Novel thiosemicarbazone derivatives containing indole fragment as potent and selective anticancer agent

Article abstract of DOI:10.1016/j.ejmech.2019.111764

Potent and safe anticancer drugs research and development are still on the way to human health. In this report, a series of novel thiosemicarbazone derivatives containing indole fragment were designed and synthesized. Most compounds exhibited excellent antiproliferative activity against PC3, MGC803 and EC109 cell lines with low micromolar IC₅₀ (0.14–12μM). Especially, compound 5j can selectively inhibit PC3 cells in three tested tumor cells with IC₅₀ value of 0.14 μM, which may be attributed to a synergistic effect after introducing indole fragment into the TSC structure. Meanwhile, compound 5j displayed more selectivity in PC3 cells toward two normal WPMY-1 and GES-1 cell lines, compared to those of 3-AP and DPC. We also found that 5j can effectively inhibit PC3 cell proliferation, colonization and induce apoptosis. What's more, 5j may significantly suppress migration and invasion by blocking the EMT process but had no effect on cell cycle. Collectively, our findings indicate that 5j with structure of thiosemicarbazone containing indole may serve as a useful anticancer lead for further optimization and development.

Full text of DOI:10.1016/j.ejmech.2019.111764

European Journal of Medicinal Chemistry 184 (2019) 111764
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel thiosemicarbazone derivatives containing indole fragment as
potent and selective anticancer agent
Zhangxu He 1, Hui Qiao 1, Feifei Yang, Wenjuan Zhou, Yunpeng Gong, Xinhui Zhang,
,
,
, 2
Haojie Wang, Bing Zhao, Liying Ma** 2, Hong-min Liu*** 2, Wen Zhao*
State Key Laboratory of Esophageal Cancer Prevention and Treatment, Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of
China, Co-innovation Center of Henan Province for New Drug R & D and Preclinical Safety, School of Pharmaceutical Sciences, Zhengzhou University,
Zhengzhou, Henan, 450001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Potent and safe anticancer drugs research and development are still on the way to human health. In this
report, a series of novel thiosemicarbazone derivatives containing indole fragment were designed and
synthesized. Most compounds exhibited excellent antiproliferative activity against PC3, MGC803 and
Received 3 July 2019
Received in revised form
3 October 2019
Accepted 3 October 2019
Available online 7 October 2019
EC109 cell lines with low micromolar IC50 (0.14e12
m
M). Especially, compound 5j can selectively inhibit
M, which may be attributed to a synergistic
PC3 cells in three tested tumor cells with IC50 value of 0.14
m
effect after introducing indole fragment into the TSC structure. Meanwhile, compound 5j displayed more
selectivity in PC3 cells toward two normal WPMY-1 and GES-1 cell lines, compared to those of 3-AP and
DPC. We also found that 5j can effectively inhibit PC3 cell proliferation, colonization and induce
apoptosis. What’s more, 5j may significantly suppress migration and invasion by blocking the EMT
process but had no effect on cell cycle. Collectively, our findings indicate that 5j with structure of thi-
osemicarbazone containing indole may serve as a useful anticancer lead for further optimization and
development.
Keywords:
Thiosemicarbazone
Selectivity
Proliferation
Migration
Apoptosis
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
inhibition of tumor cell invasion and migration [9e11], chelating
metal properties [12e14], interfering DNA synthesis [15,16], ROS
Despite continued research efforts, cancer possessing extremely
high fatality rate and low cure rate has posed the serious threat to
human health [1]. To combat with cancer, efforts need to be
concentrated on the design and synthesis of highly efficient anti-
tumor agents with low toxicity to normal cells and tissues [2].
Thiosemicarbazone (TSC) as the privileged pharmacophore has
received extensive attention from chemists and biologists because
of possessing potential biological activity, including overcoming
multidrug resistance, antituberculosis, antiviral, antifungal, anti-
malarial, and, most intriguingly, antineoplastic activity [3e8].
Antitumor activity of TSCs is mainly due to several factors, such as
generation [16], and affecting several crucial proteins [10]. To date,
several TSC related drug candidates have been in clinical trial. 3-AP
(Fig. 1), a metal chelating agent, is currently in phase II clinical trial.
Compared to 3-AP, COTI-2 and DPC (Fig. 1), another TSC-containing
drugs, improved the activity and selectivity toward tumor cells
[7,12,13]. Although this family of compounds was extensively re-
ported, highly selective TSCs with strong cytotoxicity against cancer
cells and less side effect remain to be discovered (see Fig. 2).
Recently, we have reported a series of TSC derivatives like 5n
(TS-1) that showed acceptable antiproliferative activity and could
suppress migration of MGC803 cells to some extent [17]. In addi-
tion, compounds containing indole fragment displayed extensive
biological activity of which inhibition of tumor cells metastasis
stood out [18e22]. For example, Panobinostat, approved in 2015,
shows the ability to inhibit invasion and metastasis and signifi-
cantly antiproliferative effect [23,24]. Inspired by biological profile
of indole and in continuation with our previous work on the
identification of novel TSC derivatives, we herein report the design
and synthesis of novel TSC derivatives containing indole and their
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: maliying@zzu.edu.cn (L. Ma), liuhm@zzu.edu.cn (H.-m. Liu),
zhaowen100@139.com (W. Zhao).
1
Both authors contribute equally to this work.
These senior authors contribute equally to this work.
2
https://doi.org/10.1016/j.ejmech.2019.111764
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
Z. He et al. / European Journal of Medicinal Chemistry 184 (2019) 111764
Fig. 1. Representative compounds with TSC structure in the clinical trial.
Fig. 2. Design of novel thiosemicarbazone derivatives containing indole.
underlying mechanisms of antiproliferative activity (Fig. 2).
2. Results and discussion
in 65e85% yields. The compounds 4a-f or 5a-v could be derived
from a Schiff base condensation reaction between 3a-k and
appropriate aldehyde or ketone with the presence of acetic acid in
ethanol, yields ranging from 45 to 85% [26,27].
The full characterization of all thiosemicarbazones was
permitted by 1H NMR, 13C NMR and high-resolution mass (HRMS).
The compounds 4a-f and 5a-v exhibited in 1H NMR spectra at
2.1. Chemistry
The desired TSC derivatives were provided by a synthetic three-
step route as shown in Scheme 1. At first, various aliphatic and
aromatic amines 1a-k was reacted with carbon disulfide under
d
6.75e8.60 ppm matching to protons correlated to the phenyl
group, moreover a singlet peak in the range of 7.25e8.25 ppm
d
attributed to azomethine group (-CH]N-), which was comparable
to the literature involving TSCs [28]. In addition, NeH indole ring
basic
condition,
followed
by
the
addition
of
4-
dimethylaminopyridine (DMAP) and di-tert-butyl decarbonate
(Boc2O) to provide the corresponding products 2a-k in 48e66%
yields [25]. Then, after treatment of 2a-k with an excess of hydra-
zine hydrate in dichloromethane, compounds 3a-k were afforded
showed a singlet peak in the range of
hydrazine proton displayed chemical shifts at
region. Finally, the other signals related to thiosemicarbazone, such
d
10.75e11.20 ppm and NeH
d
8.60e11.75 ppm
Scheme 1. Reagents and conditions: (a) (1) CS2, triethylamine, EtOH, rt; (2) Boc2O, DMAP, EtOH, rt; (b) hydrazine hydrate, dichloromethane, rt; (c) appropriate aldehyde or ketone,
acetic acid, EtOH, rt.

Z. He et al. / European Journal of Medicinal Chemistry 184 (2019) 111764
3
as the protons of alkane occurred between
the 13C NMR spectra, the chemical shift of C]N group occurred in
the range of 140.22e153.84 ppm and C]S group appeared at the
region of 175.93e177.90 ppm, both in accordance with the reports
[28e30]. These data markedly confirmed the formation of 4a-f and
5a-v.
The compounds 4a-f and 5a-v could exist in either E or Z
isomeric form because of azomethine (-CH]N-). We continued
exploring the predominant isomers of our derivatives. From 1H
NMR spectra, it is possible to detect a single peak as eCH]N- signal
d
2.25 and 4.80 ppm. In
(IC50 ¼ 0.28 0.04
m
M) vs 5j (IC50 ¼ 0.14 0.02
m
M). Interestingly,
when the 4-position of the pyridine ring was other substitutes
(hydroxyl, methoxy, chlorine, bromine), the activity decreased
compared to 5c against PC3 cells. These results implied that the
presence of the methyl group at the pyridine ring was critical for
cytotoxic activity. 5k-m were prepared by changing the position of
the methyl group on the pyridine ring. The results presented that
the C-2, C-3, and C-4 on the pyridine ring had slight effect on the
activity toward PC3 cells. However, the activity of compounds was
significantly decreased toward PC3, MGC803 and EC109 cells when
methyl group was added to the 5-position of pyridine ring (5j, 5k or
d
d
between
d 7.25 and 8.25 ppm. As the most outstanding compound,
5j was chosen to explain the isomerism. A single spot was observed
in thin layer chromatography (TLC) and the purity of 5j was 99.04%,
determined by reverse-phase high-performance liquid chroma-
tography (HPLC) analysis (See supplementary material). The above
results showed the presence of a single isomeric form. In order to
further confirm the stereochemistry more precisely, the NOESY
experiment of 5j was performed. The NOESY spectra clearly dis-
played the spatial correlation between azomethine (-CH]N-) at
5l vs 5m). Furthermore, 5l (IC50 ¼ 0.33 0.03
m
M) had better anti-
proliferative activity than 5j (IC50 ¼ 2.94 0.47
mM) for
MGC803 cells, but 5j showed 21 times better selectivity between
PC3 and MGC803 cells. By comparing the activity of 5n and 5c on
PC3 cells, the methyl group at the R3 position was not conducive to
increase activity. Similarly, 5o (IC50 ¼ 0.72 0.14
mM) and 5j
(IC50 ¼ 0.14 0.02 M), 5p and 5c also displayed this phenomenon
m
toward PC3 cells. The activity of 5r-v almost disappeared toward
three tumor cells after replacing the pyridyl at the R2 position with
phenyl, suggesting that pyridine ring, present in majority of our
compounds, similar to 3-AP, may improve the metal chelation
property to further increase their toxicity, which was in accordance
to the literature [5,7]. The selectivity index of all compounds be-
tween PC3 and MGC803 or EC109 cell lines was also calculated and
the results indicated most derivatives could selectively inhibit PC3
cell lines as shown Table 2 [35]. What’s more, the compounds with
selectivity were also evaluated against PC3 cell lines for 24 h and
48 h in Table 3.
d
7.25 ppm and the hydrogen of ¼ NeNH at
d 11.71 ppm, which
existed only in the E isomer because of the appropriate intra-
molecular HeH distance (See supplementary material). These re-
sults indicated that the E isomer was generated, which was
consistent with NOESY spectrum of E-thiosemicarbazone displayed
in the literature [28,31,32].
2.2. Evaluation of biological activity
2.2.1. Anti-proliferative activity
Vandresen et al., reported that TSC derivatives exhibited
considerable inhibitory effects on human prostate cancer cells
(PC3), gastric cancer cells (MGC803) and esophageal cancer cells
(EC109) [17,33,34]. Therefore, we evaluated the antiproliferative
activity of synthesized compounds 4a-f and 5a-v toward the three
cell lines by using the MTT assay. 3-AP and DPC were used as the
positive controls. Based on previous work, 4a-c were firstly syn-
thesized. As Table 1 showed that the length of the carbon chain at
the R1 position had a weak effect on the activity against PC3 cells.
However, the compounds displayed better selectivity for PC3
comparing to MGC803 cells with the carbon chain extended, the
selectivity index 1 (SI1) of 4a, 4b and 4c were 5.69, 9.42 and 16.6
respectively [35]. The antiproliferation activity of compounds 4d-f
towards PC3 cells suggested that when R1 position was a hydro-
philic heterocycle, the compounds displayed weak inhibitory ac-
tivity against the three cells screened. To our delight, 4e
Based on these data, we try to explore the efficacy of all com-
pounds in human normal prostate cell (WPMY-1) and human
gastric mucosal epithelial cells (GES-1). The results were shown in
Table 1. Surprisingly, most of derivatives displayed obvious selec-
tivity between cancer cells and normal cells. Especially, 5j sug-
gested more selectivity in PC3 cells toward two normal cell lines,
compared to those of 3-AP and DPC as shown in Table 4. What’s
more, 5j with an IC50 value of 0.14 mM against PC3 cells displayed
about 4-fold more potent than 3-AP (IC50 ¼ 0.56 0.03
m
of
M) and
equivalent
inhibitory
activity
with
that
DPC
(IC50 ¼ 0.12 0.02
mM). The compound 5j also showed significant
advantages in terms of activity and selectivity compared to
cisplatin. This series of TSC derivatives containing indole scaffold
may have great reference value for the future development of
anticancer drugs. Fig. 3 shows the summary illustration for
structure-activity relationship (SAR) study of target derivatives.
(IC50 ¼ 0.60 0.22
mM) with the indole fragment had more mark-
edly antiproliferative effect, comparing to 4c (IC50¼0.98 0.10
mM)
2.2.2. Clone assay
toward PC3 cells.
As shown in Fig. 4A, compound 5j, with excellent anti-
proliferative activity and selectivity, inhibited PC3 cells prolifera-
tion in a time- and concentration-dependent manner. Favorable
anti-proliferative activity of compound 5j against PC3 cells led us to
further explore the effect on cell colony formation. Colony forma-
tion assay represents the ability of cancer cells to grow and form
foci [36]. The results displayed that 5j was able to inhibit the colony
formation of PC3 cells dose-dependently from Fig. 4B and C. What’s
more, as the concentration of 5j increased, the colonies by the
compound-added group became smaller and fewer, compared with
the control group. Evidently, colony formation was almost
completely suppressed at 200 nM.
Compounds 5a-v were synthesized to improve antiproliferative
activity and selectivity based on 4e’s indole group. As shown in
Table 1, the antitumor activity of compounds 5a and 5b bearing a
methyl group and methoxy group respectively in indole ring
showed no obvious difference with their parent compound 4e to-
ward PC3 cell lines. What’s more, compounds with an electron-
withdrawing substitute, namely, 5c (IC50 ¼ 0.28 0.04
mM) or 5d
(IC50 ¼ 0.35 0.02 M), suggested better potency than 4e with an
m
electron-donating substitute. These results suggested that
electron-withdrawing groups at the 5-position of the indole may be
more favorable for inhibitory activity than electron-donating
groups. In addition, the results exhibited after methyl was added
at the 2-position of the indole like 5e, the activity was inferior than
4e toward PC3 cells. To investigate the effect of R2 position on ac-
tivity, 5f-j were synthesized by introducing different substitutes at
the 4-position of pyridine ring. When the C-4 of pyridine ring was
methyl, the activity had been slightly improved, such as 5c
2.2.3. Cell apoptosis assay
Apoptosis as programmed cell death can control most cell death
in biological processes [37]. To investigate the ability of compound
5j in apoptosis, Hoechst 33342 staining was performed using PC3
cells. After 48 h treatment with compound 5j, the typical changes in

