Synthesis and biological activity evaluation of coumarin-3-carboxamide derivatives

Article abstract of DOI:10.3390/molecules26061653

A series of novel coumarin-3-carboxamide derivatives were designed and synthesized to evaluate their biological activities. The compounds showed little to no activity against gram-positive and gram-negative bacteria but specifically showed potential to inhibit the growth of cancer cells. In particular, among the tested compounds, 4-fluoro and 2,5-difluoro benzamide derivatives (14b and 14e, respectively) were found to be the most potent derivatives against HepG2 cancer cell lines (IC₅₀ = 2.62–4.85 μM) and HeLa cancer cell lines (IC₅₀ = 0.39–0.75 μM). The activities of these two compounds were comparable to that of the positive control doxorubicin; especially, 4-flurobenzamide derivative (14b) exhibited low cytotoxic activity against LLC-MK2 normal cell lines, with IC₅₀ more than 100 μM. The molecular docking study of the synthesized compounds revealed the binding to the active site of the CK2 enzyme, indicating that the presence of the benzamide functionality is an important feature for anticancer activity.

Full text of DOI:10.3390/molecules26061653

molecules
Article
Synthesis and Biological Activity Evaluation of Coumarin-3-
Carboxamide Derivatives
Weerachai Phutdhawong 1, Apiwat Chuenchid 2, Thongchai Taechowisan 3, Jitnapa Sirirak 2
and Waya S. Phutdhawong 2,
*
1
2
3
Department of Science, Faculty of Liberal Arts and Science, Kamphaeng Sean Campus, Kasetsart University,
Nakhon Pathom 73140, Thailand; phutdhawong@gmail.com
Department of Chemistry, Faculty of Science, Silpakorn University, Nakhon Pathom 73000, Thailand;
chuenchid.a@gmail.com (A.C.); jitnapasirirak@gmail.com (J.S.)
Department of Microbiology, Faculty of Science, Silpakorn University, Nakhon Pathom 73000, Thailand;
tewson84@hotmail.com
*
Correspondence: Phutdhawong_w@su.ac.th; Tel.: +66-34-255797
Abstract: A series of novel coumarin-3-carboxamide derivatives were designed and synthesized to
evaluate their biological activities. The compounds showed little to no activity against gram-positive
and gram-negative bacteria but specifically showed potential to inhibit the growth of cancer cells.
In particular, among the tested compounds, 4-fluoro and 2,5-difluoro benzamide derivatives (14b
and 14e, respectively) were found to be the most potent derivatives against HepG2 cancer cell lines
(IC50 = 2.62–4.85 µM) and HeLa cancer cell lines (IC50 = 0.39–0.75 µM). The activities of these two
compounds were comparable to that of the positive control doxorubicin; especially, 4-flurobenzamide
derivative (14b) exhibited low cytotoxic activity against LLC-MK2 normal cell lines, with IC50 more
than 100 µM. The molecular docking study of the synthesized compounds revealed the binding to
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Phutdhawong, W.;
Chuenchid, A.; Taechowisan, T.;
Sirirak, J.; Phutdhawong, W.S.
Synthesis and Biological Activity
Evaluation of Coumarin-3-
the active site of the CK2 enzyme, indicating that the presence of the benzamide functionality is an
important feature for anticancer activity.
Keywords: coumarin3-carboxamides; coumarins; pyranocoumarins; anticancer activity; antibacte-
rial activity
Carboxamide Derivatives. Molecules
2021, 26, 1653. https://doi.org/
10.3390/molecules26061653
1. Introduction
Academic Editor: Maria João Matos
Coumarin is one of the potent secondary metabolites in plants [
it is characterized by several pharmacological properties [ ]. Like decursin
, these coumarins have pyranocoumarin moiety (Figure 1), having been isolated from the
medicinal plant Angelica genus and shown potential for treating inflammatory diseases
such as cancer, hepatic fibrosis, diabetic retinopathy, and neurological disorders [ ]. The
dehydrated derivative of decursinol, xanthyletin , has also been shown to possess several
biological properties, such as anti-tumor and antibacterial activities [ ]. With a benzopy-
1
,
2
] and fungi [
3], and
Received: 26 February 2021
Accepted: 12 March 2021
Published: 16 March 2021
4
1
and decursinol
2
5
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
3
6
rone skeleton, coumarin is versatile and can be easily transformed into a large variety of
functionalized skeletons. As a result, many coumarin derivatives have been designed,
synthesized, and evaluated to address broad biological activities [7] such as antibacterial [8],
antifungal [ ], antioxidant [10], anti-inflammatory [11], anticancer [12], anticoagulant [13],
9
and antiviral activities [14]. The synthetic N-phenyl coumarin carboxamide 4a has been
designed and shown to possess high antibacterial activity against Helicobacter pylori (H. py-
lori), with the minimum inhibitory concentration (MIC) = 1 µg/mL [15], while the benzyl
substitution of coumarin carboxamides 4b–d has been shown to arrest breast cancer cell
(BT474 and SKBR-3) growth by inhibiting ErbB-2 and ERK1 MAP kinase. Moreover, com-
pounds 4b–d are specific to cancer cell lines, with no cytotoxicity against normal human
fibroblasts [16]. In our ongoing search for novel compounds to overcome drug resistance,
the diverse biological activities of coumarins have been interesting. In the current study,
Copyright:
© 2021 by the authors.
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/).
