Potent hydrazone derivatives targeting esophageal cancer cells

Article abstract of DOI:10.1016/j.ejmech.2018.02.033

Hydrazone and their derivatives are a series of highly active molecules, which are widely used as lead compounds for the research and development of new anti-cancer drugs. In this study, 20 compounds were synthesized, based on this scaffold and their in vitro cytotoxicity against 6 cancer cell lines, including EC9706, SMMC-7721, MCF7, PC3, MGC-803 and EC109 was tested. Among them, compound 6p, showed strong anti-proliferative activities on esophageal carcinoma cells: EC9706 and EC109 with IC₅₀ values of 1.09 ± 0.03 and 2.79 ± 0.45 μM, respectively. 6p also significantly induces both EC9706 and EC109 cell cycle arrest at G0/G1 phase and cell apoptosis, as well as intracellular ROS accumulation, which could be markedly reversed caspase or ROS inhibitor: NAC. Meanwhile, treatment of compound 6p results in significant declined mitochondria membrane potential, increases in the expression of P53 and bax, as well as decrease in Bcl-2. 6p also activates caspase-8/9/3, PARP and Bid, indicating that 6p induces cancer cell apoptosis via the death receptor-mediated extrinsic pathway and the mitochondria-mediated intrinsic pathway. Further studies also proved that 6p does not show obvious side effects at cellular and in vivo levels. Our findings suggested that hydrazone derivative: compound 6p may serve as a lead compound for further optimization against esophageal cancer cells.

Full text of DOI:10.1016/j.ejmech.2018.02.033

Accepted Manuscript
Potent hydrazone derivatives targeting esophageal cancer cells
Ling-Yu Li, Jia-Di Peng, Wenjuan Zhou, Hui Qiao, Xin Deng, Zhou-Hua Li, Ji-Deng Li,
Yun-Dong Fu, Song Li, Kai Sun, Hong-Min Liu, Wen Zhao
PII:
S0223-5234(18)30163-6
10.1016/j.ejmech.2018.02.033
EJMECH 10210
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 September 2017
Revised Date: 19 November 2017
Accepted Date: 10 February 2018
Please cite this article as: L.-Y. Li, J.-D. Peng, W. Zhou, H. Qiao, X. Deng, Z.-H. Li, J.-D. Li, Y.-D. Fu,
S. Li, K. Sun, H.-M. Liu, W. Zhao, Potent hydrazone derivatives targeting esophageal cancer cells,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.02.033.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Potent hydrazone derivatives targeting esophageal cancer cells
#
#
Ling-yu Li , Jia-di Peng , Wenjuan Zhou , Hui Qiao, Xin Deng, Zhou-hua Li,
Ji-deng Li, Yun-dong Fu, Song Li, Kai Sun*, Hong-Min Liu* and Wen Zhao*
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, P. R. China
#
:
Both authors contribute equally to this work.
*
*
:These senior authors contribute equally to this work.
Corresponding Authors: Zhao Wen, Ph.D, Liu Hong-Min, Ph.D and Sun Kai, Ph.D
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, No. 100 Kexue
Avenue, Zhengzhou, Henan 450001, PR China.
E-mail address: zhaowen100@139.com, liuhm@zzu.edu.cn, kaisun@zzu.edu.cn
1

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Abstract：
Hydrazone and their derivatives are a series of highly active molecules, which are widely
used as lead compounds for the research and development of new anti-cancer drugs. In this
study, 20 compounds were synthesized, based on this scaffold and their in vitro cytotoxicity
against 6 cancer cell lines, including EC9706, SMMC-7721, MCF7, PC3, MGC-803 and
EC109 was tested. Among them, compound 6p, showed strong anti-proliferative activities on
esophageal carcinoma cells: EC9706 and EC109 with IC50 values of 1.09±0.03 and
2
.79±0.45µM, respectively. 6p also significantly induces both EC9706 and EC109 cell cycle
arrest at G0/ G1 phase and cell apoptosis, as well as intracellular ROS accumulation, which
could be markedly reversed caspase or ROS inhibitor: NAC. Meanwhile, treatment of
compound 6p results in significant declined mitochondria membrane potential, increases in
the expression of P53 and bax, as well as decrease in Bcl-2. 6p also activates caspase-8/9/3,
PARP and Bid, indicating that 6p induces cancer cell apoptosis via the death
receptor-mediated extrinsic pathway and the mitochondria-mediated intrinsic pathway.
Further studies also proved that 6p does not show obvious side effects at cellular and in vivo
levels. Our findings suggested that hydrazone derivative: compound 6p may serve as a lead
compound for further optimization against esophageal cancer cells.
Key words: Hydrazone compounds, cytotoxicity, cell cycle arrest, apoptosis
2

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1
. Introduction
Malignant tumors remain the leading cause of morbidity and mortality all over the world[1]
which keeps an urgent need to search for new anticancer drugs with high efficacy and low side
effects. Hydrazone scaffold is regarded as a privileged structure, due to their broad applications
from medicinal agents, agrochemicals to functional materials. The scaffold, represented by the
fusion between the amide and the imine subunits, provides pharmacophoric points to include
hydrogen-bond acceptor and a donor sites and to perform interaction with a wide range of
amino-acid residues[2]. The hydrazone derivatives have been exhibited diverse pharmacological
activities, such as anticancer[3, 4], antibacterial[5], anti-inflammatory[6], antiviral[7], as
illustrated by the structurally diverse prototypes described in Fig. 1.
On the other hand, sulfonamide derivatives also display extensive biological properties, which
are very popular among pharmaceutical chemists working on the design and synthesis of
biologically active compounds in the pharmaceutical and agrochemical industry. This attractive
scaffold displays a wide variety of pharmacological activities like anticancer[8], antibacterial[9],
anti-inflammatory[10], herbicide[11]， the description of the different structures is shown in Fig.
2
.
Fig. 1. Pharmaceutically important hydrazone derivatives.
Fig. 2. Pharmaceutically important sulfonamide derivatives.
The study of new hybrid systems in which hydrazone and sulfonamide are combined comprise
an interesting field of anticancer research[4, 12-15]. We have previously reported several
,2,3-triazole–pyrimidine hydrazone derivatives with good antiproliferative activity[16]. hese
1
T
findings encouraged us to investigate the potential synergistic effect of hydrazone and
3

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sulfonamide scaffolds. Herein, our group focused on the hydrazine-based synthesis, cytotoxic
evaluation and mechanism exploration. In this study, twenty hydrazone derivatives were designed
and synthesized. All of these compounds were evaluated for their potential cytotoxic effects
against several human cancer cell lines, including esophageal cancer cells EC9706 and EC109,
liver carcinoma cells SMMC-7721, prostate cancer cells PC3, breast cancer cells MCF7 and
gastric cancer cells MGC803. Among them, the possible mechanism of action for the highly
potential compound: 6p were also evaluated.
2. Results and discussion
2.1. Chemistry
The general route for the synthesis of hydrazone derivatives was illustrated in Scheme 1.
3
-(chlorosulfonyl)benzoic acid 2 were prepared by heating benzoic acid 1 and chlorosulfonic
acid[17], which then reacted with morpholine in the presence of potassium carbonate gave
compound 3, Compound 4 were obtained by refluxing 3 in methanol. The key intermediates 5
were conveniently produced by adding hydrazine hydrate to 4 in methanol[4]. Targeted
compounds 6a~t were readily obtained by refluxing 5 with the substituted aromatic aldehyde or
1
ketone in ethanol[18]. Finally, all the hydrazone derivatives were fully characterized by H NMR,
1
3
C NMR and HRMS.
Scheme 1. Reagents and conditions: (a) chlorosulfonic acid, 120 ºC, 4h;（b）morpholine, K CO3,
2
THF , RT, 12 h; (c) conc H SO CH OH, reflux, 10h; (d) CH OH, NH NH ·H O, RT, 5 h; (d)
2
4,
3
3
2
2
2
EtOH, reflux, 4-6 h.
2
2
.2. Evaluation of biological activity
.2.1 Antiproliferative activity
All synthesized compounds were evaluated for their in vitro cytotoxicity on six different cancer
cell lines, including EC9706 and EC109, SMMC-7721, PC3, MCF7 and MGC803, using MTT
assay. The well-known anticancer drug 5-fluorouracil (5-Fu) was employed as the positive control.
The results were shown in Table 1.
4

