Synthesis and biological evaluation of amino chalcone derivatives as antiproliferative agents

Article abstract of DOI:10.3390/molecules25235530

Chalcone is a common scaffold found in many biologically active compounds. The chalcone scaffold was also frequently utilized to design novel anticancer agents with potent biological efficacy. Aiming to continue the research of effective chalcone derivatives to treat cancers with potent anticancer activity, fourteen amino chalcone derivatives were designed and synthesized. The antiproliferative activity of amino chalcone derivatives was studied in vitro and 5-Fu as a control group. Some of the compounds showed moderate to good activity against three human cancer cells (MGC-803, HCT-116 and MCF-7 cells) and compound 13e displayed the best antiproliferative activity against MGC-803 cells, HCT-116 cells and MCF-7 cells with IC₅₀ values of 1.52 μM (MGC-803), 1.83 μM (HCT-116) and 2.54 μM (MCF-7), respectively which was more potent than the positive control (5-Fu). Further mechanism studies were explored. The results of cell colony formatting assay suggested compound 10e inhibited the colony formation of MGC-803 cells. DAPI fluorescent staining and flow cytometry assay showed compound 13e induced MGC-803 cells apoptosis. Western blotting experiment indicated compound 13e induced cell apoptosis via the extrinsic/intrinsic apoptosis pathway in MGC-803 cells. Therefore, compound 13e might be a valuable lead compound as antiproliferative agents and amino chalcone derivatives worth further effort to improve amino chalcone derivatives’ potency.

Full text of DOI:10.3390/molecules25235530

Article
Synthesis and Biological Evaluation of Amino
Chalcone Derivatives as Antiproliferative Agents
Chao-Fan Lu 1,†, Sheng-Hui Wang 1,†, Xiao-Jing Pang 1,2,†, Ting Zhu 2, Hong-Li Li 1,
Qing-Rong Li 1, Qian-Yu Li 1, Yu-Fan Gu 1, Zhao-Yang Mu 1, Min-Jie Jin 1, Yin-Ru Li 1,
Yang-Yang Hu 3, Yan-Bing Zhang 2, Jian Song 1,2,* and Sai-Yang Zhang 1,2,4,
*
1
Department of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, China;
chaofanlu@stu.zzu.edu.cn (C.-F.L.); thekingwinglory@outlook.com (S.-H.W.);
summer_pxj@163.com (X.-J.P.); Hollyly1@outlook.com (H.-L.L.); 15318989671@163.com (Q.-R.L.);
15318989671@163.com (Q.-Y.L.); 18838078036@163.com (Y.-F.G.); QQ2743757146@163.com (Z.-Y.M.);
Agile321@163.com (M.-J.J.); 202012402016083@gs.zzu.edu.cn (Y.-R.L.)
2
Department of Pharmaceutical Sciences, Institute of Drug Discovery & Development,
Key Laboratory of Advanced Drug Preparation Technologies (Ministry of Education),
Zhengzhou University, Zhengzhou 450001, China; 17671140381@sina.cn (T.Z.);
zhangyb@zzu.edu.cn (Y.-B.Z.)
3
4
Faculty of Science, The University of Melbourne, Melbourne VIC 3010, Australia;
YANGYANGH1@student.unimelb.edu.au
Henan Institute of Advanced Technology, Zhengzhou University, Zhengzhou 450001, China
* Correspondence: mumuandzz@163.com (J.S.); saiyangz@zzu.edu.cn (S.-Y.Z.)
† These authors contributed equally to this work.
Received: 29 September 2020; Accepted: 24 November 2020; Published: 25 November 2020
Abstract: Chalcone is a common scaffold found in many biologically active compounds. The
chalcone scaffold was also frequently utilized to design novel anticancer agents with potent
biological efficacy. Aiming to continue the research of effective chalcone derivatives to treat cancers
with potent anticancer activity, fourteen amino chalcone derivatives were designed and
synthesized. The antiproliferative activity of amino chalcone derivatives was studied in vitro and 5-
Fu as a control group. Some of the compounds showed moderate to good activity against three
human cancer cells (MGC-803, HCT-116 and MCF-7 cells) and compound 13e displayed the best
antiproliferative activity against MGC-803 cells, HCT-116 cells and MCF-7 cells with IC50 values of
1.52 μM (MGC-803), 1.83 μM (HCT-116) and 2.54 μM (MCF-7), respectively which was more potent
than the positive control (5-Fu). Further mechanism studies were explored. The results of cell colony
formatting assay suggested compound 10e inhibited the colony formation of MGC-803 cells. DAPI
fluorescent staining and flow cytometry assay showed compound 13e induced MGC-803 cells
apoptosis. Western blotting experiment indicated compound 13e induced cell apoptosis via the
extrinsic/intrinsic apoptosis pathway in MGC-803 cells. Therefore, compound 13e might be a
valuable lead compound as antiproliferative agents and amino chalcone derivatives worth further
effort to improve amino chalcone derivatives’ potency.
Keywords: chalcone; synthesis; antiproliferative; cell apoptosis
1. Introduction
Chalcone is a common scaffold found in many biologically active compounds [1]. Natural
chalcone products and synthetic chalcone derivatives have shown many interesting pharmacological
activities including anti-bacterial [2–4], anti-malarial [5–7], anti-fungal [8–10], anti-HIV [11–13], anti-
Molecules 2020, 25, 5530; doi:10.3390/molecules25235530
www.mdpi.com/journal/molecules

