (s, 1H), 10.47 (d, J ¼ 7.9 Hz, 1H),
7.92 (d, J ¼ 8.8 Hz, 2H), 7.72 (d, J ¼ 8.8 Hz, 2H), 5.51 (dt, J ¼ 10.9,
8.6 Hz, 1H), 4.40 (t, J ¼ 8.3 Hz, 1H), 4.35e4.27 (m, 1H), 4.26 (s, 2H),
2.65e2.53 (m, 1H), 2.50 (m, 2H), 2.30e2.13 (m, 1H), 1.31 (t,
(s, 1H), 8.04 (d, J ¼ 8.7 Hz, 2H), 7.65 (d, J ¼ 8.8 Hz, 2H), 4.37 (q,
J ¼ 7.1 Hz, 2H), 4.21 (s, 2H), 1.39 (t, J ¼ 7.1 Hz, 3H); 13C NMR
(100 MHz, CDCl3, d, ppm): 165.99, 164.08, 140.71, 130.83, 126.96,
J ¼ 7.1 Hz, 3H); 13C NMR (100 MHz, DMSO-d6,
d, ppm): 198.01,
119.17, 61.03, 42.88, 14.33.
174.04, 166.59, 165.75, 143.71, 130.77, 124.82, 118.91, 65.98, 60.92,
55.02, 40.18, 28.36, 14.68; HRMS (ESI) Calcd for C16H18N2NaO5S2
[MþNa]þ: 405.0555, found: 405.0552.
4.2.5. N-(4-butylphenyl)-2-chloroacetamide (3r)
Yield 69%, light blue solid; 1H NMR (400 MHz, CDCl3,
d, ppm):
8.21 (s, 1H), 7.43 (d, J ¼ 8.4 Hz, 2H), 7.16 (d, J ¼ 8.4 Hz, 2H), 4.17 (s,
4.3.5. (S)-2-((4-butylphenyl) amino)-2-oxoethyl (2-
oxotetrahydrofuran-3-yl) carbamodithioate (4r)
2H), 2.59 (t, J ¼ 7.8 Hz, 2H), 1.67e1.49 (m, 2H), 1.48e1.23 (m, 2H),
0.92 (t, J ¼ 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3,
d, ppm): 162.75,
139.08, 133.23, 127.99, 119.23, 41.86, 34.05, 32.56, 21.24, 12.90.
Yield 71%, yellow solid, m.p.: 69-70108-109 ꢀC; 1H NMR
(400 MHz, DMSO-d6,
d
, ppm): 10.43 (d, J ¼ 7.0 Hz, 1H), 10.17 (s, 1H),
7.45 (d, J ¼ 8.4 Hz, 2H), 7.09 (d, J ¼ 8.3 Hz, 2H), 5.49 (dt, J ¼ 11.1,
8.7 Hz, 1H), 4.37 (t, J ¼ 8.6 Hz, 1H), 4.31e4.22 (m, 1H), 4.17 (s, 2H),
2.61e2.52 (m, 1H), 2.51e2.47 (m, 2H), 2.26e2.15 (m, 1H), 1.57e1.39
(m, 2H), 1.36e1.17 (m, 2H), 0.85 (t, J ¼ 7.3 Hz, 3H); 13C NMR
4.3. Procedure for the synthesis of analogs 4n-r
Compounds 4a-m were synthesized previously in our group
[23], so only the spectra data of compounds 4n-r were given below.
(100 MHz, DMSO-d6, d, ppm): 198.10, 174.06, 165.71, 137.86, 137.06,
To the mixture of compound
1
(0.4 g, 2.91 mmol) and
128.95, 119.59, 65.98, 54.97, 40.13, 34.70, 33.68, 28.37, 22.15, 14.26;
HRMS (ESI) Calcd for C17H22N2NaO3S2 [MþNa]þ: 389.0970, found:
389.0972.
Na3PO4$12H2O (1.104 g, 2.91 mmol) in acetone (10 mL) was added
CS2 (0.22 mL, 3.64 mmol). 30 min later, acylated aniline (3n-r,
2.91 mmol) was added to the mixture slowly, the mixture was
stirred at room temperature for another 0.5 h. Upon completion
(monitored by TLC), the solvent was removed under reduced
pressure, the residue was extracted with dichloromethane, washed
with water, brine, dried over anhydrous Na2SO4 and concentrated
under reduced pressure to give the residue, which was purified
using column chromatography to obtain analogs 4n-r.