4
Z. He et al. / European Journal of Medicinal Chemistry 184 (2019) 111764
Table 1
The antiproliferative activity of compounds 4a-f and 5a-v against the tested cancer cell lines.
Compd.
R1
R2
R3
IC50
PC3
(m
M)a
MGC803
EC109
WPMY-1
GES-1
4a
4b
4c
4d
4e
H
H
H
H
H
1.75 0.24
0.97 0.01
0.98 0.10
19.53 1.29
0.60 0.22
9.96 0.99
3.02 0.48
0.67 0.18
0.32 0.05
9.14 0.96
16.28 1.21
22.31 1.35
7.78 0.89
7.15 0.85
5.57 0.74
24.26 1.38
4.47 0.65
2.87 0.46
1.17 0.72
9.43 0.87
7.47 0.87
3.12 0.49
0.82 0.08
13.20 1.12
9.88 0.99
4f
H
H
8.66 0.94
0.93 0.03
12.24 1.01
2.81 0.44
15.73 1.19
12.21 1.09
7.61 0.75
4.84 0.26
9.24 0.97
5.21 0.72
5a
5b
5c
5d
5e
H
H
H
H
0.56 0.03
0.28 0.04
0.35 0.02
1.07 0.03
3.74 0.57
0.54 0.28
1.49 0.17
4.20 0.62
3.10 0.49
2.56 0.41
5.04 0.70
3.51 0.54
2.23 0.35
2.16 0.26
0.52 0.29
3.99 0.42
3.32 0.52
2.38 0.37
2.05 0.31
4.81 0.68
5f
H
H
0.34 0.25
0.67 0.18
0.89 0.05
7.08 0.85
6.44 0.81
9.32 0.97
1.17 0.07
4.75 0.44
3.47 0.54
4.11 0.61
5g
5h
5i
H
H
H
0.52 0.28
0.51 0.03
0.14 0.02
6.86 0.84
2.23 0.35
2.94 0.47
10.64 1.03
8.23 0.91
3.99 0.60
2.17 0.07
2.13 0.33
9.85 0.26
3.75 0.57
1.58 0.20
9.23 0.96
5j
5k
5l
H
H
H
0.16 0.02
0.19 0.02
1.09 0.03
2.24 0.35
0.33 0.03
10.77 1.03
0.97 0.01
4.14 0.62
>20
1.01 0.04
1.19 0.07
9.52 0.60
1.62 0.21
1.99 0.30
8.53 0.81
5m
5n
-CH3
0.40 0.03
2.82 0.45
2.57 0.41
2.06 0.31
1.88 0.27

Z. He et al. / European Journal of Medicinal Chemistry 184 (2019) 111764
5
Table 1 (continued )
Compd.
R1
R2
R3
IC50
PC3
(m
M)a
MGC803
EC109
WPMY-1
GES-1
5o
-CH3
0.72 0.14
2.48 0.39
5.52 0.74
1.06 0.02
2.21 0.35
5p
5q
-CH3
-CH3
0.71 0.15
1.53 0.18
>20
>20
2.69 0.43
2.51 0.39
3.52 0.55
0.51 0.02
15.94 1.20
9.36 0.97
5r
5s
5t
H
H
H
19.33 1.23
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
5u
5v
-CH3
-CH3
e
18.87 1.27
>20
>20
>20
>20
11.66 1.06
>20
>20
>20
>20
Cisplatin
e
e
5.44 0.21
1.82 0.21
4.21 0.23
17.25 0.09
19.02 0.07
a
Inhibitory activity was assayed by exposure various concentrations of the tested compounds for 72 h to substance and expressed as concentration required to inhibit
tumor cell proliferation by 50% (IC50). Data are presented as the means SDs. All experiments were carried out at least three independent times.
apoptotic morphology were observed, including chromatin
shrinkage, cell rounding and formation of apoptotic bodies (Fig. 5A)
[38]. In addition, Annexin V-FITC/PI double staining flow cytometry
method was also used. We found that compound 5j can induce PC3
cell apoptosis dose-dependently (Fig. 5B). Apoptosis induction is
mediated via the intrinsic pathway with the elevation of total
reactive oxygen species [39]. In order to further explore the po-
tential mechanism, we examined the intracellular ROS, using the
reactive oxygen assay. The results indicated the compound 5j can
induce apoptosis, which was not affected by reactive oxygen
mechanism (supporting information).
concentration-dependently.
In addition, the further transwell assay (Fig. 6B) demonstrated
that compound 5j could inhibit the migration ability of PC3 cells in
a dose-dependent manner. The expression of mesenchymal cells’
biomarkers N-Cadherin and epithelial cells’ biomarkers E-Cadherin
and
Blot analysis (Fig. 6C) suggested that 5j up-regulated the expression
of E-Cadherin, while N-Cadherin and -catenin were down-
regulated. These results indicated that the compound 5j may
b-catenin are important indicator of cell migration. Western
b
inhibit migration of PC3 cells in
manner by blocking the EMT process.
a concentration-dependent
The expression of apoptosis-related proteins was also detected.
As shown in Fig. 5C, the expression of pro-apoptotic proteins Bax
was significantly increased, while anti-apoptotic protein Bcl-2 was
down-regulated. What’s more, we evaluated the effect of com-
pound 5j on the activity of caspase 3/9 and PARP. The compound 5j
had down-regulated the pro-caspase9 and PARP, meanwhile the
expression of cleaved caspase 9/3 and cleaved PARP were signifi-
2.2.5. Cell cycle analysis
Cell cycle arrest is a marker closely related to cell proliferation
inhibition [41]. To better explain the antiproliferative activity of
compound 5j against PC3 cells, cell-cycle analysis was carried out
by flow cytometry (Fig. 7). After treatment of PC3 cells with 5j for
48 h at indicated concentrations (0, 100, 200, 400 nM), we found
that 5j had almost no effect on cell cycle (Fig. 7A), which was
significantly different from that of the TSC compound TS-1 reported
previously, what led to S-phase arrest pattern toward MGC-
803 cells in a dose-dependent manner [41]. To explore the impact
of time on PC3 cell cycle, the time of 5j and PC3 cells co-incubation
was extended to 72 h. Unfortunately, PC3 cell cycle was not affected
by 5j (Fig. 7B). The effect of compounds 5j and TS-1 on the cell cycle
of MGC803 and PC3 cells was also evaluated respectively. The re-
sults showed that 5j didn’t arrest the cell cycle of MGC803 cells and
TS-1 caused an obvious S arrest in a dose-dependent manner with
concomitant decrease in terms of the number of cells in other
phases of PC3 cell cycle (Fig. 7C and D). We speculated that struc-
tural difference in drugs may lead to changes in biological function.
Jamerson et al., reported a series of thiosemicarbazone derivatives
containing indole but no pyridyl groups, of which the representa-
tive compound 2b could effectively induce G2/M arrest [28]. In
cantly
increased
concentration-dependently.
Furthermore,
expression level of P53 was not significantly changed after adding
different concentrations of compound 5j. These results collectively
indicated the compound 5j could induce PC3 cell apoptosis at least
partially via the mitochondrial death pathway.
2.2.4. Cell migration assay
The tumor cell invasion and metastasis are undoubtedly the
great challenge for the treatment of tumors. Recently, publication
has showed that abnormal epithelial-mesenchymal transition
(EMT) activation is closely related to the invasion of prostate cancer
cells [40]. In consideration of the outstanding anti-proliferative
activity of 5j against PC3 cells, we investigated whether com-
pound 5j can effectively inhibit the migration and invasion of PC3
cells. As shown in Fig. 6A, treatment of PC3 cells with 5j at indicated
concentrations significantly suppressed the wound healing

