Synthesis and biological evaluation of sulforaphane derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2013.03.045

A series of sulforaphane derivatives were synthesized and evaluated in vitro for their cytotoxicity against five cancer cell lines (HepG2, A549, MCF-7, HCT-116 and SH-SY5Y). The pharmacological results showed that many of the derivatives displayed more potent cytotoxicity than sulforaphane (SFN). Furthermore, SFN and derivative 85 could induce cell cycle arrest at S or G2/M phase and cell apoptosis. SFN and 85 exhibited time- and dose-dependent activation on Nrf2 transcription factor, and 85 acted as a more potent Nrf2 inducer than SFN.

Full text of DOI:10.1016/j.ejmech.2013.03.045

European Journal of Medicinal Chemistry 64 (2013) 529e539
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of sulforaphane derivatives as
potential antitumor agents
*
*
Kun Hu, Yan-jie Qi, Juan Zhao, He-fei Jiang, Xin Chen , Jie Ren
School of Pharmaceutical Engineering & Life Science, Changzhou University, 1 Gehu Road, Changzhou, Jiangsu 213164, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of sulforaphane derivatives were synthesized and evaluated in vitro for their cytotoxicity against
five cancer cell lines (HepG2, A549, MCF-7, HCT-116 and SH-SY5Y). The pharmacological results showed
that many of the derivatives displayed more potent cytotoxicity than sulforaphane (SFN). Furthermore,
SFN and derivative 85 could induce cell cycle arrest at S or G2/M phase and cell apoptosis. SFN and 85
exhibited time- and dose-dependent activation on Nrf2 transcription factor, and 85 acted as a more
potent Nrf2 inducer than SFN.
Received 26 November 2012
Received in revised form
19 March 2013
Accepted 24 March 2013
Available online 6 April 2013
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Sulforaphane
Cytotoxicity
Cell cycle arrest
Apoptosis
Nrf2
1. Introduction
carcinogen activation. In recent years, many studies have showed
that SNF could provide protection against tumor development
Isothiocyanates are a class of compounds which are abundant in
many cruciferous vegetables, such as broccoli, cauliflower, Brussels
sprouts, and cabbage [1,2]. There is increasing epidemiological and
clinical evidence that people that intake large amounts of vegeta-
bles of the Brassica family, particularly broccoli and other crucif-
erous vegetables are less likely to develop certain types of cancers
[3e5], and thus, many of such analogues containing the iso-
thiocyanates motif have been synthesized for potential medical
applications [6]. The isothiocyanates, are hydrolysis products of
glucosinolates that are not the putative bioactive compounds in
cruciferous vegetables, and the hydrolytic reaction is catalyzed by
the endogenous myrosinase which is released by disruption of the
plant cell during harvesting, processing, or chewing [7,8].
Sulforaphane (SFN), one of the naturally occurring iso-
thiocyanates, which was first discovered by scientists at Johns
Hopkins University in 1992 [9], has attracted considerable research
interests due to its important biological and pharmacological
properties. SFN is an effective anticancer agent with the ability for
both preventing and fighting many types of cancers [10,11]. Early
research mainly focused on the “blocking activity” of SFN via Phase
2 enzyme induction, as well as inhibition of enzymes involved in
during the post-initiation phase. The mechanisms of SFN sup-
pression effects, including cell cycle arrest and apoptosis, have also
been investigated [3,8]. Additionally, pharmacological administra-
tion of SFN might be a promising therapeutic approach for cancers
treatment. For example, according to a report from the US National
Cancer Institute, SFN is considered to be one of the 40 most
promising anticancer agents [12e14].
During the past years, a few methods for preparing SFN and its
derivatives have been reported in the literature [15e17], and
structural modifications have been envisioned to increase their
cytotoxicity, especially for their specificity toward tumor cells [18e
20]. To the best of our knowledge, there were few reports regarding
SFN derivatives that modified the side chain (the methyl group). In
our present study, we have designed and synthesized a series of
SFN derivatives with different side chains and various lengths of
spacers between the isothiocyanato and sulfoxide groups. We
evaluated their anticancer activity against several cancer cell lines
in vitro, and elucidated the possible mechanism of cell growth in-
hibition by these SFN derivatives.
2. Chemistry
In order to investigate the structureeactivity relationship of the
SFN derivatives, we replaced the methyl group in SFN (77) with
several different alkyl and aryl groups. At the same time, we
* Corresponding authors. Tel./fax: þ86 519 86334598.
E-mail addresses: xinchen@cczu.edu.cn (X. Chen), renjie2006@163.com (J. Ren).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.045

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K. Hu et al. / European Journal of Medicinal Chemistry 64 (2013) 529e539
changed the length of spacer between the isothiocyanato and
sulfoxide groups. The SFN derivatives (78e97) were obtained
through the synthetic route shown in Scheme 1. Firstly, potassium
phthalimide (1) was refluxed with different alkyl dibromides in
acetone, affording mono-bromides 2e5. Secondly, 2e5 were
refluxed with potassium thioacetate (KSAc) in THF to give thio-
acetates 6e9 [21,22]. Thiols 10e13 were obtained by acidic hydro-
lysis of 6e9 in MeOH. In the following step, 10e13 were alkylated
with various alkyl bromides in the presence of NaH (method A),
affording thio-ethers 14e34. While for phenyl group as the sub-
stituent (for synthesizing compound 21), Pd2(dba)3 was employed
as a catalyst for the alkylation of phenyl bromide (method B) [23].
However, when the same protocol was used for introducing other
cyclic and heterocyclic groups, no desired products were obtained.
After 14e34 were refluxed with 80% hydrazine hydrate in MeOH for
4 h, amines 35e55 were produced. By following the known pro-
cedure (treated with carbon disulfide, triethylamine and MsCl) [4],
isothiocyanates 56e76 were obtained in satisfactory yields. Finally,
the target compounds 77e97 were obtained by oxidizing 56e76
with 1 M equiv of MCPBA. When 64 was oxidized by 4 M equiv of
MCPBA, sulfone 98 was obtained in 52% yield. The structures of all
the final products 77e98 were confirmed by their MS, 1H NMR and
13C NMR spectral properties.
3. Results and discussion
3.1. In vitro cytotoxicity
The synthesized SFN derivatives 64, 78e98 were screened for
in vitro cytotoxicity against five cancer cell lines, including HepG2
(Liver hepatocellular carcinoma), A549 (human lung adenocarci-
noma), MCF-7 (human breast adenocarcinoma), HCT-116 (human
colon cancer cell line) and SH-SY5Y (human neuroblastoma cell
line), by the standard MTT assay, and using 5-Fu as a positive
control. Antitumor potency of the compounds was indicated by IC50
values that were calculated by linear regression analysis of the
concentrationeresponse curves obtained for each compound, and
the results were summarized in Table 1.
As shown in Table 1, all the derivatives exhibited more potent
inhibitory activity against HepG2, A549, MCF-7, HCT-116 and SH-
SY5Y than SFN, but for A549, HCT-116 and SH-SY5Y cell lines,
most of the compounds showed weaker inhibitory activity than 5-
Fu. Among the derivatives, 81 and 82 possess significant cytotox-
icity against HepG2 with the IC50 values at 2.05
and 85 had stronger inhibitory activity against MCF-7 with the IC50
values at 3.66 M and 3.30 M respectively; derivative 91 with the
IC50 value at 5.64 M against A549, and derivative 87 with the IC50
mM and 2.16 mM; 82
m
m
m
Scheme 1. Synthesis of SFN derivatives (77e97). Reagents and conditions: (a) alkyl dibromide, acetone, reflux, 10 h; (b) KSAc, THF, reflux, 3e5 h; (c) conc. HCl, MeOH, reflux, 3e4 h;
(d) method A: NaH, ReBr, THF, rt, 1e2 h; method B: Pd2(dba)3, DPPF, toluene, bromobenzene, DIPEA, reflux, 2 h; (e) 80% hydrazine hydrate, MeOH, reflux, 3e4 h; (f) (1) Et3N, CS2,
THF, 0 ꢀC to rt, 90 min; (2) MsCl, 0 ꢀC to rt, 30 min; (g) 85% MCPBA, DCM, 0 ꢀC, 1 h.

K. Hu et al. / European Journal of Medicinal Chemistry 64 (2013) 529e539
531
Table 1
The cytotoxicity of sulforaphane derivatives.
Compd.
IC50
(m
M)
R
n
X
HepG2
A549
MCF-7
HCT-116
SH-SY5Y
Vero
64
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
SFN (77)
5-Fu
12.56
5.35
3.91
5.93
2.05
2.16
3.64
6.34
2.85
3.18
4.62
6.97
5.35
3.82
3.86
9.84
7.14
3.91
3.69
6.17
7.82
8.49
14.05
24.96
51.34
10.16
10.74
9.56
10.41
9.09
10.11
23.8
7.41
14.35
32.55
10.21
8.33
8.41
5.64
38.28
8.26
5.96
5.84
8.82
3.66
4.45
7.05
3.3
7.77
8.89
9.33
6.89
4.14
5.68
7.92
7.93
3.83
6.78
5.49
9.76
7.55
17.66
10.14
41.45
3.85
3.95
2.81
3.74
2.06
3.14
4.53
3.66
2.83
5.46
8.02
3.72
3.21
3.47
15.64
15.92
5.19
3.42
11.57
9.46
6. 28
11.59
3.24
20. 71
9.81
7.66
6.47
9.42
5.7
7.14
8.75
5.21
7.23
2.79
5.6
7.83
5.31
7.79
8.89
18.05
7.01
8.62
7.63
7.82
4. 78
13.27
0.97
24. 95
7.9
Benzyl
Ethyl
Propyl
n-Butyl
Iso-propyl
Iso-butyl
Cyclopentyl
Phenyl
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
3
3
4
4
2
2
S
S]O
S]O
S]O
S]O
S]O
S]O
S]O
S]O
S]O
S]O
S]O
S]O
S]O
S]O
S]O
S]O
S]O
S]O
S]O
S]O
5.64
5.15
10.4
4.98
4.6
4.33
4.85
3.36
3.71
6.82
7.65
6.22
5.25
10.14
8.25
5.85
10.28
4.33
7.31
7.17
13.72
9.71
Benzyl
p-Nitrobenzyl
p-Methoxybenzyl
Phenethyl
2-Pyridinylmethyl
2-Thienylmethyl
2-Furanylmethyl
Benzyl
Iso-propyl
Benzyl
Iso-propyl
Benzyl
11.82
16.7
6.21
7.75
14.69
11.99
8.89
21.99
8.71
Iso-propyl
Benzyl
Methyl
O]S]O
S]O
value at 2.79
m
M against SH-SY5Y. Compared to 5-Fu, 80, 82 and 86
4-carbon spacer (85 and 81). The cytotoxicity of the SFN derivatives
with different spacers has the following tendency: 3-carbon
spacer < 6-carbon spacer < 5-carbon spacer < 4-carbon spacer.
