Synthesis and biological activities of dithiocarbamates containing 2(5H)-furanone-piperazine

Article abstract of DOI:10.1016/j.ejmech.2018.05.056

A series of new dithiocarbamates containing a 2(5H)-furanone-piperazine group was synthesized. These compounds show good in vitro cytoxic activity. Among them, compound 6c exhibits the best inhibitory activity against HeLa cell lines with an IC₅₀ of 0.06 ± 0.01 μM for 72 h, and it has good inhibitory activity against SMMC-7721 cell lines with an IC₅₀ of 0.006 ± 0.04 μM for 72 h, but the toxicity was lower against LO2 cell lines with an IC₅₀ of 45.76 ± 0.01 μM. The result showed that compound 6c is far more cytoxic towards cancer cell lines than towards benign cell lines compared with cytosine arabinoside (ARA) in vitro.

Full text of DOI:10.1016/j.ejmech.2018.05.056

Accepted Manuscript
Synthesis and biological activities of dithiocarbamates containing 2(5H)-furanone-
piperazine
Meng-Xue Wei, Jiao Zhang, Fu-Li Ma, Ming Li, Jia-Ying Yu, Wei Luo, Xue-Qiang Li
PII:
S0223-5234(18)30481-1
10.1016/j.ejmech.2018.05.056
EJMECH 10467
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 January 2018
Revised Date: 30 May 2018
Accepted Date: 31 May 2018
Please cite this article as: M.-X. Wei, J. Zhang, F.-L. Ma, M. Li, J.-Y. Yu, W. Luo, X.-Q. Li, Synthesis
and biological activities of dithiocarbamates containing 2(5H)-furanone-piperazine, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.05.056.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Synthesis and biological activities of dithiocarbamates containing 2(5H)-furanone-piperazine
,
†
†
Meng-Xue Wei * , Jiao Zhang , Fu-Li Ma, Ming Li, Jia-Ying Yu, Wei Luo, Xue-Qiang Li *
State Key Laboratory of High-efficiency Utilization of Coal and Green Chemical Engineering,
School of Chemistry and Chemical Engineering, Ningxia University, Yinchuan 750021, China
*
Corresponding authors.
E-mail addresses: weimengxue@nxu.edu.cn (M.-X. Wei), lixq@nxu.edu.cn (X.-Q. Li)
†
These authors contribute equally to this work.
Abstract
A series of new dithiocarbamates containing a 2(5H)-furanone-piperazine group was synthesized. These
compounds show good in vitro cytoxic activity. Among them, compound 6c exhibits the best inhibitory activity
against HeLa cell lines with an IC50 of 0.06 ± 0.01 µM for 72 h, and it has good inhibitory activity against
SMMC-7721 cell lines with an IC of 0.006 ± 0.04 µM for 72 h, but the toxicity was lower against LO2 cell lines
50
with an IC of 45.76 ± 0.01 µM. The result showed that compound 6c is far more cytoxic towards cancer cell lines
50
than towards benign cell lines compared with cytosine arabinoside (ARA) in vitro.
Keywords
2
(5H)-Furanone; Dithiocarbamates; Synthesis; Biological activities; SMMC-7721 cell lines
1
.
Introduction
Cancer is a disease during which malignant tumors might occur, and it seriously endangers human life and
health. According to the survey, about 60 million individuals are diagnosed with cancer every year [1]. The number
of new cancer cases has reached 368.2 million in China, with 2,229 deaths in 2013. Lung cancer, liver cancer,
stomach cancer, esophageal cancer, and colorectal cancer are the five leading causes of cancer death, accounting for
about 60% of all cancer deaths [2]. Each year, more than 300,000 women die of cervical cancer, which is the third
most common cancer in women worldwide [3]. Therefore, there is an urgent need for developing new anticancer
agents [4-9].
Dithiocarbamic acid ester is an easily accessible moiety in organic synthesis [10,11]. It has been widely used
in the synthesis and development of new drugs. Dithiocarbamics exhibit a wide range of biological activities
[12-15], such as anticancer [16], antitumor [17], antibacterial [18]. On the other hand, 2(5H)-furanone derivatives
are widely found in natural products and have been used in cancer treatment due to the notable bioactivity [19-23].
