Fluoroalkane thioheterocyclic derivatives and their antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2015.02.031

Two series of novel trifluorobutenyl derivatives of heterocyclic with convenient and efficient synthesis methods and their antitumor activity on three cell lines have been reported for the first time. The derivatives were synthesized by the nucleophilic substitution between 4-bromo-1,1,2-trifluorobutene-1-ene and commercially available nitrogen-containing heterocycles with sulfydryl or monosubstituted malononitrile. The twenty-four new compounds were characterized by ¹HNMR, ¹³CNMR and HR-MS. Totally, thirty-seven compounds were evaluated for the antitumor activity on three cancer cell lines (SH-SY5Y, MCF-7 andHepG2) using conventional MTT assay. The pharmacological results indicated that the compounds 3c, 3h, 4c, 8, 9, 10 and 11 showed potent to moderate antitumor activity against three cancer cell lines, with IC₅₀ values ranging between 0.4 μ M and 41.5μ M. Eventhough they had less active than the reference compound taxol against MCF-7 and HepG2 lines, but they were better than the reference compound noscapine against SH-SY5Y cells, especiallythe compound 3h with a IC₅₀ value of 0.4 μM.

Full text of DOI:10.1016/j.ejmech.2015.02.031

Accepted Manuscript
Fluoroalkane thioheterocyclic derivatives and their antitumor activity
Guangxiao Li, Qian Sun, Dongling Wang, Ying Xu, Jingjing Zhuang, Qian Zhang,
Dequn Sun
PII:
S0223-5234(15)00127-0
10.1016/j.ejmech.2015.02.031
EJMECH 7713
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 August 2014
Revised Date: 29 January 2015
Accepted Date: 19 February 2015
Please cite this article as: G. Li, Q. Sun, D. Wang, Y. Xu, J. Zhuang, Q. Zhang, D. Sun, Fluoroalkane
thioheterocyclic derivatives and their antitumor activity, European Journal of Medicinal Chemistry (2015),
doi: 10.1016/j.ejmech.2015.02.031.
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*Graphical Abstract (for review)
Click here to download Graphical Abstract (for review): Graphical abstracts.pdf
ACCEPTED MANUSCRIPT
IC50 (µM)
Compd.
SH-SY5Y
7.0±0.5
HepG2
4.7±0.1
3.71±0.2
6.9±1.9
6.1±1.0
MCF-7
8.6±0.7
7.2±0.4
12.2±0.2
8.4±0.2
3c
4c
8
2.64±0.1
2.1±0.1
9
6.0±0.2

*Highlights (for review)
ACCEPTED MANUSCRIPT
Highlights



Two series of novel trifluorobutenyl derivatives of heterocyclic
with convenient and efficient synthesis methods have been reported for
the first time.
The thirty-seven compounds were evaluated for the antitumor
activity on three cancer cell lines (SH-SY5Y, MCF-7 and HepG2)
using conventional MTT assay.
The compounds 3c, 3h, 4c, 8, 9, 10 and 11 showed considerablly
good antitumor activity, with IC50 values ranging between 0.4 μM and
41.5 μM.

*Manuscript
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ACCEPTED MANUSCRIPT
Fluoroalkane thioheterocyclic derivatives and their antitumor activity
Guangxiao Li†, Qian Sun†, Jingjing Zhuang, Dongling Wang, Ying Xu, Qian Zhang and Dequn Sun *
Marine College, Shandong University at Weihai, No.180, Wenhua West Road, Weihai 264209, P. R. China;
* Corresponding author: dequn.sun@sdu.edu.cn (Dequn Sun);
†
These two authors contributed equally to this work.
Abstract
Two series of novel trifluorobutenyl derivatives of heterocyclic with convenient
and efficient synthesis methods and their antitumor activity on three cell lines have
been reported for the first time. The derivatives were synthesized by the nucleophilic
substitution between 4-bromo-1,1,2-trifluorobutene-1-ene and commercially available
nitrogen-containing heterocycles with sulfydryl or monosubstituted malononitrile.
1
The twenty-four new compounds were characterized by HNMR, 13CNMR and
HR-MS. Totally, thirty-seven compounds were evaluated for the antitumor activity on
three cancer cell lines (SH-SY5Y, MCF-7 and HepG2) using conventional MTT assay.
The pharmacological results indicated that the compounds 3c, 3h, 4c, 8, 9, 10 and 11
showed potent to moderate antitumor activity against three cancer cell lines, with IC50
values ranging between 0.4 µM and 41.5 µM. Even though they had less active than
the reference compound taxol against MCF-7 and HepG2 lines, but they were better
than the reference compound noscapine against SH-SY5Y cells, especially the
compound 3h with a IC50 value of 0.4 ꢀM.
Keywords: heterocyclic, trifluorobutenyl derivatives, nucleophilic substitution,
oxidation, monosubstituted malononitrile, antitumor activity
1. Introduction
Nitrogen-containing heterocycles with sulfur atom are an important class of
compounds in medicinal chemistry. Especially, thiazole and its derivatives, such as
Vitamin B1, Penicillin [1] and coenzyme cocarboxylase [2], play a significant role.
1

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Benzothiazole derivatives are known for different biological properties, including
antitubercular [3], antimalarial [4], anticonvulsant [5], antiheꢀmintic [6], analgesic [7],
anti-inflammtory [8], antidiabetic [9] and antitumor activities [10]. Benzimidazole is
structural isostere of benzothiazole, and the key building block for a variety of
compounds, which has crucial role in the functions of biologically important
molecules, such as anticancer [11], antimicrobial [12], proton pump inhibitor [13],
antiviral [14].
Fluorine, characterized by its small size and high electronegativity, often
furnishes organic molecules with unequalled chemical and biological properties
including stability, lipophilicity, and bioavailability [15]. The trifluorobutenyl
derivatives are of great interest in medicinal chemistry [16]. However, the synthesis of
trifluorobutenyl derivatives normally required reflux conditions, longer reaction times
and other strict conditions.
On the other hand, the sulfonic acid functional group has important application
in synthetic molecules with important biological and pharmacological activities [17].
The formation of carbon-sulfur bonds need various catalysis which is limited by their
use of a strong base and high temperature [18]. In addition, some traditional oxidants
such as trifluoroperacetic acid [19], an MeNO2 solution in dilute HNO3/H2SO4 [20],
iodic acid [21], and other hypervalentiodine reagents [22] are applied frequently to the
oxidation of the sulfides. However, most of these reagents perform the disadvantages
of low effective oxygen content, environmentally unfavorable byproducts and high
cost. Therefore it is highly desirable to develop a simple, less-expensive, safer, and
highly efficient method.
Herein we report two convenient and eco-friendly methods for the synthesis of
trifluorobutenyl derivatives (Scheme I and Scheme II). The compounds of 17, 18 and
19 were synthesized according to the literature procedures [23,24]. The antitumor
activity of all the compounds was evaluated in vitro using the MTT assay against the
cancer cell lines (SH-SY5Y, MCF-7 and HepG2).
2

