Design, synthesis and biological evaluation of novel thiosemicarbazone-indole derivatives targeting prostate cancer cells

Article abstract of DOI:10.1016/j.ejmech.2020.112970

To discover novel anticancer agents with potent and low toxicity, we designed and synthesized a range of new thiosemicarbazone-indole analogues based on lead compound 4 we reported previously. Most compounds displayed moderate to high anticancer activities against five tested tumor cells (PC3, EC109, DU-145, MGC803, MCF-7). Specifically, the represented compound 16f possessed strong antiproliferative potency and high selectivity toward PC3 cells with the IC₅₀ value of 0.054 μM, compared with normal WPMY-1 cells with the IC₅₀ value of 19.470 μM. Preliminary mechanism research indicated that compound 16f could significantly suppress prostate cancer cells (PC3, DU-145) growth and colony formation in a dose-dependent manner. Besides, derivative 16f induced G1/S cycle arrest and apoptosis, which may be related to ROS accumulation due to the activation of MAPK signaling pathway. Furthermore, molecule 16f could effectively inhibit tumor growth through a xenograft model bearing PC3 cells and had no evident toxicity in vivo. Overall, based on the biological activity evaluation, analogue 16f can be viewed as a potential lead compound for further development of novel anti-prostate cancer drug.

Full text of DOI:10.1016/j.ejmech.2020.112970

Journal Pre-proof
Design, synthesis and biological evaluation of novel thiosemicarbazone-indole
derivatives targeting prostate cancer cells
Zhang-Xu He, Jin-Ling Huo, Yun-Peng Gong, Qi An, Xin Zhang, Hui Qiao, Fei-Fei
Yang, Xin-Hui Zhang, Le-Min Jiao, Hong-Min Liu, Li-Ying Ma, Wen Zhao
PII:
S0223-5234(20)30942-9
DOI:
https://doi.org/10.1016/j.ejmech.2020.112970
EJMECH 112970
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 June 2020
Revised Date: 21 October 2020
Accepted Date: 25 October 2020
Please cite this article as: Z.-X. He, J.-L. Huo, Y.-P. Gong, Q. An, X. Zhang, H. Qiao, F.-F. Yang, X.-H.
Zhang, L.-M. Jiao, H.-M. Liu, L.-Y. Ma, W. Zhao, Design, synthesis and biological evaluation of novel
thiosemicarbazone-indole derivatives targeting prostate cancer cells, European Journal of Medicinal
Chemistry, https://doi.org/10.1016/j.ejmech.2020.112970.
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Graphical abstract

Design, synthesis and biological evaluation of novel thiosemicarbazone-indole
derivatives targeting prostate cancer cells
#
#
Zhang-Xu He , Jin-Ling Huo , Yun-Peng Gong, Qi An, Xin Zhang, Hui Qiao, Fei-Fei
Yang, Xin-Hui Zhang, Le-Min Jiao, Hong-Min Liu*, Li-Ying Ma*and Wen Zhao*
State Key Laboratory of Esophageal Cancer Prevention and Treatment; Key
Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China;
School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan
4
50001, P. R. China
#
:
Both authors contribute equally to this work.
*
*
: These senior authors contribute equally to this work.
Corresponding Authors: Zhao Wen, Ph.D, Ma Li-Ying, Ph.D and Liu Hong-Min,
Ph.D
E-mail address: zhaowen100@139.com, maliying@zzu.edu.cn, liuhm@zzu.edu.cn
1

Abstract
To discover novel anticancer agents with potent and low toxicity, we designed and
synthesized a range of new thiosemicarbazone-indole analogues based on lead
compound 4 we reported previously. Most compounds displayed moderate to high
anticancer activities against five tested tumor cells (PC3, EC109, DU-145, MGC803,
MCF-7). Specifically, the represented compound 16f possessed strong
antiproliferative potency and high selectivity toward PC3 cells with the IC value of
5
0
0
.054 μM, compared with normal WPMY-1 cells with the IC value of 19.470 μM.
50
Preliminary mechanism research indicated that compound 16f could significantly
suppress prostate cancer cells (PC3, DU-145) growth and colony formation in a
dose-dependent manner. Besides, derivative 16f induced G1/S cycle arrest and
apoptosis, which may be related to ROS accumulation due to the activation of MAPK
signaling pathway. Furthermore, molecule 16f could effectively inhibit tumor growth
through a xenograft model bearing PC3 cells and had no evident toxicity in vivo.
Overall, based on the biological activity evaluation, analogue 16f can be viewed as a
potential lead compound for further development of novel anti-prostate cancer drug.
Keywords: prostate cancer, thiosemicarbazone, proliferation, cell cycle, apoptosis.
2

1
. Introduction
Development of potent anticancer agents with low toxicity and high selectivity are
urgently necessary for tumor treatment [1-3]. Metal chelation is a promising strategy
in developing chemotherapy drugs due to the elevated requirement of cancer cells for
critical metals (Fe, Cu, and Zn) needed in growth and proliferation [4-6]. Several
metal chelators (Fig. 1), including NNS, ONS, NNN, ONN or hydroxamic acid donor
chelators, displayed potent antiproliferative potency [4, 6-13]. In particular,
thiosemicarbazones (TSC) derivatives featuring the NNS donor revealed extensive
and long-term pharmacological effects [14-19], especially antitumor activity [20-22].
Anticancer activity of TSCs was connected with the perturbation of intracellular metal
homeostasis, the formation of redox active complexes, which resulted in the
generation of reactive oxygen species (ROS) [5, 23-27], or the effect on several
crucial proteins, such as ribonucleotide reductase, PARP-1, MEK and EGFR [28-31].
In addition, several TSC-chelators targeting DNA synthesis [27, 32, 33] and cell-cycle
inhibition [23, 34, 35] also revealed potentially antiproliferative activity. Considering
the key role of TSCs in cancer treatment, several novel thiosemicarbazone ligands had
been identified and investigated in clinical trials, including 3-AP, DPC and COTI-2
(Fig. 1) [10]. Although this series of agents were generally explored, the side effects
had partly limited their clinical utility [8]. Thus, the development of novel TSCs with
potent and less toxicity were urgently needed.
Fig. 1. Representative compounds with diverse chelate fragments. The donor atoms were
highlighted with blue.
3