4.4. Procedure for the synthesis of compounds 10a-k and 14
The solution of compounds 7 (0.5 g, 3.699 mmol) and 8a-k or 12
(3.792 mmol) in EtOH (25 mL) was added to the aqueous NaOH
solution (0.185 g NaOH, 25 mL H2O) dropwise. The reaction mixture
was stirred at room temperature for 4e12 h. Upon completion of
the reaction, the mixture was poured into 25 mL ice water, filtrated
and washed with water and dried over MgSO4 to obtain com-
pounds 9a-k and 13 without further purification. To the mixture of
9a-k or 13 (3.399 mmol) and K2CO3 (0.564 g, 4.079 mmol) in
acetone (10 mL) was added chloroacetyl chloride (0.461 g,
4.079 mmol) dropwise. The resulting solution was stirred at room
temperature for 1e1.5 h. Upon completion, the mixture was poured
into 20 mL of ice water. The precipitate formed was filtrated,
washed with water and dried under vacuum to obtain 10a-k and 14
without further purification.
4.3.1. (S)-2-((4-fluorophenyl) amino)-2-oxoethyl (2-
oxotetrahydrofuran-3-yl)carbamodithioate (4n)
Yield 69%, yellow solid, m.p.: 137e138 ꢀC; 1H NMR (400 MHz,
DMSO-d6,
d
, ppm): 10.47 (d, J ¼ 7.9 Hz, 1H), 10.35 (s, 1H), 7.68e7.55
(m, 2H), 7.21e7.10 (m, 2H), 5.52 (dt, J ¼ 11.1, 8.7 Hz, 1H), 4.41 (t,
J ¼ 8.3 Hz, 1H), 4.35e4.24 (m, 1H), 4.21 (s, 2H), 2.64e2.53 (m, 1H),
2.28e2.18 (m, 1H); 13C NMR (100 MHz, DMSO-d6,
d, ppm): 198.06,
174.06, 165.89, 159.70, 157.32, 135.80, 135.78, 121.35, 121.27, 115.94,
115.72, 65.98, 54.98, 40.02, 28.36; HRMS (ESI) Calcd for
C
13H13FN2NaO3S2 [MþNa]þ: 351.0249, found: 351.0251.
4.4.1. (E)-2-chloro-N-(4-(3-(4-chlorophenyl) acryloyl) phenyl)
acetamide (10a)
Yield 77%, yellow solid, m.p.: 199e200 ꢀC; 1H NMR (400 MHz,
4.3.2. (S)-2-((4-hydroxyphenyl) amino)-2-oxoethyl (2-
oxotetrahydrofuran-3-yl) carbamodithioate (4o)
CDCl3,
d
, ppm): 8.43 (s, 1H), 8.06 (d, J ¼ 8.7 Hz, 2H), 7.77 (d,
Yield 64%, brown solid, m.p.: 86e88 ꢀC; 1H NMR (400 MHz,
J ¼ 15.7 Hz, 1H), 7.73 (d, J ¼ 8.7 Hz, 2H), 7.58 (d, J ¼ 8.5 Hz, 2H), 7.50
(d, J ¼ 15.7 Hz, 1H), 7.40 (d, J ¼ 8.5 Hz, 2H), 4.23 (s, 2H); 13C NMR
DMSO-d6,
d
, ppm): 10.46 (d, J ¼ 8.0 Hz, 1H), 10.03 (s, 1H), 9.22 (s,
1H), 7.35 (d, J ¼ 8.9 Hz, 2H), 6.69 (d, J ¼ 8.9 Hz, 2H), 5.51 (dt, J ¼ 11.2,
8.7 Hz, 1H), 4.40 (t, J ¼ 8.3 Hz, 1H), 4.33e4.27 (m, 1H), 4.15 (s, 2H),
2.67e2.52 (m, 1H), 2.33e2.13 (m, 1H); 13C NMR (100 MHz, DMSO-
(100 MHz, CDCl3, d, ppm): 188.60, 164.02, 143.32, 140.81, 136.50,
134.57, 133.37, 129.98, 129.62, 129.28, 122.07, 119.41, 42.88; HRMS
(ESI) Calcd for C17H23N2O3S2 [MþH]þ: 334.0402, found: 334.0397.
d6, d, ppm): 198.12, 174.06, 165.24, 153.95, 131.02, 121.35, 115.56,
65.98, 54.94, 40.19, 28.37; HRMS (ESI) Calcd for C13H14N2NaO4S2
4.4.2. (E)-2-chloro-N-(4-cinnamoylphenyl) acetamide (10b)
Yield 72%, yellow solid, m.p.: 157e158 ꢀC; 1H NMR (400 MHz,
[MþNa]þ: 349.0293, found: 349.0296.
