Coumarin sulfonamides and amides derivatives: Design, synthesis, and antitumor activity in vitro

Article abstract of DOI:10.3390/molecules26040786

Coumarins possesses immeasurable antitumor potential with minimum side effects depending on the substitutions on the basic nucleus, which exhibits great prospects for antitumor drug development. In an attempt to develop novel antitumor candidates, a series of coumarin sulfonamides and amides derivatives were designed and synthetized. The majority of these derivatives showed good cytotoxic activity against MDA-MB-231 and KB cell lines, among which compound 9c was the most potent against MDA-MB-231 cells, with IC50 value of 9.33 μM, comparable to 5-fluorouracil. Further investigation revealed that compound 9c had versatile properties against tumors, including inhibition of cell migration and invasion as well as inducing apoptosis. Reactive oxygen species (ROS) assay and western blotting analysis suggested that compound 9c promoted cancer cell apoptosis by increasing ROS levels and upregulating the expression of caspase-3 in MDA-MB-231 cells. These results indicated that compound 9c could be promising lead compound for further antitumor drug research.

Full text of DOI:10.3390/molecules26040786

molecules
Article
Coumarin Sulfonamides and Amides Derivatives: Design,
Synthesis, and Antitumor Activity In Vitro
Jing Zhang 1, Yaling Tan 1, Guorong Li 1, Lexian Chen 1, Minyi Nie 1, Zhaohua Wang 2 and Hong Ji 1,
*
1
Key Laboratory of Molecular Target & Clinical Pharmacology and the State Key Laboratory of Respiratory
Disease, School of Pharmaceutical Sciences & the Fifth Affiliated Hospital, Guangzhou Medical University,
Guangzhou 511436, China; ZhangJingde163yx@163.com (J.Z.); tanyalinglove@163.com (Y.T.);
13423686258@163.com (G.L.); chenlexian666@163.com (L.C.); nieminyi6@163.com (M.N.)
School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou 511436, China;
gy_wzhh@163.com
2
*
Correspondence: hji@gzhmu.edu.cn; Tel.: +86-20-37103256
Abstract: Coumarins possesses immeasurable antitumor potential with minimum side effects de-
pending on the substitutions on the basic nucleus, which exhibits great prospects for antitumor drug
development. In an attempt to develop novel antitumor candidates, a series of coumarin sulfon-
amides and amides derivatives were designed and synthetized. The majority of these derivatives
showed good cytotoxic activity against MDA-MB-231 and KB cell lines, among which compound
9c was the most potent against MDA-MB-231 cells, with IC50 value of 9.33 µM, comparable to
5-fluorouracil. Further investigation revealed that compound 9c had versatile properties against
tumors, including inhibition of cell migration and invasion as well as inducing apoptosis. Reactive
oxygen species (ROS) assay and western blotting analysis suggested that compound 9c promoted
cancer cell apoptosis by increasing ROS levels and upregulating the expression of caspase-3 in
MDA-MB-231 cells. These results indicated that compound 9c could be promising lead compound
for further antitumor drug research.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Zhang, J.; Tan, Y.; Li, G.;
Chen, L.; Nie, M.; Wang, Z.; Ji, H.
Coumarin Sulfonamides and Amides
Derivatives: Design, Synthesis, and
Antitumor Activity In Vitro. Molecules
2021, 26, 786. https://doi.org/
10.3390/molecules26040786
Keywords: coumarin sulfonamides; coumarin amides; MDA-MB-231; antitumor
1. Introduction
Cancer is a widespread and lethal disease characterized by uncontrolled growth of
abnormal cells. It has been expected to be the leading cause of death in the future [1,2].
Academic Editors: Maria João Matos
and Claudiu T. Supuran
Although cancer research resulted in a range of innovative and promising therapeutic
approaches, the existing drugs for cancer treatment are frequently associated with several
drawbacks such as severe side effects, multidrug resistance, poor bioavailability. Therefore,
there is an urgent need for the development of more effective and safer anticancer agents.
Coumarins are a wide family of secondary metabolites found in various species of
Received: 17 January 2021
Accepted: 28 January 2021
Published: 3 February 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
plants but also fungi and microorganisms [
effects, especially anticancer activity [ ]. Many coumarins and their derivatives have
attracted considerable attention as cancer chemopreventive agents and also as cancer thera-
peutics [ 14]. Additionally, they have several attractive features, such as low molecular
3,4], which exhibit diverse pharmacological
5–7
8–
weight, simple structure, high bioavailability, high solubility in most of the organic solvents,
and low toxicity, which ensure them a prominent role as lead compounds in drug research
and development [15].
Copyright:
© 2021 by the authors.
The promising biological profile of coumarins as antitumor agents and their easy
synthetic modifications paved the way for design and synthesis of various coumarin deriva-
tives [16,17]. In the last decade, coumarin derivatives have been increasingly reported to
possess antitumor activity through different pharmacological mechanisms, among which
coumarin sulfonamides and amides derivatives have attracted great interest because of
their potency. Sabt et al. reported a series of novel coumarin-6-sulfonamides as antipro-
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
liferative agents [18] and found that compound
I
(Figure 1) was able to induce apoptosis
Molecules 2021, 26, 786. https://doi.org/10.3390/molecules26040786
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 786
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in HepG2 cells by the upregulation of Bax and downregulation of Bcl-2, besides boosting
caspase-3 levels. Yu et al. identified compound II as a potent inhibitor of the Hsp90
protein-folding machinery [19]. Compound III was founded to be a promising LOX and
hCA IX inhibitor [20].
O
CH3
NH
CH3
H
N
N
N
O
O
O
CH3
O
O
O
S
S
O
N
N
H3C
H3CO
HO
H
H
O
O
O
O
O
OH
Ⅰ
Ⅱ
Ⅲ
Figure 1. Chemical structures of compounds I–III.
In order to discover more potent and selective antitumor agents for further develop-
ment and as a continuation of our previous studies [21], we designed and synthesized
coumarin derivatives bearing sulfonamide and amide moieties. Their cytotoxic activity
was screened, and the antitumor effects of the most potent compound on MDA-MB-231
cell line were further investigated.
2. Results
2.1. Chemical Synthesis
The synthesis of coumarin sulfonamides derivatives 7a
Scheme 1. Compound were obtained by the sulfonylation of the commercially available
aromatic amine with methyl 2-chlorosulfonylacetate in triethylamine at room tempera-
with bromide in anhydrous DMF
–7b and 9a–9c were outlined in
3
1
2
ture. The alkylation of 2,4-dihydroxybenzaldehyde
4
5
◦
at 110 C provided compound
6
. The condensation of compound 3a with substituted
in anhydrous ethanol in the presence of piperidine gave coumarin sul-
7b. The reaction of compound and 4-(diethylamino) salicylaldehyde in
the same condition afforded the desired compound . The coumarin amides derivatives
12a 12c were synthesized as described in Scheme 2. The ethyl coumarin-3-carboxylate
11 was prepared by the condensation of 4-(diethylamino) salicylaldehyde with ethyl
malonate 10 in anhydrous ethanol in the presence of piperidine. The ammonolysis of
salicylaldehyde
fonamides 7a
6
–
3
8
9
–
8
compound 11 with aromatic amines
compound 12.
1 in anhydrous ethanol under reflux led to desired
2.2. Cytotoxicity
The in vitro cytotoxicity of the synthesized compounds (Table 1) was evaluated using
MTT assay against three human cancer cell lines MDA-MB-231 (human breast cancer
cell line), KB (human oral epidermoid carcinoma cell line), and HCT-116 (human colon
cancer cell line). 5-Fluorouracil was included as positive control. As shown in Table 2, the
majority of the compounds exhibited good inhibitory activities against MDA-MB-231 and
KB cell lines, while weak cytotoxicity was observed against HCT-116 cell line except for
compounds 9a and 9b. Almost all the compounds were more active than 5-fluorouracil
against KB cells. In particular, compounds 9b
effects than the rest of the compounds. The most potent compound 9c with the IC50 values
of 9.33 and 13.66 M against MDA-MB-231 and KB cells, respectively, was chosen for
further investigation.
, 9c and 12b showed stronger inhibitory
µ

