Synthesis and anti-cancer activity of naphthopyrone derivatives

Article abstract of DOI:10.1016/j.tetlet.2021.153111

Coumarins are naturally evolving molecules that have a wide variety of activities. In medical chemistry and chemical biology, their structural and physicochemical properties make them a suitable scaffold. Herein, we designed and synthesized novel coumarin analogues-7-O-substituted pyridyl-4-methyl naphthopyrone derivatives and initially evaluated their anti-cancer activity. RTCA profiling reveals that compounds 6, 7 and 8, in particular 7, possess stronger in vitro cytotoxicity for A549 cells. All synthesized compounds have been characterized by 1H NMR spectra, elemental analysis, and mass spectrometry.

Full text of DOI:10.1016/j.tetlet.2021.153111

Tetrahedron Letters 73 (2021) 153111
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and anti-cancer activity of naphthopyrone derivatives
Ming Wang a,b,1, Shikun Jiang a,b,1, Minrui Liu
a,b,1
, Ling Xu a,b, Suresh Narva a,b,2
,
a,b
c,
⇑
a,b,
⇑
Annoor Awadasseid , Yanling Wu , Wen Zhang
a
Lab of Chemical Biology and Molecular Drug Design, College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310014, China
Institute of Drug Development & Chemical Biology, Zhejiang University of Technology, Hangzhou 310014, China
Lab of Molecular Immunology, Virus Inspection Department, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Coumarins are naturally evolving molecules that have a wide variety of activities. In medical chemistry
and chemical biology, their structural and physicochemical properties make them a suitable scaffold.
Herein, we designed and synthesized novel coumarin analogues-7-O-substituted pyridyl-4-methyl naph-
thopyrone derivatives and initially evaluated their anti-cancer activity. RTCA profiling reveals that com-
pounds 6, 7 and 8, in particular 7, possess stronger in vitro cytotoxicity for A549 cells. All synthesized
compounds have been characterized by 1H NMR spectra, elemental analysis, and mass spectrometry.
Ó 2021 Elsevier Ltd. All rights reserved.
Received 18 February 2021
Revised 15 April 2021
Accepted 19 April 2021
Available online 22 April 2021
Keywords:
Synthesis
Naphthopyrone derivatives
Anti-cancer activity
Apoptosis
RTCA
Introduction
Coumarins are well known as compounds containing a benzo-
-pyrone mother structure, which are considered secondary
a
metabolites in many higher plant species, mostly in leaves, seeds,
and roots, especially in the Rutaceae and Umbrelliferae plants
[
[
1,2]. The first naturally occurring coumarin was isolated in 1820
3]. These phytochemical derivatives, some interesting oxygen-
containing heterocyclic compounds, are essential fluorescent fluo-
rophores [4] and excellent DNA intercalators [5]. The biological
activity of such naturally occurring compounds has become an
appealing point of the study. It seems to be a potential resource
for developing anti-cancer agents due to their different effects on
diseases and less damage to normal cells [6]. For instance, a cou-
marin-xanthoxyletin can induce S phase arrest and apoptosis in
human gastric adenocarcinoma SGC-7901 cells. Rasul et al.
observed its inhibitory effects on cells associated with DNA dam-
age [7]. The ability to induce selective DNA damage at the telom-
⇑
Corresponding authors at: Lab of Chemical Biology and Molecular Drug Design,
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou
3
Scheme 1. Synthesis of the naphthopyrone derivatives 6–11. Reagents and
conditions: (a) ethyl acetoacetate, 80% (v/v) H SO , room temperature (r.t.), 24 h;
b) 2-chloro-5-nitropyridine, K CO , acetone, reflux, 7 h; (c) zinc powder, CH OH, r.
t., 1 h; (d) 3-bromopropionyl chloride, Et3N, CH Cl , ice bath, 0.5 h; (e) ammonia
derivatives, K2CO3, KI, MeCN, 12 h; (f) HCl gas, CH Cl , r.t., 0.5 h.
10014, China.
2
4
E-mail addresses: ylwu@cdc.zj.cn (Y. Wu), wzhang63@zjut.edu.cn (W. Zhang).
These authors contributed equally to this work.
Present address: The University of Houston, Houston, Texas 77204, USA.
(
2
3
3
1
2
2
2
2
2
https://doi.org/10.1016/j.tetlet.2021.153111
040-4039/Ó 2021 Elsevier Ltd. All rights reserved.
0

M. Wang, S. Jiang, M. Liu et al.
Tetrahedron Letters 73 (2021) 153111
Scheme 2. The proposed mechanism for the formation of naphthopyrone derivatives 6a-11a starting from the compound 4.
Fig. 1. The cell index curve of A549 cells exposed to different concentrations of compounds 6 (a)-11 (f). ‘‘Control” and ‘‘DMSO” denotes RPMI1640 as a negative control and
.5% DMSO solution in RPMI 1640 as a control, respectively. Each trace is typical of 3 replicates.
0
eric level and promote apoptosis and senescence of tumor cells is
experimentally proven [8]. Coumarins exhibit anti-cancer biologi-
cal properties, highlighting great therapeutic importance in medic-
inal chemistry because they display some fascinating
pharmacological properties. To date, lots of studies have indicated
that coumarins possess a variety of
2