In our previous studies, we designed and synthesized a range of 2(5H)-furanone derivatives containing
piperazine-sulfonamide [24,25]. These compounds exhibit remarkable anti-HeLa activity, leading to an IC of 0.02
5
0
µM for 24 h under the standard MTT assay [14].
In this continuing study, we report a convenient one-pot method to synthesize dithiocarbamic acid ester
derivatives containing a 2(5H)-furanone-piperazine group. The subsequent biological studies show that these
compounds showed higher cytoxic activity against some cancer cell lines than against benign cell lines.
2
.
Results and discussion
1

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2
.1. Chemistry
The enantiomerically pure 5-(S)-5-alkoxy-4-piperazineyl-3-bromo-2(5H)-furanones (5a, 5b) were prepared by
the procedure shown in Scheme 1 [25]. Reaction of furfural 1 with Br /H O afforded mucobromic acid 2 by
2
2
sequential dibromination and oxidation. Acetalation of 2 with chiral alcohols gave rise to γ-substituted butenolides
3
. Multiple recrystallization processes was used for 3 to provide the enantiomerically pure 5-(S)-alkoxy-3,
-dibromo-2(5H)-furanone compounds 4 [26,27]. Following the protocol we previously reported, 4 was
4
transformed into compound 5 [25] by treatment with piperazine and triethylamine through a Michael
addition-elimination process.
Synthesis of dithiocarbamate-contained 2(5H)-furanone-piperazines 6 is shown in Scheme 2. This
three-component reaction was simply performed by mixing compounds 5 and carbon disulfide followed by addition
of electrophiles such as allyl bromide, benzyl bromide or methyl chloroacetate. The reactions were completed
within 1.5 to 3.5 h, affording 50-75% yields of compounds 6. We also tested other halogenated electrophiles such
as 1-bromopropane, n-butyl bromide, iso-butyl bromide and epichlorohydrin under the otherwise identical reaction
conditions, however no expected products were observed. The weaker electrophilicity of these reagents and the
poor stability of the desired products were proposed to account for the observed coupling efficiency.
Scheme 1. Synthesis of compounds 5
Scheme 2. Synthesis of compounds 6a-f
2

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2
.2. Biological evaluation
The newly synthesized compounds 6a-c, vincristine (VCR) and cytosine arabinoside (ARA) were screened for
in vitro cytoxic activity against two cancer cell lines, including human cervical carcinoma cell lines (HeLa), human
liver cancer cell lines (SMMC-7721) and were assessed for toxicities against human normal liver cell lines (LO2)
by the standard MTT assay in vitro. The cytotoxicity in vitro of the tested compounds were shown in Table 1 in the
form of the IC , which were carried out using SPSS 22.0 software and descriptive data were expressed as mean ±
50
standard deviation. To our delight, compounds 6a-c displayed good inhibition activities on these two cancer cell
lines (Table 1). Among the tested compounds, compound 6c possessed stronger inhibitory activity against HeLa
cell lines with the IC value of 0.06 ± 0.01 µM at 72 h, making it more potent than the clinical chemotherapy drugs
5
0
cytarabine [28] (IC = 0.95 µM) and etoposide (IC = 7.20-7.23 µM) [29], while compound 6b was the most potent
5
0
50
to SMMC-7721 cell lines, with an IC of 0.004 ± 0.05 µM, showing potent higher inhibitory activity in vitro than
5
0
cytosine arabinoside (ARA) (0.013 ± 0.09), although lower than vincristine (VCR). As for LO2 cell lines, except
for compound 6a, the newly synthesized compound 6b and 6c showed lower toxicities than vincristine (VCR) and
cytosine arabinoside (ARA). As a whole, compounds 6b and 6c showed higher cytoxic activity against some cancer
cell lines than against benign cell lines compared with cytosine arabinoside (ARA) in vitro.
