molecules
Article
Synthesis and Evaluation of New Pyrazoline
Derivatives as Potential Anticancer Agents in
HepG-2 Cell Line
Weijie Xu 1,† , Ying Pan 1,† , Hong Wang 1,† , Haiyan Li 2 , Qing Peng 3 , Duncan Wei 1 , Cheng Chen 1
and Jinhong Zheng 1, *
1
2
3
*
†
Department of Chemistry, Shantou University Medical College, Shantou 515041, Guangdong, China;
[email protected] (W.X.); [email protected] (Y.P.); [email protected] (H.W.);
[email protected] (D.W.); [email protected] (C.C.)
Department of Pharmacology, Shantou University Medical College, Shantou 515041, Guangdong, China;
[email protected]
Department of Hepatobiliary Surgery II, Zhujiang Hospital of Southern Medical University,
Guangzhou 510280, Guangdong, China; [email protected]
Correspondence: [email protected]; Tel.: +86-754-8890-0499; Fax: +86-754-8855-7562
These authors contributed equally to this work.
Academic Editor: Maria Emília de Sousa
Received: 28 February 2017; Accepted: 9 March 2017; Published: 16 March 2017
Abstract: Cancer is a major public health concern worldwide. Adverse effects of cancer treatments
still compromise patients’ quality of life. To identify new potential anticancer agents, a series of novel
pyrazoline derivatives were synthesized and evaluated for cytotoxic effects on HepG-2 (human liver
hepatocellular carcinoma cell line) and primary hepatocytes. Compound structures were confirmed
by 1 H-NMR, mass spectrometry, and infrared imaging. An in vitro assay demonstrated that several
compounds exerted cytotoxicity in the micromolar range. Benzo[b]thiophen-2-yl-[5-(4-hydroxy3,5-dimethoxy-phenyl)-3-(2-hydroxy-phenyl)-4,5-dihydo-pyrazol-1-yl]-methanone (b17) was the
most effective anticancer agent against HepG-2 cells owing to its notable inhibitory effect on
HepG-2 with an IC50 value of 3.57 µM when compared with cisplatin (IC50 = 8.45 µM) and low
cytotoxicity against primary hepatocytes. Cell cycle analysis and apoptosis/necrosis evaluation using
this compound revealed that b17 notably arrested HepG-2 cells in the G2 /M phase and induced
HepG-2 cells apoptosis. Our findings indicate that compound b17 may be a promising anticancer
drug candidate.
Keywords: pyrazoline; anticancer activity; HepG-2 cells; apoptosis
1. Introduction
Worldwide, liver cancer is the third most common cause of cancer deaths. It is the fifth and
seventh most common cancer in men and women, respectively [1]. Cancer is a complex disease caused
by various factors, such as high stress, bad dietary habits, aging, and smoking; uncontrolled, rapid,
and pathological proliferation of abnormally transformed cells is a direct cause of a large group of
diseases [2–4]. Great progress has been made in medical treatments, but cancer is still a major cause of
mortality. Resistance to chemotherapeutic agents, lack of selectivity, and serious adverse effects are the
primary challenges in the fight against cancer [2–6]. Therefore, new anticancer agents are continually
developed and tested to selectively destroy tumor cells or at least limit their proliferation.
Pyrazolines are a class of compounds exhibiting a wide spectrum of activities. Several pyrazolines
act as anticancer agents [7,8], tubulin assembly inhibitors [9]. Pyrazoloacridine (PZA) (I) (Figure 1) is a
new anticancer drug currently undergoing Phase II clinical trials [10–12]. Doramapimod (BIRB-796)
Molecules 2017, 22, 467; doi:10.3390/molecules22030467
www.mdpi.com/journal/molecules
Molecules 2017, 22, 467
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(II) is a selective p38α mitogen-activated protein kinase (MAPK) inhibitor undergoing Phase III
clinical
trials Axitinib
[13,14]. Axitinib
(AG013736)
(III), a endothelial
vascular endothelial
growth
factor(VEGFR)
receptor inhibitor,
(VEGFR)
trials [13,14].
(AG013736)
(III), a vascular
growth factor
receptor
inhibitor,
used in
clinical treatment,
is exploited
by Pfizer
[15,16]. Pazopanib
(GW786034)
(IV), inhibitor,
a VEGFR
used in clinical
treatment,
is exploited
by Pfizer [15,16].
Pazopanib
(GW786034)
(IV), a VEGFR
inhibitor,
is
exploited
by
GlaxoSmithKline
[17,18].
Tozasertib
(VX-680,
MK-0457)
(V)
is
an
Aurora
kinase
is exploited by GlaxoSmithKline [17,18]. Tozasertib (VX-680, MK-0457) (V) is an Aurora kinase inhibitor
0 -hydroxymethyl-2’-furyl)-1-benzyl indazole (YC-1) (VI) is a hypoxia-inducible
inhibitor
[19,20]
and
3-(5
[19,20] and 3-(5′-hydroxymethyl-2’-furyl)-1-benzyl indazole (YC-1) (VI) is a hypoxia-inducible factor
factor
inhibitor
as awell
as inhibitor
a VEGF [21,22].
inhibitor
[21,22].
They are anticancer
promising drug
anticancer
drug
(HIF)-1(HIF)-1
inhibitor
as well as
VEGF
They
are promising
candidates.
candidates.
Recent
studies
report
that
a
large
number
of
sulfonamide
derivatives
and
heterocyclic
Recent studies report that a large number of sulfonamide derivatives and heterocyclic compounds
compounds
show
antitumor
activity aand
displaychemical
a common
chemical
motif of aromatic/heterocyclic
show antitumor
activity
and display
common
motif
of aromatic/heterocyclic
sulfonamide.
sulfonamide.
The
compounds’
antitumor
action
is
attributed
to
varying
mechanisms,
such
as cell cycle
The compounds’ antitumor action is attributed to varying mechanisms, such as cell cycle perturbation,
perturbation,
disruption
of
microtubule
assembly,
and
functional
suppression
of
the
transcriptional
disruption of microtubule assembly, and functional suppression of the transcriptional activator nuclear
activator
nuclear
factorthat
(NF)-Y
that
binds gene
to DNA
and regulates
gene or
expression
by
factor (NF)-Y
(a protein
binds(a
to protein
DNA and
regulates
expression
by promoting
suppressing
promoting
or
suppressing
transcription)
[23–26].
In
this
report,
we
describe
the
synthesis
of
new
transcription). [23–26]. In this report, we describe the synthesis of new pyrazoline derivatives (b1–19)
pyrazoline
derivatives
(b1–19)
with
substituted
sulfonyl
moieties,
rings,
other
closely
with substituted
sulfonyl
moieties,
heterocyclic
rings,
or other
closelyheterocyclic
related variants
inor
their
molecular
related
variants
in their
We show
synthesis
evaluation
of a new
class of
structures.
We show
themolecular
synthesisstructures.
and evaluation
of athe
new
class ofand
pyrazolines
acting
as potential
pyrazolines
acting
as
potential
antitumor
agents
against
a
human
liver
hepatocellular
carcinoma
cell
antitumor agents against a human liver hepatocellular carcinoma cell line (HepG-2) and primary
line
(HepG-2)We
and
primary
Weof
also
outapoptosis,
an analysis
of necrosis
the cell cycle,
apoptosis,
hepatocytes.
also
carriedhepatocytes.
out an analysis
thecarried
cell cycle,
and
processes
using
and
necrosis
processes
using
the
most
effective
compound,
b17.
the most effective compound, b17.
Figure 1. Structures of pyrazoloacridine (PZA) (I); doramapimod (BIRB-796) (II); axitinib (AG013736)
Figure 1. Structures of pyrazoloacridine (PZA) (I); doramapimod (BIRB-796) (II); axitinib (AG013736)
(III); pazopanib (GW786034) (IV); tozasertib (VX-680) (V); and 3-(5’-hydroxymethyl-2’-furyl)-1-benzyl
(III); pazopanib (GW786034) (IV); tozasertib (VX-680) (V); and 3-(5’-hydroxymethyl-2’-furyl)-1-benzyl
indazole (YC-1) (VI).
indazole (YC-1) (VI).
2. Results and Discussion
2. Results and Discussion
2.1. Synthesis
Derivatives
2.1.
Synthesis of
of New
New Pyrazoline
Pyrazoline Derivatives
The synthesis
synthesisofofnew
new
pyrazoline
derivatives
(b1–19)
carried
out according
to theshown
steps
The
pyrazoline
derivatives
(b1–19)
waswas
carried
out according
to the steps
shown
in
Scheme
1.
First,
chalcone
derivatives
(a1–5)
were
obtained
via
the
base-catalyzed
Claisenin Scheme 1. First, chalcone derivatives (a1–5) were obtained via the base-catalyzed Claisen-Schmidt
Schmidt condensation
with corresponding
ketones
and aldehydes.
Chalcone ring-closure
reactions
condensation
with corresponding
ketones and
aldehydes.
Chalcone ring-closure
reactions were
carried
were
carried
out
with
hydrazine
or
phenylhydrazine
with
tetrabutylammonium
bromide
(TBAB)
as a
out with hydrazine or phenylhydrazine with tetrabutylammonium bromide (TBAB) as a catalyst
catalyst
to
obtain
compounds
b1–4,
b18,
and
b19.
Next,
compound
b1
was
treated
with
corresponding
to obtain compounds b1–4, b18, and b19. Next, compound b1 was treated with corresponding
acyl chlorides
chlorides or
sulfonyl chlorides
solvent and
and pyridine
pyridine as
catalyst to
to
◦ C with
acyl
or sulfonyl
chlorides at
at 80
80 °C
with ethanol
ethanol as
as aa solvent
as aa catalyst
1
yield compounds
compounds b5–17.
b5–17. Compounds
elucidated by
by infrared
infrared (IR),
(IR), 1 H-NMR,
H-NMR, and
and mass
mass
yield
Compounds b1–19
b1–19 were
were elucidated
spectrometry
imaging.