Molecules 2021, 26, 786
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O
S
O
NH2
HN
OCH3
O
S
(CH3CH2)3N
rt, 3h
O
OCH3
+
Cl
O
O
R1
R1
2
1
3
1a: R1=H
1b: R1=Br
1c: R1=OCH3
3a: R1=H
3b: R1=Br
3c: R1=OCH3
O
O
H
NaHCO3, DMF
H
R2Br
+
110℃
HO
OH
R2O
OH
5
6
4
6a: R2=CH2CH3
5a: R2=CH2CH3
6b: R2=CH2CH2CH3
5b: R2=CH2CH2CH3
O
S
O
O
S
O
H
HN
OCH3
+
O
N
piperidine, EtOH
H
O
O
87℃
R2O
O
R2O
OH
7
6
3a
7a: R2=CH2CH3
7b: R2=CH2CH2CH3
6a: R2=CH2CH3
6b: R2=CH2CH2CH3
O
S
O
O
H
HN
OCH3
O
R1
O
piperidine, EtOH
S
N
+
H
O
87℃
(CH3CH2)2N
OH
(CH3CH2)2N
O
O
R1
9
8
3
3a: R1=H
9a: R1=H
3b: R1=Br
3c: R1=OCH3
9b: R2=Br
9c: R3=OCH3
Scheme 1. Synthesis of compounds 7a–7b and 9a–9c.
O
O
O
O
piperidine, EtOH
OCH2CH3
rt
H
OCH2CH3
+
H3CH2CO
(CH3CH2)2N
OH
(CH3CH2)2N
O
O
8
10
11
O
O
EtOH
reflux
N
R3
OCH2CH3
H2N
R3
H
+
(CH3CH2)2N
O
O
O
O
(CH3CH2)2N
1
11
12
12a: R3=H
12b: R3=Br
1a: R1=H
1b: R1=Br
12c: R3=OCH3
1c: R1=OCH3
Scheme 2. Synthesis of compounds 12a–12c.

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Table 1. The structures of coumarin sulfonamides and coumarin amides derivatives.
Compd
7a
Structure
Compd
9c
Structure
O
O
S
OCH3
S
O
O
N
N
H
H
O
O
(CH3CH2)2N
O
CH3CH2O
O
O
O
S
O
O
N
N
7b
9a
9b
12a
12b
12c
H
H
(CH3CH2)2N
O
O
CH3CH2CH2O
O
O
O
O
Br
S
O
O
N
N
H
H
(CH3CH2)2N
O
(CH3CH2)2N
O
O
S
O
O
Br
OCH3
N
H
N
H
O
O
(CH3CH2)2N
O
(CH3CH2)2N
O
Table 2. Inhibition of cancer cells proliferation by compounds 7a–7b, 9a–9c and 12a–12c.
IC50 (µM)
Compd
MDA-MB-231
KB
HCT-116
7a
7b
9a
9b
9c
12a
12b
12c
5-Fu
>100
>100
>100
79.46 ± 1.62
20.21 ± 0.42
34.94 ± 2.83
10.78 ± 0.13
13.66 ± 1.33
>100
14.22 ± 1.64
97.14 ± 2.43
94.15 ± 2.76
>100
>100
15.51 ± 1.59
17.26 ± 2.00
>100
17.13 ± 0.68
9.33 ± 1.81
13.33 ± 1.26
11.63 ± 0.98
17.01 ± 1.04
8.59 ± 0.52
>100
>100
>100
15.28 ± 0.31
In addition, it was observed that among coumarin sulfonamides 7a
diethylaminocoumarin sulfonamides 9a 9c exhibited higher activity than 7-alkoxycoumarin
sulfonamides 7a 7b, which implied the diethylamino group at C-7 position improved
potency. Coumarin sulfonamides 9a 9c had better cytotoxicity compared to amides deriva-
tives 12a 12c, suggesting the sulfonamide moiety at C-3 position of the coumarin core is
favored to the activity. Furthermore, the IC50 values of compounds 9a 9c and 12a 12c
–7b and 9a–9c, 7-
–
–
–
–
–
–
indicated that substituents on the benzene ring in the sulfonamides or amides had little
impact on the potency.
2.3. Inhibition of Wound Healing in MDA-MB-231 Cells by Compound 9c
The wound healing assay is a classic and common method used to investigate the
effect of antitumor drugs on tumor migration. We next assessed the effect of compound 9c
on the migration of MDA-MB-231 cells with a wound-healing assay. The results showed
that the scratch gaps in compound 9c (10 and 20 µM) treated MDA-MB-231 cells were
wider than the control with increased treating time (Figure 2). The cell migration was
inhibited, and the wound area was increased compared with the control in a concentration-
dependent manner.

