Synthesis and Biological Activity of 2-Arylidene- N -(quinolin-6-yl)hydrazine-1-carboxamides

Article abstract of DOI:10.1155/2020/2189743

A series of 2-arylidene-N-(quinolin-6-yl)hydrazine-1-carboxamides 5a-5o were synthesized and characterized. The synthesized compounds (5a-5o) were screened in vitro against three breast cancer cell lines: SKBR3, MDA-MB-231, and MCF-7 cancer cell lines by the MTT assay. According to MTT results, compounds 5k and 5l showed better antiproliferative activities over MCF-7 cell lines with IC50 values of 8.50 and 12.51 μM. Colony formation assay indicated 5k/5l treatment obviously inhibited the growth of MCF-7 cells and 5k/5l-induced cell cycle was arrested in the G2-M phase. Moreover, 5k/5l significantly increased the level of cleaved PARP and induced the apoptosis in MCF-7 cells. In addition, compared to Hela cells, MCF-7 cells were more sensitive to 5k/5l treatment.

Full text of DOI:10.1155/2020/2189743

Hindawi
Journal of Chemistry
Volume 2020, Article ID 2189743, 9 pages
https://doi.org/10.1155/2020/2189743
Research Article
Synthesis and Biological Activity of 2-Arylidene-N-(quinolin-6-yl)
hydrazine-1-carboxamides
1
2
2
1
1
Shengxian Zhao, Yin Cao, Zhenzhen Cui, Jiayun Zhang, Zhixiang Pan,
and Hongyu Hu
1,3
1
College of Science and Technology, Ningbo University, Cixi 315302, China
School of Pharmaceutical Sciences and the Key Laboratory for Chemical Biology of Fujian Province, Xiamen University,
South Xiang-An Road, Xiamen 361102, China
2
3
Xingzhi College, Zhejiang Normal University, Lanxi 321100, China
Correspondence should be addressed to Hongyu Hu; huhongyu22@126.com
Received 27 May 2020; Revised 27 July 2020; Accepted 3 August 2020; Published 20 August 2020
Academic Editor: Ales Imramovsky
Copyright © 2020 Shengxian Zhao et al. +is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
A series of 2-arylidene-N-(quinolin-6-yl)hydrazine-1-carboxamides 5a–5o were synthesized and characterized. +e synthesized
compounds (5a–5o) were screened in vitro against three breast cancer cell lines: SKBR3, MDA-MB-231, and MCF-7 cancer cell
lines by the MTTassay. According to MTTresults, compounds 5k and 5l showed better antiproliferative activities over MCF-7 cell
lines with IC50 values of 8.50 and 12.51 μM. Colony formation assay indicated 5k/5l treatment obviously inhibited the growth of
MCF-7 cells and 5k/5l-induced cell cycle was arrested in the G2-M phase. Moreover, 5k/5l significantly increased the level of
cleaved PARP and induced the apoptosis in MCF-7 cells. In addition, compared to Hela cells, MCF-7 cells were more sensitive to
5
k/5l treatment.
pharmacologically important structural scaffold displaying a
wide range of biological activities [6–10]. Neratinib (Fig-
ure 1) is a quinoline derivative which was approved by FDA
for the treatment of HER-2 breast cancer in patients [11].
+e presence of urea functionality plays an important role in
a drug’s aqueous solubility and permeability due to its dual
nature as a hydrogen bond donor and acceptor; it also
stabilizes the binding of compounds in the pocket [12].
Sorafenib, which is a urea derivative, has been used in the
treatment of various cancers. Although it has shown limited
efficacy in breast cancer as a single agent, sorafenib com-
bined with other drugs in the treatment of breast cancer
could be worth looking forward to [13]. +e aromatic urea-
quinazolines [14] which integrate the structural features of
gefitinib and sorafenib and the thiourea derivatives [15] have
already showed significant antibreast cancer activities.
Recently, we investigated various urea/thiourea deriva-
tives as potential antitumor agents [16–20]. It was found in
our previous studies that indole-substituted urea derivatives
1
. Introduction
Breast cancer is one of the most common malignant tumors
threatening the health of females all over the world, causing
2
3% of the total cancer cases and 14% of the cancer deaths
[
1]. It has become the fastest growing cancer in China, with
an average annual growth rate of more than 3% [2]. Breast
cancer is a heterogeneous disease that shows remarkably
different biological characteristics and clinical behaviors, it
can be classified into five main subtypes: luminal, basal-like,
human epidermal growth factor receptor-2 (HER-2), HER-2
expression, and normal breast-like subtypes [3]. Triple-
negative breast cancer (TNBC) patients have the worst
prognosis and distant metastasis-free survival among all
major subtypes of breast cancer [4]. +e standard treatment
options for breast cancer include surgery, radiation, and/or
anticancer drugs. Despite a number of active agents available
today, it is still a huge clinical demand to discover more
effective agents [5]. Quinoline ring system is the

2
Journal of Chemistry
EtO
N
O
N
N
H
CN
Cl
HN
O
N
Figure 1: Structure of neratinib.
showed good antitumor activity [20]. A new class of urea
derivatives 5a–5o (Figure 2) was designed by replacing the
parent nucleus with a quinoline ring instead of a indole ring.
