Synthesis, antiproliferative activities, and DNA binding of coumarin-3-formamido derivatives

Article abstract of DOI:10.1002/ardp.202000236

Ten coumarin-3-formamido derivatives, N-benzyl-coumarin-3-carboxamide (2), N-fluorobenzyl-coumarin-3-carboxamide (3–5), N-methoxybenzyl-coumarin-3-carboxamide (6–8), N-((1-methyl-1H-imidazol-5-yl)methyl)-coumarin-3-carboxamide (9), N-(thiophen-2-ylmethyl)-coumarin-3-carboxamide (10), and N-(furan-2-ylmethyl)-coumarin-3-carboxamide (11), were synthesized and characterized. Compound 5 crystallizes in a monoclinic system P2₁/c space group with four chemical formulas in a unit cell; molecules of compound 5 are self-assembled into a two-dimensional supramolecular structure by intermolecular hydrogen bonds and C?C π stacking. The potential anticancer effects of these compounds on HeLa (cervical carcinoma), MCF-7 (breast), A549 (lung), HepG2 (liver), and human umbilical vein (HUVEC) cells were examined. Compared with compounds 1–8 and 10–11, compound 9 exhibits potent in vitro cytotoxicity against HeLa cells and lower cytotoxicity against normal cells. Therefore, further in-depth investigations of compound 9 were performed. Absorption titration experiments and fluorescence spectroscopy studies suggested that compound 9 binds to DNA through the intercalation mode.

Full text of DOI:10.1002/ardp.202000236

Received: 9 July 2020
Revised: 25 August 2020
Accepted: 23 September 2020
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DOI: 10.1002/ardp.202000236
F U L L P A P E R
Synthesis, antiproliferative activities, and DNA binding of
coumarin‐3‐formamido derivatives
Jiuzhou Shi1 | Wen Lu1
| Jichao Chen1 | Lu Sun1 | Shilong Yang2
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Mengyi Zhou2 | Li Xu1,3 | Ying Ma1 | Long Yu1
1College of Science, Nanjing Forestry
University, Nanjing, Jiangsu, China
Abstract
2The Advanced Analysis and Testing Center,
Ten coumarin‐3‐formamido derivatives, N‐benzyl‐coumarin‐3‐carboxamide (2),
N‐fluorobenzyl‐coumarin‐3‐carboxamide (3–5), N‐methoxybenzyl‐coumarin‐3‐car-
boxamide (6–8), N‐((1‐methyl‐1H‐imidazol‐5‐yl)methyl)‐coumarin‐3‐carboxamide (9),
N‐(thiophen‐2‐ylmethyl)‐coumarin‐3‐carboxamide (10), and N‐(furan‐2‐ylmethyl)‐
coumarin‐3‐carboxamide (11), were synthesized and characterized. Compound 5
crystallizes in a monoclinic system P21/c space group with four chemical formulas in a
unit cell; molecules of compound 5 are self‐assembled into a two‐dimensional su-
pramolecular structure by intermolecular hydrogen bonds and C⋯C π stacking. The
potential anticancer effects of these compounds on HeLa (cervical carcinoma), MCF‐7
(breast), A549 (lung), HepG2 (liver), and human umbilical vein (HUVEC) cells were
examined. Compared with compounds 1–8 and 10–11, compound 9 exhibits potent in
vitro cytotoxicity against HeLa cells and lower cytotoxicity against normal cells.
Therefore, further in‐depth investigations of compound 9 were performed. Absorption
titration experiments and fluorescence spectroscopy studies suggested that com-
pound 9 binds to DNA through the intercalation mode.
Nanjing Forestry University, Nanjing, Jiangsu,
China
3Co‐Innovation Center for Sustainable
Forestry in Southern China, Nanjing Forestry
University, Nanjing, Jiangsu, China
Correspondence
Wen Lu and Li Xu, College of Science, Nanjing
Forestry University, Nanjing, 210037 Jiangsu,
China.
Email: luwen@njfu.edu.cn (W. L.) and
xuliqby@njfu.edu.cn (L. X.)
Funding information
The Innovation Fund for Young Scholars of
Nanjing Forestry University,
Grant/Award Number: CX2017017; National
Key Research and Development Program of
China, Grant/Award Number:
2017YFD060070602; Jiangsu College
Students' Innovation and Entrepreneurship
Training Project (SPITP),
Grant/Award Number: 201910298098Y; State
Key Laboratory for Chemistry and Molecular
Engineering of Medicinal Resources (Guangxi
Normal University), Grant/Award Number:
CMEMR2017‐B06
K E Y W O R D S
antitumor, coumarin derivatives, crystal structure, DNA bonding
rings, which can introduce a variety of functional groups,[13] so it is
widely used in biomedicine, functional materials, and optoelectronic
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1
INTRODUCTION
Currently, cancer is the second leading cause of death, after cardiovas-
cular diseases, worldwide.[1] Deaths from cancer worldwide are projected
to reach 13 million in 2030.[2,3] Chemotherapy is an effective method for
treating cancer.[4–6] However, due to the resistance of tumor cells to
multiple drugs, the effectiveness of drugs is greatly reduced, resulting in
treatment failure. Therefore, overcoming the multidrug resistance of tu-
mor cells[7–9] and searching for new antitumor drugs with high efficiency
and low toxicity are important issues to be solved urgently.[10–12]
Coumarin, also known as 1,2‐benzopyrone, is a large conjugated
structure with strong modifiability formed by benzene and pyrone
materials.[14] In addition, coumarin compounds have a variety of
biological activities such as anti‐inflammatory,[15,16] antiviral,[17] anti-
bacterial,[18] antioxidant,[19] and antitumor activities[20–22] (Coumarin 1,
Coumarin 2, Coumarin 3, Figure 1).
