Synthesis, computational study and cytotoxicity of 4-hydroxycoumarin-derived imines/enamines

Article abstract of DOI:10.1007/s11030-020-10086-2

Abstract: In this study, we applied a direct condensation between 3-acetyl-4-hydroxy-2H-chromen-2-one and different amines (anilines and benzyl amines) in order to synthesize some coumarin-based imines/enamines (3a–o) as cytotoxic agents. All the compounds were characterized by means of FT-IR, NMR, mass spectroscopy and elemental analyses. Since the title compounds can exist as different forms including (s-cis)-imine and (s-trans)-imine or (E and Z)-enamines, their conformational and geometrical aspects were investigated computationally by DFT method. The optimized geometry parameters, ΔE, ΔG, ΔH, Mulliken atomic charge, HOMO and LUMO energy, and NBO analysis suggested that these compounds can exist predominantly in (E)-enamine form. All the synthesized compounds (3a–o) were evaluated in vitro for their cytotoxic activities against cancer cell lines (MCF-7 and A549) and normal cell line (BEAS-2B) using the MTT assay. The 4-hydroxybenzyl derivative 3k was found to be the most potent cytotoxic agent with no selectivity, similar to doxorubicin. However, the 4-chlorobenzyl analog 3o could be considered as an equipotent compound respect to doxorubicin with higher selectivity. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s11030-020-10086-2

Molecular Diversity
https://doi.org/10.1007/s11030-020-10086-2
ORIGINAL ARTICLE
Synthesis, computational study and cytotoxicity
of 4‑hydroxycoumarin‑derived imines/enamines
Samaneh Vaseghi1 · Mohammad Yousef2 · Mohammad Shokrzadeh3 · Zinatossadat Hossaini4 ·
Zahra Hosseini‑khah5 · Saeed Emami6
Received: 14 October 2019 / Accepted: 8 April 2020
© Springer Nature Switzerland AG 2020
Abstract
In this study, we applied a direct condensation between 3-acetyl-4-hydroxy-2H-chromen-2-one and diferent amines (anilines
and benzyl amines) in order to synthesize some coumarin-based imines/enamines (3a–o) as cytotoxic agents. All the com-
pounds were characterized by means of FT-IR, NMR, mass spectroscopy and elemental analyses. Since the title compounds
can exist as diferent forms including (s-cis)-imine and (s-trans)-imine or (E and Z)-enamines, their conformational and
geometrical aspects were investigated computationally by DFT method. The optimized geometry parameters, ΔE, ΔG, ΔH,
Mulliken atomic charge, HOMO and LUMO energy, and NBO analysis suggested that these compounds can exist predomi-
nantly in (E)-enamine form. All the synthesized compounds (3a–o) were evaluated in vitro for their cytotoxic activities against
cancer cell lines (MCF-7 and A549) and normal cell line (BEAS-2B) using the MTT assay. The 4-hydroxybenzyl derivative
3k was found to be the most potent cytotoxic agent with no selectivity, similar to doxorubicin. However, the 4-chlorobenzyl
analog 3o could be considered as an equipotent compound respect to doxorubicin with higher selectivity.
Graphic abstract
Keywords Anticancer · 2H-chromen-2-one · Coumarin · Schif bases · Computational study · DFT
Introduction
Electronic supplementary material The online version of this
According to World Health Organization (WHO) reports,
cancers will cause more than 13 million deaths in 2030.
These reports also indicate that 20% of people under age
75 will struggle with cancer in their lifetime [1, 2]. Nowa-
days, chemotherapy is the essential approach for the cancer
article (https://doi.org/10.1007/s11030-020-10086-2) contains
supplementary material, which is available to authorized users.
* Saeed Emami
sdemami12@gmail.com; semami@mazums.ac.ir
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

Molecular Diversity
treatment [3], even though it faces disadvantages such as
drug resistance and drug-induced toxicity [4, 5]. In this situ-
ation, fnding new anticancer agents with high potency and
proper therapeutic index is urgently required.
catalyst. All the compounds were characterized by means
of FT-IR, NMR, mass spectroscopy and elemental analy-
ses, and the related data are presented in Experimental sec-
tion. For clear interpretation of 1H and 13C NMR spectra,
the atom numbering of the general structure is illustrated
in Fig. 2.
Chromone derivatives including coumarin (2H-chromen-
2-one, 2H-1-benzopyran-2-one) ones possess unique bio-
logical properties such as anti-infammatory [6], antioxidant
[7], antinociceptive [8], hepatoprotective [9], antithrombotic
[10, 11], antiviral [12, 13], antimicrobial [14, 15], antitu-
berculosis [16], anti-carcinogenic [17], antihyperlipidemic
[18] and anticholinesterase [19] activity. Therefore, apply-
ing coumarin backbone to synthesize novel derivatives for
further screening as novel therapeutic agents is the driving
force for diverse investigations.
At the frst glance, it is expected that the fnal products
from the reaction of anilines or benzylamines with the com-
pound 2 be an imine. However, as depicted in Fig. 1, the
imine prototype can exist as (s-cis)-imine or s-trans forms.
Further tautomeric transformation of each imine form results
in (E)- or (Z)-enamines with an intramolecular hydrogen
bond between NH and carbonyl group of either ketone or
lactone functional group. Based on the obtained data from
1H NMR and FT-IR in some cases, we suspected that the
fnal products are preferentially in the enamine form. In par-
Among coumarin derivatives, 4-hydroxycoumarin ana-
logs and complexes can inhibit cell proliferation [20, 21].
Moreover, diferent studies suggested that 3-substituted chr-
omones and coumarins are potential scafolds for the design
and synthesis of anticancer agents [22, 23]. In particular,
Schif bases of 3-substituted coumarin have proved to have
antioxidant [24], antifungal [25] and anticancer [26] proper-
ties. A series of coumarin Schif bases containing sulfona-
mide moieties were synthesized and evaluated as cytotoxic
agents against B16-F10 (B16 melanoma) and MCF-7 (breast
cancer) cell lines [27]. Furthermore, coumarin Schif bases
of phosphorohydrazine were assessed as alkylating agents
against L1210 and P388 cell lines of leukemia [28].
In the present study, we synthesized some Schif bases
of 3-acetyl-4-hydroxycoumarin (3-acetyl-4-hydroxy-2H-
chromen-2-one) as cytotoxic agents and characterized them
by means of NMR, IR, MS and elemental analyses. Moreo-
ver, to get more insights into the electronic structures of the
title compounds together with fnding more stable tautom-
ers and conformers, the full geometry optimizations were
applied using density functional theory (DFT) calculations.
Thus, we report here, the synthesis, characterization, com-
putational studies and cytotoxic activity of coumarin-based
imines/enamines 3a–o.
1
ticular, in the H NMR spectra of benzyl compounds 3k,
3m and 3n, the benzylic CH2 was split into a doublet by
coupling with NH group. Furthermore, the wave number
of ketone functional group in 3 was much lower than that
of normal ones, which is mainly due to the hydrogen bond
between this functional group and NH. However, as inter-
preted in Experimental section, the rest of compounds can
exist in both enamine and imine forms, and consequently,
we performed the computational study to investigate which
one is the predominant form.
Computational study
The structural analysis
In order to identify the most stable forms of imines and
enamines among several possibilities, all the geometries
were optimized in the solvent media. The optimized struc-
tures have resulted in three stable conformers including
(E)-enamine, (Z)-enamine and (s-cis)-imine-1 forms as
exemplifed in Fig. 3 for compound 3a. Interestingly, the
(s-trans)-imine and (s-cis)-imine-2 forms mostly convert to
(s-cis)-imine-1 and (Z)-enamine counterparts, respectively.
Therefore, the equilibrium is toward (s-cis)-imine-1 rather
than (s-trans)-imine, and (Z)-enamine is more stable than
(s-cis)-imine-2.
Results and discussion
Chemistry
The thermodynamic parameters of both isomers of
enamine (E and Z) and (s-cis)-imine-1 rotamer of imine are
reported in Table 1. The values of electronic energy, ΔE,
are (EE −EZ), and also enthalpy and Gibbs free energy (ΔH
and ΔG) values are gained from (enamine H/G−imine H/G)
energies, which indicate more stability of (E)-enamine over
(s-cis)-imine-1.
The synthesis of title compounds 3a–o is illustrated in Fig. 1.
4-Hydroxy-2H-chromen-2-one (1) was reacted with acetic
acid as an acetyl source and solvent in the presence of phos-
phorus oxychloride to give 3-acetyl compound 2 [29]. Com-
pound 2 as a ketone can react with various amines to form a
wide range of anilino or benzylamino derivatives (3a–i and
3j–o, respectively). All the anilino and benzylamino adducts
of 3-acetyl-4-hydroxy-2H-chromen-2-one were readily pro-
vided in refux condition in ethanol as solvent without any
For further investigation, the Gibbs free energy (ΔG)
and also enthalpy energy (ΔH) of all three structures were
determined (Table 1). The calculated values of ΔH and ΔG
1 3