Target compounds 5a–5o were synthesized and evaluated
for their anticancer activity on three breast cancer cell lines:
SKBR3, MDA-MB-231, and MCF-7 by MTT assay. +e
compounds 5k and 5l exhibited remarkable activities with
IC values of 8.50 and 12.51 μM over MCF-7 cell lines. On
column (ethyl acetate : petroleum ether � 1 : 4) to afford the
product
3
(226.3 mg, 85.6%) as off-yellow solid.
1
°
164.6–167.6 C. H NMR (600 MHz, DMSO-d ) δ: 10.64 (brs,
6
1H), 8.79 (dd, J � 1.3, 4.0 Hz, 1H), 8.27 (d, J � 7.7 Hz, 1H),
8.18 (brs, 1H), 8.02 (d, J � 9.0 Hz, 1H), 7.85 (dd, J � 2.3,
9.0 Hz, 1H), 7.50-7.42 (m, 3H), 7.33-7.24 (m, 3H); 1 C NMR
3
(150 MHz, DMSO-d ) δ: 152.3, 150.9, 149.5, 145.1, 137.1,
6
135.8, 130.2, 129.9, 129.3, 128.8, 127.5, 126.0, 122.4, 122.3;
5
0
ꢀ
the basis of the results, further structural optimization and
research will be carried out.
HRMS, m/z calcd. for C H N O Na [MꢀNa] : 287.0791;
16
12 2 2
found: 287.0801.
2
. Experimental
2
.3. Synthesis of N-(Quinolin-6-yl)hydrazine Carboxamide 4.
A mixture of 3 (264.3 mg, 1 mmol) and hydrazine hydrate
solution (80%, 76.9 mmol, 3 mL) in ethanol (95%, 10 mL)
was heated to reflux. +e progress of the reaction was
monitored by TLC. After completion of the reaction, the
reaction mixture was cooled down to room temperature, and
a white solid was precipitated out. +e reaction mixture was
filtered and dried, affording the product 4 (187.6 mg, 92.8%)
All chemicals were analytically pure and purchased from
commercial sources. All melting points were determined on a
WRS-1B digital melting point apparatus (uncorrected). All new
1
13
compounds were characterized by H NMR, C NMR, and
HRMS. NMR spectra were recorded in CDCl or DMSO-d on
3
6
a Bruker AV 600 MHz instrument. HRMS spectra were ob-
tained on an Agilent 6230 mass spectrometer. Human breast
cell lines SKBR3, MDA-MB-231, and MCF7 were purchased
from Shanghai Cell Bank, Chinese Academy of Sciences.
1
°
as white solid. m.p. 200.5–200.8 C. H NMR (600 MHz,
DMSO-d ) δ: 9.04 (brs, 1H), 8.74 (dd, J � 1.7, 4.2 Hz, 1H),
6
8
.33-8.13 (m, 2H), 7.95-7.91 (m, 1H), 7.91-7.87 (m, 1H), 7.67
13
(
brs, 1H), 7.44 (dd, J � 4.2, 8.3 Hz, 1H), 4.50 (brs, 2H);
C
2
.1. Synthesis of 6-Aminoquinoline 2 [21]. +e solution of 6-
NMR (150 MHz, DMSO-d ) δ: 157.9, 148.6, 144.5, 138.5,
6
nitroquinoline (174.2 mg, 1 mmol) in methanol (10 mL) was
treated with Pd/C (10%, 14.0 mg). +e reaction mixture was
vigorously stirred under an atmosphere of hydrogen at room
temperature. +e progress of the reaction was monitored by
TLC. After completion of the reaction, the reaction mixture
was then filtered and concentrated in vacuo to give the crude
product. +e crude product was purified by column (ethyl
acetate : petroleum ether � 1 : 4) to afford the product 2
135.4, 129.7, 129.0, 123.8, 122.0, 113.2; HRMS, m/z calcd. for
ꢀ
C H N O [MꢀH] : 203.0927, found: 203.0930.
10
11 4
General Procedure for the Synthesis of 2-Arylidene-N-
(quinolin-6-yl)hydrazine-1-carboxamides 5a–5o. A mixture of
4 (202.2 mg, 1 mmol) and aldehyde (1 mmol) in absolute al-
cohol (15 mL) was heated to reflux. +e progress of the reaction
was monitored by TLC. After completion of the reaction, the
reaction mixture was cooled down to room temperature, and a
white solid was precipitated out. +e reaction mixture was
filtered and dried, affording the products 5a–5o.
1
°
(
131.1 mg, 91.0%) as white solid. m.p. 116.8–119.0 C. H
NMR (600 MHz, CDCl ) δ: 8.64 (d, J � 4.0 Hz, 1H), 7.89 (t,
3
J � 9.7 Hz, 2H), 7.25 (t, J � 4.8 Hz, 1H), 7.14 (dd, J � 9.0 Hz,
2
.6 Hz, 1H), 6.88 (d, J � 2.5 Hz, 1H), 3.84 (brs, 2H).
2
.3.1. 2-Benzylidene-N-(quinolin-6-yl)hydrazine-1-carboxamide
(
5a). Product 5a was obtained as white solid, yield 87.9%.