These results indicated that the introduction of branched alkyl and
benzyl groups at the side chain might facilitate increasing the
cytotoxic activities of the SFN derivatives, especially for those with
a 4-carbon spacer.
possess more potent inhibitory effects against HCT-116 with the
IC50 values at 2.81 M, 2.06 M and 2.83 M respectively, reflecting
m
m
m
the selectivity for a particular human HCT-116 cancer cell type.
From Table 1, the derivatives with branched alkyl groups (81, 82)
at side chains were more cytotoxic toward HepG2 cell growth
in vitro than the derivatives with straight-chain alkyl groups (78,
79, and 80), cycloalkyl groups (83), phenyl groups (84, 86e88) and
heterocyclic groups (89e91). In contrast, the derivatives with het-
erocyclic side chains (89e91) were more potent for inhibiting A549
cell growth than those with alkyl groups (78e83) and aryl groups
(84, 86e88). For MCF-7, benzyl group substituted compounds (85,
94) possess stronger inhibitory activity than other derivatives. The
derivatives with branched alkyl groups (78e82) and heterocyclic
groups (89e91) were more potent in inhibiting HCT-116 cell
growth than those with other substituted groups. For SH-SY5Y,
benzyl group substituted derivative (87) has the best activity with
3.2. Effect of SFN and derivative 85 on cell cycle arrest in HepG2
To assess whether cell growth inhibition induced by SFN and the
most active derivative 85 is mediated via alternations in cell cycle
progression, DNA cell cycle analysis was performed. As shown in
Fig. 1, compared with the control, SFN and 85 treatment resulted in
a dose-dependent accumulation of cells in the S phase accompa-
nied by a decrease in G0/G1 phase (Fig. 1A). At the same time, the
G2/M phase arrest could also be observed in the cells treated with
85. The percentage of cells in the S fraction was increased by 5.9%
the IC50 value at 2.79 mM. A comparison of activity of benzyl de-
rivatives 85e88 against SH-SY5Y indicates that an electron-
donating group at para position of benzyl could significantly
improve the activity, while an electron with drawing group has a
detrimental effect. Compound (64) without sulfoxide group
showed weaker inhibitory activity than most of the derivatives, and
compound (98) with sulfone group showed better inhibitory ac-
tivity against HepG2, A549, MCF-7, HCT-116 and SH-SY5Y than SFN.
The results indicate that replacing the sulfoxide group with a sul-
fone group might be a general approach to boost the biological
activity of this type of agents.
It is interesting to note that the cytotoxicity of the SFN de-
rivatives is related to the length of the spacers between the iso-
thiocyanato and sulfoxide groups: the derivatives with a shorter
spacer (3-carbon spacer; such as 92 and 93) and with a longer
spacer (6-carbon spacer; such as 96 and 97) have weaker cytotox-
icity against most of the tested cancer cell lines than those with a
and 9.11% when treated with 20
mM of SFN and 85, respectively,
compared to a vehicle control. The results indicated that SFN and 85
could induce S or G2/M phase cell cycle arrest, and 85 has more
potent inhibitory activity than SFN.
3.3. Effect of SFN and 85 on cell apoptosis in HepG2
In order to evaluate whether the SFN derivatives-mediated in-
hibition in cell growth is due to the induction of apoptosis,
Annexin-V/PI staining was used to detect the apoptotic ratio of SFN
and 85 treated cells. As shown in Fig. 2, after treatment with 20 mM
of SFN and 85 in HepG2 cells for 48 h, the apoptotic cells repre-
sented 5.09% and 25.57% of the total cells, respectively, whereas the
control had only 0.43% apoptotic cells. The results indicated a
greater induction of apoptosis upon treatment with 85 than SFN.

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K. Hu et al. / European Journal of Medicinal Chemistry 64 (2013) 529e539
Fig. 1. Effects of SFN and 85 on cell cycle of HepG2 cells. Cells were treated with SFN (5, 10, 20 mM) and 85 (5, 10, 20 mM) for 48 h, and the DNA content of 10,000 events was analyzed
by flow cytometry. The profiles showed the cell cycle (A and C) and the proportions (%) in each phase (B and D) of HepG2 cells treated with SFN and 85, respectively. *p < 0.05,
**p < 0.01 compared to control.
3.4. Effect of SFN and 85 on Nrf2 transcription factor in HepG2
dependent activation on Nrf2 transcription factor. Furthermore,
85 is a more potent Nrf2 inducer than SFN. The present study might
provide valuable clues in the search for more potent and selective
antitumor agents based on the scaffold of natural occurring iso-
thiocyanates, such as SNF.
Nrf2/ARE pathway plays an important role in chemical pre-
vention. It is of great significance to develop new Nrf2 targeted
inducers for preventive treatment of tumor and multi-organs dis-
eases. SFN has been found to be the strongest natural Nrf2 inducer.
To investigate whether the new SFN derivatives possessed more
potent Nrf2 activation effect, we carried out the Nrf2 activation
assay by TransAM Nrf2 Kit. As shown in Fig. 3, SFN and 85 exhibited
a time- and dose-dependent activation on Nrf2 transcription factor.
Furthermore, 85 acted as a more potent Nrf2 inducer than SFN.
5. Experimental protocols
5.1. Synthesis
Reagents and solvents were purchased from commercial sour-
ces. Solvents were dried and purified using standard techniques. All
reactions involving air or moisture sensitive reagents were per-
formed under nitrogen atmosphere. NMR (1H and 13C) spectra were
recorded on a Bruker AVANCE III; 400 and 500 Hz spectrometer.
Mass spectra were recorded on a Shimadzu VG-Autospec-3000
mass spectrometer.
4. Conclusions
We have synthesized 22 SNF derivatives, and evaluated their
antitumor activities against five cell lines (HepG2, A549, MCF-7,
HCT-116 and SH-SY5Y). All the derivatives exhibited more potent
inhibitory activity against HepG2, A549, MCF-7, HCT-116 and SH-
SY5Y than SNF, and most of them displayed selective cytotoxicity
toward HepG2 cell line. Our results indicate that the introduction of
different groups other than methyl group, especially a branched
alkyl or benzyl group, at side chain of SNF might contribute to
higher cytotoxic activity of the SNF derivatives. The mechanism
studies showed that the cytotoxic activity of these derivatives could
be attributed to the induction of cell cycle arrest and apoptosis in
cancer cells. SFN and derivative 85 exhibited time- and dose-
5.1.1. General procedures for preparation of 2e5
Acetone (150 mL) and dibromoalkyl (30 mol) were added to a
250 mL three-necked round-bottom flask fitted with a mechanical
stirrer and reflux condenser. Potassium phthalimide (11.85 g,
10 mol) was added slowly over a 15-min period, and then the re-
action mixture was heated under reflux for 10 h. The reaction
mixture was filtered via suction, and the acetone was removed via
rotary evaporation. The crude product was purified by flash

K. Hu et al. / European Journal of Medicinal Chemistry 64 (2013) 529e539
533
Fig. 2. Effects of SFN and 85 on cell apoptosis of HepG2 cells. (A and B) Induction of apoptosis were measured by Annexin-V/PI double-staining assay after treatment with SFN (5, 10
and 20 M) and 85 (5, 10 and 20 M) for 48 h. (C) The profiles showed the apoptotic proportion of HepG2 cells treated with SFN and 85, respectively. *p < 0.05, **p < 0.01 compared
to control.
m
m
chromatography on silica gel (EtOAc:petroleum ether ¼ 1:14) to
afford 2e5 as white solid.
5.1.1.4. 2-(6-Bromobutyl)isoindoline-1,3-dione (5). Yield 71%; 1H
NMR (CDCl3, 400 MHz): 1.33e1.41 (m, 2H), 1.45e1.53 (m, 2H),
d
1.66e1.73 (m, 2H), 1.82e1.89 (m, 2H), 3.39 (t, 2H, J ¼ 6.4 Hz), 3.68 (t,
5.1.1.1. 2-(4-Bromobutyl)isoindoline-1,3-dione (3). Yield 75%; 1H
2H, J ¼ 7.2 Hz), 7.72 (dd, 2H, J ¼ 2.8, 5.2 Hz), 7.83 (dd, 2H, J ¼ 2.8,
NMR (CDCl3, 500 MHz):
d
1.84e1.94 (m, 4H), 3.45 (t, 2H, J ¼ 6.0 Hz),
5.2 Hz); 13C NMR (CDCl3, 100 MHz):
d 25.02, 26.71, 27.40, 31.61,
32.60, 36.84, 122.17, 131.18, 132.86, 167.40.
3.73 (t, 2H, J ¼ 6.5 Hz), 7.73 (dd, 2H, J ¼ 3.0, 5.5 Hz), 7.85 (dd, 2H,
J ¼ 3.0, 5.0 Hz); 13C NMR (CDCl3, 125 MHz):
d 27.27, 29.89, 32.73,
36.99, 123.27, 132.11, 133.99, 168.36.
5.1.2. General procedures for preparation of 6e9
To a stirred solution of 2e5 (5 mmol) in THF (60 mL) was added
potassium thioacetate (15 mmol), and the mixture was refluxed for
3e4 h. And the solvent was evaporated to dryness in vacuo, and
then added water (30 mL), and the organic materials were
extracted with EtOAc (3 ꢁ 30 mL). The combined extracts were
washed with water and brine, dried over anhydrous MgSO4, and
concentrated in vacuo. The crude product was purified by flash
chromatography on silica gel (EtOAc:petroleum ether ¼ 1:8) to give
compounds 6e9 as white powder.
5.1.1.2. 2-(3-Bromobutyl)isoindoline-1,3-dione (2). Yield 68%; 1H
NMR (CDCl3, 400 MHz):
2.23e2.30 (m, 2H), 3.42 (t, 2H, J ¼ 6.8 Hz),
d
3.84 (t, 2H, J ¼ 6.8 Hz), 7.73 (dd, 2H, J ¼ 3.2, 5.2 Hz), 7.85 (dd, 2H,
J ¼ 3.2, 5.2 Hz); 13C NMR (CDCl3, 100 MHz):
d 29.79, 31.67, 36.76,
123.33, 132.05, 134.06, 168.23.