Thus,
the
synthetic
procedure
was
shown
to
be
versatile
and
applicable
to
spectrometry imaging. Thus, the synthetic procedure was shown to be versatile and applicable to the
the
preparation of many derivatives. Compounds a1–5 are shown in Table 1; compounds b1–19 are
shown in Table 2.
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preparation of many derivatives. Compounds a1–5 are shown in Table 1; compounds b1–19 are shown
in Table
2.
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Scheme 1. General synthesis of compounds b1–19. Reagents and conditions: (i) substituted phenylethanone
Scheme 1. General synthesis of compounds b1–19. Reagents and conditions: (i) substituted phenylethanone
(0.01 mol), substituted benzaldehyde (0.012 mol), piperidine (1 mL), 160 °C, 20 min or 30% NaOH,
(0.01 ethanol,
mol), substituted
benzaldehyde (0.012 mol), piperidine (1 mL), 160 ◦ C, 20 min or 30% NaOH,
r.t., 24 h; (ii) ethanol, hydrazine hydrate, reflux, 4 h; (iii) substituted benzoyl chloride or
ethanol,
r.t., 24 benzenesulfonyl
h; (ii) ethanol, chloride,
hydrazine
reflux,
4 h; 1(iii)
substituted
benzoyl chloride or
substituted
80 hydrate,
°C, ethanol,
pyridine,
h; (iv)
ethanol, phenylhydrazine,
◦
substituted
benzenesulfonyl
chloride,
80reflux,
C, ethanol,
pyridine, 1 h; (iv) ethanol, phenylhydrazine,
tetrabutylammonium
bromide
(TBAB),
1 h.
Scheme 1.
1. General
General synthesis
synthesis
of reflux,
compounds
b1–19. Reagents
Reagents and
and conditions:
conditions: (i)
(i) substituted
substituted phenylethanone
phenylethanone
tetrabutylammonium
bromide
(TBAB),
1 h.b1–19.
Scheme
of
compounds
b1–19.
Scheme
1.
General synthesis
of
compounds
Reagents and
conditions: (i)
substituted phenylethanone
(0.01 mol),
mol), substituted
substituted
benzaldehyde
(0.012
mol),
piperidine a1–5.
(1 mL),
mL), 160
160 °C,
°C, 20
20 min
min or
or 30%
30% NaOH,
NaOH,
(0.01
benzaldehyde
(0.012
mol),
piperidine
(1
Table
1. Structure
of the
compounds
(0.01
mol), substituted
benzaldehyde
(0.012
mol),
piperidine
(1
mL), 160
°C, 20
min or
30% NaOH,
ethanol, r.t.,
r.t., 24
24 h;
h; (ii)
(ii) ethanol,
ethanol, hydrazine
hydrazine hydrate,
hydrate, reflux,
reflux, 44 h;
h; (iii)
(iii) substituted
substituted benzoyl
benzoyl chloride
chloride or
or
ethanol,
ethanol, r.t., 24 h;Table
(ii) ethanol,
hydrazine
hydrate,
reflux, 4 h; a1–5.
(iii) substituted benzoyl chloride or
1. Structure
of2°C,
theethanol,
compounds
substituted
benzenesulfonylRchloride,
chloride,
80
pyridine,
1 h;
h;
(iv) ethanol,
ethanol,
phenylhydrazine,
Compounds
1
R
3
4 (iv)
R5 phenylhydrazine,
substituted
benzenesulfonyl
80
°C, ethanol,
ethanol,Rpyridine,
pyridine,
phenylhydrazine,
substituted
benzenesulfonyl
chloride,
80
°C,
11Rh;
(iv) ethanol,
tetrabutylammonium
bromide
(TBAB),
reflux, 11 h.
h.
tetrabutylammonium
bromide
(TBAB),
reflux,
a1
3
–OH
–OH
H
–OCH
tetrabutylammonium
bromide
(TBAB),
reflux,
1 h.–OCH3
Compounds
R
R
R3 3
1
2
a2
–OH
–OCH
HR4 –NOR2 5
–OCH3
Table 1.
1. Structure
Structure of
of the
the compounds
compounds a1–5.
a1–5.
Table
Table
1. Structure
compounds
a1–5.
a1a3
–OCH
–OH of the
–OCH
–OH H H
–Br
–OH
–OCH
33
–OH
3
Compounds
R111–OH
R222 –OCH
R3333
R444 –NO
R2555 2
a2a4 Compounds
–OCH
–OH R
–OCH
–NO
Compounds
R
R
R
R
R
–Br 3 R
3R
H HR
R
1
2
3
4
5
a1 –OCH
3
–OH
–OCH
3 –OH
–OH
H
–OCH
a3a5
–Br
–OH
–OCH
H
a1
3
–OH
–OCH
3
–OH
H
–OCH
3–OCH
–OCH
3–OH–OCH
3 3 33 H –OH –CHH
3
33
a1
–OCH
a2 –Br –OCH
3
–OH –OCH
–OCH
H
–NO2222
–OCH
a4
–OH
HH
–NO
3 3333
a2
333
–OH
–OCH
–NO
2
a3
–Br
–OH
–OCH
3
–OH
H 3
a5
–OCH
–OCH
H
–CH
3 –Br
3 b1–19.
a3–OCH
–OCH
333
–OH
H
Table
2. Structure
of the3–OH
compounds
a4
a4
a4
–Br
–Br
–Br
–OH
–OH
–OH
–OCH333
–OCH
–OCH
3
H
H
H
–NO222
–NO
–NO
2
a5
–OCH333 R–OCH
–OCH
3
–OCH
3
H
–CH333 R6
Compounds
R1 a5
R2 –OCH
3
R
4
RH
5
333
–OCH
333
–CH
3
Table
2. Structure
of3 the compounds
b1–19.
b1
–OH
–OCH3
–OH
H
H
–OCH3
Table 2.
2. Structure
Structure of
of the
the compounds
compounds b1–19.
b1–19.
Table
Table
of3 the
compounds
b1–19.
b2
–OH2. Structure
–OCH
H
–NO2
H
–OCH3
CompoundsCompounds
R1
R1 2
R3
R4 R4
R5R5
R
R11–OH
R222–OCH
R333 –OH
R6666
b3 Compounds
Compounds
R
R
R
R
4
R
5
R
–Br
3
H
H
R1
R2
R3
R44
R55
R
6
b1 –Br
3
–OH
–OCH
3–OH–OH
H
H
–OCH
b1b4
–OCH
–OH
–OCH
HH
3
–OH
–OCH3–OCH
3
HH
b1
333
–OH
333 H –OH –NO2
H
–OCH
b2
3
–OH
–OCH333 H H
H –NO
–NO
H
–OCH
b2
–OCH
–OH
–OCH3–OCH
H
3
2 2222
b2
333
–OH
–NO
–OCH
3
O H
b3
–Br
–OH
–OCH
3
–OH
H
H
b3b5
–Br
–OH
–OCH
–OH
H
H
b3 –OCH3 –Br–OH –OH
333–OH
–OH H H
S H
–OCH3–OCH
3
b4–Br
–Br
–OH
–OCH333 H H
H
–NO
H
b4
–OH
–OCH3–OCH
b4
–Br
–OH
–OCH
–NO
2 2222
O H
H
b4
–Br
–OH
H –NO
–NO
3
O
b5
b6
b5
–OCH
b5
3
b5
3
–OCH
–OH
–OCH
333
–OCH
–OH
–OCH333–OH–OH
–OH
–OCH3–OCH
–OH
–OCH
–OH
–OH
3
H
HH
H
H
b6
b7
b6
b6
–OCH
b6
3
3
–OCH
–OCH
–OH
333
–OCH
–OH
–OCH333–OH–OH
–OH
–OH
–OCH
–OCH3–OCH
–OH
–OH
3
H
H
HH
H
b7
b8
b8
b9
–OCH3
–OCH3
b7
b7
–OCH3
–OCH3
b8
b8
–OCH3
–OCH3
b9
b9
b9
b9
–OCH3
b10
b10
b10
b10
–OCH3
–OCH3
–OH
–OH
–OCH333
–OCH
–OH3
–OH
–OCH333
–OCH
–OH3
–OH
–OCH333
–OCH
–OCH
–OH3
–OH
–OCH333
–OCH
–OH3
–OCH3
–OCH3
–OH
–OH
–OH
–OCH3
–OH
–OH
–OCH3–OCH333–OH–OH
–OCH3
–OH
–OH
–OCH3
–OH
–OH
–OCH3–OCH333–OH–OH
–OCH3
–OH
–OH
–OCH333
–OH
–OH
–OCH
–OH
–OH
–OH
3–OH
–OCH3–OCH
–OCH3
–OH
–OH
–OCH3
–OH
–OH
–OCH3–OCH333–OH–OH
H
HH
H
H
HH
H
H
H
HH
H
H
HH
O
ON
OO
2
S
S
OS
S
O
O
SO
O
O2N
N
O O
O
O22N
N
O2
O
O
O
S
S
S
S
O
O
O
O
O
S
OO
O
O
O
NO2
NO2
NO
NO
NO222
S
S
O SS
O
O
O
S O
O
O
OO
O
S
S
S
S
O O
O
O
O
S O
O
O
O O
S
S
S
S
O
O
O
O2N
O
O O
ON
N
O 22N
O O22N
O
O
O
NO2
NO2
NO
NO
NO222
CH3
CH3
CH
CH
CH333
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Table 2. Cont.