1
°
2
.2. SynthesisofPhenylQuinolin-6-ylcarbamate3. A mixture
m.p. 217.22–17.5 C. H NMR (600 MHz, DMSO-d ) δ: 10.97
6
of 2 (144.2 mg, 1 mmol) and triethylamine (203.8 mg,
(s, 1H), 9.29 (s, 1H), 8.79 (dd, J � 1.7, 4.22 Hz, 1H), 8.36 (d,
J � 2.4 Hz, 1H), 8.29 (dd, J � 1.0, 8.7 Hz, 1H), 8.11 (dd, J � 2.4,
9.0 Hz, 1H), 8.04 (s, 1H), 8.00 (d, J � 9.0 Hz, 1H), 7.94-7.89
2
mmol) in dichloromethane (6 mL) was degassed and
flushed with nitrogen. +e reaction mixture was cooled
1
3
°
down to 5 C, a dichloromethane (6 mL) solution of phenyl
(m, 2H), 7.50-7.45 (m, 3H), 7.44-7.41 (m, 1H); C NMR
chloroformate (156.6 mg, 1 mmol) was added dropwise, and
the reaction was stirred overnight. +e reaction mixture was
washed by water (20 mL) twice, dried over sodium sulfate,
filtered, and evaporated. +e crude product was purified by
(150 MHz, DMSO-d ) δ: 153.7, 149.1, 145.0, 141.7, 137.7,
6
135.7, 134.8, 130.0, 129.5, 129.1, 128.8, 127.6, 124.9, 122.1,
ꢀ
115.4; HRMS, m/z calcd. for C H N ONa [MꢀNa] :
17
14 4
313.1060; found: 313.1062.

Journal of Chemistry
3
N
δ: 159.5, 159.2, 156.8, 153.3, 145.2, 142.1, 139.7, 138.1, 131.3,
1
29.6, 127.7, 124.5, 122.4, 120.6, 119.6, 116.5, 114.9; HRMS,
O
ꢀ
m/z calcd. for C H N O [MꢀH] : 307.1190, found:
17
15 4 2
307.1193.
N
R
N
H
N
H
5
a–5o
2
.3.6. 2-(4-Hydroxybenzylidene)-N-(quinolin-6-yl)hydra-
Figure 2: Structures of target compounds 5a–5o.
zine-1-carboxamide (5f). Product 5f was obtained as white
1
°
solid, yield 85.7%. m.p. 266.9–269.7 C. H NMR (600 MHz,
DMSO-d ) δ: 10.76 (s, 1H), 9.91 (s, 1H), 9.19 (s, 1H), 8.77
6
(
dd, J � 1.7, 4.0 Hz, 1H), 8.35 (d, J � 2.2 Hz, 1H), 8.26 (d,
J � 7.7 Hz, 1H), 8.09 (dd, J � 2.4, 9.0 Hz, 1H), 7.98 (d,
J � 9.0 Hz, 1H), 7.93 (s, 1H), 7.73 (d, J � 8.6 Hz, 2H), 7.46 (dd,
2
.3.2. 2-(2-Methylbenzylidene)-N-(quinolin-6-yl)hydrazine-1-
carboxamide (5b). Product 5b was obtained as white solid,
13
1
°
J � 4.2, 8.25 Hz, 1H), 6.86 (d, J � 8.6 Hz, 2H); C NMR
yield 82.6%. m.p. 215.1–216.1 C. H NMR (600 MHz,
(
150 MHz, DMSO-d ) δ: 159.4, 153.7, 149.0, 144.8, 142.0,
DMSO-d ) δ: 10.91 (s, 1H), 9.24 (s, 1H), 8.78 (d, J � 3.1 Hz,
6
6
1
37.8, 135.7, 129.5, 129.3, 128.8, 125.9, 124.8, 122.1, 116.0,
1
1
H), 8.35 (d, J � 1.5 Hz, 1H), 8.33 (s, 1H), 8.27 (d, J � 8.3 Hz,
ꢀ
1
15.1; HRMS, m/z calcd. for C H N O [MꢀH] : 307.1190,
H), 8.22-8.18 (m, 1H), 8.08 (dd, J � 1.9,9.0 Hz, 1H), 7.98 (d,
17 15
4 2
found: 307.1187.
J � 9.0 Hz, 1H), 7.47 (dd, J � 4.1, 8.1 Hz, 1H), 7.31-7.25 (m,
1
3
2
H), 7.25-7.21 (m, 1H), 2.42 (s, 3H); C NMR (150 MHz,
DMSO-d ) δ:153.6, 149.1, 144.9, 140.2, 137.7, 136.7, 135.7,
6
2
.3.7. 2-(4-(Dimethylamino)benzylidene)-N-(quinolin-6-
1
1
3
32.7, 131.1, 129.7, 129.5, 128.8, 126.5, 126.5, 124.8, 122.1,
ꢀ
yl)hydrazine-1-carboxamide (5g). Product 5g was ob-
15.3, 19.4; HRMS, m/z calcd. for C H N ONa [MꢀNa] :
1
8
16 4
1
°
tained as white solid, yield 83.4%. m.p. 195.1–196.9 C. H
NMR (600 MHz, DMSO-d ) δ: 10.65 (s, 1H), 9.15 (s, 1H),
27.1216, found: 327.1215.
