Synthesis and anticancer activities of thiosemicarbazones derivatives of thiochromanones and related scaffolds

Article abstract of DOI:10.1007/s00044-020-02503-w

A series of novel thiosemicarbazone analogs (4a–t, 6a–j) were synthesized and evaluated for their cytotoxic activities. The obtained results showed that thiochromanone-based thiosemicarbazones substituted primarily at the C-8 position exhibited higher cytotoxicity than the corresponding 1,1-dioxo-thiochromanone-, benzothiazepine-, and 1,1-dioxo-benzothiazepine-based analogs. Significantly, compound 4c (8-fluoro thiochromanone thiosemicarbazone) was found to be the most active and exhibited potent cytotoxicity against the MCF-7, SK-mel-2, and DU145 cancer cell lines, with IC₅₀ values of 0.42, 0.58, and 0.43 μM, respectively. In addition, the mechanism of compound 4c induced MCF-7 cell apoptosis was preliminarily investigated through cell cycle, Annexin V-FITC/PI staining, and ROS assays, indicating that compound 4c may exert its anticancer property through ROS-mediated apoptosis.

Full text of DOI:10.1007/s00044-020-02503-w

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02503-w
ORIGINAL RESEARCH
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Synthesis and anticancer activities of thiosemicarbazones
derivatives of thiochromanones and related scaffolds
1,2
Rongkai Pan1,2 Guobi Li1,2 Wenyi Su1 Xiumei Song3 Jincheng Li1 Shenggui Liu1,2
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Jiangli Song
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Received: 11 September 2019 / Accepted: 7 January 2020
© Springer Science+Business Media, LLC, part of Springer Nature
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Abstract
A series of novel thiosemicarbazone analogs (4a–t, 6a–j) were synthesized and evaluated for their cytotoxic activities. The
obtained results showed that thiochromanone-based thiosemicarbazones substituted primarily at the C-8 position exhibited
higher cytotoxicity than the corresponding 1,1-dioxo-thiochromanone-, benzothiazepine-, and 1,1-dioxo-benzothiazepine-
based analogs. Significantly, compound 4c (8-fluoro thiochromanone thiosemicarbazone) was found to be the most active
and exhibited potent cytotoxicity against the MCF-7, SK-mel-2, and DU145 cancer cell lines, with IC50 values of 0.42, 0.58,
and 0.43 μM, respectively. In addition, the mechanism of compound 4c induced MCF-7 cell apoptosis was preliminarily
investigated through cell cycle, Annexin V-FITC/PI staining, and ROS assays, indicating that compound 4c may exert its
anticancer property through ROS-mediated apoptosis.
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Keywords Thiosemicarbazone Thiochromanone Benzothiazepine Cytotoxic activity Apoptosis
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Introduction
and molecularly targeted therapy (Ocana et al. 2011;
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Sharma et al. 2011; Prat and Baselga 2008; Lee et al. 2018).
Although many novel methods have been developed for the
treatment of cancer over the past few years, in most cases
chemotherapy is still the leading choice.
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Cancer, as a second leading cause of death, is a serious
health problem characterized by uncontrolled growth and
spread of abnormal cells. Over 11 million people worldwide
are diagnosed with cancer, among which seven million
deaths are reported annually (Rawat 2008). According to
GLOBALCAN survey conducted in 2018 (Bray et al.
2018), cancer incidence and death rates have risen in recent
years, despite all the advances achieved in the field of
cancer therapy, including classical cytotoxic chemotherapy
Cisplatin is one of the most widely used chemotherapeutic
drugs and is utilized to treat various solid tumors, such as
testicular, ovarian, lung, head, and neck cancers (Wang and
Lippard 2005; Kelland 2007; Hannon 2007). The anticancer
activity of this drug is effectuated by damaging the DNA of
cancer cells. (Achkar et al. 2018; Petrović and Todorović
2016). However, drug resistance and grave side effects, such
as nephrotoxicity, ototoxicity, hepatotoxicity, and gastro-
intestinal toxicity, limit the application and efficacy of
cisplatin-based therapy (Ho et al. 2016; Dasari and Tchoun-
wou 2014; Sheth et al. 2017; Lu and Cederbaum 2006; Işeri
et al. 2007). Therefore, the development of new anticancer
drugs with high efficacy and low side effects is still urgently
needed in academic and industrial cancer research.
Thiosemicarbazone and its derivatives are bioactive
heterocycles featuring a wide spectrum of biological activ-
ity, especially antitumor activity (Taşdemir et al. 2015;
Czubatka-Bieńkowska et al. 2017; Kılıç-Cıkla et al. 2016;
Bisceglie et al. 2015; Merlot et al. 2013), as they are capable
of selectively killing tumor cells. Examples of
thiosemicarbazone-derived compounds include triapine®, a
chemical that has already been tested in clinical trials
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02503-w) contains supplementary
material, which is available to authorized users.
* Shenggui Liu
lsgui@sohu.com
1
School of Chemistry & Chemical Engineering, Lingnan Normal
University, Cunjin Road 29, Chikan District, Zhanjiang 524048
Guangdong, PR China
2
Key Laboratory of Clean Energy Materials Chemistry of
Guangdong Higher Education Institute, Cunjin Road 29, Chikan
District, Zhanjiang 524048 Guangdong, PR China
3
Industrial Technology Research Institute, Lingnan Normal
University, Cunjin Road 29, Chikan District, Zhanjiang 524048
Guangdong, PR China

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(Wadler et al. 2004; Mackenzie et al. 2007; Ma et al. 2008),
as well as di-2-pyridylketone 4,4-dimethyl-3-thiosemi-
carbazone (Dp44mT) and di-2-pyridylketone 4-cyclohexyl-
4-methyl-3-thiosemicarbazone (DpC), both of which are di-
2-pyridylketone thiosemicarbazone (DpT) analogs that
exhibit significant, selective inhibitory effects against the
proliferation of tumor cells (Whitnall et al. 2006; Yuan et al.
2004). Currently, DpC is being used in clinical trials for the
treatment of advanced human tumors (Guo et al. 2016). The
antitumor activity of thiosemicarbazone-derived com-
pounds is triggered by the coordination of the thiosemi-
carbazone moiety to metals such as palladium and platinum.
This results in the generation of thiosemicarbazones metal
complexes that affect critical molecular targets in tumors
(Quiroga and Ranninger 2004). Moreover, thiosemicarba-
zone derivatives function as cathepsin L inhibitors by
attacking the thiolate group of a Cys residue at the active
site of an enzyme, thereby inhibiting the proliferation of
tumors (Parker et al. 2015; Chavarria et al. 2012). Thiose-
micarbazones were also reported to induce the apoptosis of
cancer cells via mitochondrial pathway (Zhao et al. 2017;
Qi et al. 2018). Consequently, these compounds have
attracted a lot of attention for use in the development of new
cancer drugs, and they have been utilized in diverse phar-
maceutical applications (Li et al. 2010).
In recent years, numerous thiochromanones-derived com-
pounds were synthesized and their antitumor activities reported
(Cao et al 2019; Deng et al. 2018; He et al 2019; Pati et al.
2018; De Siqueira et al. 2019; Subasi et al. 2020). Some of
these structurally modified chemicals demonstrated strong
inhibitory effects against various cancer cells (Demirayak et al.
2017). In particular, two thiochromanone thiosemicarbazone
compounds functionalized primarily at the C-6 position were
identified as potent inhibitors of cathepsin L (Song et al. 2012).