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a
Table 1. Synthesis and Cytotoxic activities (IC ) of hydrazone derivatives against six cancer cell lines .
50
b
IC50(µM)
1
2
Comp.
R
R
EC9706
>100
SMMC-7721
>100
PC3
>100
MCF7
>100
MGC803
>100
EC109
>100
6
6
6
6
6
a
b
c
d
e
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
H
3
3
3
3
3
3
3
3
3
3
Ph
3-NH
3
-Ph
>100
>100
>100
>100
>100
>100
4-F-Ph
2-OH-Ph
>100
>100
81.32±1.91
2.81±0.45
>100
>100
>100
>100
1.22±0.08
>100
1.37±0.13
>100
5.40±0.73
>100
1.22±0.08
>100
5.45±0.74
>100
3-OH-Ph
6
f
4-OH-Ph
71.37±1.85
3.51±0.55
24.66±1.39
2.21±0.34
3.52±0.55
>100
88.49±1.94
>100
36.22±1.55
>100
>100
71.37±1.85
9.09±0.95
17.98±1.25
2.26±0.35
0.53±0.02
>100
88.34±1.95
21.36±1.33
5.28±0.72
3.27±0.51
5.06±0.70
>100
6
6
g
h
2,5-diOH-Ph
2-OH-5-Br-Ph
2-OH-5-F -Ph
>100
2.41±0.38
1.15±0.06
2.33±0.36
>100
6.89±0.83
4.41±0.64
2.33±0.36
>100
7.83±0.89
6.65±0.82
1.01±0.01
>100
6
6
i
j
3
2-OH-3-NH -Ph
6
k
H
H
>100
>100
>100
>100
>100
>100
>100
>100
6
l
44.28±1.64
88.13±1.94
81.72±1.91
55.73±1.75
6
m
6
n
o
p
q
H
H
H
H
H
H
H
-
2-OH-Ph
19.64±1.29
0.92±0.03
1.09±0.03
15.43±1.19
2.15±0.33
29.67±1.47
2.44±0.38
5.13±0.71
12.07±1.08
27.07±1.48
3.95±0.59
6.99±0.84
6.82±0.83
9.94±0.99
7.31±0.86
13.70±1.14
13.83±1.14
14.02±1.14
5.80±0.76
15.28±1.18
10.52±1.02
7.23±0.86
9.70±0.98
6.27±0.80
29.13±1.46
3.52±0.55
5.14±0.71
19.28±1.29
16.45±1.22
26.64±1.43
16.03±1.21
18.75±1.27
25.29±1.40
4.04±0.60
2.07±0.31
17.98±1.25
10.99±1.04
14.84±1.17
17.76±1.25
9.24±0.97
14.37±1.16
0.73±0.14
2.79±0.45
16.48±1.22
2.84±0.45
11.69±1.07
10.29±1.01
6.02±0.78
6
2-OH-4-diethylamino-Ph
2-OH-4-MeO-Ph
2-OH-3-EtO- Ph
2-OH-5-Cl -Ph
2-OH-5-Br-Ph
2-OH-3,5-diBr-Ph
-
6
6
6
r
6
s
6
t
5
-Fu
a
Cell lines were treated with different concentrations of the compounds for 72h. Cell viability was measured by MTT assay as
described in the experimental section.
b
IC50 values are indicated as the mean±SD of three independent experiments.
In order to achieve substituent diversity and explore the structure-activity relationship, we
introduced different hydrazone substituents to the scaffold. As shown in Table 1, the majority of
5

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the synthesized compounds exhibited moderate to potent activity against all the cancer cell lines.
From the biological data of compounds 6a, 6b, 6c and 6d, we concluded that the substituent on the
phenyl group had a remarkable effect on their cytotoxic activity. Comparing compound 6d or 6e
with meta or para-position, compound 6f with hydroxy group positioned at ortho-position was
much more powerful for the inhibitory activity The contribution of ortho-position at benzene was
consistent with the SAR results drawn from compound 6b vs 6j. The importance of the aromatic
ring of hydrazone scaffold in the anticancer activity was also explored. Replacing the phenyl
scaffold of compound 6a with heterocyclic scaffolds (6k, 6l and 6m), furan group (6k) and
thiophene group (6l) gave no cytotoxic activity (IC50 > 100µM), with slight improvement of the
inhibitory activity with pyridine (6m). The results revealed that the aromatic rings had a slight
effect on the activity. At the same time, we introduced an additional polar functional group, such
as halogen atoms (Cl, Br, F), amino, methoxy and ethoxy. Comparing compound 6h to 6i and 6r
to 6s indicate that the anticancer activity was prone to be sensitive to the position of halogen at
1
phenyl ring. In addition, the replacement of hydrogen atom with -CH at R exerted positive effect
3
on the inhibitory potency (6d vs 6n and 6h vs 6s), which indicated that methyl was slightly
favorable for the anticancer activity. Among them, compound 6p exhibited excellent growth
inhibition activity against EC9706 and EC109, with IC50s of 1.09±0.03µM and 2.79±0.45µM
respectively. Therefore, to have a better understanding of the mechanism of action of cytotoxic
activity of these series of compounds, we selected compound 6p and esophageal carcinoma cell
lines including EC9706 and EC109 for our further study.
2
.2.2 Compound 6p suppressed the proliferation of EC9706 and EC109 cells
We firstly, investigated the impacts of 6p (concentrations ranging from 0.16-20µM) on cell
viability,on EC9706 and EC109 cells by MTT assay for 24h, 48h and 72h, respectively. The
results showed that 6p inhibited EC9706 and EC109 cell proliferation dose- and time-dependently,
with IC value at 24h, 48h, 72h for EC9706 were > 20, 7.51±0.88, 1.09±0.03µM, respectively, for
5
0
EC109 were > 20, 11.34±1.06 and 2.79±0.45µM, respectively (Fig. 3A and 3B). We then
analyzed the effect of 6p on the cell proliferation by colony formation assay (Fig. 3C). The results
showed that the cancer cells treated with 1µM or 2µM 6p formed much fewer and smaller
colonies, compared with the control cells (Fig. 3D and 3E). Cell cycle arrest was an important
6