6
8
.76 (dd, J � 1.6, 4.13 Hz, 1H), 8.34 (d, J � 2.0 Hz, 1H), 8.26
2
.3.3. 2-(3-Methylbenzylidene)-N-(quinolin-6-yl)hydrazine-1-
(d, J � 8.1 Hz, 1H), 8.08 (dd, J � 2.2, 9.0 Hz, 1H), 7.96 (d,
carboxamide (5c). Product 5c was obtained as white solid,
J � 9.2 Hz, 1H), 7.88 (s, 1H), 7.69 (d, J � 8.8 Hz, 2H), 7.46 (dd,
1
13
°
yield 84.0%. m.p. 202.0–204.0 C. H NMR (600 MHz,
J � 4.0, 8.2 Hz, 1H), 6.74 (d, J � 8.8 Hz, 2H), 2.97 (s, 6H);
C
DMSO-d ) δ: 10.96 (s, 1H), 9.29 (s, 1H), 8.87-8.69 (m, 1H),
NMR (150 MHz, DMSO-d ) δ: 153.7, 151.6, 149.0, 144.8,
6
6
8
8
7
.36 (brs, 1H), 8.32-8.25 (m, 1H), 8.10 (d, J � 8.1 Hz, 1H),
.02-7.92 (m, 2H), 7.72–7.64 (m, 2H), 7.53-7.45 (m, 1H),
.32 (t, J � 7.4 Hz, 1H), 7.21 (d, J � 7.2 Hz, 1H), 2.36 (brs, 3H);
142.6, 137.9, 135.6, 132.0, 129.5, 128.9, 124.8, 122.3, 122.1,
114.9, 112.1, 40.3; HRMS, m/z calcd. for C H N ONa
19
19 5
ꢀ
[MꢀNa] : 356.1482, found: 356.1494.
1
3
C NMR (150 MHz, DMSO-d ) δ: 153.6, 148.9, 144.5, 141.9,
6
1
1
38.3, 137.8, 136.1, 134.7, 130.7, 129.2, 129.0, 128.8, 127.9,
25.1, 124.9, 122.1, 115.4, 21.4; HRMS, m/z calcd. for
2.3.8. 2-(4-Fluorobenzylidene)-N-(quinolin-6-yl)hydrazine-1-
ꢀ
C H N O [MꢀH] : 305.1397, found: 305.1398.
carboxamide (5h). Product 5h was obtained as white solid,
1
8
17 4
1
°
yield 80.5%. m.p. 207.0–208.5 C. H NMR (600 MHz,
DMSO-d ) δ: 10.96 (s, 1H), 9.29 (s, 1H), 8.77 (d, J � 2.6 Hz,
6
2
.3.4. 2-(4-Methylbenzylidene)-N-(quinolin-6-yl)hydrazine-1-
1
H), 8.33 (d, J � 1.8 Hz, 1H), 8.27 (d, J � 8.1 Hz, 1H), 8.08 (dd,
carboxamide (5d). Product 5d was obtained as white solid,
J � 2.0, 9.0 Hz, 1H), 8.00 (s, 1H), 7.99-7.95 (m, 3H), 7.47 (dd,
1
°
yield 74.9%. m.p. 207.5–208.7 C. H NMR (600 MHz,
13
J � 4.1, 8.2 Hz, 1H), 7.29 (t, J � 8.7 Hz, 2H); C NMR
DMSO-d ) δ: 10.89 (s, 1H), 9.24 (s, 1H), 8.77 (dd, J � 1.7,
6
(
150 MHz, DMSO-d ) δ: 164.1, 162.5, 153.7, 147.0 (d,
6
4
1
(
.2 Hz, 1H), 8.34 (d, J � 2.4 Hz, 1H), 8.27 (d, J � 8.3 Hz,
J � 321.8 Hz), 140.5, 137.7, 135.7, 131.4 (d, J � 1.5 Hz), 129.7
H), 8.09 (dd, J � 2.3, 9.1 Hz, 1H), 8.00-7.96 (m, 2H), 7.78
(
d, J � 3.8 Hz), 129.5, 128.8, 124.9, 122.1, 116.1 (d,
d, J � 8.1 Hz, 2H), 7.47 (dd, J � 4.0, 8.3 Hz, 1H), 7.25 (d,
J � 10.5 Hz), 115.0; HRMS, m/z calcd. for C H FN O
13
17 14
4
J � 8.1 Hz, 2H), 2.34 (s, 3H); C NMR (150 MHz, DMSO-
ꢀ
[
MꢀH] : 309.1146; found: 309.1134.
d ) δ:153.7, 149.1, 144.9, 141.8, 139.7, 137.7, 135.7, 132.1,
6
1
29.7, 129.5, 128.8, 127.6, 124.9, 122.1, 115.4, 21.5; HRMS,
ꢀ
m/z calcd. for C H N O [MꢀH] : 305.1397, found:
1
8
17
4
2
.3.9. 2-(4-Methoxybenzylidene)-N-(quinolin-6-yl)hydrazine-1-
3
05.1401.