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Compounds
R1
R2
R3
R4
R5
b11
b11
b11
b11
b11
b11
b11
–OCH
3
3
–OCH
–OCH
–OCH
–OCH33333333
–OCH
–OCH
–OH
–OH
–OCH
3
–OH
–OH
–OCH
–OH
–OH
–OCH
–OH
–OH
–OCH33333333
–OH
–OH
–OH
–OH
–OCH
–OH
–OCH–OCH
–OH
3
H
H
H
H
H
HH
b12
b12
b12
b12
b12
b12
b12
–OCH3
3
–OCH
–OCH
–OCH
–OCH33333333
–OCH
–OCH
–OH
–OH
–OCH
3
–OH
–OH
–OCH
–OH
–OH
–OCH
–OH
–OH
–OCH333333
–OH
–OH
–OH
–OH
–OCH–OCH
3–OCH33–OH–OH
H
H
H
H
H
HH
b13
b13
b13
b13
b13
b13
–OCH3
b13
–OCH
–OCH
33333
–OCH
–OCH
333
–OCH
–OH
3
–OCH
–OH
–OCH
3
–OH
–OH
–OCH
–OH
–OH
–OCH
–OH
–OH
–OCH33333
–OH
–OH
–OH
–OCH–OCH
–OH
–OH
3–OCH333–OH
H
HH
H
b14
b14
b14
b14
b14
–OCH3
b14
b14
–OCH
33333
–OCH
–OCH
–OCH
–OH
333
–OCH
3
–OCH
–OH
–OCH
3
–OH
–OH
–OCH
–OH
–OH
–OCH
–OH
–OH
–OCH33333333–OH
–OH
–OCH–OCH
–OH
–OH
3–OCH
–OH
–OH
H
H
H
HH
H
b15
b15
b15
b15
–OCH3
b15
b15
b15
–OCH
33333
–OCH
–OCH
–OH
–OCH
333
–OCH
3
–OCH
–OH
–OCH33333–OH
–OH
–OH
–OCH
–OH
–OH
–OH
–OCH–OCH
–OH
–OCH3333
–OH
3–OCH
–OH
–OH
–OH
–OCH
–OH
H
H
H
HH
H
b16
b16
–OCH3
b16
b16
b16
b16
b16
–OCH
–OH
33333
–OCH
–OCH
–OCH
333
–OCH
3
–OCH
–OH
3
–OH
–OCH–OCH
–OH
–OH
–OH
–OCH
–OH
3–OCH
–OH
–OCH33333333–OH
–OH
–OH
–OCH
–OH
–OH
–OCH
–OH
H
HH
H
H
b17
b17
–OCH3
b17
b17
b17
b17
b17
–OCH
–OH
–OCH
33333
–OCH
–OCH
–OCH
333
–OCH
3
–OH
3
–OH
–OCH–OCH
–OH
–OH
–OH
–OCH
–OH
3–OCH
–OH
–OCH33333333–OH
–OH
–OH
–OCH
–OH
–OH
–OCH
–OH
H
HH
H
H
b18
b18
–OCH3
b18
b18
b18
b18
b18
–OCH
–OH
–OCH
33333
–OCH
–OCH
–OCH
333
–OCH
3
–OH
3
–OH
–OCH–OCH
–OH
–OCH
–OH
–OH
–OCH
–OH
3–OCH
–OH
–OCH33333333–OH
–OH
–OH
–OH
–OH
–OCH
–OH
H
HH
H
H
H
b19
b19
b19
b19
–OCH3
b19
b19
b19
–OCH
–OCH33333 –H –H
–H
–OCH
33333
–OCH
33333
–OCH
–OCH
–OCH
–H
–OCH
–OCH
–OCH
3–OCH
–OCH
–OCH3333
–H
–OCH
333 3 –OCH
333
–OCH
–H
–OCH
–OCH
–OCH
–H
–OCH
3
3
H
H
H
H
H
H
H
H
H
R6
O
O
O
O
O
O
O
O
of
14
of
14
4444of
14
of
14
of14
14
444of
of
14
NO
NO
NO
NO
NO
22222
NO
NO
NO2222
O
O
O
O
O
O
OO
NO
NO
NO
NO
NO
22222
NO
NO
NO2222
NO
NO
NO
NO
NO22222
NO
NO2222
O
O
O
O
O
OO
NO
NO
2
NO
NO
NO2222
NO
NO2222
O
O
O
O
O
OO
O
O
O
O
O
OO
SS
S
S
SSS
O
O
O
O
O
OO
O
O
O
O
O
OO
O
O
O
O
O
O
OO
SS
S
S
S
SSS
H
–CH
3
–CH
–CH
–CH
3 333333
–CH
–CH
–CH33
2.2.
MTT
Assay
2.2.
2.2.
MTT
Assay
2.2.MTT
MTTAssay
Assay
To
identify
the
most
promising
antitumor
agent
among
the
synthesized
pyrazoline
derivatives
To
identify
the
most
promising
antitumor
agent
among
the
synthesized
pyrazoline
derivatives
To
identify
the
most
promising
antitumor
agent
among
the
synthesized
pyrazoline
derivatives
Toidentify
identifythe
themost
mostpromising
promisingantitumor
antitumoragent
agentamong
amongthe
thesynthesized
synthesizedpyrazoline
pyrazolinederivatives
derivatives
To
To
identify
the
most
promising
antitumor
agent
among
the
synthesized
pyrazoline
derivatives
(b1–19),
their
cytotoxic
effects
were
tested
on
HepG-2
cells;
cisplatin
was
used
as
aapositive
positive
control.
(b1–19),
their
cytotoxic
effects
were
tested
on
HepG-2
cells;
cisplatin
was
used
as
a
control.
(b1–19),
their
cytotoxic
effects
were
tested
on
HepG-2
cells;
cisplatin
was
used
as
positive
control.
To identify
the
most
promising
antitumor
agent
among
the
synthesized
pyrazoline
derivatives
(b1–19),
their
cytotoxic
effects
were
tested
on
HepG-2
cells;
cisplatin
was
used
as
a
positive
control.
(b1–19),
their
cytotoxic
effects
were
tested
on
HepG-2
cells;
cisplatin
was
used
as
a
positive
control.
(b1–19),
their
cytotoxic
effects
were
tested
on displayed
HepG-2 cells;
cisplatin
wasthan
used50asµM
a positive
control.
We
found
that
compounds
b5,
b9,
and
b14–18
IC
50
values
lower
against
HepG-2
50
50
We
found
that
compounds
b5,
b9,
and
b14–18
displayed
IC
50
lower
than
50
µM
against
HepG-2
We
found
that
compounds
b5,
b9,
and
b14–18
displayed
IC
50values
values
lower
than
50
µM
against
HepG-2
We
found
that
compounds
b5,
b9,
and
b14–18
displayed
IC
50
values
lower
than
50
µM
against
HepG-2
We
found
that
compounds
b9,
and
displayed
50
lower
50
µM
HepG-2
(b1–19), their cytotoxic
effects
were b5,
tested
onb14–18
HepG-2
cells;IC
cisplatin
wasthan
used
as against
aagainst
positive
control.
50values
We
found
that
compounds
b5,
b9,
and
b14–18
displayed
IC
50
values
lower
than
50
µM
HepG-2
50
value
of
3.57
µM
at
cells
at
48
h.
The
most
effective
cytotoxic
agent
was
compound
b17,
with
an
IC
50
50
50
of
3.57
µM
at
cells
at
48
h.
The
most
effective
cytotoxic
agent
was
compound
b17,
with
an
IC
50value
value
of
3.57
µM
at
cells
at
48
h.
The
most
effective
cytotoxic
agent
was
compound
b17,
with
an
IC
50value
valueof
of3.57
3.57µM
µMat
at
cellsat
at48
48h.
h.The
Themost
mosteffective
effectivecytotoxic
cytotoxicagent
agentwas
wascompound
compoundb17,
b17,with
withan
anIC
IC50
cells
50
50
value
of
3.57
µM
at
cells
at
48
h.
The
most
effective
cytotoxic
agent
was
compound
b17,
with
an
IC
We found that compounds
b5,
b9,
and
b14–18
displayed
IC
values
lower
than
50
µM
against
HepG-2
50
50
value
of
8.45
µM
at
48
hh(Table
(Table
3).
The
pyrazoline
48
h;
cisplatin,
the
positive
control,
had
an
IC
50
50
50
of
8.45
µM
at
48
h
3).
The
pyrazoline
48
h;
cisplatin,
the
positive
control,
had
an
IC
50value
value
of
8.45
µM
at
48
(Table
3).
The
pyrazoline
48
h;
cisplatin,
the
positive
control,
had
an
IC
50value
value
of
8.45
µM
at
48
h
(Table
3).
The
pyrazoline
48
h;
cisplatin,
the
positive
control,
had
an
IC
50
of
8.45
µM
at
48
h
(Table
3).
The
pyrazoline
48
h;
cisplatin,
the
positive
control,
had
an
IC
50 value of 8.45 µM at 48 h (Table 3). The pyrazoline
48
h;most
cisplatin,
the
positive
control,
had an
IC50
derivatives
also
displayed
dose-dependent
and
time-dependent
trends.
We
selected
the
three
most
cells at 48 h. The
effective
cytotoxic
agent
was
compound
b17,
with
IC50 the
value
ofmost
3.57 µM
derivatives
also
displayed
dose-dependent
and
time-dependent
trends.
We
selected
three
derivatives
also
displayed
dose-dependent
and
time-dependent
trends.
We
selected
the
three
most
derivatives
alsodisplayed
displayed
dose-dependent
andtime-dependent
time-dependent
trends.
Wean
selected
thethree
three
most
derivatives
also
dose-dependent
and
trends.
We
selected
the
most
derivatives
also
displayed
dose-dependent
and
time-dependent
trends.
