Synthesis and biological evaluation of disulfides as anticancer agents with thioredoxin inhibition

Article abstract of DOI:10.1016/j.bioorg.2021.104814

Altered redox homeostasis as a hallmark of cancer cells is exploited by cancer cells for growth and survival. The thioredoxin (Trx), an important regulator in maintaining the intracellular redox homeostasis, is cumulatively recognized as a promising target for the development of anticancer drugs. Herein, we synthesized 72 disulfides and evaluated their inhibition for Trx and antitumor activity. First, we established an efficient and fast method to screen Trx inhibitors by using the probe NBL-SS that was developed by our group to detect Trx function in living cells. After an initial screening of the Trx inhibitory activity of these compounds, 8 compounds showed significant inhibition activity against Trx. We then evaluated the cytotoxicity of these 8 disulfides, compounds 68 and 69 displayed high cytotoxicity to HeLa cells, but less sensitive to normal cell lines. Next, we performed kinetic studies of both two disulfides, 68 had faster inhibition of Trx than 69. Further studies revealed that 68 led to the accumulation of reactive oxygen species and eventually induced apoptosis of Hela cells via inhibiting Trx. The establishment of a method for screening Trx inhibitors and the discovery of 68 with remarkable Trx inhibition provide support for the development of anticancer candidates with Trx inhibition.

Full text of DOI:10.1016/j.bioorg.2021.104814

Bioorganic Chemistry 110 (2021) 104814
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and biological evaluation of disulfides as anticancer agents with
thioredoxin inhibition
,b,*
Xiangxu Weia, Miao Zhonga, Song Wanga, Lexun Lia, Zi-Long Songa, Junmin Zhanga
,
,
*
Jianqiang Xuc, Jianguo Fanga
a State Key Laboratory of Applied Organic Chemistry & College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China
b School of Pharmacy, Lanzhou University, Lanzhou 730000, China
c School of Life and Pharmaceutical Sciences & Panjin Institute of Industrial Technology, Dalian University of Technology, Panjin 124221, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Altered redox homeostasis as a hallmark of cancer cells is exploited by cancer cells for growth and survival. The
thioredoxin (Trx), an important regulator in maintaining the intracellular redox homeostasis, is cumulatively
recognized as a promising target for the development of anticancer drugs. Herein, we synthesized 72 disulfides
and evaluated their inhibition for Trx and antitumor activity. First, we established an efficient and fast method to
screen Trx inhibitors by using the probe NBL-SS that was developed by our group to detect Trx function in living
cells. After an initial screening of the Trx inhibitory activity of these compounds, 8 compounds showed signif-
icant inhibition activity against Trx. We then evaluated the cytotoxicity of these 8 disulfides, compounds 68 and
69 displayed high cytotoxicity to HeLa cells, but less sensitive to normal cell lines. Next, we performed kinetic
studies of both two disulfides, 68 had faster inhibition of Trx than 69. Further studies revealed that 68 led to the
accumulation of reactive oxygen species and eventually induced apoptosis of Hela cells via inhibiting Trx. The
establishment of a method for screening Trx inhibitors and the discovery of 68 with remarkable Trx inhibition
provide support for the development of anticancer candidates with Trx inhibition.
Thioredoxin
Disulfide
Oxidative stress
Apoptosis
Anticancer
1. Introduction
prevent carcinogenesis. However, once the malignant phenotype has
been established, the high concentration of Trx may accelerate the
Cancer is still considered one of the most fatal diseases in the world
[1]. Currently, chemotherapy is the most common and effective cancer
therapy strategy, but the efficacy of chemotherapy is still limited due to
the side effects of drugs and the increasing tolerance of treatment. The
major problem now is the lack of effective anticancer medicines [2,3].
Therefore, it is imperative to discover highly effective lead molecules for
anticancer drugs.
development of cancer cells, which promotes tumor growth and pre-
vents cancer cell apoptosis [11]. Importantly, many primary tumor cell
lines including cervical carcinomas [12], prostate cancer [13], breast
cancer [14], colorectal cancer [15], pancreatic cancer [16], hepatocel-
lular carcinoma [17], oral squamous cell carcinoma [18], gastric car-
cinoma [19], leukemia [20], and lung cancer [21] have high-level Trx.
Researchers demonstrated that Trx overexpression could increase the
production of vascular endothelial growth factors and promote tumor
angiogenesis [22]. Trx is also implicated in highly invasive and meta-
static tumor activity both in vitro and in vivo [23,24]. In addition, the
resistance of tumor cells to various chemotherapy drugs is associated
with high-level Trx [9,25]. Collectively, these studies suggest that Trx
plays a positive functional role in promoting cancer cell growth. Thus,
Thioredoxin (Trx), a ubiquitous redox regulator, is a key component
of the intact thioredoxin system. Trx system and glutathione (GSH)
system are predominant in regulating the intracellular redox homeo-
stasis [4–6]. Several studies have shown that the function of Trx in
cancer cells depends on the stage of cancer development [7–10]. In the
early stage, Trx can resist the oxidative stress caused by carcinogens to
Abbreviations: DCFH-DA, 2′, 7′-dichlorfluorescein diacetate; DCM, dichloromethane; DHE, dihydroethidium; DMSO, dimethyl sulfoxide; DTT, DL-dithiothreitol;
GSH, glutathione; MTT, 3-(4, 5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromid; PX-12, 2-[(1-Methylpropyl) dithio]-1H- imidazole; ROS, reactive oxygen
species; SAR, structure-activity relationships; Trx, thioredoxin; TrxR, thioredoxin reductase.
* Corresponding authors at: State Key Laboratory of Applied Organic Chemistry & College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou
730000, China (J. Zhang).
E-mail addresses: zhangjunmin@lzu.edu.cn (J. Zhang), fangjg@lzu.edu.cn (J. Fang).
https://doi.org/10.1016/j.bioorg.2021.104814
Received 11 October 2020; Received in revised form 19 February 2021; Accepted 6 March 2021
Available online 10 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

X. Wei et al.
Bioorganic Chemistry 110 (2021) 104814
Trx may be regarded as a potential target in the development of anti-
cancer drugs [26]. Once Trx is inhibited by small molecules, it will
inevitably produce a large amount of reactive oxygen species (ROS),
induce oxidative stress, and eventually promote cancer cell death
[6,27,28].
[40], compounds 15–53 were synthesized according to the method
described by Stellenboom, N. et al. [41], compounds 54–72 were syn-
thesized according to the method described by Khosla, C. et al. [31]. All
compounds were characterized by 1H NMR, 13C NMR with a Bruker
AMX spectrometer at 400 and 100 MHz, respectively. MS spectra were
obtained using a Hewlett-Packard 5988A spectrometer or a Shimadzu
LC-MS-2020 system.
Intriguingly, as far as the redox cycle of the entire Trx system is
concerned, thioredoxin reductase (TrxR) takes electrons from NADPH
and transfers them to Trx, Trx then directly interacts with various redox-
active proteins through free thiol and disulfide exchange reactions
[29,30]. Therefore, it is valuable to design and synthesize a series of
small molecule disulfides to inhibit Trx through free thiol and disulfide
exchange reactions. Existing structure-activity relationships (SAR) evi-
dence suggests that half of the active molecule should be aromatic
groups with electron-withdrawing substituents. It will bring substantial
improvements in increasing activity [26,31]. An excellent example is
PX-12 (2-[(1-Methylpropyl) dithio]-1H-imidazole), which has been
clinically evaluated in cancer patients [32-34]. Khosla C. et al. developed
another agent 2-(Cyclohexyldisulfanyl)-5- nitro-1H-benzo[d]imidazole,
which could oxidize extracellular Trx via selective disulfide bond ex-
change [31]. It has been confirmed that these reported compounds
primarily act on the conserved active site of Trx Cys32/Cys35, and also
react slowly and irreversibly with the non-catalytic site Cys73 of Trx.
[31,35-37].
2.2. Screening and SAR studies
As shown in Fig. 1, all compounds were screened by the small
molecule probe NBL-SS [39]. The concentrations of compounds were
100 μM, 20 μM, and 10 μM, respectively. We set the fluorescence in-
tensity at F0 when the fluorescence of the probe was released fully. In the
experiment, the fluorescence intensity was measured as F. When F/F0
reached 100%, which indicated that the Trx was not inhibited by the
compounds. As shown in Fig. 1 A, when the concentration of the com-
pounds was 100 μM, most of the compounds were able to inhibit E.coli
Trx and the fluorescence of the probe NBL-SS could not be released. To
screen out more ideal compounds, the concentration of compounds was
reduced to 20 μM to further narrow the range of compounds (Fig. 1 B).
Finally, when the concentration of the compound was set to 10 μM, we
found that compounds 4, 5, 6, 19, 22, 24, 68, and 69 exhibit apparent
oxidation capacity toward E. coli Trx (Fig. 1 C). Then, these 8 com-
pounds and PX-12 were analyzed for cytotoxicity against HeLa, HepG2,
BEAS-2B, and HEK-293T by using the MTT assay. Under this condition,
the concentrations that inhibit the cell proliferation to 50% of the con-
trol (IC50 values) are summarized in Table 1. Compounds 68 and 69
displayed significant cytotoxicity toward HeLa cells and exhibited
higher cytotoxicity than PX-12. In contrast, normal cell lines, including
BEAS-2B and HEK-293T cells, were less sensitive to 68 and 69. It shows
that the compounds have great selectivity for tumor cell lines. Next, we
chose these two compounds for further studies.
To develop highly effective small molecule lead compounds as
anticancer candidates with Trx inhibition, we established a molecular
library with 72 disulfides. In recent years, researchers have developed
many small molecular probes of protein thiols [38]. In view of this, we
established an efficient and fast method to screen Trx inhibitors by using
the probe NBL-SS developed by our group [39]. After an initial screening
of all compounds, we evaluated the cytotoxicity of these disulfides and
performed their kinetic studies. A preliminary description and summary
of SAR are helpful to further optimize disulfides as anticancer candidates
with Trx inhibition. Compound 68 displayed the greatest potency as an
inhibitor for Trx among all compounds. Therefore, 68 was selected for
the follow-up studies. Further research found that 68 might inhibit Trx
by targeting the sulfhydryl groups, leading to the accumulation of ROS
and promoting the apoptosis of tumor cells. We expect the lead com-
pound 68 to be used as an anticancer candidate with Trx inhibition.
A preliminary SAR showed that the symmetric disulfides in the
screened compounds have a good oxidation effect on protein, but they
have low cytotoxicity to cancer cells and could not kill cancer cells well.
In contrast, asymmetric disulfides not only have a good oxidation effect
on protein, but also have high cytotoxicity. Especially when half of the
molecule is aromatic groups with electron-withdrawing substituents,
those compounds are worthy of further study. Intriguingly, we found
that those molecules might also relieve the stench of the disulfide.
2. Results and discussion
2.1. Chemistry
2.3. Oxidation capacity of compounds to Trx
In this study, all compounds were synthesized through the routes
outlined in Scheme 1–3, and all of them were obtained through the
corresponding thiols or thiophenols, respectively. Compounds 1–14
were synthesized according to the method described by Sun, L. et al.