Fig. 2. Rationale for the design of novel thiosemicarbazone derivatives.
Our group had previously reported that compound 1 (Fig. 2) was a potent
anticancer agent toward MGC803 cells with the IC50 value of 4.01 μM and
significantly suppressed cancer cell migration and invasion in vivo and in vitro, of
which the TSC moiety attached to pyrimidine skeleton brought about our interest due
to its potential anticancer activity [36]. Thus, a series of phenyl-substituted TSC
derivatives (series 1) were synthesized, and most of them exhibited strong biological
activity against MGC803 cells, more active in comparison to compound 1, but
moderate selectivity between MGC803 and normal cells [37, 38]. Given improved
cytotoxicity mentioned above, a series of TSC compounds (series 2) featuring various
indole moieties were obtained and evaluated toward three cancer cell lines (MGC803,
PC3, EC109). In contrast, compared with series 1, most analogues tethering the
indole moiety showed more active against PC3 cells with the IC50 value of low
micromolar range and higher selectivity. The preliminary structure-activity
4

relationship (SAR) indicated that the number of carbon between indole and
thiosemicarbazone might play an important role on potency [39]. We speculated that
opportunities for optimization could be provided by changing various linker types. In
current work, we firstly carried out systematic SAR study after exploring the effect of
linker on antiproliferative activity. Also, several other donor chelators were combined
with indole moiety, reported as a privileged motif [40], to exam potential inhibitory
activity on cancer cells (Fig. 2). Herein, we designed and synthesized a novel series of
TSC derivatives as potent anti
-prostate cancer agents based on our previous work, and
explored their potential mechanism.
2
. Results and discussion
2
.1 Chemistry
The overall synthetic routes of the title compounds were illustrated in Schemes 1-7.
Compound 4 was efficiently synthesized from commercially available 1 following our
previously reported methods (Scheme 1) [39]. The materials 2-(1H-indol-3-yl)
ethan-1-amine 1 was firstly reacted with carbon disulfide in the presence of
triethylamine
, which was reacted with hydrazine hydrate to furnish compound 3. Finally,
compound 4 was afforded via the acid-catalyzed Schiff base condensation of 3 and
, followed by the addition of DMAP and Boc O to give the intermediate
2
2
5
-methylpicolinaldehyde in 69% yields. In Scheme 2, amide 6 was obtained from
-aminocyclohexane-1-carboxylic acid 5 by reaction with EDCI, HOBT and raw
4
material 1, then provided TSC compound 9 according to our literature methods in
2% yields [39]. Intermediates 13a-f were efficiently synthesized from the
3
corresponding amines through two steps (Scheme 3). Specifically, substituted amines
were reacted with 4-nitrobenzoic acid or 6-nitronicotinic acid in the presence of EDCI
and HOBT in dichloromethane to yield 12a-f, which was then reacted with H in the
2
presence of palladium to afford the corresponding 13a-f. Intermediates 13a-f were
further converted to compounds 16a-s through a similar reaction of Scheme 1a, b and
c in 35-65% yields. In Scheme 4, intermediate 20 was also synthesized from raw
material 17 as described for the synthesis of Scheme 1a, b and c. The hydrolysis
5

reaction of compound 20 with sodium hydroxide resulted in compound 21 in 41%
yields, which was further converted to the desired derivative 22 by condensation with
2
-(1H-indol-3-yl) ethan-1-amine in 46% yields. Similarly, condensation of
commercially available 10e with 4-(methoxycarbonyl) benzoic acid 23 yielded 24 in
8% yield, which was further reacted with hydroxylamine in the presence of sodium
4
hydroxide to afford compound 25 in 56% yields (Scheme 5) [41]. In Scheme 6,
commercially available 26 was reacted with 2-acetylpyridine, in the presence of a
catalytic amount of acetic acid (AcOH), via condensation reaction, leading to the
formation of intermediate 27 in 53% yield, which was then transformed into
compound 28 through under the catalysis of EDCI and HOBT in 50% yields. In
Scheme 7, raw material 23 and 10e were converted to the intermediate 29 using EDCI
and HOBT in dichloromethane. The compound 29 was reacted with hydrazine hydrate
to afford 30 in 65% yield. Finally, compound 31 was provided from a condensation
reaction between 30 and pyridoxal hydrochloride in 41% yield.
Scheme 1. Reagents and conditions: (a) (1) CS , TEA, EtOH, rt; (2) Boc O, DMAP, EtOH, rt; (b)
2
2
hydrazine hydrate, DCM, rt; (c) 5-methylpicolinaldehyde, acetic acid, EtOH, rt.
Scheme 2. Reagents and conditions: (a) EDCI, HOBT, DCM, rt; (b) (1) CS , TEA, EtOH, rt; (2)
2
Boc O, DMAP, EtOH, rt; (c) hydrazine hydrate, DCM, rt; (d) 5-methylpyridine-2-carbaldehyde,
2
acetic acid, EtOH, rt.
6

COOH
NO2
O
O
O
R1
R1
R1
R1 NH2
a
c
N
+
X
N
b
N
H
H
H
NCS
X
NH2
X
X
NO2
1
0a-e
11a-b
12a-f
13a-f
14a-f
O
O
R1
R1
d
N
e
N
S
S
H
H
N
R2
NH2
X
N
N
X
N
H
N
H
H
R3
H
1
5a-f
16a-s
H
H
H
H
N
H
N
N
N
N
R1=
Cl
O
O
N
N
OH
O
Cl
Br
R2=
N
N
N
N
O
N
R3= H, -CH3
Scheme 3. Reagents and conditions: (a) EDCI, HOBT, DCM, rt; (b) H , Pt-C, MeOH, rt; (c) (1)
2
CS , TEA, EtOH, rt; (2) Boc O, DMAP, EtOH, rt; (d) hydrazine hydrate, DCM, rt; (e) appropriate
2
2
aldehyde or ketone, acetic acid, EtOH, rt.
O
O
O
O
a
O
H
H
N
O
H
H
O
b
c
O
N
N
N
NCS
NH2
N
NH2
S
S
N
1
7
18
1
9
20
O
HN
O
d
HO
H
H
e
N
N
N
H
H
N
H
N
N
N
S
N
S
N
2
2
2
1
Scheme 4. Reagents and conditions: (a) (1) CS , TEA, EtOH, rt; (2) Boc O, DMAP, EtOH, rt; (b)
2
2
hydrazine hydrate, DCM, rt; (c) 5-methylpyridine-2-carbaldehyde, acetic acid, EtOH, rt; (d)
NaOH, H O, 80℃ ; (e) EDCI, HOBT, DCM, rt.
2
Scheme 5. Reagents and conditions: (a) EDCI, HOBT, DCM, rt; (b) NH OH, NaOH, THF/MeOH
2
(1:1), 0°C to rt.
7