CDCl3,
d
, ppm): 8.47 (s, 1H), 8.06 (d, J ¼ 8.7 Hz, 2H), 7.82 (d,
4.3.3. (S)-2-((4-acetylphenyl) amino)-2-oxoethyl (2-
oxotetrahydrofuran-3-yl) carbamodithioate (4p)
J ¼ 15.7 Hz, 1H), 7.73 (d, J ¼ 8.7 Hz, 2H), 7.65 (dd, J ¼ 6.7, 2.8 Hz, 2H),
7.53 (d, J ¼ 15.7 Hz, 1H), 7.42 (dd, J ¼ 4.9, 1.7 Hz, 3H), 4.22 (s, 2H). 13
C
Yield 70%, yellow solid, m.p.: 157e158 ꢀC; 1H NMR (400 MHz,
NMR (100 MHz, CDCl3, d, ppm): 188.97, 164.06, 144.87, 140.75,
DMSO-d6,
d
, ppm): 10.64 (s, 1H), 10.49 (d, J ¼ 8.0 Hz, 1H), 7.94 (d,
134.86, 134.73, 130.61, 129.97, 128.99, 128.48, 121.68, 119.41, 42.90;
HRMS (ESI) Calcd for C17H14ClNNaO2 [MþNa]þ: 322.0611, found:
322.0614.
J ¼ 8.8 Hz, 2H), 7.73 (d, J ¼ 8.8 Hz, 2H), 5.53 (dt, J ¼ 11.1, 8.7 Hz, 1H),
4.41 (t, J ¼ 8.4 Hz, 1H), 4.34e4.31 (m, 1H), 4.28 (s, 2H), 2.67e2.55
(m, 1H), 2.53 (s, 3H), 2.36e2.15 (m, 1H); 13C NMR (100 MHz, DMSO-
d6,
d
, ppm): 198.02, 196.98, 174.05, 166.60, 143.70, 132.29, 130.01,
4.4.3. (E)-N-(4-(3-(4-bromophenyl)acryloyl)phenyl)-2-
chloroacetamide (10c)
118.79, 65.99, 55.03, 40.18, 28.37, 26.91; HRMS (ESI) Calcd for
C
15H16N2NaO4S2 [MþNa]þ: 375.0449, found: 375.0452.
Yield 75%, yellow solid, m.p.: 208e210 ꢀC; 1H NMR (400 MHz,

J.-L. Ren et al. / European Journal of Medicinal Chemistry 93 (2015) 321e329
327
DMSO-d6,
d
, ppm): 10.70 (s, 1H), 8.19 (d, J ¼ 8.6 Hz, 2H), 7.99 (d,
6H), 3.91 (s, 3H); 13C NMR (100 MHz, CDCl3,
d
, ppm): 189.03, 164.13,
J ¼ 15.6 Hz, 1H), 7.86 (d, J ¼ 8.3 Hz, 2H), 7.80 (d, J ¼ 8.8 Hz, 2H), 7.70
153.51, 145.07, 140.74, 140.53, 134.77, 130.34, 129.94, 121.08, 119.44,
105.75, 61.02, 56.27, 42.91; HRMS (ESI) Calcd for C20H21ClNO5
[MþH]þ: 390.1108, found: 390.1111.
(d, J ¼ 16.0 Hz, 1H), 7.67 (d, J ¼ 8.8 Hz, 2H), 4.34 (s, 2H); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 187.95, 165.71, 143.42, 142.59, 134.55,
133.18, 132.34, 131.20, 130.49, 124.34, 123.22, 119.23, 44.10; HRMS
(ESI) Calcd for C17H14BrClNO2 [MþH]þ: 377.9896, found: 377.9897.
4.4.10. (E)-2-chloro-N-(4-(3-(p-tolyl)acryloyl)phenyl)acetamide
(10j)
4.4.4. (E)-2-chloro-N-(4-(3-(4-fluorophenyl)acryloyl)phenyl)
acetamide (10d)
Yield 69%, yellow solid, m.p.: 185e187 ꢀC; 1H NMR (400 MHz,
DMSO-d6,
d
, ppm): 10.68 (s, 1H), 8.18 (d, J ¼ 8.7 Hz, 2H), 7.89 (d,
Yield 70%, yellow solid, m.p.: 162e164 ꢀC; 1H NMR (400 MHz,
J ¼ 15.6 Hz, 1H), 7.80 (d, J ¼ 2.8 Hz, 2H), 7.77 (d, J ¼ 2.2 Hz, 2H), 7.71
DMSO-d6)
d
10.72 (s, 1H), 8.19 (d, J ¼ 8.7 Hz, 2H), 7.98 (dd, J ¼ 7.6,
(d, J ¼ 15.6 Hz, 1H), 7.28 (d, J ¼ 8.0 Hz, 2H), 4.33 (s, 2H), 2.36 (s, 3H);
5.9 Hz, 2H), 7.92 (d, J ¼ 15.7 Hz, 1H), 7.80 (d, J ¼ 8.7 Hz, 2H), 7.74 (d,
13C NMR (100 MHz, DMSO-d6,
d, ppm): 188.03, 165.69, 144.06,
J ¼ 15.6 Hz, 1H), 7.31 (t, J ¼ 8.3 Hz, 2H), 4.34 (s, 2H); 13C NMR
143.25, 141.07, 133.41, 132.52, 130.37, 130.01, 129.33, 121.35, 119.23,
44.10, 21.56; HRMS (ESI) Calcd for C18H17ClNO2 [MþH]þ: 314.0948,
found: 314.0950.