Molecules 2021, 26, 786
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(a)
(b)
Figure 2. Compound 9c inhibited wound healing of MDA-MB-231 cells. (a) The wound was captured
at different time points (0, 24, and 48 h). (b) Quantification of the cell scrape results (n = 6) by using
One-Way ANOVA followed by post-hoc Tukey’s test. *** (p < 0.001), ** (p < 0.01), * (p < 0.05), ns
(not significant).
2.4. Inhibition of Compond 9c on the Invasion of MDA-MB-231 Cells
Transwell invasion assay was used to investigate the effect of compound 9c on the
invasion of MDA-MB-231 cells. Treatment of compound 9c significantly reduced (p < 0.01)
the number of invaded cells at the concentration of 20 µM (Figure 3).
2.5. Compound 9c Induces Apoptosis in MDA-MB-231 Cells
To further evaluate whether compound 9c can induce apoptosis, the Annexin V-
FITC/PI double-staining assay was applied with flow cytometer. After treatment with
compound 9c for 48 h, MDA-MB-231 cells were dyed by Annexin V-FITC and showed
green fluorescence, which indicates the early phase of apoptosis. The cells dyed by Annexin
V-FITC and PI showed red fluorescence inside with green fluorescence outside, which
corresponded to the late phase of apoptosis (Figure 4a). The cells treated with compound
9c (10 µM) had stronger fluorescence intensity compared with the control group, which
indicated that compound 9c promoted apoptosis of MDA-MB-231 cells. Meanwhile, as
shown in Figure 4b,c, when treated with different concentrations of compound 9c, the
percentages of apoptotic MDA-MB-231 cells increased from 3.85% in control cells to 7.72%,
20.11%, 40.71%, 53.50%, and 57.46%, respectively. The results implied that compound 9c
could induce the apoptosis of MDA-MB-231 cells in a concentration-dependent manner.

Molecules 2021, 26, 786
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(a)
(b)
Figure 3. Compound 9c inhibited invasion of MDA-MB-231 cells. (
a) Microscope images of the MDA-MB-231 cells that
invaded through the matrigel after exposure to different concentrations of compound 9c for 48 h. Scale bar: 400
) Quantification of the cell invasion results (n = 3) by using Kruskal–Wallis test followed by post-hoc Dunn’s test.
** (p < 0.01), ns (not significant).
µm.
(
b
(a)
(b)
Figure 4. Cont.

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(c)
Figure 4. Compound 9c induced apoptosis of MDA-MB-231 cells. (
morphology. Scale bar: 2 m. ( ) Dot plot depicting MDA-MB-231 cells treated with different concentrations of compound
9c for 48 h. ( ) Quantification of the cell apoptosis results (n = 3) by using One-Way ANOVA followed by post-hoc Tukey’s
test. *** (p < 0.001), ** (p < 0.01), ns (not significant).
a) Effect of compound 9c on MDA-MB-231 cells apoptosis
µ
b
c
2.6. Compound 9c Increases ROS Production in MDA-MB-231 Cells
We utilized the 20,70-dichlorofluorescin diacetate (DCFH-DA) fluorescence probe to
further evaluate the changes in intracellular ROS level after treating MDA-MB-231 cells
with compound 9c. Intracellular ROS can oxidize 20,70-dichlorofluorescin (DCFH) into the
fluorescent 20,70-dichlorofluorescein (DCF) whose intensity is proportional to the amount
of intracellular ROS. After these cells were subjected to various doses of compound 9c (10,
20, 30, and 40 µM), the fluorescence intensity increased obviously (Figure 5), revealing that
compound 9c induced intracellular ROS production in concentration-dependent manner.
(a)
(b)
Figure 5. Compound 9c increased ROS production of MDA-MB-231 cells. (
a) MDA-MB-231 cells were treated with different
concentrations of compound 9c for 48 h. ( ) Quantification of the ROS production (n= 3) by using One-Way ANOVA
b
followed by post-hoc Tukey’s test. *** (p < 0.001), ** (p < 0.01), ns (not significant).
2.7. Western Blot Analysis
Next, Western blot analysis was applied to understand the molecular mechanism of
apoptosis induced by compound 9c. The protein expression levels of caspase-3 and Bcl-2
were detected since caspase-3 played a crucial role in apoptotic pathways by cleaving a
variety of key cellular proteins, and Bcl-2 is the well-known anti-apoptotic marker. After
exposure of MDA-MB-231 cells to compound 9c, the expression level of caspase-3 was
dramatically increased, while no obvious effect on the expression of Bcl-2 was observed
(Figure 6).