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indication for inhibition of proliferation [19], therefore we performed cell cycle analysis (Fig. 3F).
We found that the percentage of cells in G0/G1 phase in EC9706 was increased to 1.07, 1.22 and
1
.27-folds at 2µM, 4µM and 8µM 6p treatment, respectively, compared with control group. The
percentage of cells in G2/M phase was decreased to 0.28, 0.30, 0.26-folds, respectively, compared
with control group. At the 2µM, the percentage of cells in S phase was increased to 1.54 folds.
Similarly, 6p treatment also resulted in the G0/G1 phase arrest in EC109 cells dose-dependently.
(
Fig. 3G and 3H).
To further confirm the G0/G1 arrest effects of compound 6p, we investigated the changes in the
expression levels of G0/G1 checkpoint related proteins by western blot[20]. After 24h treatment
by compound 6p at 2, 4 and 8µM, the expression of P27 was significantly increased
to1.3,1.40,1.70-folds and 1.2,1.4,1.9-folds in EC9706 and EC109 cells, respectively (Fig. 3I and
3
J), indicating that compound 6p inhibits the proliferation of EC9706 and EC109 cells by
inducing cell cycle arrest in G0/G1 phase.
2
.2.3 Compound 6p induced EC9706 and EC109 cell apoptosis
To further explore the detail cytotoxic effect of 6p on EC9706 and EC109 cells, we determined
the effect of 6p on cell apoptosis, using Hoechst 33342 staining. After 48 h treatment with 6p, the
number of apoptotic morphological changes, including cell rounding, chromatin condensation, and
formation of apoptotic bodies under fluorescence microscope, was significantly increased
dose-dependently in both EC9706 and EC109 cells.(Fig. 4A). Annexin V-FITC/PI double staining
and quantifying by flow cytometry for apoptotic analysis were also performed (Fig. 4B). The
results showed that, after 48h incubation, the percentage of the apoptotic cells of EC9706 was up
to 23%, 50% and 64% at 2, 4 and 8µM 6p treatment, respectively. The percentage of the apoptotic
cells of EC109 up to 24%, 44% and 73% dose-dependently(Fig. 4C and 4D).
Caspase inhibitor Z-VAD-FMK was then employed to investigate the association between
caspase activity and 6p-induced cell apoptosis(Fig. 4E). We found that in EC9706 cells,
pretreatment with Z-VAD-FMK significantly reversed the apoptosis from 57.3% to 42.5% and
from 64.1% to 52.0% in 4µM and 8µM 6p treatment, respectively. Similar changes were also
occurred in EC109 cells(Fig. 4F and 4G).
2
.2.4 Measurement of intracellular ROS induced by compound 6p
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A high level of Reactive Oxygen Species (ROS) can inhibit tumor growth through the persistent
activation of cell-cycle inhibitors[21, 22]. In order to explore the mechanism of 6p-induced
apoptosis, we detected the intracellular ROS, using the Reactive Oxygen Assay Kit(Fig. 5A). The
results showed that in EC9706 and EC109 cells, 24h 6p treatment at 2µM, 4µM, 8µM could
remarkably increase intracellular ROS content to 1.16, 1.45, 1.95-folds and 1.81, 2.05, 2.21-folds,
respectively, compared to the controls(Fig. 5B and 5C). An antioxidant NAC(N-acetyl-L-cysteine),
were also used, in combination with 6p to further identify the mechanism. We found that treatment
with NAC partially reduced the ROS and rescued 6p-induced cell apoptosis. In EC9706 cells,
5
mM NAC pretreatment attenuated ROS level to 20% and 30% (Fig. 5D and 5E), and cell
apoptosis to 10% and 20% (Fig 5G and 5H), respectively, compared to those in 2 and 4µM 6p
treatment only. Similar changes occurred in EC109 cells (Fig. 5D and 5F, Fig. 5G and 5I).
2
.2.5 Mitochondria membrane potential and Western blot analysis
Mitochondria play an important role in the induction and control of apoptosis. A variety of key
events in apoptosis focus on mitochondria, including loss of mitochondrial transmembrane
potential, the release of ROS, participation of pro- and anti-apoptotic Bcl-2 family proteins[23, 24].
We then measured the mitochondrial membrane potential by the JC-1 fluorescent dye, as shown in
Fig. 6A. The microscopic image showed a decrease in red-fluorescent aggregates and an increase
in green-fluorescent monomer, after treated by 6p dose- and time-dependently, indicating that
mitochondrial membrane potential was remarkably reduced with 6p treatment.
Apoptosis is mediated by proteolytic enzymes called caspases[25]. Caspase activation is an
important hallmark of apoptosis. Two major apoptotic pathway include the death
receptor-mediated extrinsic pathway and the mitochondria-mediated intrinsic pathway[26, 27]. To
further investigate the molecular mechanism of compound 6p, we examined the activation levels
of caspase cascade by western blotting (Fig. 6B). We found that in both EC9706(Fig. 6C) and EC
1
09(Fig. 6D) cells, the cleaved caspase-8/9/3 and cleaved PARP were significantly increased dose
dependently after 48h 6p treatment. These findings indicated that compound 6p could induce the
activation of both the death receptor-mediated extrinsic pathway and the mitochondria-mediated
intrinsic pathway in these two EC cells.
In order to illustrate the mechanism of the pro-apoptotic effect of compound 6p, we
8

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investigated the expression level of mitochondria–related apoptotic proteins. We found that in both
EC9706 and EC 109 cells, the anti-apoptotic protein, Bcl-2, was downregulated and the
pro-apoptotic protein, Bax, P53 were upregulated dose dependently after 48h treatment of 6p.
These results explained the activation of caspase-9. Considering the activation of caspase-8,
caspase-8 can cleave the protein BID, then cleaved BID(tBID) activates the effector proteins
Bax[28, 29], therefore, the analysis of Bid was performed. We found that the expression levels of
tBid in both EC9706 and EC 109 cells, were obviously increased by the treatment of 6p, while the
protein level of Bid was declined. These findings revealed that compound 6p could activate
caspase-9 through the activation of the death receptor-mediated extrinsic pathway.
2
.2.6 Effect of 6p treatment on antioxidant family protein Prxs
Peroxiredoxins (Prxs I to VI) are a family of anti-oxidants that protect cells from metabolically
produced reactive oxygen species (ROS). They are found in a wide variety of species and play a
crucial role in peroxide detoxification. Therefore，we investigated the expression of Prx I-VI in
EC9706 and EC109 cells after the treatment with compound 6p(Fig. 7A).The results showed that
the expression of Prxs I-VI are not changed in EC9706(Fig. 7B) and EC109 cells(Fig. 7C), which
indicate that the anti-oxidants protein family Prxs may be not associated with the anti-apoptotic
effects of compound 6p.
2
.2.7 Safety and selectivity of compound 6p
In order to evaluate the safety and anti-cancer cell selectivity of 6p, the cytotoxicity of 6p
against normal gastric cell line: GES-1 cells (Table 2) was further investigated. Our results
showed that the IC value of 6p in GES-1 cells was 7.2 fold higher, compared that in MGC-803
5
0
cells, which indicated the better selectivity of 6p in cancer cells. We had also investigated the
cytotoxicity of the intermediate 5, whose structure includes hydrazine compound, against EC9706
and EC109 cells in 72 h treatment. IC (µM) values were over 100µM in those two test cell lines.
5
0
Our findings indicate the cytotoxicity of 6p against those cancer cell lines may be not related to
the possible metabolism structure of 6p: hydrazine. Besides that, we tested the safety of 6p in vivo
using acute toxicity experiment. Mice were given maximum dose of 6p (2g/kg) and kept alive
during 2 weeks’ observation. No dead mice were found at the end of the experiment. We then
executed the mice and took the organs to evaluate the pathological alteration of 6p at the
organizational level. Histopathological examination revealed no signs of toxicity to the liver,
kidney, lung, heart and spleen in the 6p treated mice, when compared to the controls (Fig. 8). In
addition, no obvious adverse effects with regard to general health, including：body weight (data
not shown) and other physical phenotypes were observed. These results suggested that 6p has no
9

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obvious side effects at cellular and in vivo levels.
Table 2. Cytotoxic activities (IC ) of 6p against the normal gastric cell line: GES-1 cells.
50
Times
24h
48h
72h
a
IC (µM)
> 80
> 80
14.40±1.16
50
a
IC50 values are indicated as the mean±SD of three independent experiments.
3
. Conclusion
In summary, 20 hydrazone derivatives were synthesized and evaluated. Most of them showed
strong anti-proliferative activities against six cancer cell lines (EC9706, SMMC-7721, MCF7,
PC3, MGC-803, EC109). Particularly, compound 6p, exhibited strong cytotoxicity, which could
arrest cell cycle at the G0/G1 stage and induce apoptosis. Compound 6p was also proved to
increase ROS production and decreased MMP via the death receptor-mediated extrinsic pathway
and the mitochondria-mediated intrinsic pathway, without significant side effects. Our findings
strongly suggest that compound 6p may serve as a potential candidate for developing novel
anti-cancer agents.
4
. Experimental section
4
.1. General procedures
Reagents and solvents were purchased from commercial sources and were used without further
purification. The reaction process was monitored by TLC with silica gel plates (thickness 250µm,
Indicator F-254). The target analogues were purified by column chromatography with silica gel
(300–400 meshes). Melting points were determined on an X-5 micromelting apparatus and are
1
13
uncorrected. HNMR and CNMR spectra were recorded on a Bruker 400 MHz and 100 MHz
spectrometer, respectively. High resolution mass spectra (HRMS) were recorded on a Waters
Micromass Q-T of Micromass spectrometer by electrospray ionization (ESI).
4
4
.2. Experimental Procedures and Analytical Data
.2.1. Preparation of 3-(chlorosulfonyl)benzoic acid (2)
The benzoic acid 1 (12.20 g, 10 mmol) was added to chlorosulfonic acid (20 mL, 30 mmol)
with stirring. The mixture was heated at 120 ºC for 4 h. After cooling to rt, the mixture was poured
into ice-water (100 mL), the formed precipitate was collected by filtration and washed with
ice-water. The separated solid was dissolved with ethyl acetate (50 mL × 3). The combined
organic extracts were washed with saturated brine (50 mL × 2) and dried over anhydrous sodium
sulfate overnight. After removal of the solvent, the residue was loaded onto a silica gel column
and eluted with PE/EA (1:1, v/v) to afford compound 2 (11.93 g) in 54% yield as white solid. H
1
0