We
selected
the
three
most
effective
compounds,
b15–17,
along
with
cisplatin
to
generate
growth-inhibitory
curves
(Figure
2).
effective
compounds,
b15–17,
along
with
cisplatin
to
generate
growth-inhibitory
curves
(Figure
2).
effective
compounds,
b15–17,
along
with
cisplatin
to
generate
growth-inhibitory
curves
(Figure
2).
at 48 h; cisplatin,
the
positive
control,
had
an
IC
value
of
8.45
µM
at
48
h
(Table
3).
The
pyrazoline
effective
compounds,
b15–17,
along
with
cisplatin
to
generate
growth-inhibitory
curves
(Figure
2).
50
effective
effective compounds,
compounds, b15–17,
b15–17,along
along with
with cisplatin
cisplatin to
togenerate
generate growth-inhibitory
growth-inhibitory curves
curves(Figure
(Figure2).
2).
2.2. MTT
MTTAssay
Assay
2.2. MTT Assay2.2.
derivatives also displayed
dose-dependent
and
time-dependent
trends.
Weprimary
selected
the three most
Table
3.
The
cytotoxic
effects
of
the
compounds
b1–19
on
HepG-2
cell
line
and
primary
hepatocytes.
Table
3.
The
cytotoxic
effects
of
the
compounds
b1–19
on
HepG-2
cell
line
and
hepatocytes.
Table
3.
The
cytotoxic
effects
of
the
compounds
b1–19
on
HepG-2
cell
line
and
primary
hepatocytes.
Table3.
3.The
Thecytotoxic
cytotoxiceffects
effectsof
ofthe
thecompounds
compoundsb1–19
b1–19on
onHepG-2
HepG-2cell
cellline
lineand
andprimary
primaryhepatocytes.
hepatocytes.
Table
Table
3.
The
cytotoxic
effects
of
the
compounds
b1–19
on
HepG-2
cell
line
and
primary
hepatocytes.
effective compounds, b15–17, along with cisplatin
to generate growth-inhibitory curves
(Figure 2).
−1
−1
−1
−1
−1
−1
−1
−1
−1
−1
IC
50
/μmol·L
50
/μmol·L
IC
50
50
50
50
IC
/μmol·L
/μmol·L
IC
IC
50
/μmol·L
50
/μmol·L
IC
−1
−1
IC50
50
/μmol·L−1−1
50
/μmol·L−1−1
IC50
Compounds
Compounds
−1
−1
IC
/μmol·L
/μmol·L
IC
Compounds
Compounds
Compounds
Compounds
50
50
IC50Primary
50
/μmol·LHepatocytes
50
/μmol·LHepatocytes
IC50Primary
Compounds HepG-2
Compounds HepG-2
HepG-2
Primary
Hepatocytes
HepG-2
Primary
Hepatocytes
Compounds
Compounds
HepG-2
Primary
Hepatocytes
HepG-2
Primary
Hepatocytes
Compounds
Compounds
HepG-2 Primary
PrimaryHepatocytes
Hepatocytes
HepG-2 Primary
PrimaryHepatocytes
Hepatocytes
HepG-2
HepG-2
HepG-2
Primary
Hepatocytes
HepG-2
Primary
Hepatocytes
Table 3. The cytotoxic
effects>50
of the compounds
b1–19 on HepG-2
cell line
and primary —
hepatocytes.
b1
b12
>50
—
>50
—
b1
b12
—
>50
b1
b12
>50
—
>50
—
b1
b12
>50
—
>50
—
b1
b12
>50
—
>50
—
b1
b12
>50
—
>50
—
b2
b13
>50
—
>50
—
b2
b13
>50
—
>50
—
b2
b13
>50
—
>50
—
b2
b13
>50
—
>50
—
b2
b13
>50
—
>50
—
b2
b13
>50
—
>50
—
b3
b14
>50
—
17.99
±1.37
1.37
22.65
±1.21
1.21
−1
b3
b14
>50
—
17.99
22.65
b3
b14
>50
—
17.99
1.37
22.65
1.21
IC50 /µmol
b3
b14
>50 ·L−1
—
17.99±±±±1.37
1.37 IC50 /µmol
22.65±±·±±L
1.21
b3
b14
>50
—
17.99
22.65
b3
b14
>50
—
17.99
±
1.37
22.65
±±±1.21
1.21
Compounds
Compounds
b4
b15
>50
—
4.51
±
1.49
19.24
0.08
b4
b15
>50
—
4.51
±
1.49
19.24
±
0.08
b4
b15
>50
—
4.51
±
1.49
19.24
0.08
b4
b15
>50
—
4.51±±1.49
1.49
19.24±±0.08
0.08
b4
b15
>50
—
4.51
19.24
b4
b15
>50
—±2.21
4.51
±±1.27
1.49
19.24
±±1.72
0.08
HepG-2
Primary
Hepatocytes
HepG-2
Primary
Hepatocytes
b5
b16
28.76
±1.32
1.32
35.13
2.21
4.61
1.27
20.73
1.72
b5
b16
28.76
35.13
4.61
20.73
b5
b16
28.76
1.32
35.13
2.21
4.61
1.27
20.73
1.72
b5
b16
28.76±±±±1.32
1.32
35.13±±±±2.21
2.21
4.61±±±±1.27
1.27
20.73±±±±1.72
1.72
b5
b16
28.76
35.13
4.61
20.73
b5
b16
28.76
±
1.32
35.13
±
2.21
4.61
±
1.27
20.73
±
1.72
b6
b17
>50
—
3.57
±
1.39
33.47
±
2.33
b6
b17
>50
—
3.57
±
1.39
33.47
±
2.33
b6
b17
>50
—
3.57
±± 1.39
33.47
±± 2.33
b6
b17
>50
—
3.57
1.39
33.47
2.33
b1
>50
—
b12
>50
—
b6
b17
>50
—
3.57
33.47
±±2.33
b6
b17
>50
—
3.57±±±±±1.39
1.39
33.47
2.33
b7
b18
>50
—
28.47
1.34
50.71
±3.21
3.21
b7
b18
>50
—
28.47
1.34
50.71
b7
b18
>50
—
28.47
1.34
50.71
3.21
b7
b18
>50
—
28.47
±1.34
1.34
50.71±±±±3.21
3.21—
b2
>50
—
b13
>50
b7
b18
>50
—
28.47
±
50.71
b7
b18
>50
—
28.47
± 1.34
50.71—
± 3.21
b8
b19
>50
—
>50
—
b8
b19
>50
—
>50
b8
b19
>50
—
>50
—
b8
b19
>50
—
>50
—
b8
b19
>50
>50
—
b3
>50
— 29.66—
b14
17.99
±1.05
1.37
22.65 ± 1.21
b8
b19
>50
—
>50
—
b9
Cisplatin
12.01
±±1.83
1.83
±±2.43
2.43
8.45
±±1.05
—
b9
Cisplatin
12.01
±
29.66
±
8.45
±
—
b9
Cisplatin
12.01
1.83
29.66
2.43
8.45
1.05
—
b9
Cisplatin
12.01±±±1.83
1.83 — 29.66
29.66±±±2.43
2.43
8.45±±
1.05
—
b9
Cisplatin
12.01
8.45
—
b4
>50
b15
4.51
1.49
19.24 ± 0.08
b9
Cisplatin
12.01
1.83
29.66
2.43
8.45
±±1.05
1.05
—
b10
>50
—
b10
>50
—
b10
>50
—
b10
>50 35.13 ± 2.21 —
—
>50
b5
28.76b10
±
1.32
b16
4.61
±
1.27
20.73 ± 1.72
b10
>50
—
b11
>50
—
b11
>50
—
b11
>50
—
b11
>50
—
b11
>50
—
b6
>50
—
b17
3.57 ± 1.39
33.47 ± 2.33
b11
>50
—“—”: Not determined.
“—”:
Not
determined.
“—”:
“—”: Not
Not determined.
determined.
“—”:
b7
>50
—
b18
28.47 ± 1.34
50.71 ± 3.21
“—”:Not
Notdetermined.
determined.
b8
b9
b10
b11
>50
12.01 ± 1.83
>50
>50
—
29.66 ± 2.43
—
—
b19
Cisplatin
“—”: Not determined.
>50
8.45 ± 1.05
—
—
Molecules 2017, 22, 467
Molecules 2017, 22, 467
5 of 14
5 of 14
Figure2.2. Inhibitory
Inhibitoryeffects
effectsof
ofcompounds
compoundsb15–17
b15–17and
andcisplatin
cisplatinon
onHepG-2
HepG-2cells
cellsafter
after24
24hhand
and48
48h.h.
Figure
2.3. MTS Assay
2.3. MTS Assay
According to the results of MTT (thiazolyl blue tetrazolium bromide) assay on HepG-2 cell line,
According to the results of MTT (thiazolyl blue tetrazolium bromide) assay on HepG-2 cell line, the
the cytotoxicity of compounds b5, b9, and b14–18 were evaluated against primary hepatocytes using
cytotoxicity of compounds b5, b9, and b14–18 were evaluated against primary hepatocytes using the
the MTS assay. (Isolated primary hepatocytes were unstable. Using the MTS assay, the solubilization
MTS assay. (Isolated primary hepatocytes were unstable. Using the MTS assay, the solubilization steps
steps were eliminated because the MTS formazan product is soluble in tissue culture medium. It can
were eliminated because the MTS formazan product is soluble in tissue culture medium. It can avoid
avoid further damage to cells.) Compounds b5, b9, and b14–18 showed lower cytotoxicological activity
further damage to cells.) Compounds b5, b9, and b14–18 showed lower cytotoxicological activity
against primary hepatocytes. The IC50 value of compound b17 against primary hepatocytes was 33.47 µM
against primary hepatocytes. The IC50 value of compound b17 against primary hepatocytes was
at 48 h. In terms of their anticancer potential, compound b17 can be considered as the most promising
33.47 µM at 48 h. In terms of their anticancer potential, compound b17 can be considered as the most
anticancer agent against HepG-2 due to its low cytotoxicity against primary hepatocytes (Table 3).
promising anticancer agent against HepG-2 due to its low cytotoxicity against primary hepatocytes
(Table 3).