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Figure 6. Compound 9c affected caspase-3 and Bcl-2 expression levels of MDA-MB-231 cells. MDA-
MB-231 cells were seeded in 6-well plates and treated with different concentrations of compound 9c
for 48 h. The protein expression levels of caspase-3 and Bcl-2 were evaluated by western blot,
served as a loading control.
β-actin
3. Discussion
Coumarins and their derivatives have been regarded as an important family of an-
ticancer lead compounds, which exhibit diverse effects such as anti-proliferation, anti-
invasion, anti-metastasis, anti-angiogenesis and inducing apoptosis. The various mecha-
nisms of their anticancer action have been widely explored [22–28], including inhibition of
carbonic anhydrase, PI3K/AKT/mTOR signaling pathway, microtubule polymerization,
monocarboxylate transporters, hypoxia-inducible factor-1, acting on apoptosis proteins,
inhibiting tumor multidrug resistance, and regulating ROS.
In this study, coumarin sulfonamide 9c with the best antiproliferative activity against
MDA-MB-231 and KB cell lines were obtained by modification of the basic structure of
coumarin. The effects of compound 9c on the migration and invasion of MDA-MB-231
cells have been further investigated since the ability of cancer cells to migrate and invade
into the surrounding tissue is the hallmark of tumor metastasis [29]. Treatment using
compound 9c has shown significant reduction in the percentage of migration and invasion
of MDA-MB-231 cells in a concentration-dependent manner.
Apoptosis, an immunogenic programmed cell death, eliminates unhealthy, unwanted
cells and maintains surrounding tissue [30]. In cancer, evasion of cell death is one of the ma-
jor mechanisms for abnormal cells to transform themselves into malignant ones [31]. Thus,
drugs that can induce apoptosis, specifically in malignant and metastatic cells, would be the
most promising cancer treatment candidates. Annexin V-FITC/PI double-staining assay
combined with flow cytometry analysis confirmed that compound 9c induced apoptosis in
MDA-MB-231 cells significantly in a concentration-dependent manner. Most antitumor
reagents that can induce apoptosis, including many first-line chemotherapy drugs such as
cisplatin, are known to provoke oxidative stress by generating excess ROS, which suggests
that abnormal ROS generation is closely associated with apoptosis [32]. Our results of
ROS assay showed that ROS levels in MDA-MB-231 cells were significantly increased after
treatment with compound 9c.
The activation of the key apoptotic protein caspase-3 [33] can cleave regulatory pro-
teins essential for cell survival and maintenance as well as poly (ADP-ribose) polymerase
(PARP), which is involved in DNA repair and programmed cell death, thereby promoting
apoptosis. Bcl-2 as an anti-apoptotic protein has been shown to enhance cell survival by in-
hibiting apoptosis and enhance the sensitivity of tumor cells to chemotherapy drugs [34–36].
Western blotting analysis demonstrated that the expression of caspase-3 was markedly
upregulated, whereas little changes in the expression level of Bcl-2 were observed after
treating with compound 9c. These results suggested that compound 9c induced apop-
tosis by increasing ROS levels and upregulating caspase-3 expression in MDA-MB-231
cancer cells.

Molecules 2021, 26, 786
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However, the anticancer mechanism of coumarin sulfonamides needs to be further
elucidated. Their anticancer potential, security and pharmacokinetic properties in vivo
were also left to be investigated. Anyway, this study offers a start point for further elabora-
tion to develop anticancer reagents with coumarin sulfonamide scaffold. More researches
which link the anticancer effect, safety, and bioavailability with structure modification are
expected to accelerate the development of coumarin derivatives for cancer treatment.
4. Materials and Methods
4.1. Chemical
4.1.1. Syntheses
All melting points were taken on an X-6 microscope melting point instrument (Bjfuka,
Beijing, China) and are uncorrected. Infrared spectra were determined with a TENSORII
spectrometer (Bruker, Karlsruhe, Germany). NMR spectra were measured using 300 MHz
Bruker AV III spectrometers (Bruker, Karlsruhe, Germany) and 400 MHz JNM-ECZ400S/L1
spectrometers (Varian INOVA, Palo Alto, CA, USA) with tetramethylsilane (TMS) as an
internal standard. The following abbreviations were used to designate the multiplicities:
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, brs = broad singlet. Cou-
pling constant (J) is reported in Hertz (Hz). HR-MS spectra were obtained on Finnigan
LCQ and Micromass Auto Spec Ultima-Tof spectrometer (Thermo, Boston, Massachusetts,
USA). Analytic thin-layer chromatography (TLC) was performed on precoated silica gel
60 F254 plates (Qingdao Haiyang, Qingdao, Shandong Province, China) using petroleum
ether-ethyl acetate as solvent system. All the chemicals were of analytic grade. The de-
tailed preparation procedures of intermediates, byproducts, and target compounds are
described below.
4.1.2. General Procedure for the Synthesis of Compounds 3a–3c
Methyl 2-chlorosulfonylacetate
2 (500 mg, 2.90 mmol) was added dropwise to a
solution of aromatic amines (4.35 mmol) in Et3N (10 mL). The reaction mixture was
1
stirred at room temperature for 3 h. After completion of the reaction, a solution of 10% HCl
was added slowly to the mixture to adjust the pH to 7. The mixture was then extracted
with DCM (12 mL
×
3). The combined organic layer was dried over anhydrous Na2SO4
and evaporated in a vacuum to give a crude residue which was purified by column
chromatography (cyclohexane: ethyl acetate = 15:1–1:1) to yield compounds 3a
1H-NMR spectra of compounds 3a–3c are in the Supplementary Materials.
–3c. The
1
Methyl 2-(N-phenylsulfamoyl)acetate (3a) [37] Yellow solid, yield: 76.3%. H-NMR
(300 MHz, CDCl3) 7.42–7.30 (m, 4H, -C6H5), 7.29–7.21 (m, 1H, -C6H5), 7.05 (brs, 1H,
δ
-NH-), 3.96 (s, 2H, -CH2-COO-), 3.82 (s, 3H, -OCH3). HR-MS (ESI): calcd for C9H12NO4S
[M + H]+ 230.0487, found 230.0481.
1
Methyl 2-(N-(4-bromophenyl)sulfamoyl)acetate (3b) [37]. Yellow solid, yield: 86.2%. H-
NMR (300 MHz, CDCl3) δ 7.52–7.46 (m, 2H, -C6H5), 7.25–7.18 (m, 2H, -C6H5), 3.94 (s, 2H,
-CH2-COO-), 3.82 (s, 3H, -OCH3). HR-MS (ESI): calcd for C9H11BrNO4S [M + H]+ 307.9592,
found 307.9603.
Methyl 2-(N-(4-methoxyphenyl) sulfamoyl) acetate (3c) [37]. Yellow solid powder, yield:
1
88.2%. H-NMR (300 MHz, CDCl3)
δ
7.30–7.25 (m, 2H, -C6H5), 6.91–6.86 (m, 2H, -C6H5),
6.82 (brs, 1H, -NH-), 3.91 (s, 2H, -CH2-COO-), 3.83 (s, 3H, -OCH3), 3.80 (s, 3H, -OCH3).
HR-MS (ESI): calcd for C10H14NO5S [M + H]+ 260.0593, found 260.0602.
4.1.3. General Procedure for the Synthesis of Compounds 6a–6b
2,4-dihydroxybenzaldehyde
4 (200 mg, 1.45 mmol) and NaHCO3 (240 mg, 2.89 mmol)
were dissolved in anhydrous DMF (2 mL), followed by the addition of the bromide
5
(1.45 mmol). The reaction mixture was stirred at 110 ◦C. After completion of the reaction,
the reaction mixture was diluted with DCM (20 mL) and washed with water (10 mL) and
saturated brine (10 mL). The organic layer was dried over anhydrous Na2SO4 and evapo-
rated in a vacuum to give a crude residue which was purified by column chromatography

