New mild and simple approach to isothiocyanates: A class of potent anticancer agents

Article abstract of DOI:10.3390/molecules22060773

In our current work, acetyl chloride-mediated synthesis of phenethyl isothiocyanate (PEITC) derivatives proves to be convenient and provides the expected products at good to excellent yields. Biological evaluation and structure-activity relationship analysis found that the novel compound 7 showed the best anticancer activity against human cancer cell line Panc1 and HGC27 compared with PEITC. Compounds 6 and 7 induced more apoptosis in pancreatic cancer cells but less toxicity in non-cancer cells. Further biological study demonstrated that 7 substantially increased intracellular reactive oxygen species (ROS) and depleted glutathione (GSH), leading to an oxidative stress to kill cancer cell.

Full text of DOI:10.3390/molecules22060773

molecules
Article
New Mild and Simple Approach to Isothiocyanates:
A Class of Potent Anticancer Agents
Bingling Luo 1,2,†, Jiankang Wang
1,2,†
, Xiaobing Li , Wenhua Lu , Jing Yang , Yumin Hu ,
1
1
1
1
Peng Huang 1 and Shijun Wen 1,2,*
,2
1
Sun Yat-Sen University Cancer Center; State Key Laboratory of Oncology in South China;
Collaborative Innovation Center for Cancer Medicine; Sun Yat-Sen University, 651 Dongfeng East Road,
Guangzhou 510060, China; luobl@sysucc.org.cn (B.L.); wangjk6@mail2.sysu.edu.cn (J.W.);
lixiaobing0629@126.com (X.L.); luwenh@sysucc.org.cn (W.L.); yangjing@sysucc.org.cn (J.Y.);
huym@sysucc.org.cn (Y.H.); huangpeng@sysucc.org.cn (P.H.)
2
School of Pharmaceutical Sciences, Sun Yat-sen University, 132 Waihuan East Road,
Guangzhou 510006, China
*
Correspondence: wenshj@sysucc.org.cn; Tel.: +86-20-3994-3091
†
The authors contributed equally to this work.
Academic Editors: Grzegorz Wegrzyn, Luciano Saso, László Dux and Tamás Csont
Received: 18 March 2017; Accepted: 27 April 2017; Published: 1 June 2017
Abstract: In our current work, acetyl chloride-mediated synthesis of phenethyl isothiocyanate (PEITC)
derivatives proves to be convenient and provides the expected products at good to excellent yields.
Biological evaluation and structure-activity relationship analysis found that the novel compound
showed the best anticancer activity against human cancer cell line Panc1 and HGC27 compared with
PEITC. Compounds and induced more apoptosis in pancreatic cancer cells but less toxicity in
non-cancer cells. Further biological study demonstrated that substantially increased intracellular
7
6
7
7
reactive oxygen species (ROS) and depleted glutathione (GSH), leading to an oxidative stress to kill
cancer cell.
Keywords: isothiocyanate; anticancer; reactive oxygen species; glutathione
1
. Introduction
Isothiocyanates are important natural compounds found in cruciferous vegetables such as
watercress and broccoli, and they have shown cancer prevention effects [
also reported that these natural isothiocyanates can induce apoptosis of cancer cells, although its
mechanism is still under debate [ ]. Among these reported natural compounds, one of the most
studied is phenethyl isothiocyanate (PEITC). Packham’s group found that PEITC could target mTORC1
activity in leukemia cells [ ], and Chung and his co-workers reported that isothiocyanates selectively
depleted mutant p53, compared with wild type p53 [ ]. In our previous study, PEITC showed a great
selectivity in killing oncogenically transformed cells over normal cells through a reactive oxygen
species (ROS)-mediated mechanism [10 12], indicating that PEITC could hold therapeutic promise
1,2]. Meanwhile, it is
3–6
7,8
9
–
for the treatment of cancer patients. Indeed, PEITC has been registered for clinical trials for cancer
prevention and treatment (ClinicalTrials.gov Identifiers: NCT00005883, NCT00691132, NCT01790204).
Due to the potential cancer preventive and therapeutic benefits of PEITC and other isothiocyanates,
both biological evaluation and chemical synthesis of these unique compounds have attracted more
interest [9,13].
Apart from their potential anticancer effect, isothiocyanates are also valuable intermediates to
obtain thioureas. The unique pharmacological properties and structural features of isothiocyanates
attract the interest of chemists, and a number of their synthetic routes have been developed.
Molecules 2017, 22, 773; doi:10.3390/molecules22060773
www.mdpi.com/journal/molecules