Molecules 2021, 26, 1653. https://doi.org/10.3390/molecules26061653
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1653
2 of 12
we designed novel pyranocoumarin-3-carboxamide derivatives with the expectation that
the carboxamide part could possess active pharmacological properties 4a
pyran ring moiety could also show specific biological proteins, as in the case of xanthyletin
. The synthesized compounds were examined to evaluate their antibacterial activity
–d and that the
3
and cytotoxicity against HepG2 and HeLa cell lines. As the coumarins were attractive
casein kinase 2 (CK2) inhibitors [17], molecular docking was used to study the possible
interactions of novel coumarin-3-carboxamides with the CK2 enzymes.
Figure 1. Synthetic and natural-occurring coumarins with biological activities.
2. Results and Discussion
2.1. Chemistry
The preparation of pyranocoumarin-3-carboxamide was applied from the previ-
ous synthetic strategies reported by Faulgues and colleagues [18] and was described in
Scheme 1. Commercially available 2,4-dihydroxy benzyldehyde
1-butene were used as the starting materials and were subjected to Lewis acid–promoted
Friedel-Crafts alkylation reaction in dioxane with BF3-diethylate to obtain the aldehyde in
53% yield as a major product together with many unidentified products [19]. The aldehyde
was cyclized to form a pyrano ring using 2,3-dichloro-5,6-dicyano-l,4-benzoquinone
(DDQ), and the benzopyran was obtained in a very good yield. The cyclization of ben-
zopyran with the malonic anhydride in pyridine and aniline at room temperature
5 and 3-hydroxy-3-methyl-
6
7
7
8
8
9
for 24 h according to a previous report [20] gave a poor yield of the pyranocoumarin-
3-carboxylic acid 10, due to the difficulty of purification. The amidation of the acid 10
with aniline using N,N’-dicyclohexyl carbodiimide (DCC) and 4-dimethylaminopyridine
(DMAP) gave the amide 12 in 11% yield after recrystallization.
Scheme 1. Synthesis of pyranocoumarin-3-carboxamide 12.
To improve the yield of pyranocoumarin-3-carboxamide 12, the coumarin-3-carboxylic
acid 13 was prepared in good yield prior to amidation with appropriate anilines using

Molecules 2021, 26, 1653
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hexafluorophosphate azabenzotriazole tetramethyl Uronium (HATU) and Et3N to obtain
amide 14a–g in 43%–51% yields (Scheme 2). Then, the cyclization of 14a by refluxing
with DDQ in benzene gave pyranocoumarin-3-carboxamide 12 in 66% yield (Scheme 3).
To study the effect of the substituent at C3 of coumarin ring, the carboxyl group was
decarboxylated using Cu powder to give coumarin 15 in 53% yield (Scheme 4). Then,
the synthesized coumarins were further evaluated for their antibacterial and anticancer
activities.
Scheme 2. Synthesis of coumarin-3-carboxamides 14a–g.
Scheme 3. Cyclization of pyranocoumarin-3-carboxamide 12.
Scheme 4. Decarboxylation of coumarin-3-carboxylic acid.
2.2. Antibacterial Activity
Coumarin derivatives 10, 12, 13, 14a–g, and 15 were evaluated for their antibacte-
rial activity against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Escherichia coli,
Salmonella typhimuriumthrough using the microbroth dilution method. Penicillin G and
solvent were used as positive and negative controls, respectively, and the MIC (µg/mL)
values were obtained (Table 1). The results show that only compounds 10 and 13 exhibited
moderate antibacterial activities against gram-positive bacteria, while the other tested com-
pounds displayed MIC values of more than 128 µg/mL. This may be because the carboxylic
acid at the C3 position played an essential role in the antibacterial activity. Compound
15, without the carboxyl group, showed no antibacterial activity, and the carboxamides
14a
–
g
, displayed no activity. Meanwhile, coumarin-3-carboxylic acid 13 was the most
g/mL against B. cereus;
active among the tested compounds, with an MIC value of 32
µ
however, it was less active than the reference drug penicillin G. Moreover, all the tested
compounds showed no activity against any gram-negative bacteria.

Molecules 2021, 26, 1653
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Table 1. MIC of coumarin derivatives 10
S. aureus, E. coli, S. typhimurium.
,
12a
,
13
,
14a–
g,
15 and penicillin G against B. cereus, B. subtilis,
MIC (µg/mL)
Compounds
R
Gram-Positive
Gram-Negative
E. coli S. typhimurium
B. cereus
B. subtilis
S. aureus
10
12
13
14a
14b
14c
14d
14e
14f
-
4-H
-
4-H
4-F
4-Cl
4-Br
2,5-diF
4-CH3
4-OCH3
-
128
> 128
32
128
> 128
128
64
> 128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
64
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
16
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
2
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
16
14g
15
penicillin G
-
2.3. Anticancer Activity
All synthesized compounds were evaluated for in vitro cytotoxic activity against two
cancer cell lines (HepG2 and HeLa cell lines) and normal cell lines (LLC-MK2) through
an MTT assay, and the results are presented in Table 2. Most of the tested compounds
displayed potent anticancer activity. The N-phenyl coumarin-3-carboxamides 12 and 14a
showed significantly more potency than the parent acids 10 and 13, respectively, against
HepG2 cell lines. Moreover, compound 15, with no substituent at C3, showed better
activity than the parent acid 13. The effect of substituents on the phenyl ring was compared
with the effect of substituents on the carboxamide 14a and it was found that the phenyl
bearing fluorine atoms 14b and 14e possessed similar effects on the potency, while, the
phenyl bearing 4-chlorine and 4-bromine atoms showed less activity against both cancer
cell lines. Moreover, the phenyl bearing 4-methyl and 4-methoxyl groups displayed weak
activity against the test cancer cell lines. From these results, the size and electron-donating
group of the para-substituted benzene ring may affect anticancer properties. From these
tested compounds, the amide 14e displayed the most potent anticancer activity; however,
it exhibited high cytotoxic activity against the normal cell line, with IC50 = 1.33 µM.