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O
S
F
F
F
ii
R1
F
3a,3b,3d
F
F
i
R1 SH
1
R1
S
2a-2i
O
F
F
iii
R1
S
F
O
4a-4f
2a
F
F
F
iv
Cl R1
S
5a
Cl
Cl
O
F
F
Cl F
F
S
N
ii
S
Cl
Cl
8
F
F
Cl F
F
S
S
S
Cl
O
S
Cl F
F
S
N
N
iii
Cl
6
iv
F
F
F
S
N
O
9
S
ii
O
S
F
Cl F
S
N
Cl F
F
Cl
S
N
2c
F
Cl
10
O
S
iii
7
F
Cl F
F
S
Cl
N
O
11
R1:
H
N
HS
S
S
g =
h =
a =
d =
N
N
N
N
N
F
F
H
N
O
O
S
N
e =
b =
c =
N
N
O
S
N
f =
i =
N
N
N
Scheme 1. Synthesis of compounds 2-11. Reagents and conditions: i: K2CO3,CH3CN; ii: H2O2
(1equiv), CH3COOH, 0 ; iii: H2O2 (3equiv), CH3COOH, 20-30 ; iv: NCS,CCl4
3

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Scheme 2. Synthesis of compounds 13-16. Reagents and conditions: v: CH2(CN)2, C2H5OH; vi:
NaBH4, CH3OH; vii: K2CO3, DMF, Ar; viii: CH3MgI, CuI, THF 0 .
S
S
N
S
N
S
N
N
S
H
S
S
S
N
N
17
18
19
Scheme 3. The compounds of 17, 18 and 19
2. Results and Discussion
2.1 Chemistry
In the scheme ꢁ, the commercially available nitrogen-containing heterocycles
with sulfydryl 1 served as convenient starting materials that were alkylated with
4-bromo-1,1,2-trifluorobutene-1-ene. Thiol 1 was converted to the corresponding
potassium salt in the presence of K2CO3, and the resulting salt was subjected to a
nucleophilic substitution upon 4-bromo-1,1,2-trifluorobutene-1-ene to obtain thioether
compounds 2. It has been reported that use of strong bases and reaction temperatures
over 55-60。C led to concurrent formation of small amounts of N-alkylated products
[25]. Therefore the S-alkylation reaction of hetaryl thiols was carried out with good
yield of 60-80% at room temperature in the present of K2CO3. The bonds of
heterocycle were not easily broken because K2CO3 was slightly soluble in organic
solvent.
The compounds 2 were selective oxidized by hydrogen peroxide in CH3COOH
or introduced chlorine atom by NCS. Although water was also used as the oxidation
reaction solvent, but further oxidation of the product sulfoxide 3 is difficult under the
conditions [26]. The oxidation reaction (ii and iii) illustrate that hydrogen peroxide,
without the help of a catalyst, could slowly oxidize thioanisole to the sulfoxide 3 at
0°C and to sulfones 4 at room temperature with an excellent selectivity in CH3COOH.
Hydrogen peroxide is the best fit oxidant due to its effective oxygen content, low cost,
safety in storage and operation and, mainly, the environmentally friendly character. It
gave the expected result with good yield of 50-70%.
In the scheme ꢁꢁ, the first step was a Knoevenagel condensation between
malononitrile and an aromatic aldehyde or ketone 12 at room temperature in the
4

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absence of catalysts to afford the arylmethylene malononitriles 13, which was isolated
in a practically pure form without further purification. The reaction is usually
catalyzed by various amines or their corresponding ammonium salts [27]. But the
method (v) we adopted was achieved at room temperature without catalysts. The
reaction was effective in less ethanol and had a good yield of 70-80%. The
intermediates 13 were reduced in a second step to afford the desired monosubstituted
malononitrile 14. The reaction (vi) was carried in ice bath which was mild enough to
tolerate a sensitive thiazole ring. The monosubstituted malononitrile 14 reacted with
4-bromo-1,1,2-trifluorobutene-1-ene in the present of KCO3 in DMF to furnish the
corresponding compounds 15. The ice bath and argon atmosphere were applied to
protect the sensitive thiazole ring
2.2 In vitro antitumor activity
The antitumor activity of synthesized trifluorobutenyl derivatives was assessed
in vitro by the MTT assay against three human cancer cell lines, human breastcancer
cell（MCF-7）, human hepatoma cell（HepG2） and human neuroblastoma cell
（SH-SY5Y）. It has been reported that noscapine, an current clinically used
antitussive drug which is used for brain tumor in drug development could cross the
blood-brain barrier and inhibit glioblastoma growth [28]. We used noscapine as the
positive control medicine in inducing SH-SY5Y cells apoptosis. Besides, the
proverbial anticancer drug taxol has been reported to induce HepG2 cells apoptosis
and was used to treat breast cancer, thus taxol serves as the positive control medicine
[29,30]. The antitumor activities were expressed in terms of IC50 (ꢀM) and
summarized in Table 1.
As shown in Table 1, among the 37 synthesized compounds，most of them
showed no obvious antitumor activity against three cancer cell lines, while
compounds 3c, 3h, 4c, 8, 9, 10, 11 showed excellent antitumor activity with IC50
values ranging from 0.4 µM to 41.5 µM. Compound 13b exhibited moderate
antitumor activity against these three cell lines. Compound 18 showed some activity
on HepG2 and MCF-7 cells, while compound 19 had certain potent antitumor activity
against SH-SY5Y cell line. Comparing with reference compound taxol against
MCF-7 and HepG2 lines （IC50 = 0.045 µM, IC50 = 0.032 µM，respectively),
compounds 3c, 3h, 4c, 8, 9, 10, 11 present lower activities. However, their antitumor
5