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The reported methods include using toxic thiophosgene [14], or less toxic but expensive thiophosgene
0
surrogate reagents such as 1,1 -thiocarbonyldiimidazole [15], to incorporate one carbon atom and
one sulfur atom. The alternative incorporation of these two atoms employs carbon disulfide
through the decomposition of the in situ-formed dithiocarbamate salt intermediates [16
In this strategy, several reagents have been reported, for example, dicyclohexylcarbodiimide [16],
-chloro-1,3-dimethylimidazo-liniumchloride [17], and the more recently reported di-tert-butyl
–18].
2
dicarbonate [18] and tosyl chloride [19]. It is of importance to develop novel and potent isothiocyanates
via the optimization of PEITC, while few analogues of PEITC have shown outstanding anticancer
activities regarding their in vitro IC50 values. In this paper, we report a concise method to prepare
isothiocyanates and conduct a biological evaluation of these compounds. Finally, we find that
compound
ROS levels.
7 showed the best anti-proliferative activity via modulating the increase of intracellular
2
. Results and Discussion
2
.1. Chemistry
During our initial study, the preparation of polar isothiocyanates using tosyl chloride did not
encounter any problems. However, in the synthesis of less polar isothiocyanates, a slight excess of tosyl
chloride was always difficult to remove from the products by flash chromatography. This problem
hampered our progress in obtaining a variety of compounds, thus it is of high interest to find
an alternative concise method of doing so. To our best knowledge, there is not yet a generally
conceptualized mechanism for the decomposition of dithiocarbamic acid salt mediated by the previously
reported reagents. Clearly, the in situ formed dithiocarbamate salt is a potential nucleophile which could
react with a nucleophilic receptor, i.e., an electrophile, to form an intermediate A (Scheme 1). Mediated
by bases such as triethylamine, an isothiocyanate can be formed after one molecular hydrogen sulfide
is eliminated from A and transported to the electrophile. Thus, we envisioned that some electrophiles
might be able to mediate the production of isothiocyanates (Scheme 1).
Scheme 1. The potential mechanism for the synthesis of isothiocyanates.
To validate our hypothesis that other electrophiles can replace tosyl chloride, a series of
conventional reagents including acetyl chloride (AcCl), tosyl chloride (TsCl), dimethyldichlorosilane
(
SiMe Cl ), benzoyl chloride (BzCl) and sulfonyl chloride (SO Cl ) were screened for the synthesis of
2 2 2 2
PEITC from phenethylamine (Table 1). Indeed, all reagents resulted in the expected product in modest
to good yields, ensuring that the decomposition of the dithiocarbmate was mediated by its nucleophilic
attack on an oncoming electrophile in step 2 (Scheme 1). While the excess tosyl chloride cannot be
cleared after the reaction work-up, causing an issue with the purification of low polar isothiocyanates
by flash chromatography, acetyl chloride could avoid such a problem and also provided excellent
yields. Thus, acetyl chloride was employed to prepare various isothiocyanates in this current work.
It is noteworthy that analytic grade tetrahydrofuran (THF) and triethylamine might be used in the late
synthesis of some isothiocyanates although the reactions were performed under anhydrous conditions
in the screening study. It was also found that triethylamine could be replaced by inorganic bases such
as potassium carbonate (data not shown).

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Table 1. Synthesis of phenethyl isothiocyanate mediated by various reagents.
1
2
) CS , Et N
NCS
NH2
2
3
) reagents
PEITC
Entry
1
2
3
4
5
Reagent
Yield
AcCl
94%
TsCl
70%
SiMe Cl
BzCl
42%
SO Cl
80%
2
2
2
2
79%
2 3
Reagents and Conditions: (1) phenylethylamine (1 equiv.), CS (1.2 equiv.), and Et N (3 equiv.) in anhydrous THF,
◦
◦
C–r.t.; (2) reagents (1.2 equiv.) was added, 0 C–r.t.
0
During our study, the preparation of isothiocyanates
problems when tosyl chloride was applied. However, the purification of the low polar isothiocyanates
encountered difficulty in the full removal of excess tosyl chloride from the compounds. This problem
was successfully avoided when acetyl chloride was employed (Figure 1). The remaining isothiocyanates
20 were also easily prepared at excellent yields, similar to or better than the yields obtained with tosyl
1 and 2 actually did not encounter any
3–5
6–
chloride used, as exemplified in the synthesis of 4, 6 and 7.
Figure 1. Structures of isothiocyanates and their preparation using acetyl chloride. Note: Unless
a
otherwise stated, all the yields are isolated yields. The number in the brackets indicates the yield
when prepared with tosyl chloride.

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2
.2. Biology
To design isothiocyanates with better bioactivity is a challenge, because the mechanism of their
inhibition of tumor cell growth is still debated and their exact targets remain unclear. Based on our
experience, we envisioned that aromatic rings and a chain with reasonable length might be essential
to maintain bioactivity. The substituents on the aromatic ring, varying in space size and chemical
properties, might influence the compound’s selectivity to bind to certain targets. Thus, isothiocyanates
1
and 2 (Figure 1) were firstly designed and they were prepared from a phenylalanine. Then, 3–5 were
obtained, identified by another benzene ring tethered through an oxygen or sulfur atom on either
the meta- or para-position of PEITC. Such structural variation would justify whether a bulky group
could increase its selectivity or binding affinity to certain targets. Furthermore, compounds 6 and 7
were designed and synthesized because of the possibility that such functional amides and esters might
result in an improvement of PEITC bioavailability and the formation of potential hydrogen bonds with
some potential targets.
In our preliminary study, isothiocyanates 1–7 were screened against two types of malignant cancer
cell lines, Panc 1 (pancreatic cancer) and HGC 27 (gastric cancer), since these pancreatic and gastric
cancers are fatal and have a poor prognosis (Table 2) [20–23].
Table 2. Anti-proliferative activities of isothiocyanates against two types of human cancer cell lines,
Panc1 (pancreatic cancer) and HGC27 (gastric cancer), measured by MTS assays.
IC50 (µM)
Compound
Panc1
HGC27
PEITC
10.80 ± 0.25
15.80 ± 1.06
10.87 ± 0.47
14.24 ± 0.50
6.12 ± 0.13
8.05 ± 0.36
4.55 ± 0.32
2.04 ± 0.21
5.78 ± 0.56
3.52 ± 0.11
2.68 ± 0.21
5.08 ± 0.02
8.92 ± 0.43
5.65 ± 0.16
2.25 ± 0.21
3.76 ± 0.05
6.24 ± 0.13
4.70 ± 0.27
2.78 ± 0.72
2.14 ± 0.79
4.06 ± 0.57
2.95 ± 0.41
8.79 ± 0.72
1.49 ± 0.06
2.73 ± 0.29
3.44 ± 0.23
2.67 ± 0.37
1.09 ± 0.07
0.46 ± 0.02
0.74 ± 0.01
0.84 ± 0.06
0.93 ± 0.41
1.47 ± 0.10
0.72 ± 0.21
0.99 ± 0.15
0.62 ± 0.04
1.68 ± 0.00
2.37 ± 0.43
0.77 ± 0.18
0.34 ± 0.00
0.72 ± 0.08
0.82 ± 0.06
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
0
1
2
3
4
5
6
7
8
9
0
Compared to PEITC, the new synthetic isothiocyanates
especially against Panc 1, implying that extra carboxylate function groups next to the isothiocyanate
do not contribute to the biological activity of PEITC. In isothiocyanates , additional bulky benzene
ring did not increase their anticancer activity much compared to PEITC. However, the carboxylate
functional groups in isothiocyanates and substantially improved their ability to kill cancer cells.
In particular, compound with the para amide on the benzene ring of PEITC showed the best potency
with 5 or 6 fold increasement. Based on the structure of , the subsequent isothiocyanates 20 were
1 and 2 showed worse or similar bioactivity
3–5
6
7
7
7
8–
designed and prepared in order to study their structure and activity relationship. Among the designed
compounds, the carboxylate functional groups remained unchanged because they might contribute to