5.1.1.3. 2-(5-Bromobutyl)isoindoline-1,3-dione (4). Yield 72%; 1H
NMR (CDCl3, 400 MHz): 1.48e1.54 (m, 2H), 1.68e1.73 (m, 2H),
d
1.87e1.93 (m, 2H), 3.39 (t, 2H, J ¼ 6.8 Hz), 3.70 (t, 2H, J ¼ 7.2 Hz),
7.72 (dd, 2H, J ¼ 3.2, 5.2 Hz), 7.85 (dd, 2H, J ¼ 3.2, 5.2 Hz); 13C NMR
5.1.2.1. S-(4-(1,3-Dioxoisoindolin-2-yl)butyl)ethanethioate
Yield 96%; 1H NMR (CDCl3, 500 MHz):
1.61e1.66 (m, 2H), 1.72e
1.77 (m, 2H), 2.31 (s, 3H), 2.91 (t, 2H, J ¼ 7.0 Hz), 3.69 (t, 2H,
(7).
(CDCl3, 100 MHz):
133.92, 168.39.
d
25.42, 27.74, 32.22, 33.31, 37.68, 123.22, 132.17,
d

534
K. Hu et al. / European Journal of Medicinal Chemistry 64 (2013) 529e539
Fig. 3. Effects of SFN and 85 on TransAMÔ Nrf2 activation of HepG2 cells. (A) Nrf2 activation was measured by TransAM Nrf2 Kit after treatment with SFN and 85 (20
times (2, 4 and 8 h). (B) Nrf2 activation was measured after treatment with different concentrations of SFN and 85 (5, 10 and 20 M) for 6 h.
mM) for different
m
J ¼ 7.0 Hz), 7.72 (dd, 2H, J ¼ 3.0, 5.5 Hz), 7.85 (dd, 2H, J ¼ 3.0, 5.5 Hz);
13C NMR (CDCl3, 125 MHz):
26.93, 27.70, 28.52, 30.60, 37.42,
123.24, 132.17, 133.93, 168.35, 195.60.
5.1.3.3. 2-(5-Mercaptopentyl)isoindoline-1,3-dione (12). Yield 92%;
1H NMR (CDCl3, 400 MHz):
d
d
1.32 (t, 1H, J ¼ 7.6 Hz), 1.41e1.48 (m,
2H), 1.62e1.73 (m, 4H), 2.52 (dd, 2H, J ¼ 7.6, 14.8 Hz), 3.69 (t, 2H,
J ¼ 7.2 Hz), 7.71 (dd, 2H, J ¼ 3.2, 5.2 Hz), 7.84 (dd, 2H, J ¼ 3.2, 7.2 Hz);
5.1.2.2. S-(3-(1,3-Dioxoisoindolin-2-yl)propyl)ethanethioate
(6).
13C NMR (CDCl3, 100 MHz):
123.20, 132.18, 133.90, 168.41.
d 24.40, 25.58, 28.07, 33.50, 37.81,
Yield 75%; 1H NMR (CDCl3, 400 MHz):
d 1.94e2.01 (m, 2H), 2.32 (s,
3H), 2.90 (t, 2H, J ¼ 7.2 Hz), 3.76 (t, 2H, J ¼ 6.8 Hz), 7.72 (dd, 2H,
J ¼ 3.2, 5.2 Hz), 7.85 (dd, 2H, J ¼ 3.2, 5.2 Hz); 13C NMR (CDCl3,
5.1.3.4. 2-(6-Mercaptohexyl)isoindoline-1,3-dione (13). Yield 95%;
1H NMR (CDCl3, 400 MHz):
1.25e1.47 (m, 5H), 1.57e1.72 (m, 4H),
100 MHz):
195.37.
d
26.42, 28.67, 30.58, 36.91,123.29,132.11,133.99,168.30,
d
2.51 (dd, 2H, J ¼ 7.6, 14.4 Hz), 3.68 (t, 2H, J ¼ 7.2 Hz), 7.72 (dd, 2H,
J ¼ 4.0, 8.0 Hz), 7.85 (dd, 2H, J ¼ 4.0, 8.0 Hz); 13C NMR (CDCl3,
5.1.2.3. S-(5-(1,3-Dioxoisoindolin-2-yl)pentyl)ethanethioate
(8).
100 MHz): d 23.48, 25.29, 26.85, 27.45, 32.82, 36.89, 122.16, 131.19,
Yield 87%; 1H NMR (CDCl3, 400 MHz):
d
1.37e1.45 (m, 2H), 1.58e
132.85, 167.41.
1.73 (m, 4H), 2.85 (t, 2H, J ¼ 7.2 Hz), 3.68 (t, 2H, J ¼ 7.2 Hz), 7.70e
7.72 (dd, 2H, J ¼ 3.6, 5.2 Hz), 7.83e7.85 (dd, 2H, J ¼ 3.2, 4.8 Hz); 13C
5.1.4. General procedures for preparation of 14e34
NMR (CDCl3, 100 MHz):
d
26.00, 28.11, 28.88, 29.11, 30.59, 37.78,
Method A: ReBr (18 mmol) was added to a mixture containing
sodium hydride (30 mmol) and 10e13 (15 mmol) in dry THF (50 mL).
The mixture was stirred at room temperature for 1e2 h. The reaction
mixturewascooled to0 ꢀC andwater(20 mL)wasadded. Thesolution
was extracted with EtOAc (3 ꢁ 30 mL). The organic layer was sepa-
rated, dried with anhydrous MgSO4, and evaporated under vacuum.
The crude product was purified using flash chromatography on silica
gel (EtOAc:petroleum ether ¼ 1:10) to give compounds 14e34.
Method B: To a solution of [Pd2(dba)3] (0.10 mmol) and DPPF
(1,10-bis(diphenylphosphino)ferrocene) (0.20 mmol) in toluene
(20 mL) were added bromobenzene (10 mmol), DIPEA (11 mmol)
and 11 (10 mmol) at rt. The solution was stirred under reflux for 2 h
then cooled to rt. The reaction was quenched by addition of H2O
and extracted with EtOAc (3 ꢁ 20 mL), and the combined organic
layers were washed with brine, dried over anhydrous Na2SO4, and
filtered. The filtrate was concentrated in vacuo and the residue was
purified by flash chromatography on silica gel to afford 21.
123.20, 132.21, 133.88, 168.39, 195.75.
5.1.2.4. S-(6-(1,3-Dioxoisoindolin-2-yl)hexyl) ethanethioate (9).
Yield 97%; 1H NMR (CDCl3, 400 MHz):
d 1.35e1.40 (m, 4H), 1.53e
1.58 (m, 2H), 1.65e1.69 (m, 2H), 2.30 (s, 3H), 2.85 (t, 2H,
J ¼ 7.2 Hz), 3.67 (t, 2H, J ¼ 7.6 Hz), 7.70 (dd, 2H, J ¼ 2.8, 5.2 Hz),
7.84 (dd, 2H, J ¼ 2.8, 5.2 Hz); 13C NMR (CDCl3, 100 MHz):
d 25.35,
27.29, 27.41, 28.00, 28.33, 29.58, 36.89, 122.15, 131.21, 132.83,
167.39, 194.80.
5.1.3. General procedures for preparation of 10e13
A solution of 6e9 (10 mmol) in anhydrous methanol (30 mL)
was first degassed and refilled with N2. Concentrated HCl (4 mL)
was added to the solution, and the resulting mixture was refluxed
for 5 h under N2 atmosphere until no more starting material could
be detected by TLC. The reaction mixture was quenched with water
(20 mL) and extracted with EtOAc (3 ꢁ 30 mL). The organic layer
was dried over anhydrous MgSO4 and concentrated in vacuo to give
crude product, which was purified by flash chromatography on
silica gel (EtOAc:petroleum ether ¼ 1:12) to give compounds 10e13
as white powder.
5.1.4.1. 2-(4-(Methylthio)butyl)isoindoline-1,3-dione (14). Yield 64%;
1H NMR (CDCl3, 400 MHz):
d 1.59e1.68 (m, 2H), 1.76e1.83 (m, 2H),
2.08 (s, 2H), 2.53 (t, 2H, J ¼ 6.8 Hz), 3.71 (t, 2H, J ¼ 7.2 Hz), 7.72 (dd, 2H,
J¼ 3.2,5.6Hz), 7.84(dd,2H, J¼ 3.2, 5.6Hz);13CNMR(CDCl3,100MHz):
d
15.51, 26.37, 27.71, 33.64, 37.50, 123.21, 132.12, 133.92, 168.38.
5.1.3.1. 2-(4-Mercaptobutyl)isoindoline-1,3-dione (11). Yield 94%;
1H NMR (CDCl3, 500 MHz):
d
1.26 (t, 1H, J ¼ 7.0 Hz), 1.63e1.69 (m,
5.1.4.2. 2-(4-(Ethylthio)butyl)isoindoline-1,3-dione (15). Yield 65%;
1H NMR (CDCl3, 400 MHz):
2H), 1.77e1.83 (m, 2H), 2.55e2.59 (dd, 2H, J ¼ 7.0 Hz), 3.70 (t, 2H,
J ¼ 7.0 Hz), 7.72 (dd, 2H, J ¼ 3.0, 5.5 Hz), 7.84 (dd, 2H, J ¼ 3.0, 5.5 Hz);
d
1.24 (t, 3H, J ¼ 7.2 Hz), 1.62e1.67 (m,
2H), 1.76e1.83 (m, 2H), 2.49e2.58 (m, 4H), 3.71 (t, 2H, J ¼ 6.8 Hz),
13C NMR (CDCl3, 125 MHz):
132.11, 133.95, 168.39.
d 24.09, 27.31, 31.10, 37.33, 123.24,
7.71 (dd, 2H, J ¼ 3.2, 5.2 Hz), 7.84 (dd, 2H, J ¼ 3.2, 5.2 Hz); 13C NMR
(CDCl3, 100 MHz):
d 14.78, 25.91, 26.85, 27.82, 31.05, 37.51, 123.21,
132.12, 133.92, 168.40.