Scheme 6. Reagents and conditions: (a) 2-acetylpyridine, acetic acid, EtOH, 80°C; (b) 2-(2
methyl-1H-indol-3-yl)ethan-1-amine, EDCI, HOBT, DCM, rt.
-
Scheme 7. Reagents and conditions: (a) EDCI, HOBT, DCM, rt; (b) hydrazine hydrate, MeOH,
0℃ ; (c) pyridoxal hydrochloride, acetic acid, EtOH, 80°C.
8
2
.2 Evaluation of biological activity
2
.2.1 Antiproliferative activity
The in vitro anticancer activities of all synthesized analogues were examined
against a panel of cell lines, including PC3 (human prostate cancer cells), EC109
(
human esophageal cancer cells), DU-145 (human prostate cancer cells), MGC803
human gastric cancer cells), MCF-7 (human breast cancer cells) and
(
WPMY-1(normal human prostate cells) by using the MTT assay [39]. 3-AP and DPC
were used as positive controls. Generally, the prostate cancer cells (PC3, DU-145)
revealed more sensitive to most derivatives in comparison to other cancer cells. To
investigate the importance of the linker that attached to indole and TSC moiety, we
performed further modifications of the linker by introducing various amide groups
into compound 4. As shown in Table 1, compared with compound 4, compound 9 with
a cyclohexyl suggested weak inhibitory activity against PC3 cells. Surprisingly,
conversion of the cyclohexyl in compound 9 into a phenyl group resulted in 16a,
which had an IC value of 0.241 μM in PC3 cells, about 2.5 times more potent than
5
0
8

compound 4 (IC = 0.601 ± 0.042 μM) with less toxicity against normal prostate cells
5
0
WPMY-1. Besides, changing the linker from the phenyl to pyridyl afforded derivative
6b, which showed comparable or decreased activity against five tested cancer cell
1
lines as well as obvious toxicity in comparison to compound 16a. Further,
replacement of phenyl in 16a with benzyl provided compound 22, which was inferior
to 16a against PC3 and EC109 cells. These results indicated that the benzamide
fragment as the linker was preferred.
Table 1
The anticancer activity of synthetic derivatives against the tested cell lines.
a
IC50 (μM)
Comp.
structure
PC3
EC109
DU-145
MGC803
MCF-7
WPMY-1
4
9
0.601±0.042
4.512±0.930
3.128±0.210
5.655±1.194
9.095±0.96
2.328±0.367
2.223±0.345
0.241±0.050
0.512±0.120
14.120±1.150
3.938±0.585
12.515±1.097
2.217±0018
1.458±0.156
6.149±0.312
4.576±0.660
7.015±0.846
7.590±0.880
8.129±0.910
7.107±0.852
8.317±0.920
1.565±0.195
12.476±1.210
2.501±0.398
1
6a
6b
1
2
2
1.424±0.153
18.500±1.267
1.204±0.258
4.847±0.685
8.623±0.936
1.093±0.039
a
Inhibitory activity was assayed by exposure various concentrations of the tested compounds for
2 h to substance and expressed as concentration required to inhibit tumor cell proliferation by
0% (IC ). Data are presented as the means ± SDs. All experiments were carried out at least three
7
5
50
independent times.
Next, compounds 16c-f were synthesized to explore the effect of substituents in the
indole group. As shown in Table 2, no matter an electron-withdrawing (-Cl) or
-
donating group (-CH , -OCH ) substitution at indole 2-position, the title compounds
3 3
1
6c-e displayed increased inhibitory activity toward PC3 cells as compared to no
substitution compound 16a, suggesting that substituent at indole 2-position was well
tolerated. Among these, analogue 16e revealed promising potency on PC3 with an
IC50 of 0.061 μM, about 4 times more potent than 16a. Surprisingly, introduction a
9

methyl group into 8-position of the indole resulted in compound 16f (IC = 0.054 ±
5
0
0
.012 μM), which significantly improved potency on PC3 cells comparing to 16a
(
IC = 0.241 ± 0.050 μM) with less toxicity toward normal prostate cell WPMY-1.
5
0
These results showed that substituents on the indole ring were critical for
antiproliferative activity.
Table 2
The anticancer activity of derivatives 16a-q against the tested cell lines.
O
R1
N
H
S
R2
N
N
H
N
H
R3
a
IC50 (μM)
1
2
3
Comp.
R
R
R
PC3
EC109
DU-145
MGC803
MCF-7
WPMY-1
1
6a
6c
H
H
H
H
H
H
H
H
H
H
H
H
H
0.241±0.050
0.121±0.032
0.081±0.012
0.061±0.020
0.054±0.012
0.133±0.014
2.485±0.395
0.093±0.053
0.091±0.024
1.501±0.176
0.084±0.030
1.601±0.742
0.322±0.020
3.938±0.585
2.399±0.380
1.632±0.213
5.746±0.242
4.942±0.694
5.027±0.701
>20
1.458±0.156
0.925±0.031
1.064±0.016
3.217±0.159
1.439±0.258
4.128±0.294
15.248±1.059
4.218±0.176
3.627±0.248
17.021±1.112
4.085±0.314
5.655±0.257
1.902±0.036
7.015±0.846
7.107±0.852
12.476±1.210
1
2.671±0.427
3.115±0.493
10.424±1.018
4.724±0.674
5.346±0.728
>20
7.275±0.862
4.017±0.604
5.326±0.706
5.334±0.728
6.491±0.812
>20
7.092±0.707
10.903±0.950
18.095±1.207
19.470±1.242
12.251±1.011
>20
1
6d
1
6e
HN
1
6f
1
6g
6h
HN
1
O
1
6i
6j
9.116±0.960
3.458±0.539
16.159±0.755
5.490±0.740
16.563±1.219
13.879±1.142
8.924±0.951
8.645±0.937
>20
1.956±0.291
10.307±1.013
>20
>20
1
13.117±1.046
>20
1
6k
HN
HN
1
6l
6.326±0.801
>20
6.282±0.798
>20
>20
1
6m
3.220±0.508
1.780±0.250
1
6n
>20
>20
1
0