carboxamide (5i). Product 5i was obtained as white solid,
1
°
yield 79.0%. m.p. 188.0–189.1 C. H NMR (600 MHz,
DMSO-d ) δ: 10.81 (s, 1H), 9.22 (s, 1H), 8.77 (dd, J � 1.3,
2
.3.5. 2-(2-Hydroxybenzylidene)-N-(quinolin-6-yl)hydrazine-1-
6
3
.8 Hz, 1H), 8.33 (d, J � 1.8 Hz, 1H), 8.27 (d, J � 7.9 Hz, 1H),
carboxamide (5e). Product 5e was obtained as white solid,
1
°
8.08 (dd, J � 1.9, 9.0 Hz, 1H), 7.99-7.94 (m, 2H), 7.84 (d,
yield 80.9%. m.p. 210.5–211.6 C. H NMR (600 MHz,
J � 8.6 Hz, 2H), 7.47 (dd, J � 4.2, 8.2 Hz, 1H), 7.01 (d,
DMSO-d ) δ: 11.09 (brs, 1H), 9.62 (brs, 1H), 9.02 (d,
6
13
J � 8.6 Hz, 2H), 3.81 (s, 3H); C NMR (150 MHz, DMSO-d )
J � 4.8 Hz, 1H), 8.83 (d, J � 8.4 Hz, 1H), 8.58 (d, J � 2.0 Hz,
6
δ: 160.9, 153.7, 149.0, 144.9, 141.6, 137.8, 135.7, 129.5, 129.2,
1
1
(
H), 8.34 (s, 1H), 8.27 (d, J � 8.4 Hz, 1H), 8.15 (d, J � 9.2 Hz,
H), 7.99 (brs, 1H), 7.83 (dd, J � 5.0, 8.4 Hz, 1H), 7.32-7.18
1
28.8, 127.4, 124.8, 122.1, 115.2, 114.6, 55.7; HRMS, m/z
ꢀ
m, 1H), 6.96-6.83 (m, 2H); 13C NMR (150 MHz, DMSO-d )
calcd. for C H N O [MꢀH] : 321.1346, found 321.1349.
18 17 4 2
6

4
Journal of Chemistry
1
°
2
.3.10. 2-(3-Bromobenzylidene)-N-(quinolin-6-yl)hydra-
white solid, yield 79.4%. m.p. 205.4–206.0 C. H NMR
zine-1-carboxamide (5j). Product 5j was obtained as white
(600 MHz, DMSO-d ) δ: 10.93 (s, 1H), 9.10 (s, 1H), 8.77 (d,
6
1
°
solid, yield 81.2%. m.p. 237.5–239.9 C. H NMR (600 MHz,
J � 2.8 Hz, 1H), 8.31 (d, J � 2.0 Hz, 1H), 8.26 (d, J � 8.1 Hz,
1H), 8.00–8.06 (m, 1H), 7.92–7.99 (m, 2H), 7.83 (s, 1H), 7.46
(dd, J � 4.0, 8.3 Hz, 1H), 7.00 (d, J � 3.3 Hz, 1H), 6.64 (dd,
DMSO-d ) δ: 11.21 (s, 1H), 9.34 (s, 1H), 8.78 (dd, J � 1.6,
6
4
.1 Hz, 1H), 8.42 (dd, J � 1.5, 7.9 Hz, 1H), 8.38 (s, 1H), 8.33
13
(
d, J � 2.4 Hz, 1H), 8.28 (d, J � 7.9 Hz, 1H), 8.08 (dd, J � 2.3,
J � 1.7, 3.2 Hz, 1H); C NMR (150 MHz, DMSO-d ) δ:153.4,
6
9
1
.1 Hz, 1H), 7.98 (d, J � 9.0 Hz, 1H), 7.67 (dd, J � 0.9, 8.1 Hz,
150.1, 149.1, 145.0, 144.9, 137.6, 135.7, 132.0, 129.6, 128.8,
13
H), 7.51-7.44 (m, 2H), 7.34 (dt, J � 1.7, 7.6 Hz, 1H);
C
124.6, 122.1, 115.1, 112.6, 112.5; HRMS, m/z calcd. for
ꢀ
NMR (150 MHz, DMSO-d ) δ: 153.5, 149.2, 145.0, 139.9,
C H N O Na [MꢀNa] : 303.0852; found: 303.0861.
6
15 12 4 2
1
1
37.5, 135.7, 133.5, 133.4, 131.6, 129.5, 128.8, 128.4, 128.3,
25.0, 123.5, 122.1, 115.7; HRMS, m/z calcd. for
ꢀ
C H BrN O [MꢀH] : 369.0346, found: 369.0350.
1
7
14
4
2
.3.15. 2-(@iophen-2-ylmethylene)-N-(quinolin-6-yl)hy-
drazine-1-carboxamide (5o). Product 5o was obtained as
1
°
white solid, yield 77.9%. m.p. 214.7–217.3 C. H NMR
2
.3.11. 2-(3,4-Dimethoxybenzylidene)-N-(quinolin-6-yl)
(
600 MHz, DMSO-d ) δ: 11.04 (s, 1H), 9.32 (brs.,1H), 9.03-
6
hydrazine-1-carboxamide (5k). Product 5k was obtained
1
8.85 (m, 1H), 8.65 (d, J � 8.3 Hz, 1H), 8.46 (d, J � 1.8 Hz, 1H),
°
as white solid, yield 85.4%. m.p. 191.1–193.1 C. H NMR
8
1
7
.23 (s, 1H), 8.15 (dd, J � 2.0, 9.0 Hz, 1H), 8.08 (d, J � 9.0 Hz,
(
600 MHz, DMSO-d ) δ: 10.92 (s, 1H), 9.38 (brs, 1H), 8.83
6
H), 7.72 (dd, J � 4.6, 8.3 Hz, 1H), 7.66 (d, J � 5.0 Hz, 1H),
(
d, J � 2.9 Hz, 1H), 8.44 (brs, 1H), 8.40 (d, J � 7.9 Hz, 1H),
1
3
.47 (d, J � 3.3 Hz, 1H), 7.13 (dd, J � 3.8, 4.9 Hz, 1H);
C
8
.12 (d, J � 8.4 Hz, 1H), 8.03 (d, J � 9.0 Hz, 1H), 7.97 (s, 1H),
.60 (brs, 1H), 7.54 (dd, J � 4.0, 7.9 Hz, 1H), 7.27 (d,
NMR (150 MHz, DMSO-d ) δ: 159.5, 153.2, 146.4, 140.1,
6
7
1
39.3, 139.0, 137.5, 130.3, 129.3, 128.7, 128.2, 126.7, 126.1,
J � 7.9 Hz, 1H), 6.99 (d, J � 8.1 Hz, 1H), 3.89 (s, 3H), 3.80 (s,
ꢀ
1
3
122.3, 114.9; HRMS, m/z calcd. for C H N OS [MꢀH] :
15
13 4
3
1
1
H); C NMR (150 MHz, DMSO-d ) δ: 153.7, 150.8, 149.5,
6
297.0805, found: 297.0811.