5.1.3.2. 2-(3-Mercaptopropyl)isoindoline-1,3-dione (10). Yield 60%;
1H NMR (CDCl3, 400 MHz):
d
1.59 (t, 1H, J ¼ 8.4 Hz), 1.97e2.04 (m,
5.1.4.3. 2-(4-(Propylthio)butyl)isoindoline-1,3-dione (16). Yield 53%;
2H), 2.56 (dd, 2H, J ¼ 7.2, 14.8 Hz), 3.82 (t, 2H, J ¼ 6.8 Hz), 7.72 (dd,
1H NMR (CDCl3, 500 MHz):
d
0.97 (t, 3H, J ¼ 7.5 Hz), 1.55e1.66 (m,
2H, J ¼ 3.2, 5.2 Hz), 7.85 (dd, 2H, J ¼ 3.2, 5.2 Hz); 13C NMR (CDCl3,
4H),1.76e1.82 (m, 2H), 2.47 (t, 2H, J ¼ 7.0 Hz), 2.54 (t, 2H, J ¼ 7.5 Hz),
100 MHz):
d 21.90, 32.74, 36.46, 123.28, 132.09, 134.01, 168.38.
3.71 (t, 2H, J ¼ 7.0 Hz), 7.71 (dd, 2H, J ¼ 3.0, 5.5 Hz), 7.84 (dd, 2H,

K. Hu et al. / European Journal of Medicinal Chemistry 64 (2013) 529e539
535
J ¼ 3.0, 5.5 Hz); 13C NMR (CDCl3, 125 MHz):
d
13.50, 22.99, 26.94,
J ¼ 7.2 Hz), 3.70 (t, 2H, J ¼ 6.8 Hz), 7.19 (t, 2H, J ¼ 7.2 Hz), 7.29 (d, 2H,
J ¼ 7.2 Hz), 7.70 (dd, 2H, J ¼ 2.8, 5.2 Hz), 7.83 (dd, 2H, J ¼ 2.4, 4.8 Hz);
27.81, 31.51, 34.21, 37.53, 123.22, 132.13, 133.92, 168.42.
13C NMR (CDCl3,100 MHz):
d 25.87, 26.77, 30.73, 32.64, 35.39, 36.47,
5.1.4.4. 2-(4-(Butylthio)butyl)isoindoline-1,3-dione (17). Yield 55%;
122.20, 125.29, 127.43, 127.47, 131.15, 132.89, 139.60, 167.36.
1H NMR (CDCl3, 400 MHz):
d
0.90 (t, 3H, J ¼ 7.6 Hz), 1.36e1.43 (m,
2H), 1.51e1.67 (m, 4H), 1.76e1.83 (m, 2H), 2.47e2.56 (m, 4H), 3.70
(t, 2H, J ¼ 7.2 Hz), 7.71 (dd, 2H, J ¼ 2.8, 5.2 Hz), 7.84 (dd, 2H, J ¼ 2.8,
5.1.4.13. 2-(4-((Pyridin-2-ylmethyl)thio)butyl)isoindoline-1,3-dione
(26). Yield 60%; 1H NMR (CDCl3, 400 MHz):
d 1.58e1.65 (m, 2H),
5.2 Hz); 13C NMR (CDCl3, 100 MHz):
d
13.67, 22.01, 26.96, 27.82,
1.71e1.77 (m, 2H), 2.53 (t, 2H, J ¼ 7.2 Hz), 3.66 (t, 2H, J ¼ 7.2 Hz),
3.82 (s, 2H), 7.11e7.14 (m, 1H), 7.36 (d, 1H, J ¼ 7.6 Hz), 7.61e7.65 (m,
1H), 7.70 (dd, 2H, J ¼ 3.2, 5.6 Hz), 7.84 (dd, 2H, J ¼ 3.2, 5.6 Hz), 8.49
31.59, 31.81, 31.87, 37.55, 123.21, 132.18, 133.90, 168.39.
5.1.4.5. 2-(4-(Isopropylthio)butyl)isoindoline-1,3-dione
(18). Yield
(t, 1H, J ¼ 4.0 Hz); 13C NMR (CDCl3, 100 MHz):
d 26.55, 27.74, 31.17,
37.49, 38.23, 121.84, 123.00, 123.21, 132.15, 133.90, 136.67, 149.23,
158.96, 168.36.
51%; 1H NMR (CDCl3, 400 MHz):
d
1.24 (d, 6H, J ¼ 6.8 Hz), 1.59e1.66
(m, 2H), 1.76e1.83 (m, 2H), 2.56 (t, 2H, J ¼ 7.2 Hz), 2.85e2.95 (m, 1H),
3.70 (t, 2H, J ¼ 7.2 Hz), 7.71 (dd, 2H, J ¼ 3.2, 5.2 Hz), 7.84 (dd, 2H,
J ¼ 3.2, 5.2 Hz); 13C NMR (CDCl3, 100 MHz):
d
23.41, 23.45, 27.04,
5.1.4.14. 2-(4-((Thiophen-2-ylmethyl)thio)butyl)isoindoline-1,3-
27.92, 29.94, 34.78, 37.53, 123.20, 132.13, 133.90, 168.39.
dione (27). Yield 70%; 1H NMR (CDCl3, 400 MHz):
d 1.56e1.64 (m,
2H), 1.73e1.80 (m, 2H), 2.53 (t, 2H, J ¼ 7.2 Hz), 3.68 (t, 2H,
J ¼ 7.2 Hz), 3.90 (s, 2H), 6.86e6.91 (m, 2H), 7.15e7.17 (m, 1H), 7.71
(dd, 2H, J ¼ 3.2, 5.6 Hz), 7.83 (dd, 2H, J ¼ 3.2, 5.6 Hz); 13C NMR
5.1.4.6. 2-(4-(Isobutylthio)butyl)isoindoline-1,3-dione (19). Yield 57%;
1H NMR (CDCl3, 400 MHz):
d
0.97 (d, 6H, J ¼ 6.8 Hz), 1.60e1.66 (m,
2H),1.73e1.81 (m, 3H), 2.38 (d, 2H, J ¼ 6.8 Hz), 2.53 (t, 2H, J ¼ 7.2 Hz),
(CDCl3, 100 MHz): d 26.46, 27.78, 30.63, 31.10, 37.51, 123.23, 124.80,
3.70 (t, 2H, J ¼ 7.2 Hz), 7.71 (dd, 2H, J ¼ 3.2, 5.6 Hz), 7.84 (dd, 2H,
125.97, 126.65, 126.88, 132.18, 133.93, 142.18, 168.37.
J ¼ 3.2, 5.6 Hz); 13C NMR (CDCl3, 100 MHz):
d 22.04, 26.99, 27.80,
28.63, 32.20, 37.53, 41.51, 123.20, 132.14, 133.91, 168.39.
5.1.4.15. 2-(4-((Furan-2-ylmethyl)thio)butyl)isoindoline-1,3-dione
(28). Yield 56%; 1H NMR (CDCl3, 400 MHz):
d 1.57e1.63 (m, 2H),
5.1.4.7. 2-(4-(Cyclopentylthio)butyl)isoindoline-1,3-dione
(20).
1.73e1.80 (m, 2H), 2.54 (t, 2H, J ¼ 7.2 Hz), 3.67 (t, 2H, J ¼ 6.8 Hz),
3.69 (s, 2H), 6.15 (d,1H, J ¼ 2.8 Hz), 6.26 (s, 1H), 7.32 (s, 1H), 7.71 (dd,
2H, J ¼ 3.2, 5.2 Hz), 7.84 (dd, 2H, J ¼ 3.2, 5.2 Hz); 13C NMR (CDCl3,
Yield 50%; 1H NMR (CDCl3, 400 MHz):
d 1.44e1.83 (m, 10H), 1.93e
1.99 (m, 2H), 2.57 (t, 2H, J ¼ 7.2 Hz), 3.03e3.10 (m, 1H), 3.71 (t, 2H,
J ¼ 6.8 Hz), 7.71 (dd, 2H, J ¼ 3.2, 5.6 Hz), 7.84 (dd, 2H, J ¼ 3.2, 5.6 Hz);
100 MHz):
d 26.52, 27.75, 28.33, 31.24, 37.49, 107.33, 110.37, 123.21,
13C NMR (CDCl3, 100 MHz):
d
24.81, 27.07, 27.92, 31.31, 33.87, 37.55,
132.16, 133.92, 142.04, 151.82, 168.37.
43.83, 123.20, 132.16, 133.90, 168.40.
5.1.4.16. 2-(3-(Benzylthio)propyl)isoindoline-1,3-dione
(29). Yield
5.1.4.8. 2-(4-(Phenylthio)butyl)isoindoline-1,3-dione (21). Yield 58%;
53%; 1H NMR (CDCl3, 400 MHz):
d 1.89e1.97 (m, 2H), 2.43 (t, 2H,
1H NMR (CDCl3, 400 MHz):
d
1.64e1.71 (m, 2H), 1.80e1.87 (m, 2H),
J ¼ 7.2 Hz), 3.73 (t, 2H, J ¼ 7.2 Hz), 3.76 (s, 2H), 7.17e7.28 (m, 5H), 7.71
2.95 (t, 2H, J ¼ 7.2 Hz), 3.69 (t, 2H, J ¼ 7.2 Hz), 7.12e4 (t, 1H,
J ¼ 7.6 Hz), 7.23 (d, 2H, J ¼ 7.6 Hz), 7.31 (d, 2H, J ¼ 7.6 Hz), 7.71 (dd,
2H, J ¼ 3.2, 5.2 Hz), 7.83 (dd, 2H, J ¼ 3.2, 5.2 Hz); 13C NMR (CDCl3,
(dd, 2H, J ¼ 3.2, 8.4 Hz), 7.84 (dd, 2H, J ¼ 3.2, 8.4 Hz); 13C NMR (CDCl3,
100 MHz): d 28.05, 28.40, 36.01, 37.13, 123.23, 126.93, 128.46, 128.86,
132.17, 133.92, 138.13, 168.28.
100 MHz): d 26.30, 27.60, 33.23, 37.40, 123.21, 125.96, 128.86, 129.40,
132.11, 133.91, 136.29, 168.35.
5.1.4.17. 2-(3-(Isopropylthio)propyl)isoindoline-1,3-dione
(30).
Yield 50%; 1H NMR (CDCl3, 400 MHz):
d
1.25 (d, 6H, J ¼ 6.8 Hz),
5.1.4.9. 2-(4-(Benzylthio)butyl)isoindoline-1,3-dione (22). Yield 69%;
1.93e2.00 (m, 2H), 2.57 (t, 2H, J ¼ 7.6 Hz), 2.89e2.94 (m,1H), 3.79 (t,
2H, J ¼ 7.2 Hz), 7.71 (dd, 2H, J ¼ 3.2, 5.2 Hz), 7.83 (dd, 2H, J ¼ 3.2,
1H NMR (CDCl3, 400 MHz):
d 1.54e1.62 (m, 2H), 1.71e1.78 (m, 2H),
2.45 (t, 2H, J ¼ 7.2 Hz), 3.66 (t, 2H, J ¼ 6.8 Hz), 3.68 (s, 2H), 7.17e7.28
5.2 Hz); 13C NMR (CDCl3, 100 MHz):
37.31, 123.23, 132.14, 133.94, 168.34.
d 23.38, 27.88, 28.66, 34.83,
(m, 5H), 7.70 (dd, 2H, J ¼ 3.2, 4.8 Hz), 7.83 (dd, 2H, J ¼ 3.2, 4.8 Hz); 13C
NMR (CDCl3, 100 MHz):
d 26.47, 27.75, 30.82, 36.29, 37.49, 123.20,
126.90, 128.45, 128.80, 132.11, 133.91, 138.47, 168.36.