(100 MHz, DMSO-d6) d 187.98,165.71, 165.08, 162.60, 143.35, 142.74,
133.27, 131.94, 131.91, 131.68, 131.60, 130.43, 122.32, 119.23, 116.50,
116.28, 44.09; HRMS (ESI) Calcd for
318.0697, found: 318.0699.
C
17H14ClFNO2 [MþH]þ:
4.4.11. (E)-2-chloro-N-(4-(3-(3, 4-difluorophenyl)acryloyl)phenyl)
acetamide (10k)
4.4.5. (E)-2-chloro-N-(4-(3-(2-chlorophenyl)acryloyl)phenyl)
acetamide (10e)
Yield 57%, yellow solid, m.p.: 187e189 ꢀC; 1H NMR (400 MHz,
Yield 65%, yellow solid, m.p.: 167e168 ꢀC; 1H NMR (400 MHz,
DMSO-d6,
d
, ppm): 10.70 (s, 1H), 8.29 (s, 1H), 8.21 (d, J ¼ 8.7 Hz, 2H),
8.06 (d, J ¼ 15.6 Hz, 1H), 7.88 (d, J ¼ 8.4 Hz, 1H), 7.79 (d, J ¼ 8.8 Hz,
DMSO-d6,
J ¼ 5.0 Hz, 2H), 7.81 (d, J ¼ 8.8 Hz, 2H), 7.63e7.55 (m, 1H), 7.52e7.42
(m, 2H), 4.34 (s, 2H). 13C NMR (100 MHz, DMSO-d6,
, ppm): 187.84,
d, ppm): 10.71 (s,1H), 8.21 (dd, J ¼ 9.1, 5.1 Hz, 3H), 8.02 (d,
2H), 7.73 (d, J ¼ 8.6 Hz, 1H), 7.70 (d, J ¼ 15.7 Hz, 1H), 4.33 (s, 2H); 13
C
NMR (100 MHz, DMSO-d6, d, ppm): 187.81, 165.73, 143.53, 141.16,
d
136.15, 133.09, 133.04, 132.29, 131.46, 130.60, 130.56, 129.59, 124.49,
165.74, 143.56, 138.53, 134.79, 132.99, 132.85, 132.37, 130.60, 130.49,
129.02, 128.15, 125.17, 119.26, 44.10; HRMS (ESI) Calcd for
119.22, 44.10; HRMS (ESI) Calcd for
336.0603, found: 336.0606.
C
17H13Cl3NO2 [MþH]þ:
C
17H14Cl2NO2 [MþH]þ: 334.0402, found: 334.0406.
4.4.6. (E)-2-chloro-N-(4-(3-(3-chlorophenyl)acryloyl)phenyl)
acetamide (10f)
4.4.12. 2-Chloro-N-(4-((2E, 4E)-5-phenylpenta-2, 4-dienoyl)
phenyl)acetamide (14)
Yield 68%, yellow solid, m.p.: 143e145 ꢀC; 1H NMR (400 MHz,
Yield 56%, earthy yellow solid, m.p.: 165e168 ꢀC; 1H NMR
CDCl3,
3H), 7.64 (s, 1H), 7.58e7.46 (m, 2H), 7.42e7.32 (m, 2H), 4.22 (s, 2H);
13C NMR (100 MHz, CDCl3,
, ppm): 187.46, 163.05, 142.05, 139.90,
d
, ppm): 8.46 (s, 1H), 8.06 (d, J ¼ 8.7 Hz, 2H), 7.82e7.68 (m,
(400 MHz, CDCl3,
d
, ppm): 8.50 (s, 1H), 8.01 (d, J ¼ 8.7 Hz, 2H), 7.71
(d, J ¼ 8.6 Hz, 2H), 7.61 (ddd, J ¼ 14.9, 7.0, 3.3 Hz, 1H), 7.50 (d,
d
J ¼ 7.0 Hz, 2H), 7.41e7.30 (m, 3H), 7.09 (d, J ¼ 14.8 Hz,1H), 7.05e7.00
(m, 2H), 4.22 (s, 2H); 13C NMR (100 MHz, CDCl3,
d, ppm): 187.93,
135.69, 133.99, 133.40, 129.37, 129.21, 129.00, 126.91, 125.80, 121.81,
118.42, 41.87; HRMS (ESI) Calcd for
334.0402, found: 334.0403.