However, there are very few reports about thiochromanone-
based thiosemicarbazones modified at the C-8 position. Based
on the previous studies, we evaluated the cytotoxic activities of
compounds 4a–e against different types of cathepsin L-secreting
tumor cells during cancer progression (Gocheva et al. 2006;
Joyce et al. 2004; Chen and Platt 2011; Benítez-Bribiesca et al.
1995; Friedrich et al. 1999). To further explore the antitumor
effects of thiochromanone-based thiosemicarbazone derivatives
functionalized at the C-8 position, their cytotoxic activities were
compared with those of 1,1-dioxothiochromanone, benzothia-
zepine, and 1,1-dioxo benzothiazepine analogs.
(thiochromanones, 1,1-dioxothiochromanones, benzothiaze-
pines, and 1,1-dioxobenzothiazepines) with thiosemicarbazone
or its derivatives in dry methanol under refluxing conditions, as
reported in the literature (Song et al. 2012; Song et al. 2013).
First, an SN2 reaction between thiophenol and 3-
bromopropionic acid was carried out to produce carboxylic
acids that were later cyclized in the presence of PPA, resulting
in the formation of ketones (thiochroman-4-ones and ben-
zothiazepines). The oxidation of thiochroman-4-ones and
benzothiazepines led to the generation of 1,1-dioxothiochro-
manones and 1,1-dioxobenzothiazepines compounds, respec-
tively. All the synthesized thiosemicarbazones (Fig. 1) were
purified through silica gel column chromatography or recrys-
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1
tallization. Further, characterizations were done by H NMR,
13C NMR, and HRMS. More details are presented in
Scheme 1.
In vitro evaluation of the cytotoxic activity
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In vitro cytotoxicity assessments of the synthesized thio-
semicarbazones (4a–t, 6a–j) were carried out against the
MCF-7, SK-mel-2, and DU145 cancer cell lines, as well as
the human embryonic lung fibroblasts (HELF) normal cell
line, using the CCK-8 assay (Wang et al. 2018). The IC50
(μM) values summarized in Table 1 showed that, in general,
the investigated compounds exhibited various degrees of
cytotoxicity toward cancer cells. Of these compounds, four
compounds (compounds 4a, 4b, 4c, and 4q) showed better
activities than the positive control cisplatin. In particular,
compound 4c displayed remarkable cytotoxic effects
against all the tested cancer cell lines, with IC50 values
<1 μM. Other compounds exhibited significant cytotoxicity
against one or two types of cancer cells, with IC50 values in
the range of 0.11–6.74 μM. Specifically, compounds 4e, 4p,
6b, and 6c are active against two cancer cell lines, whereas
compounds 4r, 4t, 6a, and 6d are only active against one
type of cancer cells. Notably, 1,1-dioxo-thiochromanone
thiosemicarbazone analog (compounds 6a–e) did not exhi-
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Q1
bit appreciable cytotoxicity against HELF cells (IC50
40 μM).
>
Structure–activity relationships (SARs) of all compounds
indicated that all the substituents at the C-8 position of
thiochromanone thiosemicarbazone analogs (compounds
4a–e) displayed potent activity against at least one type of
cancer cells, except for compound 4d. The substitution of
protons at the terminal amino group by CH3 or Ph groups
greatly reduced the cytotoxic effects of the analogs (com-
pounds 4f–j and 4k–o) against all three cancer cell lines.
This indicated that the hydrogen atoms of the terminal
amino group conferred enhanced cytotoxic activity, com-
pared with methyl and phenyl substituents. Increasing the
heteroatom-alkyl ring size (thiochromanone thiosemicarba-
zones, compounds 4p–t) and changing the sulfide
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Results and discussion
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Synthesis
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As shown in Scheme 1, 30 thiosemicarbazone derivatives were
prepared
by
condensation
of
different
ketones

Medicinal Chemistry Research
Scheme 1 Synthesis of
compounds 4a–t and 6a–j.
Reaction conditions and
reagents: a n = 1, NaOH/
Na2CO3/H2O, 100 °C, 1.5 h; n
= 2, K2CO3/DMF, R.T., 15 h; b
n = 1, PPA, 80 °C, 2 h; n = 2,
110 °C, 2 h; c
Thiosemicarbazide TsOH/
MeOH, reflux, 10 h, 12–89%; d
H2O2 (35% w/w)/AcOH, reflux,
2 h; e Thiosemicarbazide TsOH/
MeOH, reflux, 10 h, 38–72%
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substituent at position-1 to sulfone (thiochromanone and
benzothiazepine thiosemicarbazones, compounds 6a–e and
6f–j) also decreased the cytotoxicity of the investigated
compounds against the cancer cell lines. Overall, the results
suggested that the cytotoxic effects of thiochromanone-
based thiosemicarbazone compounds substituted at the C-8
position (compounds 4a–e) were greater than those corre-
sponding to other analogs. Interestingly, p-electro-donating
groups (CH3, OCH3) in benzothiazepine thiosemicarbazone
analogs (compounds 4p, 4q) produced greater cytotoxic
effects than the electron-withdrawing counterparts (F, Cl,
Br) (compounds 4r, 4s, 4t). Meanwhile, the electron-
withdrawing fluorine (F) substituent in thiochromanone
thiosemicarbazone analogs (compound 4c) yielded
enhanced cytotoxic activity compared with the p-electron-
donating counterparts (CH3, OCH3) (compounds 4a, 4b).
Fig. 1 The structures of compounds 4a–t and 6a–j
compound 4c was chosen for the assessment of induced
MCF-7 cell apoptosis. For this purpose, MCF-7 cells treated
with compound 4c were labeled with Annexin V-FITC/PI
and analyzed by flow cytometry in order to detect the
apoptotic cells. The results illustrated in Fig. 2 showed that
the percentage of apoptosis increased from 4.02 to 17.3%
and from 2.6 to 31.1% with increasing concentration of
compound 4c, during the early (Q3 area) and late (Q2 area)
stages, respectively. This indicated that compound 4c
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Compound 4c induces cell apoptosis in MCF-7 cells
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Considering that it had shown the highest cytotoxic activity
among all investigated thiosemicarbazone derivatives,

Medicinal Chemistry Research
Table 1 In vitro cytotoxic
activity using CCK-8 assay of
compounds 4a–t and 6a–j
Comp.