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inflammatory [14–16] and anti-cancer [17–26] activities. Especially, chalcone compounds as a class of
anticancer agents have exhibited promising therapeutic efficacy and clinical potentials for the
treatment of human tumors. In fact, many groups have reported various chalcone derivatives with
potent anticancer activity. (E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-
en-1-one 1 [22] displayed remarkable antiproliferative activities against and was identified as a
tubulin inhibitor. (E)-3-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one 2 exhibited
the antiproliferative activity against K562 cell line with an IC50 of 4.5 μM [23]. Compound 3 [24]
named millepachine showed inhibitory effect in several human cancer cells, especially in HepG2 cells
with an IC50 of 1.51 μM, and induced G2/M arrest by inhibiting CDK1 activity and causing apoptosis
via ROS-mitochondrial apoptotic pathway. Moreover, the chalcone scaffold was also frequently
utilized to design novel anticancer agents with potent biological efficacy. Pyridyl-indole based
heteroaryl chalcone 4 [25] containing a sulfonamide group exhibited significant inhibition of hCA IX
activity (IC50 = 0.13 μM) and MCF-7 cells (IC50 = 12.2 μM). Sorafenib analogues bearing chalcone unit
5 [26] showed well anticancer activity against MCF-7 cells (IC50 = 3.88 μM) and PC-3 cells (IC50 = 3.15
μM) and potent activity on VEGFR-2/KDR kinase (IC50 = 0.72 μM). Therefore, chalcone might be a
valuable lead scaffold to design novel anticancer agents and there is an urgent need to discover more
effective compounds to treat cancers. In this work, we continued with our efforts on chalcone
derivatives to discover potent anticancer agents for the treatment of human cancers (Figure 1).
Figure 1. Structures of chalcone derivates as antitumor agents previously reported.
The combinations of chalcone scaffold with other anticancer fragment by the molecular
hybridization strategy are a common and effective methods to design novel anticancer chalcone
derivatives. Recently, our group also has reported several series of novel chalcone derivatives by the
molecular hybridization strategy that exhibited potent antiproliferative activity against human
cancer cells [27–30]. Chalcone-dithiocarbamate 6 exhibited the inhibitory activity against MGC-803
cells (IC50 = 1.74 μM). Chalcone-1,2,3-triazole-azole 7 displayed the good inhibitory activity against
MGC-803 cells (IC50 = 4.26 μM). The modification of amino groups usually leads to better antitumor
activity [31,32]. For example, novel 4-substituted coumarin derivative 9 were optimized and
synthesized form 4-((3-amino-4-methoxyphenyl) (methyl)amino)-2H-chromen-2-one
8
[32].
Compound 9 exhibited more potent antiproliferative activity against SKVO3 cells (IC50 = 3.5 nM) then
compound 8 (IC50 = 23.4 nM). In this work, as the continuation of our studies on novel chalcone
derivatives as cancer agents, the modification and optimization of amino group of (E)-1-(4-
aminophenyl)-3-(3,4,5-trimethoxyphenyl) prop-2-en-1-one 10 was explored. Eleven amino chalcone
derivatives were designed, synthesized and tested its antiproliferative activity against MGC-803 cells,
HCT-116 cells and MCF-7 cells (Figure 2).

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O
O
O
O
O
OH
O
N
S
N
O
N
N
S
O
O
N
H
O
S
N
S
N
6
7
O
O
O
Cl
N
O
N
H
N
NH2
O
O
O
8
9
Anticancer pharmacophore
fragment
Cl
n=0,3
n=0,1,2,3
O
O
O
R1
O
phenyl ring
O
O
NH2
N
R2
3-pyridine ring
2-thiophene ring
H
11a-11n
10
Figure 2. Reported and proposed anticancer chalcone and coumarin derivatives.
2. Results and Discussion
2.1. Chemistry
Target amino chalcone derivatives were synthesized by outlined procedures in Scheme 1.
Commercially available aldehydes 11a–h reacted with 4-aminoacetophenone to afford compounds
12a–h. Compounds 12a–h then reacted with substituted acyl chloride intermediates in DCM to give
compounds 13a–n. Characterization of compounds 13a–n was carried out by means of NMR and
HREI-mass spectra which were showed in the Supplementary Materials.
Scheme 1. Synthesis of compounds 13a–n.