Table 1 Cytoxic activities against HeLa, SMMC-7721 and LO2 cell lines with compounds 6a-c, VCR or ARA for 72 h.
a
IC50 (µM) on cell lines
Compounds
HeLa
SMMC-7721
0.006 ± 0.04
0.004 ± 0.05
0.006 ± 0.04
0.003 ± 0.03
0.013 ± 0.09
LO2
6
6
a
0.32 ± 0.07
0.11 ± 0.02
b
0.26 ± 0.01
0.37 ± 0.02
45.76 ± 0.01
0.20 ± 0.01
0.17 ± 0.04
6
c
0.06 ± 0.01
VCR
ARA
-
-
a
IC50 values represent the compound concentration (µM) required to inhibit tumor cell proliferation by 50%, and were calculated using concentrations in
triplicate and experiment was repeated, only values with a standard deviation < 10% mean were considered.
HeLa = human cervical carcinoma cell lines. SMMC-7721 = human liver cancer cell lines. LO2 = human normal liver cell lines.
VCR = vincristine. ARA = cytosine arabinoside.
As shown in Fig. 1, it is clear that the inhibitory rate increased with the increased concentration as for HeLa
cell lines. In SMMC-7721 cell lines, the compounds 6a-c, VCR and ARA displayed similar anti-proliferative
activities at different concentrations for 72 h. However since the tested compounds showed lower toxicity in vitro
to LO2 cell lines at 0.01 µg/mL for 72 hours, and the cell inhibition rate data were negative, so the data were not
involved in drawing Fig. 1. Furthermore, in order to investigate whether compound 6b could induce cell apoptosis,
human liver cancer cell lines (SMMC-7721) treated with different concentrations of compound 6b were subjected
to Annexin V-FITC/PI. From Fig. 2, the percentage of early and late apoptotic cells increased (lower right quadrant
and upper right quadrant), as the concentration of compound 6b increased. The average of cell apoptosis including
the percentage of early and late apoptotic cells increased (lower right quadrant and upper right quadrant) reached
2
5.80%, when the compound 6b concentration was 5 µg/mL through three parallel experimental data. The results
showed that compound 6b could induce the apoptosis of SMMC-7721 cell lines in an appropriate concentration
3

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dependent manner.
Fig. 1. The inhibitory effect of compounds 6a-c, vincristine (VCR) and cytosine arabinoside (ARA) with various concentrations on
HeLa, SMMC-7721 and LO2 cell lines.
Fig. 2. The flow cytometric analysis of SMMC-7721 cell lines using different concentrations of compound 6b (0.1, 0.5, 5 µg/mL) and
each concentration was detected three times in parallel. The percentage of cells positive for AV and/or PI is reported in the quadrants
including cells in the lower left quadrant: live cells; Lower right quadrant: early apoptotic cells; Upper right quadrant: late apoptotic
cells; Upper left quadrant: necrotic cells.
3
.
Conclusion
In summary, a series of new chiral dithiocarbamates containing 2(5H)-furanone-piperazine group has been
4

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synthesized. The cytoxic activity against cervical cancer cell lines (HeLa), human liver cancer cell lines
(SMMC-7721) and in vitro cytotoxicity against human normal liver cell lines (LO2) has been evaluated. All of the
tested compounds exhibited good cytoxic activity. Compound 6c, with an IC of 0.06 ± 0.01 µM, was found to be
5
0
the most active against HeLa cell lines, and the compound 6b was the most active derivative with high
anti-proliferative action against SMMC-7721 cell lines (0.004 ± 0.05 µM) and has lower toxicity than cytosine
arabinoside (ARA) against LO2 cell lines in vitro. Especially compound 6c is far more cytoxic towards cancer cell
lines than towards benign cell lines compared with cytosine arabinoside (ARA) in vitro. All of these results provide
a reference for further research on anti-tumor drugs.
4
.
Experimental
.1. Chemistry
Thin-layer chromatography (TLC) was carried out on silica GF254 plates and 200-300 mesh (Qingdao
4
Haiyang Chemical Co., Ltd, China). All of the melting points were determined on a WRS-1B digital melting point
1
apparatus and are uncorrected. IR spectra were recorded on a Spectrum Two spectrometer as KBr discs. H NMR
13
and C NMR spectra were obtained with a Bruker Avance III 400 MHz spectrometer in chloroform (CDCl ) and
3
tetramethylsilane (TMS). All chemicals were used as received without further purification, unless otherwise
indicated.
