Identification and biological evaluation of novel benzothiazole derivatives bearing a pyridine-semicarbazone moiety as apoptosis inducers via activation of procaspase-3 to caspase-3

Article abstract of DOI:10.1039/c8md00624e

Three series of compounds were designed, synthesized and evaluated for their in vitro anticancer activity against a procaspase-3 over-expression cancer cell line (U937) and a procaspase-3 no-expression cancer cell line (MCF-7) to rule out off-target effects. Biological evaluation led to the identification of a series of benzothiazole derivatives bearing a pyridine-semicarbazone moiety, 8j and 8k, with promising anticancer activity and remarkable selectivity. Further mechanism studies revealed that compounds 8j and 8k could induce apoptosis of cancer cells by activating procaspase-3 to caspase-3, and compound 8k exhibited the strongest procaspase-3 activation activity. Structure-activity relationships (SARs) revealed that the presence of benzothiazole and an N,N,O-donor set is crucial for the anticancer activity and selectivity, and reducing the electron density of the N,N,O-donor set results in a dramatic decline in the anticancer activity and selectivity. Furthermore, toxicity evaluation (zebrafish) in vivo and metabolic stability studies (human, rat and mouse liver microsomes) were performed to provide reliable guidance for further PK/PD studies in vivo.

Full text of DOI:10.1039/c8md00624e

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DOI: 10.1039/C8MD00624E
Identification and biological evaluation of novel benzothiazole
derivatives bearing a pyridine-semicarbazone moiety as apoptosis
inducers via activation of procaspase-3 to caspase-3
a*
a
b
a
c
a
a
d*
Junjie Ma , Xin Ni , Yali Gao , Kun Huang , Jiaan liu , Yu Wang , Roufen Chen , Cuifang Wang
Three series of compounds were designed, synthesized and evaluated for their in vitro anticancer
activity against a procaspase-3 over-expression cancer cell line (U937) and a procaspase-3
no-expression cancer cell line (MCF-7) to rule out off-target effects. Biological evaluation led to the
identification of a series of benzothiazole derivatives bearing pyridine-semicarbazone moiety, 8j
and 8k, with promising anticancer activity and remarkable selectivity. Further mechanism studies
revealed that compounds 8j and 8k could induce apoptosis of cancer cells by activating
procaspase-3 to caspase-3, and compound 8k exhibited the strongest procaspase-3 activation
activity. Structure-activity relationships (SARs) revealed that the presence of benzothiazole and
N,N,O-donor set are crucial for the anticancer activity and selectivity, and reducing the electron
density of N,N,O-donor set results in a dramatic decline in the anticancer activity and selectivity.
Furthermore, toxicity evaluation (Zebrafish) in vivo and metabolic stability (human, rat and mouse
liver microsomes) were performed to provide a reliable guidance for further PK/PD studies in vivo.
a
School of medicine, Huaqiao University, Quanzhou, 362000, China. E-mail: majunjie3612@hqu.edu.cn.
b
Pharmacy Department, the Second Affiliated Hospital of Fujian Medical University. Quanzhou, 362000, China.
c
Department of Chemistry, University of Massachusetts-Amherst, Amherst, Massachusetts 01003, United States.
d
College of Oceanology and Food Science, Quanzhou Normal University, Quanzhou 362000, China. E-mail:
wlycf@163.com.
*
corresponding author
1
. Introduction
overcoming
the
mutation
of
the
Caspase-3 plays a significant role in the
middle/upstream apoptotic protein.
apoptotic cascade as the most common and
important downstream effector protein. The
conversion of zymogen procaspase-3 to
caspase-3 can catalyze the hydrolysis of
hundreds of protein substrates, leading to
PAC-1 (Fig. 1) was reported as the first
procaspase-3 activator that induced apoptosis
by selectively activating procaspase-3 to
caspase-3 via chelating the inhibitory
endogenous metal ion zinc, which inhibits the
1
-3
procaspase-310,
11,
apoptosis . One of the hallmarks of cancer is
the ability to avoid apoptosis4, 5. Studies have
shown that zymogen procaspase-3 is
overexpressed in a variety of human tumors
activation
Structure−activity
revealed that
of
relationships
(SARs)
the
ortho-hydroxy
N-acylhydrazone moiety of PAC-1 is
6
-9
owning to inhibition of apoptosis . Therefore,
identification of small molecules that induce
the apoptosis of tumor cells by directly
activating downstream over-expression of
procaspase-3 to caspase-3 has become an
effective strategy for improving selectivity and
responsible for strong procaspase-3 activity
1
2
and participates in chelating with zinc . In our
previous studies, a series of benzothiazole
derivatives bearing the ortho-hydroxy
N-carbamoylhydrazone moiety were reported
as procaspase-3 activators13, 14, however, due to
1

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DOI: 10.1039/C8MD00624E
the presence of lipophilic groups (eg.
properties, including antibacterial, antiviral,
substituted benzyloxy or heteroaryloxy
groups), these compounds exhibited high
lipophilicity (Clog P=6.13-9.67) and were not
suitable for further development (Fig. 1).
Pyridine-semicarbazone derivatives (Fig. 1)
have long attracted much attention as an
important class of compounds due to their
remarkable biological and pharmacological
antineoplastic
and
antimalarial15-17
.
Additionally, the pyridine-semicarbazone
moiety could chelate with metal ions (Zn, Pd,
Fe, Co etc.) based on the N,N,O-donor set,
which have been widely used as important
structural components in modern drug design
for
the
treatment
of
cancer18-21
.
Fig. 1. Procaspase-3 activators and pyridine-semicarbazone derivatives.
2
2
Inspired by the above findings, we
effects . In addition, these compounds with
high selectivity and promising anticancer
activity were selected for further mechanism
studies, including flow cytometric assay for
apoptosis and caspase-3 and procaspase-3
activity evaluation. Furthermore, the promising
compounds were further selected for in vivo
toxicity evaluation and apoptotic study in
zebrafish embryos. Finally, metabolic stability
was performed to assess the half-life in vitro
and provide a guidance for further PK/PD
studies in vivo.
designed a series of benzothiazole derivatives
bearing
a pyridine-semicarbazone moiety
(series A). In addition, the importance of the
benzothiazole and N,N,O-donor set were
addressed with the replacement of a benzene
group to get series B and series C, respectively
(Fig. 2). All target compounds were evaluated
for their in vitro anticancer activities against a
procaspase-3 over-expression cancer cell line
(U937, human lymphoma) and a procaspase-3
no-expression cancer cell line (MCF-7, human
breast adenocarcinoma) to rule out off-target
2

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Fig. 2. Design of target compounds.
2
2
. Results and discussion
.1 Chemistry
ammonium thiocyanate and bromine to
produce 3a-c in a satisfactory yield. Treatment
of 3a-c or 2a-c with phenyl chloroformate
though a N-acylation reaction yielded 4a-c or
5a-c. The key intermediate semicarbazides
6a-c or 7a-c were prepared via hydrazinolysis
of 4a-c or 5a-c with 80% hydrazine
monohydrate. Subsequently, 6a-c or 7a-c were
reacted with corresponding aldehydes or
ketones via a condensation reaction in the
presence of catalytic amounts of acetic acid to
obtain target the compounds 8a-l, 9a-h and
10a-e.
The preparation of the target compounds
is illustrated in Scheme 1. The commercially
available starting material 4-nitrobenzyl
bromide, was reacted with various small
molecule
secondary
dimethylamine
amines
and
(4-methylpiperidine,
diethylamine) via a nucleophilic substitution
reaction to provide intermediates 1a-c, which
were reduced in the presence of 80% hydrazine
and FeCl 6H O to obtain the substituted
3
·
2
anilines 2a-c. 2a-c was further reacted with
3