ACCEPTED MANUSCRIPT
potencies against SH-SY5Y cells were far better than the reference compound
noscapine（IC50 = 80.3 µM）, especially the compound 3h (IC50 = 0.4 ꢀM). In addition,
compound 19 seemed to have equipotent antitumor activity against SH-SY5Y cells
(IC50 = 69.1 ꢀM) compared to the control noscapine (IC50 = 80.3 µM). And
compound 18 showed moderate activity on HepG2 cells (IC50 = 55.0 ꢀM) and MCF-7
cells (IC50 = 89.2 µM).
The benzothiazole derivatives 3c, 4c, 8, 9, 10, 11 which have sulfones,
sulfoxides exhibited good antitumor activities against SH-SY5Y, HepG2 and MCF-7
cells. In addition, compounds 4c, 9, 11 with sulfone group present better biological
properties than compounds 3c, 8, 10 which have sulfoxide group. While,
benzothiazole derivatives 2c, 6, 7 without sulfone or sulfoxide groups showed no
antitumor activity against three cell lines with IC50＞120 µM. It also should be noted
that compounds 3h, one of benzoxazole derivative containing a sulfoxide group
presented excellent potency against all tested cell lines. While 2h, a benzoxazole
derivative without sulfone or sulfoxide group had no toxicity towards three cancer
cells; Compound 17 with poor solubility also had no inhibitory activity. From the
above aspects, it could conclude that the introduction of a disulfide bond, S-C bond
and S-N bond function on the benzothiazole or benzoxazole might produced inactive
compounds 2c, 2h, 6, 7, 17 (IC50＞120 µM), while the introduction of sulfone or
sulfoxide function might favors the anticancer activity as shown by compounds 3c,
3h,4c, 8, 9, 10, 11. The influence of substituents on benzothiazole group on the
antitumor activity might in the order, sulfone > sulfoxide > S-N bond >other groups.
In addition, thiazole compound 13b also displayed some extent of antitumor activity .
Table 1.
In vitro cytotoxic of compounds 2a-19 against three cancer cell lines
IC50 (µM) a
Compd.
SH-SY5Y
HepG2
MCF-7
2a
2b
2c
＞120 b
＞120
＞120
＞120
＞120
＞120
＞120
＞120
＞120
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2d
2e
2f
＞120
＞120
＞120
＞120
＞120
＞120
＞120
＞120
＞120
2g
2h
2i
＞120
＞120
＞120
＞120
＞120
＞120
＞120
＞120
＞120
3a
3b
3c
3d
3h
4a
4b
4c
4d
4e
4f
＞120
＞120
＞120
＞120
＞120
＞120
7.0±0.5
＞120
4.7±0.1
＞120
8.6±0.7
＞120
0.4±0.9
＞120
5.6±0.2
＞120
4.5±0.1
＞120
＞120
＞120
＞120
2.64±0.1
＞120
3.71±0.2
＞120
7.2±0.4
＞120
＞120
＞120
＞120
＞120
＞120
＞120
5a
6
＞120
＞120
＞120
＞120
＞120
＞120
7
＞120
＞120
＞120
8
2.1±0.1
6.0±0.2
41.5±0.4
4.5±0.2
＞120
6.9±1.9
6.1±1.0
13.5±3.3
4.5±0.2
＞120
12.2±0.2
8.4±0.2
17.1±0.1
10.1±0.5
＞120
9
10
11
13a
7

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13b
14a
68.8±0.5
＞120
105.4±3.7
＞120
109.8±2.5
＞120
14b
＞120
＞120
＞120
15a
＞120
＞120
＞120
15b
＞120
＞120
＞120
16b
＞120
＞120
＞120
17
＞120
＞120
＞120
18
＞120
55.00±2.6
＞120
89.2±3.2
＞120
19
69.1±0.8
c
Taxol
Noscapine
NT
0.045
0.032
80.3±2.2
NT
NT
a The cell were continuously treated with compounds for 48 h;b All data are expressed as means ± SD
from three separate determinations. IC50 values were given only if they were less than 120 ꢀM.c NT
means not tested.
Among above compounds, seven compounds (3c, 3h, 4c, 8, 9, 10, 11) gave
promising IC50 values on three cell lines. The dose-responsive curves (Figure 1) further
indicated that inhibition rates of these compounds depended upon their concentrations.
3h, 4c, 8, 10 displayed better selectivity than 3c, 9, 11 among SH-SY5Y, HepG2 and
MCF-7 cells, especially at the concentrations around the IC50 values. It is a
remarkable fact that at the concentration of 1ꢀM, compounds 4c, 8 did not lead to
antitumor activity on MCF-7 cells, but the inhibition rates on SH-SY5Y cells were all
about 50%. When the concentration of 3h reached to 5ꢀM, the inhibition rate on
MCF-7 cells was only 20%, while the inhibition rates on SH-SY5Y and HepG2 cells
were about 60%. When the concentration was 10ꢀM, compound 10 showed better
activity on HepG2 cells compared with SH-SY5Y and MCF-7 cells. Since the seven
compounds (3c, 3h, 4c, 8, 9, 10, 11) showed good anti-tumor activity, especially 3h,
4c, 8, 10 present better selectivity than compounds 3c, 9, 11 among these three cells,
they worthy further research.
8

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Compound 3c
120
100
80
60
40
20
0
Compound 3h
120
100
80
60
40
20
0
SH-SY5Y
HepG2
MCF-7
SH-SY5Y
HepG2
MCF-7
0
0.05 0.1
1
5
10 20 40 80 120
0
1
5
10
20
40
80
120
Concerntration (ꢀM)
Concentration (ꢀM)
Compound 8
100
90
80
70
60
50
40
30
20
10
0
Compound 4c
100
SH-SY5Y
HepG2
MCF-7
SH-SY5Y
Hep2
MCF-7
90
80
70
60
50
40
30
20
10
0
0
1
5
10
20
40
80
120
0
0.01
0.1
1
5
20
50
Concerntration (ꢀM)
Concentration (ꢀM)
Compound 10
100
Compound 9
SH-SY5Y
120
100
80
60
40
20
0
90
80
70
60
50
40
30
20
10
0
HepG2
MCF-7
SH-SY5Y
HepG2
MCF-7
0
10
20
40
80
120
0
1
5
10
20
40
80
120
Concentration (ꢀM)
Concentration (ꢀM)
Compound 11
100
90
80
70
60
50
40
30
20
10
0
SH-SY5Y
HepG2
MCF-7
0
0.05
0.5
5
10
20
80
120
Concentration (ꢀM)
Fig. 1. The proliferation inhibition of compounds 3c, 3h, 4c, 8, 9, 10,11 assayed by MTT.
9