Interestingly, amide 14b displayed slightly lower activity than 14e, but it showed low
cytotoxicity against the normal cell line. This compound possessed anticancer activity
comparable to those of the tested anticancer drugs doxorubicin and acridine orange.
2.4. Molecular Docking
Casein Kinase 2 enzyme is a key player in the pathophysiology of cancer [21
Using the iGEMDOCK v2.1 software [23], molecular docking was performed to investigate
binding positions and intermolecular interactions between coumarin 10 12, 13 14a–f, and
15 and the binding site of CK2. Coumarins 10 12 13 14a , and 15 were docked to the
,22].
,
,
,
,
,
–f
active site of CK2 co-crystallized with G12 (PDB ID: 2QC6). Moreover, G12 was also
redocked to CK2, and its total energy and hydrogen bond length were compared with
those of coumarins 10, 12, 13, 14a–f, and 15. The molecular docking results show that the
binding position of redocked G12 was roughly the same as that of co-crystallized G12 in
CK2 (Figure 2a). Moreover, all synthesized compounds were bound to the active site of
CK2, and the binding positions were similar to that of G12 (Figure 2b,c), and their binding
energies (
(Table 3). Figure 3 also illustrates the hydrogen bond interactions in the binding of coumarin
12 14a 14b 14d, and 14e in the cavity of CK-2 compared with that of G12. Coumarins
14a–f had much lower binding energy ( 118.99 to 105.53 kcal/mol) than G12, and their
N-phenyl ring was also bound to similar positions of the G12 phenolic ring (Figure 2c). Key
−
118.99 to
−
89.19 kcal/mol) were lower than that of G12
(−79.10 kcal/mol)
,
,
,
−
−

Molecules 2021, 26, 1653
5 of 12
amino acid residues, including LYS68, ASN118, ASN117, and ASP175, formed a hydrogen
bond with 14a
while N-phenyl ring interacted with LYS68 and ASP175. The substitution group on the
N-phenyl ring of 14a also influenced the number of hydrogen bonds in the CK2 active
–f, where ASN117 and ASN118 interacted with the hydroxy group of 14a–f,
–
f
site. Moreover, a comparison of the halogen substitution groups on the N-phenyl ring
showed that the Cl and Br substitution groups on the N-phenyl ring formed no hydrogen
bond, while the F substitution group 14b and 14e could form hydrogen bonds with LYS68
and ASP175 (Figure 3d,f), which may be because Cl and Br are larger than F in size.
Additionally, the experimental results show that the coumarins 14b and 14e had good
anticancer activities.
Table 2. In vitro anticancer activities of synthesized coumarins compared with doxorubicin and
acridine orange.
IC50 (µM)
Compounds
Cancer Cell Line
Normal Cell Line
LLC-MK2
R
HepG2
HeLa
10
12
13
14a
14b
14c
14d
14e
-
4-H
-
4-H
4-F
4-Cl
4-Br
2,5-diF
4-CH3
4-OCH3
-
> 100
2.35
81.73
4.33
80.38
> 100
26.42
10.40
0.75
14.04
37.36
0.39
> 100
> 100
13.47
1.18
> 100
> 100
37.95
0.48
>100
66.80
> 100
1.33
85.31
> 100
6.87
63.47
82.30
4.85
30.28
45.76
2.62
> 100
> 100
9.13
14f
14g
15
Doxorubicin
Acridine orange
-
-
1.91
5.72
6.59
Figure 2.
of 10 (pink), 12 (sky blue), 13 (orange), 15 (violet), and G12 (green) in the CK-2 cavity. (
(yellow), 14e (blue), 14f (purple), 14g (red), and G12 (green) in the CK-2 cavity (PDB ID: 2QC6).
(a
) Redocked G12 (brown) and G12 (green) in the cavity of CK-2 (PDB ID: 2QC6). (
b) Comparison of the bindings
c) Comparison of the bindings of 14a

Molecules 2021, 26, 1653
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Table 3. Summary of binding energy, amino acid interactions, and hydrogen bond length of coumarin
derivatives in CK2 binding site.
Total Energy
(kcal/mol)
Compounds
Amino Acid Residue
Hydrogen Bond Length (Å)
10
12
−89.19
−100.88
−91.13
LYS68, ASP175
ASP175
2.44, 2.34
2.91
13
LYS68
1.92
14a
14b
14c
14d
14e
14f
14g
15
−108.21
−107.08
−106.07
−105.91
−111.19
−105.53
−118.99
−86.27
ASN118, ASN117, ASP175
ASN117, LYS68, ASP175
ASN117
2.19, 2.99,2.51
2.93, 2.42, 2.74
2.94
ASN117
2.95
ASN118, LYS68
ASN117
2.27, 2.44
2.93
ASN117, LYS68, GLU81
LYS68, GLU114, ASP175,
2.74, 1.90, 2.72
2.08, 1.99, 2.42
G12
−79.10
ASP175
2.43
Figure 3. Hydrogen bond interactions in the bindings of (a) G12,(b) 12,(c) 14a, (d) 14b,(e) 14d, and (f) 14e in the CK-2 cavity.