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Scheme 1. Reagents and conditions: (i) Acetonitrile, amine, rt, 3 h; (ii) 80% hydrazine monohydrate, FeCl
activated carbon, ethanol, 65 °C to 78 °C, 5 h; (iii) Br , NH SCN, HOAc, 10 °C to rt, 4-6 h; (iv) Phenyl
, 0°C to rt, 4-6 h; (v) 80% hydrazine monohydrate, 1,4-dioxane, 80 °C, 6 h. (vi)
3 2
·6H O,
2
4
chloroformate, pyridine, CH
2 2
Cl
HOAc, ethanol, reflux.
2
.2 In vitro anticancer activity and SARs
All the target compounds were evaluated
μM and 6.6 μM as compared to other
compounds.
for their in vitro anticancer activities against a
Due to the potent anticancer activity and
high selectivity of compounds 8a-l (series A),
further investigations were carried out. The
results revealed that compounds possessing a
4-methylpiperidine group (8i-k) showed
enhanced activity compared to the compounds
possessing a dimethylamino or diethylamino
group (8a-g). Furthermore, the IC50 values of
8a-c, 8e-g and 8i-k revealed that the
introduction of methyl or 2-pyridyl in the R
group had a stronger contribution to the
potency than hydrogen. It was worth noting
that replacement of the 2-pyridyl (8b, 8f, 8j)
with 2-pyrazinyl (8d, 8h, 8l) led to a dramatic
decline in selectivity. This may be due to the
fact that the electron density of pyrazine is
lower than that of pyridine, which reduces the
electron density of the N,N,O-donor set, and
further weakenes the chelating ability to zinc
ion, leding to a remarkable change in
procaspase-3 over-expression cancer cell line
(U937) and a procaspase-3 no-expression
cancer cell line (MCF-7) by using PAC-1 as
positive control. As shown in Table 1, all the
target compounds showed more preferable
Clog P values ranging from 1.18 to 4.35,
compared
derivatives in our previous studies. In addition,
most of the compounds possessing
to
reported
benzothiazole
a
benzothiazole (series A) showed potent
anticancer activity and high selectivity against
U937, however, no obvious anticancer activity
and selectivity were observed in these
compounds possessing a benzene group (series
B), indicating that the benzothiazole was more
helpful for anticancer activity and selectivity
than benzene. Among them, the promising
compounds 8j and 8k showed the highest
selectivity and potency with IC values of 5.2
5
0
4

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selectivity. Thus, it was concluded that electron
density of N,N,O-donor set was crucial for the
selectivity.
Table 1. Anticancer activity and selectivity for target compounds 8a-l and 9a-h (series A and series B).
R1
N
R1
N
O
N
N
R2
O
S
X
R2
N
X
N
N
N
H
H
N
H
X=C or N
N
N
X=C or N
R
H
R
9
a-h
8
a-l
Series B
Series A
a
R1
N
R2
N
IC50 (μM)
Comp.
X
R
H
Selectivityc
Clog Pd
U937b
MCF-7b
8
a
C
C
23.2±0.6
20.1±0.4
>40
1.6
2.0
2.82
2.14
8
b
N
N
N
-CH
3
>40
>40
>40
8
c
C
N
N
7.8±0.5
>40
5.1
-
2.64
1.18
8
d
-CH
3
8
e
N
C
C
-H
24.8±0.8
15.6±0.5
>40
>40
1.6
2.6
3.88
3.19
8
f
N
N
N
-CH
3
8
g
C
N
9.1±0.2
>40
4.3
3.70
8
h
N
C
C
-CH
-H
3
>40
>40
>40
-
2.24
4.53
3.85
8
i
N
N
21.5±0.9
5.2±0.7
1.6
2.3
8
j
-CH
3
12.4±0.6
8
k
N
N
C
N
6.6±0.2
>40
6.0
4.35
8
l
N
C
-CH
-H
3
>40
>40
11.8±0.3
>40
<1
-
2.89
1.92
9
a
N
N
9
b
C
C
C
N
>40
>40
>40
>40
>40
>40
-
-
-
2.70
3.26
3.76
9
c
N
N
-CH
3
9
d
N
9
e
N
N
C
C
-H
>40
>40
>40
>40
-
-
3.62
3.91
9
f
-CH
3
9
g
N
N
C
N
N
>40
>40
-
4.41
9
h
-CH
3
>40
>40
>40
-
2.95
5.20
PAC-1
6.0±0.5
6.6
a
IC50: The biological data are generated from three independent experiments.
b
U937: procaspase-3 over-expression cancer cells; MCF-7: procaspase-3 no-expression cancer cells.
cSelectivity is expressed as a ratio of MCF-7/U937.
d
Clog P values were calculated by using Chemdraw software.
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In order to address the importance of the
Whereas, compounds 10a, 10c, 10d and 10e
DOI: 10.1039/C8MD00624E
N,N,O-donor set, the anticancer activity and
selectivity of compounds 10a-e (series C) were
determined. As shown in Table 2, due to the
absence of the N,N,O-donor set, compounds
displayed more potent activities with IC50
values of 12.3 μM, 12.1 μM, 13.1 μM and 13.3
μM against the procaspase-3 no-expression
cancer cell line (MCF-7). These results
indicated that the presence of the N,N,O-donor
set is crucial for selectivity.
1
0a-e showed insufficient anticancer activity
(> 40 μM) against the procaspase-3
over-expression cancer cell line (U937).
Table 2. Anticancer activity for target compounds10a-e (series C).
R1
N
O
R3
Ar
N
R2
N
N
H
H
R
10a-e
Series C
a
R1
N
R2
IC50±SD (μM)
U937b
Compd.
Ar
X
R
R
3
Selectivityc Clog Pd
MCF-7b
12.3±0.7
>40
S
1
0a
N
C
C
-CH
-CH
4-F
-
>40
>40
<1
-
4.83
5.35
3
3
N
S
1
0b
N
N
1
0c
N
C
C
C
-
-
-
>25
>25
12.1±0.6
13.1±0.8
<1
<1
5.69
6.75
1
0d
N
1
0e
N
>25
13.3±0.9
>40
<1
7.41
5.20
PAC-1
6.0±0.5
6.6
a
IC50: The biological data are generated from three independent experiments.
b
U937: procaspase-3 over-expression cancer cell line; MCF-7: procaspase-3 no-expression cancer cell line
cSelectivity is expressed as a ratio of MCF-7/U937.
d
Clog P values were calculated by using Chemdraw software.
2
.3 Apoptosis study
and flow cytometry. As shown as Fig. 3,
Treatment U937 cells with different
concentrations of 8j and 8k for 24 h and 48 h
caused significant dose- and time-dependent
increase in the population of both early and late
apoptotic cells compared to the control cells,
suggesting that 8j and 8k could induce cancer
cells apoptosis in a dose- and time-dependent
manner.
In order to determine whether the
inhibitory effects of 8j and 8k were dependent
on inducing cancer cells apoptosis, treatment
of DMSO was using as negative control, U937
cells were incubated with different
concentrations of 8j and 8k for 24 h and 48 h,
the percentages of apoptotic cells were
determined by FITC-Annexin V/PI staining
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Fig.3. Apoptosis in U937 cells by the treatment with 8j and 8k for 24 h and 48 h.
2
.4 Caspase-3 activation study
Because of their high selectivity and
potent anticancer activity, compounds 8b, 8c,
g, 8j, 8k, 10a, 10c, 10d and 10e were selected
reads. As shown in Table 3 and Fig. 4,
compared to PAC-1 (100%), compounds 8b,
8c, 8g, 8j and 8k showed strong caspase-3
activation activity (> 60%). Among them,
compound 8j showed a comparable activation
activity (99%) with PAC-1, while compounds
8c, 8g and 8k induced stronger caspase-3
activation activity than PAC-1 with the degrees
of 104%, 119% and 114%, respectively.
Compounds 10a, 10c, 10d and 10e induced
lower than 60% activation of caspase-3 due to
the absence of the N,N,O-donor set.
8
for further investigation. By using PAC-1 and
DMSO as positive and negative control, the
ability of these compounds to activate
executioner caspase-3 in whole cells was
evaluated. Cells were treated with these
compounds for 0 or 16 h, lysed, and cleavage
of the fluorescent caspase-3 substrate
Ac-DEVD-AFC was analyzed via kinetic
Table 3. Caspase-3 activation for PAC-1 and selected compounds in U937 at 30 μM.
Compd.
PAC-1
Caspase-3 activationa
Compd.
10a
10c
10d
10e
Caspase-3 activationa
100±4
68±4
104±12
119±6
40±9
28±2
20±3
58±8
7±1
8
b
8
c
8
g
8
j
99±10
DMSO
8
k
114±10
aThe biological data are generated from three independent experiments.
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Fig.4. Caspase-3 activation activity by PAC-1 (control) and selected compounds in U937 at 30 μM.
2
.5 In vitro procaspase-3 activation assay
Zebrafish
Zebrafish (Danio rerio) have been
In order to further verify that the strong
caspase-3 activation activity was caused by the
activation of procaspase-3, By using PAC-1
and DMSO as positive and negative control,
the promising compounds 8j and 8k were
selected to determine procaspase-3 activation
activity. As shown as Table 4, compared to
PAC-1, compounds 8j and 8k displayed potent
procaspase-3 activation activities at 10 μM
with degrees of 77.8% and 92.1%, respectively,
suggesting that apoptosis of cancer cells
induced by activating procaspase-3 to
caspase-3 might be a potential mechanism for
antitumor effects of these target compounds.
Taken together, these above findings
identified compounds 8j and 8k, which could
be developed as promising candidates as
procaspase-3 activators.
considered as a suitable animal model for drug
discovery due to their high fertility, high
homology with human DNA, rapid
2
3
development, transparency , and have been
widely used in high-quality in vivo validation
2
4
of drug targets and toxicological studies . In
this study, survival rates of zebrafish embryos
exposed to various concentrations (5, 10, 20
and 40 uM) of compounds 8j and 8k were
determined at 24, 48 and 72 h post fertilization
(hpf), and a no-treatment control was also
included. As shown in Fig.5 (A and B), no
obvious toxic effects were observed for 8j and
8k at the concentrations of 5, 10 and 20 uM for
24, 48, and 72 hpf, indicating that 8j and 8k
have low toxicity at these concentrations,
which is far greater than the half maximal
inhibitory concentration (IC ) of 8j and 8k in
5
0
Table 4. procaspase-3 activity of selected compounds
the anticancer effect. After 48 hpf exposure of
zebrafish to 40 uM of 8k, moderate toxic
effects were observed; however, after a longer
exposure (72 hpf), 40 uM of 8j led to the death
of most embryos, and 40 uM of 8k led to the
death of almost all embryos.
8
j, 8k and PAC-1 at 10 μM in vitro.
Compd.
Procapase-3 activity (%)
77.8±5.0
8
8
j
k
92.1±3.5
PAC-1
100±7.6
2
.6 In vivo toxicity evaluation in the
8