48.1, 143.3, 142.1, 138.2, 137.3, 129.0, 128.3, 127.6, 125.6,
22.1, 121.9, 115.1, 111.8, 109.6, 56.1, 56.0; HRMS, m/z calcd.
ꢀ
for C H N O Na [MꢀNa] : 373.1271; found: 373.1278.
1
9
18 4 3
2
.4. Antitumor Activities. +e in vitro antiproliferation
activity of target compounds 5a–5o against three human
breast cancer lines including SKBR3, MDA-MB-231, and
MCF-7 cancer cell lines was measured by the MTT col-
orimetric method. In all experiments, cells in good
condition and logarithmic growth phase were taken and
digested by 0.25% trypsin. +e adherent cells were de-
2
.3.12. 2-(2,4-Dimethoxybenzylidene)-N-(quinolin-6-yl)
hydrazine-1-carboxamide (5l). Product 5l was obtained as
1
°
white solid, yield 90.1%. m.p. 195.–195.7 C. H NMR
(
600 MHz, DMSO-d ) δ: 10.88 (s, 1H), 9.37 (brs, 1H), 8.92
6
(
d, J � 3.7 Hz, 1H), 8.59 (d, J � 8.3 Hz, 1H), 8.49 (d, J � 2.0 Hz,
1
1
6
H), 8.28 (s, 1H), 8.23 (dd, J � 1.8, 9.0 Hz, 1H), 8.18-8.13 (m,
∗
4
tached and counted, a 2–4 10 cells/mL suspension was
prepared and inoculated into a 96-well cell culture plate
with 180 μL a well, placed in a constant-temperature
carbon dioxide incubator and cultured for 24 h, then was
replaced with the dimethyl sulfoxide (DMSO) solution of
target compound (20 μL) a well, and added 10 vol% serum
culture medium (80 μL), and cultured for 48 h. MTT
H), 8.07 (d, J � 9.0 Hz, 1H), 7.68 (dd, J � 4.6, 8.3 Hz, 1H),
13
.61 (dd, J � 2.2, 4.2 Hz, 2H), 3.85 (s, 3H), 3.82 (s, 3H);
C
NMR (150 MHz, DMSO-d ) δ: 162.5, 159.2, 153.6, 146.6,
6
1
1
40.6, 139.7, 139.0, 137.6, 129.3, 127.8, 126.7, 126.3, 122.2,
15.5, 115.0, 106.7, 98.3, 56.1, 55.8; HRMS, m/z calcd. for
ꢀ
C H N O Na [MꢀNa] : 373.1271; found: 373.1278.
1
9
18 4 3
(
20 μL) was added to each well and reacted for 4 h. +e
supernatant was removed and DMSO (150 μL per well)
was added to dissolve MTT. +e plate was shaken on a
platform rocker for five minutes, and the optical density
2
.3.13. 2-(Pyridin-2-ylmethylene)-N-(quinolin-6-yl)hydrazine-1-
carboxamide (5m). Product 5m was obtained as white solid,
1
°
yield 78.0%. m.p. 212.5–214.3 C. H NMR (600 MHz,
(
OD) of each well was measured on an enzyme-linked
immunosorbent assay (ELISA) meter at the wavelength of
70 nm. +e inhibition activity of cell proliferation was
DMSO-d ) δ: 11.14 (s, 1H), 9.34 (s, 1H), 9.05 (d, J � 1.7 Hz,
6
1
1
8
1
H), 8.78 (dd, J � 1.7, 4.2 Hz, 1H), 8.59 (dd, J � 1.6, 4.7 Hz,
H), 8.36 (td, J � 1.7, 8.0 Hz, 1H), 8.33 (d, J � 2.2 Hz, 1H),
.28 (d, J � 8.3 Hz, 1H), 8.08 (dd, J � 2.3, 9.0 Hz, 1H), 8.03 (s,
5
determined. Dissolvent control group was used as the
negative control group, and target compounds 5a–5o
were used as the positive control group. +e inhibition
rate of target compounds on tumor cell growth was
calculated according to the following formula: inhibition
rate% � (OD value of the negative control−OD value of
tested compound)/OD value of the negative con-
trol×100%. +e IC50 value of target compounds was
calculated based on the inhibition rates of different
concentrations.