Molecules 2017, 22, 773
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the increment of inhibitory effects, likely by either increasing bioavailability or by forming hydrogen
bonds with potential targeted macromolecules.
Compounds
a slightly lower anticancer activity compared to
was better. Compound 10, having an acetamide replace the ethyl amide in 7, also showed similar
potency to , further confirming the importance of the carboxylate function groups in the para-position.
The length variation of chains linking the benzene ring and isothiocyanate function group provided
compounds 11 13. The subsequent biological evaluation demonstrated that a longer or shorter chain
of diminished its potency, while a two-carbon linker provided the best potency. This finding implied
that the chain length might be crucial for maintaining a balance between rigidity and flexibility to
gain more potency. Thus, more structural modifications were performed on the skeleton of ; varying
the amine motif provided compounds 14 20, with the ethyl amine in replaced by diethyl amine,
morpholine, piperazine, pyridine-2-amine, aniline, and other amines (Figure 1). The variation of the
amine motif in did not gain more potency to inhibit cancer growth in the newly prepared compounds
20. Thus, compound was selected for further biological study, using PEITC and for comparison.
Pancreatic cancer is one of the most fatal human cancers which remains a challenging health
problem due to its high metastases [24]. Our further biological evaluation of compound focused
on the human pancreatic cancer cell lines Panc1 and Capan2 (Figure 2). Compound showed better
anti-proliferative capability in a dose-dependent manner compared to PEITC and , as shown in
Figure 2a. In the colony formation experiments, substantially inhibited Panc1 growth even at 1
concentration (Figure 2b). Compared to PEITC, and induced higher apoptosis of Panc1 and Capan2
after 72 h treatment at 10 M concentration (Figure 3a). However, and caused similar or less
8
and
9
with respective meta-positioned ester and amide functional groups showed
6
and , implying that substituents on the para-position
7
7
–
7
7
–
7
7
1
4
–
7
6
7
7
6
7
µM
6
7
µ
6
7
cytotoxicity to non-cancerous cells, as evidenced by the cell viability of E6E7 that is an immortalized
human pancreatic ductal epithelium cell line (Figure 3b).
(a)
Panc1
Capan2
6
6
7
7
PEITC
PEITC
Concentration (μM)
Concentration (μM)
(
b)
Panc1
Capan2
Control
Control
1μM 6
1μM 6
1μM 7
1μM 7
1
μM
1 μM
PEITC
PEITC
Figure 2. The cellular toxicity of
6 and 7 on the human pancreatic cancer cell lines Panc1 and Capan2.
(
a) MTS assay after 72 h treatment; (b) 14-day colony formation (triplicate wells).

Molecules 2017, 22, 773
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(a)
Control
6 (10μM)
13.54%
7 (10μM)
38.12%
PEITC (10μM)
13.25%
(
b)
6.70%
6
7
E6E7
Panc1
PEITC
6.30%
39.25%
64.13%
27.87%
Capan2
Concentration (μM)
Figure 3.
(a) The apoptosis induction caused by
6
and
7 in pancreatic cancer cells. Percentage numbers
show annexin-V/PI positive cell fraction; (b) E6E7 cells were treated with various agents for 72 h before
MTS assay.
ROS play important roles in the regulation of cell survival and cell death. In general, a moderate
ROS level promotes cell proliferation, whereas an excessive increase of ROS can induce cell death
by causing cellular redox imbalance [25
modulation pathway, the effect of and 7 on intracellular ROS levels was finally tested. Indeed,
significantly increased the ROS level as PEITC, as shown in Figure 4a. Intracellular glutathione
GSH) is a very important endogenous antioxidant to defend high oxidative stress in malignant cancer
cells [27 28]. Thus, we also tested whether our synthetic isothiocyanates and could deplete GSH
was the most significant,
,26]. Since PEITC killed malignant cancer cells via the ROS
6
7
(
,
6
7
in Panc1 and Capan2 cells (Figure 4b,c). As expected, GSH depletion by
7
followed by 6 and PEITC, consistent to their anticancer activity.
Figure 4. Compounds
hydrogen peroxide (6 h); (
with compounds at a concentration of 10 µM.
6
and
7
induced a redox imbalance in pancreatic cancer cells. ( ) The content of
a
b
,
c) the content of cellular GSH in Panc1 ( ) and Capan2 (c) treated for 2 h
b