16o
16p
16q
H
0.454±0.051
0.707±0.032
0.947±0.086
14.339±1.157
5.997±0.778
6.480±0.812
5.217±0.054
4.018±0.052
5.884±0.413
11.101±1.045
6.240±0.795
4.016±0.604
10.154±1.007
6.416±0.807
5.381±0.371
1.780±0.250
3.552±0.551
4.510±0.654
-CH
3
3
HN
-CH
O
a
Inhibitory activity was assayed by exposure various concentrations of the tested compounds for
2 h to substance and expressed as concentration required to inhibit tumor cell proliferation by
0% (IC ). Data were presented as the means ± SDs. All experiments were carried out at least
7
5
50
three independent times.
With the improved potency and our better SAR understanding, we next focused on
2
the effect of R on activity. Our previous results indicated that the position of
substituent in the pyridine ring was important for activity with a relative order of 4 >
2
> 3 > 5-position [39]. Thus, derivatives 16g-o were synthesized to further explore
the effect of the substituent in 4-position of pyridyl, and the results were shown in
Table 2. Analogues (16f, 16l) with electron-donating substituents displayed better
potency than that of compound 16j with no substituent. A similar trend was also
observed (16g vs. 16e, 16i). The hydroxyl group was an exception to this trend and
significantly decreased anticancer activity relative to its parental compound (16g vs.
1
6h, 16j vs. 16k). In contrast, electron-withdrawing substituents (-Br, -Cl) at
4
-position on the pyridine ring were detrimental to anticancer activity toward PC3
cells (16j vs. 16n, 16o). The above results suggested that electronic effect on the
pyridine ring was important for potency. In addition, changing methoxy of compound
1
6l into a larger ester group (16m) significantly decreased biological activity,
suggesting that the increase of steric hindrance at 4-position of pyridine ring may be
3
detrimental to target PC3 cells. Replacement hydrogen (16j) at the R position with
methyl (16p) was not conducive to improve activity toward five cancer cells.
Similarly, derivatives 16g and 16q also exhibited this trend, and the compound 16q
with the IC50 of 0.947 μM was about 7 folds less potent than 16g (IC = 0.133 ±
50
0
.014 μM).
Several derivatives containing ONS, NNN, ONN and hydroxamic acid chelate
1
1

fragments exhibited potential anticancer activity [4, 6-11, 42]. Thus, we replaced NNS
with these donor chelators to further investigate antiproliferative activity and as was
shown in table 3. Replacement of NNS in compound 16f with ONS fragment (16r,
1
6s) led to obviously decreased potency, which may be attributed to their different
ability to chelate iron [10]. In addition, when changing NNS to hydroxamic acid or
ONN group (25 or 31) at compound 16f, the activity was completely abrogated.
Compound 28 containing the NNN donor showed moderate inhibitory activity toward
five cancer cells; however, its toxicity against WPMY-1 cells stood out. These results
indicated the NNS donor played a critical role on inhibiting cancer cells growth and
reducing toxicity.
Generally, this series of TSC derivatives were more selective to prostate cancer
cells over other cancer cells, which might be associated with multiple factors. Firstly,
types of the linker were essential for selectivity. Particularly, introduction of
benzamide group into compound 4 significantly increased selectivity (4 vs. 16a).
Substituents in the indole ring and pyridine ring also affected the selectivity profile
(16a vs. 16f, 16f vs. 16m). Besides, compounds featuring an NNS fragment showed
better selectivity than those with other chelate fragments (16f vs. 16s or 28). More
importantly, in comparison to positive control 3-AP and DPC, most of the synthesized
thiosemicarbazone derivatives exhibited comparable activities against PC3 cells, as
well as lower toxicity toward normal WPMY-1 cells. Given markedly inhibitory
activity and high selectivity in prostate cancer cells, we next explored the potential
antitumor mechanism of compound 16f using PC3 and DU-145 cells.
Table 3
The anticancer activity of synthetic derivatives against the tested cell lines.
a
IC50 (μM)
Comp.
structure
PC3
EC109
DU-145
MGC803
MCF-7
WPMY-1
H
N
O
1
6f
N
S
N
0.054±0.012
4.942±0.694
1.439±0.258
4.724±0.674
5.334±0.728
19.470±1.242
H
N
N
N
H
H
1
2

1
6r
6s
5.321±0.263
16.987±0.178
>20
9.499±0.978
14.546±0.064
18.516±0.125
>20
>20
>20
>20
>20
2.870±0.458
>20
HN
O
N
S
O
H
N
1
>20
N
N
NH
H
H
2
5
8
1
>20
>20
>20
>20
2
0.406±0.091
0.664±0.178
2.052±0.145
4.219±0.625
0.871±0.062
>20
0.450±1.349
3
>20
>20
>20
>20
>20
3
-AP
-
-
0.560±0.031
0.122±0.024
ND
ND
ND
ND
ND
ND
ND
ND
4.963±0.020
0.711±0.152
DPC
a
Inhibitory activity was assayed by exposure various concentrations of the tested compounds for
2 h to substance and expressed as concentration required to inhibit tumor cell proliferation by
0% (IC ). Data were presented as the means ± SDs. All experiments were carried out at least
7
5
50
three independent times.
2
.2.2 Effect of compound 16f on growth inhibition in human prostatic cancer cell lines
PC3 and DU-145.
MTT assay was used to determine the cell viability for 24 h, 48 h and 72 h on PC3
and DU-145 cell lines, respectively. As shown in Fig. 3A, compound 16f evidently
inhibited the proliferation of PC3 and DU-145 cells in a dose- and time-dependent
manner. Studies have revealed that iron chelators could form redox-active iron
complexes, inducing cytotoxicity via Fenton chemistry [43]. To confirm whether
these TSCs derivatives can act as iron chelators, we tested the cell viability of PC3
cells treated with IC50 concentrations of represented compounds in the presence of
3
+
3+
Fe by MTT assay. We can observe that the addition of Fe resulted in more potent
activity in comparison to compounds treatment alone, indicating that these
compounds could be regarded as potential iron chelators (Fig. S1). To further evaluate
the antiproliferative activity of compound 16f, colony assay was performed. The
1
3

results revealed that compound 16f could obviously decrease colony formation both
in PC3 and DU-145 cells, especially in the high dose groups, compared to the control
groups (Fig. 3B, Fig. 3C).
Fig. 3. A. Cell viability in PC3 and DU-145 cells treated with serial concentrations of compound
1
6f for 24 h, 48 h and 72 h, respectively, by MTT assay. B. Representative pictures of colony
formation stained by crystal violet in PC3 and DU-145 cells treated with indicated concentrations
of compound 16f for 6 days. C. The absorbance of crystal violet in PC3 and DU-145 cells
measured at 570 nm. The data was presented as Mean ± SD. Three individual experiments were
performed. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control (Con) group.
2
.2.3 Effect of compound 16f on cell cycle in PC3 and DU-145 cells.
Next, cell cycle assay was carried out to explore whether the antiproliferative
activity of compound 16f was related to cell cycle arrest in PC3 and DU-145 cells.
PC3 and DU-145 cells treated with indicated concentrations of compound 16f for 48 h
were incubated in PI staining buffer, and they were analyzed through flow cytometry.
The results demonstrated that compound 16f could induce G1/S cycle arrest as the
concentrations of compound 16f increased. The percentages of cells in the G1/S phase
1
4

were increased from 19.23% to 19.98%, 29.04% and 49.02%, respectively in PC3
cells, compared to that in the control group. Similarly, compound 16f also triggered
G1/S cycle arrest in DU-145 cells dose-dependently (Fig. 4A, Fig. 4B). In addition, as
an inhibitor of the cyclin-dependent kinase, P27 reflects the G1/S restriction [44].
Therefore, we further tested the protein levels of P27 in the PC3 and DU-145 cells
treated with different concentrations of compound 16f for 48 h by western blot. As
expected, the results displayed that P27 was significantly increased in PC3 and
DU-145 cells affected by compound 16f (Fig. 4C).
Fig. 4. A. Representative pictures of cell cycle in PC3 and DU-145 cells treated with compound
1
6f at different concentrations for 48 h. B. Quantitative analysis of the percentages of cells cycle
distribution in PC3 and DU-145 cells affected by compound 16f. C-D. The western blot analysis
and quantitation of P27 protein in PC3 and DU-145 cells affected by indicated concentrations of
compound 16f for 48 h. The data was presented as Mean ± SD. Three individual experiments were
performed. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control (Con) group.
1
5