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2.2. Antiproliferative Activity and Structure Activity Relationship Analysis
The in vitro antiproliferative activities of new target compounds 13a–n were evaluated against
four human cancer cell lines (MGC-803, HCT-116 and MCF-7) using MTT assay and 5-Fu as a positive
drug. The following Table 1 depicted the results of in vitro antiproliferative activity.
Compounds 13a–g were synthesized and evaluated against MGC-803, HCT-116 and MCF-7
cells. In this series of compounds, we first explored the importance of the substituent groups of R2 on
the antiproliferative activities of compounds with a 3,4,5-trimethoxyphenyl group of R1. As shown in
Table 1, most of the compounds 13a–g exhibited potent inhibitory efficacy against MGC-803, HCT-
116 and MCF-7 cells with IC50 values less than 10 μM than the positive drug 5-Fu. The inhibitory
efficacy of compounds 13a–g varies with its substituent groups of R2. Compound 13e with a
chloropropyl group of R2 displayed most the potent in vitro antiproliferative activity with IC50 values
of 1.52 μM (MGC-803), 1.83 μM (HCT-116) and 2.54 μM (MCF-7), respectively. Compared compound
13c, 13d, 13f and 13e, proper carbon liner length of R2 group enhanced anticancer activity. Compound
13g with a vinyl group of R2 also showed potent antiproliferative activity against three human cancer
cells. With compound 13e in hand, we started to focus our attention on the R1 moiety of compounds
with a chloropropyl group of R2. Most of the target compounds exhibited weaker antiproliferative
activity compared to compounds with a 3,4,5-trimethoxyphenyl group of R1 and the positive drug 5-
Fu. Compared compounds 13h, 13i, 13j, 13l and 13e, Compounds with electron-donating groups on
phenyl group of R1 showed improved inhibitory efficacy then compounds with an unsubstituted
group and electron-withdrawing groups. What’s more, compounds 13m–n, with heterocyclic groups
of R1 didn’t showed improved inhibitory activity against three human cancer cells.
Table 1. In vitro antiproliferative activities of compounds 10a–p against human cancer cells.
IC50 (μmol/L) a
Compounds
MGC-803
HCT-116
MCF-7
13a
13b
13c
13d
13e
13f
3.81 ± 0.22 4.012 ± 0.31 3.56 ± 0.17
4.08 ± 0.24 6.72 ± 0.28 3.11 ± 0.34
1.88 ± 0.22 2.83 ± 0.03 3.12 ± 0.01
1.64 ± 0.18 2.40 ± 0.26 2.12 ± 0.13
1.52 ± 0.12 1.83 ± 0.20 2.54 ± 0.18
3.01 ± 0.11 4.28 ± 0.32 4.45 ± 0.11
1.83 ± 0.20 1.12 ± 0.11 2.06 ± 0.21
22.1 ± 0.75 13.1 ± 0.51 22.2 ± 0.83
16.8 ± 0.82 13.1 ± 0.61 17.6 ± 0.51
13g
13h
13i
13j
>40
>40
>40
>40
>40
>40
>40
13k
13l
16.3 ± 0.65
10.1 ± 0.73
13m
13n
5-Fu
5.41 ± 0.30 6.12 ± 0.41 6.62 ± 0.48
>40 12.5 ± 0.28 21.2 ± 1.12
6.82 ± 1.12 14.4 ± 1.73 12.1 ± 1.28
a In vitro antiproliferative activity was assayed by exposure for 48 h.
Notably, compounds 13e exhibited highest activity against three test human cancer cells.
Therefore, compounds 13e also were evaluated against non-cancer cell lines GES-1ꢀcells. As shown
in Table 2, Compounds 13e exhibited weaker activity against GES-1ꢀcells with an IC50 value of 8.22ꢀμM
than compounds 13e. The selectivity of compounds 13e between MGC-803 cells and GES-1ꢀcells 5.4-
fold selectivity.

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Table 2. In vitro anti-proliferative activity of 13e against gastric cancer cells (MGC-803) and non-
cancer cell lines (GES-1).
IC50 (μM) a
Fold selectivity
Compd.
MGC-803 GES-1
A
13g
1.52
6.82
8.22ꢀ
8.22ꢀ
5.4
1.2
5-Fu
a In vitro antiproliferative activity was assayed by exposure for 48 h.
Aꢀ = ꢀIC50 (GES-1)/IC50 (MGC-803)
Based on the above preliminary results of in vitro antiproliferative activity, the structure-activity
relationships were summarized (Figure 3). 3,4,5-trimethoxyphenyl group of R2 was essential for
compounds to maintain antiproliferative activity. Proper carbon liner length enhanced anticancer
activity.
3,4,5-trimethoxyphenyl group
was essential for compounds to
maintain antiproliferative activity.
Cl > H
O
O
O
Cl
n
O
N
H
O
Proper carbon liner length
enhanced anticancer activity.
Figure 3. Summary of the structure-activity relationships.
2.3. Compound 13e Inhibited Cell Viability against Gastric Cancer Cell MGC-803 Cells
Since gastric cancer cell line MGC-803 cells was more sensitive to compound 13e, MGC-803 cells
were selected to do further study. The cell viabilities of MGC-803 cells after the treatment with
different concentrations of compound 13e for 48 h were presented in Figure 4A, as the concentration
rise, cell viability decreased obviously. These gave compound 13e an IC50 of 1.52 μmol/L against
MGC-803 cells. The trends of cell growth were curved with results of cell viabilities after compound
13e treatment. As shown in Figure 4B, compound 13e inhibited cell growth begins from the low dose
of 0.75 μmol/L after treatment for 72 h. We also tested the inhibition activity of compound 13e on
normal gastric epithelial cell GES-1. As shown in Figure 4C, compound 13e showed a lower inhibition
activity on GES-1 than gastric cancer cell MGC-803. Compound 13e exhibited a certain selective
inhibitory effect on cancer cells in the concentration range below 2 μmol/L. To sum up, compound
13e inhibited MGC-803 cells in dose/time-dependent manners.
2.4. Compound 13e Inhibited Proliferation of MGC-803 Cells
To check the effect of compound 13e on cell proliferation, cell colony formatting assay was
performed. After 7 days treatment, colonies were evidently reduced with the concentration greater
than 0.5 μmol/L compared to them of control (Figure 5A,B). 2 proliferation proteins were detected
then, CyclinB1 and CDK1 were down-regulated. Beside the activity on cell apoptosis, compound 13e
inhibited cell proliferation of MGC-803 cells as well.