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3
. Materials and Methods
3
.1. General Procedure Exemplified by the Synthesis of PEITC
All reactions were performed under argon, unless indicated otherwise. Column chromatography
was performed using silca gel (200–300 mesh). NMR spectra were recorded on a Bruker 400 MHz
1
spectrometer (Rheinstetten, Germany). H-NMR spectra were referenced internally to either CDCl
3
(δ
7.26 ppm) or DMSO-d (
δ
2.50 ppm). 13C-NMR spectra were referenced to CDCl (
δ
77.16 ppm,
6
3
the middle peak) or DMSO-d6 (δ 39.52 ppm, the middle peak). Chemical shifts (δ) were reported as
part per million (ppm), and coupling constants (J) reported in Hertz (Hz). Mass spectra were recorded
with an Agilent LC-MS 6120 spectrometer (Santa Clara, CA, USA). High-resolution mass spectrometry
1
(
HRMS) was carried out using a Shimadzu LCMS-IT-TOF spectrometer (Kyoto, Japan). H-NMR
spectrum and 13C-NMR spectrum can be found at Supplementary Materials.
To a solution of phenethylamine (200 mg, 1.65 mmol) and Et N (0.64 mL, 4.95 mmol) in anhydrous
3
THF (2.5 mL) cooled with an ice bath, a solution of CS (0.12 mL, 1.98 mmol) was slowly dropped in.
2
The reaction solution was stirred at room temperature for 0.5 h, after which AcCl (0.14 mL, 1.98 mmol)
◦
was dropped in at 0 C, and after 5 min the mixture was warmed to room temperature for 15–30 min.
When the starting amine was finished, as verified by checking thin layer chromatography (T.L.C.),
1
M HCl (aq., 2 mL) was added to quench the reaction. The solution was extracted by EtOAc three times.
All organic phases were combined and washed with brine, dried over Na SO , and finally filtered and
2
4
concentrated under reduced pressure. The residue was purified by column chromatography on silica
gel (EtOAc/petroleum = 1/1) to provide phenethyl isothiocyanate (PEITC) (253 mg, 94%).
3
.2. Characterization of Synthetic Isothiocyanates
1
Phenethyl isothiocyanate/PEITC. Yellow liquid, yield: 94%, H-NMR (400 Hz, CDCl ):
δ
7.25–7.40
3
(m, 5H), 3.75 (t, 2H, J = 6.8 Hz), 3.02 (t, 2H, J = 6.8 Hz).
1
Methyl 2-isothiocyanato-3-phenylpropanoate (1). Pale yellow liquid, yield: 91%, H-NMR (400 Hz, CDCl ):
3
δ
7.30–7.38 (m, 3H), 7.21–7.26 (m, 2H), 4.48 (dd, 1H, J = 4.8, 8.0 Hz), 3.81 (s, 3H), 3.25 (dd, 1H, J = 4.8,
1
4.0 Hz), 3.13 (dd, 1H, J = 8.0, 14.0 Hz).
1
Isopropyl 2-isothiocyanato-3-phenylpropanoate (
7.22–7.35 (m, 5H), 5.06 (m, 1H), 4.40 (dd, 1H, J = 5.2, 8.0 Hz), 3.22 (dd, 1H, J = 4.8, 13.6 Hz), 3.12
dd, 1H, J = 8.0, 13.6 Hz), 1.26 (s, 3H), 1.25 (S, 3H); 13C-NMR (100 Hz, CDCl ):
167.5, 135.3, 129.5,
28.8, 127.7, 70.8, 61.0, 39.8, 21.8. HRMS calcd for C H NO S : 248.0751, found 248.0747.
2). Yellow liquid, yield: 87%, H-NMR (400Hz, CDCl ):
3
δ
(
1
δ
3
−
2
13
14
1
(
4-(Isothiocyanatomethyl)phenyl)(phenyl)sulfane (3). Pale yellow liquid, yield: 84%, H-NMR (400 Hz,
7.14–7.32 (m, 9H), 4.59 (s, 2H) ppm; 1 C-NMR (100 Hz, CDCl ):
3
δ
137.2, 134.9, 133.0 132.0
30.9, 129.5, 127.8, 127.7, 48.5 ppm. HRMS calcd for C H NS : 256.0260, found 256.0266.
CDCl3):
1
δ
3
−
14
10
2
1
(
δ
3-(Isothiocyanatomethyl)phenyl)(phenyl)sulfane (4). Yellow liquid, yield: 68%, H-NMR (400 Hz, CDCl ):
3
13
7.44–7.19 (m, 9H), 4.68 (s, 2H) ppm; C-NMR (100 Hz, CDCl3):
δ
137.9, 135.5, 132.1, 130.1, 129.8, 129.6,
29.1, 128.4, 127.9, 127.0, 125.2, 48.5 ppm. HRMS calcd for C H NS : 256.0260, found 256.0255.
−
1
14
10
2
1
1
δ
-(Isothiocyanatomethyl)-3-phenoxybenzene (
5). Pale yellow liquid, yield: 82%, H-NMR (400 Hz, CDCl ):
3
7.32–7.39 (m, 3H), 7.15 (t, 1 H, J = 7.2 Hz), 6.96–7.06 (m, 5H), 4.67 (s, 2H) ppm; 13C-NMR (100 Hz,
CDCl ): δ 158.1, 156.7, 136.3, 130.5, 130.0, 123.9, 121.5, 119.4, 118.5, 117.1, 48.5 ppm.
3
1
Methyl4-(2-isothiocyanatoethyl)benzoate (
6
). White solid, yield: 85%, H-NMR (400 Hz, CDCl ):
δ
8.01
3
(
d, 2H, J = 8.0 Hz), 7.29 (d, 2H, J = 8.0 Hz), 3.91 (s, 3H), 3.75 (t, 2H, J = 6.8 Hz), 3.03 (t, 2H, J = 6.8 Hz) ppm;
13
◦
C-NMR (100 Hz, CDCl3):
δ
+
166.9, 142.4, 130.2, 129.3, 129.0, 52.2, 46.0, 36.5 ppm; m.p. 119.9–123.1 C;
MS (ESI, m/z) 222.0 (M + H ).
1
N-Ethyl-4-(2-isothiocyanatoethyl)benzamide (
7
): White solid, yield: 83%, H-NMR (400 Hz, CDCl ):
δ
7.67
3
(d, 2H, J = 8.0 Hz), 7.21 (d, 2H, J = 8.0 Hz), 6.05 (brs, 1H), 3.68 (t, 2H, J = 6.4 Hz), 3.43 (t, 2H, J = 6.4 Hz),