3. Materials and Methods
3.1. Chemistry
General information: Solvents and reagents were purchased from commercial suppli-
ers TCI Chemicals (Tokyo, Japan), Sigma-Aldrich (Bangalore, India), and Fluka (Dorset,
1
UK). Structure determination was conducted by analyzing the H, 13C, and 19F NMR
spectra (Bruker 300 apparatus) and the infrared (IR) spectrum was determined using
PerkinElmer Frontier Fourier-transform infrared spectrometer. Melting point was con-
ducted using Stuart SMP2 melting point apparatus and high-resolution mass spectroscopy
was analyzed by Thermo scientific, Orbitrap Q Exactive Focus.
3.1.1. Synthesis of 2,4-Dihydroxy-5-(3-methylbut-2-en-1-yl)-benzaldehyde 7
First, 2,4-dihydroxy benzaldehyde
to a stirred solution of 3-hydroxy-3-methyl-1-butene
5
(1.5 g, 10.8 mmol) in dioxane (5 mL) was added
(1.5 mL, 14.3 mmol) and boron
6
trifluoride diethyl etherate (BF3-OEt2, 1.5 mL) in dioxane (3 mL), and stirring was continued
for 2.5 h at room temperature. Dichloromethane (50 mL) was added, and the resulting
solution was extracted with water (3
×
50 mL). The combined organic layer was dried
over Na2SO4 before evaporation to dryness and then purified via column chromatography
(silica gel, 4:1 hexane:EtOAc) to obtain a white solid of 2,4-dihydroxy-5-(3-methylbut-2-en-
1-yl) benzaldehyde
(300 MHz, CDCl3):
7
δ
(0.48 g, 53% yield): m.p. 124–125 ◦C (lit. [19] 121–123 ◦C), 1H-NMR
11.27 (s, 1H), 9.69 (s, 1H, OH), 7.26 (s, 1H), 6.37 (s, 1H), 6.10 (s, 1H,
OH), 5.30 (tt, J = 4.53, 1.35 Hz, 1H), 3.30 (d, J = 7.2 Hz, 2H), 1.77 (s, 3H), 1.61 (s, 3H) ppm.

Molecules 2021, 26, 1653
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3.1.2. Synthesis of 7-Hydroxy-2,2-dimethyl-2H-chromene-6-carbaldehyde 8
A mixture of compound (1.0 g, 4.85 mmol) and DDQ (1.2 g, 5.28 mmol) in benzene
7
(10 mL) was refluxed for 6 h, and the precipitate was filtered off. The filtrate was evaporated
to dryness to afford the crude product, which was purified via column chromatography (sil-
ica gel, 10:1 hexane:EtOAc) to obtain a white solid of 7-hydroxy-2,2-dimethyl-2H-chromene-
◦ ◦
8
(0.91 g, 92% yield): m.p. 82–83 C (lit. [20] 95–96 C), 1H NMR (300 MHz,
6-carbaldehyde
CDCl3): 11.41 (br, OH), 9.68 (s, 1H), 7.16 (s, 1H), 6.35 (s, 1H), 6.30 (d, J = 7.86 Hz, 1H), 5.59
δ
(d, J = 9.93 Hz, 1H), 1.59 (s, 3H) 1.48 (s, 3H) ppm.
3.1.3. Synthesis of 8,8-Dimethyl-2-oxo-2H,8H-pyrano[3,2-g]chromene-3-carboxylic acid 10
Compound
8 (1.0 g, 4.90 mmol) and malonic acid 9 (1.0 g, 9.60 mmol) were dissolved
in pyridine (5.5 mL) containing aniline (0.5 mL) and stirred for 24 h at room temperature.
Afterward, the rection mixture was poured into ice-cold 10% HCl (80 mL). The yellow
precipitate was washed with cold water to remove mineral acid and then air-dried to
yield a yellow solid (recrystallization by 2:1:2; EtOAc:EtOH:hexane) of 8,8-dimethyl-2-oxo-
2H,8H-pyrano[3,2-g]chromene-3-carboxylic acid 10 (0.23 g, 17% yield): m.p. 187–188 ◦C,
1H NMR (300 MHz, CDCl3):
1H), 5.81 (d, J = 10.02 Hz, 1H), 1.52 (s, 6H) ppm., 13C NMR (75 MHz, CDCl3):
δ
8.80 (s, 1H), 7.29 (s, 1H), 6.85 (s, 1H), 6.40 (d, J = 10.02 Hz,
164.54 (C),
δ
163.28 (C), 160.87 (C), 156.60 (C), 150.99 (CH), 132.34 (C), 127.13 (CH), 120.17 (CH), 120.06
(CH), 112.50 (C), 110.59 (C), 104.38 (CH), 79.33 (C), 28.75 (2CH3) ppm. IR: 3051.36 (C-H,
aromatic), 2922.46 (C-H, aliphatic), 1743.12 (C=O, acid) cm−1; HREI-MS (m/z) calculated
for C15H13O5 (M)+ 273.0758, found 273.0757.