Molecular Diversity
Fig. 1 Synthesis of the anilino and benzylamino adducts of 3-acetyl-4-hydroxy-2H-chromen-2-one. The products 3a–o can exist in diferent tau-
tomeric forms
are in agreement with ΔE results, indicating the stability
of (E)-enamine over other isomers. The obtained stability
order for the mentioned compounds is: (E)-enamine>(Z)-
enamine>(s-cis)-imine-1. The data suggest that the forma-
tion of (E)-enamine is favorable and more stable than the Z
ones. The stronger intramolecular hydrogen bond between
NH and oxygen of carbonyl group in (E)-enamine than
hydrogen bond with carbonyl of lactone in (Z)-enamine can
be responsible for this assumption (Fig. 3). On the other
hand, due to the repulsion between electron pairs on adjacent
oxygen and nitrogen atoms, the optimization cycles lead to
formation of (s-cis)-imine-1 conformer rather than s-trans
conformer in the imine tautomer. In addition, the interaction
of hydrogen atoms of the methyl group with the oxygen lone
pairs in the (s-cis)-imine-1 conformer can lead to the forma-
tion of an unconventional C–H…O hydrogen bond [30, 31].
1 3

Molecular Diversity
reactions or interactions with other species were evaluated
by NBO calculations in the solvent media. The band gap
energy [Eg (EHOMO − ELUMO)] was also calculated which
can be used for predicting the most reactive site and also
provides explanation for several types of reaction in con-
jugated systems [32]. The Eg represents the intermolecular
charge transfer from an electron donor to electron acceptor
group [33]; hence, it can determine the stability, reactivity,
polarizability and chemical hardness–softness of diferent
Fig. 2 Atom numbering of the general structure of 3a–o used for
interpretation of 1H and 13C NMR data
Table 1 Computational parameters including ΔE, ΔH and ΔG
obtained for the synthesized compounds 3a–o
To further comprehend the optimized parameters of
the three species [(E)-enamine, (Z)-enamine and (s-cis)-
imine-1], the critical bond lengths (L1, L2 and L3) and also
dihedral angles (D) were computed for all compounds, as
defned for compounds 3a (C=N‒Carom=Carom connected
atoms) and 3k (N‒CH2‒Carom=Carom connected atoms) in
Fig. 4. The obtained values for bond lengths (Ǻ) and dihe-
dral angles (°) of compounds 3a–o are listed in Table 2. In
enamine form of all compounds (3a–o), L2 bond possesses
features of a double bond rather than a single bond sug-
gesting this bond is shorter when compared with the cor-
responding bond in imine form. The dihedral angles for aryl
derivatives 3a–i and benzyl analogs 3j–o were in the range
of 47.6–58.5 and 66.9–92.5 degree, respectively (Table 2).
To that end, it can be concluded that all the compounds are
non-planar. From these observations, imine C=N bond,
through tautomeric transformation, turns into NH, which
can form the hydrogen bond with the oxygen atom of either
ketone or ester functional group.
Compound ΔE (kcal/mol)a
ΔH (kcal/mol) ΔG (kcal/
(E)-enam-
ine−(s-cis)-
imine-1
mol)
Enamine (E)-enam-
(E)-enam-
ine−(s-cis)-
imine-1
(EE −EZ) ine−(s-cis)-
imine-1
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
−1.513
−1.305
−1.530
−1.539
−0.898
−1.543
−1.541
−1.547
−1.617
−0.699
−1.970
−1.925
−1.970
−2.727
−2.039
−27.229
−21.528
−27.563
−27.757
−27.348
−26.386
−25.676
−25.66
−24.513
−29.153
−21.692
−29.506
−28.218
−26.286
−27.566
−25.516
−21.061
−26.537
−26.915
−25.218
−24.944
−24.255
−23.640
−22.666
−28.377
−24.922
−28.713
−28.223
−25.738
−27.771
−22.557
−21.372
−25.208
−22.285
−26.167
−22.666
−21.288
−23.587
−23.240
−27.059
−20.553
−27.833
−24.536
−24.501
−24.747
Moreover, the highest-occupied molecular orbit-
als (HOMO) and lowest-unoccupied molecular orbitals
(LUMO) energies for both imine and enamine due to the
importance of frontier orbitals nature in governing chemical
aEnergy values calculated in DMSO (as solvent) at room temperature
Fig. 3 The optimized structures
for compound 3a: a) (E)-
enamine; b) (Z)-enamine; c)
(s-cis)-Imine-1
1 3