2.4. Cell Cycle Analysis
2.4. Cell
Analysis
To Cycle
determine
whether compound b17 would affect the cell cycle in HepG-2 cells, we examined cell
cycleToprogression
using
flow
cytometryb17
(Figure
3).affect
HepG-2
were in
treated
with
compound
b17 at
determine whether
compound
would
the cells
cell cycle
HepG-2
cells,
we examined
concentrations
of 0.9 µM,
2.7 flow
µM, and
4.5 µM for
24 h. 3).
A notable
decrease
in cells
in the
G1 and
S phases
cell
cycle progression
using
cytometry
(Figure
HepG-2
cells were
treated
with
compound
was
observed.
At
4.5
µM,
up
to
83.01%
of
the
cells
were
arrested
in
the
G
2/M phase (the data shown
b17 at concentrations of 0.9 µM, 2.7 µM, and 4.5 µM for 24 h. A notable decrease in cells in the G1
in Figure
3 is was
about
living cells
exclusion
of deadof
cells).
Thesewere
findings
indicate
that
and
S phases
observed.
Atafter
4.5 µM,
up to 83.01%
the cells
arrested
in the
G2compound
/M phase
b17
may
be
a
potent
anticancer
agent.
(the data shown in Figure 3 is about living cells after exclusion of dead cells). These findings indicate
that compound b17 may be a potent anticancer agent.
Molecules 2017, 22, 467
Molecules 2017, 22, 467
6 of 14
6 of 14
Figure 3. Effects of compound b17 on cell cycle progression in HepG-2 cells. Cells were treated with
Figure 3. Effects of compound b17 on cell cycle progression in HepG-2 cells. Cells were treated with
b17 at concentrations of 0 µM, 0.9 µM, 2.7 µM, and 4.5 µM for 24 h.
b17 at concentrations of 0 µM, 0.9 µM, 2.7 µM, and 4.5 µM for 24 h.
2.5. Annexin-V Assay
2.5. Annexin-V Assay
A biparametric cytofluorimetric analysis was performed to determine the mode of cell death
A biparametric cytofluorimetric analysis was performed to determine the mode of cell death
induced by compound b17 using propidium iodide (PI) and fluorescent immunolabeling of the
induced by compound b17 using propidium iodide (PI) and fluorescent immunolabeling of the protein
protein annexin V. HepG-2 cells were treated with compound b17 at concentrations of 0.9 µM,
annexin V. HepG-2 cells were treated with compound b17 at concentrations of 0.9 µM, 2.7 µM, and
2.7 µM, and 4.5 µM for 12 h. The percentage of cell apoptosis was 0.3% at 0 µM for compound b17,
4.5 µM for 12 h. The percentage of cell apoptosis was 0.3% at 0 µM for compound b17, and 7.6%, 8.7%,
and 7.6%, 8.7%, and 10.2% at 0.9 µM, 2.7 µM, and 4.5 µM, respectively (Figure 4). Thus, we conclude
and 10.2% at 0.9 µM, 2.7 µM, and 4.5 µM, respectively (Figure 4). Thus, we conclude that compound
that compound b17 can induce apoptosis.
b17 can induce apoptosis.
Molecules 2017, 22, 467
Molecules 2017, 22, 467
7 of 14
7 of 14
Figure
Compoundb17
b17induced
induced apoptosis
apoptosis in
in HepG-2
HepG-2 cells
Figure
4. 4.Compound
cells at
at concentrations
concentrationsofofcontrol,
control,0.90.9µM,
µM,
2.7
µM,
and
4.5
µM
in
a
12
h
exposure.
PI:
propidium
iodide.
2.7 µM, and 4.5 µM in a 12 h exposure. PI: propidium iodide.
3. Experimental Section
3. Experimental Section
Chemical
Reagentsand
andEquipment
Equipment
3.1.3.1.
Chemical
Reagents
reagentswere
werepurchased
purchasedfrom
fromcommercial
commercial suppliers.
suppliers. Melting
AllAll
reagents
Melting points
pointswere
weredetermined
determinedusing
using
an Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp, Loughborough, UK). IR spectra
an Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp, Loughborough, UK). IR spectra
were recorded on a Bruker Tensor 27 Fourier IR spectrometer (Bruker, Karlsruhe, Germany). 1H-NMR
were recorded on a Bruker Tensor 27 Fourier IR spectrometer (Bruker, Karlsruhe, Germany). 1 H-NMR
spectra were recorded on a Bruker Avance 300 spectrometer (Bruker) using tetramethylsilane (TMS)
spectra were recorded on a Bruker Avance 300 spectrometer (Bruker) using tetramethylsilane (TMS)
as the internal standard. Mass spectra were recorded on a SCIEX Triple Quad 6500+ LC/MS/MS
as the internal standard. Mass spectra were recorded on a SCIEX Triple Quad 6500+ LC/MS/MS
system (SCIEX, Los Angeles, CA, USA). HRMS (high-resolution mass spectrometry) was performed
system (SCIEX, Los Angeles, CA, USA). HRMS (high-resolution mass spectrometry) was performed
on a Thermo Scientific Q Exactive (Thermo, Waltham, MA, USA).
on a Thermo Scientific Q Exactive (Thermo, Waltham, MA, USA).
3.2. General Procedures for the Synthesis of Compounds a1–5
3.2. General Procedures for the Synthesis of Compounds a1–5
Compounds a1–4 were obtained by a mixture of substituted phenylethanone (0.01 mol), substituted
Compounds a1–4 were obtained by a mixture of substituted phenylethanone (0.01 mol),
benzaldehyde (0.012 mol), and piperidine (1 mL) as a catalyst, and stirred at 160 °C for 20 min. Next,
substituted
benzaldehyde
(0.012 mol),
and(30
piperidine
(1 mL)and
as astirred
catalyst,
and
stirred
at 160 ◦ C for
30% aqueous
sodium hydroxide
solution
mL) was added
for 15
min
[27]. Compound
Molecules 2017, 22, 467
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20 min. Next, 30% aqueous sodium hydroxide solution (30 mL) was added and stirred for 15 min [27].
Compound a5 was prepared by appropriate substituted phenylethanone (0.01 mol), substituted
benzaldehyde (0.012 mol), and 30% aqueous sodium hydroxide solution (10 mL) in ethanol (30 mL),
and stirred at 30 ◦ C for 24 h. The progress of the reaction was checked by thin-layer chromatography
(TLC). Upon completion, the reaction mixture was poured onto crushed ice, followed by neutralization
with HCl [28]. The precipitated solid was filtered, washed with water, and dried by vacuum pump.
The product was finally crystallized from ethanol.
3.3. General Procedures for the Synthesis of Compounds b1–19
Hydrazine hydrate (0.04 mol, 1.96 g) was added to a solution of compound a1 (0.01 mol,
3.00 g) in ethanol (10 mL). The mixture was refluxed under stirring for 4 h, and the reaction
mixture was subsequently poured onto crushed ice; the precipitate was filtered out, and product
b1 was later crystallized from ethanol. Compounds b2–4 were synthesized following this procedure.
Compounds b5–17 were prepared by treating compound b1 (0.01 mol, 3.14 g) with corresponding
substituted benzoyl chloride or substituted benzenesulfonyl chloride (0.015 mol) at 80 ◦ C in ethanol
as solvent with pyridine as catalyst for 1 h to yield the final N-substituted targeted compounds.
The N-phenyl-substituted pyrazolines b18 and b19 were prepared by direct cyclization of a1 and a5,
respectively, with phenylhydrazine in the presence of TBAB as a catalyst [29].
4-[5-(2-Hydroxyphenyl)-3,4-dihydro-2H-pyrazol-3-yl]-2,6-dimethoxy-phenol (b1). Yield: 70.62%; M.p.
140.3–140.7 ◦ C. IR (KBr) νmax (cm−1 ): 3277 (aromatic –OH), 2994, 2946 (aliphatic C–H asymmetric),
1618, 1498, 1461, 1424 (C=N and C=C), 1352, 1258, 1210, 1130 (C–N). 1 H-NMR (400 MHz, δ ppm,
DMSO-d6 ): 2.96–3.03 (1H, m, pyrazoline C4 –HA ), 3.54–3.58 (1H, m, pyrazoline C4 –HB ), 3.76 (6H, s,
–OCH3 ), 4.74–4.81 (1H, m, pyrazoline C5 –Hx ), 6.70 (2H, s), 6.88–6.93 (2H, m), 7.24 (1H, t, J = 8.00 Hz),
7.31 (1H, d, J = 8.00 Hz), 7.78 (1H, s, –NH–), 8.31 (1H, s, –OH), 11.21 (1H, s, –OH). MS (ESI) (m/z):
315 [M + H]+ .
2,6-Dimethoxy-4-[5-(4-nitrophenyl)-3,4-dihydro-2H-pyrazol-3-yl]-phenol (b2). Yield: 71.22%; M.p.
140.7–141.1 ◦ C. IR (KBr) νmax (cm−1 ): 3319 (aromatic –OH), 2971, 2844 (aliphatic C–H asymmetric),
1614, 1464, 1430, 1407 (C=N and C=C), 1340, 1223, 1166, 1117 (C–N). 1 H-NMR (400 MHz, δ ppm,
DMSO-d6 ): 2.89–2.96 (1H, m, pyrazoline C4 –HA ), 3.43–3.50 (1H, m, pyrazoline C4 –HB ), 3.74 (6H, s,
–OCH3 ), 4.85–4.91 (1H, m, pyrazoline C5 –Hx ), 6.65 (2H, s), 7.82 (2H, d, J = 8.00 Hz ), 8.19 (1H, s, –NH–),
8.22 (2H, d, J = 8.00 Hz), 8.32 (1H, s, –OH). MS (ESI) (m/z): 344 [M + H]+ .