Holmgren A. has proved that the fluorescence of E. coli Trx could
decrease 2-fold when the reduced E. coli Trx is oxidized enzymatically or
chemically [42]. Likewise, our results showed that when compound 68
Scheme 1. General synthetic procedures for compounds 1–14.
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Bioorganic Chemistry 110 (2021) 104814
Scheme 2. General synthetic procedures for compounds 15–53.
reacted with E. coli Trx, the fluorescence of Trx also could reduce 2-fold
(Fig. S1). Then we determined the change of fluorescence intensity of
E. coli Trx reacted with 68 and 69, respectively. E. coli Trx was oxidized
by 68 faster than it was oxidized by 69 (Fig. 2A, B). Therefore, we fitted
the change of fluorescence intensity of E. coli Trx to the second-order
rate equation for kinetic studies. Then, we determined the reaction
rate constants of the compounds with E. coli Trx, and the second-order
rate constants of 68 and 69 are 1290 (M s)-1 and 910 (M s)-1, respec-
tively. The response between Trx and 68 was faster than 69. The details
of the calculation are shown in Fig. S2 and Tables S1-S2. Next, we
determined the optimal concentration and time of the reaction between
68 and Trx via the changes in fluorescence intensity of the redox state of
E.coli Trx. As shown in Fig. 2C, the fluorescence intensity decreased with
time-dependent and concentration-dependent, and the optimal con-
centration and time were also determined. After E.coli Trx was incubated
with 68, the redox state of E.coli Trx can be determined by probe Naph-
EA-mal developed previously by our group [43]. Obviously, the fluo-
rescence intensity decreased with time-dependent and concentration-
dependent (Fig. 2D, E). Through the above experiments, we found
that the reaction between Trx and 68 might be stoichiometric, and it was
verified by HPLC (Fig. S3).
2.4. Cytotoxicity of compound 68
Since compound 68 can oxidize Trx faster than 69, we chose com-
pound 68 for further studies. We measured the cytotoxicity of 68 by
3

X. Wei et al.
Bioorganic Chemistry 110 (2021) 104814
Scheme 3. General synthetic procedures for compounds 54–72.
Fig. 1. Screening of disulfides against E.coli Trx with
NBL-SS. Reduced Trx (10 µM) was incubated with
different concentrations of compounds (A) 100 µM,
(B) 20 µM, and (C) 10 µM at 37 ◦C in 96-well plates for
30 min. The E-Trx control group has reduced E-Trx
and NBL-SS (5 µM), and the DMSO control has
reduced E-Trx, DMSO and NBL-SS (5 µM). The probe
NBL-SS was then added and incubated for 5 min, and
the fluorescence intensity was monitored with
a
Microplate Reader (FlexStation 3, USA). Results are
expressed as means
experiments.
± SD of three independent
4

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Bioorganic Chemistry 110 (2021) 104814
2.5. Imaging of Trx activity in live cells
Table 1
Cytotoxicity of 4, 5, 6, 19, 22, 24, 68, 69 and PX-12.
M)a
The probe NBL-SS was designed in our lab could be applied to image
Trx activity in live cells and in vivo conveniently [39]. HeLa cells were
seeded in 12-well plates. After 24 h, the cells were treated with 68 (10
µM) for different time. Then NBL-SS (5 µM) was added and continued
incubated for 5 min at 37 ◦C. The blank group was only incubated with
68 (10 µM) without the addition of the probe NBL-SS (5 µM). The cells
were visualized and photographed under a fluorescent microscope. The
appearance of red fluorescence indicated the activity of Trx in cells. As
shown in Fig. 4A, we did not detect a bright red fluorescence signal in
the blank group, while fluorescence in the cells treated by the 68 became
weaker as time increased. In addition, we performed a quantitative
analysis of fluorescence signal (Fig. 4B). The fluorescence intensity
decreased with the incubation time. It proved that Trx could be inhibited
effectively by compound 68 in the HeLa cells.
Compd.
IC50
(μ
HeLa
HepG2
HEK-293 T
BEAS-2B
4
24.5 ± 0.6
19.1 ± 1.0
17.1 ± 0.8
16.5 ± 2.2
16.6 ± 2.7
15.3 ± 1.8
9.3 ± 1.1
32.2 ± 2.5
22.5 ± 2.1
33.4 ± 0.9
21.7 ± 2.1
24.0 ± 1.6
25.2 ± 2.1
24.6 ± 2.0
28.8 ± 1.3
29.0 ± 1.7
44.3 ± 1.8
22.7 ± 1.4
33.8 ± 0.6
22.2 ± 0.5
19.5 ± 0.5
22.9 ± 0.8
33.3 ± 1.8
29.0 ± 2.4
22.4 ± 0.9
35.7 ± 0.3
29.3 ± 1.4
32.2 ± 2.8
34.1 ± 2.2
35.4 ± 2.9
28.6 ± 2.2
31.8 ± 2.6
24.4 ± 0.9
38.0 ± 2.3
5
6
19
22
24
68
69
PX-12
12.3 ± 0.8
13.1 ± 1.6
a
The data were obtained by the MTT assay after a 72 h treatment.
MTT assay for different time (Fig. 3A). To confirm the results of the MTT
assay, we further employed the trypan blue exclusion assay to assess cell
viability. The results of the trypan blue exclusion assay and MTT assay
are powerful consistent (Fig. 3B). In addition, the cytotoxicity of com-
pound 68 showed a significant concentration and time dependence
(Fig. A & B).
2.6. Alteration of Trx redox status
As mentioned above, 68 acted as an inhibitor of Trx. Therefore,
detecting the redox status of Trx in cells could directly prove that Trx
Fig. 2. Oxidation capacity of compounds to E.coli Trx.
(A) Time-dependent fluorescence intensity change of
Trx (8.93 µM and 9.17 µM, respectively) with 68
(10.0 µM). (B) Time-dependent fluorescence intensity
change of Trx (8.93 µM and 9.17 µM, respectively)
with 69 (10.0 µM). (C) Time- and dose-dependent
fluorescence intensity change of 68 in response to
Trx (10 µM). (D) The dose-dependent and (E) the
time-dependent of 68 (100 µM) in response to Trx
(100 µM), Trx redox states were determined by probe
Naph-EA-mal and were separated by SDS-PAGE.
Fig. 3. Cytotoxicity of compound 68. (A) Dose- and
time-dependent cytotoxicity of 68 towards HeLa cells.
The HeLa cells were incubated with varying concen-
trations of 68 for 48 h or 72 h. The cell viability was
assessed by the MTT method. (B) Cytotoxicity of 68
towards HeLa cells for 72 h. The cell viability was
determined by the trypan blue exclusion assay. Data
are expressed as mean ± SE from triplicates. ** P <
0.01 vs. the control groups in (A) and (B). (For
interpretation of the references to colour in this figure
legend, the reader is referred to the web version of
this article.)
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X. Wei et al.
Bioorganic Chemistry 110 (2021) 104814
Fig. 4. Imaging Trx activity in HeLa cells.
(A) Imaging the cellular Trx activity by NBL-
SS (5 µM). HeLa cells were treated with 68
(10 µM) at different times, and the activity of
Trx was stained by the probe NBL-SS for 5
min, the blank control was not stained by
NBL-SS. The bright-field pictures (top panel)
and the fluorescence pictures (bottom panel)
were acquired by an inverted fluorescence
microscope. Scale bars: 20 μm. The fluores-
cence intensity analysis of (A) by ImageJ was
shown in (B).
activity has changed. Then we used the PAO-sepharose drop-down assay
developed by our group to determine the redox status of Trx in HeLa
cells were treated with 68 [44,45]. This assay has been further verified
in previous studies by our group [46]. As shown in Fig. 5A, when the
compound 68 activated caspase-3 in a concentration-dependent
manner, indicating that compound 68 induced apoptosis in HeLa cells.
Then, we applied the Annexin VFITC/PI double staining assay to
quantify the apoptotic population by flow cytometer. The dot plots and
the quantification results were shown in Fig. 7A & B. As the concen-
tration of compound 68 enhanced, the number of apoptotic cells
increased significantly. All these results indicated that 68 killed HeLa
cells mainly by inducing apoptosis.
cells were treated with 68 (40 μM), the oxidized Trx increased signifi-
cantly. Compared to the control group (non-68 treated cells), Trx was
mainly presented in the reduced form. Then the oxidized and reduced
Trx was quantified by Image J, and the ratio of reduced Trx to oxidized
Trx decreased significantly (Fig. 5C). As shown in Fig. 5B, the total
expression of Trx decreased significantly, indicating that Trx was irre-
versibly inhibited by 68 [47]. These results showed that Trx was
inhibited by compound 68 irreversibly.
Trx plays an important role in many physiological and pathological
processes including multiple stages of cancer. Compared to normal cell
lines, tumor cell lines usually harbor elevated ROS as a result of their
uncontrolled proliferation and high metabolic rate. To maintain the
redox balance, cancer cells also upregulate the antioxidant system to
eliminate ROS. Many studies have shown that many tumor cell lines
with a high level of ROS and Trx [49]. Trx overexpressed in cancer cells
is inhibited by inhibitors, and a large amount of ROS would futher
accumulate in the cells, leading to the death of cancer cells [27,28,50].
Therefore, the development of anticancer drugs with Trx inhibition is a
promising tumor treatment strategy.
2.7. Induction of ROS in cells
Trx system plays an important role in maintaining the intracellular
redox homeostasis and resisting oxidative stress. Thus, Trx is inhibited
by 68 might disturb the cellular redox balance and cause the accumu-
lation of ROS in cells. The production of ROS in cells was detected by
DCFH-DA. A weak fluorescence signal in the control group was detected
indicating the presence of low-level ROS. After the HeLa cells were
treated by the 68, a bright green fluorescence became brighter as the
concentration of 68 increased (Fig. 6A). Because of the limitation of
DCFH-DA [48], we used another probe DHE to further detect ROS in
cells. Unsurprisingly, 68-treated cells promoted the fluorescence in a
dose-dependent manner (Fig. 6B). In addition, we performed a quanti-
tative analysis of fluorescence signal (Fig. 6C and D). Taken together,
compound 68 promoted the production of ROS in HeLa cells.
Disulfide bond has good reactivity and biological activity and is an
important functional group in various chemical and biological prepa-
rations. The exchange reaction between free thiol and disulfide bond
plays a very important role in the redox cycle of the Trx system [29,30].
To develop an anticancer candidate with Trx inhibition, we synthe-
sized a number of disulfides. Herein, we established a method for
screening Trx inhibitors by using NBL-SS, a highly selective fluorescent
probe for Trx developed by our group. This method reduced the work-
load of screening inhibitors greatly (Fig. 1). Combined with previous
SAR studies [26,31], our studies showed that the symmetric disulfides
can oxidize pure E.coli Trx, but they have low cytotoxicity. However,
asymmetric disulfides not only oxidized Trx well but also had high
cytotoxicity. We found that half of the molecule is an aromatic group
with electron-withdrawing substituents showing better cytotoxicity.
Therefore, compound 68 is an ideal molecule for further investigation.