Molecular Diversity
Fig. 4 The bond length (L1, L2 and L3) and dihedral angle (D) schematic of (E)-enamine for 3a (calculated for C=N‒Carom=Carom) and 3k (cal-
culated for N‒CH2‒Carom=Carom), as representative compounds
Table 2 The optimized
No.
Enamine bond length (Ǻ)
(s-cis)-Imine-1
(E)-enamine
dihedral
parameters of bond length
(Ǻ) and dihedral angles (°) of
compounds 3a–o
bond length (Ǻ)
E
Z
angles (°)
L1
L2
L3
L1
L2
L3
L1
L2
L3
D
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
1.457
1.456
1.456
1.456
1.457
1.458
1.458
1.458
1.459
1.456
1.456
1.456
1.457
1.457
1.457
1.424
1.421
1.425
1.425
1.425
1.423
1.422
1.422
1.420
1.426
1.427
1.427
1.426
1.426
1.425
1.340
1.344
1.339
1.339
1.340
1.341
1.342
1.342
1.344
1.333
1.332
1.332
1.333
1.334
1.334
1.469
1.471
1.468
1.468
1.468
1.469
1.470
1.470
1.471
1.466
1.466
1.465
1.466
1.468
1.466
1.423
1.418
1.423
1.424
1.424
1.421
1.420
1.420
1.419
1.425
1.425
1.426
1.424
1.425
1.424
1.342
1.348
1.341
1.341
1.341
1.343
1.344
1.345
1.347
1.334
1.334
1.333
1.335
1.335
1.335
1.368
1.375
1.369
1.369
1.368
1.369
1.368
1.368
1.369
1.368
1.371
1.371
1.371
1.371
1.371
1.502
1.492
1.501
1.501
1.501
1.501
1.501
1.501
1.500
1.505
1.502
1.495
1.495
1.495
1.495
1.278 49.85
1.285 58.52
1.279 52.22
1.279 52.15
1.278 49.59
1.278 51.10
1.278 48.55
1.278 48.24
1.278 47.56
1.275 89.57
1.277 88.80
1.278 92.53
1.278 89.50
1.278 66.89
1.278 89.42
chemicals. To that end, enamines of 3a–o in (E)-form are
more stable that the other isomers due to the larger energy
gap. Moreover, the (s-cis)-imine-1 forms of 3a–o are soft
molecules due to smaller Eg which is why they undergo tau-
tomeric transformation (Table 3). The HOMO and LUMO
contours of 3a at 0.02 au are shown in Fig. 5 as an example.
Besides, to investigate the possibility of hydrogen bond-
ing in enamine tautomers and imine conformers, the Mul-
liken atomic charges for oxygen atoms [of ketone functional
group in (E)-enamine and hydroxyl in (s-cis)-imine-1], nitro-
gen atom [of amine in (E)-enamine and (s-cis)-imine-1]
and hydrogen atom [of amine in (E)-enamine and hydroxyl
in (s-cis)-imine-1] were calculated by NPA analysis. The
obtained results confrmed the feasibility of formation of
hydrogen bond in (E)-enamine and (s-cis)-imine-1; however,
due to the larger negative charge of ketone functional group
in (E)-enamine in comparison with nitrogen atom in (s-cis)-
imine-1, hydrogen bonding is more favorable in (E)-enamine
(Table 3).
The molecular electrostatic potential (MEP) surfaces of
the studied structures are shown in Fig. 6. As it is shown,
MEP can be related to electron density and indicates electro-
philic or nucleophilic properties of diferent sites through-
out the whole molecule. Figure 6 shows MEP of 3a as an
example, in which the imine forms obviously hold positive
charge at their hydroxyl group and also negative charge at
their lactone group, while the enamine ones possess negative
charge at their lactone and ketone groups.
1 3

Molecular Diversity
Table 3 Band gap energy (Eg)
calculation and Mulliken atomic
charge for compounds 3a–o
Compound Eg (eV)
Mulliken atomic charge
Enamine
Imine
(E)-enamine
(s-cis)-imine-1
E
Z
s-cis
O
H
N
O
H
N
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
−4.3485 −4.228 −3.960 −0.591 0.406 −0.715 −0.608 0.426 −0.455
−4.3977 −4.202 −3.547 −0.598 0.418 −0.749 −0.615 0.427 −0.555
−4.2097 −4.078 −3.709 −0.592 0.405 −0.714 −0.607 0.425 −0.462
−4.1743 −4.044 −3.680 −0.592 0.405 −0.714 −0.607 0.425 −0.461
−4.1014 −4.017 −3.756 −0.593 0.405 −0.719 −0.609 0.424 −0.461
−4.4070 −4.279 −4.057 −0.590 0.407 −0.717 −0.608 0.426 −0.456
−4.3645 −4.251 −4.138 −0.590 0.408 −0.720 −0.609 0.427 −0.453
−4.3400 −4.227 −4.110 −0.590 0.408 −0.721 −0.604 0.427 −0.465
−4.3539 −4.258 −4.272 −0.588 0.410 −0.724 −0.609 0.429 −0.451
−4.7142 −4.590 −4.589 −0.586 0.408 −0.623 −0.611 0.426 −0.400
−4.6103 −4.504 −3.829 −0.587 0.411 −0.621 −0.607 0.432 −0.450
−4.5439 −4.439 −3.937 −0.586 0.407 −0.621 −0.604 0.423 −0.400
−4.7148 −4.588 −4.431 −0.587 0.407 −0.623 −0.604 0.423 −0.405
−4.7224 −4.594 −4.535 −0.588 0.416 −0.604 −0.604 0.423 −0.409
−4.7148 −4.588 −4.478 −0.587 0.408 −0.625 −0.605 0.424 −0.406
(an antitumor agent) was used as the standard drug. Fur-
thermore, the parent compound 2 was included in cytotoxic
assay for comparison. The obtained IC50 values are listed
Natural bond orbital analysis of (E)‑
and (Z)‑enamines
in Table 5.
The natural bond orbital (NBO) analysis of E and Z iso-
mers of N-benzyl enamines (3k–o) was performed at
B3LYP/6–31G(d) level. Hydrogen bond forms due to the
interaction of the donor lone pair atoms (LP) and the accep-
tor antibonding orbitals (BD*). The strength of such inter-
action can be evaluated with E(2). We investigated inter-
action of C=O…HN, to evaluate the stability of (E)- and
(Z)-enamine isomers. The E(2) values of (E)-enamines are
greater than those of (Z)-enamines (Table 4), suggesting that
the intramolecular electrostatic hydrogen bond interaction
in the (E)-forms is higher than that of the (Z)-ones (Fig. 7).
In summary, the computational data suggest that the (E)-
enamine form is more stable than (Z)-enamine. This may
be due to the higher electron density of ketone carbonyl in
comparison with lactone carbonyl, which leads to stronger
hydrogen bond with NH. Furthermore, the (s-cis)-imine-1
is rapidly converted to (E)-enamine form due to the prompt
transition of a proton from OH to nitrogen atom regarding
to the higher basic nature of N atom.
As evidenced from data, most of the compounds showed
signifcant cytotoxic activity against cancer cell lines with
IC50 values in the range of 1.41–50.0 µg/mL. While the par-
ent carbonyl compound (2) showed mild activity against
A549 cells (IC50 =29.75 µg/mL), the aniline-derived com-
pounds 3d and 3h, and benzyl amine derivatives 3k, 3m
and 3o had IC50 values less than 10 µg/mL, exhibiting bet-
ter activities. Among them, 3k and 3m displayed the most
cytotoxic activity against A549, with IC50 values of 6.08 and
6.29 µg/mL, respectively. Compounds 3a, 3d and 3k–n had
IC50s<5 µg/mL, showing high activity against MCF-7 supe-
rior to that of doxorubicin (8.87 µg/mL). In particular, the
activity of 3k was sixfold greater than that of doxorubicin
against MCF-7. Furthermore, all compounds with the excep-
tion of 3b, 3c, 3 h and 3i were signifcantly more potent than
parent compound 2 against MCF-7 cell line.
The cytotoxicity evaluation against normal cell line
BEAS-2B revealed that doxorubicin had no selectivity for
cancer cells as its IC50 values against cancer cells were close
to the value toward normal cell line. On the other hand, the
mother coumarin 2 showed equal toxicity against normal and
cancer cell lines. The comparison of the IC50 values against
MCF-7 and BEAS-2B cell lines indicated that compounds
3a, 3d, 3k and 3m–o display signifcant selectivity toward
MCF-7 to some extent. However, most of the compounds
showed low or no selectivity against A549 over BEAS-2B.
It is worth to note that compound 3o displayed high toxicity
Cytotoxic activity
All the synthesized compounds 3a–o were evaluated in vitro
for their cytotoxic activities against two cancer cell lines
including MCF-7 (breast cancer cell line) and A549 (ade-
nocarcinomic human alveolar basal epithelial cells) and a
normal cell line namely BEAS-2B (human normal bron-
chial epithelium cell line) using MTT assay. Doxorubicin
1 3