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Figure 4. (A–C) MGC-803 cells inhibition activity of compound 13e in vivo. (A) Cell viabilities of
MGC-803 cells, MGC-803 cells were treated with indicated concentrations of compound 13e for 48 h;
(B) Growth curves of MGC-803 cells after the treatment with indicated concentrations of compound
13e for different hours. (C) Cell viabilities of MGC-803 cells and GES-1 cells, cells were treated with
indicated concentrations of compound 13e for 48 h. The results shown were representative of three
independent experiments.
Figure 5. Compound 13e inhibited cell proliferation. (A,B) The colony formation of MGC-803 cells
after the treatment with indicated concentrations of compound 13e for 7 days; (C) Cell proliferation
related proteins in MGC-803 cells of compound 13e after the treatment with indicated concentrations
of 13e for 24 h.

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2.5. Compound 13e Induced Cell Apoptosis in MGC-803 Cells
To detect the mechanism of compound 13e on inhibiting MGC-803 cells, treated/untreated cells
were captured with a microscope. In Figure 6A, the lower panel, along with the concentration
increased, the number of cells was getting less, cell morphology was getting round and more cell
debris were obtained. Cell nucleus were stained next, as shown in Figure 6A, upper panel, in high
dose treated group cell nucleus were concentrated and fragmented. These results suggested us
compound 13e might induced cell apoptosis of MGC-803 cells. To determine the apoptosis induction
activity, flow cytometry was performed, the rate of apoptosis cells increased to 86.7% after 48 h
6μmol/L treatment from less than 10% in the control group (Figure 6B,C). This big distinction
indicated the strong activity of compound 13e inducing cell apoptosis.
Figure 6. Compound 13e induced cell apoptosis. (A) Cell nucleus (upper panel) and morphology
(lower panel) of compound 13e treated (48 h) or untreated MGC-803 cells; (B) Compound 13e induced
apoptosis of MGC-803 cells. After incubated with compound 13e for 48 h, MGC-803 cells were
detected by Annexin V/PI with flow cytometric analysis. The Q3 represents live cells, Q4 represents
early/primary apoptotic cells, Q2 represents late/secondary apoptotic cells and Q1 represents cells
necrosis. (C) The percentage of apoptosis (early and late apoptosis) cells increased dependently with
various concentrations of compound 13e. Date are represented as mean ± SD of three independent
experiments.
2.6. Compound 13e Induced Cell Apoptosis via the Extrinsic/Intrinsic Apoptosis Pathway
Cell apoptosis could be induced through extrinsic or intrinsic apoptosis pathway.
Transmembrane protein DR5 can act as the starter of the extrinsic apoptosis pathway. Figure 7A
exhibited that DR5 was up regulated after 48 h treatment, and its downstream Caspase8 was cleaved
(activated). The activation of Caspase8 led to Bid cleavage, the increase of t-Bid. As the result, the
intrinsic apoptosis pathway was activated. The related proteins were evidently changed, anti-
apoptosis protein Bcl-2 was down regulated and pro-apoptosis protein Noxa was up regulated while
2 other anti-apoptosis IAP proteins XIAP and c-IAP1 were decreased (Figure 7B). what’s next, the

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downstream of extrinsic/intrinsic apoptosis pathway Caspase12 was cleaved (activated), 2 Caspase
executers Caspase3/7 were cleaved (activated). The substrate of Caspase executers PARP was cleaved
as well. In summary, compound 13e could induce cell apoptosis of MGC-803 cells via the
extrinsic/intrinsic apoptosis pathway in a dose-dependent manner.
Figure 7. Compound 13e regulated apoptosis related proteins. (A,B) The expression of cell apoptosis
related proteins were detected by western blotting. MGC-803 cells were incubated with various
concentrations compound 13e (0 μmol/L, 1.5 μmol/L, 3 μmol/L, 6 μmol/L) for 48 h. Date are
represented as mean ± SD of three independent experiments.
3. Materials and Methods
All the chemical reagents were purchased from commercial suppliers (Energy chemical
Compony and Zhengzhou HeQi Compony). Melting points were determined on an X-5 micromelting
apparatus. NMR spectra data was recorded with a Bruker spectrometer. HRMS spectra data was
obtained using a Waters Micromass spectrometer.
3.1. Synthesis of Compounds 12a–h
A solution of commercially available aldehydes 12a–h (1.0 mmol), NaOH (2.0 mmol) and 4-
aminoacetophenone (1.0 mmol) were added into 20 mL
EtOH at 25 °C. After 8 h, adding 20mL water. And then, the reaction mixture was evaporated to
give crude products. Crude products were purified to get compounds 12a–h by column
chromatography.
3.2. Synthesis of Compounds 13a–n
A solution of commercially available aldehydes 12a–h (1.0 mmol), acyl chloride derivatives (1.5
eq) and 0.75 mmol triethylamine (1.5 eq) were added into 10 mL DCM at 25 °C. After 4 h, organic
phases were evaporated to get crude products and then were purified to give targeted compounds
13a–n by column chromatography.
(E)-N-(4-(3-oxo-3-(3,4,5-trimethoxyphenyl) prop-1-en-1-yl) phenyl) acetamide (13a), Light yellow powder,
Yield, 52%, m.p. 163–164 °C.1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.16 (d, J = 8.8 Hz, 2H),
7.812 (d, J = 15.5 Hz, 1H), 7.712 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H), 3.87 (s, 6H),
3.72 (s, 3H), 2.11 (s, 3H).13C NMR (101 MHz, DMSO- d6) δ 187.40, 168.125, 153.07, 143.78, 143.67,
1312.512, 132.16, 130.31, 1212.86, 121.012, 118.21, 106.41, 60.11, 56.012, 24.17. HR-MS (ESI): Calcd,
C20H21NO5, [M+H]+: 356.1492, found: 356.1498.
(E)-N-(4-(3-oxo-3-(3,4,5-trimethoxyphenyl) prop-1-en-1-yl)phenyl) pentanamide (13b), Light yellow
powder, Yield, 55%, m.p. 146–147 °C.1H NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1H), 7.127 – 7.86 (m,
2H), 7.65 (d, J = 15.5 Hz, 1H), 7.512 – 7.52 (m, 2H), 7.43 (d, J = 15.5 Hz, 1H), 6.128 (s, 2H), 3.62 (s, 6H),