5.1.4.18. 2-(5-(Benzylthio)pentyl)isoindoline-1,3-dione
(31). Yield
85%; 1H NMR (CDCl3, 400 MHz):
d
1.38e1.43 (m, 2H), 1.56e1.69 (m,
5.1.4.10. 2-(4-((4-Nitrobenzyl)thio)butyl)isoindoline-1,3-dione (23).
4H), 2.40 (t, 2H, J ¼ 7.2 Hz), 3.66 (t, 2H, J ¼ 7.2 Hz), 3.69 (s, 2H), 7.20e
7.30 (m, 5H), 7.71 (dd, 2H, J ¼ 3.2, 5.2 Hz), 7.84 (dd, 2H, J ¼ 3.2,
Yield 65%; 1H NMR (CDCl3, 400 MHz):
d 1.57e1.63 (m, 2H), 1.72e
1.79 (m, 2H), 2.46 (t, 2H, J ¼ 7.2 Hz), 3.69 (t, 2H, J ¼ 6.8 Hz), 3.76 (s,
2H), 7.47 (d, 2H, J ¼ 8.0 Hz), 7.70e7.73 (dd, 2H, J ¼ 3.2, 8.0 Hz), 7.83e
7.85 (dd, 2H, J ¼ 3.2, 7.6 Hz), 8.16 (d, 2H, J ¼ 8.0 Hz); 13C NMR (CDCl3,
5.2 Hz); 13C NMR (CDCl3,100 MHz):
d 26.05, 28.18, 28.76, 31.10, 36.26,
37.81, 123.18, 126.88, 128.45, 128.82, 132.15, 133.88, 138.56, 168.41.
100 MHz):
d
25.35, 26.64, 30.09, 34.81, 36.32, 122.21, 122.74, 128.57,
5.1.4.19. 2-(5-(Isopropylthio)pentyl)isoindoline-1,3-dione
Yield 51%; 1H NMR (CDCl3, 400 MHz):
(32).
131.03, 132.90, 132.98, 145.38, 145.96.
d
1.24 (d, 6H, J ¼ 6.4 Hz),1.41e
1.48 (m, 2H),1.59e1.73 (m, 4H), 2.51 (t, 2H, J ¼ 7.6 Hz), 2.86e2.93 (m,
1H), 3.68 (t, 2H, J ¼ 7.2 Hz), 7.71 (dd, 2H, J ¼ 3.2, 5.2 Hz), 7.84 (dd, 2H,
5.1.4.11. 2-(4-((4-Methoxybenzyl)thio)butyl)isoindoline-1,3-dione (24).
Yield 68%; 1H NMR (CDCl3, 400 MHz):
d
1.57e1.63 (m, 2H), 1.71e1.78
J ¼ 3.2, 5.2 Hz); 13C NMR (CDCl3, 100 MHz):
d 23.44, 26.26, 28.23,
29.42, 30.31, 34.80, 37.88, 123.18, 132.22, 133.87, 168.41.
(m, 2H), 2.44 (t, 2H, J ¼ 6.8 Hz), 3.67 (t, 4H, J ¼ 7.2 Hz), 3.78 (s, 3H),
6.82 (d, 2H, J ¼ 8.4 Hz), 7.20 (d, 2H, J ¼ 8.4 Hz), 7.71 (dd, 2H, J ¼ 3.2,
4.8 Hz), 7.84 (dd, 2H, J ¼ 3.2, 4.8 Hz); 13C NMR (CDCl3, 100 MHz):
5.1.4.20. 2-(6-(Benzylthio)hexyl)isoindoline-1,3-dione
(33). Yield
d
26.51, 27.79, 30.72, 35.65, 37.51, 55.26, 113.88, 123.19, 129.85, 130.41,
77%; 1H NMR (CDCl3, 400 MHz):
d 1.25e1.42 (m, 4H), 1.51e1.58 (m,
132.13, 133.90, 158.58, 168.36.
2H), 1.61e1.69 (m, 2H), 2.39 (t, 2H, J ¼ 7.2 Hz), 3.66 (t, 4H, J ¼ 7.2 Hz),
7.20e7.30 (m, 5H), 7.70 (dd, 2H, J ¼ 2.8, 5.6 Hz), 7.83 (dd, 2H, J ¼ 2.8,
5.1.4.12. 2-(4-(Phenethylsulfinyl)butyl)isoindoline-1,3-dione
(25).
5.6 Hz); 13C NMR (CDCl3, 100 MHz):
d 25.43, 27.33, 27.43, 28.03,
30.25, 35.29, 36.91, 122.14, 125.84, 127.42, 127.80, 131.19, 132.82,
137.62, 167.39.
Yield 55%; 1H NMR (CDCl3, 400 MHz):
d
1.59e1.68 (m, 2H), 1.75e
1.82 (m, 2H), 2.54e2.60 (m, 2H), 2.75 (t, 2H, J ¼ 8.0 Hz), 2.86 (t, 2H,

536
K. Hu et al. / European Journal of Medicinal Chemistry 64 (2013) 529e539
5.1.4.21. 2-(6-(Isopropylthio)hexyl)isoindoline-1,3-dione
(34).
1.24 (d, 6H, J ¼ 6.8 Hz),
5.1.5.7. Cyclopentyl(4-isothiocyanatobutyl)sulfane (62). Yield 49%;
Yield 50%; 1H NMR (CDCl3, 400 MHz):
d
1H NMR (CDCl3, 400 MHz):
d 1.48e1.86 (m, 10H), 1.96e2.02 (m, 2H),
1.32e1.47 (m, 4H), 1.54e1.72 (m, 4H), 2.51 (t, 2H, J ¼ 7.6 Hz), 2.86e
2.92 (m, 1H), 3.68 (t, 2H, J ¼ 7.2 Hz), 7.70 (dd, 2H, J ¼ 2.8, 5.6 Hz),
2.58 (t, 2H, J ¼ 7.2 Hz), 3.05e3.12 (m, 1H), 3.55 (t, 2H, J ¼ 6.4 Hz); 13C
NMR (CDCl3, 100 MHz):
44.74, 130.26.
d 24.80, 26.59, 29.07, 30.96, 33.86, 43.85,
7.83 (dd, 2H, J ¼ 2.8, 5.6 Hz); 13C NMR (CDCl3, 100 MHz):
d 25.50,
27.48, 27.55, 28.67, 29.43, 33.77, 36.95, 122.14, 131.20, 132.82,
167.40.
5.1.5.8. (4-Isothiocyanatobutyl)(phenyl)sulfane (63). Yield 65%; 1H
NMR (CDCl3, 400 MHz):
d
1.74e1.86 (m, 4H), 2.95 (t, 2H, J ¼ 7.2 Hz),
5.1.5. General procedures for preparation of 56e76
3.53 (t, 2H, J ¼ 6.0 Hz), 7.09e7.36 (m, 5H); 13C NMR (CDCl3,
N-Cinnamylphthalimide 14e34 (10 mmol) was mixed with 80%
hydrazine hydrate (25 mmol) and MeOH (60 mL), and the mixture
was stirred at 70 ꢀC for 3e4 h. After the solvent was removed by a
rotary evaporator, the solid residue was treated with 1 N NaOH
(30 mL), and the resulting mixture was extracted with EtOAc
(4 ꢁ 30 mL) and dried over anhydrous MgSO4. Evaporation of the
solvent under reduced pressure afforded the crude product 35e55,
which were used for next step without purification.
100 MHz):
d 26.08, 28.86, 33.09, 44.67, 126.28, 129.03, 129.60,
130.50, 135.95.
5.1.5.9. Benzyl(4-isothiocyanatobutyl)sulfane (64). Yield 65%; 1H
NMR (CDCl3, 400 MHz):
d
1.62e1.79 (m, 4H), 2.43 (t, 2H, J ¼ 6.8 Hz),
3.47 (t, 2H, J ¼ 6.0 Hz), 3.71 (s, 2H), 7.22e7.34 (m, 5H); 13C NMR
(CDCl3,100 MHz): d 25.95, 28.88, 30.28, 36.20, 44.63, 127.06,128.54,
128.84, 130.25, 138.25.
Et3N (16 mmol) was added to a solution of amine 35e55
(5 mmol) in anhydrous THF (30 mL), and the mixture was cooled to
0 ꢀC in an ice bath. Carbon disulfide (5.5 mmol) was then added to
the reaction mixture slowly. When the addition was completed, the
ice bath was removed and the mixture was stirred at room tem-
perature for 90 min. The reaction mixture was cooled to 0 ꢀC and
MsCl (5.5 mmol) was added in one portion. The ice bath was
removed and the mixture was stirred at room temperature for
30 min. The resulting yellow suspension was diluted with DCM
(40 mL), washed successively with 1 M HCl (2 ꢁ 15 mL), water
(15 mL), and brine (20 mL), and then dried with anhydrous MgSO4.
The solvent was concentrated in vacuo and the crude product was
purified by flash chromatography (EtOAc:petroleum ether ¼ 1:10)
to give a light-yellow oil 56e76.
5.1.5.10. (4-Isothiocyanatobutyl)(4-nitrobenzyl)sulfane
(65). Yield
62%; 1H NMR (CDCl3, 400 MHz):
d 1.66e1.79 (m, 4H), 2.45 (t, 2H,
J ¼ 7.2 Hz), 3.52 (t, 2H, J ¼ 6.0 Hz), 3.78 (s, 2H), 7.50 (d, 2H, J ¼ 8.4 Hz),
8.19 (d, 2H, J ¼ 8.4 Hz); 13C NMR (CDCl3, 100 MHz):
d 25.94, 28.91,
30.63, 35.67, 44.65, 123.85, 128.33, 129.66, 146.11, 147.07.
5.1.5.11. (4-Isothiocyanatobutyl)(4-methoxybenzyl)sulfane
(66).