R
X
Y
n
Cytotoxicity, IC50 [μM]a
MCF-7
SK-mel-2
DU145
HELF
4a
4b
4c
4d
4e
4f
8-CH3
8-OCH3
8-F
S
H
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
1
1
1
1
1
2
2
2
2
2
1.78 0.04
0.47 0.04
0.42 0.07
22.51 2.79
8.63 0.41
>40
1.40 0.07
0.38 0.02
0.58 0.03
>40
1.31 0.04
1.50 0.07
0.43 0.10
>40
>40
S
H
27.75 0.86
32.36 1.03
14.19 0.48
13.03 0.21
34.03 0.89
38.74 0.47
>40
S
H
8-Cl
S
H
8-Br
S
H
0.35 0.08
>40
0.11 0.04
>40
8-CH3
8-OCH3
8-F
S
CH3
CH3
CH3
CH3
CH3
Ph
Ph
Ph
Ph
Ph
H
4g
4h
4i
S
32.83 0.38
>40
>40
>40
S
>40
>40
8-Cl
S
>40
>40
>40
>40
4j
8-Br
S
>40
>40
>40
>40
4k
4l
8-CH3
8-OCH3
8-F
S
6.08 0.07
>40
17.96 0.49
>40
>40
7.83 0.01
37.0 0.33
16.08 0.26
8.69 0.34
19.13 0.44
23.75 0.33
21.96 0.45
26.22 0.71
18.1 1.51
12.28 0.31
>40
S
>40
4m
4n
4o
4p
4q
4r
S
18.25 0.50
13.50 0.36
19.79 0.33
2.68 0.03
1.14 0.12
13.25 0.02
8.62 0.56
23.96 3.61
6.06 0.29
3.07 0.26
1.95 0.16
>40
>40
>40
8-Cl
S
16.74 0.17
17.18 0.30
3.09 0.10
0.52 0.08
11.14 1.84
>40
>40
8-Br
S
>40
9-CH3
9-OCH3
9-F
S
2.81 0.05
1.63 0.24
2.85 0.16
>40
S
H
S
H
4s
9-Cl
S
H
4t
9-Br
S
H
13.2 2.07
6.74 0.25
1.30 0.07
1.50 0.08
>40
0.67 0.06
5.84 0.09
8.94 1.27
4.99 0.41
1.71 0.11
9.11 2.26
26.25 1.34
>40
6a
6b
6c
6d
6e
6f
8-CH3
8-OCH3
8-F
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
H
H
>40
H
>40
8-Cl
H
>40
8-Br
H
>40
>40
>40
9-CH3
9-OCH3
9-F
H
24.66 0.63
>40
23.00 1.17
>40
>40
6g
6h
6i
H
>40
H
>40
>40
>40
>40
9-Cl
H
20.19 1.31
33.66 0.54
3.60 0.36
>40
>40
>40
6j
9-Br
H
22.64 0.38
12.60 0.50
14 0.30
1.80 0.24
35.78 0.29
nd
Cisplatinb
nd not determined
aIC50 value corresponding to the compound concentration required to inhibit tumor cells
proliferation by 50%
bCisplatin was used as positive control
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induced the apoptosis of MCF-7 cells in a concentration-
dependent manner.
cycle arrest at G2 phase. Therefore, we concluded that
compound 4c could affect the cell cycle of MCF-7 in a
concentration-dependent way.
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To investigate the mechanism of compound 4c cyto-
toxicity toward MCF-7 cells, the cell cycle analysis was
performed. As illustrated in Fig. 3, MCF-7 cells treated for
72 h with various concentrations of compound 4c (0, 0.5, 1,
2 μM) exhibited significant changes in the rates of pro-
liferation recorded during the three phases. Compared with
the control group, the proportion of 4c-treated cells in the
G2 stage increased from 4.67 to 11.64%, while the pro-
portion of cells in the S stage decreased from 37.26 to
23.79%. This suggested that compound 4c induced cell
The mechanisms of compound 4c-induced MCF-7 cell
apoptosis were investigated by examining the role of
intracellular reactive oxygen species (ROS). The ROS
levels in MCF-7 cells treated with various concentrations
(0, 0.125, 0.25, 0.5 μM) of compound 4c were determined
using fluorescence measurements. As illustrated in Fig. 4,
the fluorescence intensity of 2,7-dichlorofluorescein (DCF)
gradually increased with increasing concentration of com-
pound 4c (0–0.5 μM), which suggested that compound 4c

Medicinal Chemistry Research
Fig. 2 Annexin V-FITC/PI
staining assay of apoptotic
MCF-7 cells after 72 h with
different concentrations of
compound 4c (0, 0.5, 1.0,
2.0 μM)
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induced the production of ROS in MCF-7 cells in a
concentration-dependent manner.
tumor cell lines, with compound 4c showing the greatest
activity [IC50 0.42 μM (MCF-7), 0.58 μM (SK-mel-2), and
0.43 μM (DU145)]. The effects of the thiochromanone
scaffold and C-8 substitutions in enhancing the cytotoxic
activity of thiosemicarbazones against cancer cells were
further confirmed by SAR analyses. Based on mechanistic
evaluations, compound 4c arrested the cell cycle of MCF-7
cells at the G2/M phase and induced apoptosis by elevating
the intracellular ROS levels in a dose-dependent manner.
Therefore, thiochromanone-based thiosemicarbazones are
potential anticancer agents with ROS-mediated cytotoxic
effects against human breast cancer MCF-7 cells.
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ROS plays an important role in signaling and cancer cell
apoptosis. Specially, the overproduction of ROS can trigger
DNA damage and promote caspase-9 dependent apoptosis
(Thannickal and Fanburg 2000; Vurusaner et al. 2012;
Wang and Choudhary 2011). Anticancer drugs that induce
cellular apoptosis, such as cisplatin and quinones drugs, are
generally associated with increased levels of ROS (Tian
et al. 2012; Kalai Selvi et al. 2017; Chen et al. 2019).
Therefore, the increased ROS expressions in compound 4c-
treated MCF-7 cancer cells constitute a direct indication of
the compound’s effect in triggering apoptosis.
Material and methods
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Conclusion
Synthesis
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Thiosemicarbazone compounds primarily substituted at the
C-8 position were synthesized and assessed in terms of
cytotoxic activities. Compared with the reference cisplatin
drug, 12 of the 30 prepared compounds (4a–c, 4e, 4p–r, 4t,
6a–d) demonstrated strong cytotoxicity against several
All the reactions were performed in an inert atmosphere of
nitrogen gas. The chemicals and reagents used were
obtained from various suppliers (Sigma Aldrich Chem. Co.,
Energy Chemical Reagent Company, J&K Scientific
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Medicinal Chemistry Research
Fig. 3 Effect on the cell cycle of
the MCF-7 cells treated by
compound 4c for 72 h
Fig. 4 The intracellular ROS
level of MCF-7 cells treated by
compound 4c for 24 h (a)
analyzed by flow cytometry (b)
analyzed by fluorescence reader
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Company, Guangzhou Chemical Reagent Company). Thin
layer chromatography plates (glass plates pre-coated with
silica gel HSGF254, 0.2 0.03 mm thickness) and the silica
gel (200–400 mesh, 60 Å) used in manual flash column
chromatography analyses were both purchased from Yantai
Jiangyou Company. The former was used to monitor the
progress of the synthesis reactions, whereas the latter served
to purify the reaction intermediates and products. All
purified species were characterized by H NMR (Bruker
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operating at 400 MHz), 13C NMR (Bruker operating at
100 MHz), and high-resolution mass spectrometry (HRMS)
equipped with an electrospray ionization source (Thermo
Scientific LTQ Orbitrap Discovery Mass Spectrometer).
Deuterated CDCl3 (with 0.03% TMS as internal standard),
acetone-d6, and DMSO-d6 were used as common solvents
for the NMR studies.

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General procedure for thiochroman-4-one
thiosemicarbazone derivatives and 3,4-dihydro-1-
benzothiepin-5-one thiosemicarbazone derivatives
4(a–t)
8-Chlorothiochroman-4-one thiosemicarbazone (4d)
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Yellow solid; yield 38%; ¹H NMR (DMSO-d6, 400 MHz):
δ = 10.34 (1H, brs), 8.41 (2H, m), 8.09 (1H, s), 7.45 (1H, d,
J = 7.78 Hz), 7.13 (1H, t, J = 8.00 Hz), 3.06 (4H, m); 13C
NMR (DMSO-d6,100 MHz): δ = 179.54 (C, C=S), 144.91
(C, C=N), 135.88 (C, C-9), 133.56 (C, C-7), 131.16 (C, C-
10), 129.93 (C, C-8), 126.51 (C, C-5), 125. 75(C, C-6), 27.2
(CH2, C-2), 25.16 (CH2, C-3); HRESIMS m/z (pos):
272.0081 C10H11ClN3S2 (calcd. 272.0077).
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A solution of 0.212 mmol 7-methylthiochroman-4-one in
8 mL anhydrous methanol was refluxed for 15 min, fol-
lowed by the addition of thiosemicarbazide (0.545 mmol)
and p-toluene sulfonic acid catalyst. The mixture was
refluxed again for another 10 h, and the resulting precipitate
was collected by filtration and washed with methanol. After
drying under high vacuum, the thiosemicarbazone deriva-
tives were obtained.