M. Wang, S. Jiang, M. Liu et al.
Tetrahedron Letters 73 (2021) 153111
improve hydrophilicity and the interaction of compounds with
their targets, such as DNA, through the cationic functional side
chain groups directing toward DNA grooves and then interacting
with negatively charged phosphate groups [21]. The naphthopy-
rone derivatives were achieved through reactions: firstly, we used
the Pechmann condensation reaction to synthesize the backbone 2
in high yield. Secondly, 7-hydroxy naphthopyranone was subjected
to nucleophilic substitution reaction to obtain compound 3.
Thirdly, compound 4 was synthesized through 3 being reduced
with zinc dust in methanol, and subsequent acylation process gave
rise to acrylamide derivatives 5. Lastly, acrylamide derivatives
were reacted with dimethylamine, diaethylamin, diethanolamine,
pyrolidine, piperidine, and morpholine, respectively, to obtain cor-
responding naphthopyrone precursors 6a-11a. Then 6a-11a ere
followed by reacting with hydrochloride gas in dichloromethane
solution to afford the target compounds 6–11. The kind of naph-
thopyranone derivatives comprises three parts: naphthopyranone,
pyridine ring, and positively charged nitrogen-containing side
chain.
Fig. 2. Apoptosis of A549 cells by RTCA in the presence/absence of naphthopyrone
derivatives 6–11. Cell killing rates are calculated based on areas under concentra-
tion–time curve (AUC) (Fig. 1). ‘‘Control” and ‘‘DMSO” denotes RPMI1640 as a
negative control and 0.5% DMSO solution in RPMI 1640 as a control, respectively.
Results and discussion
A549 cells were treated with compounds 6–11 with 0, 0.5, 1, 2, 4 and 8 lM for 48 h,
respectively. Experiments were completed in 3 replicates.
Synthesis
pharmacological activities, such as: anti-inflammatory [9],
antimicrobial [10], anti-viral [11], anti-oxidant [12], antinocicep-
tive [13], anti-tumor [14], antiasthmatic, antidepressant [15],
anti-HIV [16], antituberculosis [17], anti-Alzheimer [18], anti-
influenza [19], antihyperlipidemic [20]. Thus, a large number of
samples are required in the development of drug research and
application.
However, obtaining many coumarins only from natural biolog-
ical resources will result in severe resource scarcity and environ-
mental problems. Therefore, getting them by synthesis is the
preferred method to solve the sample source’s question; what is
more, the synthesis of active natural products and structure opti-
mization can obtain more valuable medicines. In addition, nitro-
gen-containing heterocycles represent a key structural motif in
heterocyclic chemistry and occupy a prominent position for
research with ample opportunities to synthesize novel drugs.
Hence, in this work, we introduce a substituted pyridine ring at
the 7-hydroxy position of naphthopyrone (also named: benzo-
coumarin), which is coumarin analogues having a good embedding
activity for DNA. Six naphthopyrone-pyridine conjugates 6–11
Naphthopyrone derivatives have recently been synthesized in
our laboratory and start from 1,5-dihydroxynaphthalene and ethyl
acetoacetate through a microwave-assisted Pechmann condensa-
tion to obtain 2 according to literature [22]. Then, after nucle-
ophilic substitution reaction took place between 7-hydroxy
naphthopyranone (2) and 2-chloro-5-nitropyridine to afford 3, 3
was turned into amino compound 4 in the presence of activating
zinc dust. The next step was bimolecular elimination between 4
and 3-bromopropionyl chloride to get an acrylamide derivative
(5) in the presence of an organic base. Subsequently, 1,4-conjugate
additions of secondary amines to the electron-poor alkene (5) pro-
duced precursors 6a-11a. The mechanism for the formation of
naphthopyrone coumarins 5, 6a-11a (Scheme 2) was summarized.
Finally, the target compounds (6–11) were conveniently prepared
by salifying with dry hydrogen chloride gas in dichloromethane.
These compounds were characterized (Electronic Supplementary
Information (ESI), S1.1–1.7, Figs. S1-S12).
At the beginning of the experiment, we attempted another syn-
thetic route to get target compounds 6–11 under examined differ-
ent conditions (Scheme S1, Table S1), but failed, because
nucleophilic substitution reaction of 2 to electron-poor compounds
15–20 did not occur under our experimental conditions. This is
(Scheme 1) with positive nitrogen-containing side chains were
designed and synthesized. These side chains can effectively
Table 1
The killing effect and IC50 value (
lM) of compounds 6–11 on A549 cells. The values are expressed as mean ± SD (triplicates).
a
CompoundConcentration
6AUC-48 (Cell
killing )
7AUC-48(Cell
killing)
8AUC-48(Cell killing) 9AUC-48(Cell
10AUC-48(Cell
killing)
11AUC-48(Cell
killing)
b
(l
M)
killing)
DMSO (Control)
139.9 ± 2.6
124.7 ± 2.3
96.0 ± 1.7
78.3 ± 1.4
(18.4 ± 0.6)
53.1 ± 1.2
(44.7 ± 0.4)
34.9 ± 0.5
(63.4 ± 1.7)
30.2 ± 0.4
(68.5 ± 1.5)
18.2 ± 0.2
(81.0 ± 1.6)
1.49 ± 0.27
123.6 ± 2.3
111.4 ± 2.1
(9.87 ± 0.10)
88.4 ± 1.5 (28.5 ± 0.3) 49.4 ± 1.1
(28.8 ± 0.7)
60.0 ± 1.1 (51.5 ± 0.4) 52.8 ± 1.0
(23.9 ± 0.4)
56.4 ± 0.6 (54.4 ± 0.3) 39.9 ± 0.6
(42.5 ± 0.5)
69.4 ± 1.6
50.2 ± 1.2
(27.7 ± 0.4)
70.1 ± 1.3
69.1 ± 1.1
(1.43 ± 0.03)
56.7 ± 1.3
(19.1 ± 0.4)
43.1 ± 0.7
(38.5 ± 0.5)
44.2 ± 0.9
(36.9 ± 0.6)
34.5 ± 0.8
(50.8 ± 1.2)
7.79 ± 0.62
67.2 ± 1.4
51.2 ± 1.0
(23.8 ± 0.4)
49.1 ± 0.9
(26.9 ± 0.3)
45.2 ± 0.7
(32.7 ± 0.4)
41.1 ± 0.6
(38.8 ± 0.5)
49.8 ± 0.8
(25.9 ± 0.4)
65.0 ± 2.6
0.5
1.0
2.0
4.0
8.0
(
10.9 ± 0.5)
108.7 ± 2.1
22.3 ± 0.7)
75.4 ± 1.1
46.1 ± 0.6)
50.5 ± 1.0
63.9 ± 1.4)
33.2 ± 0.5
76.3 ± 1.3)
2.57 ± 0.12
(
(
(
44.3 ± 0.4 (64.2 ± 1.2) 38.6 ± 0.9
(44.3 ± 0.8)
3.06 ± 0.45
(
IC50c
18.2 ± 1.0
a
AUC-48 (compound): area under concentration–time curve 48 h after adding of compounds (Fig. 1).
Cell killing: 100% Â [AUC-48 (Control) – AUC-48 (compound)]/AUC-48 (Control). The Cell killing rate of cells was 0 for the negative control group. AUC-48 (Control): area
b
under concentration–time curve 48 h after addition of 0.5% DMSO solution in RPMI 1640 [23].
c
IC50: Drug concentration producing 50% cancer cell death.
3