Molecules 2021, 26, 786
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(cyclohexane: ethyl acetate = 200:1) to yield compounds 6a
–
6b. The 1H-NMR spectra of
compounds 6a–6b are in the Supplementary Materials.
1
4-Ethoxy-2-hydroxybenzaldehyde (6a) [38]. Light yellow liquid, yield: 51.2%. H-NMR
(300 MHz, CDCl3) δ 11.48 (s, 1H, -CHO), 9.70 (s, 1H, -OH), 7.41 (d, J = 8.7 Hz, 1H, -C6H5),
6.52 (dd, J = 8.7, 1.8 Hz, 1H, -C6H5), 6.40 (d, J = 2.4 Hz, 1H, -C6H5), 4.08 (q, J = 7.0 Hz, 2H,
-OCH2CH3), 1.43 (d, J = 7.2 Hz, 3H, -OCH2CH3). HR-MS (ESI): calcd for C9H11O3 [M + H]+
167.0708, found 167.0704.
1
2-hydroxy-4-propoxybenzaldehyde (6b) [38] Light yellow liquid, yield: 57%. H-NMR
(300 MHz, CDCl3) δ 11.48 (s, 1H, -CHO), 9.70 (d, J = 0.4 Hz, 1H, -OH), 7.41 (d, J = 8.7 Hz,
1H, -C6H5), 6.55–6.51 (m, 1H, -C6H5), 6.41 (d, J = 2.2 Hz, 1H, -C6H5), 3.97 (t, J = 7.2 Hz,
2H, -OCH2CH2CH3), 1.84 (m, 2H, -OCH2CH2CH3), 1.04 (t, J = 7.0 Hz, 3H, -OCH2CH2CH3).
HR-MS (ESI): calcd for C10H13O3 [M + H]+ 181.0865, found 181.0862.
4.1.4. General Procedure for the Synthesis of Compounds 7a–7b
Under a N2 atmosphere, compound 3a (0.388 mmol, 89 mg) and compound 6 (0.466 mmol)
were dissolved in anhydrous ethanol (2 mL), and piperidine (1.4 mmol, 0.1 mL) was added
dropwise. The resulting mixture was stirred at 87 ◦C. After completion of the reaction, the
solvent was removed under reduced pressure and water (10 mL) was added to the residue
and then extracted with DCM (10 mL
×
3). The combined organic layers were dried over
anhydrous Na2SO4 and evaporated in a vacuum to give a crude residue which was purified
by column chromatography (cyclohexane: ethyl acetate = 100:1–5:1) to yield compounds
7a–7b. The NMR spectra of compounds 7a–7b are in the Supplementary Materials.
7-Ethoxy-2-oxo-N-phenyl-2H-chromene-3-sulfonamide (7a). Light yellow solid, yield:
50.2%. mp: 204–206◦C; IR (KBr)
CDCl3)
ν
max (cm−1) 3288, 1722, 1595, 1494; 1H-NMR (300 MHz,
8.35 (s, 1H, -C6H5), 7.45 (d, J = 8.7 Hz, 1H, -C6H5), 7.30 (d, J = 9.2, 7.2 Hz,
δ
4H, -C6H5), 7.13 (d, J = 6.6 Hz, 1H, -C6H5), 6.88 (d, J = 7.7 Hz, 1H, -C6H5), 6.80 (s, 1H,
-C6H5), 4.27 (q, J = 7.2 Hz, 2H, -OCH2CH3), 1.46 (t, J = 7.2 Hz, 3H, -OCH2CH3). 13C-NMR
(75 MHz, DMSO-d6) δ 164.50 (-COO-), 156.85 (-CH=C), 155.11 (-CH=C), 149.06 (-CH=C),
137.61 (-CH=C), 132.19 (-CH=C), 129.28 (-CH=C), 124.30 (-CH=C), 120.75 (-CH=C), 120.20
(-CH=C), 114.06 (-CH=C), 110.55 (-CH=C), 100.94 (-CH=C), 64.60 (-OCH2CH3), 14.28
(-OCH2CH3). HR-MS (ESI): calcd for C17H16NO5S [M + H]+ 346.0749, found 346.0755.
7-propoxy-2-oxo-N-phenyl-2H-chromene-3-sulfonamide (7b). Light yellow solid, yield: 66.5%.
mp: 200–202◦C; IR (KBr) max (cm−1) 3314, 1717, 1615, 1546; 1H-NMR (300 MHz, CDCl3)
ν δ
8.34 (s, 1H, -C6H5), 7.45 (d, J = 8.8 Hz, 1H, -C6H5), 7.24 (dd, J = 9.4, 7.6 Hz, 4H, -C6H5), 7.13 (t,
J = 7.4 Hz, 1H, -C6H5), 6.90 (d, J = 8.7 Hz, 1H, -C6H5), 6.81 (s, 1H, -C6H5), 4.00 (t, J = 6.5 Hz,
2H, -OCH2CH2CH3), 1.85 (dd, J = 14.0, 7.0 Hz, 2H, -OCH2CH2CH3), 1.05 (t, J = 7.4 Hz, 3H,
-OCH2CH2CH3). 13C-NMR (75 MHz, DMSO-d6)
δ 164.62 (-COO-), 156.81 (-CH=C), 155.09
(-CH=C), 149.03 (-CH=C), 137.17 (-CH=C), 132.14 (-CH=C), 129.13 (-CH=C), 124.05 (-CH=C),
120.75 (-CH=C), 119.92 (-CH=C), 113.98 (-CH=C), 110.52 (-CH=C), 100.91 (-CH=C), 70.19
(-OCH2CH2CH3), 21.70 (-OCH2CH2CH3), 10.21 (-OCH2CH2CH3). HR-MS (ESI): calcd for
C18H18NO5S [M + H]+ 360.0906, found 360.0897.
4.1.5. General Procedure for the Synthesis of Compounds 9a–9c
Under a N2 atmosphere, compound
3 (1 mmol) and 4-(diethylamino) salicylaldehyde
8
(1.2 mmol, 232mg) were dissolved in anhydrous ethanol and piperidine (1.4 mmol,
0.1 mL) was then added dropwise. The resulting mixture was heated under reflux with
stirring for 30 min. The solvent was removed under reduced pressure and water (10 mL)
was added to the residue, and then extracted with DCM (10 mL
organic layers were dried over anhydrous Na2SO4 and evaporated in a vacuum to give
a crude residue which was purified by column chromatography (cyclohexane: ethyl
×
3). The combined
acetate = 10:1) to yield compounds 9a–9c. The NMR spectra of compounds 9a–9c are in the
Supplementary Materials.
7-(Diethylamino)-2-oxo-N-phenyl-2H-chromene-3-sulfonamide (9a). White solid, yield:
61.3%. mp: 211–213 ◦C. IR (KBr) max (cm−1) 3273, 2974, 1708, 1614; 1H-NMR (300 MHz,
ν