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3. Conclusions
In summary, thirty-seven trifluorobutenyl derivatives have been synthesized for
bioactivity assay. The synthesis strategy have the advantages with neutral reaction
conditions and simple methodology. The biological activity evaluation indicated that
seven of them have good anti-tumor activity compared to noscapine and taxol. And it
was worth noting that the benzothiazole and benzoxazole derivatives with sulfones
and sulfoxides (3c, 3h, 4c, 8, 9, 10, 11 for example) exhibited good antitumor
activities against SH-SY5Y, HepG2 and MCF-7 cells. Even though they have less
activity than the reference compound taxol against MCF-7 and HepG2 lines, however
they all showed higher activity than the reference compound noscapine on SH-SY5Y
cells, especially the compound 3h has excellent antitumor activity (IC50 = 0.4 ꢀM) to
SH-SY5Y cells, which revealed that this compound might be a good leading for
anti-brain tumor. Besides, the seven title compounds (3c, 3h, 4c, 8, 9, 10, 11) showed
good anti-tumor activity, especially 3h, 4c, 8, 10 present better selectivity than
compounds 3c, 9, 11 among these three cells, they have the potential to be leadings
for anti-cancer agents and worthy further research.
4. Experimental Section
4.1. General Information
Melting points were determined in open capillary tubes and were uncorrected.
The products were purified by column chromatography by using silica gel (200-300
mesh).1H NMR spectra were run on a Varian-400 at room temperature with TMS as
an internal standard and CDCl3 as solvents. Mass spectra were recorded with a JEOL
MS-D 300 mass spectrometer. The reactions were monitored by TLC with ultraviolet
(UV) light; analytical thin-layer chromatography was carried out on silica gel GF254
.
All raw materials were purchased from commercial sources. Reagents were all
analytically or chemically pure. All the solvents and liquid reagents were dried by
standard methods in advance or distilled before use. We designed and efficiently
synthesized the compounds according to literature procedures.
4.2. General Procedure
10

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4.2.1 General Procedure for the Synthesis of compounds 2a-2i
Compound
1
(1 equiv), potassium carbonate (1.1 equiv) and
4-bromo-1,1,2-trifluorobutene-1-ene (1.1 equiv) are stirred in appropriate solvent at
room temperature. After the reaction reagent was completed, it is filtered and the
solvent is distilled off. The residue is dissolved in dichloromethane and washed with
5% aqueous solution of solution hydroxide and water in this order. It is dried over
unhydrous sodium sulfate and purified by column chromatography to obtain the
compounds 2a-2i.
2-((3,4,4-trifluorobut-3-en-1-yl)thio)thiazole (2a). Yield: 69.3%; 1H-NMR (400 MHz,
CDCl3), δ 2.79 (d, J = 22 Hz, 2H, CH2), 3.04 (t, J = 7.2 Hz, 2H, CH2-S), 7.24 (d, J =
3.2 Hz, 1H, CH-S), 7.68(d, J = 3.2 Hz, 1H, CH-N).
5-(pyridin-4-yl)-2-((3,4,4-trifluorobut-3-en-1-yl)thio)thiazole (2b). Yield: 69.6%; mp
35-36℃; 1H-NMR (400 MHz, CDCl3), δ 2.89 (td, J = 4 Hz, J = 22 Hz, 2H, CH2),
3.49 (t, J = 7.2 Hz, 2H, CH2-S), 7.60 (s, 1H, CH-N), 7.74 (d, J = 1.6 Hz, 1H, CH),
7.75 (d, J = 1.6 Hz, 1H, CH), 8.65 (d, J = 1.6 Hz, 1H, CH-N), 8.67 (d, J = 1.6 Hz, 1H,
CH-N). 13C-NMR (400 MHz, CDCl3), δ 26.3, 29.8, 116.0, 116.0, 120.3, 127.0, 140.6,
150.3, 150.4, 152.6, 153.6, 164.4. MS (ESI): m/z [M+H]+ calcd for C12H9F3N2S2:
302.0159. found: 303.0329.
1
2-((3,4,4-trifluorobut-3-en-1-yl)thio)benzothiazole (2c). Yield: 76.9%; H-NMR (400
MHz, CDCl3), δ 2.89 (m, 2H, CH2), 3.54 (t, J = 6.8 Hz, 2H, CH2-S), 7.31 (t, J = 3.2
Hz, H, CH), 7.42 (t, J = 3.2 Hz, H, CH), 7.76 (d, J = 8.4 Hz, H, CH), 7.87 (d, J = 8.4
Hz, H, CH).
2-((3,4,4-trifluorobut-3-en-1-yl)thio)-1H-benzoimidazole (2d). Yield: 76.9%; mp
124-125℃; 1H-NMR (400 MHz, CDCl3), δ 2.82 (m, 2H, CH2), 3.51 (t, J = 6.8 Hz, 2H,
CH2-S), 7.23 (m, 2H, CH2), 7.51 (m, 2H, CH2).
5-(difluoromethoxy)-2-((3,4,4-trifluorobut-3-en-1-yl)thio)-1H-benzoimidazole (2e).
Yield: 65.6%; mp 35-36℃; 1H-NMR (400 MHz, CDCl3), δ 2.82 (m, 2H, CH2), 3.49 (t,
J = 6.8 Hz, 2H, CH2-S), 6.49 (t, J = 74 Hz, 1H, CH-O) 7.03 (m, H, CH), 7.28 (d, J =
17.2 Hz, 1H, CH) ,7.45 (d, J = 8.4Hz, 1H, CH) 13C-NMR (400 MHz, CDCl3). δ 26.3,
11