8-Bromothiochroman-4-one thiosemicarbazone (4e)
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White solid; yield 37%; ¹H NMR (DMSO-d6, 400 MHz): δ
= 10.33 (1H, brs), 8.44 (1H, d, J = 7.6 Hz), 8.40 (1H, brs),
8.08 (1H, brs), 7.59 (1H, d, J = 7.6 Hz), 7.06 (1H, t, J =
7.6 Hz), 3.05 (4H, m); 13C NMR (DMSO-d6, 100 MHz): δ
= 179.54 (C, C=S), 145.16 (C, C=N), 137.72 (C, C-9),
133.87 (C, C-7), 133.23 (C, C-10), 127.07 (C, C-5), 126.21
(C, C-6), 121.98 (C, C-8), 27.27 (CH2, C-2), 25.67 (CH2, C-
3); HRESIMS m/z (pos): 315.9574 C10H11BrN3S2 (calcd.
315.9572).
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320
271
8-Methylthiochroman-4-one thiosemicarbazone (4a)
1
272
273
274
275
276
277
278
279
280
Yellow solid; yield: 51%; H NMR (DMSO-d6, 400 MHz):
δ = 10.23 (1H, brs), 8.34 (1H, brs), 8.23 (1H, d, J =
8.00 Hz), 7.9 (1H, brs), 7.19 (1H, d, J = 8.00 Hz), 7.04 (1H,
t, J = 8.00 Hz), 3.02 (4H, brs), 2.2 (3H, brs); 13C NMR
(DMSO-d6,100 MHz): δ = 179.39 (C, C=S), 146.42 (C,
C=N), 136.42 (C, C-9), 135.16 (C, C-8), 131.47 (C, C-7),
130.68 (C, C-10), 125.54 (C, C-5), 124.68 (C, C-6), 27.99
(CH2, C-2), 25.18 (CH2, C-3), 20.28 (C, CH3); HRESIMS
m/z (pos): 252.0629 C11H14N3S2 (calcd. 252.0624).
8-Methylthiochroman-4-one 4-methyl-thiosemicarbazone
(4f)
321
322
281
8-Methoxythiochroman-4-one thiosemicarbazone (4b)
White solid; yield: 62.5%; 1H NMR (DMSO-d6, 400 MHz):
δ = 10.24 (1H, s), 8.50 (1H, d, J = 4.5 Hz), 8.23 (1H, d, J
= 7.4 Hz), 7.19 (1H, d, J = 7.0 Hz), 7.06 (1H, t, J =
7.7 Hz), 3.04–3.01 (7H, m), 2.24 (3H, s); 13C NMR
(DMSO-d6, 100 MHz): δ = 179.11 (C, C=S), 146.26 (C,
C=N), 136.40 (C,C-9), 135.24 (C,C-8), 131.54 (C,C-7),
130.64 (C, C-10), 125.46 (C, C-5), 124.65 (C, C-6), 31.63
(CH3), 28.01 (CH2, C-2), 25.19 (CH2, C-3), 20.30 (CH3, N-
CH3); HRESIMS m/z (pos): 266.0783 C12H16N3S2 (calcd.
266.0780).
323
324
325
326
327
328
329
330
331
332
282
283
284
285
286
287
288
289
290
291
White solid; yield 76.73%; 1H NMR (DMSO-d6,
400 MHz): δ = 10.27 (1H, brs), 8.34 (1H, s), 8.0 (1H, d, J
= 8.12 Hz), 7.98 (1H, brs), 7.07(1H, t, J = 8.04 Hz), 6.95
(1H, d, J = 8.3 Hz), 3.81 (3H, s), 2.96 (4H, m); 13C NMR
(DMSO-d6, 100 MHz): δ = 179.40 (C, C=S), 155.41 (C, C-
8), 145.88 (C, C=N), 131.93 (C, C-10), 126.16 (CH, C-6),
125.02 (C, C-9), 119.89 (C, C-5), 111.00 (C, C-7), 56.45
(CH3, O-CH3), 27.37 (CH2, C-2), 24.35 (CH2, C-3);
HRESIMS m/z (pos): 268.0577 C11H14N3OS2 (calcd.
268.0573).
8-Methoxythiochroman-4-one 4-methy-thiosemicarbazone
(4g)
333
334
292
8-Fluorothiochroman-4-one thiosemicarbazone (4c)
White solid; yield 74.69%; 1H NMR (DMSO-d6,
400 MHz): δ = 10.28 (1H, s), 8.50 (1H, d, J = 4.5 Hz), 8.02
(1H, dd, J = 8.1, 1.0 Hz), 7.11 (1H, t, J = 8.1 Hz), 6.96 (1H,
d, J = 7.2 Hz), 3.82 (3H, s), 3.04 (3H, d, J = 4.6 Hz), 2.96
(4H, s); 13C NMR (DMSO-d6, 100 MHz): δ = 179.10 (C,
C=S), 155.44 (C, C-8), 145.69 (C, C=N), 131.98 (C, C-
10), 126.10 (C, C-6), 124.98 (C, C-9), 119.82 (C, C-5),
110.94 (C, C-7), 56.45 (CH3, O-CH3), 31.64 (CH3, N-CH3),
27.37 CH2, C-2), 24.35 (CH2, C-3); HRESIMS m/z (pos):
282.0733 C12H16N3OS2 (calcd. 282.0729).
335
336
337
338
339
340
341
342
343
344
1
293
294
295
296
297
298
299
300
301
White solid; Yield: 49%; H NMR (DMSO-d6, 400 MHz):
δ = 10.35 (1H, brs), 8.41 (1H, s), 8.28 (1H, d, J = 7.88 Hz),
8.09 (1H, s), 7.17 (2H, m), 3.05 (4H, m); 13C NMR
(DMSO-d6, 100 MHz): δ = 179.57 (C, C=S), 158.47 (C, C-
8, d, J = 238 Hz), 144.36 (C, C=N), 133.41 (C, C-10),
125.70 (C, C-6, d, J = 9.0 Hz), 124.42 (C, C-5, d, J =
19.0 Hz), 123.61 (C, C-9, d, J = 3.0 Hz), 115.45 (C, C-7, d,
J = 21.0 Hz), 27.50 (CH2, C-2), 24.52 (CH2, C-3); HRE-
SIMS m/z (pos): 256.0378 C10H11FN3S2 (calcd. 256.0373).

Medicinal Chemistry Research
345
346
8-Fluorothiochroman-4-one 4-methyl-thiosemicarbazone
(4h)
8-Methoxythiochroman-4-one 4-phenyl-thiosemicarbazone
(4l)
391
392
347
348
349
350
351
352
353
354
355
356
White solid; yield: 46.5%; 1H NMR (DMSO-d6, 400 MHz):
δ = 10.37 (1H, s), 8.57 (1H, d, J = 4.5 Hz), 8.44 (1H, dd, J
= 8.1, 1.0 Hz), 7.61 (1H, d, J = 7.8 Hz), 7.10 (1H, t, J =
7.9 Hz), 3.09-3.00 (7H, m); 13C NMR (DMSO-d6,
100 MHz): δ = 179.17 (C, C=S), 158.51 (C, C-8, d, J =
237.6 Hz), 144.19 (C, C=N), 133.45 (C, C-10), 125.68 (C,
C-6, d, J = 8.4 Hz), 124.39 (C, C-5, d, J = 19.4 Hz), 123.47
(C, C-9, d, J = 2.86 Hz), 115.53 (C, C-7, d, J = 21 Hz),
31.66 (CH3), 27.52 (CH2, C-2), 24.53 (CH2, C-3); HRE-
SIMS m/z (pos): 270.0532 C11H13FN3S2 (calcd. 270.0529).