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Fig. 5. (A) Survival rates of zebrafish embryos exposed to various concentrations of 8i and 8j over 3 days
post-fertilization (72 hpf). Data were expressed as means ± standard deviation from three independent experiments
(Twenty-four embryos for each group). (B) Gross morphological phenotypes in zebrafish embryos at 72 hpf: treated
with 20 uM of 8j (left), untreated (middle), and treated with 20 uM of 8k (right).
2
.7 8j and 8k induced apoptosis in zebrafish
embryos
In order to further study whether
very specific accumulation of dying cells in the
head and cranial neural crest at 24 hpf.
Meanwhile, treatment of zebrafish with 8j and
8k significantly increased the mRNA
expression of proapoptotic protein, Bax
(Fig.6B, p<0.01), β-actin was detected as an
internal control.
compounds 8j and 8k could induce apoptosis
through other pro-apoptotic proteins, we firstly
used in vivo labeling with Acridine Orange to
visualize apoptotic cells in live zebrafish
embryos. As shown in Fig.6A, we observed a
Fig.6. 8j and 8k induced apoptosis in the zebrafish. (A) Acridine orange staining for apoptotic cells (the dotted
boxed area) at 24 hpf on control, 8j and 8k–treated embryos. (B) The expression of pro-apoptotic (bax) gene
analyzed by real-time quantitative RT-PCR.
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2
.8 In vitro metabolic stability study
Using ketanserin as reference compound,
metabolism (T1/2 = 30-120 min) in human and
DOI: 10.1039/C8MD00624E
rat liver microsomes with T1/2 values of 77.93
metabolic stability of compounds 8j and 8k
was evaluated in vitro by using the liver
microsome assay against human, rat and mouse
liver microsomes. As shown in Table 5,
compounds 8j and 8k exhibited moderate
min, 95.55 min, 79.06 min, and 73.87 min,
respectively. Compound 8j displayed better
stability in mouse microsomes than 8k, which
was susceptible to metabolism with a T1/2 of
16.72 min (T1/2 < 30 min).
Table 5. In vitro metabolic half-Life (T1/2) [min] and intrinsic clearance (Clint) of 8j and 8k.
Species
rat
Compd.
Index
human
36.07
48.19
77.93
22.31
79.06
21.99
mouse
20.46
266.76
53.63
101.76
16.72
326.43
T
1/2 (min)
14.40
172.52
95.55
25.99
73.87
33.62
ketanserin
Clint (mL/min/kg)
1/2 (min)
Clint (mL/min/kg)
1/2 (min)
Clint (mL/min/kg)
T
8
j
T
8
k
3
. Conclusions
In summary,
benzothiazole derivatives
chemical structures of this series of compounds
provide new template for the further design of
potential procaspase-3 activators, and the more
preferable Clog P values bring a promising
future for further development. Further
investigations on the PK/PD in vivo are
ongoing.
a
novel series of
bearing
a
pyridine-semicarbazone moiety were identified
as apoptosis inducers. Among them,
compounds 8j and 8k displayed excellent
anticancer activity against procaspase-3
over-expression cancer cells (U937) with the
IC50 values of 5.2 μM and 6.6 μM. Further
mechanism studies showed that 8j and 8k
could inhibit proliferation of cancer cells by
inducing apoptosis by activating procaspase-3
to caspase-3, and compound 8k exhibited the
strongest procaspase-3 activation activity with
degree of 92.1% at 10 μM. Toxicity evaluation
in zebrafish embryos suggested that 8j and 8k
showed weak toxicity. Furthermore, metabolic
stability study indicated that 8j and 8k
exhibited moderate metabolism in vitro against
human and rat liver microsomes. The analysis
of SARs indicated that the presence of the
benzothiazole and N,N,O-donor set are crucial
for anticancer activity and selectivity, and
reducing the electron density of the
N,N,O-donor set causes dramatic decline in
selectivity. In addition, incorporation of a
4. Experimental section
4.1. Chemistry
The H and 13C NMR spectroscopy were
performed using Bruker ARX-400, 400MHz
spectrometers (Bruker Bioscience, Billerica,
MA, USA) with tetramethylsilane (TMS) as an
internal standard. High resolution mass
spectrometry was performed on an Agilent
Accurate-Mass UHPLC-QTOF 1290-6545 in
ESI mode or EI mode. Melting points were
determined on a Melting Point XT5B apparatus
(KeYi, Beijing). Column chromatography was
run on silica gel (200-300 mesh) from Qingdao
Ocean Chemicals (Qingdao, Shandong,
China). Unless otherwise noted, all materials
were obtained from commercially available
sources and were used without further
purification.
1
4.1.1. Synthesis of key intermediate
semicarbazides 6a-c or 7a-c
4
-methylpiperidine group and introduction of
either a methyl or pyridyl in the R group can
enhance the anticancer activity. The novel
The preparation of key intermediate
semicarbazides 7a-c have been illustrated in
1
0