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3.47 (d, J = 2.1 Hz, 3H), 2.12 (t, J = 7.4 Hz, 2H), 1.34 (dd, J = 14.12, 7.6 Hz, 2H), 1.012 (dd, J = 14.12, 7.4
Hz, 2H), 0.66 (t, J = 7.3 Hz, 3H).13C NMR (101 MHz, DMSO-d6) δ 187.37, 171.122, 153.07, 143.78, 143.72,
1312.512, 132.10, 130.32, 1212.85, 121.08, 118.26, 106.41, 60.012, 56.08, 36.22, 27.04, 21.712, 13.612. HR-
MS (ESI): Calcd, C23H27NO5, [M+H]+: 398.1962, found: 398.1958.
(E)-2-chloro-N-(4-(3-oxo-3-(3,4,5-trimethoxyphenyl) prop-1-en-1-yl) phenyl) acetamide (13c), Light yellow
powder, Yield, 58%, m.p. 182–183 °C.1H NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H), 8.20 (d, J = 8.8
Hz, 2H), 7.120 (d, J = 15.5 Hz, 1H), 7.80 (d, J = 8.8 Hz, 2H), 7.612 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H), 4.33
(s, 2H), 3.87 (s, 6H), 3.72 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 188.02, 165.71, 153.59, 144.50, 143.26,
140.20, 133.42, 130.77, 130.41, 121.56, 119.22, 106.97, 60.61, 56.61, 44.09. HR-MS (ESI): Calcd,
C20H20ClNO5, [M+H]+: 390.1108, found: 390.1103.
(E)-3-chloro-N-(4-(3-oxo-3-(3,4,5-trimethoxyphenyl) prop-1-en-1-yl) phenyl) propanamide(13d), Light
yellow powder, Yield, 41.8%, m.p. 183–184 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 1H), 8.18 (d,
J = 8.8 Hz, 2H), 7.120 (d, J = 15.5 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.612 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H),
3.124–3.86 (m, 8H), 3.72 (s, 3H), 2.120 (dd, J = 8.0, 4.4 Hz, 2H). HR-MS (ESI): Calcd, C21H22ClNO5,
[M+H]+: 404.1265, found: 404.1268. 13C NMR (151 MHz, DMSO-d6) δ 187.96, 169.14, 153.59, 144.39,
143.75, 140.17, 132.98, 130.80, 130.40, 121.59, 118.93, 106.97, 60.61, 56.62, 41.04, 40.40. HR-MS (ESI):
Calcd, C21H22ClNO5, [M+H]+: 404.1259, found: 404.1268.
(E)-4-chloro-N-(4-(3-oxo-3-(3,4,5-trimethoxyphenyl) prop-1-en-1-yl) phenyl) butanamide (13e), Light
yellow powder, Yield, 51.2%, m.p. 170–171 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H), 8.21 (d,
J = 8.8 Hz, 2H), 7.124 (d, J = 15.5 Hz, 1H), 7.84 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 15.5 Hz, 1H), 7.27 (s, 2H),
3.121 (d, J = 2.12 Hz, 8H), 3.76 (s, 3H), 2.60 (t, J = 7.3 Hz, 2H), 2.15–2.07 (m, 2H). 13C NMR (151 MHz,
DMSO-d6) δ 187.93, 171.36, 153.59, 144.31, 144.05, 142.38, 132.74, 131.59, 130.36, 118.86, 113.29, 106.96,
60.62, 56.62, 45.44, 34.00, 28.18. HR-MS (ESI): Calcd, C22H24ClNO5, [M+H]+: 418.1416, found: 418.1417.