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NMR (400 MHz, DMSO-d ) δ 12.27 (s, 1H), 8.20 (s, 1H), 7.89 (d, J = 7.7 Hz, 1H), 7.83 (d, J =
6
1
3
7
1
4
.7 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H). C NMR (101 MHz, DMSO-d ) δ 166.92, 148.50, 130.27,
6
29.73, 129.31, 128.14, 126.45.
.2.2. Preparation of 3-(morpholinosulfonyl)benzoic acid (3)
The compound 2 (441 mg, 2 mmol) was added to morpholine (174 µL, 2 mmol) in THF (10
mL) in presence of K CO (553 mg, 4 mmol). The resulting reaction mixture was stirred at room
2
3
temperature over night. After finishing of the reaction from TLC, the residue was concentrated
under vacuum and then dissolved in ethyl acetate. The organic layer was washed with water (2 ×
1
0 mL) and brine (2 × 20 mL) and then dried over anhydrous sodium sulfate overnight. The
solvent was removed by rotary evaporation and the residue was purified by column
chromatography, using PE/EA (3:1, v/v) as eluent to give compound 3 (184 mg) in 34% yield as
white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.84 – 7.80 (m, 1H), 7.80 – 7.74 (m, 2H), 7.72 (s,
1
H), 3.65 – 3.62 (m, 4H), 2.93 – 2.86 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 167.31, 136.82,
1
34.89, 131.70, 129.89, 128.55, 125.91, 65.25, 45.83.
4
.2.3. Preparation of methyl 3-(morpholinosulfonyl)benzoate (4)
The compound 3 (271 mg, 1 mmol) dissolved in methanol (5 mL) and was added the catalytic
amount of concentrated H SO . The resulting reaction mixture was heated to 80°C for 10h. After
2
4
completion of the reaction, the reaction mixture was cooled to rt, 10 mL of water was then added
and the solution was adjusted to pH 7 with sodium bicarbonate saturated solution. The mixture
was extracted with ethyl acetate (20 mL × 3). The combined extracts were dried over anhydrous
sodium sulfate overnight. The solvent was removed by rotary evaporation and the residue was
purified by column chromatography, using PE/EA (2:1, v/v) as eluent to give compound 4 (234
1
mg) in 82% yield as white solid. H NMR (400 MHz, CDCl ) δ 8.40 (s, 1H), 8.30 (d, J = 7.8 Hz,
3
1
H), 7.95 (d, J = 7.8 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H), 3.97 (s, 3H), 3.79 – 3.72 (m, 4H), 3.08 –
1
3
3
.00 (m, 4H). C NMR (101 MHz, CDCl ) δ 165.41, 136.11, 133.92, 131.79, 131.51, 129.47,
3
1
28.81, 66.07, 52.69, 46.01.
4
.2.3. Preparaton of 3-(morpholinosulfonyl)benzohydrazide (5)
The compound 4 (285 mg, 1 mmol) was reacted with hydrazine hydrate (295 µL, 3 mmol) in
methanol (5 mL). The mixture was stirred at rt for 5 h. The reaction progress was monitored by
TLC until it was completed. The solvent was removed by rotary evaporation. A crude solid was
obtained. The product was purified by recrystallization from Methanol. A white solid powder of
11

ACCEPTED MANUSCRIPT
1
compound 5 (257 mg) was obtained with a yield of 90 %. H NMR (400 MHz, DMSO-d ) δ 10.10
6
(s, 1H), 8.19 – 8.11 (m, 2H), 7.92 – 7.83 (m, 1H), 7.77 (t, J = 7.7 Hz, 1H), 4.61 (s, 2H), 3.69 –
1
3
3
.57 (m, 4H), 2.94 – 2.83 (m, 4H). C NMR (101 MHz, DMSO-d ) δ 164.15, 134.89, 134.41,
6
1
31.63, 129.96, 129.78, 125.95, 65.23, 45.88.
4
.2.3. General procedure for the preparation of compounds 6a-t
Appropriate aromatic aldehyde or ketone (1eq) and compound 5 (1eq) were dissolved in ethanol,
and the reaction mixture was then refluxed for 4-6 h. Following cooling, the solvents were
removed and the crude product was purified by column chromatography, using
dichloromethane/methanol (97:3, v/v) as eluent to give final compounds 6a−t.
4
.2.3.1. (E)-3-(morpholinosulfonyl)-N'-(1-phenylethylidene)benzohydrazide (6a)
1
White solid, yield: 71 %; m.p.: 106 – 109 °C. H NMR (400 MHz, DMSO-d ) δ 11.08 (s, 1H),
6
8
2
1
.24 (s, 1H), 8.15 (s, 1H), 8.03 – 7.73 (m, 4H), 7.41 (d, J = 37.8 Hz, 3H), 3.69 – 3.56 (m, 4H),
1
3
.97 – 2.84 (m, 4H), 2.39 (s, 3H). C NMR (101 MHz, DMSO-d ) δ 162.58, 156.77, 137.90,
6
35.34, 134.76, 132.59, 130.26, 129.67, 128.36, 126.84, 126.49, 65.23, 45.85, 14.88. HR-MS
+
(
ESI): Calcd. C H N O S, [M+H] m/z: 388.1326, found: 388.1328.
1
9
21
3
4
4
.2.3.2. (E)-N'-(1-(3-aminophenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide (6b)
1
White solid, yield: 80 %; m.p.: 128 – 131 °C. H NMR (400 MHz, DMSO-d ) δ 10.98 (s, 1H),
6
8
5
.23 (s, 1H), 8.14 (s, 1H), 7.92 (s, 1H), 7.83 (d, J = 7.1 Hz, 1H), 7.25 – 6.77 (m, 3H), 6.69 (s, 1H),
1
3
.67 (s, 2H), 3.74 – 3.53 (m, 4H), 3.01 – 2.76 (m, 4H), 2.31 (s, 3H). C NMR (101 MHz,
DMSO-d ) δ 162.48, 157.48, 147.10, 138.62, 135.42, 134.74, 132.54, 130.22, 129.72, 128.87,
6
1
26.80, 116.25, 115.58, 112.49, 65.24, 45.85, 14.94. HR-MS (ESI): Calcd. C H N O S,
19 22 4 4
+
[
M+Na] m/z: 425.1254, found: 425.1264.
4
.2.3.3. (E)-N'-(1-(4-fluorophenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide (6c)
1
White solid, yield: 75 %; m.p.: 212 – 215 °C. H NMR (400 MHz, DMSO-d ) δ 11.08 (s, 1H),
6
8
1
1
.23 (s, 1H), 8.14 (s, 1H), 7.93 (s, 2H), 7.83 (d, J = 7.7 Hz, 1H), 7.67 (s, 1H), 7.29 (s, 1H), 7.17 (s,
1
3
H), 3.73 – 3.53 (m, 4H), 3.04 – 2.79 (m, 4H), 2.38 (s, 3H). C NMR (101 MHz, DMSO-d ) δ
6
62.59, 155.81, 135.31, 134.42, 134.39, 132.61, 129.70, 128.81, 126.84, 115.36, 115.15, 65.25,
+
4
5.85, 14.88. HR-MS (ESI): Calcd. C H FN O S, [M+Na] m/z: 428.1051,found: 428.1058.
1
9
20
3
4
4
.2.3.4. (E)-N'-(1-(2-hydroxyphenyl)ethylidene)-3-(morpholinosulfonyl)benzohy-drazide (6d)
1
White solid, yield: 77 %; m.p.: 231 – 233 °C. H NMR (400 MHz, DMSO-d ) δ 13.25 (s, 1H),
6
1
1.62 (s, 1H), 8.29 (d, J = 7.7 Hz, 1H), 8.20 (s, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.86 (t, J = 7.8 Hz,
H), 7.66 (d, J = 7.7 Hz, 1H), 7.33 (t, J = 7.3 Hz, 1H), 6.98 – 6.88 (m, 2H), 3.72 – 3.58 (m, 4H),
1
1
2