Yield 61%; 1H NMR (CDCl3, 400 MHz):
d 1.64e1.79 (m, 4H), 2.42 (t,
2H, J ¼ 7.2 Hz), 3.49 (t, 2H, J ¼ 6.0 Hz), 3.67 (s, 2H), 3.80 (s, 3H), 6.85
(d, 2H, J ¼ 8.4 Hz), 7.23 (d, 2H, J ¼ 8.4 Hz); 13C NMR (CDCl3,
100 MHz): d 26.04, 28.98, 30.24, 35.61, 44.70, 55.35, 114.00, 128.38,
129.94, 130.22, 158.72.
5.1.5.12. (4-Isothiocyanatobutyl)(phenethyl)sulfane (67). Yield 63%;
5.1.5.1. (4-Isothiocyanatobutyl)(methyl)sulfane (56). Yield 48%; 1H
1H NMR (CDCl3, 400 MHz):
d
1.67e1.81 (m, 4H), 2.54(t, 2H, J ¼ 6.8 Hz),
2.78 (t, 2H, J ¼ 7.6 Hz), 2.89 (t, 2H, J ¼ 8.0 Hz), 3.52 (t, 2H, J ¼ 5.6 Hz),
7.20e7.32 (m, 5H); 13C NMR (CDCl3, 100 MHz):
25.92, 27.94, 30.42,
NMR (CDCl3, 400 MHz): d 1.70e1.86 (m, 4H), 2.11 (s, 3H), 2.54 (t, 2H,
J ¼ 6.8 Hz), 3.56 (t, 2H, J ¼ 6.4 Hz); 13C NMR (CDCl3, 100 MHz):
d
d
15.45, 25.86, 28.88, 33.32, 44.74, 130.39.
32.64, 35.35, 43.70, 125.38, 127.48, 127.50, 129.46, 139.44.
5.1.5.2. Ethyl(4-isothiocyanatobutyl)sulfane (57). Yield 54%; 1H
5.1.5.13. 2-(((4-Isothiocyanatobutyl)thio)methyl)pyridine
(68).
NMR (CDCl3, 400 MHz):
d
1.26 (t, 3H, J ¼ 7.6 Hz), 1.69e1.86 (m, 4H),
Yield 64%; 1H NMR (CDCl3, 400 MHz):
d 1.65e1.80 (m, 4H), 2.53 (t,
2.51e2.58 (m, 4H), 3.55 (t, 2H, J ¼ 6.0 Hz); 13C NMR (CDCl3,
2H, J ¼ 7.2 Hz), 3.49 (t, 2H, J ¼ 6.4 Hz), 3.83 (s, 2H), 7.16e7.19 (m,1H),
100 MHz):
d 14.75, 25.91, 26.35, 28.96, 30.72, 44.73, 130.19.
7.37 (d, 1H, J ¼ 7.6 Hz), 7.64e7.69 (m, 1H), 8.52e8.54 (m, 1H); 13C
NMR (CDCl3, 100 MHz):
d 26.08, 28.87, 30.67, 38.10, 44.64, 121.97,
5.1.5.3. (4-Isothiocyanatobutyl)(propyl)sulfane (58). Yield 47%; 1H
NMR (CDCl3, 500 MHz):
0.99 (t, 3H, J ¼ 5.6 Hz), 1.55e1.65 (m, 2H),
123.07, 130.19, 136.77, 149.26, 158.77.
d
1.69e1.75 (m, 2H), 1.79e1.85 (m, 2H), 2.48e2.57 (m, 4H), 3.5 (t, 2H,
5.1.5.14. 2-(((4-Isothiocyanatobutyl)thio)methyl)thiophene
(69).
J ¼ 6.5 Hz); 13C NMR (CDCl3, 125 MHz):
d
13.50, 22.95, 26.44, 28.97,
Yield 51%; 1H NMR (CDCl3, 400 MHz):
d 1.42e1.81 (m, 4H), 2.52 (t, 2H,
31.16, 34.19, 44.74, 130.24.
J ¼ 6.8 Hz), 3.50 (t, 2H, J ¼ 6.4 Hz), 3.93 (s, 2H), 6.93 (d, 2H, J ¼ 5.2 Hz),
7.21 (d, 1H, J ¼ 4.4 Hz); 13C NMR (CDCl3, 100 MHz):
d 25.91, 28.91,
30.53, 30.56, 44.65, 124.96, 126.11, 126.70, 130.47, 141.90.
5.1.5.4. Butyl(4-isothiocyanatobutyl)sulfane (59). Yield 50%; 1H
NMR (CDCl3, 400 MHz):
0.92 (t, 3H, J ¼ 7.2 Hz), 1.38e1.46 (m, 2H),
d
1.53e1.60 (m, 2H), 1.68e1.85 (m, 4H), 2.49e2.57 (m, 4H), 3.56 (t,
5.1.5.15. 2-(((4-Isothiocyanatobutyl)thio)methyl)furan
(70). Yield
2H, J ¼ 6.0 Hz); 13C NMR (CDCl3, 100 MHz):
d
13.69, 22.02, 26.46,
50%; 1H NMR (CDCl3, 400 MHz):
d
1.64e1.81 (m, 4H), 2.53 (t, 2H,
J¼ 6.8Hz), 3.51(t, 2H, J ¼ 6.4 Hz), 3.72 (s, 2H), 6.18e6.19(m,1H), 6.31e
6.32 (m, 1H), 7.36e7.37 (m, 1H); 13C NMR (CDCl3, 100 MHz):
26.07,
28.31, 28.93, 30.85, 44.69, 107.51, 110.47, 130.54, 142.16, 151.63.
29.00, 31.25, 31.77, 31.87, 44.76, 130.44.
d
5.1.5.5. Isopropyl(4-isothiocyanatobutyl)sulfane (60). Yield 43%; 1H
NMR (CDCl3, 400 MHz):
d
1.27 (d, 6H, J ¼ 6.8 Hz), 1.68e1.86 (m, 4H),
2.57 (t, 2H, J ¼ 6.8 Hz), 2.88e2.95 (m,1H), 3.55 (t, 2H, J ¼ 6.4 Hz); 13C
5.1.5.16. Benzyl(3-isothiocyanatopropyl)sulfane (71). Yield 44%; 1H
NMR (CDCl3, 100 MHz):
44.71, 130.05.
d
23.36, 23.39, 26.57, 29.05, 29.58, 34.85,
NMR (CDCl3, 400 MHz):
d
1.84e1.91 (m, 2H), 2.52 (t, 2H, J ¼ 6.8 Hz),
3.59 (t, 2H, J ¼ 6.4 Hz), 3.71 (s, 2H), 7.25e7.34 (m, 5H); 13C NMR
(CDCl3, 100 MHz):
128.81, 137.98.
d 28.02, 29.35, 36.38, 43.70, 127.19, 128.63,
5.1.5.6. Isobutyl(4-isothiocyanatobutyl)sulfane (61). Yield 45%; 1H
NMR (CDCl3, 400 MHz):
0.99 (d, 6H, J ¼ 6.8 Hz), 1.68e1.85 (m, 5H),
d
2.40 (d, 2H, J ¼ 6.8 Hz), 2.54 (t, 2H, J ¼ 7.2 Hz), 3.55 (t, 2H,
5.1.5.17. Isopropyl(3-isothiocyanatopropyl)sulfane (72). Yield 56%;
1H NMR (CDCl3, 400 MHz):
1.28 (d, 6H, J ¼ 6.8 Hz), 1.92e1.99 (m,
2H), 2.65 (t, 2H, J ¼ 6.8 Hz), 2.89e2.95 (m, 1H), 3.66 (t, 2H,
J ¼ 6.0 Hz); 13C NMR (CDCl3, 100 MHz):
d
22.05, 26.52, 28.65, 29.00,
d
31.89, 41.54, 44.77, 130.50.

K. Hu et al. / European Journal of Medicinal Chemistry 64 (2013) 529e539
537
J ¼ 6.4 Hz); 13C NMR (CDCl3, 100 MHz):
d
23.40, 27.19, 30.00, 35.15,
5.1.6.5. 1-(Isopropylsulfinyl)-4-isothiocyanatobutane
(81). Yield
43.88, 130.92.
82%; 1H NMR (CDCl3, 400 MHz):
d
1.28 (d, 3H, J ¼ 6.8 Hz), 1.32 (d,
3H, J ¼ 6.8 Hz),1.83e2.00 (m, 4H), 2.58e2.67 (m, 2H), 2.76e2.82 (m,
5.1.5.18. Benzyl(5-isothiocyanatopentyl)sulfane (73). Yield 47%; 1H
1H), 3.60 (t, 2H, J ¼ 6.4 Hz); 13C NMR (CDCl3, 100 MHz):
d 14.72,
NMR (CDCl3, 400 MHz):
d
1.42e1.69 (m, 6H), 2.42 (t, 2H, J ¼ 6.8 Hz),
15.97, 20.46, 20.50, 29.14, 44.70, 47.60, 50.56, 130.73; MS (ESI, m/z):
3.47 (t, 2H, J ¼ 6.8 Hz), 3.70 (s, 2H), 7.22e7.34 (m, 5H); 13C NMR
[M þ 1]þ205.9.
(CDCl3, 100 MHz):
d 25.76, 26.97, 28.44, 29.58, 36.47, 44.95, 127.01,
128.53, 128.86, 130.18, 138.54.
5.1.6.6. 1-(Isobutylsulfinyl)-4-isothiocyanatobutane (82). Yield 83%;
1H NMR (CDCl3, 400 MHz):
d
1.09 (d, 3H, J ¼ 5.2 Hz), 1.11 (d, 3H,
5.1.5.19. Isopropyl(5-isothiocyanatopentyl)sulfane (74). Yield 55%;
J ¼ 5.2 Hz), 1.83e2.00 (m, 4H), 2.18e2.29 (m, 1H), 2.36e2.42 (m,
1H NMR (CDCl3, 400 MHz):
d
1.26 (d, 6H, J ¼ 6.8 Hz), 1.42e1.75 (m,
1H), 2.63e2.75 (m, 3H), 3.60 (t, 2H, J ¼ 6.4 Hz); 13C NMR (CDCl3,
6H), 2.54 (t, 2H, J ¼ 7.2 Hz), 2.88e2.94 (m, 1H), 3.52 (t, 2H,
100 MHz): d 20.16, 21.71, 22.88, 23.96, 29.11, 44.69, 51.89, 62.16,
J ¼ 6.8 Hz); 13C NMR (CDCl3, 100 MHz):
d
23.48, 26.00, 29.05, 29.69,
131.04; MS (ESI, m/z): [M þ 1]þ 220.0.
30.25, 34.95, 45.00, 130.00.