6
Z. He et al. / European Journal of Medicinal Chemistry 184 (2019) 111764
Table 2
Table 2 (continued)
The selectivity index of all compounds between PC3 and MGC803 or EC109 cell lines.
Compd.
R1
R2
R3
Selectivity index
a
b
SI1
SI2
5o
-CH3
3.44
7.67
R1
R2
R3
Selectivity index
5p
5q
-CH3
-CH3
28.17
10.42
28.17
6.12
Compd.
a
b
SI1
SI2
4a
4b
4c
4d
4e
H
H
H
H
H
5.69
1.73
7.37
5.68
1.24
7.45
9.42
5r
5s
5t
H
H
H
1.03
1.00
1.00
1.03
1.00
1.00
16.60
1.14
12.97
5u
5v
-CH3
-CH3
1.06
1.00
1.06
1.00
4f
H
H
1.41
3.02
1.82
5a
13.13
a
The selectivity index (SI1) was calculated as IC50 (MGC803)/IC50 (PC3).
The selectivity index (SI2) was calculated as IC50 (EC109)/IC50 (PC3).
5b
5c
5d
5e
H
H
H
H
6.68
1.93
4.26
3.93
5.54
9.14
14.40
3.28
b
Table 3
Inhibitory activity of selective compounds against PC3 cell lines for 24, 48 and 72 h.
Compd.
IC50 (m
M)a
24 h
48 h
72 h
5f
5h
5l
5m
5p
>20
>20
7.51 0.87
15.23 1.18
>20
4.21 0.62
6.01 0.78
1.78 0.25
5.15 0.71
6.48 0.81
0.34 0.25
0.52 0.28
0.19 0.02
1.09 0.03
0.71 0.15
5f
5g
5h
5i
H
H
H
H
H
2.62
18.94
13.91
20.46
16.14
28.50
a
Inhibitory activity was assayed by exposure various concentrations of the tested
compounds for 24 h, 48 h and 72 h to substance and expressed as concentration
required to inhibit tumor cell proliferation by 50% (IC50). Data are presented as the
means SDs. All experiments were carried out at least three independent times.
10.57
13.19
4.37
addition, 3-AP, consist only of thiosemicarbazone and pyridine,
resulted in an extensive G1/S-phase cell cycle arrest. Our thio-
semicarbazone derivative 5j contains both hydrazine and pyridyl
group, which may cause 5j to lose the regulatory function of the cell
cycle. The detail mechanism needs to be further examined in our
future study.
5j
21.00
5k
5l
H
H
H
14.00
1.74
9.88
6.06
3. Discussion and conclusions
In the current investigation, a series of novel thiosemicarbazone
derivatives containing indole fragment were designed and syn-
thesized. Most of the compounds displayed potent antiproliferative
activity against PC3, MGC803 and EC109 cell lines. The SAR study
showed that both the electron-withdrawing group at the 5-
position of the indole and electron-donating substitute at the 4-
position of the pyridine ring were critical for antiproliferative ac-
tivity and selectivity. The representative compound 5j exhibited
highly potent antiproliferative activity toward the tested cancer cell
lines and less toxicity against the normal WPMY-1 and GES-1 cell
lines than 3-AP, cisplatin and DPC.
21.79
18.35
5m
5n
-CH3
7.05
6.43

Z. He et al. / European Journal of Medicinal Chemistry 184 (2019) 111764
7
Table 4
Inhibitory activity of compounds 5j, 3-AP and DPC against PC3 and two normal cell lines.
Comp.
PC3
IC50
WPMY-1
IC50
M)a
GES-1
IC50
(m
M)a
(
m
Fold selectivity
(
m
M)a
Fold selectivity
5j
3-AP
DPC
0.14 0.02
0.56 0.03
0.12 0.02
5.44 0.21
9.85 0.26
4.96 0.02
0.71 0.15
17.25 0.09
70.4
8.9
5.9
12.29 1.09
5.60 0.40
0.44 0.03
19.02 0.07
87.8
10
3.7
3.5
Cisplatin
3.2
a
Inhibitory activity was assayed by exposure various concentrations of the tested compounds for 72 h to substance and expressed as concentration required to inhibit
tumor cell proliferation by 50% (IC50). Data are presented as the means SDs. All experiments were carried out at least three independent times.
Fig. 3. The summary for SAR studies of the indicated compounds in Scheme 1.
Fig. 4. (A) PC3 cells were treated with compound 5j at the indicated concentrations for 24 h, 48 h and 72 h. Cell viability was determined by MTT. (B) The colony formation of PC3
cells after treated with compound 5j for 7 days. (C) The rate of inhibition was quantified by measuring the absorbance of 570 nm. The dates were represented as the Mean SD. All
experiments were performed at least three times. *p < 0.5, **p < 0.01, ***p < 0.001 compared with the control.
Compound 5j was chose for further exploring the mechanisms
underlying antiproliferative activity. Colony formation assay
showed that 5j can inhibited colony formation concentration-
dependently. What’s more, we certified that compound 5j could
induced apoptosis by Hoechst 33342 staining and Annexin V-FITC/
PI dual staining assays. Extrinsic pathway and intrinsic pathway as
the core pathways involve in inducing apoptosis. Extrinsic pathway
mentions DR-mediated pathway and the intrinsic pathway is a

8
Z. He et al. / European Journal of Medicinal Chemistry 184 (2019) 111764
Fig. 5. Compound 5j induced PC3 cell apoptosis and possible mechanisms. (A) PC3 cells were treated with compound 5j for 48 h and then stained with Hoechst 33342 to reflect
changes in cell morphology. (B) Apoptotic PC3 cells were analyzed with Annexin V-FITC/PI double staining. Quantitative analysis of apoptosis rates by flow cyometry. (C) Expression
of apoptosis-related proteins including Pro-caspase9, PARP, Cleaved caspase9/3, Cleaved PARP, Bax, Bcl-2 and P53. The GAPDH was used as a control. The dates were represented as
the Mean SD. All experiments were performed at least three times. *p < 0.5, **p < 0.01, ***p < 0.001 compared with the control.
mitochondrial-mediated pathway [42]. Western blot analysis
indicated that treatment of PC3 cells with 5j caused an increase of
cleaved PARP and cleaved caspases 3 and 9, suggesting that 5j
might induce apoptosis at least partially via the mitochondrial
death pathway. In addition, apoptosis is mediated mainly via the
intrinsic pathway with the elevation of total reactive oxygen spe-
cies [39], but the level of total ROS wasn’t affected by compound 5j.
The molecular mechanism how compound 5j targets and induces
apoptosis warrants to be further investigated.
Micromass Q-T of Micromass spectrometer by electrospray ioni-
zation (ESI). The purity of the compound 5j was determined by
reverse-phase high-performance liquid chromatography (HPLC)
analysis. The signal was monitored at 287 nm with a UV detector. A
flow rate of 1.0 mL/min was used with a mobile phase of MeOH in
H2O (90:10, v/v).
4.2. General procedure for the synthesis of compounds(2a-k)
Collectively, 5j can markedly inhibit colony formation, induce
apoptosis and suppress migration and invasion. Our findings sug-
gest 5j with structure of thiosemicarbazone containing indole may
serve as a useful anticancer lead for further optimization and
development.
One pot method was adopted for synthesis of 2a-k. Firstly, in a
50 ml round-bottom flask, appropriate primary amine 1a-k
(3.125 mmol), triethylamine (6.25 mmol) and CS2 (6.25 mmol) were
dissolved in ethanol (15 mL). The reaction mixture was stirred for
3 h at room temperature. Upon completion of the reaction, di-tert-
butyl decarbonate (8.5 mmol), dissolved in absolute ethanol, was
added followed by the immediate addition of a catalytic amount of
4-dimethylaminopyridine (0.425 mmol). After the reaction mixture
was kept for further 30 min at room temperature, the solvent was
distilled off under reduced pressure, and the residue was purified
by column chromatography (petroleum ether/ethyl acetate ¼ 4:1)
to afford the corresponding 2a-k in 48e66% yield.
4. Experimental section
4.1. General
Reagents and solvents were purchased from commercial sour-
ces and used without further purification. Thin-layer chromatog-
raphy (TLC) was carried out on the glass pane that painted with
silica gel, visualized by 254 nm ultraviolet lamp. Melting points
were determined on a WRS-1A digital melting point apparatus and
are uncorrected. 1H NMR and 13C NMR spectra were recorded on a
Bruker 400 MHz and 100 MHz spectrometer respectively. High
resolution mass spectra (HRMS) were recorded on a Waters
4.3. General procedure for the synthesis of compounds(3a-k)
To a solution of 2a-k (10 mmol) in dichloromethane (25 mL) was
injected hydrazine hydrate (30 mmol) portion-wise. After the
mixture was stirred at room temperature for 2 h, the formed

Z. He et al. / European Journal of Medicinal Chemistry 184 (2019) 111764
9
Fig. 6. Compound 5j suppressed the migration of PC3 cells. (A) Wound healing assay. (B) PC3 cells were treated with compound 5j at the indicated concentrations for 48 h, then
used transwell assay to reflect the anti-migration ability of compound 5j. Quantitative analysis of cell numbers. (C) Expression of N-Cadherin, E-Cadherin and -catenin were
b
quantified after treatment of compound 5j for 48 h. The dates were represented as the Mean SD. All experiments were performed at least three times. *p < 0.5, **p < 0.01,
***p < 0.001 compared with the control.
precipitation was filtered, and the left residue was washed with
dichloromethane to give compounds 3a-k as solid in 65e85% yield.
4.3.4. N-(2-morpholinoethyl) hydrazinecarbothioamide(3d)
White solid, yield 78%. 1H NMR (400 MHz, DMSO‑d6,
d
, ppm)
d
8.63 (s, 1H, eNHe), 7.83 (s, 1H, eNHe), 4.45 (s, 2H, eNH2), 3.56
(dd, J ¼ 8.6, 4.5 Hz, 6H, eCH2-), 2.44 (t, J ¼ 6.5 Hz, 2H, eCH2-), 2.39
(s, 4H, eCH2-). 13C NMR (100 MHz, DMSO‑d6,
d, ppm) d 181.05,
4.3.1. N-Phenylhydrazinecarbothioamide(3a)
White solid, yield 80%. 1H NMR (400 MHz, DMSO‑d6,
d
, ppm)
9.69 (s, 1H, eNHe), 9.12 (s, 1H, eNHe), 7.65 (d, J ¼ 6.2 Hz, 2H,
AreH), 7.29 (t, J ¼ 7.9 Hz, 2H, AreH), 7.09 (t, J ¼ 7.3 Hz, 1H, AreH),
4.79 (s, 2H, eNH2). 13C NMR (100 MHz, DMSO‑d6,
, ppm) 179.32,
66.19, 56.99, 53.18. HR-MS (ESI), calcd. C7H16N4OS, [MþH]þm/z:
d
205.1123. found: 205.1122.
d
d
4.3.5. N-(2-(1H-indol-3-yl) ethyl) hydrazinecarbothioamide(3e)
139.25, 128.00, 124.01, 123.45. HR-MS (ESI), calcd. C7H9N3S,
White solid, yield 74%. 1H NMR (400 MHz, DMSO‑d6,
d
, ppm)
[MþNa]þm/z: 190.0415. found: 190.0416.
d
10.81 (s, 1H, eNHe), 8.62 (s, 1H, eNHe), 7.94 (s, 1H, eNHe), 7.68
(d, J ¼ 7.8 Hz, 1H, AreH), 7.34 (d, J ¼ 8.1 Hz, 1H, AreH), 7.17 (d,
J ¼ 1.9 Hz, 1H, AreH), 7.07 (t, J ¼ 7.2 Hz, 1H, AreH), 6.98 (t, J ¼ 7.4 Hz,
1H, AreH), 4.43 (s, 2H, eNH2), 3.75 (dd, J ¼ 13.9, 6.8 Hz, 2H, eCH2-),
4.3.2. N-benzylhydrazinecarbothioamide(3b)
White solid, yield 84%. 1H NMR (400 MHz, DMSO‑d6,
d, ppm)
2.93 (t, J ¼ 7.5 Hz, 2H, eCH2-). 13C NMR (100 MHz, DMSO‑d6,
d, ppm)
d
8.73 (s, 1H, eNHe), 8.28 (s, 1H, eNHe), 7.44e7.10 (m, 5H, AreH),
d
181.57, 136.77, 127.73, 123.06, 121.43, 119.07, 118.68, 112.23, 111.80,
4.72 (d, J ¼ 6.1 Hz, 2H, eNH2), 4.51 (s, 2H, eCH2-). 13C NMR
44.06, 25.77. HR-MS (ESI), calcd.
257.0837. found: 257.0836.
C
11H14N4S, [MþNa] þm/z:
(100 MHz, DMSO‑d6, d, ppm) d 181.55, 139.78, 128.06, 127.31,126.62,
46.12. HR-MS (ESI), calcd. C8H11N3S, [MþNa]þm/z: 204.0572. found:
204.0573.
4.3.6. tert-butyl 4-(2-(hydrazinecarbothioamido) ethyl) piperazine-
1-carboxylate(3f)
4.3.3. N-phenethylhydrazinecarbothioamide(3c)
White solid, yield 74%. 1H NMR (400 MHz, DMSO‑d6,
d 8.63 (s, 1H, eNHe), 7.83 (s, 1H, eNHe), 4.44 (s, 2H, eNH2), 3.55
d
, ppm)
White solid, yield 85%. 1H NMR (400 MHz, DMSO‑d6,
d
, ppm)
8.64 (s, 1H, eNHe), 7.87 (s, 1H, eNHe), 7.41e7.13 (m, 5H, AreH),
4.43 (s, 2H, eNH2), 3.67 (dd, J ¼ 14.7, 6.2 Hz, 2H, eCH2-), 2.92e2.75
(m, 2H, eCH2-). 13C NMR (100 MHz, DMSO‑d6,
, ppm) 181.13,
d
(dd, J ¼ 12.0, 6.1 Hz, 2H, eCH2-), 3.30 (s, 2H, eCH2-), 2.46 (t,
J ¼ 6.5 Hz, 2H, eCH2-), 2.41e2.29 (m, 4H, eCH2-), 1.39 (s, 9H, eCH3).
d
d
13C NMR (100 MHz, DMSO‑d6,
d, ppm) d 181.14, 153.79, 78.71, 56.51,
139.40, 128.56, 128.35, 126.06, 44.37, 35.17. HR-MS (ESI), calcd.
52.39, 28.03. HR-MS (ESI), calcd.
304.1807. found: 304.1807.
C
12H25N5O2S, [MþH]þm/z:
C9H13N3S, [MþH] þm/z: 196.0908. found: 196.0905.