13
H), 7.98 (d, J � 9.0 Hz, 1H), 7.52-7.42 (m, 2H); C NMR
(150 MHz, DMSO-d ) δ: 153.6, 150.5, 149.2, 149.1, 145.0,
6
1
1
2
38.7, 137.6, 135.7, 134.2, 130.7, 129.5, 128.7, 125.0, 124.2,
ꢀ
22.1, 115.7; HRMS, m/z calcd. for C H N O [MꢀH] :
16
14 5
92.1193; found: 292.1195.
2
.3.14. 2-(Furan-2-ylmethylene)-N-(quinolin-6-yl)hydra-
zine-1-carboxamide (5n). Product 5n was obtained as

Journal of Chemistry
5
N
N
a
N
b
O
NO2
NH2
d
N
H
O
R
1
2
3
N
N
O
O
c
NH2
N
N
H
4
N
N
H
N
H
H
5a–5o
Scheme 1: Synthesis of target compounds 5a–5o. (a) Pt/C, H
hydrate, 95% ethanol; (d) aldehydes, ethanol.
2
, MeOH; (b) phenyl chloroformate, triethylamine, DCM; (c) 80% hydrazine
2
.5. Colony Formation Assay. MCF-7 cells and Hela cells
positive control. +e concentration required for 50% inhi-
bition of cell viability (IC ) was calculated and is sum-
marized in Table 1. Among the synthesized compounds
(5a–5o), the cytotoxic activity order is as follows: com-
pounds 5h–5l bearing methoxy or halogen-substituted
were cultured in a 6-well plate (200 cells/well) and treated
with various compounds in 10% serum medium for 12 days
and then fixed with methanol and stained with 0.1% crystal
violet.
50
phenyl group ≈ compounds 5m–5o bearing pyridyl, furyl,
and thienyl group >compounds 5a–5g bearing non-
substituted or other substituted phenyl group. Compound
2
.6. Annexin V/PI Staining. Cells were cultured in a six-well
5
plate with about 1 × 10 cells in each well and treated with
5
k with the substitution of 3,4-dimethoxyphenyl and 5l with
1
0 μM 5k and 5l for 24 h. +en, the apoptosis analysis was
the substitution of 2,4-dimethoxyphenyl displayed better
antiproliferative activities against tested cancer cell lines
than other compounds, and both of them exhibited more
potential cytotoxicity on MCF-7 cell line than SKBr3 and
MDA-MB-231 cell lines with IC50 of 8.50 and 12.5 μM.
MCF-10A cells (normal breast cell line) were used to
evaluate the cytotoxicity of 5k and 5l in the normal cell line.
performed following the protocols (BD Bioscience). After
stained with FITC annexin V/PI, the apoptosis status was
analyzed using a Beckman Epics Altra Culter and data was
analyzed with EXP032 software. +e status and percentage
of cells undergoing apoptosis were defined as early apoptosis
(
(
annexin V-positive and PI-negative) and late apoptosis
annexin V-positive and PI-positive).
+
e results showed that 5k and 5l had no antiproliferative
activity on MCF-10A cells with an IC50 >100 μmol/L, in-
dicating the good selectivity of both compounds.
2
.7. Western Blot Analysis. To check the level of cleaved
PARP1 after 5k and 5l treatment, the western blot was
performed using PARP1 antibody (9542, CST, Shanghai,
China); meanwhile, the GAPDH antibody (G8795, Sigma)
was used as the control. After different concentrations of 5k/
3
.3. @e Biological Activity of 5k and 5l in MCF-7 Cells.
According to the MTT assay of this series of compounds
displayed in Table 1, compounds 5k and 5l significantly
inhibited the proliferation of MCF-7 cells. +erefore,
compounds 5k and 5l were selected for further cellular
experiments. +e MTT results showed that 5k and 5l had
antiproliferation activities with the IC50 values 8.50 and
5
l treatment for 24 h, the whole cell lysates were used for
western blot analysis. Both anti-rabbit and anti-mouse IgG
were purchased from Sigma.
3
. Results and Discussion
1
2.51 μM; so, firstly we detected their growth inhibitory
activities based on colony formation assay, which indicated
k and 5l significantly inhibited MCF-7 cell growth at the
3.1. Chemistry. +e synthetic strategy to prepare target
5
compounds is outlined in Scheme 1. +e intermediate 2 was
obtained from classical reduction of 6-nitroquinoline,
compound 3 was prepared by acylation of intermediate 2,
and derivative 4 was generated from substitution reaction
between compound 3 and 80% hydrazine hydrate solution.
Target products 5a–5o were synthesized from condensation
of derivative 4 with aldehydes. +e structures of target
concentration of 10 μM (Figure 3(a)). Besides, colony for-
mation assay was conducted on HeLa cells, the results
showed that compared with HeLa cells, 5k and 5l seemed to
have stronger antiproliferation activities on MCF-7 cells. As
acknowledged, cell cycle is essential for the normal prolif-
eration of cells. To figure out the effects of 5k and 5l on cell
cycle, flow cytometry was performed after 12 h treatment
with 5k/5l at a concentration of 10 μM. +e results revealed
that 5k/5l induced cell cycle arrest in the G2-M phase with
the ratio increasing from 6.19% to 17.40%/18.66% on MCF-7
cells, suggesting that the growth inhibitory of compounds 5k
and 5l might be related to cell cycle arrest. Interestingly,
when HeLa cells were treated with compounds 5k and 5l,
contrasting to MCF-7 cells, no significant change of cell cycle
compounds 5a–5o were characterized by 1H NMR,13C
NMR, and HRMS.