Molecules 2021, 26, 786
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DMSO-d6)
δ
10.19 (s, 1H, -NH-), 8.55 (s, 1H, -C6H5), 7.64 (d, J = 9.1 Hz, 1H, -C6H5),
7.29–7.08 (m, 4H, -C6H5), 6.98 (t, J = 7.2 Hz, 1H, -C6H5), 6.76 (dd, J = 9.1, 2.3 Hz, 1H,
-C6H5), 6.51 (d, J = 2.1 Hz, 1H, -C6H5), 3.44 (q, J = 6.9 Hz, 4H, -NCH2CH3), 1.09 (t, J = 7.0
Hz, 6H, -NCH2CH3). 13C-NMR (75 MHz, DMSO-d6)
δ 157.73 (-COO-), 155.75 (-CH=C),
152.99 (-CH=C), 148.47 (-CH=C), 137.62 (-CH=C), 132.05 (-CH=C), 129.04 (-CH=C), 123.68
(-CH=C), 119.59 (-CH=C), 114.85 (-CH=C), 110.13 (-CH=C), 105.96 (-CH=C), 96.01 (-CH=C),
44.41 (-NCH2CH3), 12.23 (-NCH2CH3). HR-MS (ESI): calcd for C19H21N2O4S [M + H]+
373.1222, found 373.1229.
N-(4-bromophenyl)-7-(diethylamino)-2-oxo-2H-chromene-3-sulfonamide (9b). White solid,
1
yield: 59.2%. mp: 192–194◦C. IR (KBr)
ν
max
(cm−1) 3267, 2976, 1711, 1613; H-NMR
(300 MHz, CDCl3) δ 8.17 (s, 1H, -C6H5), 7.38–7.33 (m, 2H, -C6H5), 7.30 (d, J = 9.0 Hz, 1H,
-C6H5), 7.14–7.07 (m, 2H, -C6H5), 6.61 (dd, J = 9.0, 2.5 Hz, 1H, -C6H5), 6.44 (d, J = 2.3 Hz,
1H, -C6H5), 3.44 (q, J = 7.1 Hz, 4H, -NCH2CH3), 1.22 (t, J = 7.1 Hz, 6H, -NCH2CH3).
13C-NMR (75 MHz, DMSO-d6)
δ 157.73 (-COO-), 155.75 (-CH=C), 152.99 (-CH=C), 148.47
(-CH=C), 137.62 (-CH=C), 132.05 (-CH=C), 129.04 (-CH=C), 123.68 (-CH=C), 119.59 (-CH=C),
114.85 (-CH=C), 110.13 (-CH=C), 105.96 (-CH=C), 96.01 (-CH=C), 44.41(-NCH2CH3), 12.23
(-NCH2CH3). HR-MS (ESI): calcd for C19H20BrN2O4S [M + H]+ 451.0327; found: 451.0334.
7-(Diethylamino)-N-(4-methoxyphenyl)-2-oxo-2H-chromene-3-sulfonamide (9c). White solid,
yield: 42.8%.mp: 198–199◦C. IR (KBr)
(300 MHz, CDCl3)
ν
(cm−1) 3271, 2928, 1796, 1615; 1H-NMR
max
δ
8.12 (s, 1H, -C6H5), 7.27 (d, J = 6.6 Hz, 1H, -C6H5), 7.16 (d, J = 8.8 Hz,
2H, -C6H5), 6.76 (d, J = 8.8 Hz, 2H, -C6H5), 6.60 (dd, J = 9.0, 2.0 Hz, 1H, -C6H5), 6.46 (s,
1H, -C6H5), 3.72 (s, 3H, -OCH3), 3.42 (q, J = 7.1 Hz, 4H, -NCH2CH3), 1.22 (d, J = 7.1 Hz,
6H, -NCH2CH3).13C-NMR (75 MHz, DMSO-d6)
δ 157.70 (-COO-), 156.37 (-CH=C), 155.92
(-CH=C), 152.90 (-CH=C), 148.20 (-CH=C), 131.95 (-CH=C), 129.97 (-CH=C), 123.14 (-CH=C),
115.03 (-CH=C), 114.22 (-CH=C), 110.0 (-CH=C)9, 105.99 (-CH=C), 96.03 (-CH=C), 55.09
(-OCH3), 44.40 (-NCH2CH3), 12.25 (-NCH2CH3). HR-MS (ESI): calcd for C20H23N2O5S [M
+ H]+: 403.1328; found: 403.1333.
4.1.6. Synthesis of Compound 11
Under a N2 atmosphere, 4-(diethylamino) salicylaldehyde
8 (1 mmol, 193 mg) and
diethyl malonate 10 (2 mmol, 320 mg) were dissolved in anhydrous ethanol (10 mL) and
piperidine (1.4 mmol, 0.1 mL) was added dropwise. The reaction mixture was stirred
at room temperature for 3 h. 10% HCl was then added into the reaction mixture to
adjust the pH to 8. The mixture was then extracted with ethyl acetate (10 mL
×
3). The
combined organic layer was dried over anhydrous Na2SO4 and evaporated in a vacuum
to give a crude residue which was purified by column chromatography (petroleum ether:
ethyl acetate = 8:1) to yield compound 11. The NMR spectra of compound 11 are in the
Supplementary Materials.
Ethyl 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylate (11) [39]. Yellow solid; yield
1
75%. H-NMR (400 MHz, CDCl3)
δ 8.40 (s, 1H, -C6H5), 7.33 (d, J = 8.9 Hz, 1H, -C6H5),
6.58 (dd, J = 8.9, 2.4 Hz, 1H, -C6H5), 6.43 (d, J = 2.3 Hz, 1H, -C6H5), 4.34 (q, J = 7.1 Hz, 2H,
-OCH2CH3), 3.42 (q, J = 7.1 Hz, 4H, -NCH2CH3), 1.37 (t, J = 7.1 Hz, 3H, -OCH2CH3), 1.21 (t,
J = 7.1 Hz, 6H, -NCH2CH3). 13C-NMR (100 MHz, CDCl3)
δ 164.37 (-COO-), 158.54 (-COO-),
152.96 (-CH=C), 149.33 (-CH=C), 131.14 (-CH=C), 109.62 (-CH=C), 108.97 (-CH=C), 107.76
(-CH=C), 96.78 (-CH=C), 61.25 (-OCH2CH3), 45.19 (-NCH2CH3), 14.49 (-OCH2CH3), 12.52
(-NCH2CH3). HR-MS (ESI): calcd for C16H20NO4 [M + H]+ 290.1392, found 290.1386.
4.1.7. General Procedure for the Synthesis of Compounds 12a–12c
Compound 11 (1 mmol, 289 mg) and aromatic amines
1 (1.2 mmol) were dissolved
in ethanol (10 mL). The reaction mixture was heated under reflux. After completion of
the reaction, the solvent was evaporated in a vacuum followed by the addition of water
(10 mL) and 10% HCl to adjust the pH to 8. The mixture was then extracted with ethyl
acetate (20 mL
×
3). The combined organic layer was dried over anhydrous Na2SO4
and evaporated in a vacuum to give a crude residue which was purified by column