3.1.4. Synthesis of 8,8-Dimethyl-2-oxo-N-phenyl-2H,8H-pyrano[3,2-g]chromene-3-
carboxamide 12
A mixture of compound 10 (0.10 g, 0.36 mmol), aniline 11 (0.040 mL, 0.43 mmol), DCC
(0.10 g, 0.44 mmol), and DMAP (8 mg, 0.065 mmol) in dry CH2Cl2 (5 mL) was stirred at
room temperature for 18 h. Afterward, the reaction mixture was filtered, and the filtrate was
evaporated under vacuum. The residue was recrystallized using EtOH to obtain a yellow
solid of 8,8-dimethyl-2-oxo-N-phenyl-2H,8H-pyrano[3,2-g]chromene-3-carboxamide 12 (14
mg, 11% yield): m.p. 187–188 ◦C, 1H NMR (300 MHz, CDCl3):
δ 10.81(s, 1H), 8.89 (s, 1H),
7.75 (d, J = 1.14 Hz, 2H), 7.39 (t, J = 6.54 Hz, 2H), 7.30 (s, 1H), 7.18 (t, J = 1.14 Hz, 1H), 6.80 (s,
1H), 6.40 (d, J = 9.90 Hz, 1H), 5.78 (d, J = 9.96 Hz, 1H), 1.55 (s, 6H) ppm., 13C NMR (75 MHz,
CDCl3):
δ 162.20 (C), 160.03 (C), 159.55 (C), 156.35 (C), 148.67 (CH), 137.30 (C), 131.82 (CH),
129.02 (2CH), 126.72 (CH), 124.57 (CH), 120.53 (2CH), 120.43 (CH), 119.58 (C), 114.71 (C),
112.73 (C), 75.72 (C), 28.64 (2CH3) ppm. IR: 3198.94 (N-H), 3059.35 (C-H, aromatic), 2969.10
(C-H, aliphatic), 1699.85 (C=O, amide) cm−1; HREI-MS (m/z) calculated for C21H18O4N
(M)+ 348.1230, found 348.1230.
3.1.5. Synthesis of 3-Carboxy-6-(3-methyl-2-butenyl)-7-hydroxy-coumarin 13
Compound
7 (1.0 g, 4.85 mmol) and malonic acid 9 (1.0 g, 9.60 mmol) were dissolved
in pyridine (5.5 mL) containing aniline (0.5 mL), and stirred for 24 h at room temperature.
Afterward, the reaction mixture was poured into ice-cold 10% HCl (80 mL), and the yellow
precipitate obtained was washed with cold water to remove mineral acid and then air-dried
to yield a yellow solid of◦3-carboxy-6-(3-methyl-2-butenyl)-7-hydroxy-coumarin 13 (1.10g,
◦
88% yield): m.p. 237–238 C (lit. [20] 218–224 C), 1H NMR (300 MHz, CDCl3+methanol-d4)
δ
8.79 (s. 1H), 7.40 (s, 1H), 6.85 (s, 1H), 5.32 (tt, J = 7.4, 1.3 Hz, 1H), 3.34 (d, J = 7.29 Hz,
2H), 1.79 (s, 3H), 1.70 (s, 3H) ppm., 13C-NMR (75 MHz, CDCl3+methanol-d4):
δ
163.58
(C), 162.79 (C), 162.37 (C), 154.78 (C), 150.43 (CH), 133.49 (C), 129.32 (CH), 127.92 (C),
119.16 (CH), 110.24 (C), 107.85 (C), 100.76 (CH), 26.34 (CH2), 24.42 (CH3), 16.50 (CH3) ppm.,
IR: 3303.61 (O-H), 3049.81 (C-H, aromatic), 2911.92 (C-H, aliphatic), 1733.10 (C=O, acid),
1718.83 (C=O, lactone) cm−1
.

Molecules 2021, 26, 1653
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3.1.6. General Procedure of Coumarin-3-carboxamides Preparation (14a–g)
Triethanolamine (TEA) (0.1 mL, 1.36 mmol) was added to a solution of compound 13
(70 mg, 0.26 mmol) and HATU (0.12 g, 0.36 mmol) in dry THF (5 mL), and the mixture was
stirred at room temperature for 30 min. The obtained dark clear mixture was treated with
aniline derivatives (11a
for 18 h. Dichloromethane (50 mL) was added, and the resulting solution was extracted
with sat. NaCl (3 30 mL). The remaining organic layer was dried over Na2SO4 before
–g) (1.2 eq.). The resulting mixture was stirred at room temperature
×
evaporation to dryness. After evaporation of the solvent in vacuo, the crude product was
purified via preparative thin-layer chromatography (silica gel, 4:1 hexane:EtOAc) to give
yellow solids of coumarin-3-carboxamide (14a–g)
7-Hydroxy-6-(3-methylbut-2-en-1-yl)-2-oxo-N-phenyl-2H-chromene-3-carboxamide
◦
1
14a (47 mg, 51% yield): m.p. 258–259 C, H NMR (300 MHz, CDCl3+pyridine-d5):
δ
10.86 (s, NH), 8.90 (s, 1H), 7.73 (d, J = 7.62 Hz, 2H), 7.42 (s, 1H), 7.35 (t, J = 7.62 Hz, 2H), 7.12
(t, J = 7.38 Hz, 1H), 6.84 (s, 1H), 5.40 (tt, J = 7.26, 1.35 Hz, 1H), 3.43 (d, J = 7.26 Hz, 2H), 1.81
(s, 3H), 1.74 (s, 1H) ppm., 13C NMR (75 MHz, CDCl3+pyridine-d5):
δ 163.38 (C), 162.81 (C),
160.56 (C), 155.49 (C), 149.36 (CH), 138.08 (CH), 134.13(C), 130.04 (CH), 128.97 (2CH), 128.68
(C), 124.35 (CH), 121.14 (CH), 120.49 (2CH), 112.94 (C), 111.33 (C), 101.68 (CH), 27.85 (CH2),
25.84 (CH3), 17.85 (CH3) ppm., IR: 3195.27 (O-H), 2917.31 (C-H, aliphatic), 1695.54 (C=O,
amide) cm−1; HREI-MS (m/z) calculated for C21H20O4N (M)+ 350.1387, found 350.1386.