2-Bromo-4-[5-(2-hydroxyphenyl)-3,4-dihydro-2H-pyrazol-3-yl]-6-methoxy-phenol (b3). Yield: 70.01%; M.p.
181.8–183.9 ◦ C. IR (KBr) νmax (cm−1 ): 3314 (aromatic –OH), 2966, 2938 (aliphatic C–H asymmetric),
1620, 1499, 1424, 1348 (C=N and C=C), 1262, 1187, 1153, 1049 (C-N). 1 H-NMR (400 MHz, δ ppm,
DMSO-d6 ): 2.98–3.05 (1H, m, pyrazoline C4 –HA ), 3.55–3.61 (1H, m, pyrazoline C4 -HB ), 3.83 (3H, s,
–OCH3 ), 4.76–4.81 (1H, m, pyrazoline C5 –Hx ), 6.88–6.93 (2H, m), 7.04 (1H, s), 7.10 (1H, s), 7.22–7.26
(1H, t, J = 8.00 Hz), 7.30 (1H, d, J = 8.00 Hz), 7.81 (1H, s, –NH–), 9.42 (1H, s, –OH), 11.15 (1H, s, –OH).
MS (ESI) (m/z): 363 [M + H]+ .
2-Bromo-6-methoxy-4-[5-(4-nitrophenyl)-3,4-dihydro-2H-pyrazol-3-yl]-phenol (b4). Yield: 76.11%; M.p.
208.6–208.9 ◦ C. IR (KBr) νmax (cm−1 ): 3401, 3325 (aromatic –OH), 2965, 2934 (aliphatic C–H
asymmetric), 1614, 1466, 1451, 1416 (C=N and C=C), 1333, 1274, 1187, 1049 (C-N). 1 H-NMR (400 MHz,
δ ppm, DMSO-d6 ): 2.91–2.98 (1H, m, pyrazoline C4 –HA ), 3.44–3.51 (1H, m, pyrazoline C4 –HB ), 3.82
(3H, s, –OCH3 ), 4.87–4.93 (1H, m, pyrazoline C5 –Hx ), 6.99 (1H, s), 7.06 (1H, s), 7.82 (2H, d, J = 8.00 Hz),
8.22 (2H, d, J = 8.00 Hz), 8.24 (1H, s, –NH–), 9.39 (1H, s, –OH). MS (ESI) (m/z): 392 [M + H]+ .
4-[2-Benzenesulfonyl-5-(2-hydroxyphenyl)-3,4-dihydro-2H-pyrazol-3-yl]-2,6-dimethoxy-phenol (b5). Yield:
72.44%; M.p. 163.1–164.4 ◦ C. IR (KBr) νmax (cm−1 ): 3447, 3194 (aromatic –OH), 2969, 2937
(aliphatic C–H asymmetric), 1619, 1461, 1355, 1219 (C=N and C=C), 1171, 1116, 1056, 1002 (C–N).
1 H-NMR (400 MHz, δ ppm, DMSO-d ): 3.27–3.32 (1H, m, pyrazoline C –H ), 3.67–3.72 (1H, m,
6
4
A
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pyrazoline C4 –HB ), 3.77 (6H, s, –OCH3 ), 4.84–4.89 (1H, m, pyrazoline C5 –Hx ), 6.70 (2H, s), 6.90 (1H, t,
J = 8.00 Hz), 6.96 (1H, d, J = 8.00 Hz), 7.35 (1H, t, J = 8.00 Hz), 7.44 (1H, d, J = 8.00 Hz), 7.66 (1H, t,
J = 8.00 Hz), 7.74 (2H, t, J = 8.00 Hz), 7.85 (2H, d, J = 8.00 Hz), 8.42 (1H, s, –OH), 10.31 (1H, s, –OH).
MS (ESI) (m/z): 455 [M + H]+ .
4-[5-(2-Hydroxyphenyl)-2-(2-nitro-benzenesulfonyl)-3,4-dihydro-2H-pyrazol-3-yl]-2,6-dimethoxy-phenol (b6).
Yield: 40.03%; M.p. 151.1–152.8 ◦ C. IR (KBr) νmax (cm−1 ): 3441, 3211 (aromatic –OH), 2930, 2837
(aliphatic C–H asymmetric), 1619, 1462, 1431, 1376 (C=N and C=C), 1304, 1180, 1113, 1008 (C–N).
1 H-NMR (400 MHz, δ ppm, DMSO-d ): 3.42–3.44 (1H, m, pyrazoline C –H ), 3.76 (6H, s, –OCH ),
6
4
A
3
3.91–3.98 (1H, m, pyrazoline C4 –HB ), 5.19–5.24 (1H, m, pyrazoline C5 –Hx ), 6.67 (2H, s), 6.91–6.98
(2H, m), 7.36 (1H, t, J = 8.00 Hz), 7.55 (1H, d, J = 8.00 Hz), 7.84 (1H, t, J = 8.00 Hz), 7.93 (1H, t,
J = 8.00 Hz), 7.99–8.03 (2H, m), 8.45 (1H, s, –OH), 10.16 (1H, s, –OH). MS (ESI) (m/z): 500 [M + H]+ .
4-[5-(2-Hydroxyphenyl)-2-(3-nitro-benzenesulfonyl)-3,4-dihydro-2H-pyrazol-3-yl]-2,6-dimethoxy-phenol (b7).
Yield: 51.37%; M.p. 131.9–133.5 ◦ C. IR (KBr) νmax (cm−1 ): 3466, 3198 (aromatic –OH), 2971, 2938
(aliphatic C–H asymmetric), 1619, 1493, 1463, 1428 (C=N and C=C), 1357, 1216, 1177, 1116 (C–N).
1 H-NMR (400 MHz, δ ppm, DMSO-d ): 3.43–3.46 (1H, m, pyrazoline C –H ), 3.72 (6H, s, –OCH ),
6
4
A
3
3.75–3.78 (1H, m, pyrazoline C4 –HB ), 4.95–4.99 (1H, m, pyrazoline C5 –Hx ), 6.61 (2H, s), 6.91 (1H, t,
J = 8.00 Hz), 6.96 (1H, d, J = 8.00 Hz), 7.35 (1H, d, J = 8.00 Hz), 7.52 (1H, d, J = 8.00 Hz), 7.92 (1H, t,
J = 8.00 Hz), 8.21 (1H, d, J = 8.00 Hz), 8.34 (1H, s), 8.45 (1H, s, –OH), 8.51 (1H, d, J = 8.00 Hz), 10.24
(1H, s, –OH). MS (ESI) (m/z): 500 [M + H]+ .
4-[5-(2-Hydroxyphenyl)-2-(4-nitro-benzenesulfonyl)-3,4-dihydro-2H-pyrazol-3-yl]-2,6-dimethoxy-phenol (b8).
Yield: 63.51%; M.p. 195.5–197.0 ◦ C. IR (KBr) νmax (cm−1 ): 3385 (aromatic –OH), 2969, 2846
(aliphatic C–H asymmetric), 1620, 1464, 1430, 1368 (C=N and C=C), 1314, 1207, 1065, 1012 (C–N).
1 H-NMR (400 MHz, δ ppm, DMSO-d ): 3.41–3.47 (1H, m, pyrazoline C –H ), 3.73 (6H, s, –OCH ),
6
4
A
3
3.74–3.79 (1H, m, pyrazoline C4–HB ), 4.90–4.95 (1H, m, pyrazoline C5 –Hx ), 6.63 (2H, s), 6.90 (1H,t,
J = 8.00 Hz), 6.95 (1H, d, J = 8.00 Hz), 7.34 (1H, t, J = 8.00 Hz), 7.51 (1H, d, J = 8.00 Hz), 8.04
(2H, d, J = 8.00 Hz), 8.41 (1H, d, J = 8.00 Hz), 8.45 (1H, s, –OH), 10.22 (1H, s, –OH). MS (ESI) (m/z):
500 [M + H]+ .
4-[5-(2-Hydroxyphenyl)-2-(toluene-4-sulfonyl)-3,4-dihydro-2H-pyrazol-3-yl]-2,6-dimethoxy-phenol (b9).
Yield: 71.43%; M.p. 194.6–194.7 ◦ C. IR (KBr) νmax (cm−1 ): 3453, 3192 (aromatic –OH), 2969, 2938
(aliphatic C–H asymmetric), 1615, 1493, 1462, 1429 (C=N and C=C), 1358, 1218, 1115, 1026 (C–N).
1 H-NMR (400 MHz, δ ppm, DMSO-d ): 2.38 (3H, s, –CH ), 3.41–3.44 (1H, m, pyrazoline C –H ),
6
3
4
A
3.67–3.75 (1H, m, pyrazoline C4 –HB ), 3.76 (6H, s, –OCH3 ), 4.77–4.83 (1H, m, pyrazoline C5 –Hx ), 6.69
(2H, s), 6.90 (1H, t, J = 8.00 Hz), 6.96 (1H, d, J = 8.00 Hz), 7.34 (1H, t, J = 8.00 Hz), 7.42–7.46 (3H, m), 7.72
(2H, d, J = 8.00 Hz), 8.41 (1H, s, –OH), 10.33 (1H, s, –OH). MS (ESI) (m/z): 469 [M + H]+ .