10
Z. He et al. / European Journal of Medicinal Chemistry 184 (2019) 111764
Fig. 7. Effect of compound 5j and TS-1 on cell cycle. (A) Cell cycle analysis of PC3 cells treatment of compound 5j for 48 h (B) Cell cycle analysis of PC3 cells treatment of compound
5j for 72 h (C) Cell cycle analysis of MGC803 cells treatment of compound 5j for 48 h (D) Cell cycle analysis of PC3 cells treatment of compound TS-1 for 48 h. The dates were
represented as the Mean SD. All experiments were performed at least three times. *p < 0.5, **p < 0.01, ***p < 0.001 compared with the control.
4.3.7. N-(2-(5-methyl-1H-indol-3-yl) ethyl)
hydrazinecarbothioamide(3g)
NMR (100 MHz, DMSO‑d6, d, ppm) d 181.03, 134.64, 127.43, 126.51,
122.64, 122.53, 118.16, 111.16, 111.01, 43.53, 25.25, 21.29. HR-MS
White solid, yield 75%. 1H NMR (400 MHz, DMSO‑d6,
d
, ppm)
(ESI), calcd. C12H16N4S, [MþH] þm/z: 249.1174. found: 249.1174.
d
10.67 (s, 1H, eNHe), 8.62 (s,1H, eNHe), 7.92 (s,1H, AreH), 7.44 (s,
1H, AreH), 7.22 (d, J ¼ 8.2 Hz,1H, eNHe), 7.10 (s, 1H, AreH), 6.89 (d,
J ¼ 8.2 Hz, 1H, AreH), 4.43 (s, 2H, eNH2), 3.72 (dd, J ¼ 13.8, 6.7 Hz,
2H, eCH2-), 2.89 (t, J ¼ 7.5 Hz, 2H, eCH2-), 2.38 (s, 3H, eCH3). 13C
4.3.8. N-(2-(5-methoxy-1H-indol-3-yl) ethyl)
hydrazinecarbothioamide(3h)
White solid, yield 72%. 1H NMR (400 MHz, DMSO‑d6,
d, ppm)

Z. He et al. / European Journal of Medicinal Chemistry 184 (2019) 111764
11
d
10.65 (s,1H, eNHe), 8.65 (s,1H, eNHe), 7.98 (s, 1H, AreH), 7.24 (s,
DMSO‑d6,
d
, ppm)
d
11.82 (s, 1H, eNHe), 9.25 (t, J ¼ 6.1 Hz, 1H,
1H, AreH), 7.21 (d, J ¼ 8.8 Hz, 1H, eNHe), 7.11 (s, 1H, AreH), 6.71
(dd, J ¼ 8.7, 2.1 Hz, 1H, AreH), 4.44 (s, 2H, eNH2), 3.76 (s, 3H, eCH3),
3.70 (d, J ¼ 6.6 Hz, 2H, eCH2-), 2.88 (t, J ¼ 7.6 Hz, 2H, eCH2-). 13C
eNHe), 8.57 (d, J ¼ 4.3 Hz, 1H, AreH), 8.29 (d, J ¼ 8.0 Hz, 1H, AreH),
8.14 (s, 1H, eCH]N-), 7.87e7.79 (m, 1H, eNHe), 7.42e7.17 (m, 6H,
AreH), 4.85 (t, J ¼ 9.4 Hz, 2H, eCH2-). 13C NMR (100 MHz, DMSO‑d6,
NMR (100 MHz, DMSO‑d6,
d
, ppm)
d
181.05, 152.89, 131.37, 127.57,
d, ppm) d 177.86, 153.29, 149.32, 142.48, 139.24, 136.41, 128.15,
123.18, 111.90, 111.45, 111.09, 100.49, 55.30, 43.44, 25.41. HR-MS
127.18, 126.72, 124.06, 120.20, 46.62. HR-MS (ESI), calcd. C14H14N4S,
(ESI), calcd. C12H16N4OS, [MþH] þm/z: 265.1123. found: 265.1125.
[MþH]þm/z: 271.1017. found: 271.1017.
4.3.9. N-(2-(5-chloro-1H-indol-3-yl) ethyl)
4.4.3. (E)-N-phenethyl-2-(pyridin-2-ylmethylene) hydrazine-1-
carbothioamide(4c)
hydrazinecarbothioamide(3i)
White solid, yield 69%. 1H NMR (400 MHz, DMSO‑d6,
d, ppm)
Yellow solid, m. p. 175.6e177.1 ꢀC, yield 65%. 1H NMR (400 MHz,
d
11.04 (s, 1H, eNHe), 8.66 (s, 1H, eNHe), 8.01 (s, 1H, AreH), 7.76 (s,
DMSO‑d6,
d
, ppm)
d
11.74 (s, 1H, eNHe), 8.73 (t, J ¼ 5.8 Hz, 1H,
1H, AreH), 7.35 (d, J ¼ 8.6 Hz, 1H, eNHe), 7.25 (s, 1H, AreH),
7.15e7.00 (m, 1H, AreH), 4.45 (s, 2H, eNH2), 3.70 (dd, J ¼ 14.0,
6.6 Hz, 2H, eCH2-), 2.90 (t, J ¼ 7.6 Hz, 2H, eCH2-). 13C NMR
eNHe), 8.57 (d, J ¼ 4.3 Hz, 1H, AreH), 8.20 (d, J ¼ 8.0 Hz, 1H, AreH),
8.10 (s, 1H, eCH]N-), 7.86 (td, J ¼ 7.7, 1.4 Hz, 1H, AreH), 7.44e7.15
(m, 6H, AreH), 3.86e3.61 (m, 2H, eCH2-), 3.01e2.85 (m, 2H, eCH2-
(100 MHz, DMSO‑d6,
d, ppm)
d
181.01, 134.69, 128.41, 124.52, 122.92,
). 13C NMR (100 MHz, DMSO‑d6,
d, ppm) d 177.20, 153.28, 149.35,
120.85, 117.93, 112.82, 111.73, 105.18, 43.49, 29.57, 25.14, 0.73. HR-
142.16, 139.15, 136.45, 128.57, 128.44, 126.19, 124.05, 120.02, 45.07,
34.77. HR-MS (ESI), calcd. C15H16N4S, [MþH]þm/z: 285.1174. found:
285.1175.
MS (ESI), calcd.
269.0626.
C
11H13ClN4S, [MþH]þm/z: 269.0628. found:
4.3.10. N-(2-(5-bromo-1H-indol-3-yl) ethyl)
hydrazinecarbothioamide(3j)
4.4.4. (E)-N-(2-morpholinoethyl)-2-(pyridin-2-ylmethylene)
hydrazine-1-carbothioamide(4d)
White solid, yield 71%. 1H NMR (400 MHz, DMSO‑d6,
d
, ppm)
White solid, m. p. 165.3e166.0 ꢀC, yield 45%. 1H NMR (400 MHz,
d
11.05 (s, 1H, eNHe), 8.65 (s, 1H, eNHe), 7.96 (d, J ¼ 38.6 Hz, 2H,
DMSO‑d6, d, ppm) d 11.74 (s, 1H, eNHe), 8.67e8.47 (m, 2H, eNHe,
AreH), 7.49e7.06 (m, 3H, eNHe, 2AreH), 4.44 (s, 2H, eNH2), 3.70
AreH), 8.17 (d, J ¼ 8.0 Hz, 1H, AreH), 8.10 (s, 1H, eCH]N-), 7.86 (td,
J ¼ 7.8, 1.3 Hz, 1H, AreH), 7.42e7.34 (m, 1H, AreH), 3.69 (dd,