Molecular Diversity
Fig. 5 The energy (eV) and some contours of HOMO and LUMO orbitals of 3a; (E)-enamine (a), (Z)-enamine (b) and (s-cis)-imine-1 (c). Posi-
tive values of the HOMOs and LUMOs contour are represented in green and pink and the negative values in yellow and blue color, respectively
1 3

Molecular Diversity
Fig. 6 Calculated molecular
electrostatic potential surfaces
(MEP) for imine and enamine
forms (3a is presented as an
example). The surfaces are
defned by the 0.0004 electrons/
b3 contour of the electron den-
sity. Color ranges, in au (blue, is
more positive than 0.050; red is
more negative than −0.050 and
green is neutral)
Table 4 NBO analysis of (E and
Z)-enamines of 3k–o by DFT
method B3LYP/6-31G(d)
Compound Donor(i) Type Acceptor(j) Type E(2) kcal/mol E(j)–E(i) (a.u.) F(i,j) (a.u.)
(E)-3k
(E)-3l
O
O
O
O
O
O
O
O
O
O
LP
LP
LP
LP
LP
LP
LP
LP
LP
LP
N–H
N–H
N–H
N–H
N–H
N–H
N–H
N–H
N–H
N–H
σ*
σ*
σ*
σ*
σ*
σ*
σ*
σ*
σ*
σ*
20.35
20.36
20.84
21.12
21.00
17.24
17.10
17.71
19.86
17.93
0.72
0.72
0.72
0.72
0.72
0.72
0.72
0.72
0.72
0.72
0.110
0.110
0.111
0.112
0.111
0.102
0.102
0.103
0.109
0.104
(E)-3m
(E)-3n
(E)-3o
(Z)-3k
(Z)-3l
(Z)-3m
(Z)-3n
(Z)-3o
Fig. 7 NBO analysis of (E)-enamine for 3k
against A549 and MCF-7 (IC50 value of 8.26 and 7.56 µg/
mL) which are very close to those of doxorubicin, while its
toxicity against the normal cell line was far more lower than
that of doxorubicin.
4-HO–Bn (3k), 4-F–Bn (3m) and 4-Cl–Bn (3o) derivatives
showed higher activity against cancer cells compared to their
corresponding anilino congeners 3c, 3f and 3g, respectively. In
the anilino series, better activities were observed with 4-MeO
and 4-Br substituents against A549, while 4-MeO and 4-Me
groups had more positive efect on activity toward MCF-7.
Among the halogenated anilines 3f–i, the 4-bromo compound
(3h) showed better activity on A549 cells and 4-fuoro-deriva-
tive (3f) exhibited higher activity against MCF-7 cell line. The
The structure–activity study of the synthesized compounds
demonstrated that the transformation of ketone in compound
2 to imine/enamine signifcantly increases the cytotoxic activ-
ity. In general, the substituted benzyl amino derivatives were
more potent than substituted aniline analogs. For example, the
1 3

Molecular Diversity
Table 5 Cytotoxicity (IC50, µg/
mL) of synthesized compounds
against diferent cell lines
R
A549
14.28
MCF -7
1.93
BEAS-2B
10.43
Compound
3a
11.07
> 50
> 50
3b
49.98
7.66
37.57
3.03
22.82
13.89
23.87
13.55
46.07
8.54
3c
3d
3e
3f
15.25
24.56
40.00
6.84
27.80
6.52
27.35
> 50
3g
3h
3i
37.20
41.07
> 50
35.28
6.09
12.46
1.41
10.15
4.08
3j
3k
9.35
6.29
4.55
2.03
3.02
2.06
4.05
3l
3m
3n
10.74
35.33
8.26
29.75
5.95
7.56
32.65
8.87
> 50
28.81
4.08
3o
2
–
–
Doxorubicin
Each experiment was conducted in triplicate, and the IC50 values were presented as mean
efect of halogen substituent depends on the type of cancer
cell line. While the 4-hydroxy derivative of aniline (3c) was
less potent than the 4-methoxy one (3d), conversely in benzyl
amines, the 4-hydroxy analog (3k) was more potent than the
4-methoxy derivative (3l). Among the benzyl amine deriva-
tives, better selectivity toward cancer cells was observed with
chloro-derivatives.
Conclusion
We have synthesized some imines/enamines 3a–o derived
from 3-acetyl-4-hydroxy-2H-chromen-2-one with good
yields as cytotoxic agents. Due to the diferent struc-
tural possibility including imine (s-cis and s-trans con-
formers) and enamine (E- and Z-stereoisomers) for these
compounds, we performed some calculations by applying
1 3