In addition, the results of the kinetic study further indicate that com-
pound 68 has excellent interaction with E. coli Trx, we thus have further
studied the intracellular behavior of compound 68.
2.8. Apoptosis induction by compound 68
Once Trx was inhibited by small molecules, it would inevitably
produce a large amount of ROS, induce oxidative stress, and eventually
promote cancer cell death. Although the above studies showed that
compound 68 could effectively inhibit Trx activity and elicit the accu-
mulation of ROS in HeLa cells, we could not confirm that compound 68
induces oxidative stress-mediated apoptosis of HeLa cells. Therefore, the
apoptosis was evaluated Annexin VFITC/PI double staining assay and
caspase-3 activity test. Herein, we investigated the activity of caspase-3
after HeLa cells were incubated with 68 for 48 h. As shown in Fig. 7C,
The effect of compound 68 on HeLa cells was demonstrated by the
following evidence. First, we imaged the activity of Trx in HeLa cells by
NBL-SS. The activity of Trx was inhibited by 68 in HeLa cells under our
Fig. 5. Alteration of Trx redox status in HeLa cells. (A) Mea-
surement of the oxidized Trx1 and reduced Trx1 by the PAO-
sepharose assay. After treatment of HeLa cells with 68 (40
µM) for 48 h, the redox states of Trx1 were determined. S,
samples in the supernatant; P, samples eluted from the PAO-
sepharose beads; R, reduced form; O, oxidized form. (B)
There was a significant change in Trx1 protein expression after
being treated with 68. After treatment of HeLa cells with 68 for
48 h, Western blot was performed to detect the expression of
Trx1 protein. (C) The ratio of reduced/oxidized Trx1 was
evaluated by ImageJ. ** P < 0.01 vs. the control groups in (C).
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X. Wei et al.
Bioorganic Chemistry 110 (2021) 104814
Fig. 6. Induction of oxidative stress by 68 in HeLa cells. HeLa cells were treated with varying concentrations of 68 for 12 h and were continued to incubate with (A)
DCFH-DA (10 μM) or (B) DHE (10 μM) for 30 min. The pictures were acquired by an inverted fluorescence microscope. Scale bars: 20 μm. The fluorescence intensity
analysis of (A) and (B) by ImageJ was shown in (C) and (D). Data are expressed as mean ± SE from triplicates. ** P < 0.01 vs. the control groups in (C) and (D).
Fig. 7. Induction of apoptosis by 68 in HeLa
cells. (A) Flow cytometric analysis of
apoptosis by Annexin V/PI double staining
assay in HeLa cells. HeLa cells were treated
with different concentrations of 68 for 48 h
and the population of live cells, apoptotic
cells, and necrotic cells were analyzed by
flow cytometry. The cells displayed four
different groups marked as follows: PI-
positively and FITC-negatively stained cells
showing necrotic cells (upper left, Q1),
double-negative (unstained) cells showing
live cells (lower left, Q3), FITC-positively
and PI-negatively stained cells showing
early apoptosis (lower right, Q4) and FITC
and PI double-stained cells showing late
apoptosis (upper right, Q2). (B) Quantifica-
tion of necrotic cells (Q1), apoptotic cells
(Q2 and Q4), and live cells (Q3) were illus-
trated. (C) Activation of caspase-3 by 68 in
HeLa cells. HeLa cells were treated with the
indicated concentrations of 68 for 48 h and
the activity of caspase-3 in the cell extracts
was determined by a colorimetric assay. Data
are expressed as mean ± SE from triplicates.
** P < 0.01 vs. the control groups in (C).
experimental conditions (Fig. 4). Since 68 could act as a Trx inhibitor by
oxidizing Trx, the redox state of Trx in the HeLa cell is then tested
(Fig. 5). These results showed that Trx was inhibited by compound 68,
and this may be similar to the established mechanism [31,35-37]. Trx
inhibition by 68 disturbs the cellular redox balance, leading to the
accumulation of ROS in cells (Fig. 6) and promoting cancer cell death
eventually (Fig. 7).
antitumor activity in HeLa cells. We established an efficient and fast
method to screen Trx inhibitors by using the probe NBL-SS and found
compound 68 had the greatest potential as a candidate for Trx in-
hibitors. In addition, studies demonstrated that 68 impinged on the Trx
system via inhibition of Trx, which induced oxidative stress and even-
tually led to apoptosis of HeLa cells. The SAR analysis would facilitate
our further optimization disulfides to develop anticancer candidates
with Trx inhibition.
3. Conclusion
In summary, 72 disulfides were synthesized and evaluated for
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Bioorganic Chemistry 110 (2021) 104814
4. Experimental section
and 1H-Benzotriazole (320 mg, 2.7 mmol) was added to a flask and
dissolved in anhydrous dichloromethane (DCM, 30 mL), then 4-Fluoro-
thiophenol as R1SH (346 mg, 2.7 mmol) was dissolved in DCM (2 mL)
and was added dropwise under Ar atmosphere at ꢀ 78 ◦C. After stirring
for 2 h with slow warming to ꢀ 20 ◦C, 1-Propanethiol (304 mg, 4 mmol)
as R2SH in DCM (2 mL) was added to the reaction mixture slowly at
ꢀ 20 ◦C and was continuously stirred for 30 min at 0 ◦C. The reaction was
then quenched with a solution of Na2S2O3 (0.50 g in 10 mL water)
together with saturated aqueous NaHCO3 (20 mL). The resulting
mixture was extracted with DCM. The organic phase was dried over
anhydrous MgSO4, filtered, evaporated under reduced pressure and
purified with column chromatography by using petroleum ether/eth-
yl acetate as eluent to give the desired product. Compounds 16–53 were
prepared by the method used for 15.
4.1. Materials and instruments
The recombinant E. coli Trx was prepared according to our estab-
lished protocol, and the purity of the protein was > 95% judged by
Coomassie-stained SDS-polyacrylamide gel electrophoresis (PAGE)
[39]. HepG2, HeLa, HEK 293T, and BEAS-2B cells were obtained from
the Shanghai Institute of Biochemistry and Cell Biology, Chinese Acad-
emy of Sciences. RPMI 1640 medium, Dulbecco’s modified Eagle me-
dium (DMEM), DL-dithiothreitol (DTT), dimethyl sulfoxide (DMSO),
were obtained from Roche (Mannheim, Germany). 3-(4, 5-Dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Amresco, Solon,
USA). The primary antibodies against Trx1 and β-actin were from San-
gon Biotech (Shanghai, China). 1H NMR and 13C NMR spectra were
recorded on a Bruker AMX spectrometer at 400 and 100 MHz in
deuterated solvents, respectively, and tetramethylsilane (TMS) was used
as a reference (chemical shifts are given in δ values, J is given in Hz).
Mass spectra were obtained using a Hewlett-Packard 5988A spectrom-
eter or a Shimadzu LC-MS-2020 system. The purity of all final com-
pounds was assessed by HPLC. All other reagents were purchased from
commercial supplies. All reactions were monitored using thin-layer
chromatography (TLC) on commercial silica-gel plates (GF254). Visu-
alization of the developed plates was performed under UV light (254
nm). Flash column chromatography was performed on silica gel
(200–300 mesh). Fluorescence studies were carried out using a Cary
Eclipse Fluorescence spectrophotometer (Agilent Technologies). The
fluorescence images of the cells were acquired on a Floid cell imaging
station (life technology) or an inverted fluorescent microscope (Leica
DMI4000). The absorbance was measured on Multiskan GO (Thermo
Scientific) or Microplate Reader (FlexStation 3, USA). The gel images
were acquired with a Gel imager (G: BOX Chemi XX9, Syngene, UK). The
data of Annexin V/PI staining were determined by a FACSCanto™ flow
cytometer (BD Biosciences, USA).
Compounds 54–72 were synthesized following the method by Kho-
sla, C. et al. [31].
Intermediate s54 was obtained from commercially available sources.
Cyclohexyl mercaptan (754 mg, 6.5 mmol) and thiourea (380 mg, 5
mmol) were dissolved in H2O-ethanol (40 mL, 1:3 mixture). Then
concentrated hydrochloric acid (2 mL) was added to the reaction
mixture carefully under an ice-water bath⋅H2O2 (30%, 0.9 mL) was
added dropwise with vigorous stirring and was continually stirred at
0
◦C for 3 h. The solvent was evaporated under reduced pressure and
collected the crude intermediates. All intermediates were prepared by
the method used for s54.
Synthesis of compound 54. 2-Mercaptobenzoxazole (332 mg, 2.2
mmol) and s54 (565 mg, 2.5 mmol) were dissolved in methanol (6 mL).
NaHCO3 (286 mg, 3.4 mmol) in water (10 mL) was added dropwise with
vigorous stirring at room temperature for 1 h. The resulting mixture was
diluted with water, and was extracted with DCM. The organic phase was
dried over anhydrous MgSO4, filtered, evaporated under reduced pres-
sure and purified with column chromatography by using petroleum
ether/ethyl acetate as eluent to give the desired product. Compounds
55–72 were prepared by the method used for 54.
Diphenyl disulfide (1). Yield: 85.7%; 1H NMR (400 MHz, CDCl3) δ
7.508–7.490 (m, 4H), 7.325–7.285 (m, 4H), 7.245–7.208 (m, 2H). 13
C
4.2. Compounds purity analysis
NMR (100 MHz, CDCl3) δ 137.16, 129.21, 127.63, 127.30. MS-APCI m/
z: 242.0 [M + Na]+. HPLC analysis: Rt = 8.11 min, purity 97.87%, 90:10
CH3OH/H2O.
The purity of all compounds was determined by HPLC. The analyses
were performed on the Shimadzu LC-MS-2020 system with a Wondasil
C18 Superb reversed-phase column (5 mm, 4.6 mm × 150 mm). The
mobile phase was methanol and water, and the flow rate was 0.6 mL/
min. All tested compounds were dissolved in methanol, and the injection
Bis(4-methoxyphenyl) disulfide (2). Yield: 82.3%; 1H NMR (400
MHz, CDCl3) δ 7.413 (d, J = 6.4 Hz, 4H), 6.852 (d, J = 8.8 Hz, 4H), 3.814
(s, 6H). 13C NMR (100 MHz, CDCl3) δ 160.04, 132.80, 128.55, 114.74.
MS-APCI m/z: 278.0 [M + H]+. HPLC analysis: Rt = 4.09 min, purity
99.49%, 90:10 CH3OH/H2O.
volume was 5 μL. The maximum absorbance range of the detection
wavelength was 210–400 nm. The purity of target compounds was
higher than 95%, except compounds 9, 64, 72, whose purity is 94.0%,
94.2% and 94.1%, respectively.
Bis(2-methoxyphenyl) disulfide (3). Yield: 90.9%; 1H NMR (400
MHz, CDCl3) δ 7.531 (d, J = 6.4 Hz, 2H), 7.187 (t, J = 1.6 Hz, 2H), 6.913
(t, J = 3.8 Hz, 2H), 6.855(d, J = 7.2 Hz, 2H). 13C NMR (100 MHz, CDCl3)
δ 156.67, 127.85, 127.64, 124.64, 121.44, 110.59, 55.98. MS-APCI m/z:
279.0 [M + H]+. HPLC analysis: Rt = 6.548 min, purity 97.61%, 90:10
CH3OH/H2O.