Molecules 2017, 22, 773
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2
.96 (t, 2H, J = 6.4 Hz), 1.18 (t, 3H, J = 7.2 Hz) ppm; 13C-NMR (100 Hz, CDCl3):
δ
167.1, 140.6, 134.0,
◦
+
1
29.1, 127.5, 46.2, 36.4, 35.1, 15.0 ppm; m.p. 89.8–92.5 C; HRMS calcd for C H N OSNa : 257.0719,
12
14
2
found 257.0722.
1
Methyl 3-(2-isothiocyanatoethyl)benzoate (8). White solid, yield: 86%, H-NMR (400 Hz, CDCl ):
3
δ
7.96–7.94 (m, 1H), 7.90 (s, 1H), 7.42 (d, 2H, J = 4.4 Hz), 3.92 (s, 3H), 3.76 (t, 2H, J = 6.8 Hz), 3.04 (t, 2H,
J = 6.8 Hz); 13C-NMR (100 Hz, CDCl ):
ppm; m.p. 86.5–90.8 C; HRMS calcd for C H NO SNa : 244.0403, found 244.0397.
δ
166.9, 137.5, 133.6, 130.8, 129.9, 129.0, 128.6, 52.3, 46.2, 36.3
3
◦
+
11
11
2
1
N-Ethyl-3-(2-isothiocyanatoethyl)benzamide (9). Yellow solid, yield: 88%, H-NMR (400 Hz, CDCl ):
3
δ
7.62–7.67 (m, 1H), 7.62 (s, 1H), 7.27–7.32 (m, 2H), 6.51 (brs, 1H), 3.65 (dd, 2H, J = 11.2, 6.4 Hz),
3
.34–3.52 (m, 2H), 2.93 (dd, 2H, J = 11.2, 6.4 Hz), 1.17 (t, 3H, J = 8.0 Hz); 13C-NMR (100 Hz, CDCl3):
δ
+
167.4, 137.5, 135.6, 131.8, 129.1, 127.6, 126.0, 46.3, 36.4, 35.1, 14.9. HRMS calcd for C H N OSNa :
12 14 2
2
57.0719, found 257.0714.
1
N-(4-(2-Isothiocyanatoethyl)phenyl)acetamide (10). White solid, yield: 100%, H-NMR (400 Hz, DMSO-d ):
6
δ
9.90 (brs, 1H), 7.52 (d, 2H, J = 8.2 Hz), 7.19 (d, 2H, J = 8.2 Hz), 3.86 (t, 2H, J = 6.4 Hz), 2.89 (t, 2H,
J = 6.4 Hz), 2.03 (s, 3H) ppm; 13C-NMR (100 Hz, DMSO-d ):
3
C H N OS : 219.0598, found 219.0602.
δ 168.1, 138.0, 132.0, 129.0, 119.0, 46.0,
4.7, 23.9 ppm; m.p. 154.0–158.1 C; MS (ESI, m/z) 221.1 (M + H ), 441.1 (2M + H ); HRMS calcd for
6
◦
+
+
−
11
11
2
1
N-Ethyl-4-(3-isothiocyanatopropyl)benzamide (11). Colorless oil, yield: 92%, H-NMR (400 Hz, CDCl ):
3
δ
7.71 (d, 2H, J = 8.0 Hz), 7.25 (d, 2H, J = 8.0 Hz), 6.10 (brs, 1H), 3.53–3.46 (m, 4H), 2.80 (t, 2H, J = 7.2 Hz),
1
1
.99–2.06 (m, 2H), 1.25 (t, 3H, J = 7.2 Hz) ppm; 13C-NMR (100 Hz, CDCl ):
27.4, 44.2, 35.0, 32.5, 31.2, 15.0 ppm. MS (ESI, m/z) 249.1 (M + H ), 497.2 (2M + H ); HRMS calcd for
δ
167.4, 143.7, 133.1, 128.7,
3
+
+
−
C H N OS : 247.0911, found 247.0914.
13
15
2
1
Methyl 4-(isothiocyanatomethyl)benzoate (12). Brown liquid, yield: 84%, H-NMR (400 Hz, CDCl ):
(
δ
8.05
d, 2H, J = 8.0 Hz), 7.38 (d, 2H, J = 8.0 Hz), 4.77 (s, 2H), 3.91 (s, 3H) ppm; 13C-NMR (100 Hz, CDCl3):
δ 166.5, 139.3, 130.3, 126.8, 52.33, 48.5 ppm.
3
1
N-Ethyl-4-(isothiocyanatomethyl)benzamide (13). White solid, yield: 86%, H-NMR (400 Hz, CDCl ):
3
δ
7.90 (d, 2H, J = 8.4 Hz), 7.37 (d, 2H, J = 8.4 Hz), 6.14 (brs, 1H), 4.76 (s, 2H), 3.54–3.47 (m, 2H), 1.26
13
(t, 3H, J = 7.2 Hz); C-NMR (100 Hz, CDCl3):
δ
166.8, 137.6, 135.0, 127.7, 127.0, 48.5, 35.1, 15.0 ppm;
◦
−
m.p. 94.6–98.7 C; HRMS calcd for C H N OS : 219.0598, found 219.0592.
11
11
2
1
N,N-Diethyl-4-(2-isothiocyanatoethyl)benzamide (14). Pale brown liquid, yield: 91%, H-NMR (400 Hz,
CDCl3): 7.32–7.34 (d, 2H, J = 8.0 Hz), 7.22–7.24 (d, 2H, J = 8.0 Hz), 3.72 (t, 2H, J = 6.8 Hz), 3.52 (brs, 2H),
.24 (brs, 2H), 2.99 (t, 2H, J = 6.8 Hz), 1.10–1.24 (m, 6H) ppm; 13C-NMR (100 Hz, CDCl3):
δ
3
δ
171.1, 138.2,
+
1
36.3, 129.0, 126.9, 46.2, 43.4, 39.4, 36.3, 28.5, 14.3, 13.0 ppm. HRMS calcd for C H N OS : 263.1213,
14
19
2
found 263.1200.
1
(
4-(2-Isothiocyanatoethyl)phenyl)(morpholino)methanone (15). Yellow liquid, yield: 87%, H-NMR (400 Hz,
CDCl ):
(
δ 7.38 (d, 2H, J = 8.0 Hz), 7.26 (d, 2H, J = 8.0 Hz), 3.73 (t, 2H, J = 6.4 Hz), 3.44–3.76 (m, 8H), 3.00
3
t, 2H, J = 6.4 Hz) ppm; 13C-NMR (100 Hz, CDCl ):
δ
170.2, 139.1, 134.5, 129.1, 127.8, 67.0, 60.4, 46.2,
3
+
+
3
6.3 ppm. MS (ESI, m/z) 277.2 (M + H ). HRMS calcd for C H N O SNa : 299.0825, found 299.0832.
14
16
2
2
1
(
(
2
4
4-(2-Isothiocyanatoethyl)phenyl)(4-methylpiperazin-1-yl)methanone (16). Brown solid, yield: 80%, H-NMR
400 Hz, CDCl3): 7.30–7.16 (m, 4H), 3.70–3.38 (m, 4H), 3.65 (t, 2H, J = 6.8 Hz), 2.92 (t, 2H, J = 6.8 Hz),
.41–2.36 (m, 4H), 2.24 (s, 3H); 13C-NMR (100 Hz, CDCl ):
170.2, 139.0, 134.8, 129.1, 127.8, 55.2, 46.2,
6.0, 36.4, 21.2 ppm; HRMS calcd for C H N OS : 290.1322, found 290.1317.
δ
δ
3
+
15
20
3
1
4
-(2-Isothiocyanatoethyl)-N-(pyridin-2-yl)benzamide (17). White solid, yield: 95%, H-NMR (400 Hz,
8.66 (s, 1H), 8.38 (d, 1H, J = 8.4 Hz), 8.29 (d, 1H, J = 4.0 Hz), 7.92 (d, 2H, J = 8.0 Hz), 7.74–7.78
m, 1H), 7.36 (d, 2H, J = 8.0 Hz), 7.06–7.09 (m, 1H), 3.78 (t, 2H, J = 7.2 Hz), 3.07 (t, 2H, J = 7.2 Hz) ppm;
CDCl3):
δ
(
1
3
C-NMR (100 Hz, CDCl3):
δ 151.6, 148.1, 141.8, 138.7, 133.4, 129.5, 127.9, 120.1, 114.3, 46.1, 36.5, 29.8