White solid; yield 76.73%;1H NMR (DMSO-d6, 400 MHz):
δ = 10.65 (1H, s), 10.07 (1H, s), 8.12 (1H, dd, J = 8.1,
0.9 Hz), 7.56 (2H, d, J = 7.5 Hz), 7.38 (2H, t, J = 7.9 Hz),
7.23 (1H, d, J = 7.4 Hz), 7.11 (1H, t, J = 8.1 Hz), 6.98 (1H,
dd, J = 8.1, 0.9 Hz,), 3.83 (3H, s), 3.05–3.00 (4H, m); 13C
NMR (DMSO-d6, 100 MHz): δ = 177.55 (C, C=S), 155.42
(C, C-8), 147.11 (C, C=N), 139.62 (C, C-1′), 131.73 (C, C-
10), 128.56 (C, C-3′), 126.43 (C, C-2′), 125.92 (C, C-6),
125.02 (C, C-5), 120.23 (C, C-9), 111.23 (C, C-7), 56.47
(C, OCH3), 27.64 (C, C-2), 24.35 (C, C-3); HRESIMS m/z
(pos): 366.0708 C17H17N3OS2Na (calcd. 366.0705).
393
394
395
396
397
398
399
400
401
402
403
357
358
8-Chlorothiochroman-4-one 4-methyl-thiosemicarbazone
(4i)
8-Fluorothiochroman-4-one 4-phenyl-thiosemicarbazone
(4m)
404
405
359
360
361
362
363
364
365
366
367
White solid; yield 56.6%; ¹H NMR (DMSO-d6, 400 MHz):
δ = 10.38 (1H, s), 8.58 (1H, d, J = 4.5 Hz), 8.41 (1H, dd, J
= 8.1, 1.2 Hz), 7.47 (1H, dd, J = 7.8, 1.2 Hz), 7.17 (1H, t, J
= 8.0 Hz), 3.08–3.02 (7H, m); 13C NMR (DMSO-d6,
100 MHz): δ = 179.17 (C, C=S), 144.73 (C, C=N), 135.86
(C, C-9), 133.63 (C, C-7), 131.23 (C, C-10), 129.90 (C, C-
8), 126.39 (C, C-5), 125.73 (C, C-6), 31.66 (C, CH3), 27.21
(CH2, C-2), 25.16 (CH2, C-3); HRESIMS m/z (pos):
286.0239 C11H13ClN3S2 (calcd. 286.0234).
White solid; yield 55.53%; ¹H NMR (DMSO-d6, 400 MHz):
δ = 10.69 (1H, s), 10.13 (1H, s), 8.38 (1H, d, J = 7.3 Hz),
7.55 (2H, d, J = 7.6 Hz), 7.39 (2H, t, J = 7.8 Hz), 7.21 (3H,
ddd, J = 14.1, 8.1, 3.6 Hz), 3.12 (4H, s); 13C NMR (DMSO-
d6, 100 MHz): δ = 177.72 (C, C=S), 159.65 (C, C-8, d, J =
237.6 Hz), 157.27 (C, C=N), 145.59 (C, C-1′), 139.51 (C,
C-10), 133.12 (C, C-4′), 128.63 (C, C-3′), 126.66 (C, C-2′),
125.78 (C, C-6, d, J = 8.4 Hz), 124.64 (C, C-5, d, J =
19 Hz), 123.92 (C, C-9), 115.74 (C, C-7, d, J = 21 Hz),
27.77 (C, C-2), 24.54 (C, C-3); HRESIMS m/z (pos):
354.0506 C16H14FN3S2Na (calcd. 354.0505).
406
407
408
409
410
411
412
413
414
415
416
368
369
8-Bromothiochroman-4-one 4-methyl-thiosemicarbazone
(4j)
370
371
372
373
374
375
376
377
378
White solid; yield 45.1%; ¹H NMR (DMSO-d6, 400 MHz):
δ = 10.37 (1H, s), 8.57 (1H, d, J = 4.5 Hz), 8.44 (1H, dd, J
= 8.1, 1.0 Hz), 7.61 (1H, d, J = 7.8 Hz), 7.10 (1H, t, J =
7.9 Hz), 3.08–3.01 (7H, m); 13C NMR (DMSO-d6,
100 MHz): δ = 179.16 (C, C=S), 144.97 (C, C=N), 137.70
(C, C-9), 133.93 (C, C-7), 133.18 (C, C-10), 126.93 (C, C-
5), 126.18 (C, C-6), 122.06 (C, C-8), 31.66 (CH3), 27.29
(CH2, C-2), 25.67 (CH2, C-3); HRESIMS m/z (pos):
329.9725 C11H13BrN3S2 (calcd. 329.9729).
8-Chlorothiochroman-4-one 4-phenyl-thiosemicarbazone
(4n)
417
418
Yellow solid; yield 95%; ¹H NMR(DMSO-d6, 400 MHz): δ
= 10.72 (1H, s), 10.15 (1H, s), 8.53 (1H, dd, J = 8.1,
1.1 Hz), 7.53 (2H, d, J = 7.5 Hz), 7.49 (1H, dd, J = 7.8,
1.3 Hz), 7.39 (2H, dd, J = 10.6, 5.1 Hz), 7.24 (1H, d, J =
7.4 Hz), 7.16 (1H, t, J = 8.0 Hz), 3.12 (4H, s); 13C NMR
(DMSO-d6, 100 MHz): δ = 177.75 (C, C=S), 146.11 (C,
C=N), 139.58 (C, C-1′), 136.10 (C, C-9), 133.37 (C, C-7),
131.16 (C, C-10), 130.18 (C, C-8), 128.59 (C, C-3′), 126.78
(C, C-2′), 126.07 (C, C-5), 125.78 (C, C-6), 27.47 (C, C-2),
25.17 (C, C-3); HRESIMS m/z (pos): 348.0390
C16H15ClN3S2 (calcd. 348.0390).
419
420
421
422
423
424
425
426
427
428
429
379
380
8-Methylthiochroman-4-one 4-phenyl-thiosemicarbazone
(4k)
381
382
383
384
385
386
387
388
389
390
Yellow solid; Yield:74.89%; 1H NMR (DMSO-d6, 400 MHz):
δ = 10.58 (1H, s), 10.06 (1H, s), 8.33 (1H, d, J = 7.5 Hz), 7.57
(2H, d, J = 7.5 Hz), 7.42–7.34 (2H, m), 7.25–7.19 (2H, m),
7.10–7.04 (1H, m), 3.12–3.05 (4H, m), 2.26 (3H, s); 13C NMR
(DMSO-d6, 100 MHz): δ = 177.57 (C, C=S), 147.64 (C,
C=N), 139.63 (C,C-9), 136.68 (C,C-1′), 135.21 (C, C-8),
131.33 (C, C-7), 130.93 (C, C-10), 128.55 (C, C-3′), 126.35
(C, C-2′), 125.87 (C, C-5, C-4′), 124.70 (C, C-6), 28.25 (C, C-
2), 25.22 (C, C-3), 20.27 (CH3); HRESIMS m/z (pos):