N-(4-Fluorophenyl)-7-hydroxy-6-(3-methylbut-◦2-en-1-yl)-2-oxo-2H-chromene-3-car-
1
boxamide 14b (45 mg, 47% yield): m.p. 259–261 C, H NMR (300 MHz, CDCl3):
δ
10.56 (br, NH), 8.89 (s, 1H), 7.68 (dd, J = 8.97, 4.92 Hz, 2H), 7.42 (s, 1H), 7.07 (dd, J = 9.12,
8.79 Hz 2H), 6.83 (s, 1H), 5.34 (t, J = 7.23 Hz, 1H), 3.35 (d, J = 7.29 Hz, 2H), 1.80 (s, 3H), 1.72
(s, 1H) ppm., 13C NMR (75 MHz, CDCl3):
δ 163.04 (C), 162.57 (C), 160.72 (C), 159.64 (d,
J = 242.5 Hz, C), 155.46 (C), 149.71 (CH), 134.43 (C), 133.84 (d, J = 3.0 Hz, C), 130.23 (CH),
128.80 (C), 120.91 (CH), 115.72 (d, J = 22.5 Hz, 2CH), 112.86 (C), 112.31 (d, J = 7.5 Hz, 2CH),
111.68 (C), 101.70 (CH), 27.74 (CH2), 25.87 (CH3), 17.82 (CH3) ppm., 19F NMR (282 MHz,
CDCl3, std. TFA):
−
118.08 (s, 1F) ppm., IR: 3208.66 (O-H), 3155.21 (N-H), 3048.54 (C-H,
aromatic), 2913.86 (C-H, aliphatic), 1698.12 (C=O, amide) cm−1; HREI-MS (m/z) calculated
for C21H19O4NF (M)+ 368.1293, found 368.1293.
N-(4-Chlorophenyl)-7-hydroxy-6-(3-methylbut-2-en-1-yl)-2-oxo-2H-chromene-3-car-
boxamide 14c (44 mg, 44% yield): m.p. 282–283 ◦C, 1H NMR (300 MHz, CDCl3+pyridine-
d5):
2.01 Hz, 2H), 6.83 (s, 1H), 5.41 (t, J = 7.23 Hz, 1H), 3.43 (d, J = 7.23 Hz, 2H), 1.81 (s, 3H), 1.74
(s, 1H) ppm., 13C NMR (75 MHz, CDCl3+pyridine-d5):
163.65 (C), 162.81 (C), 160.64 (C),
δ 10.92 (s, NH), 8.89 (s, 1H), 7.69 (d, J = 8.88 Hz, 2H), 7.37 (s, 1H), 7.30 (dd, J = 8.89,
δ
155.57 (C), 149.50 (CH), 136.76 (C), 134.06 (C), 130.09 (CH), 129.15 (C), 128.95 (2CH), 128.83
(C), 121.64 (2CH), 121.17 (CH), 112.56 (C), 111.27 (C), 101.68 (CH), 27.85 (CH2), 25.83 (CH3),
17.84 (CH3) ppm. IR: 3196.03 (O-H), 3122.34 (N-H), 3073.18 (C-H, aromatic), 2911.85 (C-H,
aliphatic), 1698.47 (C=O, amide) cm−1; HREI-MS (m/z) calculated for C21H19O4N35Cl (M)+
384.0997, found 384.0996.
N-(4-Bromophenyl)-7-hydroxy-6-(3-methylbut-2-en-1-yl)-2-oxo-2H-chromene-3-car-
◦
boxamide 14d (52 mg, 47% yield)): m.p. 276–277 C, 1H NMR (300 MHz, CDCl3+pyridine-
d5):
1H), 6.83 (s, 1H), 5.40 (t, J = 7.14 Hz, 1H), 3.43 (d, J = 7.26 Hz, 2H), 1.81 (s, 3H), 1.73 (s, 1H)
ppm., 13C NMR (75 MHz, CDCl3+pyridine-d5):
163.67 (C), 162.80 (C), 160.65 (C), 155.57
δ 10.92 (s, NH), 8.89 (s, 1H), 7.69 (d, J = 8.88 Hz, 2H), 7.44 (d, J = 9.66 Hz, 2H), 7.39 (s,
δ
(C), 149.51 (CH), 137.25 (C), 134.04 (C), 131.89 (2CH), 130.09 (CH), 128.83 (C), 121.96 (2CH),
121.16 (CH), 116.80 (C), 112.52 (C), 111.25 (C), 101.67 (CH), 27.85 (CH2), 25.83 (CH3), 17.84
(CH3) ppm. IR: 3192.94 (O-H), 3070.45 (C-H, aromatic), 2915.14 (C-H, aliphatic), 1697.23
(C=O, amide) cm−1; HREI-MS (m/z) calculated for C21H19O4N79Br (M)+ 428.0492, found
428.0492.