ACCEPTED MANUSCRIPT
1
3
2
1
4
4
.98 – 2.87 (m, 4H), 2.50 (s, 3H). C NMR (101 MHz, DMSO-d ) δ 163.12, 159.20, 158.74,
6
34.91, 134.27, 132.85, 131.47, 130.66, 129.79, 128.66, 127.05, 119.28, 118.57, 117.32, 65.26,
+
5.87, 14.31. HR-MS (ESI): Calcd. C H N O S, [M+Na] m/z: 426.1094, found: 426.1102.
1
9
21
3
5
.2.3.5. (E)-N'-(1-(3-hydroxyphenyl)ethylidene)-3-(morpholinosulfonyl)benzohydra-zide (6e)
1
White solid, yield: 76 %; m.p.: 169 – 176 °C. H NMR (400 MHz, DMSO-d ) δ 11.02 (s, 1H),
6
9
7
.57 (s, 1H), 8.23 (s, 1H), 8.14 (s, 1H), 7.93 (s, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.33 (s, 1H), 7.30 –
1
3
.03 (m, 2H), 6.83 (s, 1H), 3.73 – 3.52 (m, 4H), 3.00 – 2.81 (m, 4H), 2.34 (s, 3H). C NMR (101
MHz, DMSO-d ) δ 162.57, 157.28, 156.73, 139.25, 135.41, 134.81, 132.58, 130.23, 129.69,
6
1
29.33, 126.84, 117.56, 116.85, 113.00, 65.25, 45.85, 14.89. HR-MS (ESI): Calcd. C H N O S,
19 21 3 5
+
[
M+Na] m/z: 426.1094, found: 426.1103.
4
.2.3.6. (E)-N'-(1-(4-hydroxyphenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide (6f)
1
Yellow solid, yield: 74 %; m.p.: 220 – 224 °C. H NMR (400 MHz, DMSO-d ) δ 10.96 (s, 1H),
6
9
1
3
1
.84 (s, 1H), 8.23 (d, J = 7.6 Hz, 1H), 8.14 (s, 1H), 7.93 (d, J = 7.5 Hz, 1H), 7.81 (t, J = 7.6 Hz,
H), 7.78 – 7.43 (m, 2H), 6.89 – 6.66 (m, 2H), 3.72 – 3.54 (m, 4H), 2.98 – 2.83 (m, 4H), 2.31 (s,
1
3
H). C NMR (101 MHz, DMSO-d ) δ 162.20, 159.10, 157.70, 135.46, 134.82, 132.45, 130.11,
6
29.68, 128.66, 128.18, 126.73, 115.09, 65.25, 45.86, 14.67. HR-MS (ESI): Calcd. C H N O S,
19
21
3
5
+
[
M+Na] m/z: 426.1094, found: 426.1103.
4
.2.3.7. (E)-N'-(1-(2,5-dihydroxyphenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide (6g)
1
Yellow solid, yield: 82 %; m.p.: 272 – 274 °C. H NMR (400 MHz, DMSO-d ) δ 12.50 (s, 1H),
6
1
1.58 (s, 1H), 8.96 (s, 1H), 8.28 (d, J = 7.7 Hz, 1H), 8.19 (s, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.85 (t,
J = 7.8 Hz, 1H), 7.00 (d, J = 1.7 Hz, 1H), 6.83 – 6.72 (m, 2H), 3.70 – 3.61 (m, 4H), 2.98 – 2.87 (m,
1
3
4
H), 2.44 (s, 3H). C NMR (101 MHz, DMSO-d ) δ 163.04, 158.89, 151.43, 149.12, 134.81,
6
1
34.32, 132.84, 130.63, 129.79, 127.02, 119.27, 118.97, 117.73, 113.95, 65.24, 45.86, 14.36.
+
HR-MS (ESI): Calcd. C H N O S, [M+Na] m/z: 442.1043, found: 442.1047.
1
9
21
3
6
4
.2.3.8. (E)-N'-(1-(5-bromo-2-hydroxyphenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide
(6h)
1
Yellow solid, yield: 84 %; m.p.: 235 – 238 °C. H NMR (400 MHz, DMSO-d ) δ 13.36 (s, 1H),
6
1
1
2
1.70 (s, 1H), 8.29 (d, J = 7.7 Hz, 1H), 8.20 (s, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.85 (t, J = 7.8 Hz,
H), 7.78 (d, J = 2.1 Hz, 1H), 7.52 – 7.41 (m, 1H), 6.91 (d, J = 8.7 Hz, 1H), 3.70 – 3.59 (m, 4H),
1
3
.98 – 2.86 (m, 4H), 2.51 (s, 3H). C NMR (101 MHz, DMSO-d ) δ 163.28, 157.86, 157.64,
6
1
3

ACCEPTED MANUSCRIPT
1
1
4
34.95, 134.10, 133.79, 132.91, 130.69, 129.79, 127.08, 121.38, 119.59, 109.59, 65.26, 45.86,
+
4.44. HR-MS (ESI): Calcd. C H BrN O S, [M+Na] m/z: 504.0199, found: 504.0201.
1
9
20
3
5
.2.3.9. (E)-N'-(1-(5-fluoro-2-hydroxyphenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide
(6i)
1
Yellow solid, yield: 78 %; m.p.: 214 – 216 °C. H NMR (400 MHz, DMSO-d ) δ 13.05 (s, 1H),
6
1
1
1.67 (s, 1H), 8.30 (d, J = 6.3 Hz, 1H), 8.21 (s, 1H), 7.98 (s, 1H), 7.86 (s, 1H), 7.50 (d, J = 8.7 Hz,
1
3
H), 7.19 (s, 1H), 6.95 (s, 1H), 3.76 – 3.55 (m, 4H), 3.03 – 2.83 (m, 4H), 2.50 (s, 3H). C NMR
(
101 MHz, DMSO-d ) δ 163.24, 157.83, 155.87, 154.92, 153.56, 134.91, 134.15, 132.90, 130.73,
6
1
29.80, 127.08, 118.32, 114.51, 114.26, 65.25, 45.86, 14.43. HR-MS (ESI): Calcd. C H FN O S,
19 20 3 5
+
[
M+Na] m/z: 444.1000, found: 444.1004.
4
.2.3.10. (E)-N'-(1-(3-amino-2-hydroxyphenyl)ethylidene)-3-(morpholinosulfonyl)benzohydrazide
(6j)
1
Yellow solid, yield: 74%; m.p.: 232 – 236 °C. H NMR (400 MHz, DMSO-d ) δ 13.36 (s, 1H),
6
1
1
–
1
1
1.54 (s, 1H), 8.29 (d, J = 7.7 Hz, 1H), 8.20 (s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.85 (t, J = 7.8 Hz,
H), 6.90 (d, J = 7.7 Hz, 1H), 6.73 (d, J = 7.5 Hz, 1H), 6.66 (t, J = 7.8 Hz, 1H), 4.77 (s, 2H), 3.73
1
3
3.53 (m, 4H), 3.00 – 2.85 (m, 4H), 2.46 (s, 3H). C NMR (101 MHz, DMSO-d ) δ 163.03,
6
59.99, 145.93, 137.45, 134.93, 134.38, 132.83, 130.60, 129.79, 127.00, 118.35, 117.89, 115.85,
+
15.46, 65.26, 45.87, 14.34. HR-MS (ESI): Calcd. C H N O S, [M+Na] m/z: 441.1203, found:
1
9
22
4
5
4
41.1203.
4
.2.3.11. (E)-N'-(furan-2-ylmethylene)-3-(morpholinosulfonyl)benzohydrazide (6k)
1
Yellow solid, yield: 80 %; m.p.: 100 – 103 °C. H NMR (400 MHz, DMSO-d ) δ 12.03 (s, 1H),
6
8
.37 (s, 1H), 8.30 – 8.19 (m, 2H), 7.96 (d, J = 7.9 Hz, 1H), 7.90 – 7.80 (m, 2H), 6.98 (d, J = 3.3
1
3
Hz, 1H), 6.69 – 6.63 (m, 1H), 3.67 – 3.62 (m, 4H), 2.95 – 2.89 (m, 4H). C NMR (101 MHz,
DMSO-d ) δ 161.50, 149.20, 145.44, 138.37, 134.95, 134.44, 132.38, 130.59, 129.96, 126.45,
6
+
1
14.12, 112.25, 65.23, 45.86. HR-MS (ESI): Calcd. C H N O S, [M+Na] m/z: 386.0781, found:
1
6
17
3
5
3
86.0785.
4
.2.3.12. (E)-3-(morpholinosulfonyl)-N'-(thiophen-2-ylmethylene)benzohydrazide (6l)
1
White solid, yield: 84%; m.p.: 222 – 226 °C. H NMR (400 MHz, DMSO-d ) δ 12.05 (s, 1H),
6
8
.70 (s, 1H), 8.30 – 8.20 (m, 2H), 7.95 (d, J = 7.8 Hz, 1H), 7.84 (t, J = 7.8 Hz, 1H), 7.71 (d, J =
.0 Hz, 1H), 7.51 (d, J = 3.2 Hz, 1H), 7.20 – 7.13 (m, 1H), 3.67 – 3.61 (m, 4H), 2.95 – 2.89 (m,
5
1
4