C
17H14Cl2NO2 [MþH]þ:
163.13, 143.86, 141.07, 139.70, 135.07, 133.69, 128.80, 128.26, 127.85,
126.31, 125.88, 123.98, 118.39, 41.91; HRMS (ESI) Calcd for
C
19H17ClNO2 [MþH]þ: 326.0948, found: 326.0951.
4.4.7. (E)-2-chloro-N-(4-(3-(4-nitrophenyl)acryloyl)phenyl)
acetamide (10g)
4.5. General procedure for the synthesis of compounds 11a-g
Yield 71%, yellow solid, m.p.: 200e202 ꢀC; 1H NMR (400 MHz,
DMSO-d6,
J ¼ 8.7 Hz, 2H), 8.19e8.08 (m, 3H), 7.87e7.76 (m, 3H), 4.34 (s, 2H);
13C NMR (100 MHz, DMSO-d6,
, ppm): 187.81, 165.74, 148.47,
143.64, 141.73, 141.08, 132.89, 130.65, 130.25, 126.44, 124.37, 119.24,
44.10; HRMS (ESI) Calcd for C17H14ClN2O4 [MþH]þ: 345.0642,
found: 345.0641.
d
, ppm): 10.71 (s, 1H), 8.28 (d, J ¼ 8.7 Hz, 2H), 8.22 (d,
To the mixture of compound 1 (0.2 g, 1.454 mmol) and
Na3PO4$12H2O (0.454 g, 1.454 mmol) in acetone (10 mL) was added
CS2 (110 mL, 1.82 mmol). The mixture was stirred at room temper-
ature for 0.5 h. Then compounds 10a-g (1.454 mmol) were added to
the mixture slowly, and stirred at room temperature for another
0.5 h. Upon completion, the solvent was removed under reduced
pressure, the residue was extracted with dichloromethane, washed
with water, brine, dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The residue was purified using column
chromatography to give analogs 11a-g.
d
4.4.8. (E)-2-chloro-N-(4-(3-(3-methoxyphenyl)acryloyl)phenyl)
acetamide (10h)
Yield 73%, yellow solid, m.p.: 124e126 ꢀC; 1H NMR (400 MHz,
CDCl3,
d
, ppm): 8.51 (s, 1H), 8.05 (d, J ¼ 8.7 Hz, 2H), 7.77 (d,
J ¼ 15.7 Hz, 1H), 7.72 (d, J ¼ 8.7 Hz, 2H), 7.50 (d, J ¼ 15.7 Hz,1H), 7.34
(t, J ¼ 7.9 Hz, 1H), 7.24 (d, J ¼ 7.7 Hz, 1H), 7.18e7.12 (m, 1H), 6.97 (dd,
J ¼ 8.1, 1.9 Hz, 1H), 4.22 (s, 2H), 3.86 (s, 3H); 13C NMR (100 MHz,
4.5.1. (S, E)-2-((4-(3-(4-chlorophenyl)acryloyl)phenyl)amino)-2-
oxoethyl (2-oxotetrahydrofuran-3-yl)carbamodithioate (11a)
Yield 50%, yellow solid, m.p.: 110e111 ꢀC; 1H NMR (400 MHz,
CDCl3,
d, ppm): 189.00, 164.14, 159.97, 144.79, 140.82, 136.22,
134.65, 129.98, 121.98, 121.12, 119.44, 116.37, 113.52, 55.38, 42.92;
HRMS (ESI) Calcd for C18H17ClNO3 [MþH]þ: 330.0897, found:
330.0898.
DMSO-d6,
d
, ppm): 10.68 (s, 1H), 10.49 (d, J ¼ 8.0 Hz, 1H), 8.18 (d,
J ¼ 8.8 Hz, 2H), 7.98 (d, J ¼ 15.6 Hz, 1H), 7.94 (d, J ¼ 8.5 Hz, 2H), 7.78
(d, J ¼ 8.8 Hz, 2H), 7.72 (d, J ¼ 15.6 Hz, 1H), 7.53 (d, J ¼ 8.5 Hz, 2H),
5.53 (dt, J ¼ 11.0, 8.7 Hz, 1H), 4.41 (t, J ¼ 8.3 Hz, 1H), 4.35e4.26 (m,
3H), 2.65e2.54 (m, 1H), 2.31e2.18 (m, 1H); 13C NMR (100 MHz,
4.4.9. (E)-2-chloro-N-(4-(3-(3, 4, 5-trimethoxyphenyl)acryloyl)
phenyl)acetamide (10i)
DMSO-d6, d, ppm): 198.03, 187.89, 174.03, 166.66, 143.89, 142.40,
Yield 66%, yellow solid, m.p.: 174e175 ꢀC; 1H NMR (400 MHz,
135.44, 134.25, 132.81, 130.99, 130.50, 129.41, 123.20, 118.94, 65.99,
55.04, 40.21, 28.37; HRMS (ESI) Calcd for C22H19ClN2NaO4S2
[MþNa]þ: 497.0372, found: 497.0374.