2
.2.4 Effect of compound 16f on apoptosis in PC3 and DU-145 cells.
Moreover, Hoechst 33342 staining was used to determine the pro-apoptotic ability
of analogue 16f. As shown in Fig 5A, evident morphological changes, including cell
rounding, formation of apoptotic bodies and nuclear fragmentation were presented
and increased in the PC3 and DU-145 cells treated with compound 16f. Next, we
assessed cell apoptosis using Annexin V-FITC/PI double staining. Significant increase
of positive cells was observed in PC3 and DU-145 cells, suggesting potential
pro-apoptotic effects of compound 16f (Fig. 5B, Fig. 5C). Death receptor signaling
and mitochondrial control of apoptosis are the main pathways in the regulation of
tumor apoptosis [45]. Thus, we investigated the expression of apoptosis-related
proteins in PC3 and DU-145 cells treated with different concentrations of compound
1
6f by western blot (Fig. 5D, Fig. 5E). The results exhibited that the expression of
anti-apoptotic protein Bcl-2 was markedly reduced, while pro-apoptotic protein Bax
was increased. Simultaneously, the expression of cleaved caspase 9/3 and PARP
exhibited a significant elevation in a concentration-dependent manner. Additionally,
compound 16f promoted the expression of P53 in PC3 and DU-145 cells. Taken
together, these finding suggested that compound 16f could induce human prostatic
cancer PC3 and DU-145 cell apoptosis potentially via both the death receptor and
mitochondria-mediated
pathways.
1
6

Fig. 5. A. Hoechst 33342 staining in PC3 and DU-145 cells treated with different concentrations
of compound 16f for 72 h (changes in cell morphology marked with red arrows). B.
Representative pictures of apoptotic distributions in PC3 and DU-145 cells affected by compound
1
6f at indicated concentrations for 72 h by Annexin V-FITC/PI double staining. C. The
quantitative analysis of apoptotic rates at both early and advanced stages of PC3 and DU-145 cells
1
7

by flow cytometry. D. The western blot analysis of the apoptosis-related proteins. F. The
quantitative analysis of the protein levels. The data was presented as Mean ± SD. Three individual
experiments were performed. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control (Con)
group.
2
.2.5 Effect of compound 16f on reactive oxygen species (ROS) in PC3 and DU-145
cells.
Excess accumulation of ROS in cells can lead to oxidative stress damage and tumor
cell death, and contribute to enhancing cell cycle arrest and apoptosis [46, 47]. In
addition, the anticancer activity of TSCs was associated with their ability to generate
ROS in a Fenton and Haber-Weiss reaction [23, 30, 34]. In order to explore the
potential mechanism of compound 16f induced cell cycle arrest and apoptosis, we
detected the intracellular ROS level in PC3 and DU-145 cells treated with compound
1
6f by flow cytometry (Fig. 6A). The results suggested that compound 16f could
dose-dependently increase ROS production both in PC3 and DU-145 cells (Fig. 6B).
Importantly, pretreatment with 5 mM of NAC, a strong antioxygen, obviously
alleviated the intracellular level of ROS induced by compound 16f stimulation (Fig.
6
C, Fig. 6D). To further understand the potential mechanism underlying the
accumulation of intracellular ROS induced by compound 16f in PC3 and DU-145
cells, MAPK signaling pathway was analyzed. As shown in Fig. 6E, the MAPK
signaling pathway was activated by compound 16f. The phosphorylation levels of
ERK1/2 (Thr202/Tyr204), JNK (Thr183/Tyr185) and P38 (Thr180/Tyr182) were
significantly increased in response to compound 16f treatment. In addition, the
statistical analysis of the western blot revealed an obvious elevation in the
p-JNK/JNK, p-P38/P38 and p-ERK/ERK ratios in compound 16f treatment groups,
compared to those in the control (Con) groups (Fig. 6F). Of note, p-JNK and p-ERK
are known mediators of cell proliferation and inhibition of apoptosis [48]. Moreover,
nuclear P27, increased by compound 16f in PC3 and DU-145 cells (Fig. 4C), can
effectively regulate MAPK pathway [49]. In order to further verify whether P38
MAPK pathway participated in the compound 16f-induced apoptosis, PC3 and
1
8

DU-145 cells were pretreated with 4 mM P38 inhibitor SB203580 for 4 h, followed
by treatment with the IC concentration of compound 16f for 72 h, respectively, by
5
0
MTT assay. Expectedly, the results displayed that the P38 inhibitor (SB203580)
effectively improved the cell viability in PC3 and DU-145 cells treated with
compound 16f, respectively, suggesting that P38 MAPK pathway indeed played an
important role in the compound 16f-induced apoptosis (Fig. 6G). In general, the
above results suggested that compound 16f resulted in high levels of ROS by
activating the MAPK signaling pathway in PC3 and DU-145 cells, which may be
related to cell cycle arrest and apoptosis.
1
9

Fig. 6. A. Representative images of ROS production in PC3 and DU-145 cells treated with
analogue 16f at different concentrations for 48 h by DCFH-DA staining. B. The quantitative
analysis of ROS levels in PC3 and DU-145 cells affected by compound 16f through flow
cytometry. C. Representative images of ROS production in PC3 and DU-145 cells treated with
compound 16f for 48 h in the presence or absence of NAC (5 mM). D. The quantitative analysis of
ROS levels in PC3 and DU-145 cells affected by compound 16f for 48 h in the presence or
absence of NAC (5 mM) through flow cytometry. E. The western blot analysis of ERK1/2,
2
0