5.1.6.7. ((4-Isothiocyanatobutyl)sulfinyl)cyclopentane
(83). Yield
5.1.5.20. Benzyl(6-isothiocyanatohexyl)sulfane (75). Yield 45%; 1H
NMR (CDCl3, 400 MHz): 1.36e1.41 (m, 2H),1.54e1.70 (m, 4H), 2.42
(t, 2H, J ¼ 7.2 Hz), 3.48 (t, 2H, J ¼ 6.4 Hz), 3.70 (s, 2H), 7.21e7.33 (m,
5H); 13C NMR (CDCl3, 100 MHz):
25.11, 26.89, 27.89, 28.79, 30.20,
81%; 1H NMR (CDCl3, 400 MHz):
d 1.55e2.00 (m,11H), 2.12e2.21 (m,
d
1H), 2.64e2.67 (m, 2H), 3.01e3.0 (m, 1H), 3.60 (t, 2H, J ¼ 6.4 Hz); 13C
NMR (CDCl3, 100 MHz):
d 25.69, 26.19, 27.41, 29.15, 44.71, 49.87,
d
60.00, 130.83; MS (ESI, m/z): [M þ 1]þ 232.0.
35.39, 43.95, 125.90, 127.44, 127.80, 128.99, 137.57.
5.1.6.8. ((4-Isothiocyanatobutyl)sulfinyl)benzene (84). Yield 90%; 1H
5.1.5.21. Isopropyl(6-isothiocyanatohexyl)sulfane (76). Yield 58%; 1H
NMR (CDCl3, 400 MHz):
d 1.74e1.93 (m, 4H), 2.79e2.85 (m, 2H),
NMR (CDCl3, 400 MHz):
d
1.26 (d, 6H, J ¼ 6.8 Hz), 1.42e1.47 (m, 4H),
3.53e3.56 (m, 2H), 7.51e7.64 (m, 5H); 13C NMR (CDCl3, 100 MHz):
1.57e1.74 (m, 4H), 2.53 (t, 2H, J ¼ 7.2 Hz), 2.86e2.96 (m, 1H), 3.51 (t,
d
19.56, 28.99, 44.65, 55.97, 123.96, 129.37, 131.17 (2ꢁC), 143.52; MS
2H, J ¼ 6.8 Hz); 13C NMR (CDCl3, 100 MHz):
d
23.47, 26.23, 28.15,
(ESI, m/z): [M þ 23]þ 261.9.
29.54, 29.88, 30.37, 34.90, 45.01, 130.01.
5.1.6.9. (((4-Isothiocyanatobutyl)sulfinyl)methyl)benzene
(85).
5.1.6. General procedures for preparation of 77e97
Yield 90%; 1H NMR (CDCl3, 400 MHz):
d
1.75e1.94 (m, 4H), 2.59 (t,
2H, J ¼ 6.8 Hz), 3.54 (t, 2H, J ¼ 6.4 Hz), 4.02 (dd, 2H, J ¼ 12.8,
43.2 Hz), 7.28e7.42 (m, 5H); 13C NMR (CDCl3, 100 MHz):
20.01,
A solution of isothiocyanate 56e76 (3 mmol) in CH2Cl2 (20 mL)
was chilled to ꢂ10 ꢀC in an iceesalt bath. A solution of 85% MCPBA
(3 mmol) in CH2Cl2 (10 mL) was added dropwise over 5 min, while
the internal temperature was kept below 0 ꢀC. When the addition
was completed, the mixture was stirred at 0 ꢀC for 1 h, and then
the resulting light-yellow suspension was treated with saturated
NaHCO3 (15 mL). The organic layer was separated and the aqueous
layer was extracted with CH2Cl2 (2 ꢁ 15 mL). The combined CH2Cl2
layers were washed with saturated NaHCO3 (30 mL) and brine
(30 mL), then dried with anhydrous MgSO4 and concentrated with
a rotary evaporator. The crude product was purified by flash
chromatography (MeOH:CH2Cl2 ¼ 1:20) to give a light-yellow oil
77e97.
d
29.08, 44.64, 49.73, 58.48, 128.51, 129.08, 129.22, 129.99, 131.08; MS
(ESI, m/z): [M þ 1]þ 254.0.
5.1.6.10. 1-(((4-Isothiocyanatobutyl)sulfinyl)methyl)-4-nitrobenzene
(86). Yield 72%; 1H NMR (CDCl3, 400 MHz):
d 1.81e1.99 (m, 4H),
2.66 (t, 2H, J ¼ 7.2 Hz), 3.55 (t, 2H, J ¼ 6.4 Hz), 4.06 (dd, 2H, J ¼ 13.2,
37.2 Hz), 7.50 (d, 2H, J ¼ 8.8 Hz), 8.26 (d, 2H, J ¼ 8.4 Hz); 13C NMR
(CDCl3, 100 MHz): d 20.19, 29.00, 44.66, 50.52, 57.28, 124.06, 131.09
(2 ꢁ C), 137.21, 147.98; MS (ESI, m/z): [M þ 1]þ 299.0.
5.1.6.11. 1-(((4-Isothiocyanatobutyl)sulfinyl)methyl)-4-
methoxybenzene (87). Yield 72%; 1H NMR (CDCl3, 400 MHz):
5.1.6.1. 1-Isothiocyanato-4-(methylsulfinyl)butane (77). Yield 85%;
d
1.77e1.94 (m, 4H), 2.57 (t, 2H, J ¼ 6.8 Hz), 3.55 (t, 2H, J ¼ 6.4 Hz),
1H NMR (CDCl3, 400 MHz):
d
1.83e1.99 (m, 4H), 2.61 (s, 3H), 2.67e
3.81 (s, 3H), 3.97 (dd, 2H, J ¼ 12.8, 38 Hz), 6.91 (d, 2H, J ¼ 8.4 Hz),
2.80 (m, 2H), 3.61 (t, 2H, J ¼ 6.0 Hz); 13C NMR (CDCl3, 100 MHz):
7.21 (d, 2H, J ¼ 8.8 Hz); 13C NMR (CDCl3, 100 MHz):
d 20.00, 29.09,
d
20.11, 29.03, 38.74, 44.68, 53.51, 131.10; MS (ESI, m/z): [M þ 1]þ
44.64, 49.49, 55.32, 57.69, 114.52, 121.35, 130.87, 131.17, 159.80; MS
177.8.
(ESI, m/z): [M þ 23]þ 306.0.
5.1.6.2. 1-(Ethylsulfinyl)-4-isothiocyanatobutane (78). Yield 90%; 1H
5.1.6.12. (2-((4-Isothiocyanatobutyl)sulfinyl)ethyl)benzene
Yield 90%; 1H NMR (CDCl3, 400 MHz):
1.82e1.98 (m, 4H), 2.61e
2.77 (m, 2H), 2.82e3.18 (m, 4H), 3.57 (t, 2H, J ¼ 6.4 Hz), 7.24e7.34
(m, 5H); 13C NMR (CDCl3, 100 MHz):
19.23, 27.81, 28.07, 43.65,
(88).
NMR (CDCl3, 400 MHz):
d
1.35 (t, 3H, J ¼ 7.2 Hz), 1.83e1.99 (m, 4H),
d
2.62e2.78 (m, 4H), 3.60 (t, 2H, J ¼ 6.0 Hz); 13C NMR (CDCl3,
100 MHz):
d
6.81, 20.18, 29.10, 44.68, 45.90, 50.55, 130.95; MS (ESI,
d
m/z): [M þ 1]þ 191.9.
50.42, 53.02, 125.86, 127.56, 127.84, 130.23, 137.73; MS (ESI, m/z):
[M þ 1]þ 268.0.
5.1.6.3. 1-Isothiocyanato-4-(propylsulfinyl)butane (79). Yield 93%;
1H NMR (CDCl3, 400 MHz):
d
1.10 (t, 3H, J ¼ 7.2 Hz), 1.78e1.99 (m,
5.1.6.13. 2-(((4-Isothiocyanatobutyl)sulfinyl)methyl)pyridine(89).
6H), 2.58e2.77 (m, 4H), 3.60 (t, 2H, J ¼ 6.0 Hz); 13C NMR (CDCl3,
Yield 85%; 1H NMR (CDCl3, 400 MHz):
d 1.81e1.95 (m, 4H), 2.67e
100 MHz):
d
13.41, 16.32, 20.20, 29.13, 44.71, 51.31, 54.60, 131.19; MS
2.82 (m, 2H), 3.56 (t, 2H, J ¼ 6.4 Hz), 4.17 (dd, 2H, J ¼ 12.8, 35.6 Hz),
(ESI, m/z): [M þ 1]þ 205.9.
7.29 (t, 1H, J ¼ 4.8 Hz), 7.37 (d, 1H, J ¼ 7.6 Hz), 7.70e7.72 (m, 1H),
8.62 (d, 1H, J ¼ 4.8 Hz); 13C NMR (CDCl3, 100 MHz):
d 20.03, 29.06,
5.1.6.4. 1-(Butylsulfinyl)-4-isothiocyanatobutane (80). Yield 90%; 1H
44.61, 50.33, 59.63, 123.71, 125.36, 130.95, 136.91, 150.03, 150.54;
NMR (CDCl3, 400 MHz):
d
0.97 (t, 3H, J ¼ 7.6 Hz), 1.44e1.55 (m, 2H),
MS (ESI, m/z): [M þ 1]þ 254.0.
1.70e1.99 (m, 6H), 2.62e2.75 (m, 4H), 3.60 (t, 2H, J ¼ 6.0 Hz); 13C
NMR (CDCl3, 100 MHz):
d
13.68, 20.21, 22.08, 24.62, 29.14, 44.71,
5.1.6.14. 2-(((4-Isothiocyanatobutyl)sulfinyl)methyl)thiophene (90).
51.29, 52.43, 131.19; MS (ESI, m/z): [M þ 1]þ 220.0.
Yield 73%; 1H NMR (CDCl3, 400 MHz):
d 1.79e1.94 (m, 4H), 2.61 (t,

538
K. Hu et al. / European Journal of Medicinal Chemistry 64 (2013) 529e539
2H, J ¼ 5.2 Hz), 3.55 (t, 2H, J ¼ 6.4 Hz), 4.12 (s, 2H), 7.05 (t, 2H,
J ¼ 3.2 Hz), 7.32 (t, 1H, J ¼ 2.0 Hz); 13C NMR (CDCl3, 100 MHz):
d
1.78e1.84 (m, 2H), 1.90e1.96 (m, 2H), 2.85 (t, 2H, J ¼ 7.5 Hz), 3.54
(t, 2H, J ¼ 6.5 Hz), 4.25 (s, 2H), 7.42 (s, 5H); 13C NMR (CDCl3,
d
19.94, 29.08, 44.63, 49.74, 52.31, 126.83, 127.69, 128.79, 130.03,
125 MHz): d 19.24, 28.61, 44.45, 49.86, 59.83, 127.85, 129.22, 130.50,
131.16; MS (ESI, m/z): [M þ 1]þ 259.9.