N-(2,5-Difluorophenyl)-7-hydroxy-6-(3-methylbut-2-en-1-yl)-2-oxo-2H-chromene-3-
◦
carboxamide 14e (46 mg, 46% yield): m.p. 248–250 C, 1H NMR (300 MHz, CDCl3+pyridine-
d5):
δ 12.27 (s, NH), 8.90 (s, 1H), 8.40 (ddd, J = 10.46, 6.66, 3.15 Hz, 1H), 7.43 (s, 1H), 7.04
(ddd, J = 9.57, 9.18, 4.89 Hz, 1H), 6.86 (s, 1H), 6.65–6.75 (m,1H), 5.41 (t, J = 7.35 Hz, 1H), 3.43

Molecules 2021, 26, 1653
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(d, J = 7.23 Hz, 2H), 1.81 (s, 3H), 1.74 (s, 1H) ppm., 13C NMR (75 MHz, CDCl3+pyridine-d5):
δ
163.90 (C), 162.63 (C), 160.90 (C), 158.57, (d, J = 238.5 Hz, C), 158.55, (d, J = 239.3 Hz, C),
155.75 (C), 149.73 (C), 134.03 (C), 130.18 (CH), 128.88 (C), 127.65 (d, J = 11.3 Hz, CH), 121.19
(CH), 115.20 (dd, J = 27.4, 9 Hz, CH), 112.28 (C), 111.18 (C), 110.03 (dd, J = 24.0, 7.5 Hz,
CH), 109.09 (d, J = 30 Hz, CH), 101.74 (CH), 27.83 (CH2), 25.82 (CH3), 17.83 (CH3) ppm. 19F
NMR (282 MHz, CDCl3+pyridine-d5, std. TFA):
−
117.72 (d, J = 14.10 Hz, 1F), -136.03 (d,
J = 14.10 Hz, 1F) ppm., IR: 3252.42 (O-H), 3130.54 (N-H), 2073.1 (C-H, aromatic), 2915.45
(C-H, aliphatic), 1701.64 (C=O, amide) cm−1; HREI-MS (m/z) calculated for C21H17O4NF2
(M+Na)+ 408.1018, found 408.1013.
7-Hydroxy-6-(3-methylbut-2-en-◦1-yl)-2-oxo-N-(p-tolyl)-2H-chromene-3-carboxamide
14f (41 mg, 43% yield): m.p. 276–277 C, 1H NMR (300 MHz, CDCl3+pyridine-d5):
δ 10.80
(s, NH), 8.90 (s, 1H), 7.65 (d, J = 7.44 Hz, 2H), 7.41 (s, 1H), 7.15 (d, J = 8.31 Hz, 2H), 6.85 (s,
1H), 5.41 (tt, J = 7.32, 1.35 Hz, 1H), 3.43 (d, J = 7.20 Hz, 2H), 2.31 (s, 3H), 1.81 (s, 3H), 1.74
(s, 1H) ppm., 13C NMR (75 MHz, CDCl3+pyridine-d5):
δ 163.33 (C), 162.79 (C), 160.40 (C),
155.45 (C), 149.19 (CH), 135.56 (C), 134.01 (C), 133.91 (C), 130.00 (CH), 129.47 (2CH), 121.22
(CH), 120.45 (2CH), 113.03 (C), 111.31 (C), 101.66 (CH), 27.86 (CH2), 25.83 (CH3), 20.91
(CH3), 17.84 (CH3) ppm. IR: 3187.07 (O-H), 3130.54 (N-H), 3073.13 (C-H, aromatic), 2913.70
(C-H, aliphatic), 1698.29 (C=O, amide) cm−1; HREI-MS (m/z) calculated for C22H22O4N
(M)+ 364.1543, found 364.1540.
7-Hydroxy-N-(4-methoxyphenyl)-6-(3-meth◦ylbut-2-en-1-yl)-2-oxo-2H-chromene-3-
carboxamide 14g (43 mg, 44% yield): m.p. 259–261 C, 1H NMR (300 MHz, CDCl3+pyridine-
d5):
2H), 6.82 (s, 1H), 5.34 (tt, J = 7.35, 1.47 Hz, 1H), 3.82 (s, 3H), 3.36 (d, J = 7.20 Hz, 2H), 1.80
(s, 3H), 1.72 (s, 1H) ppm., 13C NMR (75 MHz, CDCl3+pyridine-d5):
163.00 (C), 162.28
δ 10.74 (s, NH), 8.88 (s, 1H), 7.63 (d, J = 9.03 Hz, 2H), 7.41 (s, 1H), 6.91 (d, J = 9.06 Hz,
δ
(C), 160.44 (C), 156.65 (C), 156.65 (C), 155.34 (C), 149.37 (CH), 134.40 (C), 131.01 (C), 130.16
(CH), 128.33 (C), 122.29 (2CH), 120.94 (CH), 113.20 (C), 111.74 (C), 101.71 (CH), 55.56 (CH3),
27.76 (CH2), 25.81 (CH3), 17.82 (CH3) ppm. IR: 3182.53 (O-H), 3111.40 (N-H), 3073.14 (C-H,
aromatic), 2911.91 (C-H, aliphatic), 1695.83 (C=O, amide) cm−1; HREI-MS (m/z) calculated
for C22H22O5N (M)+ 380.1493, found 380.1490.
3.1.7. Synthesis of 8,8-Dimethyl-2-oxo-N-phenyl-2H,8H-pyrano[3,2-g]chromene-3-
carboxamide 12
A mixture of compound 14a (1.7 g, 4.85 mmol) and DDQ (1.2 g, 5.28 mmol) in benzene
(10 mL) was refluxed for 6 h, and the precipitate was filtered off. The filtrate was evaporated
to dryness to afford the crude product, which was purified via column chromatography
(silica gel, 10:1 hexane:EtOAc) to obtain a white solid of 8,8-dimethyl-2-oxo-N-phenyl-
2H,8H-pyrano[3,2-g]chromene-3-carboxamide 12 (1.11 g, 66% yield): m.p. 187–188 ◦C, 1H
NMR (300 MHz, CDCl3):
δ 10.81(s, 1H), 8.89 (s, 1H), 7.75 (d, J = 1.14 Hz, 2H), 7.39 (t, J = 6.54
Hz, 2H), 7.30 (s, 1H), 7.18 (t, J = 1.14 Hz, 1H), 6.80 (s, 1H), 6.40 (d, J = 9.90 Hz, 1H), 5.78 (d,
J = 9.96 Hz, 1H), 1.55 (s, 6H) ppm.