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28.3, 106.0, 113.7, 115.5, 116.3, 118.9, 125.6, 128.0, 146.9, 150.4, 155.1; MS (ESI):
m/z [M+H]+ calcd for C12H9F5N2OS: 324.0356. found: 325.0263.
1
2-((3,4,4-trifluorobut-3-en-1-yl)thio)pyridine (2f). Yield: 68%; H-NMR (400 MHz,
CDCl3), δ 2.72 (m, 2H, CH2), 3.36 (t, J =7.2 Hz, 2H, CH2-S), 6.99 (m, 1H, CH), 7.17
(d, 1H, CH), 7.48 (m, 1H, CH), 8.42 (d, 1H, CH).
5-((3,4,4-trifluorobut-3-en-1-yl)thio)-1,3,4-thiadiazole-2-thiol (2g). Yield: 55.6%; mp
1
66-67℃; H-NMR (400 MHz, CDCl3), δ 2.78 (m, 2H, CH2), 3.33 (m, 2H, CH2-S),
11.03 (s, 1H, SH).
2-((3,4,4-trifluorobut-3-en-1-yl)thio)benzooxazole (2h). 1H-NMR (400 MHz, CDCl3),
δ 2.91 (m, 2H, CH2), 3.47 (t, J = 3.2 Hz, 2H, CH2-S), 7.28 (m, 2H, 2 CH), 7.451 (m,
1H, CH), 7.60 (m, 1H, CH)
3-methyl-6-(pyridin-4-yl)-2-((3,4,4-trifluorobut-3-en-1-yl)thio)pyrimidin-4(3H)-one
(2i). Yield: 76.5%; mp 120-121℃; 1H-NMR (400 MHz, CDCl3), δ 2.86 (m, 2H, CH2),
3.50 (t, J = 7.2 Hz, 2H, CH2-S), 3.56 (s, 3H, CH3-N), 6.75 (s, 1H, CH), 7.77 (d, J =
1.6 Hz, 1H, CH), 7.78 (d, J = 1.6 Hz, 1H, CH), 8.73 (d, J = 1.6 Hz, 1H, CH-N), 8.74
(d, J = 1.6 Hz, 1H, CH-N). 13C-NMR (400 MHz, CDCl3), δ 25.9, 28.1, 30.3, 106.1,
120.5, 127.0, 143.4, 150.5, 153.6, 156.4, 162.1, 162.2. MS (ESI): m/z [M+H]+ calcd
for C14H12F3N3OS: 327.0653. found: 328.0764
4.2.2 General Procedure for the Synthesis of compounds 3a-3d, 3h and 10.
To the solution of compounds 2 (1 equiv), 31% hydrogen peroxide water (1
equiv), acetic acid is mixture is added and stirred at ice cooling for certain hours.
When the reagent was completed, the reaction mixture is cooling to 5 and adjusted
to pH 6 by adding an appropriate amount of an aqueous solution hydroxide, diluted
with water and extracted three times with dichloromethane. The dichloromethane
layer is washed with water, 10% sodium thiosulfate and water in this order and dried
over unhydrous sodium sulfate. The solvent is distilled off and the concentrate is
purified by column chromatography to obtain the compounds 3a-3d, 3h and 10.
12

ACCEPTED MANUSCRIPT
1
2-((3,4,4-trifluorobut-3-en-1-yl)sulfinyl)thiazole (3a). Yield: 66.7%; H-NMR (400
MHz, CDCl3), δ 2.76 (m, 2H, CH2), 3.37 (m, 2H, CH2-S), 7.68 (t, J = 3.2 Hz, 1H,
CH-S), 7.99 (t, J = 3.2 Hz, 1H, CH-N). 13C-NMR (400 MHz, CDCl3), δ 18.3, 51.2,
124.1, 126.3, 145.2, 153.1, 174.5. MS (ESI): m/z [M+H]+ calcd for C7H6F3NOS2:
240.9843. found: 241.9800.
5-(pyridin-4-yl)-2-((3,4,4-trifluorobut-3-en-1-yl)sulfinyl)thiazole (3b) Yield: 65.7%;
mp 87-88℃; 1H-NMR (400 MHz, CDCl3), δ 2.83 (m, 2H, CH2), 3.44 (m, 2H, CH2-S),
7.75 (d, J = 1.6 Hz, 1H, CH), 7.76 (d, J = 1.6 Hz, 1H, CH), 8.03 (s, 1H, CH-N), 8.71
(d, J = 1.6 Hz, 1H, CH-N), 8.72 (d, J = 1.6 Hz, 1H,CH-N). 13C-NMR (400 MHz,
CDCl3), δ 18.3, 51.4, 120.6, 120.7, 126.2, 140.3, 150.8, 153.1, 155.6, 175.7. MS (ESI):
m/z [M+H]+ calcd for C12H9F3N2OS2: 318.0108. found: 319.0143
2-((3,4,4-trifluorobut-3-en-1-yl)sulfinyl)benzothiazole (3c). 1H-NMR (400 MHz,
CDCl3), δ 2.83 (m, 2H, CH2), 3.46 (m, 2H, CH2-S), 7.51 (t, J = 7.2 Hz, 1H, CH), 7.58
(t, J = 7.2 Hz, 1H, CH), 8.01 (d, J = 8.0 Hz, 1H, CH), 8.08 (d, J = 8.0 Hz, 1H, CH).
13C-NMR(400 MHz, CDCl3), δ 18.4, 51.3, 122.5, 124.3, 125.5, 126.6, 127.3, 136.2,
153.2, 154.1, 176.2. MS (ESI): m/z [M+H]+ calcd for C11H8F3NOS2: 290.9999. found:
292.0088.
2-((3,4,4-trifluorobut-3-en-1-yl)sulfinyl)-1H-benzoimidazole (3d). Yield: 69.8%; mp
1
84-85℃; H-NMR (400 MHz, CDCl3), δ 2.82 (m, 2H, CH2), 3.57 (m, 2H, CH2-S),
7.37 (m, 2H, 2CH), 7.59 (d, J = 7.6Hz, 1H, CH), 7.80 (d, J = 7.6Hz, 1H, CH), 11.87
(s, 1H, NH). 13C-NMR (400 MHz, CDCl3), δ 18.5, 49.7, 112.0, 120.3, 123.4, 124.6,
127.2, 134.2, 144.1, 151.2, 152.9. MS (ESI): m/z [M+H]+ calcd for C11H9F3N2OS:
274.0388. found: 275.0472.
2-((3,4,4-trifluorobut-3-en-1-yl)sulfinyl)benzooxazole (3h). Yield: 28.6%; mp
1
66-68℃; H-NMR (400 MHz, CDCl3), δ 2.93 (m, 2H, CH2), 3.58 (m, 2H, CH2-S),
7.48 (m, 2H, 2CH), 7.66 (d, J = 8.0 Hz, 1H, CH), 7.84 (d, J = 8.0 Hz, 1H, CH).
6-chloro-2-((3,4-dichloro-3,4,4-trifluorobutyl)sulfinyl)benzothiazole (8) Yield: 25.0%;
mp 90-92℃; 1H-NMR (400MHz, CDCl3), δ 2.86 (m, 2H, CH2), 3.58 (m, 2H, CH2-S),
7.56 (d, J =8.8 Hz, 1H, CH), 8.00 (d, J =8.8 Hz, 1H, CH), 8.02 (s, 1H, CH). 13C-NMR,
13