Amide compounds have antibacterial,[23] antitumor,[24–26] and in-
secticidal biological activities,[27] and they are widely used in medicines,
materials, and industrial solvents. Amide groups are the basic structure of
proteins, which have the advantages of low toxicity and high bioavail-
ability, so they are often used in the structural design of drug mole-
cules.[28] Wen et al.[29] synthesized a series of 4‐(5H‐pyrimido[5,4‐b]indol‐
Jiuzhou Shi and Wen Lu contributed equally to this study.
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Arch Pharm. 2020;e2000236.
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
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FIGURE 1 Coumarin derivatives with antitumor activity
2‐yl‐amino)benzamide compounds, and the results showed that these
compounds had a significant inhibitory effect on tumor cell proliferation.
Zhang et al.[30] synthesized a series of 1,3,4‐thiadiazolamide compounds
and found that some compounds had a better inhibitory effect on human
prostate cancer cells (PC‐3) than the positive control drug dacarbazine.
In this paper, a series of coumarin‐3‐formamido derivatives (com-
pounds 2–11) was synthesized by coumarin‐3‐formate and aminomethyl
benzene with different functional groups, aminomethyl thiophene, ami-
nomethyl furan, and aminomethyl imidazole (Scheme 1). Using the MTT
(3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) method
and human umbilical vein (HUVEC) cells as a normal cell line, the anti-
tumor activities of 2–11 against HeLa, MCF‐7, HepG2, and A549 cancer
cell lines under different concentrations and different action times were
investigated in vitro. This study aspires to lay a foundation for the
research and development of coumarins with a high anticancer activity
and low side effects.
substituted with fluorine, benzylamine substituted with methoxy
groups, 1‐methyl‐5‐aminomethylimidazole, 2‐thiophenemethylamine,
and 2‐furfurylamine by aminolysis of esters as reported.[31]
The structures of the synthesized compounds were confirmed by
infrared (IR; Figures S1–S11), nuclear magnetic resonance (NMR;
Figures S12–S33), and high‐resolution mass spectra (HRMS; Figures
S34–S44). The formation of compounds 2–11 was determined by the
presence of amide absorption peaks, that is, in the region of
1,648–1,662 cm−1 in IR spectra. In 1H NMR spectra, the triplet peaks
of characteristic NH (amide) were found at δ 8.90–9.19 ppm, and
double peaks of methylene (–CH2–) were observed at δ 4.52–4.72 ppm
due to the –NH bond. In the 1H NMR spectra of compounds 6–8, one
singlet at δ 3.74–3.77 ppm was assigned as a methyl proton. In the 13
C
NMR spectra of compounds 2–11, the carbonyl carbon signals of the
amide group and lactone were observed at about δ 160 ppm and about
δ 40 ppm for the methylene group.
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Structural description
2
RESULTS AND DISCUSSION
2.2
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2.1
Chemistry
Colorless needle crystals of compound 5 were obtained by slowly
evaporating the mixed solution of methanol–dichloromethane
(v/v, 2:1) at room temperature. As shown in Figure 1a, compound
5 crystallizes in a monoclinic system P21/c space group with four
Coumarin‐3‐formamido derivatives (2–11) were synthesized by
coumarin‐3‐ethyl formate (1) and benzylamine, benzylamine
SCHEME 1 The synthetic route of compounds 2–11

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chemical formulas in a unit cell. It can be seen from Figure 2a that the
dihedral angle between the pyrone and benzene rings in the cou-
marin motif is 0.40(6)°; thus, the coumarin moiety is almost coplanar.
The 4‐fluorobenzene ring is greatly distorted from the coumarin
parent plane, and the dihedral angle between the two planes is
75.092(55)°. The bond length of the C(6)–C(7) bond is 1.3458(25) Å,
which is a C═C double bond, corresponding to the C═C bond
in coumarin‐3‐formyl‐(3‐(aminomethyl)pyridine (1.34(1) Å).[32] The
bond lengths of the C(7)–C(8) and C(7)–C(16) bonds connected to
the double bond are 1.4982(25) and 1.4536(26) Å, respectively.
Electron withdrawal induces a decrease in the density of its electron
cloud, which changes its bond length (>1.40 Å).