Molecular Diversity
DFT/B3LYP-6-31G(d) method. The computational results
including optimized geometry parameters, ΔE, ΔG, ΔH,
Mulliken atomic charge, HOMO and LUMO energy, and
NBO analysis suggested that the (E)-enamine is the more
favorable tautomeric form. The in vitro evaluation of
anticancer activity for 3a–o confrmed that some of these
coumarin derivatives have high toxicity against cancer
cell lines A549 and MCF-7. The 4-hydroxybenzyl amine
derivative 3k showed the highest activity (IC50s 6.09 and
1.41 µg/mL), being equipotent or more potent than doxoru-
bicin. However, this compound had no selectivity, similar
to doxorubicin. Notably, the 4-chlorobenzyl derivative 3o
with remarkable anticancer activity displayed acceptable
selectivity toward cancer cells tested.
resulted in pure compound 2 as white needle-like crystals.
Yield 95% (3.6 g); mp 134–136 °C; 1H NMR (400 MHz,
DMSO-d6) δ: 2.79 (s, 3H, CH3), 7.30 (d, 1H, J = 8.4 Hz,
H-8), 7.35 (td, 1H, J =7.6 and 0.8 Hz, H-6), 7.70 (td, 1H,
J=8.4 and 1.6 Hz, H-7), 8.05 (dd, 1H, J=8.0 and 1.6 Hz,
H-5), 17.76 (s, 1H, OH). 13C NMR (100 MHz, DMSO-d6)
δ: 30.19, 101.93, 115.18, 117.32, 125.27, 125.67, 137.16,
154.63, 159.67, 178.36, 205.97.
General procedure for the synthesis of compounds
3a–o
To a mixture of compound 2 (1 mmol) in absolute etha-
nol (5 mL) was added appropriate amine (1 mmol). The
temperature of reaction mixture was raised until all com-
pounds were fully dissolved. Then, the resulted solution
was refuxed and monitored by TLC using ethyl acetate
and n-hexane (1:3) as the eluent. After completion of the
reaction (10–120 min), it was cool down to room tempera-
ture and the product was precipitated out as a fne pow-
der. The collected powder was recrystallized from absolute
ethanol to give pure corresponding compound 3.
Experimental
General information
All the materials including reagents and solvents were
purchased from Sigma-Aldrich or Merck companies. The
materials were used without further purifcation unless
otherwise stated. The progress and completion of reac-
tions were monitored by TLC using pre-coated silica gel
60 F254 aluminum sheets. The compound spots on TLC
were visualized using a UV lamp (254 nm). Melting points
were determined in open capillary tubes by a Bibby Stu-
art Scientifc SMP3 apparatus (Stuart Scientifc, Stone,
UK), and the uncorrected values are reported. Bruker 400
spectrometer was used to record the NMR spectra, and the
values were expressed as δ (ppm). The coupling constants
are reported in hertz (Hz), and multiplicity was defned as
singlet (s), doublet (d), triplet (t) or multiplet (m). Moreo-
ver, a HP 5937 Mass Selective Detector (Agilent Tech-
nologies, CA, USA) was used to obtain the mass spectra
of compounds.
4‑Hydroxy‑3‑(1‑(p‑tolylimino)ethyl)‑2H‑chromen‑2‑one
or 3‑(1‑(p‑tolylamino)ethylidene) chromane‑2,4‑dione
(3a) White powder; yield 98% (292 mg); mp 148–149 °C
[35].
4 ‑ H y d r ox y ‑ 3 ‑ ( 1 ‑ ( ( 2 ‑ h y d r ox y p h e n y l ) i m i n o )
ethyl)‑2H‑chromen‑2‑one or 3‑(1‑((2‑hydroxyphenyl)
amino)ethylidene)chromane‑2,4‑dione (3b) Pale yel-
low powder; yield 78% (232 mg); mp 237–239 °C; IR (KBr,
cm−1) νmax: 3410 (N–H), 3272 (O–H), 3057 (C–H Ar), 2973
(C–H), 1704 (C=O/C=N), 1562 (C=C). 1H NMR (400 MHz,
DMSO-d6) δ: 3.44 (s, 3H, CH3), 6.95 (t, 1H, J=7.6 Hz, H-5
phenyl), 7.07 (d, 1H, J= 8.0 Hz, H-3 phenyl), 7.25–7.42
(m, 4H, H-4 and H-6 phenyl, H-6 and H-8), 7.70 (t, 1H,
J=8.0 Hz, H-7), 8.02 (d, 1H, J=7.6 Hz, H-5), 10.40 (s, 1H,
OH), 15.25 (br s, 1H, OH/NH). 13C NMR (100 MHz, DMSO-
d6) δ: 20.96, 97.65, 116.85, 116.97, 119.86, 120.33, 123.64,
124.30, 126.26, 127.32, 130.03, 134.90, 151.95, 153.68,
162.06, 176.38, 180.73. MS (m/z, %): 295 (M+, 21), 280
(100), 187 (15), 160 (20), 134 (28), 121 (37). Anal. Calcd for
C17H13NO4: C, 69.15; H, 4.44; N, 4.74. Found: C, 69.07; H,
4.51; N, 4.63.
Preparation of 3‑acetyl‑4‑hydroxy‑2H‑chromen‑2‑
one (2)
Compound 2 was synthesized according to the previously
reported procedure [34]. Briefy, 4-hydroxy-2H-chromen-
2-one (1, 3 g, 18.6 mmol) was dissolved in acetic acid
(20 mL) followed by dropwise adding phosphorus oxychlo-
ride (5.6 mL) at room temperature. The resulted solution was
brought to refux temperature. After 30 min, an extra phos-
phorus oxychloride (1 mL) was added to the solution and
the solution was refuxed for another 30 min. After cooling,
the product was precipitated as a yellow powder. In order to
purify the product, it was recrystallized from ethanol which
4 ‑ H y d r ox y ‑ 3 ‑ ( 1 ‑ ( ( 4 ‑ h y d r ox y p h e n y l ) i m i n o )
ethyl)‑2H‑chromen‑2‑one or 3‑(1‑((4‑hydroxyphenyl)
amino)ethylidene)chromane‑2,4‑dione (3c) Pale yel-
low powder; yield 80% (236 mg); mp 211–214 °C; IR (KBr,
cm−1) νmax: 3236 (C–H Ar), 1682 (C=O), 1608 (C=O/C=N),
1564 (C=C). 1H NMR (400 MHz, DMSO-d6) δ: 2.59 (s, 3H,
CH3), 6.90 (d, 2H, J=8.4 Hz, H-3 and H-5 phenyl), 7.25 (d,
1 3