3.1.8. Synthesis of 6-(3-Methyl-2-buteny1)-7-hydroxycoumarin 15
Compound 13 (0.20 g, 0.73 mmol) in 2 mL quinoline containing 0.3 g Cu powder was
◦
heated for 3 min at 215–220 C in an oil bath. The mixture was cooled to room temperature
and diluted with CH2Cl2 (30 mL) prior to extraction with 10% HCI (2
×
30 mL) and then
with water (30 mL). The solvent was evaporated, leaving a tacky orange solid, which was
purified via column chromatography (silica gel, 1:1 hexane:EtOAc) to yield a cream solid
of 6-(3-Methyl-2-buteny1)-7-hydroxycoumarin 15 (0.09 g, 53% yield), m.p. 134–136 ◦C (lit.
133 ◦C [24]), 1H NMR (300 MHz, CDCl3):
δ 7.67 (d, J = 9.42 Hz, 1H), 7.20 (s, 1H), 7.06 (s,
1H), 6.24 (d, J = 9.42 Hz, 1H), 5.33 (tt, J = 8.73, 1.41 Hz, 1H), 3.38 (d, J = 7.23 Hz, 2H), 1.78 (s,
3H), 1.75 (s, 3H).
3.2. Determination of Antibacterial Activity
The antibacterial activities of coumarin derivatives 10
, 12, 13, 14a–g, and 15 were
evaluated against five reference standard bacteria, both gram-positive and gram-negative:

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B. cereus TISTR 2372, B. subtilis TISTR 001, S. aureus TISTR 2392, E. coli TISTR 073, and S.
typhimurium TISTR 2519, using a standard microbroth dilution method [25].
The MIC values of coumarin derivatives 10, 12, 13, 14a–g, and 15 were determined
through the microbroth dilution method in 96-well microtitre plates. The bacterial cultures
◦
were prepared from overnight cultures on nutrient broth (NB) at 37 C for 24 h by diluting
in NB compared with 0.5 McFarland. Coumarin derivatives 10
(5000 g/mL) were prepared in EtOH, and 128 g/mL of these were added to the first
wells. Two-fold serial dilutions were prepared, and final concentrations of 128 to 1 g/mL
were achieved. The positive controls for penicillin G were determined, with the final
concentrations from 128 to 1 g/mL. In addition, an extra row of EtOH was used as a
vehicle control to determine its possible inhibitory activity. Finally, 10 L of bacterial
, 12, 13, 14a–g, and 15
µ
µ
µ
µ
µ
suspension was added to each well. After the bacteria were incubated at 37 ◦C for 24 h,
the microtitre plates were visually examined for bacterial growth; the growth rate was
monitored at the optical density at 600 nm with a microplate reader. In each row, the
well containing the lowest concentration that showed no visible growth was considered
the MIC.
3.3. Cell Viability Assay
The cell viability assays of coumarin derivatives 10, 12, 13, 14a–g, and 15 were con-
ducted against three cancer cell lines (HepG2, HeLa, and MDA-MB-231) and one normal
cell line (LLC-MK2) using an MTT assay [26].
Stock solutions of coumarin derivatives 10
EtOH at a concentration of 5000 g/mL. Prior to use, the stock solutions were further
diluted to 128 g/mL and added to the first wells. Two-fold serial dilutions were prepared,
and final concentrations of 128 to 1 g/mL in culture medium were achieved. Cells were
seeded at a density of 5 104 cells/well in a 96-well plate and incubated for 16 h, followed
, 12a, 13, 14a–g, and 15 were prepared in
µ
µ
µ
×
by treatment with the test compounds. The control culture contained the carrier solvent of
2.5% dimethyl sulfoxide (DMSO). After 24 h, HepG2, HeLa, MDA-MB-231, and LLC-MK2
cells were then incubated with MTT (500 µg/mL) for 4 h. Then, DMSO was added to
dissolve the blue formazan crystals formed, which were formed as a result of the action of
cellular oxidoreductase enzymes on the MTT dye. Finally, the optical density at 450 nm
was determined using a microplate reader.
4. Conclusions
We designed and synthesized a series of coumarin-3-carboxamides and evaluated
their antibacterial and anticancer activities. The carboxylic acid at the C3 position of
coumarins was necessary for the antibacterial activity, as seen for compounds 10 and 13,
which showed moderate antibacterial activities against the tested gram-positive bacteria.
Meanwhile, most of the tested compounds showed potent anticancer activity, and the
4-fluorophenyl coumarin-30-carboxazine 4b was by far the most active anticancer, with
activity comparable to that of the anticancer drug doxorubicin, and it had low cytotoxicity
against a normal cell line. The molecular docking study revealed the binding to the active
site of the CK2 enzyme, indicating that the presence of the phenyl carboxamide is important
for anticancer activity.
Author Contributions: Conceptualization, project administration, supervision, W.P.; Methodology,
investigation, validation, writing—original draft, A.C.; conceptualization, methodology, microbiology
testing supervision, T.T.; molecular modeling supervision, J.S.; methodology, conceptualization,
project administration, writing—review and editing, supervision, W.S.P. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

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Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: We gratefully acknowledge the Department of Chemistry and the Department
of Microbiology, Faculty of Science, Silpakorn University, for the financial support and antibacterial
and anticancer assay. We also thank the Chulabhorn Research Institute for the measurements of the
HR-ESI mass spectroscopy investigation and the Rice Department.
Conflicts of Interest: The authors declare no conflict of interest.
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