FIGURE 2 (a) Molecular structure of compound 5. All hydrogen atoms are ignored for clarity. (b) The molecular couple formed by the
interaction of hydrogen bonds in compound 5. (c) A view of the one‐dimensional infinite chain‐like motif of 5 (hydrogen atoms, except those
forming hydrogen bonds, are omitted for clarity). (d) A view of the two‐dimensional supramolecular structure of 5 along the ab crystallographic
plane (hydrogen atoms, except those forming hydrogen bonds, are omitted for clarity)

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There are abundant C–H⋯O intermolecular hydrogen bonds in
compound 5. Although C–H⋯O hydrogen bonds are weak, these
hydrogen bonds have a certain direction, which can determine the
direction and position of each component, and play an important
role in the stacked structure of crystals.[31] The molecules of 5 are
self‐assembled into chair‐like molecular couples through C(4)–H(4)
⋯O(3)i, and C(6)–H(6)⋯O(3)i (symmetry code: 2 − x, 2 – y, z) hydro-
gen bonds (Figure 2b). Every couple connects with neighboring
couples by N(2)–H(1A)⋯O(2)ii (symmetry code: x, y, −1 + z) hydrogen
bonds along the a axis direction, forming a one‐dimensional chain
(Figure 2c). Besides the hydrogen bonds, C–C π stackings are also
found within these chains for compound 5 with distances 3.3944(28)
(C(5)⋯C(2)i) and 3.3768(28) Å (C(5)⋯C(2)i), respectively, which
containing imidazole ring (9) against HeLa cells is superior to com-
pounds 1–8 and 10–11. This observation indicates the advantage of
compound 9 over compounds 1–8 and 10–11 in practical application
as an antitumor agent. The stronger inhibitory effect of compound 9
against HeLa cells may be due to the presence of the imidazole ring in
its structure. The azole compounds are aromatic and rich in elec-
trons, so these compounds can interact with enzymes, DNA, and so
forth, thus exhibiting various biological activities such as anticancer,
antibacterial, and antiviral.[35] Zhao et al.[34] reported a series of
dehydroabietylamine derivatives. Compared with derivatives con-
taining other substituents, derivatives containing imidazole groups
showed strong cytotoxicity. For HepG2 cells, only compounds 6 and
7 have a stronger anticancer activity than that of coumarin‐3‐ethyl
connect the chains to form
a
two‐dimensional supramolecular
formate (1). For MCF‐7 cells, the IC50 value of compound 4
structure (Figure 2d). These important distances of the weak inter-
(21.68 μM) is much lower than that of compound 1; for A549 cells,
the IC50 value of compound 6 (21.66 μM) is much lower than that of
compound 1, which shows that compound 4 has a higher anti‐MCF‐7
activity than that of compound 1; and compound 6 has a higher anti‐
A549 activity than that of compound 1. In general, substituent
groups often have a significant impact on the biological activity. The
introduction of different substituent groups in the molecule can
change the electrical, steric, and hydrophobic properties of the
compound, and it can affect the biological activity of the compound
consequently.
actions in 5 are listed in Table 1.
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2.3
Antitumor activities
The cytotoxic activities of these compounds (1–11) against the
human cancer cell lines HeLa, HepG2, MCF‐7, and A549, and
the normal cell HUVEC were evaluated in vitro by MTT thiazolyl
blue colorimetry. Doxorubicin (DOX) was used as a positive
control. DOX is an antineoplastic antibiotic, which can inhibit the
synthesis of RNA and DNA, and it has a good inhibitory effect on
many kinds of tumors.[33] The results of 1–11 against the four
human tumor cells and the normal cell line were summarized as
IC50 values at 48 hr in Table 2.
According to the above results, the structure–activity relation-
ship of the coumarin‐3‐formyl derivatives can be preliminarily
inferred. When the substituent on the amino group is fluorine‐
substituted benzene, the antitumor activity is enhanced, but when
the substituent is methoxybenzene, the antitumor activity is reduced.
The introduction of thiophene and furan groups does not cause the
enhancement of the antitumor activity, but the introduction of the
imidazole group significantly enhances the antitumor activity of ethyl
coumarin‐3‐carboxylate, which may be due to the fact that imidazole
and its derivatives are commonly used as pharmaceutical raw
materials. This will provide a theoretical basis for the development of
coumarin‐based antitumor drugs.
On the basis of the IC50 values (Table 1), a histogram (Figure 3)
was drawn to compare the antineoplastic activity and cytotoxicity of
coumarin‐3‐ethyl formate (1), coumarin‐3‐formamide derivatives
(2–11), and DOX against axenic cancers cells more clearly.
As can be seen from Figure 3, the toxicity of coumarin‐3‐
formamide derivatives (2–11) is lower than DOX (4.40 0.55 μM)[34]
against HUVEC cells, but the antineoplastic activities of all com-
pounds against HeLa, HepG2, MCF‐7, and A549 cells are lower than
DOX. In fact, different compounds have diverse antineoplastic ac-
tivities against a variety of cell lines. For HeLa cells, the IC50 values of
compounds 2–11 were lower than that of coumarin‐3‐ethyl formate
(1), especially compound 9 (5.081 μM), which meant that they had a
higher anti‐HeLa activity than 1 and DOX. The results suggest that
the antiproliferative activity of coumarin‐3‐carboxamide derivatives
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2.4
DNA binding modes
DNA is an important target of antitumor drugs. DNA replication,
transcription, translation, and other biological activities will be
affected when small‐molecule drugs interact with DNA.[36,37] The
TABLE 1 Hydrogen bonds of
∠DHA (°)
D–H⋯A
d(D–H) (Å)
0.9303 (21)
0.9305 (18)
0.8598 (15)
d(H⋯A) (Å)
2.5745 (13)
2.6365 (13)
2.3353 (13)
d(D⋯A) (Å)
3.3610 (25)
3.4087 (23)
3.0209 (20)
compound 5
C(4)‐H(4)⋯O(3)i
C(6)‐H(6)···O(3)i
N(2)‐H(1A)···O(2)ii
142.556 (133)
140.802 (116)
136.904 (103)
i2 – x, 2 – y, –z.
iix, y, –1 + z.