CDCl3,
d
, ppm): 8.49 (s,1H), 8.05 (d, J ¼ 8.7 Hz, 2H), 7.73 (dd, J ¼ 12.1,
3.4 Hz, 3H), 7.40 (d, J ¼ 15.6 Hz,1H), 6.87 (s, 2H), 4.23 (s, 2H), 3.93 (s,

328
J.-L. Ren et al. / European Journal of Medicinal Chemistry 93 (2015) 321e329
4.5.2. (S, E)-2-((4-cinnamoylphenyl) amino)-2-oxoethyl (2-
oxotetrahydrofuran-3-yl)carbamodithioate (11b)
4.5.7. (S, E)-2-((4-(3-(4-nitrophenyl)acryloyl)phenyl)amino)-2-
oxoethyl (2-oxotetrahydrofuran-3-yl)carbamodithioate (11g)
Yield 30%, yellow solid, m.p.: 126e128 ꢀC; 1H NMR (400 MHz,
Yield 46%, yellow solid, m.p.: 96e97 ꢀC; 1H NMR (400 MHz,
DMSO-d6,
d
, ppm): 10.69 (s, 1H), 10.50 (d, J ¼ 7.9 Hz, 1H), 8.17 (d,
DMSO-d6,
d
, ppm): 10.71 (s, 1H), 10.50 (d, J ¼ 8.0 Hz, 1H), 8.29 (d,
J ¼ 8.8 Hz, 2H), 7.95 (d, J ¼ 15.5 Hz, 1H), 7.89 (dd, J ¼ 6.5, 2.8 Hz, 2H),
7.79 (d, J ¼ 8.8 Hz, 2H), 7.73 (d, J ¼ 15.6 Hz, 1H), 7.54e7.42 (m, 3H),
5.52 (dt, J ¼ 11.0, 8.6 Hz, 1H), 4.41 (t, J ¼ 8.4 Hz, 2H), 4.35e4.31 (m,
1H), 4.29 (s, 2H), 2.62e2.56 (m, 1H), 2.30e2.19 (m, 1H); 13C NMR
J ¼ 8.8 Hz, 2H), 8.20 (d, J ¼ 8.8 Hz, 2H), 8.17 (d, J ¼ 8.7 Hz, 2H), 8.14
(d, J ¼ 15.6 Hz, 1H), 7.82e7.78 (m, 3H), 5.52 (dt, J ¼ 11.0, 8.7 Hz, 1H),
4.41 (t, J ¼ 8.4 Hz, 1H), 4.35e4.27 (m, 3H), 2.64e2.54 (m, 1H),
2.32e2.17 (m, 1H); 13C NMR (100 MHz, DMSO-d6,
d, ppm): 198.04,
(100 MHz, DMSO-d6,
d
, ppm): 198.04, 188.06, 174.05, 166.67, 143.92,
187.81, 174.05, 166.73, 148.46, 144.10, 141.74, 141.02, 132.52, 130.65,
130.24,126.47, 124.38, 119.00, 66.01, 55.04, 40.14, 28.32; HRMS (ESI)
Calcd for C22H19N3NaO6S2 [MþNa]þ: 508.0613, found: 508.0614.
143.79, 135.22, 132.93,130.99, 130.44,130.37, 129.39, 129.27, 122.44,
119.11, 118.99, 66.01, 55.04, 40.16, 28.34; HRMS (ESI) Calcd for
C
22H20N2NaO4S2 [MþNa]þ: 463.0762, found: 63.0764.
4.6. Procedure for the synthesis of OdDHL
4.5.3. (S, E)-2-((4-(3-(4-bromophenyl)acryloyl)phenyl)amino)-2-
oxoethyl (2-oxotetrahydrofuran-3-yl)carbamodithioate (11c)
Yield 50%, yellow solid, m.p.: 167e168 ꢀC; 1H NMR (400 MHz,
To a mixture of malonic acid 15 (4.20 g, 40.36 mmol) and acetic
anhydride (4.96 mL, 52.47 mmol) was added H2SO4 (0.17 mL,
3.23 mmol), the mixture was then stirred for about 0.5 h in an ice-
water bath, followed by addition of acetone 16 (4.16 mL,
56.51 mmol). After stirring for another 4 h, the resulting solution
was then allowed to warm to room temperature and kept overnight
at the same temperature. Upon completion of the reaction, the
precipitate was filtrated, washed with ice water and dried to gain
Meldrum's acid 17. To a solution of n-decanoic acid (1.376 g,
8 mmol) in dry CH2Cl2 were added DMAP (1.024 g, 8.4 mmol), DCC
(1.816 g, 8.8 mmol) and 17 (1.152 g, 8 mmol) sequentially, and the
reaction mixture was stirred overnight at room temperature. N, N'-
dicyclohexylurea formed was removed by filtration, and the filtrate
was concentrated under vacuo to give the residue, which was then
dissolved in ethyl acetate, washed with 2 N hydrochloric acid, dried
over MgSO4 and concentrated to give the crude mixture of 19. To a
solution of 1 (0.461 g, 3.35 mmol) and 19 (1.001 g) in acetonitrile
(50 mL) was added Et3N (0.407 g, 4.02 mmol). The reaction mixture
was stirred at room temperature for 4 h and then stirred under
reflux. Upon completion of the reaction, the reaction mixture was
concentrated under vacuo to give the residue, which was then
subjected to column chromatography (petroleum ether:
acetone ¼ 10: 1) to give OdDHL.