J ¼ 12.6, 6.4 Hz, 2H, eCH2-), 3.64e3.51 (m, 4H, eCH2-), 2.55 (t,
J ¼ 6.3 Hz, 2H, eCH2-), 2.45 (s, 4H, eCH2-). 13C NMR (100 MHz,
(dd, J ¼ 13.5, 6.5 Hz, 2H, eCH2-), 2.90 (t, J ¼ 7.5 Hz, 2H, eCH2-). 13
C
NMR (100 MHz, DMSO‑d6, d, ppm) d 181.01, 134.92, 129.13, 124.36,
123.36, 120.97, 113.31, 111.66, 110.91, 43.52, 25.13. HR-MS (ESI),
calcd. C11H13BrN4S, [MþNa]þm/z: 334.9942. found: 334.9944.
DMSO‑d6, d, ppm) d 177.17, 153.23, 149.41, 142.14, 136.49, 124.10,
119.82, 66.26, 56.50, 53.21. HR-MS (ESI), calcd.
C13H19N5OS,
4.3.11. N-(2-(2-methyl-1H-indol-3-yl) ethyl)
hydrazinecarbothioamide(3k)
[MþH]þm/z: 294.1388. found: 294.1387.
White solid, yield 65%. 1H NMR (400 MHz, DMSO‑d6,
d, ppm)
4.4.5. (E)-N-(2-(1H-indol-3-yl)ethyl)-2-(pyridin-2-ylmethylene)
hydrazine-1-carbothioamide(4e)
d
10.72 (s, 1H, eNHe), 8.64 (s, 1H, eNHe), 7.92 (s, 1H, AreH), 7.56
(d, J ¼ 7.6 Hz, 1H, AreH), 7.22 (d, J ¼ 7.8 Hz, 1H, eNHe), 6.94 (dt,
J ¼ 14.5, 7.0 Hz, 2H, AreH), 4.43 (s, 2H, eNH2), 3.58 (dd, J ¼ 14.3,
6.5 Hz, 2H, eCH2-), 2.94e2.75 (m, 2H, eCH2-), 2.35 (s, 3H, eCH3).
Yellow solid, m. p. 191.0e193.2 ꢀC, yield 68%. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d 11.73 (s, 1H, eNHe), 10.86 (s, 1H, eNHe), 8.74 (t,
J ¼ 5.5 Hz, 1H, eNHe), 8.57 (s, J ¼ 4.4 Hz, 1H, AreH), 8.14 (d,
J ¼ 8.0 Hz, 1H, AreH), 8.11 (s, 1H, AreH), 7.87 (d, J ¼ 7.4 Hz, 1H,
AreH), 7.73 (d, J ¼ 7.8 Hz, 1H, AreH), 7.37 (dd, J ¼ 11.9, 7.4 Hz, 2H,
AreH), 7.22 (s, 1H, eCH]N-), 7.08 (t, J ¼ 7.4 Hz, 1H, AreH), 6.99 (t,
J ¼ 7.4 Hz, 1H, AreH), 3.86 (dd, J ¼ 11.4, 6.5 Hz, 2H, eCH2-), 3.04 (d,
13C NMR (100 MHz, DMSO‑d6,
d, ppm) d 181.03, 135.19, 132.14,
128.29, 119.89, 118.01, 117.59, 110.26, 107.39, 43.67, 24.10, 11.20. HR-
MS (ESI), calcd.
271.0996.
C
12H16N4S, [MþNa]þm/z: 271.0993. found:
J ¼ 7.4 Hz, 2H, eCH2-). 13C NMR (100 MHz, DMSO‑d6,
d, ppm)
4.4. General procedure for the synthesis of compounds(4a-f,5a-v)
d 177.57, 153.75, 149.82, 142.57, 136.97, 127.73, 124.51, 123.13, 121.46,
120.48, 119.03, 118.73, 111.85, 44.89, 25.30. HR-MS (ESI), calcd.
To the solution of 3a-k (0.64 mmol) in ethanol containing acetic
acid (0.064 mmol), appropriate aldehyde or ketone (0.77 mmol)
was added. The mixture was kept at room temperature for 2 h, the
formed precipitation was filtered, and the left residue was washed
with ethanol to give compounds 4a-f and 5a-v as solid in 45e85%
yield.
C
17H17N5S, [MþH]þm/z: 324.1283. found: 324.1284.
4.4.6. tert-butyl(E)-4-(2-(2-(pyridin-2-ylmethylene) hydrazine-1-
carbothioamido) ethyl) piperazine-1-carboxylate(4f)
Yellow solid, m. p. 155.8e158.2 ꢀC, yield 55%. 1H NMR (400 MHz,
DMSO‑d6, d, ppm) d 11.73 (s, 1H, eNHe), 8.66e8.51 (m, 2H, eNHe,
AreH), 8.17 (d, J ¼ 8.0 Hz, 1H, AreH), 8.10 (s, 1H, eCH]N-),
7.90e7.78 (m, 1H, AreH), 7.38 (dd, J ¼ 6.9, 5.4 Hz, 1H, AreH), 3.69
(dd, J ¼ 12.8, 6.4 Hz, 2H, eCH2-), 3.32 (s, 4H, eCH2-), 2.57 (d,
J ¼ 6.9 Hz, 2H, eCH2-), 2.46e2.36 (m, 4H, eCH2-),1.40 (s, 9H, eCH3).
4.4.1. (E)-N-phenyl-2-(pyridin-2-ylmethylene) hydrazine-1-
carbothioamide(4a)
White solid, m. p. 198.7e199.8 ꢀC, yield 70%. 1H NMR (400 MHz,
DMSO‑d6,
d, ppm)
d
12.03 (s, 1H, eNHe), 10.25 (s, 1H, eNHe),
13C NMR (100 MHz, DMSO‑d6,
d, ppm) d 177.19, 153.80, 153.24,
8.64e8.53 (m, 1H, AreH), 8.45 (d, J ¼ 8.0 Hz, 1H, AreH), 8.21 (s, 1H,
eCH]N-), 7.88e7.76 (m, 1H, AreH), 7.56 (d, J ¼ 7.6 Hz, 2H, AreH),
7.45e7.33 (m, 3H, AreH), 7.23 (s, J ¼ 7.4 Hz, 1H, AreH). 13C NMR
149.40, 142.14, 136.45, 124.08, 119.88, 78.72, 56.08, 52.44, 40.67,
28.04. HR-MS (ESI), calcd. C18H28N6O2S, [MþH]þm/z: 393.2072.
found: 393.2071.
(100 MHz, DMSO‑d6, d, ppm) d 176.41, 153.16, 149.32, 143.06, 138.93,
136.45, 128.08, 126.06, 125.51, 124.21, 120.58. HR-MS (ESI), calcd.
C
4.4.7. (E)-N-(2-(5-methyl-1H-indol-3-yl)ethyl)-2-(pyridin-2-
ylmethylene)hydrazine-1-carbothioamide(5a)
13H12N4S, [MþH]þm/z: 257.0861. found: 257.0862.
White solid, m. p. 177.2e179.0 ꢀC, yield 58%. 1H NMR (400 MHz,
4.4.2. (E)-N-benzyl-2-(pyridin-2-ylmethylene) hydrazine-1-
carbothioamide(4b)
DMSO‑d6, ppm) d 11.75 (s, 1H, eNHe), 10.73 (s, 1H, eNHe),
8.81e8.66 (m, 1H, eNHe), 8.57 (s, J ¼ 4.8 Hz, 1H, AreH), 8.13 (s, 1H,
Yellow solid, m. p. 180.6e190.5 ꢀC, yield 66%. 1H NMR (400 MHz,
AreH), 8.11 (s, 1H, AreH), 7.96e7.75 (m, 1H, AreH), 7.46 (d,