Molecular Diversity
2H, J=8.4 Hz, H-2 and H-6 phenyl), 7.31 (d, 1H, J=8.0 Hz,
H-8), 7.34 (t, 1H, J=7.6 Hz, H-6), 7.69 (t, 1H, J=7.6 Hz,
H-7), 8.00 (d, 1H, J = 7.6 Hz, H-5), 9.84 (br s, 1H, OH),
15.32 (s, 1H, OH/NH). 13C NMR (100 MHz, DMSO-d6)
δ: 20.88, 97.43, 116.47, 116.86, 120.31, 124.33, 126.23,
127.29, 127.42, 134.91, 153.65, 157.76, 162.08, 176.09,
180.65. MS (m/z, %): 295 (M+, 100), 278 (21), 202 (31),
187 (53), 125 (50), 123 (40), 109 (21), 93 (13). Anal. Calcd
for C17H13NO4: C, 69.15; H, 4.44; N, 4.74. Found: C, 69.11;
H, 4.57; N, 4.61.
IR (KBr, cm−1) νmax: 3411 (N–H), 3053 (C–H Ar), 2900
1
(C–H), 1718 (C=O), 1609 (C=O/C=N), 1560 (C=C). H
NMR (400 MHz, CDCl3) δ: 2.72 (s, 3H, CH3), 7.21 (d, 2H,
J = 8.4 Hz, H-2 and H-6 phenyl), 7.25–7.36 (m, 2H, H-6
and H-8), 7.50 (d, 2H, J = 8.0 Hz, H-3 and H-5 phenyl),
7.62 (t, 1H, J=8.0 Hz, H-7), 8.11 (d, 1H, J=7.6 Hz, H-5),
15.93 (s, 1H, OH/NH). MS (m/z, %): 315 ([M +2]+, 35),
313 (M+, 100), 296 (20), 187 (81), 121 (56). Anal. Calcd for
C17H12ClNO3: C, 65.08; H, 3.86; N, 4.46. Found: C, 65.01;
H, 3.91; N, 4.44.
4 ‑ H yd r ox y ‑ 3 ‑ ( 1 ‑ ( ( 4 ‑ m e t h ox y p h e ny l ) i m i n o )
ethyl)‑2H‑chromen‑2‑one or 3‑(1‑((4‑methoxyphenyl)
amino)ethylidene)chromane‑2,4‑dione (3d) Yellowish
green; yield 92% (284 mg); mp 241–242 °C [35].
3 ‑ ( 1 ‑ ( ( 4 ‑ B r o m o p h e n y l ) i m i n o ) e t h y l ) ‑ 4 ‑ h y ‑
droxy‑2H‑chromen‑2‑one or 3‑(1‑((4‑bromophenyl)
amino)ethylidene)chromane‑2,4‑dione (3h) Pale
white powder; yield 73% (260 mg); mp 175–178 °C; IR
(KBr, cm−1) νmax: 3399 (N–H), 3087 (C–H Ar), 2900
1
3‑(1‑((3,4‑Dimethoxyphenyl)imino)ethyl)‑4‑hy‑
droxy‑2H‑chromen‑2‑one or 3‑(1‑((3,4‑dimethoxyphe‑
nyl)amino)ethylidene)chromane‑2,4‑dione (3e) White
powder; yield 88% (298 mg); mp 203–205 °C; IR (KBr, cm−1)
νmax: 3384 (N–H), 3063 (C–H Ar), 2995, 2931, 2843 (C–H),
(C–H), 1708 (C=O), 1609 (C=O/C=N), 1558 (C=C). H
NMR (400 MHz, CDCl3) δ: 2.70 (s, 3H, CH3), 7.14 (d, 2H,
J=8.8 Hz, H-2 and H-6 phenyl), 7.22–7.35 (m, 2H, H-6 and
H-8), 7.59 (t, 1H, J=8.4 Hz, H-7), 7.64 (d, 2H, J=8.4 Hz,
H-3 and H-5 phenyl), 8.08 (d, 1H, J=7.6 Hz, H-5), 15.94 (s,
1H, OH/NH). MS (m/z, %): 359 ([M+2]+, 70), 357 (M+,
72), 342 (18), 277 (41), 187 (100), 121 (59). Anal. Calcd for
C17H12BrNO3: C, 57.01; H, 3.38; N, 3.91. Found: C, 56.98;
H, 3.49; N, 3.75.
1
1698 (C=O), 1606 (C=O/C=N), 1566 (C=C). H NMR
(400 MHz, CDCl3) δ: 2.72 (s, 3H, CH3), 3.93 (s, 3H, OCH3),
3.96 (s, 3H, OCH3), 6.75 (d, 1H, J=1.6 Hz, H-2 phenyl),
6.81 (dd, 1H, J=8.4 and 1.6 Hz, H-6 phenyl), 6.96 (d, 1H,
J=8.4, H-5 phenyl), 7.22–7.37 (m, 2H, H-6 and H-8), 7.61 (t,
1H, J=7.6 Hz, H-7), 8.11 (d, 1H, J=8.0 Hz, H-5), 15.73 (s,
1H, OH/NH). 13C NMR (100 MHz, CDCl3) δ: 20.92, 56.16,
56.18, 97.92, 108.91, 111.27, 116.72, 117.88, 120.16, 123.71,
126.05, 129.06, 134.21, 148.93, 149.65, 153.90, 162.55,
176.30, 181.83. MS (m/z, %): 339 (M+, 100), 324 (27), 307
(9), 218 (6), 187 (34), 138 (14), 121 (28). Anal. Calcd for
C19H17NO5: C, 67.25; H, 5.05; N, 4.13. Found: C, 67.07; H,
5.15; N, 4.08.
3‑(1‑((3,4‑Dichlorophenyl)imino)ethyl)‑4‑hy‑
droxy‑2H‑chromen‑2‑one or 3‑(1‑((3,4‑dichlorophenyl)
amino)ethylidene)chromane‑2,4‑dione (3i) Pale white
powder; yield 66% (229 mg); mp 158–160 °C; IR (KBr,
cm−1) νmax: 3054 (C–H Ar), 2900 (C–H), 1695 (C=O), 1608
(C=O/C=N), 1564 (C=C). 1H NMR (400 MHz, CDCl3) δ:
2.73 (s, 3H, CH3), 7.14 (dd, 1H, J=8.4 and 1.6 Hz, H-6 phe-
nyl), 7.25–7.36 (m, H-6 and H-8), 7.41 (d, 1H, J=1.6 Hz,
H-2 phenyl), 7.60 (d, 1H, J=8.4 Hz, H-5 phenyl), 7.63 (t,
1H, J=8.0 Hz, H-7), 8.11 (d, 1H, J=8.0 Hz, H-5), 16.07 (s,
1H, OH/NH). 13C NMR (100 MHz, CDCl3) δ: 20.88, 98.44,
116.81, 119.81, 123.88, 125.09, 126.17, 127.65, 131.40,
132.72, 133.88, 134.64, 135.70, 153.94, 162.15, 176.28,
182.24. MS (m/z, %): 351 ([M + 4]+, 20), 349 ([M + 2]+,
48), 347 (M+, 70), 346 (M+-H, 95), 324 (27), 311 (12), 187
(100), 121 (70). Anal. Calcd for C17H11Cl2NO3: C, 58.64; H,
3.18; N, 4.02. Found: C, 58.59; H, 3.35; N, 3.98.
3 ‑ ( 1 ‑ ( ( 4 ‑ F l u o r o p h e n y l ) i m i n o ) e t h y l ) ‑ 4 ‑ h y ‑
droxy‑2H‑chromen‑2‑one or 3‑(1‑((4‑fluorophenyl)
amino)ethylidene)chromane‑2,4‑dione (3f) Pale white
powder; yield 98% (291 mg); mp 148–150 °C; IR (KBr,
cm−1) νmax: 3398 (N–H), 3071 (C–H Ar), 2921 (C–H),
1
1712 (C=O), 1610 (C=O/C=N), 1568 (C=C). H NMR
(400 MHz, CDCl3) δ: 2.70 (s, 3H, CH3), 7.15–7.36 (m, 6H,
H-2, H-3, H-5 and H-6 phenyl, H-6 and H-8), 7.61 (t, 1H,
J = 8.0 Hz, H-7), 8.11 (d, 1H, J = 8.0 Hz, H-5), 15.87 (s,
1H, OH/NH). MS (m/z, %): 297 (M+, 100), 282 (26), 202
(11), 187 (61), 136 (22), 121 (43), 95 (26). Anal. Calcd for
C17H12FNO3: C, 68.68; H, 4.07; N, 4.71. Found: C, 68.61;
H, 4.14; N, 4.77.
3‑(1‑(Benzylimino)ethyl)‑4‑hydroxy‑2H‑chromen‑2‑one
or 3‑(1‑(benzylamino)ethylidene)chromane‑2,4‑dione
(3j) Pale yellow powder; yield 85% (249 mg); mp 169–
171 °C; IR (KBr, cm−1) νmax: 3385 (N–H), 3100 (C–H Ar),
2927 (C–H), 1699 (C=O), 1607 (C=O/C=N), 1568 (C=C).
1H NMR (400 MHz, DMSO-d6) δ: 2.72 (s, 3H, CH3), 4.89 (s,
2H, CH2 benzyl), 7.23–7.52 (m, 7H benzyl and H-6 and H-8),
7.64 (dt, 1H, J=8.8 and 1.6 Hz, H-7), 7.93 (dd, 1H, J=8.0
and 1.6 Hz, H-5), 14.01 (br s, 1H, OH/NH). MS (m/z, %):
3 ‑ ( 1 ‑ ( ( 4 ‑ C h l o r o p h e n y l ) i m i n o ) e t h y l ) ‑ 4 ‑ h y ‑
droxy‑2H‑chromen‑2‑one or 3‑(1‑((4‑chlorophenyl)
amino)ethylidene)chromane‑2,4‑dione (3g) Pale
yellow powder; yield 70% (219 mg); mp 153–155 °C;
1 3