4.3. Target compounds synthesis and characterization
Compounds 1–14 were synthesized following the method described
by Sun, L. et al. [40].
Bis(2-aminophenyl) disulfide (4). Yield: 88.3%; 1H NMR (400
MHz, CDCl3) δ 7.179–7.137 (m, 4H), 6.726–6.706 (m, 2H), 6.605–6.567
(m, 2H). 13C NMR (100 MHz, CDCl3) δ 148.72, 136.91, 131.70, 118.80,
118.31, 115.33. MS-ESI m/z: 248.9 [M + H]+. HPLC analysis: Rt =
10.41 min, purity 95.68%, 80:20 CH3OH/H2O.
Synthesis of compound 1. Thiophenol (440 mg, 4 mmol) was added
to a flask and dissolved in anhydrous dichloromethane (DCM, 50 mL),
sodium bicarbonate (420 mg, 5 mmol) was added to the flask with
vigorous stirring at room temperature. After stirring for 15 min, iodine
(1.5 g, 6 mmol) was added to the reaction mixture slowly and stirred for
an additional 30 min vigorously. Then, add saturated aqueous Na2S2O3
to the reaction mixture to remove excess iodine, until the solution turns
colorless. The resulting mixture was extracted with DCM. The organic
phase was dried over anhydrous MgSO4, filtered, evaporated under
reduced pressure and purified with column chromatography by using
petroleum ether/ethyl acetate as eluent to give the desired product.
Compounds 2–14 were prepared by the method used for compound 1.
Compounds 15–53 were synthesized following the method by Stel-
lenboom, N. et al. [41].
Bis(4-aminophenyl) disulfide (5). Yield: 83.9%; 1H NMR (400
MHz, CDCl3) δ 7.276 (d, J = 6.8 Hz, 4H), 6.618 (d, J = 7.2 Hz, 4H). 13
C
NMR (100 MHz, CDCl3) δ 178.07, 161.23, 132.12, 130.78, 116.23,
33.66, 32.76. MS-ESI m/z: 248.9 [M + H]+. HPLC analysis: Rt = 7.89
min, purity 99.18%, 90:10 CH3OH/H2O.
4,4′-Dithiobisbenzoic acid (6). Yield: 91.1%; 1H NMR (400 MHz,
DMSO‑d6) δ 7.932 (d, J = 8.8 Hz, 4H), 7.638 (d, J = 8.4 Hz, 4H). 13
C
NMR (100 MHz, DMSO‑d6) δ 166.732, 140.778, 130.386, 129.823,
126.114. MS-ESI m/z: 611.3 [2Mꢀ H]-. HPLC analysis: Rt = 10.18 min,
purity 95.13%, 80:20 CH3OH/H2O.
Bis(4-methylphenyl) disulfide (7). Yield: 84.6%; 1H NMR (400
Synthesis of compound 15. 1-Chlorobenzotriazole (610 mg, 4 mmol)
8

X. Wei et al.
Bioorganic Chemistry 110 (2021) 104814
MHz, CDCl3) δ 7.187 (d, J = 8.4 Hz, 4H), 7.052 (d, J = 8.0 Hz, 4H). 13
C
4-Fluorophenyl 4-methylphenyl disulfide (19). Yield: 75.5%; 1H
NMR (400 MHz, CDCl3) δ 7.473–7.438 (m, 1H), 7.394–7.361 (m, 2H),
7.129–7.109 (m, 2H), 7.094–6.972 (m, 1H), 2.326 (d, J = 4.0 Hz, 3H).
13C NMR (100 MHz, CDCl3) δ 161.26, 137.94, 133.51, 130.83, 130.01,
128.98, 128.58, 116.15, 21.21. MS-APCI m/z: 249.0 [Mꢀ H]-. HPLC
analysis: Rt = 8.44 min, purity 97.35%, 85:15 CH3OH/H2O.
4-Fluorophenyl cyclopentyl disulfide (20). Yield: 84.7%; 1H NMR
(400 MHz, CDCl3) δ 7.531–7.496 (m, 2H), 7.014 (t, J = 8.8 Hz, 2H),
3.343–3.294 (m, 1H), 1.980–1.915 (m, 3H), 1.746–1.669 (m, 5H). 13C
NMR (100 MHz, CDCl3) δ 160.87, 133.34, 133.31, 129.94, 116.20,
50.40, 32.82, 24.80. MS-APCI m/z: 229.0 [M + H]+. HPLC analysis: Rt
= 6.73 min, purity 95.58%, 85:15 CH3OH/H2O.
NMR (100 MHz, CDCl3) δ 135,729, 129.935, 128.580, 126.676. MS-
APCI m/z: 246.0 [M]+. HPLC analysis: Rt = 6.575 min, purity
99.56%, 90:10 CH3OH/H2O.
Bis(4-hydroxyphenyl) disulfide (8). Yield: 88.1%; 1H NMR (400
MHz, DMSO‑d6) δ 9.842 (s, 2H), 7.268 (d, J = 8.8 Hz, 4H), 6.754 (d, J =
8.8 Hz). 13C NMR (100 MHz, DMSO‑d6) δ 158.32, 133.10, 125.14,
116.36. MS-ESI m/z: 499.5 [2Mꢀ H]-. HPLC analysis: Rt = 10.54 min,
purity 98.89%, 75:25 CH3OH/H2O.
Bis(3-chlorophenyl) disulfide (9). Yield: 88.9%; 1H NMR (400
MHz, CDCl3) δ 7.474–7.134 (m, 8H). 13C NMR (100 MHz, DMSO‑d6) δ
138.14, 134.44, 131.56, 128.10, 126.77, 126.09. MS-APCI m/z: 285.0
[Mꢀ 2H]-. HPLC analysis: Rt = 9.60 min, purity 94.00%, 75:25 CH3OH/
H2O.
2-[2-(4-Fluorophenyldisulfanyl)] benzoic acid (21). Yield:
85.4%; 1H NMR (400 MHz, CDCl3) δ 8.158 (d, J = 6.4 Hz, 1H), 8.078 (d,
J = 7.6 Hz, 1H), 7.576 (t, J = 7.0 Hz, 1H), 7.478–7.443 (m, 2H), 7.320
(d, J = 0.8 Hz, 1H), 6.992 (t, J = 8.8 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6) δ 167.75, 139.59, 133.39, 131.69, 131.11, 129.94, 127.66,
126.12, 124.97, 116.72. MS-ESI m/z: 279 [Mꢀ H]-. HPLC analysis: Rt =
7.57 min, purity 98.63%, 85:25 CH3OH/H2O.
Bis(2-chlorophenyl) disulfide (10). Yield: 82.9%; 1H NMR (400
MHz, CDCl3) δ 7.546 (d, J = 6.4 Hz, 2H), 7.366 (d, J = 6.8 Hz, 2H), 7.226
(t, J = 7.0 Hz, 2H), 7.163 (t, J = 7.0 Hz, 2H). 13C NMR (100 MHz,
DMSO‑d6) δ 133.46, 131.69, 130.55, 129.61, 128.95, 128.15. MS-APCI
m/z: 285.0 [Mꢀ 2H]-. HPLC analysis: Rt = 3.05 min, purity 95.58%,
90:10 CH3OH/H2O.
4-Fluorophenyl isopropyl disulfide (22). Yield: 83.1%; 1H NMR
(400 MHz, CDCl3) δ 7.527–7.492 (m, 2H), 7.008 (t, J = 8.8 Hz, 2H),
3.104–3.003 (m, 1H), 1.292 (d, J = 6.8 Hz, 6H). 13C NMR (100 MHz,
CDCl3) δ 160.80, 133.52, 129.86, 115.95, 41.65, 22.46. MS-APCI m/z:
203.0 [M + H]+. HPLC analysis: Rt = 12.40 min, purity 96.41%, 80:20
CH3OH/H2O.
Bis(4-fluorodiphenyl) disulfide (11). Yield: 79.8%; 1H NMR (400
MHz, CDCl3) δ 7.400 (d, J = 8.4 Hz, 4H), 7.277 (d, J = 8.8 Hz, 4H). 13
C
NMR (100 MHz, CDCl3) δ 161.48, 132.27, 131.33, 116.54. MS-APCI m/
z: 253.0 [Mꢀ H]-. HPLC analysis: Rt = 13.24 min, purity 98.64%, 90:10
CH3OH/H2O.
Bis(1H-benzimidazol) disulfide (12). Yield: 78.9%; 1H NMR (400
2-[2-(4-Fluorophenyldisulfanyl)] methyl acetate (23). Yield:
69.6%; 1H NMR (400 MHz, CDCl3) δ 7.556–7.521 (m, 2H), 7.063–7.020
(m, 2H), 3.608 (s, 3H), 3.494 (s, 2H). 13C NMR (100 MHz, CDCl3) δ
169.40, 163.90, 131.94, 131.85, 116.18, 52.54, 40.57. MS-APCI m/z:
233.0 [M + H]+. HPLC analysis: Rt = 7.61 min, purity 96.09%, 85:15
CH3OH/H2O.
MHz, DMSO‑d6) δ 7.518 (q, J = 8.8, 4H), δ 7.203 (d, J = 2.8 Hz, 4H). 13
C
NMR (100 MHz, DMSO‑d6) δ 147.78, 140.16, 132.68, 123.07, 122.79,
155.59, 109.94. MS-ESI m/z: 298.8 [M + H]+. HPLC analysis: Rt =
13.74 min, purity 98.90%, 80:20 CH3OH/H2O.
Dibenzoxazolyl disulfide (13). Yield: 80.4%; 1H NMR (400 MHz,
DMSO‑d6) δ 7.544 (s, 2H), 7.238 (m, 2H), 2.975 (m, 1H), 1.762 (m, 1H),
3-[2-(4-Methylphenyldisulfanyl)] propionic acid (24). Yield:
70.2%; 1H NMR (400 MHz, CDCl3) δ 7.478 (d, J = 8.8 Hz, 1H), 7.396 (d,
J = 8.8 Hz, 1H), 6.873–6.822 (m, 2H), 3.086 (s, 3H), 2.725–2.649 (m,
2H), 1.738–1.674 (m, 2H), 1.013–0.940 (m, 3H). 13C NMR (100 MHz,
CDCl3) δ 159.51, 131.68, 128.56, 114.67, 55.47, 40.83, 22.15, 13.23.
MS-APCI m/z: 214.0 [M + H]+. HPLC analysis: Rt = 5.66 min, purity
98.01%, 90:10 CH3OH/H2O.
1.567 (m, 1H) 1.326 (d, J = 6.8 Hz, 3H) 0.968 (t, J = 7.6 Hz, 3H). 13
C
NMR (100 MHz, DMSO‑d6) δ 180.58, 148.58, 131.63, 125.57, 124.20,
110.91, 110.41. MS-APCI m/z: 301.0 [M + H]+. HPLC analysis: Rt =
3.82 min, purity 99.11%, 90:10 CH3OH/H2O.