12
Z. He et al. / European Journal of Medicinal Chemistry 184 (2019) 111764
J ¼ 13.8 Hz, 1H, AreH), 7.38 (dd, J ¼ 6.8, 5.4 Hz, 1H, AreH), 7.23 (s,
J ¼ 7.5 Hz, 1H, eCH]N-), 7.15 (dd, J ¼ 17.2, 6.3 Hz, 1H, AreH), 6.90
(d, J ¼ 8.2 Hz, 1H, AreH), 3.82 (dt, J ¼ 37.6, 18.8 Hz, 2H, eCH2-),
3.11e2.82 (m, 2H, eCH2-), 2.36 (s, 3H, eCH3). 13C NMR (100 MHz,
4.4.12. (E)-N-(2-(1H-indol-3-yl)ethyl)-2-((5-methoxypyridin-2-yl)
methylene)hydrazine-1-carbothioamide(5f)
Yellow solid, m. p. 226.5e228.2 ꢀC, yield 67%. 1H NMR (400 MHz,
DMSO‑d6, ppm) d 11.61 (s, 1H, eNHe), 11.07 (s, 1H, eNHe), 8.67 (dd,
DMSO‑d6,
d
, ppm)
d
177.06, 153.27, 149.33, 142.06, 136.47, 134.66,
J ¼ 15.1, 9.3 Hz, 1H, NH-), 8.29 (s, J ¼ 2.8 Hz, 1H, AreH), 8.10 (d,
J ¼ 8.9 Hz, 1H, AreH), 8.09 (s, 1H, AreH), 7.78 (d, J ¼ 1.8 Hz, 1H,
AreH), 7.47 (dd, J ¼ 8.8, 2.8 Hz, 1H, AreH), 7.37 (d, J ¼ 8.6 Hz, 1H,
eCH]N-), 7.31 (d, J ¼ 2.0 Hz, 1H, AreH), 7.07 (dd, J ¼ 8.6, 1.9 Hz, 1H,
AreH), 4.00e3.87 (m, 3H, eCH3), 3.79 (dt, J ¼ 38.6, 19.3 Hz, 2H,
eCH2-), 2.99 (dd, J ¼ 25.9, 18.4 Hz, 2H, eCH2-). 13C NMR (100 MHz,
127.48, 126.60, 124.02, 122.75, 122.59, 119.97, 118.12, 111.06, 111.00,
44.42, 24.78, 21.28. HR-MS (ESI), calcd. C18H19N5S, [MþH]þm/z:
338.1439. found: 338.1441.
4.4.8. (E)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-2-(pyridin-2-
ylmethylene)hydrazine-1-carbothioamide(5b)
DMSO‑d6, d, ppm) d 176.91, 155.84, 145.82, 142.14, 136.56, 134.72,
White solid, m. p. 118.2e120.4 ꢀC, yield 68%. 1H NMR (400 MHz,
128.45, 124.63, 123.02, 121.33, 120.94, 120.91, 117.87, 112.89, 111.58,
55.79, 44.27, 24.70. HR-MS (ESI), calcd. C18H19N5OS, [MþH]þm/z:
388.09. found:388.09.
DMSO‑d6, ppm)
d 11.75 (s, 1H, eNHe), 10.70 (s, 1H, eNHe), 8.75 (t,
J ¼ 5.7 Hz, 1H, eNHe), 8.59 (s, J ¼ 13.5 Hz, 1H, AreH), 8.14 (d,
J ¼ 8.0 Hz, 1H, AreH), 8.11 (s, 1H, AreH), 7.92e7.77 (m, 1H, AreH),
7.39 (dd, J ¼ 11.2, 5.9 Hz, 1H, AreH), 7.26 (dd, J ¼ 8.3, 5.4 Hz, 2H,
AreH), 7.16 (s, J ¼ 12.2 Hz,1H, eCH]N-), 6.73 (dd, J ¼ 8.7, 2.1 Hz,1H,
AreH), 3.85 (dd, J ¼ 14.4, 6.5 Hz, 2H, eCH2-), 3.76 (s, 3H, eCH3),
4.4.13. (E)-N-(2-(1H-indol-3-yl)ethyl)-2-((5-hydroxypyridin-2-yl)
methylene)hydrazine-1-carbothioamide(5g)
Yellow solid, m. p. 215.2e218.5 ꢀC, yield 78%. 1H NMR (400 MHz,
3.09e2.89 (m, 2H, eCH2-). 13C NMR (100 MHz, DMSO‑d6,
d, ppm)
DMSO‑d6, ppm)
d
11.56 (s, 1H, eNHe), 11.06 (d, J ¼ 14.7 Hz, 1H,
eNHe), 10.35 (s, 1H, eOH), 8.60 (t, J ¼ 5.7 Hz, 1H, eNHe), 8.11 (t,
J ¼ 8.9 Hz, 1H, AreH), 8.05 (s, 1H, AreH), 7.98 (d, J ¼ 8.7 Hz, 1H,
AreH), 7.77 (t, J ¼ 4.6 Hz, 1H, AreH), 7.37 (d, J ¼ 8.6 Hz, 1H, AreH),
7.30 (s, J ¼ 5.9 Hz, 1H, eCH]N-), 7.23 (dd, J ¼ 8.7, 2.6 Hz, 1H, AreH),
7.07 (dd, J ¼ 8.6, 1.9 Hz, 1H, AreH), 3.79 (dt, J ¼ 32.0, 15.9 Hz, 2H,
d
177.09, 153.26, 152.96, 149.33, 142.09, 136.48, 131.39, 127.61,
124.03, 123.29, 119.99, 111.98, 111.26, 111.13, 55.28, 44.30, 24.89. HR-
MS(ESI), calcd.
376.1209.
C
18H19N5OS, [MþNa]þm/z: 376.1208. found:
eCH2-), 3.14e2.83 (m, 2H, eCH2-). 13C NMR (100 MHz, DMSO‑d6,
d,
4.4.9. (E)-N-(2-(5-chloro-1H-indol-3-yl)ethyl)-2-(pyridin-2-
ylmethylene)hydrazine-1-carbothioamide(5c)
ppm) d 176.83, 154.39, 144.39, 142.57, 137.32, 134.71, 128.46, 124.64,
123.03, 122.71, 121.13, 120.90, 117.86, 112.88, 111.58, 44.26, 24.69.
White solid, m. p. 183.6e185.2 ꢀC, yield 71%. 1H NMR (400 MHz,
HR-MS (ESI), calcd. C17H17N5OS, [MþH]þm/z: 374.08. found:374.08.
DMSO‑d6, ppm)
d 11.76 (s, 1H, eNHe),11.08 (s, 1H, eNHe), 8.78 (t,
1H, eNHe), 8.56 (s, 1H, AreH), 8.15 (d, J ¼ 7.4 Hz, 1H, AreH), 8.11 (s,
1H, AreH), 7.85 (s, 1H, AreH), 7.77 (s, 1H, AreH), 7.36 (d, J ¼ 8.0 Hz,
2H, AreH), 7.30 (s, 1H, eCH]N-), 7.06 (d, J ¼ 7.6 Hz, 1H, AreH), 3.81
4.4.14. (E)-N-(2-(5-chloro-1H-indol-3-yl)ethyl)-2-((5-
chloropyridin-2-yl)methylene)hydrazine-1-carbothioamide(5h)
White solid, m. p. 236.5e236.6 ꢀC, yield 69%. 1H NMR (400 MHz,
(s, 2H, eCH2-), 3.01 (s, 2H, eCH2-). 13C NMR (100 MHz, DMSO‑d6,
d,
DMSO‑d6, ppm)
d 11.80 (s, 1H, eNHe), 11.07 (s, 1H, eNHe), 8.84 (t,
ppm) d 177.09, 153.28, 149.33, 142.16, 136.46, 134.70, 128.45, 124.65,
J ¼ 5.8 Hz, 1H, eNHe), 8.62 (s, J ¼ 2.3 Hz, 1H, AreH), 8.21 (d,
J ¼ 8.6 Hz, 1H, AreH), 8.07 (s, J ¼ 18.3 Hz, 1H, AreH), 8.01 (dd,
J ¼ 8.6, 2.4 Hz, 1H, AreH), 7.76 (d, J ¼ 1.8 Hz, 1H, AreH), 7.37 (d,
J ¼ 8.6 Hz, 1H, AreH), 7.31 (s, J ¼ 2.1 Hz, 1H, eCH]N-), 7.07 (dd,
J ¼ 8.6, 2.0 Hz, 1H, AreH), 3.82 (dd, J ¼ 14.9, 6.3 Hz, 2H, eCH2-),
124.03, 123.02, 120.90, 120.00, 117.86, 112.89, 111.53, 44.36, 24.63.
HR-MS(ESI), calcd. C17H16ClN5S, [MþNa]þm/z: 380.0713. found:
380.0710.
3.08e2.88 (m, 2H, eCH2-). 13C NMR (100 MHz, DMSO‑d6,
d, ppm)
4.4.10. (E)-N-(2-(5-bromo-1H-indol-3-yl)ethyl)-2-(pyridin-2-
ylmethylene)hydrazine-1-carbothioamide(5d)
d
177.17, 151.95, 147.84, 140.80, 136.38, 134.73, 131.11, 128.46, 124.64,
White solid, m. p. 182.6e184.2 ꢀC, yield 65%. 1H NMR (400 MHz,
123.03, 121.19, 120.90, 117.84, 112.89, 111.54, 44.39, 24.60. HR-MS
(ESI), calcd. C17H15Cl2N5S, [Mþ Na]þm/z: 414.0323. found: 414.0322.
DMSO‑d6, ppm)
d 11.77 (s, 1H, eNHe), 11.10 (s, 1H, eNHe), 8.78 (t,
J ¼ 5.7 Hz, 1H, eNHe), 8.58 (s, J ¼ 4.6 Hz, 1H, AreH), 8.15 (s,
J ¼ 8.0 Hz, 1H, AreH), 8.12 (s, 1H, AreH), 7.92 (s, 1H, AreH), 7.86 (t,
J ¼ 7.7 Hz, 1H, AreH), 7.40 (dd, J ¼ 12.7, 6.8 Hz, 1H, AreH), 7.33 (d,
J ¼ 8.6 Hz, 1H, eNHe), 7.30 (s, 1H, eCH]N-), 7.18 (d, J ¼ 8.6 Hz, 1H,
AreH), 3.82 (dd, J ¼ 14.4, 6.6 Hz, 2H, eCH2-), 3.23e2.80 (m, 2H,
4.4.15. (E)-2-((5-bromopyridin-2-yl)methylene)-N-(2-(5-chloro-
1H-indol-3-yl)ethyl)hydrazine-1-carbothioamide(5i)
White solid, m. p. 232.6e233.5 ꢀC, yield 61%. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d
11.80 (s, 1H, eNHe), 11.04 (d, J ¼ 17.2 Hz, 1H,
eCH2-). 13C NMR (100 MHz, DMSO‑d6,
d, ppm) d 177.09, 153.28,
eNHe), 8.84 (t, J ¼ 5.8 Hz, 1H, eNHe), 8.70 (s, 1H, AreH), 8.23e8.11
(m, 2H, AreH), 8.08 (s, 1H, AreH), 7.76 (d, J ¼ 1.6 Hz, 1H, AreH), 7.36
(t, J ¼ 8.0 Hz, 1H, AreH), 7.29 (t, J ¼ 11.1 Hz, 1H, eCH]N-), 7.07 (dd,
J ¼ 8.6, 1.9 Hz, 1H, AreH), 3.82 (dd, J ¼ 14.7, 6.5 Hz, 2H, eCH2-), 3.00
149.33, 142.17, 136.46, 134.93, 129.17, 124.48, 124.03, 123.42, 120.89,
120.01, 113.37, 111.46, 111.00, 44.37, 24.63. HR-MS (ESI), calcd.
C
17H16BrN5S, [MþNa]þm/z: 424.0207. found: 424.0209.
(dd, J ¼ 25.5, 18.1 Hz, 2H, eCH2-). 13C NMR (100 MHz, DMSO‑d6,
d,
4.4.11. (E)-N-(2-(2-methyl-1H-indol-3-yl)ethyl)-2-(pyridin-2-
ylmethylene)hydrazine-1-carbothioamide(5e)
ppm) d 177.13, 152.17, 150.02, 140.89, 139.13, 134.72, 128.44, 124.65,
123.02, 121.58, 120.90, 120.42, 117.85, 44.37, 24.61. HR-MS (ESI),-
Yellow solid, m. p. 108.2e109.5 ꢀC, yield 80%. 1H NMR (400 MHz,
calcd. C28H28BrClN6OS, [MþNa]þm/z: 457.9818.found: 457.9817.
DMSO‑d6, ppm)
d 11.75 (s, 1H-, eNHe), 10.77 (s, 1H-, eNHe), 8.76
(d, J ¼ 22.3 Hz, 1H, eNHe), 8.58 (s, J ¼ 3.6 Hz, 1H, AreH), 8.13 (d,
J ¼ 8.1 Hz, 1H, AreH), 8.11 (s, 1H, AreH), 7.93e7.76 (m, 1H, AreH),
7.61 (t, J ¼ 13.5 Hz, 1H, AreH), 7.40 (d, J ¼ 5.1 Hz, 1H, eNHe), 7.25 (s,
J ¼ 7.5 Hz, 1H, eCH]N-), 6.96 (dt, J ¼ 20.1, 7.1 Hz, 2H, AreH), 3.71
(d, J ¼ 6.2 Hz, 2H, eCH2-), 3.05e2.87 (m, 3H, eCH2-), 2.39 (s, 3H,
4.4.16. (E)-N-(2-(5-chloro-1H-indol-3-yl)ethyl)-2-((5-
methylpyridin-2-yl)methylene)hydrazine-1-carbothioamide(5j)
White solid, m. p. 225.6e227.1 ꢀC, yield 67%. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d 11.71 (s, 1H, eNHe), 11.09 (s, 1H, eNHe), 8.74 (d,
J ¼ 5.5 Hz,1H, eNHe), 8.41 (s,1H, AreH), 8.08 (s, 1H, AreH), 8.06 (d,
J ¼ 8.2 Hz, 1H, AreH), 7.78 (s, 1H, AreH), 7.68 (d, J ¼ 8.0 Hz, 1H,
AreH), 7.37 (d, J ¼ 8.5 Hz, 1H, AreH), 7.25 (s, 1H, eCH]N-), 7.07 (d,
J ¼ 8.4 Hz, 1H, AreH), 3.81 (d, J ¼ 7.0 Hz, 2H, eCH2-), 3.07e2.84 (m,
eCH3). 13C NMR (100 MHz, DMSO‑d6,
d, ppm) d 177.09, 153.26,
149.35, 142.03, 136.47, 135.20, 132.20, 128.37, 124.04, 119.96, 118.10,
117.53, 110.34, 107.18, 48.57, 44.38, 23.61, 11.23. HR-MS(ESI), calcd.
C
18H19N5S, [MþNa]þm/z: 360.1259. found: 360.1260.
2H, eCH2-), 2.32 (s, 3H, -CH3). 13C NMR (100 MHz, DMSO‑d6,
d,