Page 11 of 20
MedChemComm
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detail in our previous work14, 25, and the
134.60, 132.12, 127.41, 124.40, 121.83, 121.49,
DOI: 10.1039/C8MD00624E
synthesis methods would not be listed here.
The 6a-c were prepared by referring to
synthesis methods of 7a-c.
120.01, 63.76, 45.40(2C), 12.58.
4.1.2.3.2-(di(pyridin-2-yl)methylene)-N-(6-((di
methylamino)methyl)benzo[d]thiazol-2-yl)hyd
razine-1-carboxamide(8c).
4
8
.1.2. General procedure for preparation of
a-l, 9a-h and 10a-e.
Light yellow solid; Yield: 71%; mp:
A mixture of compounds 6a-c or 7a-c
156-158
°C;
HRMS
(ESI)
for
+
(
0.001 mol), appropriate aldehydes or ketones
0.0011mol) and a drop of glacial acetic acid in
C H N OS[M+H] .calcd: 431.1528, found:
2
2
21
7
1
(
432.1609; H NMR (400 MHz, DMSO) δ
12.89 (s, 1H), 11.78 (s, 1H), 8.84 (d, J = 4.7 Hz,
1H), 8.56 (d, J = 4.6 Hz, 1H), 8.25-8.08 (m,
1H), 7.99 (q, J = 7.8 Hz, 2H), 7.84 (s, 1H),
7.64-7.55 (m, 2H), 7.53-7.43 (m, 2H), 7.34 (d,
5
6
mL absolute ethanol was heated at reflux for
-12 h and cooled to room temperature, The
mixture was concentrated to get crude product,
which was purified by chromatography on
silica gel using MeOH/CH Cl to afford the
1
3
J = 8.2 Hz, 1H), 3.49 (s, 2H), 2.18 (s, 6H); C
NMR (101 MHz, DMSO) δ 159.91, 155.79,
152.40, 151.72, 148.96, 148.83, 148.13, 145.35,
137.89, 137.50, 134.34, 131.81, 127.49, 126.79,
124.90, 124.28, 123.72, 121.97, 119.77, 63.65,
45.30(2C).
2
2
compounds 8a-k, 9a-h and 10a-e.
.1.2.1.(E)-N-(6-((dimethylamino)methyl)benz
4
o[d]thiazol-2-yl)-2-(pyridin-2-ylmethylene)hy
drazine-1-carboxamide(8a).
Light yellow solid; Yield: 52%; mp:
1
42-144
°C;
HRMS
(ESI)
for
4.1.2.4.(E)-N-(6-((dimethylamino)methyl)benz
o[d]thiazol-2-yl)-2-(1-(pyrazin-2-yl)ethylidene)h
ydrazine-1-carboxamide(8d).
+
C H N OS[M+H] .calcd: 354.1263, found:
1
7
18
6
1
355.1339; H NMR (400 MHz, DMSO) δ
11.48 (s, 2H), 8.58 (d, J = 4.4 Hz, 1H),
8.56-8.46 (m, 1H), 8.06 (s, 1H), 7.87 (t, J = 7.8
Light white solid; Yield: 68%; mp: 234-236 °C;
+
HRMS (ESI) for C H N OS[M+H] .calcd:
1
7
19
7
1
Hz, 1H), 7.82 (s, 1H), 7.63 (d, J = 8.2 Hz, 1H),
369.1372, found: 370.1446; H NMR (400
MHz, DMSO) δ 11.40 (s, 1H), 10.67 (s, 1H),
9.80 (s, 1H), 8.63 (s, 2H), 7.84 (s, 1H), 7.65 (d,
J = 8.2 Hz, 1H), 7.34 (d, J = 8.2 Hz, 1H), 3.48
(s, 2H), 2.37 (s, 3H), 2.17 (s, 6H); 13C NMR
(101 MHz, DMSO) δ 159.49, 153.40, 150.48,
148.50, 147.88, 144.45, 144.04, 143.47, 134.67,
132.14, 127.39, 121.79, 120.03, 63.78,
7
.42 – 7.36 (m, 1H), 7.34 (d, J = 8.2 Hz, 1H),
1
3
3
.47 (s, 2H), 2.17 (s, 6H); C NMR (101 MHz,
DMSO) δ 159.71, 153.62, 153.34, 149.65,
1
1
4
4
48.08, 144.16, 136.94, 134.59, 132.02,
27.37, 124.59, 121.79, 121.25, 119.79, 63.76,
5.40(2C).
.1.2.2.(E)-N-(6-((dimethylamino)methyl)benz
o[d]thiazol-2-yl)-2-(1-(pyridin-2-yl)ethylidene)h 45.42(2C), 12.33.
ydrazine-1-carboxamide(8b).
4.1.2.5.(E)-N-(6-((diethylamino)methyl)benzo[
Light yellow solid; Yield: 63%; mp:
d]thiazol-2-yl)-2-(pyridin-2-ylmethylene)hydr
1
47-149
°C;
HRMS
(ESI)
for
azine-1-carboxamide(8e).
Light white solid; Yield: 48%; mp: 164-166 °C;
+
C H N OS[M+H] .calcd: 368.1419, found:
1
8
20
6
1
+
3
1
1
69.1493; H NMR (400 MHz, DMSO) δ
1.09 (s, 1H), 10.47 (s, 1H), 8.59 (d, J = 4.6 Hz,
H), 8.50 (s, 1H), 7.90-7.80 (m, 2H), 7.64 (d, J
HRMS (ESI) for C H N OS[M+H] .calcd:
1
9
22
6
1
382.1576, found: 383.1650; H NMR (400
MHz, DMSO) δ 11.49 (s, 2H), 8.58 (d, J = 4.6
Hz, 1H), 8.57-8.47 (m, 1H), 8.07 (s, 1H),
7.90-7.81 (m, 2H), 7.63 (d, J = 8.2 Hz, 1H),
7.42 – 7.33 (m, 2H), 3.61 (s, 2H), 2.50 – 2.43
(m, 4H), 0.99 (t, J = 7.2 Hz, 6H); 13C NMR
=
8.4 Hz, 1H), 7.44-7.38 (m, 1H), 7.34 (d, J =
8
6
1
.4 Hz, 1H), 3.47 (s, 2H), 2.38 (s, 3H), 2.17 (s,
H); 13C NMR (101 MHz, DMSO) δ 159.39,
55.12, 149.89, 148.86, 148.49, 140.26, 136.88,
1
1

MedChemComm
Page 12 of 20
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DOI: 10.1039/C8MD00624E
(101 MHz, DMSO) δ 159.58, 153.64, 153.34,
Hz, 6H); 13C NMR (101 MHz, DMSO) δ
1
1
4
4
49.64, 148.03, 144.13, 136.91, 135.63, 132.00,
160.81, 150.51, 150.17, 148.30, 144.50, 143.86,
27.16, 124.56, 121.44, 121.22, 119.80, 57.31,
6.52(2C), 12.08(2C).
143.55, 140.27, 132.54, 129.44, 125.39, 124.79,
120.62, 55.27, 46.12(2C), 12.54, 8.84(2C).
4.1.2.9.(E)-N-(6-((4-methylpiperidin-1-yl)met
hyl)benzo[d]thiazol-2-yl)-2-(pyridin-2-ylmeth
ylene)hydrazine-1-carboxamide(8i).
.1.2.6.(E)-N-(6-((diethylamino)methyl)benzo[
d]thiazol-2-yl)-2-(1-(pyridin-2-yl)ethylidene)h
ydrazine-1-carboxamide(8f).
Light yellow solid; Yield: 58%; mp:
Light yellow solid; Yield: 49%; mp:
1
87-189
°C;
HRMS
(ESI)
for
155-157
°C;
HRMS
(ESI)
for
+
+
C H N OS[M+H] .calcd: 396.1732, found:
C H N OS[M+H] .calcd: 408.1732, found:
2
0
24
6
21 24
6
1
1
3
1
1
7
7
2
6
4
97.1806; H NMR (400 MHz, DMSO) δ
1.07 (s, 1H), 10.47 (s, 1H), 8.60 (d, J = 4.5 Hz,
H), 8.54-8.41 (m, 1H), 7.90-7.82 (m, 2H),
.64 (d, J = 8.1 Hz, 1H), 7.44-7.38 (m, 1H),
.36 (d, J = 8.1 Hz, 1H), 3.62 (s, 2H), 2.49-
.44 (m, 4H), 2.38 (s, 3H), 0.99 (t, J = 6.8 Hz,
H).
409.1809; H NMR (400 MHz, DMSO) δ
11.57 (s, 1H), 8.59 (d, J = 4.6 Hz, 1H), 8.55 (s,
1H), 8.08 (s, 1H), 7.97-7.91 (m, 1H), 7.91-7.84
(m, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.47-7.37 (m,
2H), 3.89 (s, 3H), 3.09-2.95 (m, 2H), 2.47-2.18
(m, 2H), 1.66 (d, J = 12.6 Hz, 2H), 1.52 – 1.38
(m, 1H), 1.24 (d, J = 10.0 Hz, 2H), 0.90 (d, J =
6.4 Hz, 3H).
.1.2.7.2-(di(pyridin-2-yl)methylene)-N-(6-((di
ethylamino)methyl)benzo[d]thiazol-2-yl)hydra
4.1.2.10.(E)-N-(6-((4-methylpiperidin-1-yl)me
thyl)benzo[d]thiazol-2-yl)-2-(1-(pyridin-2-yl)e
thylidene)hydrazine-1-carboxamide(8j).
Light yellow solid; Yield: 61%; mp:
zine-1-carboxamide(8g).
Light yellow solid; Yield: 69%; mp:
1
78-180
°C;
HRMS
(ESI)
for
+
C H N OS[M+H] .calcd: 459.1841, found:
162-164
°C;
HRMS
(ESI)
for
2
4
25
7
1
+
4
1
–
60.1917; H NMR (400 MHz, DMSO-d ) δ
C H N OS[M+H] .calcd: 422.1889, found:
6
22 26
6
1
2.95 (s, 1H), 11.84 (s, 1H), 8.84 (m, 1H), 8.61
8.50 (m, 1H), 8.12 m, 2H), 7.99 (m, 2H), 7.69
423.1967; H NMR (400 MHz, DMSO) δ
11.21 (s, 1H), 10.52 (s, 1H), 8.60 (s, 1H),
8.56-8.42 (m, 1H), 8.04-7.92 (m, 1H),
7.92-7.81 (m, 1H), 7.80-7.67 (m, 1H),
7.55-7.36 (m, 2H), 4.08 (s, 2H), 3.23-2.93 (m,
2H), 2.40-2.38 (m, 2H), 2.09 (s, 3H), 1.78-1.64
(m, 2H), 1.56-1.41 (m, 1H), 1.32-1.21 (m, 2H),
0.96-0.81 (m, 3H); 13C NMR (101 MHz,
DMSO) δ 160.36, 155.07, 153.20, 150.12,
149.57, 148.87, 136.88, 132.36, 130.23, 128.93,
124.45, 123.93, 121.50, 120.35, 60.58,
52.44(2C), 32.10(2C), 29.21, 21.63, 12.64.
4.1.2.11.2-(di(pyridin-2-yl)methylene)-N-(6-((
4-methylpiperidin-1-yl)methyl)benzo[d]thiazol
-2-yl)hydrazine-1-carboxamide(8k).
(
m, 1H), 7.64-7.54 (m, 2H), 7.53-7.44 (m, 2H),
4
6
1
1
1
5
4
.12 (s, 2H), 2.87 (q, 4H), 1.18 (t, J = 7.3 Hz,
H); 13C NMR (101 MHz, DMSO) δ 160.62,
55.76, 151.67(2C), 149.00(2C), 148.85(2C),
45.64, 137.91(2C), 137.51(2C), 132.06,
28.72, 126.77, 124.93, 124.31, 123.70, 120.13,
5.78, 46.21(2C), 9.83(2C).
.1.2.8.(E)-N-(6-((diethylamino)methyl)benzo[
d]thiazol-2-yl)-2-(1-(pyrazin-2-yl)ethylidene)h
ydrazine-1-carboxamide(8h).
Light white solid; Yield: 61%; mp:
1
90-192
°C;
HRMS
(ESI)
for
+
C H N OS[M+H] .calcd: 397.1685, found:
1
9
23
7
1
3
1
8
98.1760. H NMR (400 MHz, DMSO) δ
1.72 (s, 1H), 10.89 (s, 1H), 10.11 (s, 1H),
.67-8.61 (m, 2H), 8.18 (s, 1H), 7.78 (d, J = 8.2
Yellow solid; Yield: 61%; mp: 134-136
°C;
HRMS
(ESI)
for
+
C H N OS[M+H] .calcd: 485.1998, found:
2
6
27
7
1
Hz, 1H), 7.64 (d, J = 8.2 Hz, 1H), 4.38 (s, 2H),
.20-2.97 (m, 4H), 2.39 (s, 3H), 1.26 (t, J = 7.2
486.2074; H NMR (400 MHz, DMSO) δ
3
12.88 (s, 1H), 11.79 (s, 1H), 8.83 (s, 1H), 8.56
1
2