DMSO-d6,
d
, ppm): 10.67 (s, 1H), 10.48 (d, J ¼ 7.9 Hz, 1H), 8.17 (d,
J ¼ 8.8 Hz, 2H), 7.99 (d, J ¼ 15.6 Hz, 1H), 7.86 (d, J ¼ 8.5 Hz, 2H), 7.78
(d, J ¼ 8.8 Hz, 2H), 7.71e7.66 (m, 3H), 5.52 (dt, J ¼ 11.0, 8.7 Hz, 1H),
4.41 (t, J ¼ 8.3 Hz, 1H), 4.36e4.23 (m, 3H), 2.63e2.54 (m, 1H),
2.30e2.17 (m, 1H); 13C NMR (100 MHz, DMSO-d6,
d, ppm): 198.03,
187.90, 174.03, 166.67, 143.89, 142.50, 134.57, 132.80, 132.34, 131.20,
130.50, 124.31, 123.25, 118.95, 65.99, 55.04, 40.21, 28.36; HRMS
(ESI) Calcd for C22H19BrN2NaO4S2 [MþNa]þ: 540.9867, found:
540.9871.
4.5.4. (S, E)-2-((4-(3-(4-fluorophenyl)acryloyl)phenyl)amino)-2-
oxoethyl (2-oxotetrahydrofuran-3-yl)carbamodithioate (11d)
Yield 32%, yellow solid, m.p.: 124e125 ꢀC; 1H NMR (400 MHz,
DMSO-d6,
d
, ppm): 10.66 (s, 1H), 10.48 (d, J ¼ 7.8 Hz, 1H), 8.17 (d,
J ¼ 8.8 Hz, 2H), 7.97 (dd, J ¼ 8.7, 5.7 Hz, 2H), 7.91 (d, J ¼ 15.6 Hz, 1H),
7.78 (d, J ¼ 8.8 Hz, 2H), 7.73 (d, J ¼ 15.7 Hz, 1H), 7.31 (t, J ¼ 8.8 Hz,
2H), 5.52 (dt, J ¼ 10.6, 8.7 Hz, 1H), 4.41 (t, J ¼ 8.3 Hz, 1H), 4.35e4.31
(m,1H), 4.29 (s, 2H), 2.64e2.54 (m,1H), 2.30e2.19 (m,1H); 13C NMR
(100 MHz, DMSO-d6, d, ppm): 198.04, 187.95, 174.03, 166.66, 165.06,
162.58, 143.81, 142.65, 132.91, 131.95, 131.92, 131.65, 131.57, 130.43,
122.35,118.96,116.49,116.27, 66.00, 55.05, 40.20, 28.35; HRMS (ESI)
Calcd for C22H19FN2NaO4S2 [MþNa]þ: 481.0668, found: 481.0670.
4.6.1. (S)-3-oxo-N-(2-oxotetrahydrofuran-3-yl) dodecanamide
(OdDHL)
Yield 30%, white solid; 1H NMR (400 MHz, CDCl3,
d, ppm): 7.71
4.5.5. (S, E)-2-((4-(3-(2-chlorophenyl)acryloyl)phenyl)amino)-2-
oxoethyl (2-oxotetrahydrofuran-3-yl)carbamodithioate (11e)
Yield 41%, yellow solid, m.p.: 95e96 ꢀC; 1H NMR (400 MHz,
(d, J ¼ 5.2 Hz, 1H), 4.61 (ddd, J ¼ 11.4, 8.7, 6.8 Hz, 1H), 4.48 (t,
J ¼ 8.7 Hz, 1H), 4.31e4.25 (m, 1H), 3.48 (s, 2H), 2.83e2.65 (m, 1H),
2.53 (t, J ¼ 7.4 Hz, 1H), 2.35e2.11 (m, 1H), 1.70e1.46 (m, 2H), 1.26 (s,
DMSO-d6,
d, ppm): 10.71 (s, 1H), 10.50 (d, J ¼ 7.9 Hz, 1H), 8.25e8.20
12H), 0.88 (t, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3,
d, ppm):
(m, 2H), 8.19 (d, J ¼ 8.8 Hz, 2H), 8.02 (d, J ¼ 4.0 Hz, 2H), 7.79 (d,
J ¼ 8.8 Hz, 2H), 7.60e7.55 (m, 1H), 7.52e7.42 (m, 2H), 5.52 (dt,
J ¼ 10.9, 8.7 Hz, 1H), 4.41 (t, J ¼ 8.4 Hz, 1H), 4.35e4.27 (m, 3H),
2.66e2.54 (m, 1H), 2.30e2.19 (m, 1H); 13C NMR (100 MHz, DMSO-
206.58, 174.94, 166.48, 65.92, 49.02, 48.20, 43.88, 31.85, 29.71,
29.39, 29.35, 29.24, 28.99, 23.35, 22.66, 14.11.