(E)-5-chloro-N-(4-(3-oxo-3-(3,4,5-trimethoxyphenyl) prop-1-en-1-yl) phenyl) pentanamide (13f), Light
yellow powder, Yield, 51%, m.p. 125–126 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.30 (s, 1H), 8.16 (d, J
= 8.8 Hz, 2H), 7.812 (d, J = 15.5 Hz, 1H), 7.80 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 15.5 Hz, 1H), 7.23 (s, 2H),
3.87 (s, 6H), 3.72 (s, 3H), 3.68 (t, J = 6.2 Hz, 2H), 2.42 (t, J = 6.12 Hz, 2H), 1.76 (ddd, J = 7.2, 6.5, 2.6 Hz,
4H). 13C NMR (151 MHz, DMSO) δ 187.93, 172.04, 153.59, 144.29, 144.11, 140.16, 132.71, 130.82, 130.35,
121.62, 118.83, 106.95, 60.61, 56.61, 45.55, 36.05, 32.02, 22.77.HR-MS (ESI): Calcd, C22H24ClNO5, [M+H]+:
432.1578, found: 432.1575.
(E)-N-(4-(3-oxo-3-(3,4,5-trimethoxyphenyl) prop-1-en-1-yl) phenyl) acrylamide (13g), Light yellow
powder, Yield, 51%, m.p. 178–179 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H), 8.20 (d, J = 8.8
Hz, 2H), 7.88 (d, J = 8.3 Hz, 2H), 7.612 (d, J = 15.5 Hz, 1H), 7.24 (s, 2H), 6.412 (dd, J = 17.0, 10.1 Hz, 1H),
6.33 (dd, J = 17.0, 1.8 Hz, 1H), 5.84 (dd, J = 10.1, 1.8 Hz, 1H), 3.88 (s, 6H), 3.72 (s, 3H). 13C NMR (101
MHz, DMSO) δ 187.43, 163.512, 153.08, 143.121, 143.36, 1312.62, 132.60, 131.50, 130.30, 1212.812,
127.86, 121.05, 118.71, 106.44, 60.10, 56.012. HR-MS (ESI): Calcd, C21H21NO5, [M+H]+: 368.1498, found:
368.1497.
(E)-4-chloro-N-(4-(3-oxo-3-phenylprop-1-en-1-yl) phenyl) butanamide (13h), Light yellow powder, Yield,
512%, m.p. 161–162 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.16 (d, J = 8.7 Hz, 2H), 7.121
(ddd, J = 10.5, 12.3, 2.8 Hz, 4H), 7.80 (d, J = 8.7 Hz, 2H), 7.73 (d, J = 15.6 Hz, 1H), 7.412–7.44 (m, 3H),
3.72 (t, J = 6.5 Hz, 2H), 2.55 (dd, J = 15.0, 7.7 Hz, 2H), 2.11–2.01 (m, 2H). 13C NMR (101 MHz, DMSO) δ
187.50, 170.87, 143.62, 143.212, 134.75, 132.12, 130.45, 1212.87, 128.87, 128.76, 121.126, 118.312, 44.123,
33.412, 27.612. HR-MS (ESI): Calcd, C19H18ClNO2, [M+H]+: 328.1099, found: 328.1096.
(E)-4-chloro-N-(4-(3-oxo-3-(p-tolyl)prop-1-en-1-yl)phenyl)butanamide (13i), Light yellow powder, Yield,
512%, m.p. 146–147 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.15 (d, J = 8.8 Hz, 2H), 7.125–
7.64 (m, 7H), 7.28 (d, J = 8.0 Hz, 2H), 3.72 (t, J = 6.5 Hz, 2H), 2.56 (t, J = 7.3 Hz, 2H), 2.36 (s, 3H), 2.11–
2.02 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 187.44, 170.84, 143.54, 143.34, 140.412, 132.22, 132.05,
1212.712, 1212.50, 128.712, 120.88, 118.37, 44.123, 33.412, 27.612, 21.04. HR-MS (ESI): Calcd,
C20H20ClNO2, [M+H]+: 342.1255, found: 342.1258.