Bis(5-methoxy-1H-benzimidazol) disulfide (14). Yield: 78.4%; 1H
NMR (400 MHz, CDCl3) δ 7.595 (d, J = 8.8 Hz, 2H), 7.127 (d, J = 1.2 Hz,
2H), 6.984 (d, J = 8.8 Hz, 2H), 3.884 (s, 6H). 13C NMR (100 MHz,
CDCl3) δ 156.72, 140.51, 138.93, 132.71, 116.18, 112.78, 96.99, 55.97.
MS-ESI m/z: 239.8 [M + H]+. HPLC analysis: Rt = 6.52 min, purity
97.49%, 85:15 CH3OH/H2O.
4-Methylphenyl 2-pyridyl disulfide (25). Yield: 84.9%; 1H NMR
(400 MHz, CDCl3) δ 7.477 (d, J = 8.8 Hz, 2H), 6.849 (d, J = 8.8 Hz, 2H),
3.801 (s, 3H), 2.858–2.774 (m, 1H), 1.760–1.654 (m, 1H), 1.561–1.453
(m, 1H), 1.274 (d, J = 6.8 Hz, 3H), 0.917 (t, J = 7.2 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 159.27, 131.10, 129.25, 114.59, 55.46, 48.28,
28.79, 19.97, 11.52. MS-APCI m/z: 228.0 [M + H]+. HPLC analysis: Rt
= 9.47 min, purity 97.04%, 85:15 CH3OH/H2O.
4-Fluorophenyl propyl disulfide (15). Yield: 79.1%; 1H NMR (400
MHz, CDCl3) δ 7.526–7.491 (m, 2H), 7.044–7.000 (m, 2H), 2.726 (t, J =
7.2 Hz, 2H), 1.756–1.646 (m, 2H), 0.963 (t, J = 7.2 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 161.040, 133.063, 130.345, 116.068, 41.041,
22.251, 13.201. MS-APCI m/z: 201.0 [Mꢀ H]-. HPLC analysis: Rt =
14.75 min, purity 95.08%, 80:20 CH3OH/H2O.
4-Methylphenyl propyl disulfide (26). Yield: 87.7%; 1H NMR
(400 MHz, CDCl3) δ 7.488 (d, J = 8.8 Hz, 2H), 6.837 (d, J = 8.8 Hz, 2H),
3.794 (s, 3H), 1.290 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 158.92,
130.28, 129.73, 114.45, 55.37, 48.99, 29.95. MS-APCI m/z: 229.0 [M +
H]+. HPLC analysis: Rt = 3.01 min, purity 97.73%, 90:10 CH3OH/H2O.
4-Methylphenyl isopropyl disulfide (27). Yield: 86.5%; 1H NMR
(400 MHz, CDCl3) δ 7.681–7.658 (m, 1H), 7.230 (d, J = 0.8 Hz, 1H),
7.030 (t, J = 7.4 Hz, 1H), 6.863 (d, J = 8.0 Hz, 1H), 3.891 (s, 3H), 3.678
(s, 3H), δ 2.956 (t, J = 7.2 Hz, 2H), 2.761 (t, J = 7.2 Hz, 2H). 13C NMR
(100 MHz, CDCl3) δ 172.26, 156.81, 128.29, 128.23, 124.68, 121.28,
110.76, 55.89, 51.91, 33.65, 32.82. MS-APCI m/z: 259.0 [M + H]+.
HPLC analysis: Rt = 14.59 min, purity 97.94%, 80:20 CH3OH/H2O.
4-Methylphenyl 1-methylpropyl disulfide (28). Yield: 80.3%; 1H
NMR (400 MHz, CDCl3) δ 7.649 (d, J = 8.0 Hz, 1H), 7.256 (d, J = 8.0 Hz,
1H), 6.978 (t, J = 7.6 Hz, 1H), 6.879 (d, J = 8.0 Hz, 1H), 3.895 (s, 3H),
3.634 (s, 3H), 3.542 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 170.04,
156.69, 129.35, 128.77, 127.23, 123.42, 121.77, 111.78, 56.41, 52.70,
40.50. MS-APCI m/z: 245.0 [M + H]+. HPLC analysis: Rt = 6.56 min,
purity 95.94%, 85:15 CH3OH/H2O.
4-Fluorophenyl 2-allyl disulfide (16). Yield: 90.2%; 1H NMR (400
MHz, CDCl3) δ 7.523–7.488 (m, 2H), 7.023 (t, J = 8.8 Hz, 2H),
5.852–5.748 (m, 1H), 5,180–5.129 (m, 2H), 3.358 (d, J = 7.2 Hz, 2H).
13C NMR (100 MHz, CDCl3) δ 161.163, 132.657, 131.009, 119.277,
116.084, 41.895. MS-APCI m/z: 201.0 [M + H]+. HPLC analysis: Rt =
12.10 min, purity 95.87%, 80:20 CH3OH/H2O.
4-Fluorophenyl tert-butyl disulfide (17). Yield: 75.2%; 1H NMR
(400 MHz, CDCl3) δ 7.540–7.505 (m, 2H), 6.993 (t, J = 8.8 Hz, 2H),
1.291 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 163.12, 134.17, 129.36,
116.07, 49.43, 29.96. MS-APCI m/z: 217.0 [M + H]+. HPLC analysis: Rt
= 11.86 min, purity 95.05%, 80:20 CH3OH/H2O.
4-Fluorophenyl 2-pyridyl disulfide (18). Yield: 77.3%; 1H NMR
(400 MHz, CDCl3) δ 8.489 (d, J = 4.8 Hz, 1H), 7.653–7.636 (m, 2H),
7.535–7.500 (m, 1H), 7.459–7.424 (m, 1H), 7.127–7.116 (m, 1H),
7.025–6.987 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 163.81, 159.47,
149.84, 137.54, 131.36, 130.41, 121.24, 119.87, 116.53. MS-APCI m/z:
238.0 [M + H]+. HPLC analysis: Rt = 13.85 min, purity 99.08%, 80:20
CH3OH/H2O.
4-Methylphenyl 2-allyl disulfide (29). Yield: 83.8%; 1H NMR (400
MHz, CDCl3) δ 8.454 (d, J = 4.0 Hz, 1H), 7.675–7.652 (m, 1H),
9

X. Wei et al.
Bioorganic Chemistry 110 (2021) 104814
7.599–7.595 (m, 1H), 7.538–7.515 (m, 1H), 7.239–7.214 (m, 1H) ,
7.200–2.196 (m, 1H), 7.095–7.062 (m, 1H), 6.918–6.883 (m, 1H), 3.902
(s, 3H). 13C NMR (100 MHz, CDCl3) δ 158.95, 156.58, 149.66, 137.52,
128.38, 127.81, 124.51, 121.41, 120.81, 119.70, 110.51, 55.96. MS-
APCI m/z: 250.0 [M + H]+. HPLC analysis: Rt = 6.06 min, purity
97.52%, 85:15 CH3OH/H2O.
H2O.
2-[2-(2-Methoxyphenyldisulfanyl)] methyl acetate (39). Yield:
80.6%; 1H NMR (400 MHz, CDCl3) δ 7.427 (d, J = 8.0 Hz, 2H), 7.133 (d,
J = 8.0 Hz, 2H), 5.873–5.770 (m, 1H), 5.174–5.127 (m, 2H), 3.356 (d, J
= 7.2 Hz, 2H), 2.339 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 137.30,
133.81, 132.74, 129.83, 128.89, 119.08, 41.71, 21.18. MS-APCI m/z:
197.0 [M + H]+. HPLC analysis: Rt = 8.11 min, purity 95.85%, 85:15
CH3OH/H2O.
4-Methylphenyl cyclopentyl disulfide (30). Yield: 74.1%; 1H NMR
(400 MHz, CDCl3) δ 7.715 (d, J = 6.0 Hz, 1H), 7.189 (t, J = 7.6 Hz, 1H),
6.988 (t, J = 7.6 Hz, 1H), 6.845 (d, J = 8.4 Hz, 1H), 3.898 (s, 3H),
3.108–3.007 (m, 1H), 1.313 (d, J = 2.8 Hz, 6H). 13C NMR (100 MHz,
CDCl3) δ 156.35, 127.43, 126.06, 121.20, 110.60, 55.94, 41.28, 22.53.
MS-APCI m/z: 215.0 [M + H]+. HPLC analysis: Rt = 7.85 min, purity
98.62%, 85:15 CH3OH/H2O.
2-Methoxyphenyl 2-pyridyl disulfide (40). Yield: 81.7%; 1H NMR
(400 MHz, CDCl3) δ 7.435 (d, J = 8.4 Hz, 2H), 7.123 (d, J = 8.0 Hz, 2H),
3.358–3.278 (m, 1H), 2.330(s, 3H), 1.940–1.644 (m, 8H). 13C NMR
(100 MHz, CDCl3) δ 136.70, 134.60, 129.71, 127.95, 50.25, 32.79,
24.79, 21.11. MS-APCI m/z: 225.0 [M + H]+. HPLC analysis: Rt = 4.92
min, purity 96.93%, 90:10 CH3OH/H2O.
4-Methylphenyl tert-butyl disulfide (31). Yield: 85.2%; 1H NMR
(400 MHz, CDCl3) δ 7.670 (d, J = 6.0 Hz, 1H), 7.239 (t, J = 1.6 Hz, 1H),
6.998 (t, J = 3.6 Hz, 1H), 6.878 (d, J = 0.8 Hz, 1H), 3.892 (s, 3H), 2.952
(t, J = 7.0 Hz, 2H), 2.823 (t, J = 7.0 Hz, 2H). 13C NMR (100 MHz, CDCl3)
δ 178.50, 156.92, 128.57, 128.43, 124.50, 121.32, 110.82, 55.89,
33.71, 32.34. MS-ESI m/z: 287.3[2 Mꢀ H]-. HPLC analysis: Rt = 8.31
min, purity 96.64%, 85:15 CH3OH/H2O.
2-Methoxyphenyl isopropyl disulfide (41). Yield: 59.6%; 1H NMR
(400 MHz, CDCl3) δ 7.444 (d, J = 8.4 Hz, 2H), 7.097 (d, J = 8.0 Hz, 2H),
2.316 (s, 3H), 1.297 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 136.38,
135.42, 129.62, 127.44, 49.18, 29.97, 21.12. MS-APCI m/z: 213.0 [M +
H]+. HPLC analysis: Rt = 8.65 min, purity 95.15%, 85:15 CH3OH/H2O.
3-[2-(2-Methoxyphenyldisulfanyl)] propionic acid (42). Yield:
66.7%; 1H NMR (400 MHz, CDCl3) δ 7.425 (d, J = 8.4 Hz, 2H), 7.118 (d,
J = 8.4 Hz, 2H), 2.828–2.756 (m, 1H), 2.328 (s, 3H), 2.030–1.993 (m,
2H), 1.778–1.740 (m, 2H), 1.370–1.205 (m, 6H). 13C NMR (100 MHz,
CDCl3) δ 136.57, 135.08, 129.72, 127.61, 49.82, 32.70, 26.13, 25.71,
21.14. MS-APCI m/z: 239.0 [M + H]+. HPLC analysis: Rt = 12.19 min,
purity 95.09%, 80:20 CH3OH/H2O.