Molecular Diversity
293 (M+, 100), 202 (18), 106 (87), 91 (100). Anal. Calcd for
C18H15NO3: C, 73.71; H, 5.15; N, 4.78. Found: C, 73.88; H,
5.25; N, 4.71.
162.65 (d, JC,F = 246 Hz), 162.67, 177.02, 181.75. MS
(m/z, %): 311 (M+, 71), 294 (8), 202 (45), 174 (13), 124
(21), 109 (100). Anal. Calcd for C18H14FNO3: C, 69.45; H,
4.53; N, 4.50. Found: C, 69.41; H, 4.58; N, 4.42.
4 ‑ H y d r ox y ‑ 3 ‑ ( 1 ‑ ( ( 4 ‑ h y d r ox y b e n z y l ) i m i n o )
ethyl)‑2H‑chromen‑2‑one or 3‑(1‑((4‑hydroxybenzyl)
amino)ethylidene)chromane‑2,4‑dione (3k) Pale yel-
low powder; yield 70% (216 mg); mp 217–220 °C; IR (KBr,
cm−1) νmax: 3232 (O–H), 2930 (C–H), 1669 (C=O), 1607
(C=O/C=N), 1461 (C=C). 1H NMR (400 MHz, DMSO-d6)
δ: 2.72 (s, 3H, CH3), 4.74 (d, 2H, J=4.0 Hz, CH2 benzyl),
6.83 (d, 2H, J = 8.0 Hz, H-3 and H-5 benzyl), 7.20–7.35
(m, 4H, H-2 and H-6 benzyl, H-6 and H-8), 7.63 (t, 1H,
J = 8.4 Hz, H-7), 7.91 (d, 1H, J = 7.6 Hz, H-5), 9.50 (br
s, 1H, OH/NH), 13.87 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6) δ: 19.05, 47.72, 96.64, 116.18, 116.69, 120.58,
124.10, 126.10, 126.29, 129.90, 134.48, 153.5, 157.76,
162.32, 176.25, 180.12. MS (m/z, %): 309 (M+, 43), 203
(100), 107 (55), 92 (26). Anal. Calcd for C18H15NO4: C,
69.89; H, 4.89; N, 4.53. Found: C, 70.01; H, 4.93; N, 4.48.
3 ‑ ( 1 ‑ ( ( 2 ‑ C h l o r o b e n z y l ) i m i n o ) e t h y l ) ‑ 4 ‑ h y ‑
droxy‑2H‑chromen‑2‑one or 3‑(1‑(2‑chloroben‑
zylamino)ethylidene)‑3H‑chromene‑2,4‑dione
(3n) Pale yellow powder; yield 100% (330 mg); mp 154–
155 °C; IR (KBr, cm−1) νmax: 3062 (C–H Ar), 2900 (C–H),
1
1695 (C=O), 1587 (C=O/C=N), 1465 (C=C). H NMR
(400 MHz, DMSO-d6) δ: 2.80 (s, 3H, CH3), 4.86 (d, 2H,
J = 5.6 Hz, CH2 benzyl), 7.20–7.41 (m, 5H, H-4, H-5 and
H-6 benzyl, H-6, H-8), 7.45–7.52 (m, 1H, H-3 benzyl), 7.57
(td, 1H, J= 8.0 and 1.6 Hz, H-7), 8.06 (d, 1H, J = 7.6 Hz,
5-H), 14.66 (s, 1H, OH/NH). 13C NMR (100 MHz, DMSO-
d6) δ: 18.83, 45.93, 97.66, 116.61, 120.28, 123.61, 126.04,
127.63, 128.96, 129.92, 130.17, 132.68, 133.40, 134.03,
153.75, 162.68, 177.35, 181.74. MS (m/z, %): 329
([M+2]+, 10), 327 (M+, 20), 292 (100), 202 (84), 172 (36),
140 (30), 125 (80), 89 (22). Anal. Calcd for C18H14ClNO3:
C, 65.96; H, 4.31; N, 4.27. Found: C, 66.08; H, 4.37; N,
4.22.
4 ‑ H yd r ox y ‑ 3 ‑ ( 1 ‑ ( ( 4 ‑ m e t h ox y b e n z y l ) i m i n o )
ethyl)‑2H‑chromen‑2‑one or 3‑(1‑((4‑methoxybenzyl)
amino)ethylidene)chromane‑2,4‑dione (3l) White pow-
der; yield 90% (270 mg); mp 162–163 °C; IR (KBr, cm−1)
νmax: 3414 (N–H), 2921 (C–H), 2890 (C–H), 1708 (C=O),
3 ‑ ( 1 ‑ ( ( 4 ‑ C h l o r o b e n z y l ) i m i n o ) e t h y l ) ‑ 4 ‑ h y ‑
droxy‑2H‑chromen‑2‑one or 3‑(1‑(4‑chloroben‑
zylamino)ethylidene)‑3H‑chromene‑2,4‑dione (3o) Pale
yellow powder; yield 97.7% (320 mg); mp 170–172 °C; IR
(KBr, cm−1) νmax: 3404 (N–H), 3036 (C–H Ar), 2927 (C–H),
1
1607 (C=O/C=N), 1463 (C=C). H NMR (400 MHz,
DMSO-d6) δ: 2.72 (s, 3H, CH3), 3.77 (s, 3H, OCH3), 4.79 (s,
2H, CH2), 6.99 (d, 2H, J=8.4 Hz, H-3 and H-5 benzyl), 7.25
(d, 1H, J= 8.4 Hz, H-8), 7.27 (dt, 1H, J = 8.0 and 0.8 Hz,
H-6), 7.36 (d, 2H, J = 8.8 Hz, H-2 and H-6 benzyl), 7.62
(td, 1H, J=8.4 and 1.6 Hz, H-7), 7.90 (dd, 1H, J=8.0 and
1.6 Hz, H-5), 13.90 (s, 1H, OH/NH). 13C NMR (100 MHz,
DMSO-d6) δ: 19.04, 47.54, 55.58, 96.68, 114.84, 116.69,
120.57, 124.11, 126.10, 128.14, 129.86, 134.51, 153.50,
159.49, 162.31, 176.40, 180.14. MS (m/z, %): 323 (M+, 50),
186 (9), 161 (8), 121 (100). Anal. Calcd for C19H17NO4: C,
70.58; H, 5.30; N, 4.33. Found: C, 70.69; H, 5.35; N, 4.31.
1
1715 (C=O), 1605 (C=O/C=N), 1550 (C=C). H NMR
(400 MHz, DMSO-d6) δ: 2.70 (s, 3H, CH3), 4.90 (s, 2H, CH2),
7.27 (d, 1H, J=7.6 Hz, H-8), 7.29 (t, 1H, J=7.6 Hz, H-6),
7.46 (d, 2H, J=8.4 Hz, H-2 and H-6 benzyl), 7.52 (d, 2H,
J=8.4 Hz, H-3 and H-5 benzyl), 7.64 (t, 1H, J=7.2 Hz, H-7),
7.93 (d, 1H, J=7.6 Hz, H-5), 13.99 (br s, 1H, OH/NH). 13
C
NMR (100 MHz, DMSO-d6) δ: 19.10, 47.20, 96.89, 116.69,
120.52, 124.11, 126.12, 129.42, 130.15, 133.12, 134.55,
135.52, 153.51, 162.26, 176.95, 180.24. MS (m/z, %): 329
(([M+2]+, 28), 327 (M+, 78), 202 (81), 125 (100). Anal.
Calcd for C18H14ClNO3: C, 65.96; H, 4.31; N, 4.27. Found:
C, 65.77; H, 4.30; N, 4.33.
3 ‑ ( 1 ‑ ( ( 4 ‑ F l u o r o b e n z y l ) i m i n o ) e t h y l ) ‑ 4 ‑ h y ‑
droxy‑2H‑chromen‑2‑one or 3‑(1‑((4‑fluorobenzyl)
amino)ethylidene)chromane‑2,4‑dione (3m) Pale
yellow powder; yield 97% (288 mg); mp 165–167 °C;
IR (KBr, cm−1) νmax: 3443 (N–H), 3100 (C–H Ar), 2795
Cell culturing and MTT assay
1
(C–H), 1704 (C=O), 1608 (C=O/C=N), 1511 (C=C). H
Cell viability was evaluated by MTT assay [36]. Cancer
cell lines including MCF-7 (breast cancer cells) and A549
(adenocarcinomic human alveolar basal epithelial cells),
and normal cell line BEAS-2B (human bronchial epithelium
cells) were provided from Pasture Institute, Iran. RPMI with
10% FBS was applied for cell culturing. Firstly, 104 cells
were seeded into 96-well plates in 200 µL of RPMI culture
medium. Five triplicate wells were set up for each compound
for diferent concentrations. After 24 h, 20 µL of sample
NMR (400 MHz, CDCl3) δ: 2.80 (s, 3H, CH3), 4.76 (d,
2H, J = 5.2 Hz, CH2 benzyl), 7.14 (t, 2H, J = 8.4 Hz, H-3
and H-5 benzyl), 7.20–7.42 (m, 4H, H-2, H-6 benzyl, H-6
and H-8), 7.58 (td, 1H, J = 8.4 and 1.6 Hz, H-7), 8.05 (d,
1H, J = 7.6 Hz, H-5), 14.64 (s, 1H, OH/NH). 13C NMR
(100 MHz, CDCl3) δ: 18.92, 47.56, 97.57, 116.35 (d,
JC,F =21.0 Hz), 116.63, 120.26, 123.63, 125.99, 129.15 (d,
JC,F = 8.0 Hz), 130.70 (d, JC,F = 3.0 Hz), 134.05, 153.77,
1 3