Z. He et al. / European Journal of Medicinal Chemistry 184 (2019) 111764
13
ppm)
d
176.99, 150.70, 149.53, 142.31, 136.89, 134.70, 133.70, 128.45,
4.4.21. (E)-N-(2-(5-chloro-1H-indol-3-yl)ethyl)-2-(1-(5-
124.63, 123.01, 120.90, 119.53, 117.87, 112.89, 111.55, 44.32, 24.67,
17.90. HR-MS (ESI), calcd. C18H18ClN5S, [MþH]þm/z: 372.1050.
found: 372.1051.
methylpyridin-2-yl)ethylidene)hydrazine-1-carbothioamide(5o)
Yellow solid, m. p. 190.3e191.6 ꢀC, yield 53%. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d 14.22 (s, 1H, eNHe), 11.05 (s, 1H, eNHe), 8.71 (t,
J ¼ 5.8 Hz, 1H, eNHe), 8.64 (d, J ¼ 11.7 Hz, 1H, AreH), 7.96e7.83 (m,
1H, AreH), 7.76 (t, J ¼ 2.8 Hz, 1H, AreH), 7.71e7.66 (m, 1H, AreH),
7.39e7.34 (m, 1H, AreH), 7.29 (d, J ¼ 1.9 Hz, 1H, AreH), 7.07 (dd,
J ¼ 8.6, 1.9 Hz, 1H, AreH), 3.91e3.68 (m, 2H, eCH2-), 3.06e2.88 (m,
2H, eCH2-), 2.39 (d, J ¼ 5.1 Hz, 3H, eCH3), 2.34 (d, J ¼ 5.6 Hz, 3H,
4.4.17. (E)-N-(2-(5-chloro-1H-indol-3-yl)ethyl)-2-((3-
methylpyridin-2-yl)methylene)hydrazine-1-carbothioamide(5k)
White solid, m. p. 219.2e219.5 ꢀC, yield 55%. 1H NMR (400 MHz,
eCH3). 13C NMR (100 MHz, DMSO‑d6,
d, ppm) d 176.92, 149.79,
DMSO‑d6, ppm) d 11.69 (s, 1H, eNHe), 11.06 (s, 1H, eNHe), 8.46 (d,
J ¼ 4.2 Hz, 1H, eNHe), 8.32 (s, 1H, AreH), 8.02 (t, J ¼ 5.7 Hz, 1H,
AreH), 7.73 (s, J ¼ 4.1 Hz, 1H, eCH]N-), 7.65 (d, J ¼ 7.6 Hz, 1H,
AreH), 7.36 (d, J ¼ 8.6 Hz, 1H, AreH), 7.28 (t, J ¼ 6.1 Hz, 2H, AreH),
7.05 (dd, J ¼ 8.6, 1.8 Hz, 1H, AreH), 3.86 (dd, J ¼ 13.4, 6.8 Hz, 2H,
147.96, 138.40, 137.96, 134.71, 134.65, 128.46, 124.65, 124.11, 123.03,
120.88, 117.88, 112.86, 111.57, 44.36, 24.59, 21.85, 17.91. HR-MS (ESI),
calcd. C19H20ClN5S, [MþNa]þm/z: 408.1026. found: 408.1024.
eCH2-), 2.96 (d t, J ¼ 39.6, 7.4 Hz, 2H, eCH2-), 2.37 (s, 3H, eCH3). 13
C
4.4.22. (E)-N-(2-(1H-indol-3-yl)ethyl)-2-(1-(pyridin-2-yl)
ethylidene)hydrazine-1-carbothioamide(5p)
NMR (100 MHz, DMSO‑d6, d, ppm) d 175.93, 140.22, 134.84, 131.62,
Yellow solid, m. p. 147.8e149.7 ꢀC, yield 85%. 1H NMR (400 MHz,
128.43, 124.60, 123.03, 120.95, 120.58, 117.97, 112.90, 111.60, 111.24,
108.93, 108.70, 98.06, 97.81, 48.57, 43.82, 24.90. HR-MS (ESI), calcd.
DMSO‑d6, ppm) d 10.89 (s, 1H, eNHe), 10.43 (s, 1H, eNHe), 8.70 (s,
C
18H18ClN5S, [MþH]þm/z: 372.1050. found: 372.1051.
1H, eNHe), 8.59 (s,1H, AreH), 8.21 (d, J ¼ 7.5 Hz,1H, AreH), 7.83 (d,
J ¼ 7.2 Hz, 1H, AreH), 7.75 (d, J ¼ 7.3 Hz, 1H, AreH), 7.38 (s, 2H,
AreH), 7.24 (s,1H, AreH), 7.09 (s,1H, AreH), 7.00 (s,1H, AreH), 3.90
4.4.18. (E)-N-(2-(5-chloro-1H-indol-3-yl)ethyl)-2-((4-
(d, J ¼ 5.3 Hz, 2H, eCH2-), 3.06 (s, 2H, eCH2-),
d 2.40 (s, 3H, eCH3).
, ppm) 178.51, 155.15, 148.90,
methylpyridin-2-yl)methylene)hydrazine-1-carbothioamide(5l)
13C NMR (100 MHz, DMSO‑d6,
d
d
Yellow solid, m. p. 207.7e209.6 ꢀC, yield 71%. 1H NMR (400 MHz,
148.42, 136.86, 127.68, 124.37, 123.23, 121.52, 121.03, 119.09, 118.76,
112.01, 111.81, 44.90, 25.24, 12.66. HR-MS (ESI), calcd. C18H19N5S,
[MþH]þm/z: 338.1439.found: 338.1438.
DMSO‑d6, ppm)
d 11.71 (s, 1H, eNHe), 11.06 (s, 1H, eNHe), 8.74 (t,
J ¼ 5.8 Hz, 1H, eNHe), 8.42 (s, J ¼ 5.0 Hz, AreH), 8.11 (s, 1H, AreH),
7.98 (s, 1H, AreH), 7.78 (d, J ¼ 1.5 Hz, 1H, AreH), 7.35 (dd, J ¼ 15.6,
7.7 Hz, 1H, AreH), 7.30 (s, J ¼ 1.8 Hz, 1H, eCH]N-), 7.21 (d,
J ¼ 4.8 Hz, 1H, AreH), 7.07 (dd, J ¼ 8.6, 1.8 Hz, 1H, AreH), 3.84 (dd,
J ¼ 14.9, 6.4 Hz, 2H, eCH2-), 3.11e2.92 (m, 2H, eCH2-), 2.40 (d,
4.4.23. (E)-N-(2-(1H-indol-3-yl)ethyl)-2-(1-(pyridin-2-yl)
ethylidene)hydrazine-1-carbothioamide(5q)
Yellow solid, m. p. 186.6e187.5 ꢀC, yield 72%. 1H NMR (400 MHz,
J ¼ 10.8 Hz, 3H, eCH3). 13C NMR (101 MHz, DMSO‑d6,
d, ppm)
DMSO‑d6, ppm) d 10.87 (s, 1H, eNHe), 10.34 (s, 1H, eNHe), 8.64 (t,
d
177.13, 153.04, 149.06, 147.20, 142.46, 134.72, 128.47, 124.99,
J ¼ 5.7 Hz, 1H, eNHe), 8.42 (s, 1H, AreH), 8.11 (d, J ¼ 8.2 Hz, 1H,
AreH), 7.74 (d, J ¼ 7.8 Hz, 1H, AreH), 7.71e7.60 (m, 1H, AreH), 7.36
(t, J ¼ 7.0 Hz, 1H, AreH), 7.22 (dd, J ¼ 10.4, 2.1 Hz, 1H, AreH), 7.08 (t,
J ¼ 7.5 Hz, 1H, AreH), 6.99 (t, J ¼ 7.4 Hz, 1H, AreH), 3.88 (dt, J ¼ 19.8,
10.0 Hz, 2H, 2H, eCH2-), 3.04 (dd, J ¼ 16.7, 9.3 Hz, 2H, 2H, eCH2-),
2.38 (s, 3H, eCH3), 2.34 (s, 3H, eCH3). 13C NMR (100 MHz, DMSO‑d6,
124.60, 123.01, 122.75, 120.90, 120.43, 117.91, 112.87, 111.58, 44.35,
43.01, 24.74, 20.59. HR-MS (ESI), calcd. C18H18ClN5S, [Mþ Na]þm/z:
394.0869. found: 394.0867.
4.4.19. (E)-N-(2-(5-chloro-1H-indol-3-yl)ethyl)-2-((3-
d, ppm) d 177.72, 152.13, 148.60, 148.12, 136.84, 136.31, 133.40,
methylpyridin-2-yl)methylene)hydrazine-1-carbothioamide(5m)
White solid, m. p. 187.0e188.2 ꢀC, yield 51%. 1H NMR (400 MHz,
127.21, 122.71, 121.01, 120.03, 118.60, 118.24, 111.43, 44.35, 24.76,
17.79, 12.14. HR-MS (ESI), calcd. C17H17N5S, [MþNa]þm/z: 346.1102.
found: 346.1108.
DMSO‑d6, ppm)
d 11.65 (s, 1H, eNHe), 11.00 (s, 1H, eNHe), 8.65 (t,
J ¼ 5.8 Hz, 1H, eNHe), 7.94 (s, J ¼ 37.1 Hz, 1H, AreH), 7.87 (d,
J ¼ 7.8 Hz, 1H, AreH), 7.76e7.58 (m, 2H, AreH), 7.35e7.27 (m, 1H,
AreH), 7.23 (s, J ¼ 4.8 Hz, 1H, eCH]N-), 7.17 (d, J ¼ 7.5 Hz, 1H,
AreH), 7.00 (dd, J ¼ 8.6, 1.9 Hz, 1H, AreH), 3.75 (dd, J ¼ 14.8, 6.4 Hz,
2H, eCH2-), 3.01e2.87 (m, 2H, eCH2-), 2.41 (s, 3H, eCH3). 13C NMR
4.4.24. (E)-N-(2-(1H-indol-3-yl)ethyl)-2-(4-chlorobenzylidene)
hydrazine-1-carbothioamide(5r)
White solid, m. p. 196.8e198.0 ꢀC, yield 60%. 1H NMR (400 MHz,
DMSO‑d6, d, ppm) d 11.58 (s, 1H, eNHe), 10.86 (s, 1H, eNHe), 8.63
(100 MHz, DMSO‑d6, d, ppm) d 177.07, 157.67, 152.64, 142.43, 136.69,
(t, J ¼ 5.7 Hz, 1H, eNHe), 8.04 (s, 1H, AreH), 7.75 (dd, J ¼ 22.1,
8.2 Hz, 3H, AreH), 7.49 (d, J ¼ 8.5 Hz, 2H, AreH), 7.36 (d, J ¼ 8.1 Hz,
1H, AreH), 7.22 (s, J ¼ 2.1 Hz,1H, eCH]N-), 7.08 (dd, J ¼ 11.1, 4.0 Hz,
1H, AreH), 6.99 (dd, J ¼ 10.9, 4.0 Hz, 1H, AreH), 3.84 (dd, J ¼ 14.9,
6.3 Hz, 2H, eCH2-), 3.13e2.98 (m, 2H, eCH2-). 13C NMR (101 MHz,
134.71, 128.46, 124.64, 123.34, 123.03, 120.91, 117.85, 117.10, 112.89,
111.55, 44.34, 24.63, 23.79. HR-MS (ESI), calcd. C18H18ClN5S, [Mþ
Na]þm/z: 394.0869. found: 394.0870.
4.4.20. (E)-N-(2-(5-chloro-1H-indol-3-yl)ethyl)-2-(1-(pyridin-2-
yl)ethylidene)hydrazine-1-carbothioamide(5n)
DMSO‑d6, d, ppm) d 176.90, 140.38, 136.28, 134.15, 133.17, 128.77,
128.71, 127.24, 122.66, 120.99, 118.55, 118.24, 111.53, 111.36, 44.26,
24.85. HR-MS (ESI), calcd. C18H17ClN4S, [MþH]þm/z: 357.0940.
found: 357.0939.
Yellow solid, m. p. 182.2e183.7 ꢀC, yield 61%. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d
11.07 (d, J ¼ 6.5 Hz, 1H, eNHe), 10.41 (s, 1H,
eNHe), 8.70 (t, J ¼ 5.8 Hz, 1H, NH-), 8.60 (t, J ¼ 9.3 Hz, 1H, AreH),
8.17 (t, J ¼ 19.6 Hz, 1H, AreH), 7.82 (dt, J ¼ 13.4, 3.6 Hz, 1H, AreH),
7.79e7.70 (m, 1H, AreH), 7.46e7.36 (m, 2H, AreH), 7.30 (dd, J ¼ 8.6,
2.0 Hz, 1H, AreH), 7.06 (dd, J ¼ 8.6, 1.9 Hz, 1H, AreH), 3.82 (ddd,
J ¼ 42.8, 24.5, 17.3 Hz, 2H, eCH2-), 3.02 (dd, J ¼ 16.3, 8.9 Hz, 2H,
eCH2-), 2.39 (d, J ¼ 7.8 Hz, 3H, eCH3). 13C NMR (100 MHz, DMSO‑d6,
4.4.25. (E)-N-(2-(1H-indol-3-yl)ethyl)-2-(4-methylbenzylidene)
hydrazine-1-carbothioamide(5s)
White solid, m. p. 119.1e120.6 ꢀC, yield 59%. 1H NMR (400 MHz,
DMSO‑d6, d, ppm) d 11.45 (s, 1H, eNHe), 10.86 (s, 1H, eNHe), 8.53
(t, J ¼ 5.8 Hz, 1H, eNHe), 8.02 (s, 1H, AreH), 7.73 (d, J ¼ 7.8 Hz, 1H,
AreH), 7.63 (d, J ¼ 8.1 Hz, 2H, AreH), 7.35 (d, J ¼ 8.1 Hz, 1H, AreH),
7.23 (dd, J ¼ 10.8, 5.0 Hz, 3H, 2AreH, 1-CH]N-), 7.08 (t, J ¼ 7.1 Hz,
1H, AreH), 6.99 (t, J ¼ 7.4 Hz, 1H, AreH), 3.83 (dd, J ¼ 14.9, 6.3 Hz,
2H, eCH2-), 3.08e2.96 (m, 2H, eCH2-), 2.34 (s, 3H, eCH3). 13C NMR
d
, ppm) d 177.90, 154.68, 148.48, 148.08, 136.32, 134.76, 128.44,
124.68, 123.85, 123.05, 120.92, 120.53, 117.87, 112.88, 111.57, 44.40,
24.55, 12.16. HR-MS (ESI), calcd.
372.1050. found: 372.1051.
C
18H18ClN5S, [MþH]þm/z:

14
Z. He et al. / European Journal of Medicinal Chemistry 184 (2019) 111764
(100 MHz, DMSO‑d6,
d, ppm)
d
176.74, 141.91, 139.55, 136.27, 131.44,
4.6. Cell viability assay
129.27, 127.25, 127.13, 122.64, 120.97, 118.57, 118.22, 111.58, 111.35,
44.25, 24.91, 21.02. HR-MS (ESI), calcd. C19H20N4S, [MþH]þm/z:
337.1487. found: 337.1486.
Cells in the experimental growth phase were seeded in 96-well
plates at approximately 3.8e5.0 ꢁ 103 cells per well. Incubate for
approximately 24 h, then removed the medium and added 200 ml
fresh medium with different concentrations of compound 5j per
well. After 24 h, 48 h, 72 h incubation, 20 l of MTT (3-(4,5-
4.4.26. (E)-N-(2-(1H-indol-3-yl)ethyl)-2-benzylidenehydrazine-1-
carbothioamide(5t)
m
White solid, m. p. 213.7e215.9 ꢀC, yield 63%. 1H NMR (400 MHz,
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution
(5 mg/ml) was added to each well, and cells were incubated at 37 ꢀC
DMSO‑d6, d, ppm) d 11.52 (s, 1H, eNHe), 10.86 (s, 1H, eNHe), 8.58
for 4 h in the dark. Then, the medium was removed, added 150 ml
(t, J ¼ 5.7 Hz, 1H, eNHe), 8.06 (s, 1H, AreH), 7.74 (dd, J ¼ 7.6, 4.7 Hz,
3H, AreH), 7.51e7.31 (m, 4H, AreH), 7.22 (s, J ¼ 1.8 Hz, 1H, eCH]N-
), 7.08 (t, J ¼ 7.5 Hz, 1H, AreH), 6.99 (t, J ¼ 7.4 Hz, 1H, AreH), 3.84
(dd, J ¼ 14.7, 6.4 Hz, 2H, eCH2-), 3.12e2.95 (m, 2H, eCH2-). 13C NMR
DMSO, and was shaken on the shaker for about 10e15 min until the
formazan dissolved completely. The absorbance was measures at
490 nm with a microplate reader. The date of IC50 was calculated
using SPASS 17.0 software, and the cell viability curve of compound
5j was obtained by GraphPad Prism 5.0 software.
(101 MHz, DMSO‑d6, d, ppm) d 176.87, 141.78, 136.27, 134.16, 129.75,
128.65, 127.26, 127.14, 122.65, 120.98, 118.56, 118.24, 111.57, 111.35,
44.29, 24.88. HR-MS (ESI), calcd. C18H18N4S, [MþH]þm/z: 323.1330.
found: 323.1329.
4.7. Colony formation assay
4.4.27. (E)-N-(2-(1H-indol-3-yl)ethyl)-2-(1-(p-tolyl)ethylidene)
hydrazine-1-carbothioamide(5u)
Cells in the exponential growth phase were seeded in 6-well
plate at a concentration of 1.1 ꢁ 103 per well. After approximately
24 h incubation, added 2 ml fresh medium with different concen-
trations of compound 5j per well, and incubated for 7 days. After
washing 2e3 times with PBS, cells were fixed with methanol (4 ꢀC
pre-cooling) for 1 h and stained with crystal violet for 1 h. After
that, images were photographed after drying the plate. The crystals
were dissolved in 33% acetic acid for 30 min. The absorbance was
measured at 570 nm with a microplate reader.
White solid, m. p. 190.9e191.7 ꢀC, yield 53%. 1H NMR (400 MHz,
DMSO‑d6, d, ppm) d 10.87 (s, 1H, eNHe), 10.22 (s, 1H, eNHe), 8.47
(t, J ¼ 5.7 Hz, 1H, eNHe), 7.77e7.64 (m, 3H, AreH), 7.36 (d,
J ¼ 8.1 Hz, 1H, AreH), 7.22 (dd, J ¼ 5.2, 2.7 Hz, 3H, AreH), 7.13e7.02
(m, 1H, AreH), 7.03e6.91 (m, 1H, AreH), 3.87 (dd, J ¼ 14.4, 6.4 Hz,
2H, eCH2-), 3.10e2.95 (m, 2H, eCH2-), 2.34 (s, 3H, eCH3), 2.27 (s,
3H, eCH3). 13C NMR (100 MHz, DMSO‑d6,
d, ppm) d 177.68, 147.70,
138.77, 136.33, 134.82, 128.87, 127.20, 126.36, 122.71, 121.00, 118.61,
118.23, 111.52, 111.36, 44.23, 24.82, 20.85, 13.96. HR-MS (ESI), calcd.
C
20H22N4S, [MþH]þm/z: 351.1643. found: 351.1643.
4.8. Cell cycle analysis
PC3 cells were seeded in 6-well plates at a concentration of
2.5 ꢁ 105 per well. After 24 h incubation, treated with various
concentrations of compound 5j for 48 h or 72 h. After that, collected
cells, fixed with pre-cooled 70% ethanol at ꢂ20 ꢀC overnight and
4.4.28. (E)-N-(2-(1H-indol-3-yl)ethyl)-2-(1-(4-chlorophenyl)
ethylidene)hydrazine-1-carbothioamide(5v)
White solid, m. p. 176.6e178.2 ꢀC, yield 58%. 1H NMR (400 MHz,
DMSO‑d6, d, ppm) d 10.87 (s, 1H, eNHe), 10.33 (s, 1H, eNHe), 8.51
stained with 20 mg/ml PI and 20 mg/ml RNase A. Before analyzed by
(t, J ¼ 5.6 Hz, 1H, eNHe), 7.84 (d, J ¼ 8.7 Hz, 2H, AreH), 7.72 (d,
J ¼ 7.8 Hz, 1H, AreH), 7.46 (d, J ¼ 8.7 Hz, 2H, AreH), 7.36 (d,
J ¼ 8.1 Hz, 1H, AreH), 7.23 (d, J ¼ 2.1 Hz, 1H, AreH), 7.07 (dd, J ¼ 11.1,
3.9 Hz, 1H, AreH), 6.98 (dd, J ¼ 10.9, 3.9 Hz, 1H, AreH), 3.87 (dd,
J ¼ 14.2, 6.5 Hz, 2H, eCH2-), 3.04 (t, J ¼ 7.5 Hz, 2H, eCH2-), 2.29 (s,
flow cytometer (BD Bioscience, USA), the cells were protected from
light for 30 min. Flowjo 7.6 software was used to analyze the date of
cell cycle.
3H, eCH3). 13C NMR (100 MHz, DMSO‑d6,
d, ppm) d 177.79, 146.33,
4.9. Wound healing assay
136.47, 136.34, 133.85, 128.23, 128.18, 127.19, 122.75, 121.01, 118.59,
118.24, 111.48, 111.36, 44.24, 40.14, 39.93, 39.72, 39.52, 39.31, 39.10,
38.89, 24.75, 13.91. HR-MS (ESI), calcd. C19H19ClN4S, [MþH]þm/z:
371.1097. found: 371.1098.
The cell migration ability was evaluated by wound healing assay.
PC3 cells were seeded in 6-well plates at a concentration of
3.5 ꢁ 105 per well. Until the cells density reached 90%e95%, the cell
surface was scratched by a 200 ml pipet tip. Next, the cells were
4.5. Cell culture and reagents
washed three or four times with PBS and added fresh medium with
1% FBS and different concentrations of compound 5j. After 48 h
incubation, images were photographed using an inverted
microscope.
Human prostate cancer cells (PC3), gastric cancer cells
(MGC803), esophageal cancer cells (EC109), human gastric mucosal
epithelial cells (GES-1) and human normal prostate cell (WPMY-1)
were obtained from our laboratories (originally purchased from the
CCTCC). RPMI 1640 medium was bought from Solarbio. Fetal Bovine
Serum (FBS) was purchased from Bioind (BI). All cancer cells were
cultured in RPMI 1640 medium with 10% FBS and all cancer cells
were cultured in a humidified incubator of 95% air and 5% CO2 at
37 ꢀC. Compounds were dissolved in DMSO at an initial concen-
tration of 10 mM. Hoechst 33342 was purchased from Beyotime
Biotechnology. PI and RNase A were purchased from Solarbio.
Annexin V-FITC/PI Apoptosis Detection Kit was bought from Key-
gen Biotech. The antibodies used in western blotting are as follows:
Bax (#505992, Proteintech), Bcl-2 (#15071T, CST), P53 (2527T, CST),
Caspase9 (9508T, CST), Cleaved caspase 3 (9664T, CST), PARP
(9532T, CST), GAPDH (AB-P-R001, Good Here Technology).
4.10. Transwell assay
Cells were first starved with serum-free medium for 12 h and
then harvested with trypsin. Cells were seeded in transwell 24-well
plate at a concentration of 3 ꢁ 105 per well. 20% FBS and compound
5j was added in the lower chamber, and compound 5j was added in
the upper chamber with no serum. After incubation for 48 h,
removed the medium and washed twice with PBS. Then, the cells
were fixed with pre-cooled methanol for 1 h at 4 ꢀC, stained with
crystal violet for 30 min at room temperature, and photographed
using upright microscope. The date of cell number was analyzed by
ImageJ software.

Z. He et al. / European Journal of Medicinal Chemistry 184 (2019) 111764
15
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4.11. Hoechst 33342 staining
Cells were seeded in 24-well plates at a concentration of 5 ꢁ 104
per well. After 24 h incubation, added 500 ml fresh medium con-
taining different concentrations of compound 5j for another 72 h.
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4.13. Western analysis
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10%e15% SDS-PAGE, and transferred to nitrocellulose membrane.
The nitrocellulose membrane was blocked with PBST containing 5%
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primary antibody (1:1000) overnight at 4 ꢀC, followed by incuba-
tion with secondary antibody (1:10000) for 2 h at room tempera-
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This work was supported by National Key Grant from Chinese
Ministry of Science and Technology (2016YFA0501800 by Z.W),
National Natural Science Foundation of China (NO. 81470524 and
81870297 by Z.W; NO. 81430085 by L.H-M; 81703328 by M. L-Y)
and Henan Scientific Innovation Talent Team, Department of Edu-
cation (19IRTSTHN001 by Z.W); the Starting Grant of Zhengzhou
University (Grant 32210535 by M. L-Y); Scientific Program of Henan
Province (Grant 182102310070 by M. L-Y).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111764.
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