4
.1.1 General procedures for synthesis of target compounds 6a-f
A mixture of (S)-3-bromo-5-(((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)-4-(piperazin-1-yl)furan-2(5H)
-one (5a) or (5S)-3-bromo-4-(piperazin-1-yl)-5-(((2S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl)oxy)furan-2(5H)-
one (5b) [25] (0.25 mmol), CHCl (2 mL), carbon disulfide (3 mL), and anhydrous triethylamine (0.25 mmol) was
3
stirred at room temperature. Then, halohydrocarbons (0.25 mmol) were added dropwise, and the reaction was
monitored by TLC. On completion of the reaction (1.5-3.5 h), the mixture was extracted with dichloromethane and
the organic layer was washed with saturated brine. Then, the organic layer was dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. The purification of the residue by silica gel column chromatography or
crystallization yielded the desired compounds 6a-f.
4
.1.1.1 Methyl 2-((4-((S)-4-bromo-2-(((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)-5-oxo-2,5-dihydrofuran-3-
yl) piperazine-1-carbonothioyl)thio)acetate (6a)
White solid, yield 64%, m.p. 90.5-91.6 °C; [α] +1 (c 1.00, CH Cl ); H NMR (CDCl , 400 MHz) δ: 5.85 (S,
2
5
1
D
2
2
3
1
1
1
1
7
2
H), 4.42-4.19 (m, 4H), 4.14 (s, 2H), 3.84-3.78 (m, 4H), 3.74 (S, 3H), 3.57 (td, J = 10.6, 4.5 Hz, 1H), 2.17 (d, J =
2.4, 1H), 2.08-2.04 (m, 1H), 1.63 (tdd, J = 8.2, 5.2, 2.5 Hz, 2H), 1.36 (dddd, J = 21.4, 12.6, 6.3, 3.2 Hz, 1H),
.30-1.20 (m, 1H), 1.10 (td, J = 12.4, 10.9 Hz, 1H), 1.01-0.92 (m, 1H), 0.89 (dd, J = 6.8, 2.0 Hz, 6H), 0.85-0.78 (m,
1
3
H), 0.75 (d, J = 6.9 Hz, 3H); C NMR (CDCl , 100 MHz) δ: 196.09, 168.75, 168.19, 15.78, 157.62, 98.09, 81.05,
3
-1
5.75, 52.97, 48.09, 46.67, 42.34, 38.79, 33.83, 31.64, 25.24, 22.69, 22.26, 21.17; IR (KBr) νmax/cm : 2952, 2925,
+
867, 1756, 1624, 1417, 1343, 1288, 1221, 1153, 1116, 998, 959; HRMS: m/z [M+Na] calcd for
C H BrN NaO S : 571.0912; found: 571.0909.
2
2
33
2
5 2
4
.1.1.2 Benzyl 4-((S)-4-bromo-2-(((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)-5-oxo-2,5-dihydrofuran-3-yl)
piperazine-1-carbodithioate (6b)
2
5
1
White solid, yield 50%; [α]D +2 (c 1.00, CH Cl ); H NMR (CDCl , 400 MHz) δ: 7.32 (d, J = 1.8 Hz, 2H),
2
2
3
5

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7.25 (dd, J = 6.6, 1.8 Hz, 1H), 7.23-7.20 (m, 2H), 5.77 (S, 1H), 4.50 (s, 1H), 4.30-4.05 (m, 4H), 3.79-3.71 (m, 4H),
3.51 (td, J = 10.6, 4.4 Hz, 1H), 2.13 (d, J = 12.4 Hz, 1H), 2.02 (d, J = 2.0 Hz, 1H), 1.63-1.54 (m, 2H), 1.28-1.18((m,
3H), 1.06 (td, J = 12.4, 10.8 Hz, 2H), 0.97-0.88 (m, 1H), 0.85 (dd, J = 6.8, 1.4 Hz, 6H), 0.70 (d, J = 7.0 Hz, 3H);
1
3
C NMR (CDCl , 100 MHz) δ: 197.68, 168.21, 157.63, 135.39, 129.40, 128.70, 127.78, 98.10, 81.06, 75.72, 48.10,
3
-
1
4
1
5
4
6.75, 42.35, 42.26, 33.85, 29.71, 22.71, 21.28, 21.20, 15.81; IR (KBr) νmax/cm : 3028, 2953, 2922, 2867, 1759,
+
623, 1454, 1411, 1219, 1115, 997, 958; HRMS: m/z [M+Na] calcd for C H BrN NaO S : 589.1170; found:
2
6
35
2
3 2
89.1173.