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TABLE 2 Cytotoxicity of 1–11 and DOX against axenic cancer cells and the normal cell
IC50 SEa (μM)
HeLa
HepG2
MCF‐7
A549
HUVEC
1
>150
36.38 0.43
>150
118.46 0.25
115.64 0.45
131.8 0.56*
21.68 0.08
146.37 0.67*
130.79 0.43*
>150
134.01 0.33
32.14 0.17**
103.86 0.44
73.91 0.25**
104.67 0.33
21.66 0.05***
45.85 0.20*
73.40 0.18
>150
119.1 0.25
>150
2
96.36 0.03**
83.02 0.25*
70.58 0.05
94.71 0.37**
42.36 0.03*
73.46 0.15
30.01 0.29**
5.081 0.12**
>150
3
57.12 0.28
46.03 0.03**
90.95 0.22
32.10 0.09***
35.08 0.10
40.02 0.15*
>150
135.21 0.09*
>150
4
5
75.89 0.06
78.12 0.22*
123.36 0.13
110.98 0.07*
>150
6
7
8
78.85 0.34
>150
9
10
>150
49.56 0.13*
131.96 0.26
14 1.03***
117.97 0.23
>150
>150
11
114.08 0.33
3.55 0.17b
50.67 0.27**
1.20 0.04b
153.71 0.11
4.40 0.55b
DOX[34]
3.35 0.69b
aAverage IC50 values from at least three independent experiments.
bNot significant, compounds compared with 1, respectively.
*p < .05.
**p < .01.
***p < .001.
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study on the binding mode between compounds and DNA is of
great significance to understand the mechanism of antitumor
drugs. To further investigate the antitumor mechanism of
coumarin‐3‐formamido derivatives, the interaction between
compound 9 and herring sperm DNA (HS‐DNA) was studied by
UV spectroscopy and ethidium bromide (EB)–DNA fluorescence
quenching.
2.4.1
UV‐visible absorption spectroscopy studies
UV absorption spectroscopy is one of the means to study the inter-
action between compounds and DNA. The intensity and position of
the characteristic absorption peak will change when the compound
binds to the base of DNA. Therefore, it can be determined whether
the compound has interacted with DNA according to the change of
FIGURE 3 Comparison of the
antiproliferative effects and cytotoxicity of
doxorubicin (DOX) and 1–11

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the absorption peak intensity of the compound and the displacement
of the characteristic absorption peak.[38] The interaction between
compound 9 and HS‐DNA was investigated by UV spectroscopy. As
shown in Figure 4, peaks at ~280 and ~340 nm were observed in the
spectra of compound 9, which may be attributed to the π–π transi-
tions in 9.[39] The compound absorption band exhibited hypochro-
mism without any significant shift after the addition of different
concentrations of DNA to the Tris‐HCl/NaCl buffer solution of
compound 9. The results may indicate that the binding mode
between compound 9 and DNA is intercalation.
plot (Figure 5, inset), the apparent binding constant of compound 9
was calculated to be 1.09 × 105 M−1. The results indicate a moderate
intercalative binding between compound 9 and DNA, which is
consistent with the UV results.
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3
CONCLUSION
Ten new substituted coumarin‐3‐carboxamides (2–11) have been
prepared and characterized in this study. Among them, compounds 6,
8, and 9 showed better inhibitory activities against HeLa cells,
compounds 4, 6, 7, and 8 exhibited good inhibitory effects on HepG2,
compound 4 has a good anticancer activity against MCF‐7, and
compounds 2, 6, and 7 displayed a better anticarcinogenic activity
against A549. It should be noted that the antiproliferative activity of
compound 9 against HeLa cells (IC50 = 5.08 μM) was comparable to
that of positive control DOX (IC50 = 3.55 μM), and the toxicity against
normal cells was lower than DOX. Various spectroscopic approaches
indicate that compounds 1 and 2 could effectively bind with DNA
through intercalation mode. Overall, these results indicate that
compound 9 could be used as a promising anticancer drug and
deserves further investigation.
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2.4.2
Fluorescence spectroscopy studies
A competitive binding experiment was evaluated to confirm the DNA
binding mode of compound 9. The fluorescence of the EB molecule
itself is very weak, but if DNA is present, the EB molecule can be
quickly inserted into the DNA base pair, thus emitting a strong
fluorescence. The intercalation between the compound and the base
pairs of DNA can be confirmed when the emission intensity of
DNA–EB is reduced or quenched after the addition of the com-
pound.[40] As seen in Figure 5, the emission intensity decreased ob-
viously with the addition of compound 9 to the EB–DNA solution,
suggesting that DNA‐bound EB was replaced by compound 9 and the
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EXPERIMENTAL
interaction between DNA and compound
calative mode.