ACCEPTED MANUSCRIPT
δ 28.8, 49.0, 109.8, 122.2, 125.1, 128.1, 128.4, 133.1, 137.4, 152.6, 176.4. MS (ESI):
m/z [M+H]+ calcd for C11H7Cl3F3NOS2: 394.8987. found: 395.9087.
2-((3,4-dichloro-3,4,4-trifluorobutyl)sulfinyl)benzothiazole (10). Yield: 31.5%; mp
1
70-72℃; H-NMR (400 MHz, CDCl3), δ 2.73 (m, 2H, CH2), 3.60 (m, 2H, CH2-S),
7.53 (t, J = 7.2 Hz, 1H, CH), 7.60 (t, J = 7.2 Hz, 1H, CH), 8.03 (d, J = 7.2 Hz, 1H,
CH), 8.10 (d, J = 7.2 Hz, 1H, CH); 13C-NMR(400 M Hz, CDCl3), δ 28.7, 48.7, 109.7,
122.3, 124.2, 125.2, 126.5, 127.2, 136.0, 153.9, 175.5. MS (ESI): m/z [M+H]+ calcd
for C11H8Cl2F3NOS2: 360.9376. found: 361.9370.
4.2.3 General Procedure for the Synthesis of compounds 4a-4f and 11.
To the solution of compounds 2 (1 equiv), 31% hydrogen peroxide water (3
equiv), acetic acid is mixture is added and stirred at room temperature for certain
hours. When the sulfoxide (the first product) is completed, the reaction mixture is
cooling to 5 and adjusted to pH 6 by adding an appropriate amount of an aqueous
solution hydroxide, diluted with water and extracted three times with dichloromethane.
The dichloromethane layer is washed with water, 10% sodium thiosulfate and water
in this order, and dried over unhydrous sodium sulfate. The solvent is distilled off and
the concentrate is purified by column chromatography to obtain the compounds 4a-4f.
1
2-((3,4,4-trifluorobut-3-en-1-yl)sulfonyl)thiazole (4a). Yield: 60.5%; H-NMR (400
MHz, CDCl3), δ 2.76 (m, 2H, CH2), 3.37 (m, 2H, CH2-S), 7.68 (t, J = 3.2 Hz, 1H,
CH-S), 7.99 (t, J = 3.2 Hz, 1H, CH-N), 13C-NMR (400 MHz, CDCl3), δ 18.2, 51.2,
124.2, 126.4, 145.2, 153.1, 174.4, MS (ESI): m/z [M+H]+ calcd for C7H6F3NO2S2:
256.9792. found: 257.9856.
5-(pyridin-4-yl)-2-((3,4,4-trifluorobut-3-en-1-yl)sulfonyl)thiazole (4b). Yield: 52.1%;
1
mp 78-79℃; H-NMR (400 MHz, CDCl3) ,δ 2.98 (m, 2H, CH2), 3.73 (t, J = 7.2Hz,
2H, CH2-S), 7.80 (d, J = 1.6 Hz, 1H, CH), 7.81 (d, J = 1.6 Hz, 1H, CH), 8.11 (s, 1H,
CH-N), 8.73 (d, J = 1.6 Hz, 1H, CH-N), 8.74 (d, J = 1.6 Hz, 1H, CH-N). 13C-NMR
(400 MHz, CDCl3), δ 20.3, 50.7, 120.8, 122.8, 124.9, 139.7, 150.8, 153.0, 155.6,
165.6. MS (ESI): m/z [M+H]+ calcd for C12H9F3N2O2S2: 334.0058. found: 335.0094
2-((3,4,4-trifluorobut-3-en-1-yl)sulfonyl)benzothiazole (4c). Yield: 63.9%; mp
1
52-54℃; H-NMR (400 MHz, CDCl3), δ 2.97 (m, 2H, CH2), 3.74 (t, J = 7.6 Hz, 2H,
14

ACCEPTED MANUSCRIPT
CH2-S), 7.64 (m, 2H, 2CH), 8.03 (d, J = 8.0 Hz, 1H, CH), 8.22 (d, J = 8.0 Hz, 1H,
CH). 13C-NMR(400 MHz, CDCl3), δ 20.2, 50.6, 122.5, 125.4, 125.7, 128.0, 128.5,
136.9, 152.7, 153.2, 165.1.MS (ESI): m/z [M+H]+ calcd for C11H8F3NO2S2: 306.9949.
found: 308.0064.
2-((3,4,4-trifluorobut-3-en-1-yl)sulfonyl)-1H-benzoimidazole (4d). Yield: 65.2%; mp
1
82-84℃; H-NMR (400 MHz, CDCl3), δ 2.95 (m, 2H, CH2), 3.74 (m, 2H, CH2-S),
7.43 (m, 1H, CH), 7.49 (m, 1H, CH), 7.55 (d, J = 8 Hz, 1H, CH), 7.90 (d, J = 7.6 Hz,
1H,CH), 10.66 (s, 1H, NH). 13C-NMR (400 MHz, CDCl3), δ 19.9, 50.6, 112.4, 121.8,
124.2, 124.6,126.8, 132.9, 142.9, 146.3, 152.9. MS (ESI): m/z [M+H]+ calcd for
C11H9F3N2O2S: 290.0337. found: 291.0424.
5-(difluoromethoxy)-2-((3,4,4-trifluorobut-3-en-1-yl)sulfonyl)-1H-benzoimidazole
1
(4e). Yield: 60.6%; mp 78-79℃; H-NMR (400 MHz, CDCl3), δ 2.96 (m, 2H, CH2),
3.74 (t, J = 7.6 Hz, 2H, CH2-S), 6.56 (t, J = 73.2 Hz, 1H, CH-F) 7.27 (m, 1H, CH),
7.59 (m, 1H, CH), 7.86 (m, 1H, CH), 10.81 (s, 1H, NH). 13C-NMR (400 MHz,
CDCl3), δ 19.8, 50.6, 103.3, 112.7, 118.3, 122.9, 125.1, 130.5, 133.2, 140.3, 143.1,
147.7, 152.9. MS (ESI): m/z [M+H]+ calcd for C12H9F5N2O3S: 356.0254. found:
357.0566.
1
2-((3,4,4-trifluorobut-3-en-1-yl)sulfonyl)pyridine (4f). Yield: 51.3%; H-NMR (400
MHz, CDCl3), δ 2.83 (m, 2H, CH2), 3.63 (t, J = 7.6Hz, 2H, CH2-S), 7.60 (m, 1H, CH),
8.01 (m, 1H, CH), 8.11 (d, 1H, CH), 8.76 (d, 1H, CH). 13C-NMR (400 MHz, CDCl3),
δ 20.0, 47.7, 122.1,125.8, 127.8, 138.5, 150.4, 153.1, 157.1. MS (ESI): m/z [M+H]+
calcd for C9H8F3NO2S: 251.0228. found: 252.0394.
6-chloro-2-((3,4-dichloro-3,4,4-trifluorobutyl)sulfonyl)benzothiazole
(9)
Yield:
1
29.6%; mp 118-120℃; H-NMR (400MHz, CDCl3), δ 3.00 (m, 2H, CH2), 3.84 (m,
2H, CH2-S), 7.63 (d, J =8.8 Hz, 1H, CH), 8.03 (s, 1H, CH), 8.15 (d, J =8.8 Hz, 1H,
CH). 13C-NMR, δ 30.6, 49.2, 109.3, 122.1, 125.3, 126.6, 129.3, 135.1, 137.9, 151.1,
165.3. MS (ESI): m/z [M+H]+ calcd for C11H7Cl3F3NO2S2: 410.8936. found:
411.9629.
2-((3,4-dichloro-3,4,4-trifluorobutyl)sulfonyl)benzothiazole (11). Yield: 38.5%; mp
87-88℃; 1H-NMR (400 MHz, CDCl3), δ 2.96 (m, 2H, CH2), 3.87 (m, 2H, CH2-S),
7.65 (m, 2H, 2CH), 8.04 (d, J=8.0Hz, 1H, CH), 8.25 (d, J=8.0Hz, 1H, CH), 13C-NMR
15