.1.1.3 Allyl 4-((S)-4-bromo-2-(((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)-5-oxo-2,5-dihydrofuran-3-yl)
piperazine-1-carbodithioate (6c)
White solid, yield 72 %, m.p. 163.4-164.9 °C; [α] +2.5 (c 1.00, CH Cl ); H NMR (CDCl , 400 MHz) δ:
2
5
1
D
2
2
3
5
3
1
3
7
.93-5.85 (m, 2H), 5.84 (s, 1H), 5.34 (dd, J = 2.6, 1.4Hz, 1H), 5.17 (dd, J = 10.0, 1.2Hz, 1H), 4.01-4.00 (m, 4H),
.99 (d, J = 0.8Hz, 2H), 3.84-3.80 (m, 4H), 3.58-3.57 (m, 1H), 2.18 (d, J = 12.4Hz, 1H), 2.08-2.07 (m, 1H),
.67-1.62 (m, 2H), 1.31-1.30 (m, 3H), 1.11 (td, J = 12.4, 10.8 Hz, 1H), 0.99-0.95 (m, 3H), 0.91 (dd, J = 6.8, 2.4Hz,
1
3
H), 0.76 (d, J = 6.8Hz, 3H); C NMR (CDCl , 100 MHz) δ: 197.61, 168.22, 157.64, 132.02, 119.27, 98.13, 81.11,
3
+
5.80, 48.14, 46.77, 42.39, 40.40, 33.88, 25.30, 22.74, 22.30, 21.22, 15.83; HRMS: m/z [M+Na] calcd for
C H BrN NaO S : 539.1014; found: 539.1009.
2
2
33
2
3 2
4
.1.1.4 Methyl 2-((4-((2S)-4-bromo-5-oxo-2-(((2S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl)oxy)-2,5-dihydrofuran-
3
-yl)piperazine-1-carbonothioyl)thio)acetate (6d)
2
5
1
White solid, yield 67%; [α]D +1 (c 1.00, CH Cl ); H NMR (CDCl , 400 MHz) δ: 5.77 (d, J = 7.3 Hz, 1H),
2
2
3
4
1
1
3
1
5
4
.35 (s, 3H), 4.29 (t, J = 6.5 Hz, 1H), 4.45-4.04 (m, 3H), 4.01-3.78 (m, 2H), 3.76 (s, 3H), 2.26 (tq, J = 13.2, 4.1 Hz,
1
3
H), 1.87-1.62 (m, 3H), 1.34-1.19 (m, 3H), 1.17-1.01 (m, 2H), 0.85 (dd, J = 8.4, 4.2 Hz, 9H); C NMR (CDCl ,
3
00 MHz) δ: 196.30, 168.80, 168.16, 157.12, 99.39, 97.07, 87.84, 84.48, 75.45, 49.53, 47.96, 44.94, 38.92, 37.35,
-
1
5.47, 28.56, 28.10, 26.67, 19.77, 18.91, 14.42, 13.41; IR (KBr) νmax/cm : 2954, 2877, 1748, 1624, 1433, 1329,
+
287, 1234, 1151, 1125, 1001, 952; HRMS: m/z [M+Na] calcd for C H BrN NaO S : 569.0755; found:
2
2
31
2
5 2
69.0763.