Page 13 of 20
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(s, 1H), 8.21-8.10 (m, 1H), 8.03-7.92 (m, 2H),
4.1.2.14.2-(di(pyridin-2-yl)methylene)-N-(4-((
DOI: 10.1039/C8MD00624E
7
2
.82 (s, 1H), 7.63-7.54 (m, 2H), 7.53-7.43 (m,
H), 7.36-7.29 (m, 1H), 3.52 (s, 2H), 2.84-2.71
dimethylamino)methyl)phenyl)hydrazine-1-car
boxamide(9b).
Yellow solid; Yield: 53%; mp: 110-112
(m, 2H), 2.02-1.86 (m, 2H), 1.61 – 1.50 (m,
2
0
H), 1.37-1.30 (m, 1H), 1.20-1.06 (m, 2H),
.94-0.81 (m, 3H); 13C NMR (101 MHz,
°C;
HRMS
+
(ESI)
for
C H N O
2
1
22
6
1
[M+H] .calcd: 374.1855, found: 375.1929; H
NMR (400 MHz, DMSO) δ 12.30 (s, 1H), 9.27
(s, 1H), 8.83 (d, J = 4.2 Hz, 1H), 8.55 (d, J =
4.1 Hz, 1H), 8.22 (d, J = 7.9 Hz, 1H), 8.03 –
7.91 (m, 2H), 7.58 – 7.51 (m, 3H), 7.49 (d, J =
8.0 Hz, 1H), 7.47 – 7.42 (m, 1H), 7.23 (d, J =
8.4 Hz, 2H), 3.37 (s, 2H), 2.15 (s, 6H); 13C
NMR (101 MHz, DMSO) δ 155.96, 152.74,
151.98, 148.81, 148.71, 142.43, 138.09,
137.79, 137.47, 133.43, 129.56(2C), 126.87,
124.72, 124.04, 123.78, 120.11(2C), 63.31,
45.23(2C).
DMSO)
δ
159.86, 155.78(2C), 151.71,
1
1
1
3
4
48.97(2C), 148.83(2C), 137.89(2C),
37.50(2C), 131.79, 127.62, 126.78, 124.90,
24.28, 122.08, 119.76, 62.65, 53.60(2C),
4.22(2C), 30.65, 22.24.
.1.2.12.(E)-N-(6-((4-methylpiperidin-1-yl)me
thyl)benzo[d]thiazol-2-yl)-2-(1-(pyrazin-2-yl)e
thylidene)hydrazine-1-carboxamide(8l).
Light white solid; Yield: 61%; mp:
2
35-237
°C;
HRMS
(ESI)
for
+
C H N OS[M+H] .calcd: 423.1841, found:
2
1
25
7
1
4
1
8
24.1918; H NMR (400 MHz, DMSO) δ
1.73 (s, 1H), 10.92 (s, 1H), 9.75 (s, 1H),
.67-8.61 (m, 2H), 8.15 (s, 1H), 7.78 (d, J = 8.2
4.1.2.15.(E)-N-(4-((diethylamino)methyl)phen
yl)-2-(1-(pyridin-2-yl)ethylidene)hydrazine-1-
carboxamide(9c).
Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 4.42-4.24 (m,
Yellow solid; Yield: 57%; mp: 106-108
+
2
2
1
3
1
1
1
2
4
H), 3.37-3.34 (m, 2H), 3.02-2.84 (m, 2H),
.39 (s, 3H), 1.84-1.70 (m, 2H), 1.67-1.54 (m,
H), 1.52-1.34 (m, 2H), 0.91 (d, J = 6.2 Hz,
H); 13C NMR (101 MHz, DMSO) δ 160.71,
53.12, 150.53, 150.15, 148.30, 144.45,
43.54, 132.37, 129.82, 125.09, 124.98,
24.73, 120.43, 59.47, 51.88(2C), 31.02(2C),
8.70, 21.55, 12.61.
°C; HRMS (ESI) for C H N O[M+H] .calcd:
1
9
25
5
1
339.2059, found: 340.2133. H NMR (400
MHz, DMSO) δ 9.92 (s, 1H), 8.93 (s, 1H), 8.58
(d, J = 4.2 Hz, 1H), 8.40 (d, J = 8.2 Hz, 1H),
7.86-7.80 (m, 1H), 7.56 (d, J = 8.4 Hz, 2H),
7.41-7.35 (m, 1H), 7.24 (d, J = 8.4 Hz, 2H),
3.48 (s, 2H), 2.46 (q, J = 7.2 Hz, 4H), 2.36 (s,
3H), 0.98 (t, J = 7.2 Hz, 6H); 13C NMR (101
MHz, DMSO) δ 155.43, 153.75, 148.82,
146.99, 137.86, 136.83, 134.58, 129.12(2C),
124.02, 121.06, 120.45(2C), 56.95, 46.46(2C),
12.34 , 12.14(2C).
.1.2.13.(E)-N-(4-((dimethylamino)methyl)phe
nyl)-2-(pyridin-2-ylmethylene)hydrazine-1-car
boxamide(9a).
Light yellow solid; Yield: 43%; mp:
1
02-104
°C;
HRMS
(ESI)
for
4.1.2.16.2-(di(pyridin-2-yl)methylene)-N-(4-((
diethylamino)methyl)phenyl)hydrazine-1-carb
oxamide(9d).
+
C H N O[M+H] .calcd: 297.1590, found:
1
6
19
5
1
298.1662; H NMR (400 MHz, DMSO) δ
10.98 (s, 1H), 8.99 (s, 1H), 8.57 (d, J = 4.6 Hz,
1H), 8.34 (d, J = 8.0 Hz, 1H), 8.00 (s, 1H), 7.86
Yellow solid; Yield: 51%; mp: 118-120
+
°C; HRMS (ESI) for C H N O[M+H] .calcd:
2
3
26
6
1
(
M, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.40-7.35 (m,
402.2168, found: 403.2244; H NMR (400
MHz, DMSO) δ 12.32 (s, 1H), 9.23 (s, 1H),
8.83 (d, J = 4.2 Hz, 1H), 8.55 (d, J = 4.2 Hz,
1H), 8.22 (d, J = 8.0 Hz, 1H), 8.02-7.91 (m,
2H), 7.59-7.55 (m, 1H), 7.53 (d, J = 8.4 Hz,
2H), 7.49 (d, J = 8.0 Hz, 1H), 7.47-7.42 (m,
1
H), 7.21 (d, J = 8.4 Hz, 2H), 3.34 (s, 2H), 2.14
1
3
(s, 6H); C NMR (101 MHz, DMSO) δ 153.86,
1
1
4
53.34, 149.68, 141.63, 138.12, 136.93, 133.58,
29.33(2C), 124.26, 120.62, 120.35(2C), 63.45,
5.33(2C).
1
3