9
was in an inter-
4
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Chemistry
The quenching strength of compound 9 toward EB–DNA was
calculated by the classical Stern–Volmer equation[41,42]: I0/I = 1 + KSV
[Q], where, I and I0 are the emission intensities at 340 nm in the
presence and absence of compound 9, respectively. The linear
Stern–Volmer quenching constant value is denoted as KSV and [Q] is
the concentration of the DNA (quencher). From the Stern–Volmer
4.1
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4.1.1
General
All reagents and solvents were of analytical grade and used without
further purification. DOX, EB, HS‐DNA, Dulbecco's modified Eagle's
FIGURE 4 Absorption spectra of 9
(5 × 10−5 M) in the absence and presence of
increasing amounts of herring sperm DNA
(HS‐DNA; 6.74 × 10−4) at room temperature in
Tris‐NaCl/HCl buffer (pH 7.4). The arrow
shows the absorbance change when the
HS‐DNA concentration is increased

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FIGURE 5 Emission spectra of DNA–EB in
the absence and the presence of increasing
amounts of compound 9 at room temperature
([EB] = 2 × 10−5 M, [DNA] = 1 × 10−4 M,
[9] = 1.5 × 10−5 M). EB, ethidium bromide
medium (DMEM) and MTT, fetal bovine serum, and penicillin/strep-
tomycin were purchased from Nanjing KeyGen Biotech. Co. Ltd. The
Tris‐HCl buffer solution was prepared with triple‐distilled water.
IR spectra were recorded on a Bruker Vertex 80 FT‐IR spec-
trometer with KBr discs in the 4,000−500 cm−1 range. The melting
point apparatus (Shenguan Instrument Co., Ltd, Shanghai, China) was
used for determining melting points (°C). The 1H and 13C NMR
spectra were measured on a 600 M Bruker Avance III NMR spec-
trometer (Billerica, MA). The 1H NMR and 13C NMR spectra were
recorded in CDCl3 or dimethyl sulfoxide (DMSO)‐d6 as solvent.
Chemical shifts (δ) were expressed as parts per million (ppm), relative
to the NMR solvent signals (CDCl3 7.26 and 77.00 ppm for 1H and
13C; DMSO‐d6 2.50 and 40.00 ppm for 1H and 13C NMR, respec-
tively). J values are given in hertz. HRMS were measured to de-
termine the purity of all tested compounds by LTQ Orbitrap XL mass
spectrometer (Thermo Electron). The DNA binding modes were in-
vestigated by Lambda 950 (PerkinElmer) and LS55 fluorescence
spectrophotometer (PerkinElmer). The IR, 1H NMR, 13C NMR, and
MS results are shown in Figures S1–S44.
(s, 1H, ═CH), 7.65–7.60 (m, 2H, Ar‐H), 7.36–7.32 (m, 2H, Ar‐H),
4.430–4.394 (m, 2H, –CH2), and 1.41 (t, J = 14.4 Hz, 3H, –CH3); 13C
NMR (150 M, CDCl3, 25°C, TMS, δ ppm): 163.08, 156.71, 155.18,
148.58, 134.33, 129.49, 124.84, 118.37, 117.90, 116.80, 61.99, and
14.24; HRMS (ESI) m/z [M+H]+ calculated for C12H11O4: 219.0652,
found: 219.0659.
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4.1.3
Synthesis of the substituted
coumarin‐3‐carboxamides 2–11
Compounds 2–11 were synthesized by a reaction between coumarin‐
3‐ethyl formate (1) and substituted amine, as described in Scheme 1.
Amine (6 mmol) was added to a solution of coumarin‐3‐ethyl formate
(1.09 g, 5 mmol) in 20 ml hot ethanol. The resulting mixture was re-
fluxed for 6 hr and a white precipitate was obtained. The precipitate
was isolated by vacuum filtration, then recrystallized with ethanol,
and dried under vacuum.
The InChI codes of the investigated compounds, together
with some biological activity data, are provided as Supporting
Information.
N‐Benzyl‐2‐oxo‐2H‐chromene‐3‐carboxamide (2)
Colorless solid, yield: 63.5%, M.P.: 153–154.6°C. IR (KBr) νmax: 3,330
;
(–CONH–), 3,054 (═CH, Ar‐H), 1,705 (C═O), 1,655 (C═O) cm−1 1H
NMR (600 M, DMSO‐d6, 25°C, TMS): δ = 9.13 (t, J = 11.4, 1H, –NH),
8.88 (s, 1H, ═CH), 7.99–7.98 (m, 1H, Ar‐H), 7.76–7.73 (m, 1H, Ar‐H),
7.50 (d, J = 8.4, 1H, Ar‐H), 7.45–7.43 (m, 1H, Ar‐H), 7.37–7.33 (m, 4H,
Ar‐H), 7.28–7.25 (m, 1H, Ar‐H), and 4.56 (d, J = 6.0, 2H, –CH2) ppm;
13C NMR (150 M, DMSO‐d6, 25°C, TMS): δ = 161.71, 160.81, 154.35,
148.00, 139.37, 134.54, 130.72, 128.85, 127.86, 127.42, 125.58,
119.54, 118.94, 116.60, and 43.22 ppm; HRMS (ESI) m/z [M+H]+
calculated for C17H14NO3: 280.0968, found: 280.0969.