.1.1.5 Benzyl 4-((2S)-4-bromo-5-oxo-2-(((2S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl)oxy)-2,5-dihydrofuran-3-yl)
piperazine-1-carbodithioate (6e)
White solid, yield 53%, m.p. 108.3-110.0 °C; [α]D +1 (c 1.00, CH Cl ); H NMR (CDCl , 400 MHz) δ: 7.38
2
5
1
2
2
3
(dd, J = 8.0, 1.6 Hz, 2H), 7.34 (dt, J = 8.2, 1.6 Hz, 2H), 7.82-7.31 (m, 1H), 5.75 (s, 1H), 4.58 (s, 2H), 4.16-4.19 (m,
4
3
1
2
1
4
H), 3.89-3.84 (m, 4H), 2.31-2.22 (m, 1H), 1.81 (d, J = 2.8 Hz, 2H), 1.70 (dt, J = 25.0, 4.4 Hz, 1H), 1.27-1.22 (m,
1
3
H), 1.04 (dd, J = 13.2, 3.4 Hz, 1H), 0.87-0.83 (m, 9H); C NMR (CDCl , 100 MHz) δ: 197.78, 168.19, 157.10,
3
35.41, 129.42, 128.73, 127.81, 99.35, 97.04, 87.74, 84.41, 75.36, 49.48, 47.91, 44.88, 42.32, 37.29, 35.39, 28.50,
-1
6.70, 21.12, 19.66, 18.86, 14.37, 13.37; IR (KBr) νmax/cm : 2950, 2873, 1758, 1625, 1453, 1415, 1334, 1286,
+
218, 1123, 997, 962; HRMS: m/z [M+Na] calcd for C H BrN NaO S : 587.1014; found: 587.1022.
26
33
2
3 2
.1.1.6 Allyl 4-((2S)-4-bromo-5-oxo-2-(((2S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl)oxy)-2,5-dihydrofuran-3-yl)
piperazine-1-carbodithioate (6f)
White solid, yield 75 %, m.p. 179.4-180.0 °C; [α]D +2 (c 1.00, CH Cl ); H NMR (CDCl , 400 MHz) δ:
2
5
1
2
2
3
5
.94-5.87 (m, 1H), 5.77 (d, J = 7.2, 1H) 5.35-5.29 (m, 1H), 5.18 (s 1H), 4.28-4.27 (m, 4H), 4.01 (d, J = 6.8 Hz, 2H),
6

ACCEPTED MANUSCRIPT
3
1
9
.84 (d, J = 8.7 Hz, 4H) 3.70 (q, J = 6.8 Hz, 1H), 1.80 (d, J = 2.8 Hz, 2H), 1.76-1.65 (m, 2H), 1.29-1.23 (m, 2H),
1
3
.07-1.02 (m, 1H), 0.88-0.83 (m, 9H); C NMR (CDCl , 100 MHz) δ: 197.72, 168.13, 157.15, 132.06, 119.28,
3
7.08, 84.46, 75.23, 58.50, 49.46, 47.68, 46.72, 44.85, 40.44, 37.33, 35.44, 28.09, 19.69, 18.53, 14.39, 13.40; IR
-1
+
(
KBr) νmax/cm : 2950, 2873, 1752, 1627, 1417, 1334, 1286, 1225, 1125, 1000, 960; HRMS: m/z [M+Na] calcd for
C H BrN NaO S : 537.0857; found: 537.0870.
2
2
31
2
3 2
4
4
.2. Pharmacology
.2.1 Human cervical carcinoma cell lines (HeLa)
4
Human cervical carcinoma cells (1×10 in 100 µL) were seeded on 96 plates in triplicate. Following a 24 h
culture at 37 °C, the medium was replaced with fresh medium at various concentrations (6.25, 12.5, 25, 50, 100
µg/mL) of compounds 6a-c in a final volume of 100 µL. At the same time, the drug-free medium negative control
well and the solvent control well were set up using the same volume of dimethyl sulfoxide (DMSO). Cells were
respectively incubated at 37°C for 72 h. Then, 10 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) (2 mg/mL in a phosphate buffer solution (PBS)) was added to each well and incubated for an
additional 4 h; the plates were centrifuged at 1000 r/min for 10 min before the medium was removed. MTT
formazan precipitates were dissolved in 150 µL of DMSO, shaken mechanically for 10 min and then read
immediately at 568 nm in a plate reader (Multiskan MK3, Thermo Fisher Scientific, USA).
Cell inhibition rate = [A568 (negative control well) - A568 (dosing well)]/A568 (negative control well) × 100%.