ACCEPTED MANUSCRIPT
1
3
4
1
H). C NMR (101 MHz, DMSO-d ) δ 161.44, 143.80, 138.82, 134.92, 134.50, 132.40, 131.37,
6
30.56, 129.96, 129.30, 127.91, 126.41, 65.23, 45.86. HR-MS (ESI): Calcd. C H N O S,
1
6
17
3
5
+
[
M+Na] m/z: 402.0553, found: 402.0553.
4
.2.3.13. (E)-3-(morpholinosulfonyl)-N'-(pyridin-2-ylmethylene)benzohydrazide (6m)
1
Yellow solid, yield: 79 %; m.p.: 126 – 129 °C. H NMR (400 MHz, DMSO-d ) δ 12.29 (s, 1H),
6
8
2
.64 (d, J = 4.4 Hz, 1H), 8.52 (s, 1H), 8.34 – 8.23 (m, 2H), 8.05 – 7.95 (m, 2H), 7.95 – 7.83 (m,
1
3
H), 7.52 – 7.40 (m, 1H), 3.68 – 3.61 (m, 4H), 2.96 – 2.89 (m, 4H). C NMR (101 MHz,
DMSO-d ) δ 161.87, 153.04, 149.59, 148.89, 136.94, 135.00, 134.30, 132.52, 130.76, 130.02,
6
+
1
26.59, 124.58, 120.03, 65.23, 45.87. HR-MS (ESI): Calcd. C H N O S, [M+Na] m/z: 397.0941,
1
7
18
4
4
found: 397.0950.
4
.2.3.14. (E)-N'-(2-hydroxybenzylidene)-3-(morpholinosulfonyl)benzohydrazide (6n)
1
White solid, yield: 74%; m.p.: 209 – 211 °C. H NMR (400 MHz, DMSO-d ) δ 12.33 (s, 1H),
6
1
7
2
1
1.15 (s, 1H), 8.70 (s, 1H), 8.37 – 8.22 (m, 2H), 7.97 (d, J = 7.9 Hz, 1H), 7.86 (t, J = 7.7 Hz, 1H),
.60 (d, J = 7.6 Hz, 1H), 7.38 – 7.26 (m, 1H), 6.94 (t, J = 8.3 Hz, 2H), 3.71 – 3.59 (m, 4H), 2.98 –
1
3
.87 (m, 4H). C NMR (101 MHz, DMSO-d ) δ 161.31, 157.45, 148.76, 135.02, 133.99, 132.49,
6
31.61, 130.75, 130.02, 129.24, 126.47, 119.39, 118.69, 116.41, 65.24, 45.87. HR-MS(ESI):
+
Calcd. C H N O S, [M+Na] m/z: 412.0938, found: 412.0945.
18
19
3
5
4
.2.3.15. (E)-N'-(4-(diethylamino)-2-hydroxybenzylidene)-3-(morpholinosulfonyl)benzohydrazide
(6o)
1
Yellow solid, yield: 76 %; m.p.: 241 – 243 °C. H NMR (400 MHz, DMSO-d ) δ 12.05 (s, 1H),
6
1
1
–
1
1
4
4
1.35 (s, 1H), 8.48 (s, 1H), 8.27 (d, J = 8.7 Hz, 2H), 7.95 (d, J = 7.9 Hz, 1H), 7.84 (t, J = 7.7 Hz,
H), 7.29 – 7.20 (m, 1H), 6.31 – 6.25 (m, 1H), 6.14 (d, J = 2.2 Hz, 1H), 3.68 – 3.61 (m, 4H), 3.41
1
3
3.34 (m, 4H), 2.95 – 2.88 (m, 4H), 1.11 (t, J = 7.0 Hz, 6H). C NMR (101 MHz, DMSO-d ) δ
6
60.63, 159.75, 150.63, 150.33, 134.95, 134.31, 132.30, 131.57, 130.45, 129.93, 126.32, 106.33,
+
03.74, 97.44, 65.25, 45.88, 43.79, 12.52. HR-MS (ESI): Calcd. C H N O S, [M+H] m/z:
2
2
28
4
5
61.1853, found: 461.1859.
.2.3.16. (E)-N'-(2-hydroxy-4-methoxybenzylidene)-3-(morpholinosulfonyl)benzohydrazide (6p)
1
Yellow solid, yield: 85 %; m.p.: 203 – 206 °C. H NMR (400 MHz, DMSO-d ) δ 12.23 (s, 1H),
6
1
1.49 (s, 1H), 8.60 (s, 1H), 8.28 (d, J = 9.2 Hz, 2H), 7.96 (d, J = 7.9 Hz, 1H), 7.85 (t, J = 7.7 Hz,
H), 7.48 (d, J = 8.6 Hz, 1H), 6.59 – 6.47 (m, 2H), 3.79 (s, 3H), 3.69 – 3.60 (m, 4H), 2.96 – 2.86
1
1
5