p-ERK1/2, JNK, p-JNK, P38 and p-P38 in PC3 and DU-145 cells treated with indicated
concentrations of compound 16f. F. The quantitative analysis of these protein levels in PC3 and
DU-145 cells affected by compound 16f. G. Cell viability in PC3 and DU-145 cells pretreated with
4
mM p38 inhibitor SB203580 for 4 h, respectively, followed by treatment with the IC50
concentrations of compound 16f for 72 h, respectively, by MTT assay. The data was presented
as Mean ± SD. Three individual experiments were performed. *p < 0.05, **p < 0.01, ***p < 0.001
compared with the control (Con) group, #p < 0.05, ##p < 0.01 compared with the compound 16f
group.
2
.2.6 Safety evaluation of compound 16f in vivo.
The potential side effects have limited clinical utility of thiosemicarbazone
derivatives to some extent [8]. Given highly antiproliferative activity and selectivity
toward PC3 cells, acute toxicity test was performed to evaluate the safety of
compound 16f in vivo. Mice were administrated intragastrically at a dosage of 2g/kg
at a time, and then they were fed normally and observed for two weeks. During this
time, not only the mice in the 16f treated group survived, but they maintained their
weight without any noticeable behavioral abnormalities (Fig. 7A). In addition, H&E
staining demonstrated that compound 16f showed no signs of toxicity in the heart,
liver, spleen, lung and kidney, compared to the control group (Fig. 7B). These data
revealed that compound 16f had no obvious toxicity and side effect in vivo.
Fig. 7. A. The weight gain of the mice intragastrically administrated with compound 16f or control
oil. B. The representative H&E staining of the major tissue sections from compound 16f treated
2
1

and control mice. n= 10 per group.
2
.2.7 Antitumor studies of compound 16f on xenograft model bearing PC3 cells.
Given the potent antiproliferative and pro-apoptotic activity of compound 16f toward
prostate cancer cells in vitro, we then further investigated the antitumor effect of 16f
in vivo through a xenograft model bearing PC3 cells. The corresponding solvent and
3
-AP were used as negative and positive controls, respectively. The mouse body
weight and tumor volume were measured every 2 days. The results demonstrated that
compound 16f treatment at 12.5 mg/kg and 25 mg/kg significantly alleviated the
tumor volume, compared to that in the negative control group (Fig. 8A, Fig. 8B). The
average tumor weight in compound 16f treatment group mice was decreased to 53.6%
and 43.4%, respectively (Fig. 8C). In addition, in comparison to 3-AP, compound 16f
displayed similar inhibitory effect in the same concentration. Besides, compound 16f
did not result in obvious side effects in the body weight and general health, compared
to the negative control mice (Fig. 8D). These data indicated that compound 16f could
effectively inhibit tumor growth through a xenograft model bearing PC3 cells.
2
2

Fig. 8. PC3 cells were transplanted subcutaneously to the right sides of BALB/C-NU mice and
subjected to 0.5% CMC-Na, 3-AP (10 mg/kg) and 16f (12.5 and 25 mg/kg), respectively, for 21
days. A. Images of tumors. B. Tumor volume. C. Final tumor weight. D. Body weight in each
group. The data was presented as Mean ± SD. n=9 per group. **p < 0.01, ***p < 0.001 compared
with the 0.5% CMC-Na group.
3
Conclusions
In summary, we have designed and synthesized a variety of new thiosemicarbazone
derivatives and carried out systematic SAR research. Particularly, we found that
introduction of benzamide group into the linker was beneficial to anticancer activity
and selectivity. Additionally, replacement NNS donor with other donor chelators
resulted in significantly decreased potency, suggesting that NNS donor was
responsible for antiproliferative activity. Besides, most described compounds
displayed strong biological activity against prostate cancer PC3 cells. The most
promising molecule 16f was confirmed potent activity and high selectivity toward
PC3 cells, as well as no obvious toxicity in vivo and in vitro. Preliminary mechanism
studies revealed that compound 16f effectively inhibited colony formation in PC3 and
DU-145 cells. Also, G1/S cycle arrest and apoptosis were induced by derivative 16f,
which may be linked to ROS-mediated MAPK pathways. More importantly, analogue
1
6f could significantly suppress tumor growth in the xenograft model bearing PC3
cells. Our findings highlight thiosemicarbazone derivative 16f as a promising
compound for further study regarding novel anti-prostate cancer therapies.
4
. Experimental section
4
.1 General
We determined melting points of all analogues using the WRS-1A digital melting
point apparatus. Thin-layer chromatography (TLC) was performed on the glass pane
that painted with silica gel, visualized by 254 nm ultraviolet lamp. All chemicals and
solvents were obtained from commercial sources and used without further purification.
High resolution mass spectra (HRMS) were recorded on a Waters Micromass Q-T of
2
3

Micromass spectrometer by electrospray ionizaton (ESI). NMR spectra were recorded
1
13
on a Bruker 400 MHz spectrometer (400 and 100 MHz for the H and C nuclei,
respectively) using DMSO as solvent from commercial sources. The purity of all
compounds was examined 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 H O (90:10, v/v). The data
2
in this study was analyzed by one-way analysis of variance, and the results were
expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically
significant.
4
.2 General procedure for the synthesis of compound
（4）.
To a solution of commercially available 1 (3.125 mmol) in anhydrous ethanol (18
mL) was added triethylamine (6.25 mmol) and CS (6.25 mmol). The mixture was
2
stirred at room temperature for 3h. Upon completion, then DMAP (0.425 mmol) and
Boc O (8.5 mmol) were added, and the reaction mixture was stirred at room
2
temperature for another 0.5 h. The solvent was distilled off and extracted with ethyl
acetate. The combined organics were dried and evaporated to dryness. Purification by
silica chromatography afforded intermediate 2 as a colorless liquid. A mixture of 2
(2.5 mmol) and hydrazine hydrate (7.5 mmol) in dichloromethane (15 mL) was stirred
under at room temperature for 2 h. The precipitated solid was filtered off and washed
with dichloromethane to give the title compound 3 as white solid. To the solution of 3
(0.64 mmol) in ethanol containing 5-methylpicolinaldehyde (0.77 mmol), catalytic
amount acetic acid (0.064 mmol) was added. The reaction mixture was stirred at room
temperature for 2 h. Upon completion, formed precipitation was filtered washed with
ethanol to provide derivative 4 as yellow solid in 69% yield.
4
.2.1
E)-N-(2-(1H-indol-3-yl)ethyl)-2-((5-methylpyridin-2-yl)methylene)hydrazine-1-carbo
thioamide (4)
(
o
1
Yellow solid, m. p. 178.7-180.4 C, yield 69%, purity 98.63%. H NMR (400 MHz,
DMSO-d6, ppm) δ 11.68(s, 1H), 10.86(s, 1H), 8.69 (dd, J = 20.2, 14.7 Hz, 1H), 8.41
2
4