ACCEPTED MANUSCRIPT
(400 MHz, CDCl3), δ 30.5, 49.0, 109.1, 122.3, 125.1, 125.6, 127.9, 128.4, 136.6,
152.5, 164.6. MS (ESI): m/z [M+H]+ calcd for C11H8Cl2F3NO2S2: 376.9326. found:
377.9415.
4.2.4 General Procedure for the Synthesis of compounds 5a, 6 and 7.
To a stirred solution of compounds 2 (1 equiv)in carbon tetrachloride
N-chlorosuccinimide (1.1 equiv) is added. The mixture was refluxed for appropriate
hours by heating. As soon as the reaction has reached room temperature, the mixture
is filtered and the solvent is distilled off. The concentrate is purified by column
chromatography to obtain the compounds 5a, 6 and 7.
5-chloro-2-((3,4,4-trifluorobut-3-en-1-yl)thio)thiazole (5a). Yield: 66%; 1H-NMR
(400 MHz, CDCl3), δ 2.76 (td, J = 4 Hz, J = 24.4 Hz, 2H, CH2), 3.36 (t, J = 7.2 Hz,
2H, CH2-S), 7.45 (s, 1H, CH-N).
6-chloro-2-((3,4-dichloro-3,4,4-trifluorobutyl)thio)benzothiazole (6) Yield: 27.5%;
1H-NMR (400 MHz, CDCl3), δ 2.86 (m, 2H, CH2), 3.59 (m, 2H, CH2-S), 7.38 (m, 1H,
CH), 7.73 (m, 1H, CH), 7.77 (m, 1H, CH). 13C-NMR(400 MHz, CDCl3), δ 26.3, 36.8,
110.0, 120.6, 122.3, 125.4, 126.8, 136.4, 151.6, 162.5, 165.6. MS (ESI): m/z [M+H]+
calcd for C11H7Cl3F3NS2: 378.9038. found: 379.9102.
1
2-((3,4-dichloro-3,4,4-trifluorobutyl)thio)benzothiazole (7). Yield: 33.6%; H-NMR
(400MHz, CDCl3), δ 2.91 (m, 2H, CH2), 3.61 (m, 2H, CH2-S), 7.31 (t, J = 7.2 Hz, 1H,
CH), 7.43 (t, J = 7.2 Hz, 1H, CH), 7.76 (d, J = 8.0 Hz, 1H, CH), 7.88 (d, J = 8.0 Hz,
1H, CH). 13C-NMR, δ 26.5, 37.1, 110.2, 121.2, 121.9, 124.7, 125.7, 126.3, 135.5,
153.2, 165.1. MS (ESI): m/z [M+H]+ calcd for C11H8Cl2F3NS2: 344.9427. found:
345.9492.
3.2.5 General Procedure for the Synthesis of compounds 13.
To a stirred solution of compounds 12 (1 equiv) in absolute ethyl alcohol the
malononitrile (1.1 equiv) was added. The mixture was stirred at room temperature for
times . Without any further purification, the precipitate was filtered off to give the
compounds 13.
16

ACCEPTED MANUSCRIPT
2-(thiazol-2-yꢀMethylene)malononitrile (13a). Yield: 70.3%; mp 143-145 ℃ ;
1H-NMR (400 MHz, CDCl3), δ 7.88 (d, J = 3.2 Hz, 1H, CH-S), 8.02 (s, 1H, CH), 8.24
(d, J = 2.8 Hz ,1H, CH-N)
2-(1-(thiazol-2-yl)ethylidene)malononitrile (13b). Yield: 75.7%; mp 121-122 ℃;
1H-NMR (400 MHz, CDCl3), δ 2.83 (s, 3H, CH3), 7.81 (d, J = 3.2 Hz, 1H, CH-S),
8.18 (d, J = 3.2 Hz, 1H, CH-N). 13C-NMR (400 MHz, CDCl3), δ 22.0, 83.5, 112.6,
112.8, 126.0, 145.5, 160.5, 161.3. MS (ESI): m/z [M+H]+ calcd for C8H5N3S:
175.0204. found: 176.0279.
4.2.6 General Procedure for the Synthesis of compounds 14.
To a stirred solution of compound 13 (1 equiv) in methyl alcohol a suspension of
sodium borohydride (1.1 equiv ) in methyl alcohol was added at ice bath. The reaction
mixture was stirred for another 30 min as monitored by TLC and quenched with water
10% hydrochloric acid. After evaporation of volatile material in vacuo the remaining
crude product was dissolved in dichloromethane. The organic layer was successively
washed with 10% hydrochloric acid, a saturated aqueous sodium chloride solution,
dried over anhydrous magnesium sulfate, and concentrated under reduced pressure.
The residue was subjected to column chromatography to give the compounds 14.
2-(thiazol-2-yꢀMethyl)malononitrile (14a). Yield: 57.9%; 1H-NMR (400 MHz,
CDCl3), δ 3.71 (d, J = 7.6 Hz, 2H, CH2), 4.55 (t, J = 7.2 Hz, 1H, CH), 7.38 (d, J = 3.2
Hz, 1H, CH-S), 7.81 (d, J = 3.2 Hz, 1H, CH-N).
2-(1-(thiazol-2-yl)ethyl)malononitrile (14b). Yield: 62.1%; 1H-NMR (400 MHz,
CDCl3), δ 1.78 (d, 3H, CH3), 3.84 (m, 1H, CH-CH3) 4.66 (d, 1H, J = 5.6 Hz, CH-CN),
7.38 (d, J = 3.2 Hz, 1H, CH-S), 7.80 (d, J = 3.2 Hz, 1H, CH-N). 13C-NMR (400 MHz,
CDCl3), δ 17.8, 28.6, 39.0, 111.0, 111.9, 120.1, 143.0, 167.0. MS (ESI): m/z [M+H]+
calcd for C8H7N3S: 177.0361. found: 178.0495.
4.2.7 General Procedure for the Synthesis of compounds 15.
The mixture of compounds 14 (1 equiv) and potassium carbonate (1.1 equiv)
were dissolved in DMF (N,N-Dimethylf-ormamide) was stirred at certain temperature
17