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4.1.2
Synthesis of coumarin‐3‐ethyl formate (1)
Coumarin‐3‐ethyl formate was prepared according to the procedure
of a previous study.[43] Yield, 66.8%. M.P.: 92.4–94.6°C. IR (KBr) νmax
3,060 (═CH, Ar‐H), 2,975–2,921 (–CH2, –CH3), 1,768 (C═O) cm−1; 1H
:
NMR (600 M, CDCl3, 25°C, tetramethylsilane [TMS], δ ppm): 8.52

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N‐(2‐Fluorobenzyl)‐2‐oxo‐2H‐chromene‐3‐carboxamide (3)
White needle crystals, yield: 65.8%. M.P.: 151.1–152.5°C. IR (KBr) νmax
128.90, 125.90, 125.22, 120.55, 118.71, 118.67, 116.58, 110.36,
58.41, and 55.34 ppm; HRMS (ESI) m/z [M+H]+ calculated for
:
3,342 (–CONH–), 3,033 (═CH, Ar‐H), 1,705 (C═O), 1,658 (C═O) cm−1
1H NMR (600 M, DMSO‐d6, 25°C, TMS): δ = 9.12 (t, J = 12.0 Hz,
1H, –NH), 8.87 (s, 1H, ═CH), 7.98–7.97 (m, 1H, Ar‐H), 7.76–7.73
(m, 1H, Ar‐H), 7.49 (d, J = 8.4 Hz, 1H, Ar‐H), 7.44–7.40 (m, 2H, Ar‐H),
7.34–7.31 (m, 1H, Ar‐H), 7.21–7.16 (m, 2H, Ar‐H), and 4.60
(d, J = 6.0 Hz, 2H, –CH2) ppm; 13C NMR (150 M, 25°C, DMSO‐d6, TMS):
δ = 161.81, 161.42, 160.82, 159.80, 154.37, 148.11, 134.60, 130.75,
129.98, 129.95, 129.57, 129.52, 126.04, 125.95, 125.60, 124.87,
124.85, 119.37, 118.91, 116.61, 115.68, 115.54, 37.29, and 37.26 ppm;
HRMS (ESI) m/z [M+H]+ calculated for C17H13NFO3: 298.0874, found:
298.0882.
;
C18H16NO4: 310.1074, found: 310.1076.
N‐(3‐Methoxybenzyl)‐2‐oxo‐2H‐chromene‐3‐carboxamide (7)
White needle crystals, yield: 70.5%. M.P.: 146.5–146.9°C. IR (KBr)
ν
max: 3,344 (–CONH–), 3,054 (═CH, Ar‐H), 1,701 (C═O), 1,656
(C═O) cm−1 1H NMR (DMSO‐d6, 600 MHz, 25°C, TMS): δ = 9.11
;
(t, J = 15.0 Hz, 1H, –NH), 8.89 (s, 1H, ═CH), 8.01–7.99 (m, 1H, Ar‐H),
7.78–7.75 (m, 2H, Ar‐H), 7.53–7.51 (d, J = 10.2 Hz, 1H, Ar‐H), 7.46
(t, J = 18 Hz, 1H, Ar‐H), 7.30–7.26 (m, 1H, Ar‐H), 6.97–6.95 (m, 2H,
Ar‐H), 6.87–6.84 (m, 1H, Ar‐H), 4.56 (d, J = 7.2 Hz, 2H, –CH2), and
3.77 (s, 3H, –CH3) ppm; 13C NMR (CDCl3, 150 MHz, 25°C, TMS):
δ = 161.53, 161.43, 15.91, 154.47, 148.60, 139.48, 134.11, 129.83,
129.76, 125.32, 119.95, 118.66, 118.44, 116.67, 113.25, 113.02,
55.25, and 43.82 ppm; HRMS (ESI) m/z [M+H]+ calculated for
C18H16NO4: 310.1074, found: 310.1089.
N‐(3‐Fluorobenzyl)‐2‐oxo‐2H‐chromene‐3‐carboxamide (4)
White needle crystals, yield: 60.6%, M.P.: 170.8–171.6°C. IR (KBr)
ν
max: 3,321 (–CONH–), 3,053 (═CH, Ar‐H), 1,705 (C═O), 1,655
(C═O) cm−1 1H NMR (600 M, DMSO‐d6, 25°C, TMS): δ = 9.18
;
(t, J = 12.6 Hz, 1H, –NH), 8.86 (s, 1H, ═CH), 7.96 (d, J = 7.8 Hz, 1H, Ar‐
H), 7.75–7.72 (m, 1H, Ar‐H), 7.50–7.49 (d, J = 7.8 Hz, 1H, Ar‐H),
7.44–7.41 (t, J = 15.0 Hz, 1H, Ar‐H), 7.39–7.35 (m, 1H, Ar‐H),
7.20–7.16 (m, 2H, Ar‐H), 7.08 (t, J = 10.8 Hz, 1H, Ar‐H), and 4.57
(d, J = 6.0 Hz, 2H, –CH2) ppm; 13C NMR (150 M, DMSO‐d6, 25°C,
TMS): δ = 163.51, 161.91, 160.73, 154.36, 148.00, 142.55, 142.51,
134.54, 130.75, 130.71, 130.70, 125.57, 123.80, 123.79, 119.55,
118.92, 116.58, 114.57, 114.43, 114.16, 114.03, 42.73, and
42.72 ppm; HRMS (ESI) m/z [M+H]+ calculated for C17H13NFO3:
298.0874, found: 298.0885.