MedChemComm
Page 14 of 20
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1
H), 7.24 (d, J = 8.4 Hz, 2H), 3.47 (s, 2H), 2.44
4.1.2.19.2-(di(pyridin-2-yl)methylene)-N-(4-((
DOI: 10.1039/C8MD00624E
1
3
(
q, J = 7.2 Hz, 4H), 0.97 (t, J = 7.2 Hz, 6H); C
NMR (101 MHz, DMSO) δ 155.98, 152.76,
4-methylpiperidin-1-yl)methyl)phenyl)hydrazi
ne-1-carboxamide(9g).
1
1
1
1
4
51.99, 148.78, 148.70, 142.33, 137.77, 137.73,
37.45, 134.75, 129.21(2C), 126.87, 124.70,
24.01, 123.78, 120.20(2C), 56.92, 46.47(2C),
2.13(2C).
Yellow solid; Yield: 76%; mp: 110-112
+
°C; HRMS (ESI) for C H N O[M+H] .calcd:
2
5
28
6
1
428.2325, found: 429.2398; H NMR (400
MHz, DMSO) δ 12.31 (s, 1H), 9.25 (s, 1H),
8.83 (d, J = 4.0 Hz, 1H), 8.55 (d, J = 4.0 Hz,
1H), 8.21 (d, J = 7.8 Hz, 1H), 8.04 – 7.92 (m,
2H), 7.59-7.42 (m, 5H), 7.21 (d, J = 8.0 Hz,
2H), 3.38 (s, 2H), 2.76 (d, J = 10.8 Hz, 2H),
1.88 (t, J = 10.8 Hz, 2H), 1.55 (d, J = 11.6 Hz,
2H), 1.38-1.24 (m, 1H), 1.19-1.04 (m, 2H),
.1.2.17.(E)-N-(4-((4-methylpiperidin-1-yl)me
thyl)phenyl)-2-(pyridin-2-ylmethylene)hydrazi
ne-1-carboxamide(9e).
Yellow solid; Yield: 64%; mp: 166-168
+
°
C; HRMS (ESI) for C H N O[M+H] .calcd:
2
0
25
5
1
3
51.2059, found: 352.2133; H NMR (400
1
3
MHz, DMSO) δ 11.01 (s, 1H), 9.01 (s, 1H),
0.88 (d, J = 6.4 Hz, 3H); C NMR (101 MHz,
DMSO) δ 155.97, 152.74, 151.99, 148.80,
148.71, 142.38, 137.92, 137.78, 137.46, 133.47,
129.52(2C), 126.86, 124.71, 124.02, 123.78,
120.15(2C), 62.53, 53.67(2C), 34.43(2C),
30.81, 22.30.
8
.57 (d, J = 4.5 Hz, 1H), 8.34 (d, J = 8.0 Hz,
H), 8.01 (s, 1H), 7.86 (t, J = 7.1 Hz, 1H), 7.60
1
(d, J = 8.3 Hz, 2H), 7.42 – 7.31 (m, 1H), 7.24 (d,
J = 8.1 Hz, 2H), 3.47 (s, 2H), 2.89 – 2.72 (m,
2
1
H), 2.09 – 1.88 (m, 2H), 1.62 – 1.50 (m, 2H),
.38 – 1.28 (m, 1H), 1.23 – 1.11 (m, 2H), 0.88
4.1.2.20.(E)-N-(4-((4-methylpiperidin-1-yl)me
thyl)phenyl)-2-(1-(pyrazin-2-yl)ethylidene)hyd
razine-1-carboxamide(9h).
(
d, J = 6.4 Hz, 3H); 13C NMR (101 MHz,
DMSO) δ 153.88, 153.32, 149.68, 141.69,
1
1
3
4
38.34, 136.92, 129.94, 129.70(2C), 124.26,
20.59, 120.34(2C), 62.12, 53.40(2C),
4.00(2C), 30.54, 22.18.
Light white solid; Yield: 61%; mp:
212-214 °C; HRMS (ESI) for C H N O
2
0
26
6
1
+
[M+H] .calcd: 366.2168, found: 367.2245; H
NMR (400 MHz, DMSO) δ 10.12 (s, 1H), 9.69
(d, J = 1.4 Hz, 1H), 9.05 (d, J = 11.9 Hz, 1H),
8.65-8.56 (m, 2H), 7.58 (d, J = 8.4 Hz, 2H),
7.22 (d, J = 8.4 Hz, 2H), 3.39 (s, 2H), 2.77 (d, J
= 11.4 Hz, 2H), 2.34 (s, 3H), 1.88 (t, J = 11.0
Hz, 2H), 1.56 (d, J = 11.0 Hz, 2H), 1.38-1.25
(m, 1H), 1.20-1.06 (m, 2H), 0.89 (d, J = 6.4 Hz,
3H); 13C NMR (101 MHz, DMSO) δ 153.69,
150.76, 145.05, 144.03, 143.56, 143.45, 137.93,
133.60, 129.33(2C), 120.88(2C), 62.58,
53.69(2C), 34.46(2C), 30.84, 22.32, 12.05.
4.1.2.21.(E)-N-(6-((diethylamino)methyl)benz
o[d]thiazol-2-yl)-2-(1-(4-fluorophenyl)ethylid
ene)hydrazine-1-carboxamide(10a).
.1.2.18.(E)-N-(4-((4-methylpiperidin-1-yl)me
thyl)phenyl)-2-(1-(pyridin-2-yl)ethylidene)hyd
razine-1-carboxamide(9f).
Yellow solid; Yield: 72%; mp: 170-172
+
°
C; HRMS (ESI) for C H N O[M+H] .calcd:
2
1
27
5
1
3
65.2166 found: 366.2290; H NMR (400
MHz, DMSO) δ 9.94 (s, 1H), 8.95 (s, 1H), 8.58
d, J = 4.2 Hz, 1H), 8.40 (d, J = 8.1 Hz, 1H),
(
7
7
3
3
2
0
.86 – 7.79 (m, 1H), 7.56 (d, J = 8.4 Hz, 2H),
.41 – 7.33 (m, 1H), 7.21 (d, J = 8.4 Hz, 2H),
.38 (s, 2H), 2.76 (d, J = 11.4 Hz, 2H), 2.36 (s,
H), 1.93 – 1.82 (m, 2H), 1.56 (d, J = 11.7 Hz,
H), 1.38 – 1.27 (m, 1H), 1.19 – 1.06 (m, 2H),
1
3
.89 (d, J = 6.5 Hz, 3H); C NMR (101 MHz,
DMSO) δ 155.42, 153.73, 148.82, 147.01,
Light yellow solid; Yield: 81%; mp:
1
1
3
38.03, 136.82, 133.40, 129.41(2C), 124.02,
21.06, 120.39(2C), 62.59, 53.69(2C),
4.47(2C), 30.85, 22.32, 12.35.
171-173 °C; HRMS (ESI) for C H FN OS
2
1
24
5
+
1
[M+H] .calcd: 413.1686, found: 414.1762; H
NMR (400 MHz, DMSO) δ 10.90 (s, 1H),
1
0.38 (s, 1H), 8.10 – 7.99 (m, 2H), 7.85 (s, 1H),
1
4