Molecules 2017, 22, 773
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◦
+
−
ppm; m.p. 139.6–141.3 C; MS (ESI, m/z) 284.1 (M + H ). HRMS calcd for C H N OS : 282.0707,
15
12
3
found 282.0697.
1
4
-(2-Isothiocyanatoethyl)-N-phenylbenzamide (18). White solid, yield: 91%, H-NMR (400 Hz, CDCl ):
3
δ
7.85 (d, 2H, J = 8.0 Hz), 7.64 (d, 2H, J = 8.0 Hz), 7.39–7.33 (m, 4H), 7.14–7.18 (m, 1H), 3.77 (t, 2H,
J = 6.8 Hz), 3.06 (t, 2H, J = 6.8 Hz); 13C-NMR (100 Hz, CDCl ): δ 165.5, 141.3, 138.0, 134.1, 129.4, 129.2,
3
◦
27.7, 124.8, 120.4, 46.1, 36.4 ppm; m.p. 137.0–142.9 C; HRMS calcd for C H N OSNa : 305.0719,
16 14 2
+
1
found 305.0707.
1
N-Benzyl-4-(2-isothiocyanatoethyl)benzamide (19). White solid, yield: 86%, H-NMR (400 Hz, CDCl ):
3
δ
7.67 (d, 2H, J = 8.0 Hz), 7.25–7.16 (m, 7H), 6.39 (brs, 1H), 4.54 (d, 2H, J = 5.6 Hz), 3.64 (t, 2H, J = 6.8 Hz),
2
.92 (t, 2H, J = 6.8 Hz); 13C-NMR (100 Hz, CDCl ):
δ
167.1, 140.9, 138.3, 133.5, 129.2, 128.9, 128.0,
3
◦
+
127.8, 127.6, 46.1, 44.3, 36.4 ppm; m.p. 143.0–145.8 C; HRMS calcd for C H N OSNa : 319.0876,
17
16
2
found 319.0877.
1
4
δ
2
-(2-Isothiocyanatoethyl)-N-phenethylbenzamide (20). Yellow solid, yield: 97%, H-NMR (400 Hz, CDCl ):
3
7.58 (d, 2H, J = 8.0 Hz), 7.23 (d, 2H, J = 6.8 Hz), 7.18–7.14 (m, 5H), 6.12 (brs, 1H), 3.66–3.60 (m, 4H),
.92 (t, 2H, J = 6.8 Hz), 2.85 (t, 2H, J = 6.8 Hz); 13C-NMR (100 Hz, CDCl3):
33.8, 129.1, 128.9, 128.8, 127.5, 126.7, 46.1, 41.3, 36.4, 35.8 ppm; m.p. 98.2–102.5 C; HRMS calcd for
δ 167.2, 140.8, 139.0,
◦
1
+
C H N OSNa : 333.1032, found 333.1046.
18
18
2
3
.3. Biological Cellular Assay
-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)
3
powder and DMSO were bought from Sigma. DMEM medium, RPMI-1640 medium and fetal bovine
serums were purchased from GIBCO.
3
.3.1. Cell Culture
Human cancer cell lines Panc1 and Capan2 (pancreatic cancer), HGC27 (gastric cancer), and E6E7
were obtained from Sun-Yat Sen University Cancer Center. Cells were cultured in medium
supplemented with 5–10% fetal bovine serum under the conditions of a humidified 5% carbon dioxide
incubator. Every two to three days the medium was removed, when the confluence of cells reached
about 80%, and trypsin was used to digest the cells.
3
.3.2. MTS Assay
Human cancer cells in logarithmic phase were inoculated in a 96-well plate (three wells for
each drug concentration), with 1000–10,000 cells in each well. Once the cells were adherent, 100
medium containing different concentrations of drugs were added, and after incubation for 72 h, 20
µ
L
L
µ
MTS (5 mg/mL) was added, followed by incubation for another 4 h. At the end of the incubation
period, DMSO was used to dissolve purple formazen (the production of formazen was proportional to
the number of viable cells). Then, the plate was shaken for 10–15 min and the optical density (OD)
value of the plate was detected by microreader (Thermo Scientific, Waltham, USA) at a wavelength
of 570 nm. The survival rates were expressed as follows: survival rate % = mean OD value of drug
treatment/mean OD value of control
×
100% (Experiments were done for three times independently).
3
.3.3. Colony Formation
Human cancer cells were seeded in 6-well plates at 500 cells per well. Then, cells were incubated
with the indicated agents for 14 days, followed by fixation and staining with crystal violet. The samples
were photographed and the colonies were counted.