N‐(4‐Methoxybenzyl)‐2‐oxo‐2H‐chromene‐3‐carboxamide (8)
White needle crystals, yield: 72.0%. M.P.: 146.3–146.8°C. IR (KBr)
νmax 3,344 (–CONH–), 3,054 (═CH, Ar‐H), 1,701 (C═O), 1,656
(C═O) cm−1 1H NMR (DMSO‐d6, 300 MHz, 25°C, TMS): δ = 9.03
;
(t, J = 11.4 Hz, 1H, –NH), 8.88 (s, 1H, ═CH), 7.99 (d, J = 13.8 Hz, 1H,
Ar‐H), 7.75 (d, J = 15.0 Hz, 1H, Ar‐H), 7.51–7.41 (m, 2H, Ar‐H), 7.32
(d, J = 16.8 Hz, 2H, Ar‐H), 6.92 (t, J = 17.4, 2H, Ar‐H), 4.50–4.52
(d, J = 11.4 Hz, 2H, –CH2), and 3.74 (s, 3H, –CH3) ppm; 13C NMR
(CDCl3, 150 MHz, 25°C, TMS): δ = 161.38, 161.36, 159.00, 154.43,
146.45, 134.05, 130.06, 129.80, 129.11, 125.28, 118.64, 118.49,
116.62, 114.10, 55.29, and 43.34 ppm; HRMS (ESI) m/z [M+H]+ cal-
culated for C18H16NO4: 310.1074, found: 310.1088.
N‐(4‐Fluorobenzyl)‐2‐oxo‐2H‐chromene‐3‐carboxamide (5)
Colorless needle single‐shaped crystals, yield: 64.6%, M.P.:
174.4–175.6°C. IR (KBr) νmax: 3,332 (–CONH–), 3,049 (═CH, Ar‐H),
;
1,703 (C═O), 1,655 (C═O) cm−1 1H NMR (600 M, DMSO‐d6, 25°C,
N‐[(1‐Methyl‐1H‐imidazol‐5‐yl)methyl]‐2‐oxo‐2H‐chromene‐3‐
carboxamide (9)
TMS): δ = 9.09 (t, J = 12.6 Hz, 1H, Ar‐H), 8.85 (s, 1H, ═CH), 7.97–7.94
(m, 1H, Ar‐H), 7.76–7.70 (m, 1H, Ar‐H), 7.49–7.38 (m, 4H, Ar‐H),
Light yellow solid, yield: 79%, M.P.: 173.0–173.8°C. IR (KBr) νmax
:
3,332 (–CONH–), 3,043 (Ar‐H), 1,708 (C═O), 1,648 (C═O) cm−1 1H
;
7.17–7.11 (m, 2H, Ar‐H), and 4.54 (d, J = 12.0 Hz, 2H, –CH2) ppm; 13
C
NMR (DMSO‐d6, 600 MHz, 25°C, TMS): δ = 8.91 (t, J = 10.8 Hz, 1H,
–NH), 8.86 (s, 1H, ═CH), 7.97 (t, J = 7.8 Hz, 1H, Ar‐H), 7.76–7.33
(m, 1H, Ar‐H), 7.57 (s, 1H, Ar‐H), 7.51 (d, J = 7.8 Hz, 1H, Ar‐H), 7.44
(t, J = 15 Hz, 1H, Ar‐H), 6.87 (s, 1H, Ar‐H), 4.54 (d, J = 6.0 Hz, 2H,
–CH2), and 3.63 (s, 3H, –CH3) ppm; 13C NMR (DMSO‐d6, 150 MHz,
25°C, TMS,): δ = 161.58, 160.77, 154.35, 147.88, 138.96, 134.59,
130.72, 129.10, 128.15, 125.63, 119.65, 118.93, 116.63, and
32.97 ppm; HRMS (ESI) m/z [M+H]+ calculated for C15H14N3O3:
284.1030, found: 284.1028.
NMR (150 M, DMSO‐d6, 25°C, TMS): δ = 163.06, 162.52, 161.92,
160.74, 156.44, 155.00, 154.35, 149.11, 148.02, 135.68, 135.66,
134.93, 134.55, 130.74, 130.71, 129.95, 129.90, 125.58, 125.30,
119.51, 118.92, 118.27, 118.16, 116.61, 116.59, 115.60, 115.46, and
42.51 ppm; HRMS (ESI) m/z [M+Na]+ calculated for C17H12NFNaO3:
320.0693, found: 2,320.0708.
N‐(2‐Methoxybenzyl)‐2‐oxo‐2H‐chromene‐3‐carboxamide (6)
White needle crystals, yield: 73.2%. M.P.: 176.5–177.8°C. IR (KBr)
ν
max: 3,369 (–CONH–), 3,037 (═CH, Ar‐H), 1,701 (C═O), 1,656
N‐(Thiophen‐2‐ylmethyl)‐2‐oxo‐2H‐chromene‐3‐carboxamide (10)
Colorless needle crystals, yield: 63.7%. M.P.: 176.8–177.5°C. IR (KBr)
(C═O) cm−1 1H NMR (DMSO‐d6, 600 MHz, 25°C, TMS): δ = 9.08
;
(t, J = 14.4 Hz, 1H, –NH), 8.86 (s, 1H, ═CH), 7.97–7.96 (m, 2H, Ar‐H),
7.75–7.72 (m, 3H, Ar‐H), 7.49 (d, J = 9.6 Hz, 1H, Ar‐H), 7.43 (t, J = 18,
2H, Ar‐H), 7.26–7.23 (m, 1H, Ar‐H), 6.93–6.92 (m, 2H, Ar‐H),
6.83–6.81 (m, 1H, Ar‐H), 4.53 (d, J = 7.2 Hz, 2H, –CH2), and 3.74
(s, 3H, –CH3) ppm; 13C NMR (CDCl3, 150 MHz, 25°C, TMS):
δ = 161.32, 161.25, 157.68, 154.41, 148.31, 133.93, 129.79, 129.53,
ν
max: 3,328 (–CONH–), 3,049 (═CH, Ar‐H), 1,701 (C═O), 1,651
(C═O) cm−1 1H NMR (DMSO‐d6, 600 MHz, 25°C, TMS): δ = 9.16
;
(t, J = 12.0 Hz, 1H, –NH), 8.89 (s, 1H, ═CH), 7.99–7.97 (m, 1H, Ar‐H),
7.76–7.72 (m, 1H, Ar‐H), 7.50 (d, J = 8.4 Hz, 1H, Ar‐H), 7.44–7.40
(m, 2H, thiophene‐H and Ar‐H), 7.07 (d, J = 3.6 Hz, 1H, thiophene‐H),
6.98–6.97 (m, 1H, thiophene‐H), and 4.71 (d, J = 6.0 Hz, 2H,

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SHI ET AL.