ACCEPTED MANUSCRIPT
for 1h under an argon atmosphere. Subsequently, 4-bromo-1,1,2-
trifluorobutene-1-ene (1.1 equiv) were added and the mixture was stirred 10 hours at
room temperature. After evaporation of volatile material in vacuo the remaining crude
product was dissolved in dichloromethane, washed with water and dried over
anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The
residue was subjected to column chromatography to obtain the compounds 15.
2-(thiazol-2-yꢀMethyl)-2-(3,4,4-trifluorobut-3-en-1-yl)malononitrile (15a). Yield:
29.5%; mp 49-51℃; 1H-NMR (400 MHz, CDCl3), δ 2.37 (m, 2H, CH2), 2.76 (m, 2H,
CH2), 3.72 (s, 2H, CH2), 7.34 (d, J = 3.2 Hz, 1H, CH-S), 7.87 (d, J = 3.2 Hz, 1H,
CH-N). 13C-NMR (400 MHz, CDCl3), δ 22.5, 33.0, 36.7, 39.6, 114.1, 121.2, 121.3,
126.1, 143.9, 153.2, 158.9. MS (ESI): m/z [M+H]+ calcd for C11H8F3N3S: 271.0391.
found: 272.0521.
2-(1-(thiazol-2-yl)ethyl)-2-(3,4,4-trifluorobut-3-en-1-yl)malononitrile(15b).
Yield:
1
32.5%; H-NMR (400 MHz, CDCl3), δ 1.84 (d, J = 7.2 Hz, 3H,CH3), 2.21 (m,2H,
CH2), 2.71 (m, 2H, CH2), 3.82 (q, 1H, CH), 7.42 (d, J = 3.2 Hz, 1H, CH-S), 7.84 (d, J
= 3.2 Hz, 1H, CH-N). 13C-NMR (400 MHz,CDCl3), δ 18.5, 22.7, 32.0, 42.6, 44.2,
113.5, 114.3, 120.6, 126.3, 143.3, 153.2, 165.5. MS (ESI): m/z [M+H]+ calcd for
C12H10F3N3S: 285.0548. found: 286.0622.
4.2.8 Synthesis of compound 16b.
To a stirred solution of compound 13b (200mg, 1.14mmol) and a catalytic amount
of copper (I) iodide in dry THF (6ml), a solution of methyl mangnesium iodide
(4.46ml, 1.25mmol) in diethyl ether (prepared from 0.30 g of magnesium, 10ml of
diethyl ether, and 0.86 ml of methyl iodide ) was added at ice cooling under an argon
atmosphere. The reaction mixture was stirred for another 1 h as monitored by TLC
and quenched with water 10% hydrochloric acid. After evaporation of volatile
material in vacuo the remaining crude product was dissolved in dichloromethane. The
organic layer was successively washed with 10% hydrochloric acid, a saturated
aqueous sodium chloride solution, dried over anhydrous magnesium sulfate and
concentrated under reduced pressure. The residue was subjected to column
chromatography to give the compounds 16b (91mg).
18

ACCEPTED MANUSCRIPT
2-(2-(thiazol-2-yl)propan-2-yl)malononitrile (16b). Yield: 41.7%; mp 46-48 ℃;
1H-NMR (400 MHz, CDCl3), δ 1.75 (s, 6H, 2CH3), 4.63 (s, 1H, CH-CN) 7.35 (d, J =
13
3.2 Hz, 1H, CH-S), 7.77 (d, J = 3.2 Hz, 1H, CH-N). C-NMR (400 MHz, CDCl3), δ
26.5, 34.5, 43.6, 111.5, 119.8, 119.9, 142.9, 172.1. MS (ESI): m/z [M+H]+ calcd for
C9H9N3S: 191.0517. found: 192.0684.
4.2.8 Synthesis of 2,2'-Dithiobis(benzothiazole) (17).
To a stirred solution of benzothiazole-2-thiol(334 mg, 2 mmol) in acetonitril (3
mL), the catalyst (Fe/SBA-15, 30 mg, 0.02 mmol) and hydrogenperoxide (30%
aqueous, 250 mg) were added. The reaction mixture was stirred for 15 min. After the
completion of the reaction, which is associated with precipitation of disulfide and
elimination of thiol odor. The precipitate was filtered off to give
2,2'-Dithiobis(benzothiazole) (228 mg). Yield: 68.6%; mp 177-179℃ (177-180℃
[23])
4.2.9 General Procedure for the Synthesis of compounds 18,19.
To a stirred solution of benzothiazole-2-thiol(1 equiv) and cyclohexane (2.5
equiv), the sodium hypochlorite solution was added at 45℃. After the completion of
the reaction, the reaction solution was cooled to room temperature and the products
separated out. Without any further purification, the precipitate was filtered off to give
the products.
N-cyclohexylbenzothiazole-2-sulphenamide (18). Yield: 65.7%; mp 100-102 ℃
(93-100℃[24])
N,N'-dicyclohexyl-2-Benzothiazole sulfenamide (19).Yield: 61.2%; mp 101-103℃
(102-104℃[24])
4.3. Cell Culture and Cell viability Assay
3.3.1 Cell culture. MCF-7 and SH-SY5Y cell lines were maintained in DMEM
supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin. The
HepG2 cells were maintained in MEM supplemented with 10% fetal bovine serum,
1% penicillin and streptomycin. The cells were grown at 37 °C in an environment of
5% CO2.
19

ACCEPTED MANUSCRIPT
4.3.2 Cell viability assay. Cell viability was assessed by MTT assay. After grown
overnight in 96-well plates, the cells were treated with indicated concentrations of
compounds for 48 h in DMEM (MCF-7 and SH-SY5Y) or MEM (HepG2)
supplemented with 10% fetal bovine serum. The cells were washed with PBS twice
and then incubated with serum-free DMEM containing 0.5 mg/ml MTT for 4 h. Next,
the cells were washed with PBS again and the formazan blue formed by living cells
was dissolved in DMSO. Finally, the optical density was measured at 570 nm.
Supplementary Materials
Supplementary materials can be accessed at:
Acknowledgments
The project was supported by the National High-Tech Program of China (863 Project
No. 2012AA020306).
Conflicts of Interest
The authors declare no conflict of interest.
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