ACCEPTED MANUSCRIPT
1
3
(
m, 4H). C NMR (101 MHz, DMSO-d ) δ 162.25, 161.06, 159.42, 149.37, 134.99, 134.06,
6
1
32.42, 131.00, 130.65, 129.99, 126.41, 111.72, 106.56, 101.15, 65.24, 55.31, 45.87. HR-MS
+
(
ESI): Calcd. C H N O S, [M+Na] m/z: 442.1043, found: 442.1046.
1
9
21
3
6
4
.2.3.17. (E)-N'-(3-ethoxy-2-hydroxybenzylidene)-3-(morpholinosulfonyl)benzohydrazide (6q)
1
Yellow solid, yield: 82 %; m.p.: 190 – 194 °C. H NMR (400 MHz, DMSO-d ) δ 12.31 (s, 1H),
6
1
1
2
0.77 (s, 1H), 8.71 (s, 1H), 8.30 (d, J = 11.6 Hz, 2H), 7.97 (d, J = 7.9 Hz, 1H), 7.86 (t, J = 7.7 Hz,
H), 7.18 (d, J = 6.9 Hz, 1H), 7.04 (d, J = 7.1 Hz, 1H), 6.86 (t, J = 7.9 Hz, 1H), 4.08 (q, J = 7.0 Hz,
1
3
H), 3.71 – 3.56 (m, 4H), 2.99 – 2.85 (m, 4H), 1.36 (t, J = 7.0 Hz, 3H). C NMR (101 MHz,
DMSO-d ) δ 161.29, 148.84, 147.47, 147.07, 135.04, 134.01, 132.48, 130.74, 130.02, 126.47,
6
1
20.74, 119.12, 119.00, 115.32, 65.25, 64.13, 45.88, 14.71. HR-MS (ESI): Calcd. C H N O S,
20 23 3 6
+
[
M+Na] m/z: 456.1200, found: 456.1209.
4
.2.3.18. (E)-N'-(5-chloro-2-hydroxybenzylidene)-3-(morpholinosulfonyl)benzohydrazide (6r)
Yellow solid, yield: 83%; m.p.: 157 – 160 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.40 (s, 1H),
1.17 (s, 1H), 8.68 (s, 1H), 8.29 (d, J = 11.9 Hz, 2H), 7.97 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 7.7 Hz,
H), 7.70 (d, J = 2.6 Hz, 1H), 7.39 – 7.29 (m, 1H), 6.97 (d, J = 8.8 Hz, 1H), 3.72 – 3.55 (m, 4H),
.99 – 2.85 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 161.46, 156.02, 146.25, 134.98, 133.91,
32.54, 130.99, 130.81, 130.02, 127.25, 126.50, 123.02, 120.71, 118.24, 65.23, 45.86. HR-MS
1
1
2
1
(ESI): Calcd. C18H18ClN3O5S, [M+Na]+m/z: 446.0548, found: 446.0553.
4
.2.3.19. (E)-N'-(5-bromo-2-hydroxybenzylidene)-3-(morpholinosulfonyl)benzohydrazide (6s)
White solid, yield: 78%; m.p.: 207 – 210 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.40 (s, 1H),
1.19 (s, 1H), 8.67 (s, 1H), 8.30 (d, J = 10.6 Hz, 2H), 7.97 (d, J = 7.0 Hz, 1H), 7.92 – 7.79 (m, 2H),
.45 (d, J = 7.9 Hz, 1H), 6.92 (d, J = 8.6 Hz, 1H), 3.70 – 3.60 (m, 4H), 2.99 – 2.87 (m, 4H). 13C
1
7
NMR (101 MHz, DMSO-d6) δ 161.46, 156.42, 146.08, 134.97, 133.91, 133.79, 132.54, 130.80,
30.10, 130.02, 126.49, 121.32, 118.68, 110.50, 65.23, 45.86. HR-MS (ESI): Calcd.
C18H18BrN3O5S, [M+Na]+m/z: 490.0043, found: 490.0023.
1
4
.2.3.20. (E)-N'-(3,5-dibromo-2-hydroxybenzylidene)-3-(morpholinosulfonyl)benzohydrazide (6t)
1
White solid, yield: 84%; m.p.: 246 – 250 °C. H NMR (400 MHz, DMSO-d ) δ 12.79 (s, 1H),
6
1
3
2.61 (s, 1H), 8.58 (s, 1H), 8.33 – 8.26 (m, 2H), 8.00 (d, J = 7.9 Hz, 1H), 7.93 – 7.81 (m, 3H),
1
3
.70 – 3.60 (m, 4H), 2.97 – 2.88 (m, 4H). C NMR (101 MHz, DMSO-d ) δ 161.64, 153.68,
6
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1
6
5
47.85, 135.79, 135.11, 133.35, 132.63, 132.14, 131.08, 130.11, 126.60, 120.93, 111.30, 110.48,
+
5.25, 45.86. HR-MS (ESI): Calcd. C H Br N O S, [M+Na] m/z: 567.9148, found: 567.9152.
1
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17
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5
. Materials and Methods
.1 Materials
All compounds ware dissolved in DMSO to make a 10mM stock solution. RPMI-1640 and
5
DMEM were purchased from Solarbio. MTT was also purchased from Solarbio. Fetal Bovine
Serum (FBS) was obtained from Tianhang Biotechnology Co. Annexin V-FITC/PI Apoptosis
Detection Kit was purchased from Keygen Biotech. ROS assay kit, NAC (N-acetyl-L-cysteine)
and JC-1 were purchased from Beyotime Biotechnology. Z-VAD-FMK was purchased from CST.
Bax, P53, PARP polyclonal antibodies were purchased from Proteintech; Bcl-2, Bid, and
Caspase-3/8/9 monoclonal antibodies were purchased from CST.
5.2 Cell culture
Human cancer cells EC109, EC9706, SMMC-7721, PC3, MCF7, MGC803 were obtained from
Zhengzhou University[30-32] and maintained in RPMI-1640 (Solarbio, China) and DMEM
(Solarbio) complete medium (which supplemented with 10% FBS and 100 U/ml penicillin and
1
00 g/ml streptomycin antibiotics) at 37°C in a 5% CO humidified atmosphere.
2
5.3 MTT assay
The day before treatment, cells were seeded in 96-well plates (NEST Biotechnology, China) at
3
a concentration of 2.0-4.0×10 cells per well, followed by treatment with serial dilutions of the
compound 6p (0.16µM-20µM) and incubated for 24h, 48h and 72h separately. 20 µL MTT
reagent (5mg/ml, 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide, Solarbio) was
added to each well, and cells were incubated for another 4h. Then, the medium was removed and
the formazan was dissolved in 150µL DMSO[33]. Absorbance values were measured at 490nm,
using an enzyme-linked immunosorbent assay reader (BioTek, USA) after shaking for
approximately 10 min. The cell viability rate was calculated as follows: viability rate = Abs 490
treated cells /Abs 490 control cells ×100%. The drug concentrations required to inhibit cell growth
by 50% (IC ) was determined from concentration-response curves created with SPSS 16.0
5
0
software. The results are reported as the mean ± standard deviation (SD) of three independent
experiments. The cell viability curves at different concentrations of all compounds were created
with GraphPad Prism 7.0 software.
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5.4 Colony formation assay
The day before treatment, cells were seeded in a six-well plate at a concentration of 500 cells
per well[34]. They were then treated with 0, 0.5 ,1 and 2 µM 6p for 7 days. After washing with
PBS (phosphate-buffered saline), cells were fixed with ice-cold methanol and stained with crystal
violet solution (0.5% in 20% methanol). Images were photographed after drying the plates
overnight. The crystal violet crystals were dissolved by adding 75% ethanol. The absorbance at
5
95 nm was measured by an enzyme-linked immunosorbent assay reader. Data were analyzed
using the GraphPad Prism software.
5.5 Cell cycle analysis
5
Cells were seeded at 2.0×10 cells/well in six-well plates and treated with 0 , 2, 4 and 8 µM 6p
for 24 hours, harvested with trypsin, washed twice with PBS and then fixed with ice-cold ethanol
(75%) for 1 hour at 4°C. The cells were washed twice in cold PBS and resuspended in 400 µL of
freshly prepared PBS with 20µg/mL RNase A (Beyotime, China) and 20µg/mL propidium
iodide (PI, Solarbio) [35]. After incubation at room temperature in the dark for 30 minutes, the
cells were filtered through a nylon mesh, loaded onto the FAC-SCalibur flow cytometer (BD,
USA) and analyzed, using the ModFit software.
5.6 Cell morphology analysis and Hoechst 33342 staining[36]
4
Cells were seeded at 5.0×10 cells/well in 24-well plates and incubated overnight for adherent
and treated with 6p at 0, 2, 4 and 8µM for 48h. The morphological changes were observed under
an inverted microscope. Nuclear fragmentation was visualized by Hoechst 33342 staining of
apoptotic nuclei for 30 min in the dark and examined under Nikon Eclipse TE 2000-S
fluorescence microscope.
5.7 Analysis of apoptosis by flow cytometry
5
Cells were seeded at 4.0×10 cells/well in 6-well plates and incubated for 12h until adherent.
Next, the cells were treated with 6p at 0, 2, 4 and 8µM for 48h. Then, the adherent and
non-adherent cells were harvested and the Annexin V-FITC/PI Apoptosis Detection Kit (Keygen
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Biotech,China) was used, according to the manufacturer’s protocol. Briefly, cells were washed
with ice-cold PBS, resuspended in binding buffer containing Annexin V-FITC and propidium
iodide (PI), and incubated for 15 min at room temperature in the dark prior to analysis using a
flow cytometer[37]. Cells that were Annexin V-positive only and both Annexin V- and PI-positive
were considered early and late apoptosis cells, respectively. Both the early and late apoptotic cells
were included in the total number of apoptotic cells. All experiments were performed in triplicate.
5.8 Measurement of intracellular ROS
Intracellular ROS levels were measured by flow cytometry using a cell-based ROS assay kit