(s, 1H), 8.08 (s, 1H), 8.05 (d, J = 8.1 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 7.68 (d, J =
7
.9 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.22 (s, 1H), 7.08 (t, J = 7.4 Hz, 1H), 6.99 (t, J
13
=
7.4 Hz, 1H), 3.85 (dd, J = 14.1, 6.5 Hz, 2H), 3.10 – 2.94 (m, 2H), 2.33 (s, 3H). C
NMR (100 MHz, DMSO-d6, δ, ppm) δ 177.00, 150.64, 149.53, 142.21, 136.95,
1
4
3
36.26, 133.74, 127.24, 122.65, 121.00, 119.59, 118.53, 118.26, 111.53, 111.36,
+
4.36, 24.79, 17.89. HR-MS (ESI), calcd. C H N S, [M+H] m/z: 338.1439. found:
1
8
19
5
38.1435.
.3 General procedure for the synthesis of compound（9）.
4
A mixture of raw materials 1 (20 mmol), 4-aminocyclohexane-1-carboxylic acid
20 mmol), EDCI (20 mmol) and HOBT (20 mmol) in dichloromethane (100 mL)
(
were stirred at room temperature for 6 h. Upon completion of the reaction indicated
by TLC, the mixture was extracted with DCM for two times. The combined organic
layers were evacuated to afford the residue, which was then purified by
chromatography (silica gel, 5% methanol/dichloromethane) to obtain intermediate 6
as a white solid. Compound 9 was synthesized from intermediate 6, according to our
previously reported methods in 32% yields [39].
4
.3.1
N-(2-(1H-indol-3-yl)ethyl)-4-(hydrazinecarbothioamido)cyclohexane-1-carboxamide
8)
(
o
1
Yellow solid, m. p. 160.4-161.2 C, yield 38%. H NMR (400 MHz, DMSO-d6, ppm)
δ 10.78 (s, 1H), 8.66 (s, 1H), 7.85 (s, 1H), 7.68 (s, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.33
(d, J = 8.1 Hz, 1H), 7.19 – 6.90 (m, 3H), 4.54 (s, 2H), 4.29 (s, 1H), 3.31 (s, 2H), 2.81
1
3
(
t, J = 7.1 Hz, 2H), 2.20 (s, 1H), 1.79 – 1.46 (m, 8H). C NMR (100 MHz, DMSO-d6,
δ, ppm) δ 174.35, 136.19, 127.22, 122.57, 120.85, 118.25, 118.14, 111.86, 111.31,
+
4
3
4
7.79, 41.50, 29.01, 25.21, 24.78. HR-MS (ESI), calcd. C H N OS, [M+H] m/z:
1
8
25
5
60.1858. found: 360.1850.
.3.2
(E)-N-(2-(1H-indol-3-yl)ethyl)-4-(2-((5-methylpyridin-2-yl)methylene)hydrazine-1-ca
rbothioamido)cyclohexane-1-carboxamide (9)
o
1
Yellow solid, m. p. 210.1-213.4 C, yield 32%, purity 95.05%. H NMR (400 MHz,
DMSO-d6, ppm) δ 11.66 (s, 1H), 10.80 (s, 1H), 8.41 (s, 1H), 8.15 – 7.99 (m, 3H),
2
5

7
.86 (s, 1H), 7.66 (d, J = 7.5 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 8.0 Hz,
H), 7.14 (s, 1H), 7.06 (t, J = 7.3 Hz, 1H), 6.98 (t, J = 7.3 Hz, 1H), 4.30 (s, 1H), 3.34
1
(s, 4H), 2.84 (t, J = 7.1 Hz, 2H), 2.31 (s, 4H), 1.83 (d, J = 7.2 Hz, 4H), 1.61 (d, J =
1
3
3
9.7 Hz, 4H). C NMR (100 MHz, DMSO-d6, δ, ppm) δ 175.35, 174.12, 150.47,
1
49.60, 142.64, 137.01, 136.21, 133.81, 127.46, 127.23, 122.56, 120.85, 119.67,
118.25, 118.13, 111.90, 111.32, 50.81, 28.35, 25.67, 25.31, 17.89. HR-MS (ESI),
+
calcd. C H N OS, [M+H] m/z: 463.2280. found: 463.2272.
2
5
30
6
4
.4 General procedure for the synthesis of compound（16a-s）.
To a solution of 10a-e (15 mmol) and 11a-b (15 mmol) in dry dichloromethane
100 mL) were added EDCI (15 mmol) and HOBT (15 mmol) at room temperature.
(
The reaction was monitored by TLC. After 6 h stirring at room temperature, H O was
2
added. The aqueous layer was extracted twice with dichloromethane. The combined
organic layers were concentrated and purified by flash chromatography on silica gel
(5% methanol/dichloromethane) to give the intermediates 12a-f as solid. A mixture of
1
2a-f (10 mmol) and 10% Pd/C (2.2 mmol) in methanol (70 mL) was stirred under an
atmosphere of H at room temperature for 6 h. Upon completion, the reaction mixture
2
was filtered, and the filtrate was evaporated to dryness. Then, the residue was purified
by chromatography on silica gel (10% methanol/dichloromethane) to obtain 13a-f as
solid. Derivatives 16a-s were synthesized from intermediates 13a-f according to our
previously reported methods in 35-65% yields [39].
4
.4.1 N-(2-(1H-indol-3-yl)ethyl)-4-(hydrazinecarbothioamido)benzamide (15a)
o
1
Yellow solid, m. p. 150.6-152.5 C, yield 54%. H NMR (400 MHz, DMSO-d6, ppm)
δ 10.80 (s, 1H), 9.29 (s, 1H), 8.50 (s, 1H), 7.80 (dd, J = 15.0, 8.4 Hz, 4H), 7.59 (d, J =
7
1
7
1
.8 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 1.9 Hz, 1H), 7.07 (t, J = 7.5 Hz,
H), 6.98 (t, J = 7.4 Hz, 1H), 4.89 (s, 2H), 3.54 (dd, J = 13.6, 6.9 Hz, 2H), 2.95 (t, J =
1
3
.5 Hz, 2H). C NMR (100 MHz, DMSO-d6, δ, ppm) δ 165.72, 136.20, 127.25,
27.13, 122.57, 122.15, 120.90, 118.28, 118.21, 111.91, 111.34, 40.15, 25.19.
+
HR-MS (ESI), calcd. C H N OS, [M+H] m/z: 354.1388. found: 354.1381.
1
8
19
5
4
.4.2 N-(2-(1H-indol-3-yl)ethyl)-6-(hydrazinecarbothioamido)nicotinamide (15b)
2
6