d6, d, ppm): 198.04, 187.82, 174.05, 166.72, 144.01, 138.48, 134.77,
4.7. Cell culturing
132.84, 132.62, 132.35, 130.59, 130.47, 128.99, 128.14, 125.15, 119.00,
66.01, 55.05, 40.21, 28.33; HRMS (ESI) Calcd for C22H19ClN2NaO4S2
[MþNa]þ: 497.0372, found: 497.0374.
Human cancer cell lines were maintained in minimal essential
medium supplemented with 10% fetal bovine serum (FBS) and 1%
penicillinestreptomycin in a humidified atmosphere of 5% CO2 and
95% air at 37 ꢀC. Cancer cells were maintained in RPMI1640 me-
dium. All cell lines were purchased from the China Center for Type
Culture Collection (CCTCC, Shanghai, China). For pharmacological
investigations, 10 mM stock solutions of the tested compounds
were prepared with DMSO. The highest DMSO concentration of the
medium (0.1%) did not have any substantial effect on the deter-
mined cellular functions.
4.5.6. (S, E)-2-((4-(3-(3-chlorophenyl)acryloyl)phenyl)amino)-2-
oxoethyl (2-oxotetrahydrofuran-3-yl)carbamodithioate (11f)
Yield 42%, yellow solid, m.p.: 168e169 ꢀC; 1H NMR (400 MHz,
DMSO-d6,
d
, ppm): 10.68 (s, 1H), 10.48 (d, J ¼ 7.8 Hz, 1H), 8.20 (d,
J ¼ 8.8 Hz, 2H), 8.09 (s, 1H), 8.04 (d, J ¼ 15.6 Hz, 1H), 7.84e7.80 (m,
1H), 7.78 (d, J ¼ 8.8 Hz, 2H), 7.70 (d, J ¼ 15.6 Hz, 1H), 7.54e7.44 (m,
2H), 5.52 (dt, J ¼ 10.5, 8.7 Hz, 1H), 4.41 (t, J ¼ 8.3 Hz, 1H), 4.35e4.26
(m, 3H), 2.64e2.54 (m, 1H), 2.30e2.16 (m, 1H); 13C NMR (100 MHz,
DMSO-d6,
d, ppm): 198.03, 187.87, 174.03, 166.68, 143.95, 142.15,
4.8. Cytotoxic activity assays
137.53, 134.28, 132.73, 131.14, 130.58, 130.49, 128.34, 128.32, 123.98,
118.94, 66.00, 55.05, 40.21, 28.36; HRMS (ESI) Calcd for
C
Exponentially growing cells were seeded into 96-well plates at a
22H19ClN2NaO4S2 [MþNa]þ: 497.0372, found: 497.0372.
concentration of 5 ꢁ 103 cells per well. After 24 h incubation at

J.-L. Ren et al. / European Journal of Medicinal Chemistry 93 (2015) 321e329
329
37 ꢀC, the culture medium was removed and replaced with fresh
medium containing the tested compounds at different concentra-
tions. The cells were incubated for another 72 h. Afterwards 20 L of
MTT solution (5 mg/mL) was added to all wells and incubated for
4 h at 37 ꢀC. Discarded the suspension and added 150 mL of DMSO
to each well and shook the plates to dissolve the dark blue crystals
(formazan); the absorbance was measured using a microplate
reader at a wavelength of 570 nm. Each concentration was analyzed
in triplicate and the experiment was repeated for three times. The
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For flow cytometric analysis of DNA content, 5.0 ꢁ 106 MCF-7
cells in exponential growth were treated with different concen-
trations of the test compounds for 12 or 24 h. After an incubation
period, the cells were collected, centrifuged and fixed with ice-cold
ethanol (70%). The cells were then treated with buffer containing
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We are grateful for the financial support from the Natural Sci-
ence Foundation of China (No. 21372206) and the Development
Foundation
of
Priority,
Ministry
of
Education
(No.
20134101130001).
Appendix A. Supplementary data
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