4-Methylphenyl cyclohexyl disulfide (32). Yield: 74.2%; 1H NMR
(400 MHz, CDCl3) δ 7.432 (d, J = 8.8 Hz, 2H), 6.781 (d, J = 8.4 Hz, 2H),
2.859–2.775 (m, 1H), 1.738–1.651 (m, 1H), 1.546–1.475 (m, 1H), 1.276
(d, J = 6.8 Hz, 3H), 0.919 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 155.13, 131.26, 129.40, 116.09, 48.30, 28.73, 19.90, 11.42.
MS-APCI m/z: 213.0 [Mꢀ H]-. HPLC analysis: Rt = 6.93 min, purity
98.15%, 85:15 CH3OH/H2O.
4-[2-(1-Methylpropyldisulfanyl)] phenol (43). Yield: 74.8%; 1
H
3-[2-(4-Methylphenyldisulfanyl)] methyl propionate (33).
Yield: 78.7%; 1H NMR (400 MHz, CDCl3) δ 7.433 (d, J = 8.8 Hz, 2H),
6.783 (d, J = 8.8 Hz, 2H), 3.080–3.013 (m, 1H), 1.291 (d, J = 6.8 Hz,
6H). 13C NMR (100 MHz, DMSO‑d6) δ 157.62, 131.80, 125.73, 116.21,
40.70, 22.14. MS-ESI m/z: 199.3 [Mꢀ H]-. HPLC analysis: Rt = 11.24
min, purity 90.08%, 80:10 CH3OH/H2O.
NMR (400 MHz, CDCl3) δ 7.421 (d, J = 8.4 Hz, 2H), 7.137 (d, 8.0 Hz,
2H), 3.676 (s, 3H), 2.954 (t, J = 7.2 Hz, 2H), 2.736 (t, J = 7.2 Hz, 2H),
2.337 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 137.47, 133.49, 129.93,
128.69, 51.97, 33.62, 33.06, 21.16. MS-APCI m/z: 243.0 [M + H]+.
HPLC analysis: Rt = 7.89 min, purity 98.74%, 85:15 CH3OH/H2O.
4-[2-(Isopropyldisulfanyl)] phenol (44). Yield: 65.9%; 1H NMR
(400 MHz, CDCl3) δ 7.441 (d, J = 8.4 Hz, 1H), 7.378 (d, J = 8.0 Hz, 1H),
7.149 (d, J = 8.0 Hz, 1H), 7.103 (d, J = 8.0 Hz, 1H), 3.769 (s, 1H), 3.604
(s, 3H), 3.488 (s, 1H), 2.340 (s, 3H). 13C NMR (100 MHz, CDCl3) δ
169.55, 138.05, 133.88, 129.72, 128.52, 52.49, 40.55, 21.14. MS-APCI
m/z: 229.0 [M + H]+. HPLC analysis: Rt = 10.54 min, purity 98.89%,
85:15 CH3OH/H2O.
2-[2-(4-Methylphenyldisulfanyl)] methyl acetate (34). Yield:
85.1%; 1H NMR (400 MHz, CDCl3) δ 7.425 (d, J = 8.0 Hz, 2H), 7.141 (d,
J = 8.0 Hz, 2H), 2.941 (t, J = 6.6 Hz, 2H), 2.796 (t, J = 7.0 Hz, 2H),
2.338 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 137.65, 133.33, 130.02,
128.84, 33.69, 32.63, 21.20. MS-ESI m/z: 454.5 [2 Mꢀ H]-. HPLC anal-
ysis: Rt = 6.14 min, purity 97.81%, 85:15 CH3OH/H2O.
4-Methoxyphenyl propyl disulfide (35). Yield: 72.2%; 1H NMR
(400 MHz, CDCl3) δ 8.460 (s, 2H), 7.675 (d, J = 8.0 Hz, 1H),
7.634–7.591 (m, 1H), 7.420 (d, J = 8.0 Hz, 2H), 7.110 (d, J = 7.6 Hz,
2H), 7.076 (d, J = 4.8 Hz, 1H), 2.312 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ 159.97, 149.57, 137.65, 137.30, 132.68, 129.97, 128.07,
120.85, 119.58, 21.11. MS-APCI m/z: 234.0 [M + H]+. HPLC analysis:
Rt = 3.46 min, purity 95.05%, 90:10 CH3OH/H2O.
3-[2-(4-Chlorophenyldisulfanyl)] propionic acid (45). Yield:
75.2%; 1H NMR (400 MHz, CDCl3) δ 7.465 (d, J = 8.4 Hz, 2H), 7.300 (d,
J = 8.4 Hz, 2H), 2.57 (t, J = 7.0 Hz, 2H), 2.782 (t, J = 7.0 Hz, 2H). 13
C
NMR (100 MHz, DMSO‑d6) δ 172.66, 135.63, 131.95, 129.32, 128.87,
33.45, 33.10. MS-ESI m/z: 49.3 [2 Mꢀ H]-. HPLC analysis: Rt = 6.24 min,
purity 99.01%, 85:15 CH3OH/H2O.
2-[2-(4-Chlorophenyldisulfanyl)] methyl acetate (46). Yield:
63.7%; 1H NMR (400 MHz, CDCl3) δ 7.482 (d, J = 8.8 Hz, 2H), 7.310 (d,
J = 8.4 Hz, 2H), 3.604 (s, 3H), 3.492 (s, 2H). 13C NMR (100 MHz, CDCl3)
δ 169.27, 134.70, 133.62, 130.01, 129.23, 52.55, 40.56. MS-APCI m/z:
246.0 [Mꢀ 2H]-. HPLC analysis: Rt = 3.82 min, purity 98.31%, 90:10
CH3OH/H2O.
4-Methoxyphenyl 1-methylpropyl disulfide (36). Yield: 81.6%;
1H NMR (400 MHz, CDCl3) δ 7.429 (d, J = 8.0 Hz, 2H), 7.130 (d, J = 8.0
Hz, 2H), 2.706 (t, J = 7.2 Hz, 2H), 2.335 (s, 3H), 1.743–1.652 (m, 2H),
0.964 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 137.01, 134.28,
129.82, 128.33, 40.91, 22.24, 21.15, 13.23. MS-APCI m/z: 196.0
[Mꢀ H]-. HPLC analysis: Rt = 3.54 min, purity 95.33%, 90:10 CH3OH/
H2O.
4-Chlorophenyl 2-pyridyl disulfide (47). Yield: 77.7%; 1H NMR
(400 MHz, CDCl3) δ 8.484 (d, J = 4.4 Hz, 1H), 7.650–7.588 (m, 2H),
4-Methoxyphenyl tert-butyl disulfide (37). Yield: 83.3%; 1H NMR
(400 MHz, CDCl3) δ 7.430 (d, J = 8.4 Hz, 2H), 7.115 (d, J = 8.0 Hz, 2H),
3.084–3.015 (m, 1H), 2.323 (s, 3H), 1.293 (d, J = 6.4 Hz, 6H). 13C NMR
(100 MHz, CDCl3) δ 136.73, 134.87, 129.74, 127.91, 41.51, 22.50,
21.14. MS-APCI m/z: 221.0 [M + Na]+. HPLC analysis: Rt = 13.28 min,
purity 99.10%, 80:20 CH3OH/H2O.
7.458 (d, J = 8.4 Hz, 2H), 7.260 (s, 2H), 7.116 (t, J = 5.4 Hz, 1H). 13
C
NMR (100 MHz, DMSO‑d6) δ 157.59, 149.71, 137.96, 134.41, 132.40,
129.26, 128.91, 121.63, 119.33. MS-APCI m/z: 254.0 [M + H]+. HPLC
analysis: Rt = 9.77 min, purity 99.26%, 85:15 CH3OH/H2O.
3-[2-(4-Chlorophenyldisulfanyl)] methyl propionate (48).
Yield: 82.7%; 1H NMR (400 MHz, CDCl3) δ 7.460 (d, J = 8.8 Hz, 2H),
7.297 (d, J = 8.8 Hz, 2H), 3.678 (s, 3H), 2.960 (t, J = 7.2 Hz, 2H), 2.720
(t, J = 7.2 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 171.92, 135.56,
133.00, 129.18, 128.98, 51.94, 33.50, 33.16. MS-APCI m/z: 261.0
[Mꢀ H]-. HPLC analysis: Rt = 9.31 min, purity 98.89%, 85:15 CH3OH/
H2O.
3-[2-(2-Methoxyphenyldisulfanyl)] methyl propionate (38).
Yield: 83.7%; 1H NMR (400 MHz, CDCl3) δ 7.430 (d, J = 8.4 Hz, 2H),
7.114 (d, J = 8.0 Hz, 2H), 2.863–2.779 (m, 1H), 2.326 (s, 3H),
1.747–1.678 (m, 1H), 1.548–1.480 (m, 1H), 1.284 (t, J = 6.4 Hz, 3H),
0.962 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 136.70, 134.92,
129.72, 127.93, 48.35, 28.84, 21.14, 19.97, 11.57. MS-APCI m/z: 213.0
[M + H]+. HPLC analysis: Rt = 3.74 min, purity 95.0%, 90:10 CH3OH/
2-[2-(Propionyloxydisulfanyl)] benzoic acid (49). Yield: 85.1%;
1H NMR (400 MHz, DMSO‑d6) δ 8.100 (d, J = 7.6 Hz, 1H), 7.972 (d, J =
10

X. Wei et al.
Bioorganic Chemistry 110 (2021) 104814
6.4 Hz, 1H), 7.677 (t, J = 7.0 Hz, 1H), 7.341 (t, J = 7.4 Hz, 1H), 2.872 (t,
J = 6.2 Hz, 2H), 2.594 (t, J = 8 Hz, 2H). 13C NMR (100 MHz, DMSO‑d6) δ
172.78, 167.68, 133.18, 131.68, 127.75, 125.71, 33.41, 32.66. MS-ESI
m/z: 257.2 [Mꢀ H]-. HPLC analysis: Rt = 7.85 min, purity 95.64%,
85:15 CH3OH/H2O.
75.4%; 1H NMR (400 MHz, DMSO‑d6) δ 7.491 (d, J = 3.6 Hz, 2H), 7.172
(d, J = 9.2 Hz, 2H), 1.988 (d, J = 11.2 Hz, 1H), 1.715–1.684 (m, 2H),
1.402–1.166 (m, 8H). 13C NMR (100 MHz, DMSO‑d6) δ 168.13, 132.28,
122.35, 109.51, 49.16, 31.78, 25.32. MS-ESI m/z: 265.3 [M + H]+.
HPLC analysis: Rt = 7.18 min, purity 97.86%, 80:20 CH3OH/H2O.
2-(Cyclopentyldisulfanyl)-1H-benzo[d]imidazole (60). Yield:
77.3%; 1H NMR (400 MHz, DMSO‑d6) δ 12.787 (s, 1H), 7.504 (s, 2H),
7.185 (d, J = 2.8 Hz, 2H), 2.004–1.966 (m, 1H), 1.691–1.594 (m, 8H).