350.0759 C17H17N3S2Na (calcd. 350.0756).
8-Bromothiochroman-4-one 4-phenyl-thiosemicarbazone
(4o)
430
431
White solid; yield 85%; ¹H NMR (DMSO-d6, 400 MHz): δ
= 10.71 (1H, s), 10.14 (1H, s), 8.56 (1H, dd, J = 8.1,
1.1 Hz), 7.63 (1H, dd, J = 7.8, 1.2 Hz), 7.53 (2H, d, J =
7.5 Hz), 7.39 (2H, dd, J = 10.7, 5.1 Hz), 7.23 (1H, t, J =
7.4 Hz), 7.09 (1H, t, J = 8.0 Hz), 3.11 (4H, s); 13C NMR
432
433
434
435
436

Medicinal Chemistry Research
437
438
439
440
441
442
(DMSO-d6, 100 MHz): δ = 177.76 (C, C=S), 146.34 (C,
C=N), 139.60 (C, C-9), 137.95 (C, C-1′), 133.82–133.79
(C, C-7), 133.57 (C, C-10), 128.59 (C, C-3′), 127.42 (C, C-
5), 126.69 (C, C-2′), 126.14 (C, C-4′), 121.98 (C, C-6),
100.00 (C, C-8), 27.55 (C, C-2), 25.68 (C, C-3); HRESIMS
m/z (pos): 413.9700 C16H14BrN3S2Na (calcd. 413.9705).
7.88 Hz), 2.98 (4H, m), 2.10 (2H, m); 13C NMR (DMSO-d6,
100 MHz): δ = 179.61 (C, C=S), 150.50 (C, C=N), 140.30
(C, C-10), 136.45 (C, C-8), 132.87 (C, C-11), 129.61 (C, C-
6), 129.49 (C, C-7), 126.90 (C, C-9), 40.17 (CH2, C-2),
32.77 (CH2, C-3), 27.48 (CH2, C-4); HRESIMS m/z (pos):
286.0237 C11H13ClN3S2 (calcd. 286.0234).
482
483
484
485
486
487
443
444
9-Methyl-3,4-dihydro-1-benzothiepin-5-one
thiosemicarbazone (4p)
9-Bromo-3,4-dihydro-1-benzothiepin-5-one
thiosemicarbazone (4t)
488
489
1
445
446
447
448
449
450
451
452
453
Yellow solid; yield 31.30%; ¹H NMR (DMSO-d6, 400 MHz):
δ = 10.43 (1H, brs), 8.28 (1H, s), 7.77 (1H, s), 7.51 (1H, d, J
= 6.8 Hz), 7.22 (1H, d, J = 6.8 Hz), 7.12 (1H, t, J = 7.6 Hz),
2.92 (4H, m), 2.33 (3H, s), 2.01(2H, m); 13C NMR (DMSO-
d6, 100 MHz): δ = 179.46 (C, C=S), 152.73 (C, C=N),
139.45 (C, C-10), 137.54 (C, C-9), 135.72 (C, C-8), 130.41
(C, C-11), 128.29 (C, C-6), 126.12 (C, C-7), 32.33 (CH2, C-
2), 27.74 (CH2, C-3), 26.85 (CH2, C-4), 21.20 (CH3); HRE-
SIMS m/z (pos): 266.0783 C12H17N3S2 (calcd. 266.0780).
White solid; yield 12.2%; H NMR (DMSO-d6, 400 MHz):
490
491
492
493
494
495
496
497
498
δ = 10.56 (1H, brs), 8.34 (1H, brs), 7.88 (1H, brs), 7.72 (1H,
m), 7.62 (1H, dd, J1 = 1.32 Hz, J2 = 7.88 Hz), 7.12 (1H, t, J
= 7.88 Hz), 2.97 (4H, m), 2.09 (2H, m); 13C NMR (DMSO-
d6, 100 MHz): δ = 179.62 (C, C=S), 150.60 (C, C=N),
140.32 (C, C-10), 138.08 (C, C-8), 132.98 (C, C-11), 130.11
(C, C-6), 127.21 (C, C-7), 123.91 (C, C-9), 33.14 (CH2, C-
2), 27.48 (CH2, C-3), 27.42 (CH2, C-4); HRESIMS m/z
(pos): 329.9729 C11H13BrN3S2 (calcd. 329.9729).
454
455
9-Methoxy-3,4-dihydro-1-benzothiepin-5-one
thiosemicarbazone (4q)
General procedure for thiochroman-4-one-1,1-
dioxide thiosemicarbazone derivativesand3,4-
dihydro-1-benzothiepin-5-one-1,1-dioxide
thiosemicarbazone derivatives 6(a–j)
499
500
501
502
456
457
458
459
460
461
462
463
464
465
White solid; yield 38.66%; ¹H NMR (DMSO-d6, 400 MHz):
δ = 10.47 (1H, brs), 8.29 (1H, s), 7.79 (1H, s), 7.32 (1H, m),
7.15 (1H, t, J = 8.00 Hz), 6.97 (1H, dd, J1 = 1.12 Hz, J2 =
8.20 Hz), 3.81 (3H, s), 2.96 (2H, t, J = 6.36 Hz), 2.85 (2H, t,
J = 6.12 Hz), 2.05(2H, m); 13C NMR (DMSO-d6, 100 MHz):
δ = 179.46 (C, C=S), 156.66 (C, C-9), 151.54 (C, C=N),
139.14 (C, C-11), 126.81 (C, C-7), 126.33 (C, C-6), 122.60
(C, C-10), 110.90 (C, C-8), 56.63 (C, O-CH3), 31.98 (CH2, C-
2), 27.94 (CH2, C-3), 27.65 (CH2, C-4); HRESIMS m/z (pos):
282.0732 C12H16N3OS2 (calcd. 282.0729).
A solution of 0.725 mmol 8-methylthiochroman-4-one-1,1-
dioxide in 14 mL anhydrous methanol was refluxed for
15 min, followed by the addition of thiosemicarbazide
(0.810 mmol) and p-toluene sulfonic acid catalyst. The
mixture was refluxed again for another 7 h, and the resulting
precipitate was collected by filtration and washed with
methanol. After drying under high vacuum, the thiosemi-
carbazone derivatives were obtained.
503
504
505
506
507
508
509
510
466
467
9-Fluoro-3,4-dihydro-1-benzothiepin-5-one
thiosemicarbazone (4r)
8-Methyl-1,1-dioxo-thiochroman-4-one thiosemicarbazone
(6a)
511
512
468
469
470
471
472
473
474
475
476
White solid; yield 50%; ¹H NMR (DMSO-d6, 400 MHz): δ =
10.56 (1H, brs), 8.33 (1H, brs), 7.86 (1H, s), 7.59 (1H, m),
7.22 (2H, m), 2.96 (4H, m), 2.06 (2H, m); 13C NMR (DMSO-
d6, 100 MHz): δ = 179.63 (C, C=S), 159.68 (C, C-9, d, J =
239 Hz), 150.39 (C, C=N), 140.97 (C, C-11), 127.41 (C, C-7,
d, J = 9 Hz), 126.20 (C, C-6, d, J = 3 Hz), 124.73 (C, C-10,
d, J = 18 Hz), 115.26 (C, C-8, d, J = 23 Hz), 32.30 (CH2, C-
2), 27.52 (CH2, C-3), 27.26 (CH2, C-4); HRESIMS m/z (pos):
270.0532 C11H14FN3S2 (calcd. 270.0529).
White solid; yield: 50%; 1H NMR (DMSO-d6, 400 MHz): δ
= 10.62 (1H, brs), 8.51 (1H, s), 8.43(1H, d, J = 8.00 Hz),
8.18 (1H, s), 7.50 (1H, t, J = 7.96 Hz), 7.42 (1H, d, J =
7.36 Hz), 3.67 (2H, m), 3.36 (2H, m), 2.62 (3H, s); 13C
NMR (DMSO-d6, 100 MHz): δ = 179.39 (C, C=S), 146.42
(C, C=N), 136.42 (C, C-9), 135.16 (C, C-8), 131.47 (C, C-
7), 130.68 (C, C-6), 125.54 (C, C-10), 124.68 (C, C-5),
27.98 (CH2, C-2), 25.18 (CH3), 20.28 (CH2, C-3); HRE-
SIMS m/z (pos): 284.0527 C11H14N3O2S2 (calcd. 284.0522).