Page 15 of 20
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7
.63 (d, J = 8.4 Hz, 1H), 7.36 (dd, J = 8.4, 1.3
4.1.2.24.N-(4-((diethylamino)methyl)phenyl)-2
DOI: 10.1039/C8MD00624E
Hz, 1H), 7.26 (t, J = 8.8 Hz, 2H), 3.62 (s, 2H),
-(diphenylmethylene)hydrazine-1-carboxamid
e(10d).
Light white solid; Yield: 41%; mp:
125-127 °C; HRMS (ESI) for
C H N O[M+H] .calcd: 400.2263, found:
2
7
1
1
1
1
4
.51 – 2.43 (m, 4H), 2.30 (s, 3H), 0.99 (t, J =
.2 Hz, 6H); 13C NMR (101 MHz, DMSO) δ
64.47, 162.01, 159.18, 148.29, 135.56, 134.73,
34.70, 132.09, 129.35, 129.27, 127.18, 121.48,
19.96, 115.61, 115.40, 57.30, 46.53(2C),
4.50(2C), 12.07.
+
2
5
28
4
1
401.2339; H NMR (400 MHz, DMSO) δ 9.08
(s, 1H), 8.67 (s, 1H), 7.67 – 7.61 (m, 2H), 7.61
– 7.58 (m, 1H), 7.58 – 7.53 (m, 2H), 7.46 (d, J
= 8.4 Hz, 2H), 7.41 – 7.36 (m, 3H), 7.35 – 7.30
(m, 2H), 7.22 (d, J = 8.5 Hz, 2H), 3.46 (s, 2H),
2.44 (q, J = 7.2 Hz, 4H), 0.97 (t, J = 7.2 Hz,
6H); 13C NMR (101 MHz, DMSO) δ 152.55,
148.50, 138.03, 137.83, 134.30, 132.73,
130.10(2C), 129.98, 129.49, 129.25(2C),
.1.2.22.(E)-N-(6-((4-methylpiperidin-1-yl)me
thyl)benzo[d]thiazol-2-yl)-2-(1-phenylethylide
ne)hydrazine-1-carboxamide(10b).
Light yellow solid; Yield: 76%; mp:
2
32-234
°C;
HRMS
(ESI)
for
+
C H N OS[M+H] .calcd: 421.1936, found:
2
3
27
5
1
4
1
22.2012; H NMR (400 MHz, DMSO) δ
1.07 (s, 1H), 10.47 (s, 1H), 8.08 (s, 1H), 7.96
128.97(2C),
128.74(2C),
127.51(2C),
(
d, J = 5.4 Hz, 2H), 7.78 (d, J = 8.2 Hz, 1H),
119.63(2C), 56.90, 46.45(2C), 12.12(2C).
4.1.2.25.2-(diphenylmethylene)-N-(4-((4-meth
ylpiperidin-1-yl)methyl)phenyl)hydrazine-1-ca
rboxamide(10e).
7
4
.55 (d, J = 8.2 Hz, 1H), 7.48 – 7.38 (m, 3H),
.36 (s, 2H), 3.43 – 3.34 (m, 2H), 3.02 – 2.85
(m, 2H), 2.32 (s, 3H), 1.79 (d, J = 13.6 Hz, 2H),
1
6
1
1
1
3
.59 (s, 1H), 1.42 – 1.27 (m, 2H), 0.91 (d, J =
.4 Hz, 3H); 13C NMR (101 MHz, DMSO) δ
60.76, 153.12, 150.22, 149.69, 138.17, 132.48,
30.10, 129.67, 128.72(2C), 127.00(2C),
25.03, 124.73, 120.50, 59.75, 52.12(2C),
1.26(2C), 28.63, 21.57, 14.56.
Light white solid; Yield: 43%; mp:
126-128
°C;
HRMS
(ESI)
for
+
C H N O[M+H] .calcd: 426.2420, found:
2
7
30
4
1
427.2493; H NMR (400 MHz, DMSO) δ 9.08
(s, 1H), 8.67 (s, 1H), 7.67 – 7.61 (m, 2H), 7.61
– 7.58 (m, 1H), 7.58 – 7.50 (m, 2H), 7.47 (d, J
= 8.4 Hz, 2H), 7.41 – 7.36 (m, 3H), 7.35 – 7.29
(m, 2H), 7.19 (d, J = 8.4 Hz, 2H), 3.37 (s, 2H),
2.75 (d, J = 11.4 Hz, 2H), 1.87 (t, J = 10.8 Hz,
2H), 1.55 (d, J = 11.4 Hz, 2H), 1.38 – 1.23 (m,
1H), 1.17 – 1.05 (m, 2H), 0.88 (d, J = 6.4 Hz,
3H); 13C NMR (101 MHz, DMSO) δ 152.53,
148.52, 138.20, 137.83, 133.12, 132.73,
130.09(2C), 129.99, 129.54(2C), 129.50,
4
.1.2.23.N-(4-((dimethylamino)methyl)pheny
l)-2-(diphenylmethylene)hydrazine-1-carbox
amide(10c).
Light white solid; Yield: 38%; mp:
1
23-125
°C;
HRMS
(ESI)
for
+
C H N O[M+H] .calcd: 372.1950, found:
2
3
24
4
1
3
9
7
73.2027; H NMR (400 MHz, DMSO) δ
.10 (s, 1H), 8.68 (s, 1H), 7.66 – 7.61 (m, 2H),
.61 – 7.58 (m, 1H), 7.58 – 7.53 (m, 2H), 7.48
128.97(2C),
128.74(2C),
127.51(2C),
(
d, J = 8.4 Hz, 2H), 7.42 – 7.36 (m, 3H), 7.35
7.30 (m, 2H), 7.19 (d, J = 8.4 Hz, 2H), 3.32
s, 2H), 2.13 (s, 6H); 13C NMR (101 MHz,
DMSO) δ 152.54, 148.55, 138.28, 137.82,
119.59(2C), 62.55, 53.68(2C), 34.45(2C),
30.82, 22.31.
–
(
4.2 CCK-8 Assay
U937 and MCF-7 cell lines were
purchased from cell bank of Chinese Academy
of Sciences Shanghai, China. U937 cells were
grown in Roswell Park Memorial Institute
1
1
1
33.38, 132.73, 130.10(2C), 129.99(2C),
29.51(2C), 128.97(2C), 128.75(2C),
27.51(2C), 119.56(2C), 63.45, 45.34(2C).
1
640 Medium mixed with 10% fetal bovine
serum, and MCF-7 cells were grown in
1
5