13C NMR (100 MHz, DMSO‑d6) δ 149.22, 132.29, 122.15, 109.51, 50.06,
32.19, 24.31. MS-APCI m/z: 251.0 [M + H]+. HPLC analysis: Rt = 6.09
min, purity 98.34%, 80:20 CH3OH/H2O.
2-[2-(Isopropyldisulfanyl)] benzoic acid (50). Yield: 78.7%; 1H
NMR (400 MHz, DMSO‑d6) δ 8.110 (d, J = 7.2 Hz, 1H), 7.947 (d, J = 6.0
Hz, 1H), 7.654 (t, J = 7.0 Hz, 1H), 7.315 (t, J = 7.0 Hz, 1H), 3.124–3.024
(m, 1H), 1.252 (d, J = 6.8 Hz, 6H). 13C NMR (100 MHz, DMSO‑d6) δ
167.56, 141.16, 132.88, 131.41, 127.69, 125.50, 40.57, 22.32. MS-ESI
m/z: 227.7 [Mꢀ H]-. HPLC analysis: Rt = 6.46 min, purity 96.17%,
85:15 CH3OH/H2O.
2-(tert-Butyldisulfanyl)-1H-benzo[d]imidazole
(61).
Yield:
2-Pyridyl tert-butyl disulfide (51). Yield: 69.3%; 1H NMR (400
MHz, CDCl3) δ 8.430 (d, J = 4.0 Hz, 1H), 7.795 (d, J = 8.4 Hz, 1H), 7.620
(t, J = 6.8 Hz, 1H), 7.053 (t, J = 6.2 Hz, 1H), 1.335 (s, 9H). 13C NMR
(100 MHz, CDCl3) δ 161.57, 149.12, 136.90, 120.41, 119.59, 49.30,
29.76. MS-APCI m/z: 200.0 [M + H]+. HPLC analysis: Rt = 6.89 min,
purity 95.01%, 85:15 CH3OH/H2O.
83.2%; 1H NMR (400 MHz, DMSO‑d6) δ 12.493 (s, 1H), 7.124 (d, J = 2.4
Hz, 4H), 1.326 (s, 9H). 13C NMR (100 MHz, DMSO‑d6) δ 149.74, 132.30,
122.40, 109.56, 49.27, 29.12. MS-ESI m/z: 238.9 [M + H]+. HPLC
analysis: Rt = 11.62 min, purity 97.58%, 80:20 CH3OH/H2O.
2-(Propyldisulfanyl)-1H-benzo[d]imidazole (62). Yield: 81.7%;
1H NMR (400 MHz, CDCl3) δ 7.553 (s, 2H), 7.250–7.225 (m, 2H), 2.857
2-Pyridyl 1-methylpropyl disulfide (52). Yield: 78.4%; 1H NMR
(400 MHz, CDCl3) δ 8.443 (d, J = 4.0 Hz, 1H), 7.773 (d, J = 8.4 Hz, 1H),
7.659–7.636 (m, 1H), 7.082–7.054 (m, 1H), 2.937–2.853 (m, 1H),
1.780–1.692 (m, 1H), 1.588–1.535 (m, 1H), 1.307 (d, J = 6.8 Hz, 3H),
0.986 (t, J = 4.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 161.30, 149.28,
37.11, 20.54, 119.67, 48.54, 28.86, 19.89, 11.55. MS-APCI m/z: 200.0
[M + H]+. HPLC analysis: Rt = 6.06 min, purity 98.83%, 85:15 CH3OH/
H2O.
(t, J = 7.2 Hz, 2H), 1.805–1.713 (m, 2H), 0.995 (t, J = 7.4 Hz, 3H). 13
C
NMR (100 MHz, DMSO‑d6) δ 123.07, 122.19, 118.77, 111.62, 41.00,
21.97, 13.10. MS-ESI m/z: 224.9 [M + H]+. HPLC analysis: Rt = 6.01
min, purity 96.21%, 80:20 CH3OH/H2O.
2-(1-Methylpropyldisulfanyl)-1H-benzo[d]imidazole
(63).
Yield: 75.9%; 1H NMR (400 MHz, CDCl3) δ 7.544 (s, 2H), 7.249–7.226
(m, 2H), 3.019–2.934 (m, 1H), 1.815–1.709 (m, 1H), 1.621–1.513 (m,
1H), 1.325 (d, J = 6.8 Hz, 3H), 0.969 (t, J = 7.4 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ 150.81, 139.48, 122.74, 114.75, 49.04, 28.71, 19.66,
11.48. MS-ESI m/z: 238.9 [M + H]+. HPLC analysis: Rt = 9.60 min,
purity 97.21%, 80:20 CH3OH/H2O.
2-[2-(1-Methylpropyldisulfanyl)] aniline (53). Yield: 68.3%; 1
H
NMR (400 MHz, CDCl3) δ 7.457–7.435 (m, 1H), 7.156–7.114 (m, 1H),
6.739–6.663 (m, 2H), 2.896–2.812 (m, 1H), 1.801–1.696 (m, 1H),
1.589–1.481 (m, 1H), 1.301 (d, J = 6.8 Hz, 3H), 0.908 (t, J = 7.2 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ 147.76, 34.88, 130.62, 119.74,
118.39, 115.65, 47.74, 28.67, 19.80, 1.37. MS-ESI m/z: 213.9 [M + H]+.
HPLC analysis: Rt = 3.18 min, purity 96.07%, 90:10 CH3OH/H2O.
2-(Cyclohexyldisulfanyl) benzo[d]oxazole (54). Yield: 81.3%; 1H
NMR (400 MHz, CDCl3) δ 7.672 (d, J = 8.4 Hz, 1H), 7.494 (t, J = 8.4 Hz,
1H), 7.306 (t, J = 4.0 Hz, 2H), 3.111–3.057 (m, 1H), 2.110 (d, J = 10.0
Hz, 2H), 1.605 (d, J = 10.0 Hz, 2H), 1.471–1.251 (m, 6H). 13C NMR
(100 MHz, CDCl3) δ 164.01, 152.33, 142.04, 124.61, 124.54, 119.32,
110.23, 49.96, 32.27, 25.92, 25.44. MS-APCI m/z: 267.0 [M + H]+.
HPLC analysis: Rt = 8.90 min, purity 95.12%, 80:20 CH3OH/H2O.
2-(Isopropyldisulfanyl) benzo[d]oxazole (55). Yield: 71.3%; 1H
NMR (400 MHz, CDCl3) δ 7.670 (d, J = 8.4 Hz, 1H), 7.497 (d, J = 8.8 Hz,
1H), 7.321–7.293 (m, 2H), 3.414–3.313 (m, 1H), 1.402 (d, J = 6.8 Hz,
6H). 13C NMR (100 MHz, CDCl3) δ 162.88, 152.06, 141.81, 125.63,
125.31, 119.54, 110.99, 41.96, 22.28. MS-APCI m/z: 226.0 [M + H]+.
HPLC analysis: Rt = 10.63 min, purity 97.36%, 80:20 CH3OH/H2O.
2-(tert-Butyldisulfanyl) benzo[d]oxazole (56). Yield: 58.6%; 1H
NMR (400 MHz, DMSO‑d6) δ 7.746–7.697 (m, 2H), 7.407–7.379 (m,
2H), 1.371 (s, 9H). 13C NMR (100 MHz, DMSO‑d6) δ 162.76, 151.96,
141.84, 125.60, 125.28, 119.52, 110.94, 49.86, 29.50. MS-APCI m/z:
240.0 [M + H]+. HPLC analysis: Rt = 12.20 min, purity 98.05%, 80:20
CH3OH/H2O.
2-(tert-Butyldisulfanyl)-5-(difluoromethoxy)-1H-benzo[d]imid-
azole (64). Yield: 81.7%; 1H NMR (400 MHz, CDCl3) δ 7.481 (d, J = 8.0
Hz, 1H), 7.321 (s, 1H), 7.042 (d, J = 8.8 Hz, 1H), 6.500 (t, J = 74.0 Hz,
1H), 1.373 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 152.59, 146.92,
118.97, 116.39, 115.70, 113.81, 49.88, 29.50. MS-APCI m/z: 305.0 [M
+ H]+. HPLC analysis: Rt = 7.46 min, purity 94.15%, 80:20 CH3OH/
H2O.
2-(1-Methylpropyldisulfanyl)-5-(difluoromethoxy)-1H-benzo
[d]imidazole (65). Yield: 80.2%; 1H NMR (400 MHz, CDCl3) δ 7.495
(d, J = 8.8 Hz, 1H), 7.331 (s, 1H), 7.052 (d, J = 7.2 Hz, 1H), 6.507 (t, J =
74.0 Hz, 1H), 3.029–2.945 (m, 1H), 1.818–1.712 (m, 1H), 1.632–1.524
(m, 1H), 1.333 (d, J = 6.4 Hz, 3H), 0.980 (t, J = 7.2 Hz, 3H). 13C NMR
(100 MHz, DMSO‑d6) δ 151.34, 146.91, 119.90, 117.34, 115.18, 114.78,
48.46, 28.42, 19.78, 11.48. MS-APCI m/z: 303.0 [Mꢀ H]-. HPLC anal-
ysis: Rt = 5.96 min, purity 96.68%, 80:20 CH3OH/H2O.
2-(Propyldisulfanyl)-5-nitro-1H-benzo[d]imidazole (66). Yield:
89.1%; 1H NMR (400 MHz, CDCl3) δ 8.475 (s, 1H), 8.204 (d, J = 6.8 Hz,
1H), 7.597 (d, J = 8.8 Hz, 1H), 2.897 (t, J = 7.2 Hz, 2H), 1.821–1.730
(m, 2H), 1.019 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 143.04,
137.83, 132.70, 119.39, 118.37, 109.69, 105.09, 40.96, 21.99, 13.06.
MS-APCI m/z: 270.0 [M + H]+. HPLC analysis: Rt = 9.60 min, purity
98.94%, 80:20 CH3OH/H2O.
2-(Cyclopentyldisulfanyl)-5-nitro-1H-benzo[d]imidazole (67).
Yield: 79.1%; 1H NMR (400 MHz, CDCl3) δ 8.473 (s, 1H), 8.193 (d, J =
6.8 Hz, 1H), 7.595 (d, J = 8.8 Hz, 1H), 3.501–3.471 (m, 1H),
1.784–1.630 (m, 8H). 13C NMR (100 MHz, CDCl3) δ 156.37, 143.60,
118.81, 51.08, 32.85, 24.73. MS-APCI m/z: 294.0 [Mꢀ H]-. HPLC anal-
ysis: Rt = 7.44 min, purity 96.10%, 80:20 CH3OH/H2O.
2-(Cyclopentyldisulfanyl) benzo[d]oxazole (57). Yield: 74.5%;
1H NMR (400 MHz, CDCl3) δ 7.689–7.665 (m, 1H), 7.511–7.487 (m,
1H), 7.322–7.293 (m, 2H), 3.672–3.641 (m, 1H), 2.081–2.018 (m, 2H),
1.810–1.741 (m, 4H), 1.654–1.617 (m, 2H). 13C NMR (100 MHz, CDCl3)
δ 163.88, 152.39, 142.03, 124.65, 124.57, 119.34, 110.24, 50.60,
32.76, 24.77. MS-APCI m/z: 252.0 [M + H]+. HPLC analysis: Rt = 8.13
min, purity 98.99%, 80:20 CH3OH/H2O.