Molecules 2021, 26, 786
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chromatography (petroleum ether: ethyl acetate = 5:1–1:1) to yield compounds 12a
The NMR spectra of compounds 12a–12c are in the Supplementary Materials.
–12c.
7-(diethylamino)-2-oxo-N-phenyl-2H-chromene-3-carboxamide (12a). Yellow solid; yield
◦
60%; mp: 156.1–160.0 C. IR (KBr)
ν
(cm−1) 3125, 1699, 1605. 1H-NMR (400 MHz,
max
CDCl3) 10.87 (s, 1H, -NH-), 8.80 (s, 1H, -C6H5), 7.74 (dd, J = 8.5, 0.9 Hz, 2H, -C6H5),
δ
7.47 (d, J = 9.0 Hz, 1H, -C6H5), 7.33–7.38 (m, 2H, -C6H5), 7.12 (t, J = 7.4 Hz, 1H, -C6H5),
6.67 (dd, J = 9.0, 2.5 Hz, 1H, -C6H5), 6.53 (d, J = 2.4 Hz, 1H, -C6H5), 3.47 (q, J = 7.2 Hz,
4H, -NCH2CH3), 1.25 (t, J = 7.2 Hz, 6H, -NCH2CH3). 13C-NMR (100 MHz, CDCl3)
δ
163.20 (-COO-), 161.21 (-CONH-), 157.86 (-CH=C), 152.89 (-CH=C), 148.59 (-CH=C),
138.43 (-CH=C), 131.42 (-CH=C), 129.04 (-CH=C), 124.20 (-CH=C), 120.46 (-CH=C), 110.27
(-CH=C), 108.66 (-CH=C), 96.68 (-CH=C), 45.26 (-NCH2CH3), 12.55 (-NCH2CH3). HR-MS
(ESI): calcd for C20H21N2O3 [M + H]+: 337.1552; found: 337.1561.
N-(4-bromophenyl)-7-(diethylamino)-2-oxo-2H-chromene-3-carboxamide (12b). Yellow solid;
1
yield 59%; mp: 231.5–235.6 ◦C. IR (KBr)
ν
(cm−1) 3431, 1685, 1617. H-NMR (400 MHz,
max
CDCl3) δ 8.38 (s, 1H, -C6H5), 7.48 (d, J = 8.7 Hz, 2H, -C6H5), 7.15 (d, J = 8.8 Hz, 1H, -C6H5),
7.10 (d, J = 8.7 Hz, 2H, -C6H5), 6.25 (dd, J = 8.8, 2.5 Hz, 1H, -C6H5), 6.18 (d, J = 2.5 Hz, 1H,
-C6H5), 3.40 (q, J = 7.1 Hz, 4H, -NCH2CH3), 1.21 (t, J = 7.1 Hz, 6H, -NCH2CH3). 13C-NMR
(100 MHz, CDCl3) δ 162.90 (-COO-), 159.82 (-CONH-), 156.78 (-CH=C), 150.96 (-CH=C),
147.07 (-CH=C), 132.87 (-CH=C), 131.22 (-CH=C), 121.47 (-CH=C), 107.93 (-CH=C), 102.89
(-CH=C), 96.66 (-CH=C), 43.61 (-NCH2CH3), 11.68 (-NCH2CH3). HR-MS (ESI): calcd for
C20H20BrN2O3 [M + H]+: 415.0657; found: 415.0646.
7-(diethylamino)-N-(4-methoxyphenyl)-2-oxo-2H-chromene-3-carboxamide (12c). Yellow solid;
1
yield 59%; mp: 189.8–194.0 ◦C. IR (KBr)
ν
max
(cm−1) 3260, 1687,1588. H-NMR (400 MHz,
CDCl3) δ 8.36 (s, 1H, -C6H5), 7.20 (d, J = 8.8 Hz, 2H, -C6H5), 7.14 (d, J = 8.5 Hz, 1H, -C6H5),
6.92 (d, J = 8.8 Hz, 2H, -C6H5), 6.24 (d, J = 7.6 Hz, 2H, -C6H5), 3.82 (s, 3H, -OCH3), 3.40 (q,
J = 7.1 Hz, 4H, -NCH2CH3), 1.20 (t, J = 7.1 Hz, 6H, -NCH2CH3). 13C-NMR (100 MHz, CDCl3)
δ
163.14 (-COO-), 157.48 (-CONH-), 156.78 (-CH=C), 150.87 (-CH=C), 132.70 (-CH=C), 123.34
(-CH=C), 122.45 (-CH=C), 120.53 (-CH=C), 113.52 (-CH=C), 107.84 (-CH=C), 102.85 (-CH=C),
96.90 (-CH=C), 54.52 (-OCH3), 43.62 (-NCH2CH3), 11.70 (-NCH2CH3). HR-MS (ESI): calcd for
C21H23N2O4 [M + H]+: 367.1658; found: 367.1666.
4.2. Biology
4.2.1. Cell Culture
MDA-MB-231 (ATCC® HTB-26
™ ™
), KB (ATCC® CCL-17 ) and HCT-116 (ATCC®
CCL-247 ) cells were purchased from the American Type Culture Collection (ATCC,
™
Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)
media with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin at 37 ◦C in a 5%
CO2 incubator.
4.2.2. MTT Assay
4
×
103 cells were seeded in each well of the 96-well plate in triplicates followed by
24 h of incubation. The cells were treated with various concentrations of compounds (0.78,
1.56, 3.12, 6.25, 12.50, 25.00, and 50.00 M). After a 72-h incubation, 3-(4,5-dimethylthiazol-
µ
2-yl)-2,5-diphenyltetrazolium bromide (MTT) were added, and the cells were incubated for
another 4 h prior to the addition of DMSO. Absorbance was recorded at a wavelength of
540 nm with a reference length of 655 nm, and the results shown were collected from three
independent experiments. The IC50 was determined by using GraphPad Prism software
version 7.0 and nonlinear regression (curve fit).
4.2.3. Cell Migration Assay
5
×
105 MDA-MB-231 cells per well were cultured in a 6-well plate and allowed to
reach 80% confluency for 12 h. The monolayer was then scratched using a 200 L pipet tip
µ
to produce a wound. The culture medium was removed, and each plate was washed with
phosphate buffer saline (PBS) and treated with different concentrations of compound 9c (5,