[5-(4-Hydroxy-3,5-dimethoxyphenyl)-3-(2-hydroxyphenyl)-4,5-dihydro-pyrazol-1-yl]-(2-nitrophenyl)-methanone
(b10). Yield: 71.43%; M.p. 194.6–194.7 ◦ C. IR (KBr) νmax (cm−1 ): 3532, 3190 (aromatic –OH), 2936,
2836 (aliphatic C–H asymmetric), 1666 (C=O), 1618, 1524, 1441, 1342 (C=N and C=C), 1253, 1224,
1146, 1106 (C–N). 1 H-NMR (400 MHz, δ ppm, DMSO-d6 ): 3.32–3.34 (1H, m, pyrazoline C4 –HA ), 3.78
(6H, s, –OCH3 ), 3.96–4.06 (1H, m, pyrazoline C4 –HB ), 5.56–5.60 (1H, m, pyrazoline C5 –Hx ), 6.62 (2H, s),
6.84–6.88 (2H, m), 7.29 (1H, t, J = 8.00 Hz), 7.38 (1H, d, J = 8.00 Hz), 7.70 (1H, d, J = 8.00 Hz), 7.77 (1H, t,
J = 8.00 Hz), 7.90 (1H, t, J = 8.00 Hz), 8.20 (1H, s), 8.37 (1H, s, –OH), 9.79 (1H, s, –OH). MS (ESI) (m/z):
464 [M + H]+ .
[5-(4-Hydroxy-3,5-dimethoxyphenyl)-3-(2-hydroxyphenyl)-4,5-dihydro-pyrazol-1-yl]-(3-nitrophenyl)-methanone
(b11). Yield: 70.03%; M.p. 207.7–209.3 ◦ C. IR (KBr) νmax (cm−1 ): 3212, 3078 (aromatic –OH), 2930,
2840 (aliphatic C–H asymmetric), 1667 (C=O), 1610, 1525, 1425, 1341 (C=N and C=C), 1310, 1214,
1159, 1113 (C–N). 1 H-NMR (400 MHz, δ ppm, DMSO-d6 ): 3.35–3.37 (1H, m, pyrazoline C4 –HA ), 3.75
(6H, s, –OCH3 ), 3.97–4.05 (1H, m, pyrazoline C4 –HB ), 5.63–5.67 (1H, m, pyrazoline C5 –Hx ), 6.63 (2H, s),
Molecules 2017, 22, 467
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6.90–6.95 (2H, m), 7.33 (1H, t, J = 8.00 Hz), 7.56 (1H, d, J = 8.00 Hz), 7.82 (1H, t, J = 8.00 Hz), 8.28–8.30
(1H, m), 8.40–8.42 (2H, m), 8.62 (1H, s), 10.03 (1H, s, –OH). MS (ESI) (m/z): 464 [M + H]+ .
[5-(4-Hydroxy-3,5-dimethoxyphenyl)-3-(2-hydroxyphenyl)-4,5-dihydro-pyrazol-1-yl]-(4-nitrophenyl)-methanone
(b12). Yield: 68.02%; M.p. 250.1–251.2 ◦ C. IR (KBr) νmax (cm−1 ): 3378, 3266 (aromatic –OH), 2936,
2842 (aliphatic C–H asymmetric), 1650 (C=O), 1614, 1518, 1444, 1336 (C=N and C=C), 1258, 1243,
1210, 1110 (C–N). 1 H-NMR (400 MHz, δ ppm, DMSO-d6 ): 3.40–3.46 (1H, m, pyrazoline C4 –HA ), 3.76
(6H, s, –OCH3 ), 3.98–4.05 (1H, m, pyrazoline C4 –HB ), 5.65–5.66 (1H, m, pyrazoline C5 –Hx ), 6.62 (2H, s),
6.90–6.94 (2H, m), 7.34 (1H, t, J = 8.00 Hz), 7.54 (1H, d, J = 8.00 Hz), 8.05 (2H, d, J = 8.00 Hz), 8.36 (2H, d,
J = 8.00 Hz), 8.40 (1H, s, –OH), 10.03 (1H, s, –OH). MS (ESI) (m/z): 464 [M + H]+ .
(3,5-Dinitrophenyl)-[5-(4-hydroxy-3,5-dimethoxyphenyl)-3-(2-hydroxyphenyl)-4,5-dihydro-pyrazol-1-yl]methanone (b13). Yield: 51.38%; M.p. 240.4–240.9 ◦ C. IR (KBr) νmax (cm−1 ): 3336, 3109 (aromatic
–OH), 2939, 2843 (aliphatic C–H asymmetric), 1647 (C=O), 1614, 1543, 1465, 1432 (C=N and C=C),
1341, 1219, 1112, 1031 (C–N). 1 H-NMR (400 MHz, δ ppm, DMSO-d6 ): 3.41–3.43 (1H, m, pyrazoline
C4 –HA ), 3.75 (6H, s, –OCH3 ), 3.98–4.06 (1H, m, pyrazoline C4 –HB ), 5.63–5.67 (1H, m, pyrazoline
C5 –Hx ), 6.64 (2H, s), 6.90 (1H, t, J = 8.00 Hz), 6.96 (1H, d, J = 8.00 Hz), 7.33 (1H, t, J = 8.00 Hz), 7.65
(1H, d, J = 8.00 Hz), 8.39 (1H, s, –OH), 8.97 (1H, s), 9.07 (2H, s), 10.06 (1H, s, –OH). MS (ESI) (m/z):
509 [M + H]+ .
[5-(4-Hydroxy-3,5-dimethoxyphenyl)-3-(2-hydroxyphenyl)-4,5-dihydro-pyrazol-1-yl]-naphthalen-2-yl-methanone
(b14). Yield: 63.51%; M.p. 185.5–186.5 ◦ C. IR (KBr) νmax (cm−1 ): 3421 (aromatic –OH), 2931, 2835
(aliphatic C–H asymmetric), 1635 (C=O), 1616, 1522, 1465, 1426 (C=N and C=C), 1328, 1218, 1118,
1031 (C–N). 1 H-NMR (400 MHz, δ ppm, DMSO-d6 ): 3.39–3.44 (1H, m, pyrazoline C4 –HA ), 3.75 (6H, s,
–OCH3 ), 3.98–4.05 (1H, m, pyrazoline C4 –HB ), 5.67–5.71 (1H, m, pyrazoline C5 –Hx ), 6.65 (2H, s),
6.89–6.94 (2H, m), 7.30–7.34 (1H, m), 7.49–7.52 (1H, m), 7.60–7.67 (2H, m), 7.86–7.88 (1H, m), 8.01–8.04
(2H, m), 8.07 (1H, d, J = 8.00 Hz), 8.38 (1H, s), 8.41 (1H, s, –OH), 10.13 (1H, s, –OH). MS (ESI) (m/z):
469 [M + H]+ .
[5-(4-Hydroxy-3,5-dimethoxyphenyl)-3-(2-hydroxyphenyl)-4,5-dihydro-pyrazol-1-yl]-thiophen-2-yl-methanone
(b15). Yield: 63.41%; M.p. 196.1–197.2 ◦ C. IR (KBr) νmax (cm−1 ): 3375, 3199 (aromatic –OH), 2942,
2836 (aliphatic C–H asymmetric), 1632 (C=O), 1612, 1516, 1435, 1336 (C=N and C=C), 1245, 1209,
1116, 1043 (C–N). 1 H-NMR (400 MHz, δ ppm, DMSO-d6 ): 3.32–3.33 (1H, m, pyrazoline C4 –HA ), 3.69
(6H, s, –OCH3 ), 3.93–4.00 (1H, m, pyrazoline C4 –HB ), 5.57–5.61 (1H, m, pyrazoline C5 –Hx ), 6.50 (2H, s),
6.94–6.99 (2H, m), 7.20–7.23 (1H, m), 7.33–7.37 (1H, m), 7.78–7.80 (1H, m), 7.92–7.93 (1H, m), 7.99–8.00
(1H, m), 8.37 (1H, s, –OH), 10.19 (1H, s, –OH). MS (ESI) (m/z): 425 [M + H]+ .
Furan-2-yl-[5-(4-hydroxy-3,5-dimethoxyphenyl)-3-(2-hydroxyphenyl)-4,5-dihydro-pyrazol-1-yl]-methanone
(b16). Yield: 63.41%; M.p. 201.1–203.7 ◦ C. IR (KBr) νmax (cm−1 ): 3355, 3128 (aromatic –OH), 2940,
2837 (aliphatic C–H asymmetric), 1620 (C=O), 1600, 1519, 1458, 1426 (C=N and C=C), 1340, 1248,
1209, 1117 (C–N). 1 H-NMR (400 MHz, δ ppm, DMSO-d6 ): 3.29–3.34 (1H, m, pyrazoline C4 –HA ), 3.70
(6H, s, –OCH3 ), 3.88–3.97 (1H, m, pyrazoline C4 –HB ), 5.57–5.61 (1H, m, pyrazoline C5 –Hx ), 6.51 (2H, s),
671–6.72 (1H, m), 6.93–7.00 (2H, m), 7.35 (1H, t, J = 8.00 Hz), 7.51–7.52 (1H, m), 7.64 (1H, d, J = 8.00 Hz),
7.96 (1H, s), 8.36 (1H, s, –OH), 10.47 (1H, s, –OH). MS (ESI) (m/z): 409 [M + H]+ .