131.09; MS (ESI, m/z): [M þ 23]þ 291.95.
5.1.6.15. 2-(((4-Isothiocyanatobutyl)sulfinyl)methyl)furan
(91).
5.2. Cell line and culture condition
Yield 77%; 1H NMR (CDCl3, 400 MHz):
d
1.82e1.94 (m, 4H), 2.64e
2.68 (m, 2H), 3.57 (t, 2H, J ¼ 6.4 Hz), 4.08 (dd, 2H, J ¼ 14.0, 22 Hz),
6.40e6.42 (m, 2H), 7.44 (s, 1H); 13C NMR (CDCl3, 100 MHz):
19.88,
HepG2 (Liver hepatocellular carcinoma), A549 (human lung
adenocarcinoma), MCF-7 (human breast adenocarcinoma), HCT-
116 (human colon cancer cell line), SH-SY5Y (human neuroblas-
toma cell line) and Vero (Africa green monkey kidney cell line) were
kindly provided by Shanghai Jiao Tong University. The cell was
routinely cultured in RPMI-1640 medium or DMEM, supplemented
with 10% neonatal bovine serum (NBS) or 10% fetal bovine serum
(FBS). The culture was maintained at 37 ꢀC with a gas mixture of 5%
CO2/95% air. All media were supplement with 100 U/mL penicillin
d
29.11, 44.63, 50.45, 50.76, 111.29, 111, 39, 131.08, 143.57, 143.71; MS
(ESI, m/z): [M þ 1]þ 243.9.
5.1.6.16. (((3-Isothiocyanatopropyl)sulfinyl)methyl)benzene
Yield 86%; 1H NMR (CDCl3, 400 MHz):
2.13e2.20 (m, 2H), 2.60e
2.74 (m, 2H), 3.68 (t, 2H, J ¼ 6.4 Hz), 4.04 (dd, 2H, J ¼ 12.8, 38 Hz),
(92).
d
7.29e7.42 (m, 5H); 13C NMR (CDCl3, 100 MHz):
d 23.52, 44.15, 47.23,
58.63, 128.64, 129.17, 129.27, 130.00, 132.12; MS (ESI, m/z): [M þ 1]þ
and 100 mg/mL streptomycin.
239.9.
5.3. Cell viability assay
5.1.6.17. 1-(Isopropylsulfinyl)-3-isothiocyanatopropane
82%; 1H NMR (CDCl3, 400 MHz):
1.29 (d, 3H, J ¼ 6.8 Hz), 1.34 (d, 3H,
J ¼ 6.8 Hz), 2.19e2.26 (m, 2H), 2.65e2.75 (m, 2H), 2.77e2.85 (m, 1H),
(93). Yield
d
The cytotoxic activity in vitro was measured using the MTT
assay. The MTT solution (10.0 mL/well) was added in culture media
3.68e3.82 (m, 2H); 13C NMR (CDCl3, 100 MHz):
d
14.80, 15.90, 24.01,
after cells were treated with various concentrations of compounds
for 72 h, and cells were incubated for further 4 h at 37 ꢀC. The
purple formazan crystals were dissolved in 100 mL DMSO. After
10 min, the plates were read on an automated microplate spec-
trophotometer (Bio-Tek Instruments, Winooski, VT) at 570 nm and
630 nm. Assays were performed in triplicate on three independent
experiments. The concentration required for 50% inhibition of cell
viability (IC50) was calculated using the software “DoseeEffect
Analysis with Microcomputers”. The tumor cell lines panel con-
sisted of HepG2, A549, MCF-7, HCT-116 and SH-SY5Y. In all of these
experiments, three replicate wells were used to determine each
point.
44.26, 45.19, 50.97, 53.45, 132.29; MS (ESI, m/z): [M þ 1]þ 191.9.
5.1.6.18. (((5-Isothiocyanatopentyl)sulfinyl)methyl)benzene
(94).
Yield 87%; 1H NMR (CDCl3, 400 MHz):
d
1.47e1.63 (m, 2H), 1.67e
1.86 (m, 4H), 2.56e2.60 (m, 2H), 3.51 (t, 2H, J ¼ 6.4 Hz), 4.01 (dd, 2H,
J ¼ 12.8, 48 Hz), 7.28e7.41 (m, 5H); 13C NMR (CDCl3, 100 MHz):
d
21.92, 25.87, 29.58, 44.75, 50.46, 53.48, 58.51, 128.46, 129.06,
129.75, 129.99, 130.30; MS (ESI, m/z): [M þ 1]þ 267.9.
5.1.6.19. 1-(Isopropylsulfinyl)-5-isothiocyanatopentane
(95). Yield
83%; 1H NMR (CDCl3, 400 MHz):
d
1.27 (d, 3H, J ¼ 6.8 Hz), 1.32 (d, 3H,
J ¼ 6.8 Hz), 1.53e1.90 (m, 6H), 2.56e2.66 (m, 2H), 2.75e2.80 (m, 1H),
3.55 (t, 2H, J ¼ 6.4 Hz); 13C NMR (CDCl3, 100 MHz):
d
14.77, 16.00,
5.4. Cell cycle analysis
22.36, 26.03, 29.68, 44.82, 48.29, 50.50, 53.48, 130.33; MS (ESI, m/z):
[M þ 1]þ 220.0.
The cell cycle was analyzed by flow cytometry. Briefly, cells were
treated with different concentrations of SFN and 85 for 48 h. After
incubation, a total of 1 ꢁ 108 cells were harvested from the treated
and normal samples. The cells were washed twice with PBS,
5.1.6.20. (((6-Isothiocyanatohexyl)sulfinyl)methyl)benzene
(96).
Yield 88%; 1H NMR (CDCl3, 400 MHz):
d
1.39e1.50 (m, 4H), 1.65e
1.82 (m, 4H), 2.56 (t, 2H, J ¼ 7.6 Hz), 3.49 (t, 2H, J ¼ 6.8 Hz), 3.99 (dd,
staining was carried out by incubating cells with 500 mL of 4 mg/mL
2H, J ¼ 12.8, 41.6 Hz), 7.28e7.40 (m, 5H); 13C NMR (CDCl3,
DAPI for 15 min in darkness. The cell cycle distribution was then
detected by flow cytometry using a Cell Lab Quanta SC (Beckman
Coulter, Fullerton, CA). All experiments were performed three
times.
100 MHz):
d 21.27, 25.10, 26.90, 28.57, 43.85, 49.63, 57.46, 127.33,
127.95, 128.87, 128.94 (2 ꢁ C); MS (ESI, m/z): [M þ 23]þ 303.9.
5.1.6.21. 1-(Isopropylsulfinyl)-6-isothiocyanatohexane
(97).
Yield 84%; 1H NMR (CDCl3, 400 MHz):
d
1.27 (d, 3H, J ¼ 6.8 Hz), 1.31
5.5. Annexin V-FITC/PI assay
(d, 3H, J ¼ 6.8 Hz), 1.48e1.86 (m, 8H), 2.53e2.65 (m, 2H), 2.71e2.81
(m, 1H), 3.52 (t, 2H, J ¼ 6.4 Hz); 13C NMR (CDCl3, 100 MHz):
d
14.69,
Cells in different states were observed with an annexin V-FITC/
PI double-staining kit by flow cytometry. Cells were treated with
different concentrations of SFN and 85 for 48 h. After incubation,
cells were collected and washed twice in cold PBS and resuspended
in 200 mL of binding buffer at 1 ꢁ 105 cells/mL. The samples were
16.03, 22.73, 26.21, 28.09, 29.67, 44.94, 48.41, 50.35,130.16; MS (ESI,
m/z): [M þ 23]þ 256.0.
5.1.7. Procedures for preparation of 98
A solution of 85% MCPBA (12 mmol) in CH2Cl2 (20 mL) was
added to a solution of isothiocyanate 64 (3 mmol) in CH2Cl2 (20 mL)
over 15 min at room temperature, when the addition was
completed, the mixture was stirred for 3 h, and then the resulting
white suspension was treated with saturated NaHCO3 (50 mL). The
organic layer was separated and the aqueous layer was extracted
with CH2Cl2 (2 ꢁ 20 mL). The combined CH2Cl2 layers were washed
with saturated NaHCO3 (50 mL) and brine (30 mL), then dried with
anhydrous MgSO4 and concentrated with a rotary evaporator. The
crude product was purified by flash chromatography (CH2Cl2)
to give a white solid 98. Yield 52%; 1H NMR (CDCl3, 500 MHz):
incubated with 5 mL of Annexin V-FITC and propidium iodide (PI) in
the dark for 15 min at room temperature. Finally, samples were
analyzed by flow cytometry and evaluated based on the percentage
of cells for Annexin V positive. All experiments were performed
three times.
5.6. Preparation of nuclear and cytosolic extracts
HepG2 cells grown on 100-mm Petri dishes were treated for
different hours with various concentrations of SFN and 85. There-
after, cells were washed twice with cold PBS/phosphatase

K. Hu et al. / European Journal of Medicinal Chemistry 64 (2013) 529e539
539
inhibitors, and suspended in cold buffer. Centrifuged cell suspen-
sion for 5 min at 500 rpm in a centrifuge pre-cooled at 4 ꢀC. Dis-
Province and Changzhou Key Technology R&D Program (Society
Development).
carded supernatant and gently resuspended cells in 500
hypotonic buffer by pipetting up and down several times. After
15 min on ice, 25 L of detergent was added, and vortexed for 10 s at
m
L of 1ꢁ
Appendix A. Supplementary data
m
highest setting. The supernatant, cytosolic fraction, was removed
into new microcentrifuge tubes; and the pellets were suspended
again in 50 mL of complete lysis buffer to extract nuclear proteins.
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.03.
045.
The cells were then incubated on ice with vigorous agitation every
5 min for 30 s followed by centrifugation for 10 min at 14,000 g at
4
ꢀC, and the supernatant (nuclear extract) was removed into new
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Acknowledgments
We sincerely thank the financial support from the National
Natural Science Foundation of China (grant No. 21202012), 2010
Industry for Attracting Ph. D. Scientists Program of Jiangsu