Beyotime Biotechnology, Haimen, People’s Republic of China)[30]. Cells seeded in 6-well plates
(
were treated with compound 6p(0µM, 1µM, 2µM and 4µM) for 24h and 50µg/ml Rosup for 20min.
Cells were harvested and washed twice with PBS, incubated with 10µM dichlorofluorescin
diacetate for 20 minutes at 37°C. Then analyzed by the FACSCaliber flow cytometer (BD
Biosciences, San Jose, CA, USA). Intracellular ROS levels were expressed as the average
dichlorodihydrofluorescein fluorescence intensity of the cells.
5.9 Measurement of mitochondrial membrane potential
Mitochondrial membrane potential was measured using the JC-1 dye [38] (Beyotime
Biotechnology, Haimen, People’s Republic of China), following the manufacturer’s instructions.
Cells seeded in 24-well plates were incubated with JC-1 for 20 minutes, then treated with 0µM,
2
µM, 4µM, 8µM and 16µM for 12h, 24h, 36h, separately. Images were taken using a fluorescent
microscope.
5.10 Western blot analysis
Cells seeded in 10cm dishes were treated with 0µM, 2µM, 4µM and 8µM 6p for 48h. The cells
were harvested with trypsin/EDTA, and lysed with RIPA lysis buffer. Total proteins were
extracted and separated by 8%-15% SDS-PAGE, and transferred on a nitrocellulose filter
membrane. The membranes were blocked in 5% skim milk for 2h at room temperature, and then
probed with the appropriate primary antibodies (1:1000) at 4°C overnight. After washing in PBST
(3×10min) , membranes were incubated with horseradish-peroxidase-conjugated secondary
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antibody (1:5000) at room temperature for 2h. Then,the membrane preparations were washed
three times as above and examined by enhanced chemiluminescence[39]. The films were
subsequently scanned, and the signal intensity of each band was determined using Image J
software. All experiments were performed in triplicate.
5.11 Acute toxicity test
C57/BL6 mice were divided into two groups (n=10 mice/each group). After 8 hours of
starvation treatment, compound 6p dissolved in oil (2g/kg) were given intragastrically, the mice in
control group were given oil. After 2 weeks observation, dead mice were counted and the organs
(heart, liver, spleen, lung, and kidney) were taken to fix in paraformaldehyde. At the end of the
observation , we executed all mice and took all organs, which were applied to HE staining.
5.12 Hematoxylin and Eosin (HE) staining
Organs were fixed in 4% paraformaldehyde for 48h and were embedded in paraffin and cut into
3
-µm sections.The sections were deparaffinized in xylene, rehydrated in graded ethanol, rinsed in
distilled water. Sections were stained with hematoxylin and eosin followed by dehydration in
graded alcohol and examined under a morphometric microscope.
5.13 Statistical analysis
Results were expressed as mean ± standard deviation. Statistical analysis was performed using
student t test for two group comaprision and One-Way ANOVA for more than two groups. P<0.05
was considered statistically significant.
Acknowledgement:
This work was supported by the National Key Project from Ministry of Science
&Technology (2016YFA0501800 for H.-M.L and W.Z.), the Natural Science Foundation of China
(Project No. 81470524 for W.Z. No. 81430085 for H.-M. L.).
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Fig. 3. Compound 6p inhibited the proliferation of EC9706 and EC109 cells. A and B. EC9706
and EC109 cells were treated with compound 6p for 24 h, 48 h and 72 h, respectively, the cell
viabilities were measured, using MTT assay. C. EC9706 and EC109 were treated with compound
6
p at 0.5µM, 1µM, 2µM for 7 days. The EC9706(D) and EC109(E) cells clonogenicity assay was
quantified by measuring absorbance of crystal violet. F. EC9706 and EC109 were treated with
compound 6p at 0, 2, 4 and 8µM for 24h. Quantitative analysis of the percentage of EC9706(G)
and EC109(H) cells cycle phase distribution. I. Western blot. EC9706 and EC109 were treated
with compound 6p at 0, 2, 4 and 8µM for 24h. J. Quantitative analysis of the protein P27 level.
Three individual experiments were performed for each group. The data were expressed as the
Mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control.
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Fig. 4. Compound 6p induced apoptosis in EC9706 and EC109 cells. A. Hoechst 33342 staining.
EC9706 and EC109 were treated with compound 6p at 0, 2, 4, and 8µM for 48h, then stained with
Hoechst 33342 to analysis the nuclear morphological changes (marked with white arrows,
magnification: 100×). B. EC9706 and EC109 were treated with compound 6p at 0, 2, 4, and
8
µM for 48h. EC9076(C) and EC109(D) apoptosis rates were quantified by flow cytometry. Three
individual experiments were performed for each group. The data was expressed as the Mean ± SD.
p < 0.05, ****p < 0.0001 compared with the control. E. EC9706 and EC109 were pretreated with
*
caspase inhibitor Z-VAD-FMK (40µM) for 1h, followed with compound 6p treatment for 48h.
EC9076(F) and EC109(G) cells apoptosis rates were measured by flow cytometry. Three
individual experiments were performed for each group. The data was expressed as the Mean ± SD.
*p < 0.05, ***p < 0.001 when the Z-VAD-FMK plus 6p group is compared with the 6p group.
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Fig. 5. Compound 6p induced apoptosis through ROS. A. EC9706 and EC109 cells were treated
with compound 6p at 0, 1, 2 and 4µM for 24h and thereafter loaded with 10µM DCFH-DA for
examination of ROS surge, 50µg/ml Rosup was taken as a positive control. The levels of ROS in
EC9706(B) and EC109(C) cells were quantified by flow cytometry. Three individual experiments
were performed for each group. The data was expressed as the Mean ± SD. **p < 0.01, ***p <
0
.001 compared with the control. D. EC9706and EC109 cells were pretreated with NAC (5mM)
for 1h, followed with compound 6p at 2µM, 4µM for 24h. The levels of ROS in EC9706(E) and
EC109(F) cells were quantified by flow cytometry. **p < 0.01, ***p < 0.001, ****p <
0
.0001when the NAC plus 6p group is compared with the 6p group. G. EC9706 and EC109 cells
were pretreated with NAC (5mM) for 1h, followed with compound 6p at 0, 4 and 8µM for 48h.
EC9076(H) and EC109(I) cells apoptosis rates were measured by flow cytometry. Three
individual experiments were performed for each group. The data was expressed as the Mean ± SD.
*p < 0.05, **p < 0.01, ***p < 0.001 when the NAC plus 6p group is compared with the 6p group.
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Fig. 6. The role of compound 6p on pro- and anti-apoptosis-related protein expressions. A.
EC9706 and EC109 cells were treated with 6p at 0, 2, 4, 8 and 16µM for 12, 24 and 36h,
respectively, then loaded with MMP indicator JC-1. Images were taken using a fluorescent
microscope. (magnification: 100×). B. EC9706 and EC109 were treated with compound 6p at 0,
2
, 4 and 8µM for 48h and subjected to western blot analysis, using the indicated antibodies. C and
D. Quantitative analysis of the proteins level. Three individual experiments were performed for
each group. The data was expressed as the Mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001,
*
***p < 0.0001 compared with the control.
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Fig. 7. The role of compound 6p on Prxs protein expressions. A. EC9706 and EC109 were treated
with compound 6p at 0, 2, 4 and 8µM for 48h and subjected to western blot analysis using the
indicated antibodies. B and C. Quantitative analysis of the proteins level. Three individual
experiments were performed for each group. p > 0.05.
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Fig.8. Histopathological examination of major organs in 6p (2g/kg) treated and control mice. The
mice were executed, major organs (heart, liver, spleen, lung, kidney) were collected and fixed in 4%
paraformaldehyde and paraffin embedded for H&E staining. Pictures were original captured at
2
00×magnification.
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