Benzo[b]thiophen-2-yl-[5-(4-hydroxy-3,5-dimethoxyphenyl)-3-(2-hydroxyphenyl)-4,5-dihydro-pyrazol-1-yl]methanone (b17). Yield: 66.67%; M.p. 169.2–170.1 ◦ C. IR (KBr) νmax (cm−1 ): 3392, 3262 (aromatic –OH),
2943, 2842 (aliphatic C–H asymmetric), 1690 (C=O), 1616, 1514, 1440, 1376 (C=N and C=C), 1338,
1237, 1113, 1049 (C–N). 1 H-NMR (400 MHz, δ ppm, DMSO-d6 ): 3.39–3.40 (1H, m, pyrazoline C4 –HA ),
3.70 (6H, s, –OCH3 ), 3.96–4.03 (1H, m, pyrazoline C4 –HB ), 5.63–5.67 (1H, m, pyrazoline C5 –Hx ), 6.53
(2H, s), 6.98–7.01 (2H, m), 7.35–7.39 (1H, m), 7.44–7.52 (2H, m), 7.85–7.87 (1H, m), 8.03–8.08 (2H, m),
8.37 (1H, s), 8.40 (1H, s, –OH), 10.23 (1H, s, –OH). 13 C-NMR (600 MHz, δ ppm, DMSO-d6 ): 40.41,
44.01, 56.02, 103.17, 116.72, 117.00, 119.64, 122.54, 124.85, 125.49, 126.63, 129.30, 130.80, 132.04, 132.07,
Molecules 2017, 22, 467
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135.01, 135.23, 137.92, 141.85, 148.11, 156.77, 156.86, 158.08. MS (ESI) (m/z): 475 [M + H]+ . HRMS:
m/z [M − H]¯ calcd for C26 H22 N2 O5 S: 473.1177; found: 474.1249.
4-[5-(2-Hydroxyphenyl)-2-phenyl-3,4-dihydro-2H-pyrazol-3-yl]-2,6-dimethoxy-phenol (b18). Yield: 48.46%;
M.p. 169.7–170.8 ◦ C. IR (KBr) νmax (cm−1 ): 3400 (aromatic –OH), 2939, 2842 (aliphatic C–H asymmetric),
1493, 1464, 1431 (C=N and C=C), 1373, 1245, 1113, 1026 (C–N). 1 H-NMR (400 MHz, δ ppm, DMSO-d6 ):
3.25–3.30 (1H, m, pyrazoline C4 –HA ), 3.70 (6H, s, –OCH3 ), 4.00–4.07 (1H, m, pyrazoline C4 -HB ),
5.25–5.29 (1H, m, pyrazoline C5 –Hx ), 6.63 (2H, s), 6.81 (1H, t, J = 8.00 Hz), 6.92–7.01 (4H, m), 7.23 (2H, t,
J = 8.00 Hz), 7.30 (1H, t, J = 8.00 Hz), 7.44 (1H, d, J = 8.00 Hz), 8.39 (1H, s, –OH), 10.62 (1H, s, –OH). MS
(ESI) (m/z): 391 [M + H]+ .
1-Phenyl-3-p-tolyl-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazole (b19). Yield: 50.39%; M.p. 137.9–140.3 ◦ C.
IR (KBr) νmax (cm−1 ): 2934, 2834 (aliphatic C–H asymmetric), 1495, 1462, 1418 (C=N and C=C), 1348,
1319, 1235, 1125 (C–N). 1 H-NMR (400 MHz, δ ppm, DMSO-d6 ): 2.34 (3H, s, –CH3 ), 3.09–3.15 (1H, m,
pyrazoline C4 –HA ), 3.63 (3H, s, –OCH3 ), 3.70 (6H, s, –OCH3 ), 3.83–3.91 (1H, m, pyrazoline C4 –HB ),
5.29–5.33 (1H, m, pyrazoline C5 -Hx ), 6.62 (2H, s), 6.74 (1H, t, J = 8.00 Hz), 7.04 (2H, d, J = 8.00 Hz), 7.18
(2H, t, J = 8.00 Hz), 7.25 (2H, d, J = 8.00 Hz), 7.65 (2H, d, J = 8.00 Hz). MS (ESI) (m/z): 403 [M + H]+ .
b1–19 1 H-NMR, b17 13 H-NMR and b17 HRMS are as provided as Supplementary Materials.
3.4. Pharmacology
3.4.1. Cell Culture and Treatment
Primary hepatocytes were isolated from male SD rats weighing 250–300 g using a modified in
situ collagenase perfusion method and then the cells were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM) with 2 g/L bovine serum albumin (BSA) and 50 mg/L gentamicin. HepG-2 cells
were incubated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin–streptomycin liquid. Cells were incubated at 37 ◦ C in a humidified atmosphere of 95%
air and 5% CO2 . Stock solutions of compounds and cisplatin were prepared in dimethyl sulfoxide
(DMSO), then added to fresh culture medium to obtain final concentrations.
3.4.2. MTT Assay
To evaluate the compounds’ cytotoxicity, 50 µM and 100 µM concentrations of each compound
were used to target the more effective compounds for further study. Next, compounds b14 and b18
were prepared in five different concentrations (20 µM, 40 µM, 60 µM, 80 µM, 100 µM), and compounds
b15, b16, and b17, along with cisplatin, were prepared in seven different concentrations (2.5 µM, 5 µM,
7.5 µM, 10 µM, 20 µM, 40 µM, 60 µM). A suspension of growing cells (50,000/mL) was prepared,
and 100 µL/well dispensed into 96-well plates, yielding a density of 5000 cells/well. The cells were
incubated for 24 h before adding the pyrazoline derivatives. The optimal cell number for cytotoxicity
assays was determined in preliminary experiments. At the end of the incubation period, the medium
was removed and 100 µL of different concentrations of pyrazoline derivatives were added to wells
for 24 h and 48 h. Following the exposure period, 20 µL of MTT (5 mg/mL) were added to cells
and incubated for a further 4 h at 37 ◦ C. The medium was removed and the formazan crystals were
solubilized by adding 150 µL DMSO to each well. After 10 min of shaking, the absorbance was
measured at 550 nm using a FilterMax F5 Multi-Mode Microplate Reader (Energy Chemical, Shanghai,
China). Cells were treated with each compound with four replicates per concentration, and each
experiment was conducted at least three times. Dose–inhibition rate curves and IC50 values, defined
as the drug concentrations which reduced absorbance to 50% of control values, were then generated.
3.4.3. MTS Assay
The cytotoxicological activity of compounds b5, b9, and b14–18 were evaluated against
primary hepatocytes using the MTS assay (Cell Titer 96® Aqueous Cell Proliferation Assay,
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Promega, Cat. No. G5421) as a fast and sensitive quantification of cell proliferation and viability.
The concentrations of compounds were prepared in seven different concentrations (2.5 µM, 5 µM,
10 µM, 20 µM, 40 µM, 80 µM, 160 µM). Cells were seeded into 96-well plates at a density of
26,000 cells/well. The plates were incubated for 24 h prior to any treatment. At the end of this
period, the medium was removed and 100 µL of different concentrations of pyrazoline derivatives
were added to wells for 48 h. Following the exposure period, 20 µL of the combined MTS/PMS
solution was added to cells and incubated for a further 2 h at 37 ◦ C, then the absorbance was recorded
at 490 nm using an ELISA Plate Reader (ELISA, Emeryville, CA, USA). The values of IC50 , the effective
concentration at which 50% of the primary hepatocytes were inhibited, were calculated to evaluate the
cytotoxic activities.
3.4.4. Cell Cycle Analysis
Based on the results of the cytotoxicity assay, compound b17 was selected for further mechanistic
study with HepG-2 cells. For flow cytometry analysis of DNA content, approximately 1.5 × 105
HepG-2 cells/well in exponential growth mode were plated in 6-well plates and allowed to adhere,
then treated with different concentrations of compound b17 for 24 h. After incubation, the cells were
collected, centrifuged, and fixed with ice-cold ethanol (70%). The cells were treated with RNase A and
stained with PI. Samples were analyzed on a BD Accuri C6 flow cytometer (BD, New York, NY, USA).
DNA histograms were analyzed using ModFit for Windows.
3.4.5. Annexin-V Assay
Approximately 1.5 × 105 HepG-2 cells/well were plated in 6-well plates and allowed to adhere.
After 24 h, the medium was replaced with fresh culture medium containing compound b17 at final
concentrations of 0 µM, 0.9 µM, 2.7 µM, and 4.5 µM. Cells were harvested after 12 h. The cells were
trypsinized, washed in phosphate-buffered saline (PBS), and centrifuged at 1000 rpm for 5 min.
The cells were then resuspended in 200 µL of staining solution (containing 10 µL PI and 5 µL Annexin
V–PE in binding buffer), mixed gently, and incubated for 15 min at 25 ◦ C in the dark. The samples
were later analyzed with a BD Accuri™ C6 flow cytometer.
4. Conclusions
In the present study, we synthesized 19 new pyrazoline derivatives and investigated their
antiproliferative effects on HepG-2 cells. We found that compound b17 was the most effective
anticancer agent following a 48 h exposure with an IC50 value of 3.57 µM compared with the cisplatin
value of 8.45 µM. Compound b17 was therefore selected for cell cycle analysis and apoptosis/necrosis
evaluation. We observed that HepG-2 cells treated with compound b17 could be arrested in the
G2 /M phase. In addition, compound b17 can be regarded as a potent inducer of apoptosis in the
cells. These results provide an important foundation for further development of compound b17 as
a potent antitumor agent. We also found that compounds with a heterocyclic ring, such as b15 and
b16, exhibited better pharmacological activity than most other pyrazoline derivatives synthesized
in this study. Compounds b15 and b16 will be tested further to improve characterization of their
antitumor activity.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: This study was funded by the Guangdong Natural Science Foundation of China
(No. 9351503102000001).
Author Contributions: Weijie Xu, Ying Pan, and Hong Wang designed the research. Weijie Xu performed the
synthesis work. Jinhong Zheng and Ying Pan were responsible for the direction of the biological research.
Weijie Xu, Duncan Wei, Haiyan Li, Qing Peng and Cheng Chen performed the anticancer activity assays. Weijie Xu
wrote the manuscript. All authors discussed, edited, and approved the final version.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of compounds b1–19 are available from the authors.
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