MedChemComm
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Dulbecco's Modified Eagle's Medium mixed
cells/well). Inner wells without compound
DOI: 10.1039/C8MD00624E
with 10% fetal bovine serum, all the cells were
were filled with 200 μL of media, and outer
cultured in a 5% CO incubator at 37 °C. The
wells were filled with 200 μL of sterile PBS.
2
anticancer acitivity was evaluated by CCK-8
assay. A stock solution of each test compounds
was prepared in DMSO at the concentration of
The plates were incubated at 37 °C with 5%
CO for 15 hours. Solutions of compounds
2
(seclected compounds, PAC-1 and DMSO
alone) in media were prepared at 3x final
concentrations (3% DMSO), and 50 μL of each
solution was added to the appropriate wells.
Each compound was tested in six wells per
plate. In the first plate, 50 μL of bifunctional
cell lysis/caspase activity buffer was added to
each treatment well. Fluorescence (ex. 400 nm,
em. 505 nm) was monitored on an Analyst AD
96-384 plate reader via a 60-minute kinetic
read, and the slope was used to determine
caspase activity. The second plate was
1
mM. Prior to use, the stock solution was
further diluted to a final concentration by
culture medium. The concentration of DMSO
was less than 0.01%, and the amount was
determined by preliminary testing. Cells were
4
seeded at a density of 5×10 cells/well in
9
6-well plate and incubated for 12 h, followed
by the treatment with the test compounds.
Control culture received the carrier solvent
(0.01% DMSO). After 24 h, U937 and MCF7
cells viability was measured by a Cell
Counting Kit 8 (CCK-8) assay. 10 μL of
CCK-8 solution was added into each well and
incubated at 37 °C for 2 h. Optical density at
incubated at 37 °C with 5 % CO for 16 hours,
2
at which time 50 μL of bifunctional cell
lysis/caspase activity buffer was added to each
treatment well, and fluorescence was
monitored as above. The slopes at 16 hours
were normalized to the slopes of each
compound at 0 hours (0% activity) and the
PAC-1-treated samples at 16 hours (100%
activity) to give percent caspase activity.
4
50 nm was determined with a microplate
reader.
4
.3 Apoptosis study
Apoptosis of U937 cells was detected
using a flow cytometric assay. Briefly, cells
were seeded in 6-well plates and incubated
overnight. The following day, cells were
treated with different concentrations of
compounds 8j and 8k for 24 h and 48 h. The
cells and supernatants were harvested and
washed twice with cold PBS and then
resuspended in 100µl 1× Binding Buffer. 5 µl
of FITC Annexin V and 5 µl PI were added in
each tube and the cells were then gently
vortexed incubated for 15 min at RT (25 °C) in
the dark. 400 µl of 1× Binding Buffer then
added to each tube. The stained cells were
analyzed by a flow cytometer (FACS Calibur;
BD).
4.5 Procaspase-3 activation assay
Procaspase-3 was purchased from R&D
Systems. Procaspase-3 was incubated at 100
nM, in the presence of selected compounds at
10 μM in a reaction buffer consisted of 20 mM
Tris, 300 mM NaCl, 5 mM Dithiothreitol, 5%
Sucrose and 0.05% CHAPS, pH 8.0. The
mixtures were assayed for kinetic activity by
incubation with 20 μM Ac-DEVD-AFC for 5
minutes after 4 hours of incubation at 37 °C.
Fluorescence value was read at excitation and
emission wavelengths of 400 nm and 505 nm.
The activation rate (%) was calculated using
4
.4 Caspase Activity in Cell Lysate
the
following
equation:
To each inner well of two 96-well plates
(Fcompound-Fdmso)/(FPAC-1-Fdmso)×100.
was added 100 μL of a suspension of U-937
cells in phenol red-free RPMI-1640 complete
growth media at 500,000 cells/mL (50,000
4.6 Toxicity and morphological observation
in zebrafish embryos
Wild type zebrafish (AB strain) was
1
6

Page 17 of 20
MedChemComm
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raised and maintained at 28.5 °C as described
5’-TAAACAACTCTAATCTTCAGCCGAC-
DOI: 10.1039/C8MD00624E
2
6
previously . The developmental stages were
staged in hours post-fertilization according to
morphological criteria described by Kimmel et
al. Embryos were exposed to different
concentrations of 8j and 8k (5, 10, 20 and 40
μM) prepared by diluting the stock solution of
each compound in the fish medium. A
no-treatment control was also included.
Experiments were performed in triplicates and
a total of 24 embryos each group have been
used per each treatment. The survival rate and
gross morphology of the embryos in each
system were observed every 24 h for a period
of 72 h under a stereo microscope (Olympus,
Japan). All animal procedures were performed
in accordance with the Guidelines for Care and
Use of Laboratory Animals of Huaqiao
University and approved by the Animal Ethics
Committee of School of medicine.
3’
and
reverse:
5’-AGCTTATGATGGTTCTGATGATGTC-
3’; β-actin forward:
5’-AGCACGGTATTGTGACTAACTG-3’
and
reverse:
5’-TCGAACATGATCTGTGTCATC-3’.
β-actin was detected as an internal control.
Real-time PCR analyses were performed as
described previously. The probability (P) of <
0.01 was considered statistically significant.
4.9 Metabolic stability
Experimental procedure:1. Buffer A: 1.0
L of 0.1 M monobasic Potassium Phosphate
buffer containing 1.0 mM EDTA, Buffer B: 1.0
L of 0.1 M Dibasic Potassium Phosphate buffer
containing 1.0 mM EDTA, Buffer C: 0.1 M
Potassium Phosphate buffer, 1.0 mM EDTA,
pH 7.4 by titrating 700 mL of buffer B with
buffer A while monitoring with the pH meter.
2. Reference compound (Ketanserin) and test
compounds spiking solution: 500 µM spiking
solution: add 10 µL of 10 mM DMSO stock
solution into 190 µL ACN. 1.5 µM spiking
solution in microsomes (0.75 mg/mL): add 1.5
µL of 500 µM spiking solution and 18.75 µL of
20 mg/mL liver microsomes into 479.75 µL of
Buffer C on ice. 3. Prepare NADPH stock
solution (6 mM) by dissolving NADPH into
buffer C. 4. Dispense 30 µL of 1.5 µM spiking
solution containing 0.75 mg/mL microsomes
solution to the assay plates designated for
different time points (0, 5, 15, 30, 45 min) on
ice. 5. For 0-min, add 135 µL of ACN
containing IS to the wells of 0-min plate and
then add 15 µL of NADPH stock solution (6
mM). 6. Pre-incubate all other plates at 37 °C
for 5 minutes. 7. Add 15 µL of NADPH stock
solution (6 mM) to the plates to start the
reaction and timing. 8. At 5-min, 15-min,
30-min, and 45-min, add 135 µL of ACN
containing IS to the wells of corresponding
plates, respectively, to stop the reaction. 9.
After quenching, shake the plates at the
4
.7 Acridine Orange staining in zebrafish
embryos
Embryos that developed normally and
reached the blastula stage (2 h post fertilization
hpf]) were exposed to 8j and 8k (40 μM and
0 μM ) in culture dish until 24 hpf. Live
[
6
embryos were placed in 50 μg/ml Acridine
Orange (Sigma-Aldrich) diluted in egg water
for 30 minutes at room temperature in the dark.
Embryos were then washed in egg water and
analyzed under the fluorescence microscope.
4
.8 RNA isolation and real-time PCR
Total RNAs were isolated from zebrafish
embryos or tissues of adults using SV Total
RNA Isolation System (Promega), and the
quantity and quality were determined by
agarose
electrophoresis
and
spectrophotometer. The isolated RNAs were
then reverse-transcribed by using the M-MLV
reverse transcriptase kit (Takara Biochemicals,
Dalian, China) following the manufacturer's
recommendation. The real-time SYBR Green
primers for zebrafish bax and β-actin were
chosen
as
follows:
bax
forward:
1
7

MedChemComm
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vibrator (IKA, MTS 2/4) for 10 min (600
Mod. Pathol., 2004, 17, 861-867.
DOI: 10.1039/C8MD00624E
rpm/min) and then centrifuge at 5594 g for 15
min (Thermo Multifuge × 3R). 10. Transfer 50
μL of the supernatant from each well into a
10 K.S. Putt, G.W. Chen, J.M. Pearson, J.S.
Sandhorst, M.S. Hoagland, J.-T. Kwon,
S.-K. Hwang, H. Jin, M.I. Churchwell,
M.-H. Cho, Nat. Chem. Biol., 2006, 2,
543-550.
9
6-well sample plate containing 50 μL of ultra
pure water (Millipore, ZMQS50F01) for
LC/MS analysis.
11 Q.P. Peterson, D.R. Goode, D.C. West,
K.N. Ramsey, J.J.Y. Lee, P.J.
Hergenrother, J. Mol. Biol., 2009, 388,
144-158.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was financially supported by
the National Natural Science Foundation of
China (81602970), FuJiang Province Natural
Science Foundation (2017J01143), Quanzhou
12 Q.P. Peterson, D.C. Hsu, D.R. Goode, C.J.
Novotny, R.K. Totten, P.J. Hergenrother,
J. Med. Chem., 2009, 52, 5721-5731.
13 J. Ma, G. Zhang, X. Han, G. Bao, L. Wang,
X. Zhai, P. Gong, Arch. Pharm., 2014,
347, 936-949.
High-level
Talents
Innovation
and
Entrepreneurship
Project
(2018C074R),
Promotion Program for Young and
Middle-aged Teacher in Science and
Technology Research of Huaqiao University
14 J. Ma, D. Chen, K. Lu, L. Wang, X. Han, Y.
Zhao, P. Gong, Eur. J. Med. Chem., 2014,
86, 257-269.
(
Z17X0177).
15 D. Sriram, P. Yogeeswari, K. Madhu,
Bioorg. Med. Chem. Lett., 2005, 15,
4502-4505.
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DOI: 10.1039/C8MD00624E
A series of benzothiazole derivatives bearing a pyridine-semicarbazone moiety were identified as
apoptosis inducers via activation of procaspase-3 to caspase-3.