513
514
515
516
517
518
519
520
521
477
478
9-Chloro-3,4-dihydro-1-benzothiepin-5-one
thiosemicarbazone (4s)
8-Methoxy-1,1-dioxo-thiochroman-4-one
thiosemicarbazone (6b)
522
523
479
480
481
Yellow solid; yield 30%; ¹H NMR (DMSO-d6, 400 MHz):
δ = 10.58 (1H, brs), 8.34 (1H, s), 7.88 (1H, s), 7.71 (1H, dd,
J1 = 1.16 Hz, J2 = 7.86 Hz), 7.48 (1H, m), 7.20 (1H, t, J =
White solid; yield: 64.5%; 1H NMR (DMSO-d6, 400 MHz):
δ = 10.66 (1H, brs), 8.51 (1H, s), 8.15 (2H, m), 7.54 (1H, t,
J = 8.24 Hz), 7.26 (1H, d, J = 8.36 Hz), 3.88 (3H, s), 3.57
524
525
526

Medicinal Chemistry Research
527
528
529
530
531
532
(2H, m), 3.21 (2H, m); 13C NMR (DMSO-d6, 100 MHz): δ
= 179.93 (C, C=S), 157.24 (C, C-8), 141.56 (C, C=N),
134.26 (C, C-6), 133.56 (C, C-10), 127.08 (C, C-9), 119.24
(C, C-5), 114.48 (C, C-7), 57.05 (OCH3), 49.04 (CH2, C-2),
23.8 (CH2, C-3); HRESIMS m/z (pos): 300.0474
C11H14N3O2S2 (calcd. 300.0471).
7.67 (1H, t, J = 7.6 Hz), 7.53 (1H, dd, J = 7.0, 1.6 Hz), 7.44
(1H, dd, J = 7.1, 1.6 Hz), 3.49 (2H, t, J = 6.3 Hz), 2.87 (2H,
t, J = 6.2 Hz), 2.67 (3H, s), 1.92–1.90 (2H, m); 13C NMR
(DMSO-d6, 100 MHz): δ = 179.76 (C, C=S), 151.78 (C,
C=N), 138.76 (C, C-10), 138.04 (C, C-9), 136.81 (C, C-8),
134.05 (C, C-7), 133.07 (C, C-11), 129.49 (CH, C-6), 55.10
(CH2, C-2), 28.06 (CH2, C-4), 21.25 (CH3), 18.91 (CH2, C-
3); HRESIMS m/z (pos): 320.0497 C12H15N3NaO2S2Na
(calcd. 320.0498).
572
573
574
575
576
577
578
579
580
533
534
8-Fluoro-1,1-dioxo-thiochroman-4-one thiosemicarbazone
(6c)
535
536
537
538
539
540
541
542
543
544
White solid; yield: 45%; 1H NMR (DMSO-d6, 400 MHz): δ
= 10.73 (1H, brs), 8.58 (1H, s), 8.45 (1H, d, J = 8.08 Hz),
8.27 (1H, s), 7.65 (1H, m), 7.47 (1H, d, J = 1.48 Hz), 3.76
(2H, m), 3.29 (2H, m); 13C NMR (DMSO-d6, 100 MHz): δ
= 180.01 (C, C=S), 158.48 (C, C-8, d, J = 252 Hz), 139.85
(C, C=N), 135.21 (C, C-6), 134.41 (C, C-10, d, J =
9.0 Hz), 126.65 (C, C-9, d, J = 14 Hz), 123.58 (C, C-5, d, J
= 4.0 Hz), 118.13 (C, C-7, d, J = 21.0 Hz), 48.22 (CH2, C-
2), 24.05 (CH2, C-3); HRESIMS m/z (pos): 288.0277
C10H11FN3O2S2 (calcd. 288.0271).
9-Methoxy-3,4-dihydro-1-benzothiepin-5-one-1,1-dioxide
thiosemicarbazone (6g)
581
582
White solid; yield 61.33%; 1H NMR (DMSO-d6,
400 MHz): δ = 10.64 (1H, s), 8.35 (1H, s), 7.85 (1H, s),
7.58 (1H, t, J = 8.1 Hz), 7.30 (2H, d, J = 7.7 Hz), 3.90 (3H,
s), 3.40 (2H, t, J = 6.2 Hz), 2.91 (2H, t, J = 6.5 Hz), 2.01-
1.98 (2H, m); 13C NMR (DMSO-d6, 100 MHz): δ = 179.89
(C, C=S), 158.37 (C, C-9), 149.68 (C, C=N), 138.76 (C,
C-7) 133.97 (C, C-11), 128.78 (C, C-10), 122.49 (C, C-6),
114.92 (C, C-8), 57.37 (CH3), 55.33 (CH2, C-2), 27.96
(CH2, C-4), 18.09 (CH2, C-3); HRESIMS m/z (pos):
336.0445 C12H15N3NaO3S2Na (calcd. 336.0447).
583
584
585
586
587
588
589
590
591
592
545
546
8-Chloro-1,1-dioxo-thiochroman-4-one thiosemicarbazone
(6d)
1
547
548
549
550
551
552
553
554
555
Yellow solid; yield: 50%; H NMR (DMSO-d6, 400 MHz):
9-Fluoro-3,4-dihydro-1-benzothiepin-5-one-1,1-dioxide
thiosemicarbazone (6h)
593
594
δ = 10.73 (1H, brs), 8.59 (1H, d, J = 8.0 Hz), 8.57 (1H,
brs), 8.25 (1H, brs), 7.67 (1H, d, J = 7.2 Hz), 7.58 (1H, t, J
= 8.0 Hz), 3.76 (2H, m), 3.28 (2H, m); 13C-NMR (DMSO-
d6, 100 MHz,): δ = 179.88 (C, C=S), 140.70 (C, C=N),
135.56 (C, C-9), 135.53 (C, C-6), 133.56 (C, C-7), 133.18
(C, C-8), 129.91 (C, C-10),126.73 (C, C-5), 48.42 (CH2, C-
2), 23.62 (CH2, C-3); HRESIMS m/z (pos): 303.9978
C10H11ClN3O2S2 (calcd. 303.9976).