2-(tert-Butyldisulfanyl)-5-nitro-1H-benzo[d]imidazole
(68).
Yield: 85.2%; 1H NMR (400 MHz, CDCl3) δ 8.468 (s, 1H), 1.191 (d, J =
6.8 Hz, 1H), 7.587 (d, J = 8.8 Hz, 1H), 1.398 (s, 9H). 13C NMR (100
MHz, CDCl3) δ 143.66, 118.86, 50.43, 29.63. MS-ESI m/z: 284.2 [M +
H]+. HPLC analysis: Rt = 9.60 min, purity 99.09%, 80:20 CH3OH/H2O.
2-(1-Methylpropyldisulfanyl)-5-nitro-1H-benzo[d]imidazole
(69). Yield: 76.3%; 1H NMR (400 MHz, CDCl3) δ 8.469, (s, 1H), 8.192 (d,
J = 6.4 Hz, 1H), 7.590 (d, J = 8.8 Hz, 1H), 3.071–2.987 (m, 1H),
1.809–1.740 (m, 1H), 1.633–1.561(m, 1H), 1.350 (d, J = 6.8 Hz, 3H),
2-(Isopropyldisulfanyl)-1H-benzo[d]imidazole (58). Yield:
69.7%; 1H NMR (400 MHz, DMSO‑d6) δ 7.501 (d, J = 2.4 Hz, 2H), 7.123
(d, J = 4.0 Hz, 2H), 3.370–3.269 (m, 1H), 1.286 (d, J = 6.8 Hz, 6H). 13
C
NMR (100 MHz, DMSO‑d6) δ 149.42, 132.29, 122.36, 109.52, 41.41,
21.9. MS-ESI m/z: 224.9 [M + H]+. HPLC analysis: Rt = 9.60 min, purity
98.94%, 80:20 CH3OH/H2O.
2-(Cyclohexyldisulfanyl)-1H-benzo[d]imidazole (59). Yield:
11

X. Wei et al.
Bioorganic Chemistry 110 (2021) 104814
0.996 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 156.63, 143.59,
118.82, 49.31, 28.74, 19.63, 11.45. MS-APCI m/z: 282.0 [Mꢀ H]-. HPLC
analysis: Rt = 6.61 min, purity 97.93%, 80:20 CH3OH/H2O.
[A]0ꢀ [A]0(Fre ꢀ Ft)/(Fre ꢀ Fox) and [B]0ꢀ [A]0(Fre ꢀ Ft)/(Fre ꢀ Fox) at a given
time, respectively. These results were shown in Fig. S2 & Tables S1/2.
2-(tert-Butyldisulfanyl)-5-methoxy-1H-benzo[d]imidazole (70).
Yield: 65.7%; 1H NMR (400 MHz, CDCl3) δ 7420 (d, J = 8.4 Hz, 1H),
4.7. Determination of Trx redox states by probe Naph-EA-mal [43]
7.019 (s, 1H), 6.858 (d, J = 8.8 Hz, 1H), 3.819 (s, 3H), 1.346 (s, 9H). 13
C
The stock Trx was incubated with DTT to prepare the reduced Trx.
The triploid volume of cold-acetone (acetone was put at ꢀ 20 ◦C for a
3–5 h) was added to precipitate the protein for 1 h at ꢀ 80 ◦C, and the
reaction mixture was centrifuged at 4 ◦C and removed supernatant. Then
PBS and SDS were added to thaw Trx and the Trx were incubated with
68 for 30 min, the probe Naph-EA-mal (10 mM) which is a thiol probe
for protein labeling and bioimaging was added, incubated, and shaken
every 15 min. After an hour, cold-acetone was added to precipitate the
protein for 1 h at ꢀ 80 ◦C, the reaction mixture was centrifuged at 4 ◦C
and the supernatant was removed. PBS with SDS (1%, w/v) were added
to dissolve the protein, and all samples were boiled with loading buffer
and then were analyzed by SDS-PAGE. The gel images were acquired
with a Gel imager (G: BOX Chemi XX9, UK) and stained with CB.
NMR (100 MHz, DMSO‑d6) δ 156.25, 148.73, 112.03, 55.87, 49.47,
29.58. MS-APCI m/z: 269.0 [M + H]+. HPLC analysis: Rt = 5.15 min,
purity 98.45%, 80:20 CH3OH/H2O.
2-(Propyldisulfanyl)-5-methoxy-1H-benzo[d]imidazole
(71).
Yield: 75.8%; 1H NMR (400 MHz, CDCl3) δ 7.457 (d, J = 8.8 Hz, 1H),
7.046 (d, J = 3.6 Hz, 1H), 6.893 (d, J = 8.8 Hz, 1H), 3.838 (s, 3H), 2.841
(t, J = 7.2 Hz, 2H), 1.771–1.682 (m, 2H), 0.964 (t, J = 7.2 Hz, 3H). 13
C
NMR (100 MHz, CDCl3) δ 158.64, 140.29, 132.05, 125.20, 116.73,
115.76, 96.96, 56.35, 53.88, 18.77, 13.79. MS-APCI m/z: 255.0 [M +
H]+. HPLC analysis: Rt = 3.40 min, purity 95.79%, 80:20 CH3OH/H2O.
2-(1-Methylpropyldisulfanyl)-5-methoxy-1H-benzo[d]imid-
azole (72). Yield: 77.7%; 1H NMR (400 MHz, CDCl3) δ 7.432 (d, J = 8.8
Hz, 1H), 7.025 (s, 1H), 6.875 (d, J = 8.8 Hz, 1H), 3.389 (s, 3H),
3.015–2.931 (m, 1H), 1.810–1.704 (s, 1H), 1.619–1.511 (m, 1H), 1.321
(d, J = 6.8 Hz, 3H), 0.959 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz,
CDCl3) δ 156.34, 148.31, 112.26, 97.37, 55.88, 48.40, 28.41, 19.87,
11.49. MS-APCI m/z: 269.0 [M + H]+. HPLC analysis: Rt = 3.53 min,
purity 94.10%, 80:20 CH3OH/H2O.
4.8. Cell cultures
The cell culture conditions were described in previous publications
[52,53].
4.4. Screening of disulfides against Trx by NBL-SS
4.9. Cytotoxicity assays
The E.coli Trx was reduced with excessive DTT, and then the excess
DTT was removed by a Sephadex G-25 desalting column. The concen-
tration of the E.coli Trx was quantified by a Microplate Reader using the
Bradford asaay. The reduced Trx (10 µM) was incubated with different
concentrations (100 µM, 20 µM, 10 µM) of compounds in TE buffer (pH
= 7.4) at 37 ◦C in 96-well plates for 30 min. The positive control group
only had reduced Trx and TE buffer but without tested compounds. To
eliminate the interference of DMSO, we set a blank control group where
the same amount DMSO was introduced. Then, the probe NBL-SS (5 µM)
was added into all wells quickly and incubated for another 5 min, and
the fluorescence intensity was monitored with a Microplate Reader (λex
= 610 nm; λem = 661 nm, FlexStation 3, USA).
4.9.1. MTT assay [52,53]
HeLa cells (1 × 104 cells/well) were incubated with compounds 4, 5,
6, 19, 22, 24, 68 and 69 in triplicate in a 96-well plate for the indicated
time at 37 ◦C in a final volume of 100
μL. Cells were treated only with
DMSO as a control group. At the end of the treatment with eight com-
pounds (48 h and 72 h), 10 L of MTT (5 mg/mL) was added to each well
L,
μ
and continued incubated for 4 h at 37 ◦C. An extraction buffer (100
μ
10% SDS, 5% isobutanol, 0.1% HCl) was added, and the cells were
incubated overnight at 37 ◦C. The absorbance was measured at 570 nm
on Multiskan GO (Thermo Scientific)
4.9.2. Trypan blue exclusion assay [52,53]
HeLa cells were seeded in 12-well plates and treated with different
concentrations of 68 for 72 h. Cells were treated with DMSO only as a
control, and cell viability was determined by the trypan blue exclusion
assay. After treatment, the cells were stained with trypan blue (0.4%, w/
v), and the number of viable (non-stained) and dead (stained) cells were
counted by a microscope.
4.5. Determination of fluorescence of Trx redox status
We determined the fluorescence intensity of Trx redox status at 340
nm after excitation at 280 nm. First, the fluorescence intensity of the
reduced Trx was measured in TE buffer at 25 ◦C, then the reduced Trx
and the 68 were incubated together, and the fluorescence intensity of
Trx was measured every minute in TE buffer at 25 ◦C for 30 min. The 68
was dissolved in DMSO, the reduced Trx was prepared by DTT. All the
data were determined by a Cary Eclipse Fluorescence spectrophotometer
(Agilent Technologies).
4.10. Imaging Trx activity in HeLa cells by NBL-SS
The cells were seeded in 12-well plates (2 × 104 cells/well) for 24 h
at 37 ◦C, and then, the cells were treated with indicated concentrations
of 68 (10 µM) for 6 h, 12 h, 24 h at 37 ◦C, respectively. Then NBL-SS (5
µM) in DMSO was added and continued incubated for another 5 min at
37 ◦C in the dark. After washing the cells with PBS twice to remove the
remaining NBL-SS, the fluorescence images were acquired with a Floid
cell imaging station (Life Technology).
4.6. Kinetic studies [31,35,39,51]
The reactions of compound 68, 69 (10 µM each) with Trx (<10 µM)
in TE buffer (pH = 7.4, containing 1‰ DMSO) were monitored by
measuring the fluorescence intensity at 340 nm after excitation at 280
nm. The reactions were carried out at 25 ◦C. The reduced Trx was pre-
pared by DTT. The second-order rate constants for the reactions were
determined by fitting the fluorescence intensities of the sample to the
second-order equation (1):
4.11. Determination of intracellular ROS
HeLa cells were incubated with different concentrations of 68 in 12-
well plates (1 × 104 cells/well) for 48 h, while the cells incubated only
with DMSO served as a control. After removing the medium, the cells
were continued to incubate with 2′, 7′-dichlorofluorescein diacetate
(DCFH-DA, 10 µM) or DHE (10 µM) in a fresh medium for 0.5 h in the
dark. Following incubation, the cell images were observed by a fluo-
rescence microscope.
(
)
1
[B]0[A]
[A]0[B]
ln
= k’t
(1)
[A]0 ꢀ [B]0
where [A]0 and [B]0 were the initial concentrations of Trx and com-
pounds, respectively. Inln([B]0[A]/[A]0[B]), [A] and [B] were
12

X. Wei et al.
Bioorganic Chemistry 110 (2021) 104814
4.12. PAO-Sepharose assay
Appendix A. Supplementary material
This assay was based on the binding of vicinal dithiols to phenyl-
arsine oxide (PAO). PAO beads (PAO-Sepharose) was prerpared by our
group [44,46]. Reduced Trx was captured by PAO and hence pulled
down, while oxidized forms remained in the supernatant. HeLa cells
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104814.
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111 project are acknowledged.
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