Molecules 2017, 22, 773
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3
.3.4. Apoptosis and ROS Measurement
Apoptosis was detected using Annexin V/PI staining. Cells were harvested and stained with
Annexin V and PI for 15 min, and apoptosis rates were analyzed using BD Calibur flow cytometry.
Similarly, cells were stained for 30 min with DCF-DA for ROS detection. The samples were washed
and analyzed using flow cytometry.
3
.3.5. GSH Levels
Cellular GSH contents were measured using a GSH-Glo™ Glutathione Assay kit (Promega, WI, USA)
according to the protocol provided by the manufacturer.
4
. Conclusions
A concise preparation of isothiocyanates mediated by acetyl chloride and other electrophiles
were studied. Using acetyl chloride, the expected isothiocyanates were obtained at good to excellent
yields and the limitation of the often used tosyl chloride was overcome. These factors along its
low cost would make acetyl chloride practical to prepare isothiocyanates. In our study, a series of
novel isothiocyanates were designed and conveniently prepared using our newly discovered method.
A biological evaluation on two malignant human cancer cell lines, pancreatic cancer Panc1 and gastric
cancer HGC27, was investigated. Finally, compound
the test cancer cells relative to PEITC that is one of the most studied naturally occuring isothiocyanates.
In further study, compound substantially inhibited the colony formation of pancreatic cancer cell
lines Panc1 and Capan2, and caused better apoptosis than PEITC. Compound also induced an
7 was found to have a better growth inhibition in
7
7
increase in the levels of intracellular ROS and depleted the levels of GSH, which is one of the most
recognized mechanisms of PEITC. Since several clinical trials with PEITC have been registered,
our newly discovered compound
7 could provide therapeutic promise for the treatment of malignant
tumors. Further study of the anticancer therapeutic effects of
due time.
7 is under way and will be reported in
Supplementary Materials: Supplementary data including H-NMR spectrum and 13C-NMR spectrum can be
1
available online.
Acknowledgments: We are grateful to the National Natural Science Foundation of China (81672952, 81430060),
Guangdong Science and Technology Program (2014A030313196, 2013B051000034), Guangzhou Innovation
Research Program (LCY201317), and Guangzhou Medicare Collaborative Innovation Program (201508020250).
Author Contributions: S.W., J.W. and B.L. conceived and designed the experiments; J.W., B.L. and X.L. performed
the experiments; J.W. and B.L. analyzed the data; J.W., B.L., S.W. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Ferguson, L.R.; Philpott, M. Cancer prevention by dietary bioactive components that target the immune
response. Curr. Cancer Drug Targets 2007, 7, 459–464. [CrossRef] [PubMed]
2
.
Cheung, K.L.; Khor, T.O.; Huang, M.T.; Kong, A.N. Differential in vivo mechanism of chemoprevention of
tumor formation in azoxymethane/dextran sodium sulfate mice by PEITC and DBM. Carcinogenesis 2010, 31,
8
80–885. [CrossRef] [PubMed]
3
4
.
.
Hu, R.; Kim, B.R.; Chen, C.; Hebbar, V.; Kong, A.N. The roles of JNK and apoptotic signaling pathways in
PEITC-mediated responses in human HT-29 colon adenocarcinoma cells. Carcinogenesis 2003, 24, 1361–1367.
[CrossRef] [PubMed]
Xiao, D.; Powolny, A.A.; Moura, M.B.; Kelley, E.E.; Bommareddy, A.; Kim, S.H.; Hahm, E.R.; Normolle, D.;
Van Houten, B.; Singh, S.V. Phenethyl isothiocyanate inhibits oxidative phosphorylation to trigger reactive
oxygen species-mediated death of human prostate cancer cells. J. Biol. Chem. 2010, 285, 26558–26569.
[CrossRef] [PubMed]