3.2. Antitumor Activities. +e in vitro cytotoxic activity of
target compounds 5a–5o was evaluated in three human
breast cancer cell lines including SKBR3, MDA-MB-231, and
MCF-7 by the MTT assay, and Tamoxifen was used as the

6
Journal of Chemistry
Table 1: +e cytotoxicity of target compounds 5a–5o against SKBr3, MDA-MB-231, and MCF-7 cancer cell lines.
Substituents
IC50 (μmol/L)
MDA-MB-231
Compounds
R
SKBr3
MCF-7
5
a
47.89 ± 0.44
45.55 ± 0.56
35.55 ± 0.22
34.34 ± 0.50
5
b
43.80 ± 0.12
37.34 ± 0.16
5
5
c
33.82 ± 0.33
35.35 ± 0.19
33.78 ± 0.22
35.87 ± 0.13
d
43.22 ± 0.26
34.32 ± 1.23
OH
5
5
5
e
23.12 ± 0.16
23.45 ± 0.26
23.10 ± 0.26
23.78 ± 0.27
27.78 ± 0.33
20.33 ± 0.21
27.54 ± 0.18
24.50 ± 0.23
27.51 ± 0.33
f
OH
g
N
5
5
5
5
h
i
17.10 ± 0.26
15.16 ± 0.11
17.10 ± 0.20
9.16 ± 0.11
16.33 ± 0.11
17.39 ± 0.12
17.55 ± 0.23
10.34 ± 0.36
13.51 ± 0.13
13.51 ± 0.14
14.52 ± 0.32
8.50 ± 0.26
F
OCH3
Br
j
OCH3
k
OCH3
OCH3
5
5
l
16.10 ± 0.11
15.39 ± 0.46
12.51 ± 0.33
OCH3
N
m
19.20 ± 0.11
18.30 ± 0.15
16.33 ± 0.12
O
5
n
o
16.11 ± 0.12
15.33 ± 0.26
16.51 ± 0.21
S
5
14.18 ± 0.22
7.54 ± 0.16
16.30 ± 0.34
10.68 ± 0.23
13.56 ± 0.11
9.05 ± 0.22
Tamoxifen
Con
Dox
5k
5l
MCF-7
Hela
(
a)
Figure 3: Continued.

Journal of Chemistry
7
Control (MCF-7)
G1% = 72.59%
5k (MCF-7)
5l (MCF-7)
6
5
4
3
2
1
60
50
40
30
20
10
0
600
500
400
300
200
100
0
420
350
280
210
140
70
G1% = 63.58%
G1% = 59.59%
G2% = 18.66%
G2% = 17.40%
G2% = 6.19%
0
0
64 128 192 256 320 384 448 512
DNA content
0
64 128 192 256 320 384 448 512
DNA content
0
64 128 192 256 320 384 448 512
DNA content
Control (Hela)
5k (Hela)
5l (Hela)
5
4
3
2
1
40
50
60
70
80
420
350
280
210
140
70
420
350
280
210
140
70
G1% = 68.01%
G1% = 66.12%
G1% = 66.05%
G2% = 13.14%
G2% = 10.94%
G2% = 11.78%
90
0
0
0
0
64 128 192 256 320 384 448 512
DNA content
0
64 128 192 256 320 384 448 512
DNA content
0
64 128 192 256 320 384 448 512
DNA content
(
b)
Figure 3: Effects of compounds 5k and 5l on colony formation and cell cycle disruption: (a) compounds 5k and 5l inhibited cell growth on
MCF-7 cells. (b) Compounds 5k and 5l induced G2-M phase arrest.
5k (MCF-7)
5l (MCF-7)
0
0.1 0.5
1
5
10 (µM)
IB:PARP
0
0.1 0.5
1
5
10 (µM)
IB:PARP
IB:GAPDH
IB:GAPDH
(
a)
Control (MCF-7)
5k (MCF-7)
5l (MCF-7)
PI
PI
PI
AnnexinV-FITC
Control (Hela)
AnnexinV-FITC
5k (Hela)
AnnexinV-FITC
5l (Hela)
PI
PI
PI
AnnexinV-FITC
AnnexinV-FITC
AnnexinV-FITC
(
b)
Figure 4: Apoptosis induction by compounds 5k and 5l. (a) Western blot analysis of PARP in MCF-7 cells treated with different
concentrations of compounds 5k and 5l. (b) Annexin V/PI staining of MCF-7 cells and Hela cells treated with 10 μM 5k and 5l for 24 h.

8
Journal of Chemistry
was observed in HeLa cells. +e data implied that 5k and 5l
were more sensitive to breast cancer cell lines than the Hela
cell line (Figure 3(b)).
2019KF01) and sponsored by K.C. Wong Magna Fund in
Ningbo University and Ningbo 3315 Innovative Talents
Program.
Subsequently, in order to test whether 5k and 5l could
induce apoptosis on MCF-7 cells, cleaved PARP (the mark of
apoptosis) was detected by western blot assay after 24 h
treatment with 5k and 5l on MCF-7 cells. +e results showed
that the PARP cleavage increased in a dose-dependent
manner (Figure 4(a)). Moreover, after the treatment with 5k
and 5l for 24 h, the MCF-7 or HeLa cells were doubly stained
with annexin V/PI (propidium iodide). +e results suggested
that both 5k and 5l could notably promote apoptosis.
Consistent with the abovementioned hypothesis that 5k and
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