Yellow solid; yield 60.00%; 1H NMR (DMSO-d6,
400 MHz): δ = 10.64 (1H, s), 8.41 (1H, s), 7.90 (1H, s),
7.73 (1H, td, J = 8.1, 5.2 Hz), 7.59 (1H, d, J = 7.5 Hz), 7.48
(1H, dd, J = 10.6, 8.9 Hz), 3.59 (2H, t, J = 6.2 Hz), 2.90
(2H, t, J = 6.2 Hz), 1.98 (2H, m); 13C NMR (DMSO-d6,
100 MHz) δ = 179.96 (C, C=S), 159.26 (C, C-9, d, J =
256 Hz), 149.50 (C, C=N), 139.80 (C, C-7), 135.52 (C, C-
11, d, J = 10 Hz), 127.10 (C, C-10), 126.79 (C, C-6, d, J =
11 Hz), 118.54 (C, C-8, d, J = 23 Hz), 55.54 (CH2, C-2),
27.96 (CH2, C-4), 19.14 (CH2, C-3); HRESIMS m/z (pos):
302.0432 C11H14FN3O2S2 (calcd. 302.0428).
595
596
597
598
599
600
601
602
603
604
605
556
557
8-Bromo-1,1-dioxo-thiochroman-4-one thiosemicarbazone
(6e)
558
559
560
561
562
563
564
565
566
567
White solid; yield 72%; 1H NMR (DMSO-d6, 400 MHz): δ
= 10.72 (1H, brs,), 8.64 (1H, dd, J1 = 8.2 Hz, J1 = 1 Hz),
8.57 (1H, brs), 8.25 (1H, brs), 7.84 (1H, dd, J1 = 7.8 Hz, J1
= 1 Hz), 7.48 (1H, t, J = 8.04 Hz), 3.76 (2H, t, J = 5.8 Hz),
3.28 (2H, t, J = 6.5 Hz); 13C NMR (DMSO-d6, 100 MHz):
δ = 180.03 (C, C=S), 140.69 (C, C=N), 137.09 (C, C-9),
136.60 (C, C-6), 135.86 (C, C-7), 133.44 (C, C-10), 127.45
(C, C-5), 117.11 (C, C-8), 48.19 (CH2, C-2), 23.61 (CH2, C-
3); HRESIMS m/z (pos): 347.9474 C10H11BrN3O2S2 (calcd.
347.9471).
9-Chloro-3,4-dihydro-1-benzothiepin-5-one-1,1-dioxide
thiosemicarbazone (6i)
606
607
Yellow solid; yield 22.80%; 1H NMR (DMSO-d6,
400 MHz): δ = 10.66 (1H, s), 8.40 (1H, s), 7.91 (1H, s),
7.75 (1H, dd, J = 7.5, 1.5 Hz), 7.69 (1H, dd, J = 8.0,
1.5 Hz), 7.64 (1H, t), 3.57 (2H, t, J = 6.3 Hz), 2.93 (2H, t, J
= 6.5 Hz), 2.05–1.98 (2H, m); 13C NMR (DMSO-d6,
100 MHz): δ = 179.98 (C, C=S), 149.31 (C, C=N), 140.31
(C, C-10), 137.15 (C, C-7), 133.79 (C, C-8), 133.20 (C, C-
9), 131.68 (C, C-11), 130.34 (C, C-6), 55.23 (CH2, C-2),
27.91 (CH2, C-4), 18.26 (CH2, C-3). HRESIMS m/z (pos):
318.0136 C11H14ClN3O2S2 (calcd. 318.0132).
608
609
610
611
612
613
614
615
616
617
568
569
9-Methyl-3,4-dihydro-1-benzothiepin-5-one-1,1-dioxide
thiosemicarbazone (6f)
570
571
Yellow solid; yield 26.53%; 1H NMR (DMSO-d6,
400 MHz): δ = 10.45 (1H, s), 8.34 (1H, s), 7.81 (1H, s),

Medicinal Chemistry Research
618
619
9-Bromo-3,4-dihydro-1-benzothiepin-5-one-1,1-dioxide
thiosemicarbazone (6j)
(50 mg mL−1, 37 °C) for another 30 min. Subsequently, the
cell cycle distribution was analyzed by flow cytometer.
662
663
1
620
621
622
623
624
625
626
627
628
629
Yellow solid; yield 20%; H NMR (DMSO-d6, 400 MHz):
Intracellular ROS analysis
664
δ = 10.64 (1H, s), 8.39 (1H, s), 7.91 (1H, s), 7.88 (1H, dd, J
= 8.0, 1.1 Hz), 7.78 (1H, dd, J = 7.8, 1.1 Hz), 7.54 (1H, t, J
= 7.9 Hz), 3.56 (2H, t, J = 6.3 Hz), 2.92 (2H, t, J = 6.3 Hz),
2.04–1.97 (2H, m); 13C NMR (DMSO-d6, 100 MHz): δ =
197.90 (C, C=S), 179.97 (C, C=N), 149.57 (C, C-10),
140.58 (C, C-8), 136.82 (C, C-11), 133.89 (C, C-9), 130.93
(C, C-6), 119.29 (C, C-7), 54.99 (CH2, C-2), 27.91 (CH2, C-
4), 18.26 (CH2, C-3). HRESIMS m/z (pos): 361.9630
C11H14BrN3O2S2 (calcd. 361.9627).
ROS was determined using a fluorescent 2,7-dichlorodi-
hydrofluorescein diacetate (DCFH-DA) probe. On its own,
DCFH-DA does not fluorescence. However, after entering
the cell, this compound will eventually be oxidized by ROS
to produce fluorescence DCF. The fluorescence intensity of
DCF is directly related to the level of intracellular ROS. The
tested MCF-7 cells were seeded into a 24-well plate at a
density of 1 × 105 cells/well and cultured for 24 h. After
washing with PBS three times, cells were treated with dif-
ferent concentrations of compound 4c (0, 0.125, 0.25, and
0.5 μM) for 24 h. Subsequently, 10 μM DCFH–DA was
added, and the cells were incubated in the dark for another
20 min. Later, the cells were washed twice with PBS to
remove unlabeled DCFH–DA probes, and changes in
fluorescence intensity were measured by flow cytometry.
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
630
631
Biological experiment
Cell viability assay
632
633
634
635
636
637
638
639
640
641
642
643
The viability of the cultured cells was determined by Cell
Counting Kit-8 assay (CCK-8 assay). MCF-7 cells (Eno
Gene) were seeded in a 96-well microplate at a density of
1 × 104 cells/well and cultured overnight. Thereafter, the
cells were treated with various concentration of compounds
4(a–t), 6(a–j). Seventy-two hours later, 20 μL of the water-
soluble tetrazolium (WST-8) solution (Eno Gene) was
added to each well, and the plates were incubated for
another 4 h at 37 °C in a humidified atmosphere (5% CO2).
Absorbance measurement of the wells was conducted at
450 nm using an enzyme-linked immunosorbent assay
reader (Thermo MK3).
Acknowledgements This work was financially supported by the
National Natural Science Foundation of China (No. 21502085), Spe-
cial Funds for Public Welfare Research and Capacity Building of
Guangdong Province (Nos. 2016A010103042 and 2015A020211038),
the Research Group of Rare Earth Resource Exploiting and Lumi-
nescent Materials (2017KCXTD022), and Lingnan Normal University
Science Research Foundation (No. ZL1401).
680
681
682
683
684
685
686
Compliance with ethical standards
687
Conflict of interest The authors declare that they have no conflict of
interest.
688
689
644
Apoptosis assay
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
690
691
645
646
647
648
649
650
651
652
653
MCF-7 cells were cultured at a concentration of 1 × 106 mL−1
for 24 h, and then treated with various concentrations of
compound 4c (0, 0.5, 1.0, and 2.0 μM) for 72 h. Cellular
apoptosis was assessed by double staining with Annexin V-
FITC and propidium iodide (PI), according to the protocol of
the Annexin V-FITC/PI kit. (Eno Gene). The stained cells
were analyzed using a flow cytometer (FACS Calibur, Bec-
ton-Dickinson, San Jose, CA) and the flowJo software
(version 7.6).
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