Molecules 2021, 26, 786
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10, and 20
µM) for 24 and 48 h, respectively. Images were captured at the identified time
point (0, 24, and 48 h) and processed with the ImageJ software.
4.2.4. Analysis of Cell Invasion
For the assay, 24-well transwell chambers coated with 45 µL matrigel were used. In
brief, 6
with 200
600 L DMEM containing 10% FBS. The cells were allowed to grow for 48 h followed by
treatment with different concentrations of compound 9c (5, 10, and 20 M). The invaded
×
104 MDA-MB-231 cells per well were seeded in the upper part of the insert filled
µL of DMEM without FBS, and for the lower part, the chamber was filled with
µ
µ
cells were fixed with paraformaldehyde for 2 h and stained with crystal violet solution
(0.2%) for 1 h, and non-invading cells in the top chambers of the transwell plates were
scraped away by cotton swabs. Every insert was imaged at 10
using ImageJ software.
×
magnification and analyzed
4.2.5. Apoptosis Analysis and Morphology Observation
5
×
105 MDA-MB-231 cells per well were seeded in a 6-well plate for 12 h and were
treated with different concentrations of compound 9c (5, 10, 20, 40, and 80 M) for 48 h.
After treatment, the cells were harvested, washed twice with ice-cold PBS and resuspended
µ
in 500
and 5
µ
L of 1
×
Annexin V binding buffer and then mixed with 5 µL of Annexin V-FITC
µL of PI solution at room temperature in the dark for 10 min. Then, one part of the
cells was immediately analyzed with a flow cytometer. The other part was washed twice
with PBS, and the cells were photo-graphed by fluorescence microscope.
4.2.6. Intracellular ROS Level Detection
In 6-well plates, 2
concentrations (0, 10, 20, 30, and 40
×
105 MDA-MB-231 cells were plated and treated with different
M) of compound 9c for 48 h. ROS assay kit (Beyotime)
µ
was used to measure intracellular ROS level. DCFH-DA diluted with serum-free medium
◦
with 1:1000 was added into the cells at 37 C for 20 min. The cells were collected after
washing three times with serum-free medium. Images were acquired using an inverted
fluorescence microscope, and the fluorescence intensity was detected at an excitation
wavelength of 488 nm and an emission wavelength of 525 nm with microplate reader.
4.2.7. Western Blotting
For Western blotting analyses, the collected cells were rinsed in PBS and lysed in
sodium dodecyl sulfate (SDS) lysis buffer. Equal amounts of protein were separated on 10%
SDS-polyacrylamide gels, transferred to polyvinylidene fluoride (PVDF) membranes, and
blocked with 5% nonfat dry milk in tris-buffered saline and tween 20 (TBST) for 1 h. The
membranes were then incubated with primary antibodies (caspase-3, Bcl-2 and β-actin)
overnight at 4 ◦C. After being washed three times with TBST for 10 min, the membranes
were incubated with appropriate secondary antibodies in TBST for 1 h. After several
washes of TBST, the blots were developed by enhanced chemiluminescence (ECL) solution.
4.2.8. Statistical Analysis
GraphPad Prism software version 7.0 was used to determine the differences between
groups. Values with a p-value less than 0.05 are considered as significant and statistical
significance is represented as **** (p < 0.0001), *** (p < 0.001), ** (p < 0.01), * (p < 0.05), ns
(not significant).
5. Conclusions
A novel series of coumarin sulfonamides and amides derivatives were designed and
synthesized. Compound 9c was found to be the most potent derivative, which displayed
favorable antiproliferative activities toward MDA-MB-231 cells and inhibited migration
and invasion and induced apoptosis in MDA-MB-231 cells. Furthermore, it was verified
that compound 9c promoted MDA-MB-231 cell apoptosis by increasing intracellular ROS

Molecules 2021, 26, 786
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levels and upregulating the expression of caspase-3. This work presents information that is
helpful for the design and synthesis of new coumarin derivatives as potential antitumor
drug candidates.
Supplementary Materials: The following are available online. 1H- and 13C-NMR spectra of
all compounds.
Author Contributions: Conceptualization, H.J.; methodology, J.Z., G.L., and H.J.; validation, L.C.
and M.N.; formal analysis, J.Z.; investigation, J.Z., G.L., and Y.T.; resources, Z.W.; writing—original
draft preparation, J.Z.; writing—review and editing, H.J.; supervision, H.J.; project administration,
H.J.; funding acquisition, H.J. and Z.W. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Natural Science Foundation of Guangdong Province
(2018A0303130139, 2017A030310488), the Science and Technology Planning Project of Guangdong
Province (2015A020211039), Scientific Research Project for Guangzhou Municipal Colleges and Uni-
versities (1201610139, 1201630263), Science and Technology Program of Guangzhou (201707010353),
and Educational Commission of Guangdong Province (2015KQNCX134).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in Supplementary Materials.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.
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