M. Wang, S. Jiang, M. Liu et al.
Tetrahedron Letters 73 (2021) 153111
possibly because 2-position of the pyridine cycle in 15–20 pos-
sesses less electron deficiency than the pyridine cycle in 2-
chloro-5-nitropyridine.
beneficial to the anti-cancer activity of these coumarin analogues.
Further evaluation of druggability is underway.
Declaration of Competing Interest
Cytotoxicity
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
To evaluate the cytotoxicity of naphthopyrone derivatives 6–11
to a type of human lung adenocarcinoma cell line (A549, supplied
by China General Microbiological Culture Collection Center
(
CGMCC, Beijing, China)); a real-time cell analysis (RTCA) tech-
Acknowledgments
nique was used to monitor changes of cells dynamically, including
cell number, kinetic behavior of cell apoptosis, and so on, exposed
to 0, 0.5, 1.0, 2.0, 4.0, and 8.0 lM of examined compounds. A chem-
This research was supported by the National Natural Science
Foundation of China (NSFC, grant numbers 21572207, 21877101),
the Zhejiang Leading Innovation and Entrepreneurship Team
(grant number 2018R01015) and the Zhejiang Provincial Key Disci-
pline of Chemical Biology.
ically induced decrease in a cell index (CI) value, which corre-
sponds to cell number, shape, and substrate attachment,
indicates that fewer cells interact with the microelectrodes due
to diminished viability or disruption of the cellular footprint. At
the same time, an increased CI can reflect higher cell numbers,
increased cell adhesion, or changes in cell morphology (e.g.,
increased cell/electrode contact area due to cell spreading). Based
on experimental curves (CI vs. time), IC50 of drugs inhibiting cell
lines can be calculated by RTCA system software. Therefore, this
methodology can provide useful analyses for various viable cell
activities, drug-induced cellular apoptosis, and kinetics of
cytotoxicity responses for drugs. As shown in Fig. 1, cell culture
medium alone as a negative control and 0.5% DMSO only as a ref-
erence almost did not affect the viability of tested cancer cells.
We found that cancer cells’ viability lowered and the time
required to induce the cell apoptosis decreased with increasing
compounds. Still, it was noteworthy that only little changes with
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2021.153111.
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