o
1
Yellow solid, m. p. 168.1-169.4 C, yield 43%. H NMR (400 MHz, DMSO-d6, ppm)
δ 10.81 (s, 1H), 9.51 (s, 1H), 8.83 (d, J = 30.4 Hz, 2H), 8.37 (s, 1H), 7.97 (d, J = 8.3
Hz, 1H), 7.62 (d, J = 7.7 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.19 (s, 1H), 7.07 (t, J =
7
.3 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 3.59 (d, J = 6.6 Hz, 2H), 2.96 (t, J = 7.2 Hz, 2H).
C NMR (100 MHz, DMSO-d6, δ, ppm) δ 179.49, 163.49, 143.45, 136.25, 131.08,
1
3
1
27.22, 124.50, 122.54, 121.10, 120.92, 118.38, 118.19, 111.76, 111.32, 25.29.
+
HR-MS (ESI), calcd. C H N OS, [M+H] m/z: 355.1341. found: 355.1333.
1
7
18
6
4
.4.3
15c)
White solid, m. p. 148.7-149.1 C, yield 45%. H NMR (400 MHz, DMSO-d6, ppm) δ
1.02 (s, 1H), 9.29 (s, 1H), 8.50 (s, 1H), 7.79 (t, J = 13.6 Hz, 4H), 7.63 (s, 1H), 7.35
d, J = 8.5 Hz, 1H), 7.27 (s, 1H), 7.06 (d, J = 7.4 Hz, 1H), 4.87 (s, 2H), 3.51 (d, J =
N-(2-(5-chloro-1H-indol-3-yl)ethyl)-4-(hydrazinecarbothioamido)benzamide
(
o
1
1
(
1
3
6
1
4
3
4
.1 Hz, 2H), 2.93 (t, J = 7.2 Hz, 2H). C NMR (100 MHz, DMSO-d6, δ, ppm) δ
65.70, 134.66, 128.47, 127.12, 124.59, 122.97, 120.79, 117.61, 112.87, 111.96,
+
0.13, 24.98. HR-MS (ESI), calcd. C H ClN OS, [M+H] m/z: 388.0999. found:
1
8
18
5
88.0999.
.4.4 4-(hydrazinecarbothioamido)-N-(2-(5-methyl-1H-indol-3-yl)ethyl)benzamide
15d)
White solid, m. p. 166.2-167.5 C, yield 42%. H NMR (400 MHz, DMSO-d6, ppm) δ
(
o
1
1
7
2
0.65 (s, 1H), 9.29 (s, 1H), 8.48 (s, 1H), 7.80 (dd, J = 14.8, 8.5 Hz, 4H), 7.35 (s, 1H),
.22 (d, J = 8.2 Hz, 1H), 7.09 (d, J = 20.2 Hz, 1H), 6.89 (d, J = 8.2 Hz, 1H), 4.86 (s,
13
H), 3.52 (dd, J = 13.6, 6.6 Hz, 2H), 2.92 (t, J = 7.4 Hz, 2H), 2.36 (s, 3H). C NMR
(100 MHz, DMSO-d6, δ, ppm) δ 165.63, 134.61, 127.52, 127.14, 126.53, 122.68,
1
22.46, 122.14, 117.94, 111.43, 111.04, 40.28, 25.23, 21.25. HR-MS (ESI), calcd.
+
C H N OS, [M+H] m/z: 368.1545. found: 368.1538.
19
21
5
4
.4.5 4-(hydrazinecarbothioamido)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)benzamide
(15e)
o
1
Yellow solid, m. p. 166.2-167.4 C, yield 48%. H NMR (400 MHz, DMSO-d6, ppm)
δ 10.62 (s, 1H), 9.27 (s, 1H), 8.48 (s, 1H), 7.80 (d, J = 10.8 Hz, 4H), 7.22 (d, J = 8.7
2
7

Hz, 1H), 7.10 (d, J = 30.0 Hz, 2H), 6.71 (d, J = 8.3 Hz, 1H), 4.89 (s, 2H), 3.73 (s, 3H),
13
3
.53 (d, J = 5.7 Hz, 2H), 2.92 (t, J = 6.9 Hz, 2H). C NMR (100 MHz, DMSO-d6, δ,
ppm) δ 165.64, 152.95, 131.35, 127.61, 127.12, 124.50, 123.25, 122.10, 111.96,
11.77, 111.04, 100.16, 55.28, 40.13, 25.21. HR-MS (ESI), calcd. C H N O S,
1
1
9
21
5
2
+
[
M+H] m/z: 384.1494. found: 384.1487.
4
.4.6
4-(hydrazinecarbothioamido)-N-(2-(2-methyl-1H-indol-3-yl)ethyl)benzamide
(15f)
o
1
Yellow solid, m. p. 164.9-165.7 C, yield 50%. H NMR (400 MHz, DMSO-d6, ppm)
δ 10.71 (s, 1H), 9.30 (s, 1H), 8.51 (s, 1H), 7.79 (t, J = 12.9 Hz, 4H), 7.48 (d, J = 7.5
Hz, 1H), 7.23 (d, J = 7.6 Hz, 1H), 6.95 (dt, J = 14.8, 6.7 Hz, 2H), 4.88 (s, 2H), 3.40 (d,
13
J = 6.6 Hz, 2H), 2.88 (t, J = 7.2 Hz, 2H), 2.31 (s, 3H). C NMR (100 MHz,
DMSO-d6, δ, ppm) δ 165.64, 135.20, 132.05, 128.36, 127.11, 122.06, 119.88, 118.06,
1
17.31, 110.34, 107.70, 40.31, 24.15, 11.19. HR-MS (ESI), calcd. C H N OS,
19 21 5
+
[
M+H] m/z: 368.1545. found: 368.1538.
.4.7
E)-N-(2-(1H-indol-3-yl)ethyl)-4-(2-((5-methylpyridin-2-yl)methylene)hydrazine-1-c
arbothioamido)benzamide (16a)
4
(
1
White solid, m. p. 204.1-205.4°C, yield 65%, purity 98.45%. H NMR (400 MHz,
DMSO-d6, ppm) δ 12.09 (s, 1H), 10.81 (s, 1H), 10.31 (s, 1H), 8.67 – 8.50 (m, 1H),
8.44 (s, 1H), 8.35 (d, J = 8.1 Hz, 1H), 8.20 (s, 1H), 7.85 (dd, J = 17.8, 7.3 Hz, 2H),
7.71 (dd, J = 14.6, 8.4 Hz, 3H), 7.60 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H),
7.20 (s, 1H), 7.08 (t, J = 7.4 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 3.56 (dd, J = 13.4, 6.9
1
3
Hz, 2H), 2.97 (t, J = 7.4 Hz, 2H), 2.34 (s, 3H). C NMR (100 MHz, DMSO-d6, δ,
ppm) δ 175.97, 165.64, 150.46, 149.59, 143.63, 141.40, 136.91, 136.23, 134.06,
1
31.31, 127.28, 127.07, 124.88, 122.59, 120.89, 120.21, 118.28, 118.20, 111.92,
+
1
11.35, 25.20, 17.93. HR-MS (ESI), calcd. C H N OS, [M+Na] m/z: 479.1630.
2
5
24
6
found: 479.1627.
2
8