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(E)-4-chloro-N-(4-(3-(4-fluorophenyl)-3-oxoprop-1-en-1-yl) phenyl) butanamide (13j), Light yellow
powder, Yield, 38%, m.p. 167–168 °C.1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.15 (d, J = 8.8
Hz, 2H), 8.00–7.812 (m, 3H), 7.712 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 15.6 Hz, 1H), 7.30 (t, J = 8.8 Hz, 2H),
3.72 (t, J = 6.5 Hz, 2H), 2.56 (t, J = 7.3 Hz, 2H), 2.11 – 2.02 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ
187.312, 170.86, 164.54, 162.06, 143.63, 142.03, 132.012, 131.47, 131.44, 131.14, 131.05, 1212.86, 121.87,
121.85, 118.37, 115.127, 115.76, 44.122, 33.412, 27.612. HR-MS (ESI): Calcd, C19H17ClFNO2, [M+H]+:
346.1005, found: 346.1007.
(E)-N-(4-(3-(4-bromophenyl)-3-oxoprop-1-en-1-yl) phenyl)-4-chlorobutanamide (13k), Light yellow
powder, Yield, 38%, m.p. 188–189 °C.1H NMR (400 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.16 (d, J = 8.6
Hz, 2H), 7.128 (d, J = 15.6 Hz, 1H), 7.85 (d, J = 8.4 Hz, 2H), 7.712 (d, J = 8.6 Hz, 2H), 7.68 (t, J = 11.2 Hz,
3H), 3.72 (t, J = 6.5 Hz, 2H), 2.56 (t, J = 7.3 Hz, 2H), 2.10–2.00 (m, 2H). 13C NMR (101 MHz, DMSO-d6)
δ 187.36, 170.88, 143.70, 141.812, 134.07, 132.00, 131.83, 130.68, 1212.122, 123.77, 122.76, 118.38, 44.123,
33.412, 27.68. HR-MS (ESI): Calcd, C19H17ClBrNO2, [M+H]+: 406.0204, found: 406.0199.
(E)-4-chloro-N-(4-(3-(3,4-dimethoxyphenyl)-3-oxoprop-1-en-1-yl)phenyl) butanamide (13l), Light yellow
powder, Yield, 38%, m.p. 166–167 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.16 (d, J = 8.8
Hz, 2H), 7.121 (d, J = 15.6 Hz, 1H), 7.80 (d, J = 8.8 Hz, 2H), 7.70 (d, J = 15.5 Hz, 1H), 7.46 (s, 1H), 7.33
(d, J = 6.12 Hz, 1H), 7.22 (d, J = 7.7 Hz, 1H), 3.120 (s, 3H), 3.72 (t, J = 6.5 Hz, 2H), 2.56 (t, J = 7.3 Hz, 2H),
2.112 (s, 3H), 2.10–2.03 (m, 2H). 13C NMR (101 MHz, DMSO) δ 187.48, 170.85, 157.63, 143.712, 143.55,
133.86, 132.22, 130.66, 1212.83, 128.812, 121.78, 120.126, 118.35, 1012.61, 55.47, 44.124, 33.412, 27.68,
16.17. HR-MS (ESI): Calcd, C21H22ClNO2, [M+H]+:410.1130, found: 410.0920.
(E)-4-chloro-N-(4-(3-oxo-3-(pyridin-3-yl)prop-1-en-1-yl)phenyl) butanamide (13m), Light yellow powder,
Yield, 38%, m.p. 139–140 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.312 (s, 1H), 8.612 (d, J = 4.7 Hz, 1H),
8.13 (dd, J = 23.5, 12.1 Hz, 3H), 7.121 (dd, J = 4.7, 1.1 Hz, 2H), 7.81 (d, J = 8.8 Hz, 2H), 7.71 (d, J = 15.4
Hz, 1H), 7.44 (d, J = 4.4 Hz, 1H), 3.72 (t, J = 6.5 Hz, 2H), 2.56 (t, J = 7.3 Hz, 2H), 2.10–2.02 (m, 2H). 13C
NMR (101 MHz, DMSO) δ 187.61, 170.120, 152.48, 1412.44, 143.86, 141.66, 137.712, 131.80, 131.20,
1212.122, 125.53, 124.123, 118.51, 112.120, 44.122, 33.50, 27.68. HR-MS (ESI): Calcd, C18H17ClN2O2,
[M+H]+:329.1051, found: 329.1054.
(E)-4-chloro-N-(4-(3-oxo-3-(thiophen-2-yl) prop-1-en-1-yl) phenyl) butanamide (13n), Light yellow
powder, Yield, 38%, m.p. 160–161 °C.1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.012 (d, J = 8.8
Hz, 2H), 7.120 (d, J = 15.3 Hz, 1H), 7.81–7.76 (m, 3H), 7.68 (d, J = 3.4 Hz, 1H), 7.57 (d, J = 15.3 Hz, 1H),
7.112 (dd, J = 5.0, 3.7 Hz, 1H), 3.72 (t, J = 6.5 Hz, 2H), 2.55 (t, J = 7.3 Hz, 2H), 2.10–2.02 (m, 2H). 13C
NMR (101 MHz, DMSO) δ 186.125, 170.85, 143.58, 1312.81, 136.05, 132.55, 132.00, 130.16, 1212.612,
128.65, 120.26, 118.42, 44.123, 33.412, 27.70. HR-MS (ESI): Calcd, C17H16ClNO2S, [M+H]+: 334.0663,
found: 334.0665.
3.3. Cell Culture
Cell lines used were cultured in humidified incubator at 37 °C and 5% CO . The RPMI-1640
2
medium was supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin
(0.1 mg/mL).
3.4. MTT Assay
Cell lines were seeded into 126-well plates and incubated for 24 h. Then cells were treated with
different concentrations of compounds. And after another 48 h, MTT reagent (20 μL per well) was
added and then incubated at 37 °C for 4 h. Formazan was then dissolved with DMSO. Absorbencies
of formazan solution were measured at 4120 nm. The IC50 values of tested compounds were
calculated by SPSS version 17.0.
3.5. DAPI Assay
Cells were seeded in 6-welled plate, then treated with different concentration of compounds for
48 h. The treated and untreated cells were washed with PBS buffer. Then fixed with 4%