|
–CH2) ppm; 13C NMR (DMSO‐d6, 150 MHz, 25°C, TMS): δ = 160.44,
159.68, 153.33, 147.20, 140.901, 133.58, 129.71, 126.11, 125.44,
124.75, 124.54, 118.17, 117.86, 115.56, and 37.13 ppm; HRMS (ESI)
m/z [M+H]+ calculated for C15H12NO3S: 286.0532, found: 280.0539.
4.4
DNA binding studies
|
All experiments involving DNA were performed in a buffer solution
(10 mM Tris‐HCl/50 mM NaCl; pH 7.40) at room temperature. The
UV absorption ratio (A260/A280) at 260 and 280 nm is 1.87, in-
dicating that the HS‐DNA solution is sufficiently free of protein.
The concentration of HS‐DNA was determined at 260 nm with
6,600 M−1·cm−1 as the molar absorption coefficient. The UV ab-
sorption spectra titrations were carried out at room temperature in
Tris‐HCl/NaCl buffer solution from 240 to 400 nm by increasing the
amounts of DNA relative to the compound. The fluorescence emis-
sion spectra were performed in Tris‐HCl/NaCl buffer solution by
adding different concentrations of compound 9 into the EB–DNA
solution (2 × 10−5 M EB, 1 × 10−4 M DNA).
N‐(Furan‐2‐ylmethyl)‐2‐oxo‐2H‐chromene‐3‐carboxamide (11)
White needle crystals, yield: 68.7%. M.P.: 174.0–174.9°C. IR (KBr)
ν
max: 3,351 (–CONH–), 3,054 (═CH, Ar‐H), 1,703 (C═O), 1,662
(C═O) cm−1 1H NMR (DMSO‐d6, 600 MHz, 25°C, TMS): δ = 9.02
;
(t, J = 12.0 Hz, –NH), 8.89 (s, 1H, ═CH), 8.00–7.99 (m, Ar‐H),
7.77–7.74 (m, 1H, Ar‐H), 7.60 (t, J = 1.8 Hz, Ar‐H), 7.52
(d, J = 8.4 Hz, Ar‐H), 7.46–7.43 (m, 1H, furan‐H), 6.42–6.41 (m, 1H,
furan‐H), 6.34–6.33 (m, 1H, furan‐H), and 4.55 (d, J = 6.0 Hz, 2H,
–CH2) ppm; 13C NMR (DMSO‐d6, 150 MHz, 25°C, TMS): δ = 161.58,
160.86, 154.40, 152.11, 148.21, 142.86, 134.66, 130.80, 125.64,
119.32, 118.94, 116.65, 111.01, 107.71, and 36.54 ppm; HRMS (ESI)
m/z [M+H]+ calculated for C15H12NO4: 270.0761, found: 270.0774.
ACKNOWLEDGMENTS
The authors would like to thank the advanced analysis and testing
center of Nanjing Forestry University for supporting IR, NMR, and
MS testing. This study was supported by the Innovation Fund for
Young Scholars of Nanjing Forestry University (Project Number:
CX2017017), Jiangsu College Students' Innovation and En-
trepreneurship Training Project (SPITP; Project Number:
201910298098Y), National Key Research and Development Program
of China (Project Number: 2017YFD060070602), State Key La-
boratory for Chemistry and Molecular Engineering of Medicinal Re-
sources (Guangxi Normal University; Project Number: CMEMR2017‐
B06), and the Priority Academic Program Development of Jiangsu
Higher Education Institutions.
|
4.2
Crystal structure determination
Diffraction data for compound 5 were collected on a Bruker D8
VENTURE diffractometer equipped with a graphite monochromatic
Mo Κα radiation (λ = 0.71073 Å) at 297(2) K by using ω–ϕ scan mode.
The structures were solved by direct methods using SHELXS‐2014
and refined by full‐matrix least‐squares methods on F2 using
SHELXL‐2014.[44] The non‐hydrogen atoms were refined with ani-
sotropic thermal parameters. The details of crystallographic data and
structure refinement parameters of compound 5 are summarized in
Table S1, and relevant bond lengths and angles of compound 5 are
shown in Table S2. Crystallographic data for compound 5 have been
deposited at the Cambridge Crystallographic Data Centre. The
CCDC reference number for compound 5 is 1510619.
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
ORCID
Wen Lu
http://orcid.org/0000-0001-8400-0072
|
4.3
Antitumor and cytotoxicity assay
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How to cite this article: Shi J, Lu W, Chen J, et al. Synthesis,
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