Molecules 2017, 22, 773
11 of 12
5
6
7
.
.
.
Runarsdottir, A.; Mannervik, B. A novel quasi-species of glutathione transferase with high activity towards
naturally occurring isothiocyanates evolves from promiscuous low-activity variants. J. Mol. Biol. 2010, 401,
4
51–464. [CrossRef] [PubMed]
Syed Alwi, S.S.; Cavell, B.E.; Donlevy, A.; Packham, G. Differential induction of apoptosis in human breast
cancer cell lines by phenethyl isothiocyanate, a glutathione depleting agent. Cell Stress Chaperones 2012, 17,
5
29–538. [CrossRef] [PubMed]
Yeomans, A.; Lemm, E.; Wilmore, S.; Cavell, B.E.; Valle-Argos, B.; Krysov, S.; Hidalgo, M.S.; Leonard, E.;
Willis, A.E.; Forconi, F.; et al. PEITC-mediated inhibition of mRNA translation is associated with both
inhibition of mTORC1 and increased eIF2alpha phosphorylation in established cell lines and primary human
leukemia cells. Oncotarget 2016, 7, 74807–74819. [PubMed]
8
9
1
1
1
1
.
Cavell, B.E.; Syed Alwi, S.S.; Donlevy, A.M.; Proud, C.G.; Packham, G. Natural product-derived antitumor
compound phenethyl isothiocyanate inhibits mTORC1 activity via TSC2. J. Nat. Prod. 2012, 75, 1051–1057.
[CrossRef] [PubMed]
.
Wang, X.; Di Pasqua, A.J.; Govind, S.; McCracken, E.; Hong, C.; Mi, L.; Mao, Y.; Wu, J.Y.; Tomita, Y.;
Woodrick, J.C.; et al. Selective depletion of mutant p53 by cancer chemopreventive isothiocyanates and their
structure-activity relationships. J. Med. Chem. 2011, 54, 809–816. [CrossRef] [PubMed]
0. Chen, G.; Chen, Z.; Hu, Y.; Huang, P. Inhibition of mitochondrial respiration and rapid depletion
of mitochondrial glutathione by beta-phenethyl isothiocyanate: mechanisms for anti-leukemia activity.
Antioxid. Redox Signal. 2011, 15, 2911–2921. [CrossRef] [PubMed]
1. Trachootham, D.; Zhang, H.; Zhang, W.; Feng, L.; Du, M.; Zhou, Y.; Chen, Z.; Pelicano, H.; Plunkett, W.;
Wierda, W.G.; et al. Effective elimination of fludarabine-resistant CLL cells by PEITC through
a redox-mediated mechanism. Blood 2008, 112, 1912–1922. [CrossRef] [PubMed]
2. Trachootham, D.; Zhou, Y.; Zhang, H.; Demizu, Y.; Chen, Z.; Pelicano, H.; Chiao, P.J.; Achanta, G.;
Arlinghaus, R.B.; Liu, J.; et al. Selective killing of oncogenically transformed cells through a ROS-mediated
mechanism by beta-phenylethyl isothiocyanate. Cancer Cell 2006, 10, 241–252. [CrossRef] [PubMed]
3. Mays, J.R.; Weller Roska, R.L.; Sarfaraz, S.; Mukhtar, H.; Rajski, S.R. Identification, synthesis, and enzymology
of non-natural glucosinolate chemopreventive candidates. ChemBioChem 2008, 9, 729–747. [CrossRef]
[PubMed]
1
1
4. Vermeulen, M.; Zwanenburg, B.; Chittenden, G.J.; Verhagen, H. Synthesis of isothiocyanate-derived
mercapturic acids. Eur. J. Med. Chem. 2003, 38, 729–737. [CrossRef]
5. Berglund, M.; Dalence-Guzman, M.F.; Skogvall, S.; Sterner, O. SAR studies of capsazepinoid bronchodilators
3: The thiourea part (coupling region) and the 2-(4-chlorophenyl)ethyl moiety (C-region). Bioorg. Med. Chem.
2
008, 16, 2529–2540. [CrossRef] [PubMed]
1
6. Tsogoeva, S.B.; Hateley, M.J.; Yalalov, D.A.; Meindl, K.; Weckbecker, C.; Huthmacher, K. Thiourea-based
non-nucleoside inhibitors of HIV reverse transcriptase as bifunctional organocatalysts in the asymmetric
Strecker synthesis. Bioorg. Med. Chem. 2005, 13, 5680–5685. [CrossRef] [PubMed]
1
1
1
2
2
7. Isobe, T.; Ishikawa, T. 2-Chloro-1, 3-dimethylimidazolinium chloride. 2. Its application to the construction of
heterocycles through dehydration reactions. J. Org. Chem. 1999, 64, 6989–6992. [CrossRef]
8. Munch, H.; Hansen, J.S.; Pittelkow, M.; Christensen, J.B.; Boas, U. A new efficient synthesis of isothiocyanates
from amines using di-tert-butyl dicarbonate. Tetrahedron Lett. 2008, 49, 3117–3119. [CrossRef]
9. Wong, R.; Dolman, S.J. Isothiocyanates from tosyl chloride mediated decomposition of in situ generated
dithiocarbamic acid salts. J. Org. Chem. 2007, 72, 3969–3971. [CrossRef] [PubMed]
0. Maisonneuve, P.; Lowenfels, A.B. Risk factors for pancreatic cancer: a summary review of meta-analytical
studies. Int. J. Epidemiol. 2015, 44, 186–198. [CrossRef] [PubMed]
1. Von Hoff, D.D.; Ervin, T.; Arena, F.P.; Chiorean, E.G.; Infante, J.; Moore, M.; Seay, T.; Tjulandin, S.A.; Ma, W.W.;
Saleh, M.N.; et al. Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine. N. Engl.
J. Med. 2013, 369, 1691–1703. [CrossRef] [PubMed]
2
2
2. Leung, W.K.; Wu, M.S.; Kakugawa, Y.; Kim, J.J.; Yeoh, K.G.; Goh, K.L.; Wu, K.C.; Wu, D.C.; Sollano, J.;
Kachintorn, U.; et al. Screening for gastric cancer in Asia: Current evidence and practice. Lancet Oncol. 2008
,
9
, 279–287. [CrossRef]
3. Zhang, E.B.; Han, L.; Yin, D.D.; Kong, R.; De, W.; Chen, J. C-Myc-induced, long, noncoding H19 affects
cell proliferation and predicts a poor prognosis in patients with gastric cancer. Med. Oncol. 2014, 31, 914.
[CrossRef] [PubMed]

Molecules 2017, 22, 773
12 of 12
2
4. Li, D.; Xie, K.; Wolff, R.; Abbruzzese, J.L. Pancreatic cancer. Lancet 2004, 363, 1049–1057. [CrossRef]
2
5. Raj, L.; Ide, T.; Gurkar, A.U.; Foley, M.; Schenone, M.; Li, X.; Tolliday, N.J.; Golub, T.R.; Carr, S.A.;
Shamji, A.F.; et al. Selective killing of cancer cells by a small molecule targeting the stress response to
ROS. Nature 2011, 475, 231–234. [CrossRef] [PubMed]
2
2
2
6. Trachootham, D.; Lu, W.; Ogasawara, M.A.; Nilsa, R.D.; Huang, P. Redox regulation of cell survival.
Antioxid. Redox Signal. 2008, 10, 1343–1374. [CrossRef] [PubMed]
7. Franco, R.; Cidlowski, J.A. Apoptosis and glutathione: Beyond an antioxidant. Cell Death Differ. 2009, 16,
1
303–1314. [CrossRef] [PubMed]
8. Chiang, Y.T.; Yen, Y.W.; Lo, C.L. Reactive oxygen species and glutathione dual redox-responsive micelles for
selective cytotoxicity of cancer. Biomaterials 2015, 61, 150–161. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 6, 7 are available from the authors.
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).