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paraformaldehyde for 10 min in dark. After washed with PBS buffer, cells were stained by 2 μg/mL
DAPI solution containing 0.1% triton X-100 for 30 min. Discard the solution and wash the cells with
PBS buffer. Capture the images with a fluorescence microscope.
3.6. Western Blotting Analysis
Gastric cancer cells were seeded in dishes and treated with 13e or DMSO. After 48 h, MGC-803
cells were collected and then lysed. The denatured lysates of each groups were electrophoretic
separated in SDS-PAGE. Proteins were then transferred onto PVDF membranes from gels. After
blocking for 2 h, membranes were incubated with primary antibodies conjugation. Then, the
membranes were washed and incubated with 2nd antibodies. At last, specific proteins were detected.
3.7. General Methods
In this work, some other assays including colony formation assay and cell apoptosis assay were
referred to our previous work [33–35].
4. Conclusions
Chalcone is a common scaffold found in many biologically active compounds. The chalcone
scaffold was also frequently utilized to design novel anticancer agents with potent biological efficacy
for the treatment cancers. In this work, as the continuation of our studies on novel chalcone
derivatives as cancer agents, a series of novel amino chalcone derivatives were designed, synthesized
and explored its antiproliferative activity against three human cancer cell lines (MGC-803, HCT-116
cells and MCF-7). Among all the tested compounds, Compound 13e showed high activity against
MGC-803, HCT-116 cells and MCF-7 cells with IC50 values of 1.54 μM (MGC-803), 1.83 μM (HCT-116)
and 2.54 μM (MCF-7), respectively, which was more potent than the positive control (5-Fu). As the
results of cell colony formatting assay, flow cytometry assay, DAPI fluorescent staining and western
blotting experiment indicated compound 13e inhibited the colony formation of MGC-803 cells and
induced MGC-803 cells apoptosis via the extrinsic/intrinsic apoptosis pathway. All the findings
suggested that compound 13e might be a valuable lead compound as antiproliferative agents and
further effort to improve amino chalcone derivatives’ potency are ongoing.
Supplementary Materials: The following are available online, Figure S1: 1H NMR of compound 13a (DMSO-d6,
400 MHz), Figure S2: 13C NMR of compound 13a (DMSO-d6, 400 MHz), Figure S3: HR-MS of compound 13a,
Figure S4: 1H NMR of compound 13b (DMSO-d6, 400 MHz), Figure S5: 13C NMR of compound 13b (DMSO-d6,
1
400 MHz), Figure S6: HR-MS of compound 13b,Figure S7: H NMR of compound 13c (DMSO-d6, 400 MHz),
Figure S8: 13C NMR of compound 13c (DMSO-d6, 600 MHz), Figure S9: HR-MS of compound 13c, Figure S10: 1H
NMR of compound 13d (DMSO-d6, 400 MHz), Figure S11: 13C NMR of compound 13d (DMSO-d6, 600 MHz),
Figure S12: HR-MS of compound 13d, Figure S13: 1H NMR of compound 13e (DMSO-d6, 400 MHz), Figure S14:
13C NMR of compound 13e (DMSO-d6, 600 MHz), Figure S15: HR-MS of compound 13e, Figure S16: 1H NMR of
compound 13f (DMSO-d6, 400 MHz), Figure S17: 13C NMR of compound 13f (DMSO-d6, 600 MHz), Figure S18:
HR-MS of compound 13f, Figure S19: 1H NMR of compound 13g (DMSO-d6, 400 MHz), Figure S20: 13C NMR of
compound 13g (DMSO-d6, 400 MHz), Figure S21: HR-MS of compound 13g, Figure S22: 1H NMR of compound
13h (DMSO-d6, 400 MHz), Figure S23: 13C NMR of compound 13h (DMSO-d6, 400 MHz), Figure S24: 1H NMR
of compound 13i (DMSO-d6, 400 MHz), Figure S25: 13C NMR of compound 13i (DMSO-d6, 400 MHz), Figure
S26: HR-MS of compound 13i, Figure S27: 1H NMR of compound 13j (DMSO-d6, 400 MHz), Figure S28: 13C NMR
of compound 13j (DMSO-d6, 400 MHz), Figure S29: HR-MS of compound 13j, Figure S30: 1H NMR of compound
13k (DMSO-d6, 400 MHz), Figure S31: 13C NMR of compound 13k (DMSO-d6, 400 MHz), Figure S32: HR-MS of
compound 13k, Figure S33: 1H NMR of compound 13l (DMSO-d6, 400 MHz), Figure S34: 13C NMR of compound
13l (DMSO-d6, 400 MHz), Figure S35: HR-MS of compound 13l, Figure S36: 1H NMR of compound 13m (DMSO-
d6, 400 MHz), Figure S37: 13C NMR of compound 13m (DMSO-d6, 400 MHz), Figure S38: HR-MS of compound
1
13m, Figure S39: H NMR of compound 13n (DMSO-d6, 400 MHz), Figure S40: 13C NMR of compound 13n
(DMSO-d6, 400 MHz), Figure S41: HR-MS of compound 13n.

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Author Contributions: S.-Y.Z., C.-F.L. and J.S. designed the research; S.-H.W., X.-J.P., Q.-R.L., Q.-Y.L. Y.-R.L.
and T.Z. performed the synthetic work. C.-F.L., Z.-Y.M., H.-L.L. M.-J.J. Y.-Y.H. and Y.-F.G. were responsible for
the direction of the biological research. Y.-B.Z. was also responsible for the correspondence of the manuscript.
All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: This research was funded by the National Natural Sciences Foundations of China (No.
81703541 for Sai-Yang Zhang and No. 81673322 for Yan-Bing Zhang) and China Postdoctoral Science Foundation
(No. 2018M632812 for Sai-Yang Zhang) and the Henan Scientific Innovation Talent Team, Department for
Education (No. 112ITSTHN001 for Wen Zhao, China). Henan Association of Science and Technology (No.
2020HYTP056 for Sai-Yang Zhang, China) and Science and Technology Department of Henan Province (No.
20202310144, for Sai-Yang Zhang, China).
Conflicts of Interest: The authors declare no conflict of interest.
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