4
.2.2 Human liver cancer cell lines (SMMC-7721)
4
SMMC-7721 cells (1×10 in 100 µL) were seeded on 96 plates in triplicate. Following a 24 h culture at 37 °C,
the medium was replaced with fresh medium at various concentrations (0.01, 0.1, 0.5, 2.5, 5 µg/mL) of compounds
a-c, vincristine (VCR) and cytosine arabinoside (ARA) in a final volume of 100 µL. At the same time, the
6
drug-free medium negative control well and the solvent control well were set with the same volume of dimethyl
sulfoxide (DMSO). Cells were respectively incubated at 37 °C for 72 h. Then, 10 µL of 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) (2 mg/mL in a phosphate buffer solution (PBS)) was added to each
well, incubated for an additional 4 h, centrifuged at 1000 r/min for 10 min, and then the medium was removed.
MTT formazan precipitates were dissolved in 150 µL of DMSO, shaken mechanically for 10 min and then read
immediately at 568 nm using a plate reader (Multiskan MK3, Thermo Fisher Scientific, USA).
Cell inhibition rate = [A568 (negative control well) - A568 (dosing well)]/A568 (negative control well) × 100%.
4
.2.3 Human normal liver cell lines (LO2)
4
Human normal liver cells (1×10 in 100 µL) were seeded on 96 plates in triplicate. Following a 24 h culture at
3
7 °C, the medium were replaced with fresh medium at various concentrate (0.01, 0.1, 0.5, 2.5, 5 µg/mL) of
compounds 6a-c, vincristine and cytosine arabinoside in a final volume of 100 µL. At the same time, the drug-free
medium negative control well, and solvent control well were set with the same volume of dimethyl sulfoxide
(DMSO). Cells were respectively cultivated at 37 °C for 72 h. Then 10 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) (2 mg/mL in a phosphate buffer solution (PBS)) was added to each well,
incubated for an additional 4 h, the plates were centrifuged at 1000 r/min for 10 min, then the medium was
7

ACCEPTED MANUSCRIPT
removed. MTT formazan precipitates were dissolved in 150 µL of DMSO, shaken mechanically for 10 min and
then read immediately at 568 nm in a plate reader (Multiskan MK3, Thermo Fisher Scientific, USA).
Cell inhibition rate = [A568 (negative control well) - A568 (dosing well)]/A568 (negative control well) × 100%.
4
.2.4 Flow cytometry analysis
SMMC-7721 cells were seeded in on 96 plates in triplicate. Following a 24 h culture at 37 °C, the medium
were replaced with fresh medium at various concentrate (0.1, 0.5, 5 µg/mL) of compounds 6b. At the same time,
drug-free medium negative control well and solvent control well were set with the same volume of dimethyl
sulfoxide (DMSO). Cells were respectively cultivated at 37 °C for 72 h. then 0.25% trypsin without ethylene
diamine tetra acetic acid (EDTA) was added to to each well. After trypsinization, the treated cells were stained
using Annexin V-FITC/PI apoptosis detection kit (NanJing KeyGen Biotech Co., Ltd, CHN) according to the
manufacturer’s instructions. After incubation at room temperature for 5-15 min in the dark, the apoptotic cells were
immediately analyzed by flow cytometry (cytoFlex, BECKMAN COULTER, USA).
Acknowledgments
We are thankful for the financial support from National Natural Science Foundation of China (No. 21462032),
Natural Science Foundation of Ningxia (No. NZ17001), National First-rate Discipline Construction Project of
Ningxia (No. NXYLXK2017A04), Major Innovation Projects for Building First-class Universities in China's
Western Region (No. ZKZD2017003), and College Students’ Innovative and Entrepreneurship Training Program
of Ningxia University (No. Q201710749028).
Appendix A. Supplementary data
Supplementary data related to this article can be found in the “supporting information”.
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Highlights:
1
2
3
4
. The dithiocarbamates containing a 2(5H)-furanone-piperazine were synthesized.
. The target compounds showed good cytoxic activity.
. Compound 6c is far more cytoxic against cancer cells than against benign cells.
. Compound 6b has higher cytoxic activity against SMMC-7721 cell lines than ARA.