Molecular Diversity
6. Thomas V, Giles D, Basavarajaswamy GPM, Das AK, Patel A
(2017) Coumarin derivatives as anti-infammatory and anticancer
agents. Anticancer Agents Med Chem 17(3):415–423. https://doi.
org/10.2174/1871520616666160902094739
solution (synthetic compound or doxorubicin) was added
into the wells and incubated at 37 °C under 5% CO2 for 48 h.
Then, 20 µL MTT solution (0.5 mg/mL) was added into the
wells and incubated at 37 °C for additional 4 h in darkness.
Finally, the supernatant was removed and formazan crys-
tals were dissolved by 200 μL dimethyl sulfoxide (DMSO,
Sigma-Aldrich) and the wells absorbance was measured at
570 nm by a microplate spectrophotometer (Biotek). Finally,
the IC50 values were calculated with respect to the percent-
age of inhibition at diferent concentrations by nonlinear
curve ftting.
7. Xi GL, Liu ZQ (2015) Coumarin-fused coumarin: antioxidant
story from N, N-dimethylamino and hydroxyl groups. J Agric
Food Chem 63(13):3516–3523. https://doi.org/10.1021/acs.
jafc.5b00399
8. Alonso-Castro AJ, Guzman-Gutierrez SL, Betancourt CA, Gasca-
Martinez D, Alvarez-Martinez KL, Perez-Nicolas M, Espitia-
Pinzon CI, Reyes-Chilpa R (2018) Antinociceptive, anti-infam-
matory, and central nervous system (CNS) efects of the natural
coumarin soulattrolide. Drug Dev Res 79(7):332–338. https://doi.
org/10.1002/ddr.21471
9. Tian D, Wang F, Duan M, Cao L, Zhang Y, Yao X, Tang J (2019)
Coumarin analogues from the Citrus grandis (L.) osbeck and their
hepatoprotective activity. J Agric Food Chem 67(7):1937–1947.
https://doi.org/10.1021/acs.jafc.8b06489
Computational study
A DFT method was employed to optimize all the structures,
using Becke’s three-parameter hybrid exchange functional
with the Lee–Yang–Parr gradient corrected correlation func-
tional (B3LYP hybrid functional) in Gaussian 09 program
suite [37, 38]. All the atoms were described with a split
valence Pople basis set plus difuse functions, 6-31G (d) in
the gas phase. In order to investigate the efect of solvents
(DMSO and CHCl3), the calculations were carried out by
solvation model SMD/6-31G(d) level [39]. Frequency cal-
culations were also performed on the all optimized geom-
etries to ensure that the local minima were found. Natural
bond analysis (NBO) was used at the same level of theory
to analyze the electronic properties as well as the constitu-
tion of the molecular orbital. In order to increase the single
point, the tight convergence criterion and ultrafne integral
grid were exploited.
10. Kasperkiewicz K, Ponczek MB, Budzisz E (2018) A biological,
fuorescence and computational examination of synthetic cou-
marin derivatives with antithrombotic potential. Pharmacol Rep
70(6):1057–1064. https://doi.org/10.1016/j.pharep.2018.06.002
11. Kontogiorgis C, Nicolotti O, Mangiatordi GF, Tognolini M,
Karalaki F, Giorgio C, Patsilinakos A, Carotti A, Hadjipavlou-
Litina D, Barocelli E (2015) Studies on the antiplatelet and
antithrombotic profle of anti-infammatory coumarin deriva-
tives. J Enzyme Inhib Med Chem 30(6):925–933. https://doi.
org/10.3109/14756366.2014.995180
12. Hassan MZ, Osman H, Ali MA, Ahsan MJ (2016) Therapeu-
tic potential of coumarins as antiviral agents. Eur J Med Chem
123:236–255. https://doi.org/10.1016/j.ejmech.2016.07.056
13. Tang ZH, Liu YB, Ma SG, Li L, Li Y, Jiang JD, Qu J, Yu SS
(2016) Antiviral spirotriscoumarins A and B: two pairs of oligo-
meric coumarin enantiomers with a spirodienone-sesquiterpene
skeleton from Toddalia asiatica. Org Lett 18(19):5146–5149.
https://doi.org/10.1021/acs.orglett.6b02572
14. Emami S, Foroumadi A, Faramarzi MA, Samadi N (2008) Syn-
thesis and antibacterial activity of quinolone-based compounds
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Acknowledgements This project was related to the Ph.D. thesis of SV
(Department of Chemistry, Science and Research Branch, Islamic Azad
University, Tehran, Iran). We thank Dr. Nahid Hasani for her valuable
comments and suggestions on the molecular modeling part of our work.
15. Song JL, Yuan Y, Tan HB, Huang RM, Liu HX, Xu ZF, Qiu SX
(2017) Anti-infammatory and antimicrobial coumarins from the
stems of Eurya chinensis. J Asian Nat Prod Res 19(3):222–228.
https://doi.org/10.1080/10286020.2016.1191474
16. Keri RS, Sasidhar BS, Nagaraja BM, Santos MA (2015) Recent
progress in the drug development of coumarin derivatives as
potent antituberculosis agents. Eur J Med Chem 100:257–269.
https://doi.org/10.1016/j.ejmech.2015.06.017
Compliance with ethical standards
Conflict of interest Authors declared no conficts of interest.
17. Abdel-Latif MS, Elmeleigy KM, Aly TAA, Khattab MS,
Mohamed SM (2017) Pathological and biochemical evalua-
tion of coumarin and chlorophyllin against afatoxicosis in rat.
Exp Toxicol Pathol 69(5):285–291. https://doi.org/10.1016/j.
etp.2017.01.014
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Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
Afliations
Samaneh Vaseghi1 · Mohammad Yousef2 · Mohammad Shokrzadeh3 · Zinatossadat Hossaini4 ·
Zahra Hosseini‑khah5 · Saeed Emami6
1
2
3
4
5
6
Department of Chemistry, Science and Research Branch,
Department of Chemistry, Qaemshahr Branch, Islamic Azad
Islamic Azad University, Tehran, Iran
University, Qaemshahr, Iran
Department of Chemistry, Yadegar-e Imam Khomeini
Diabetes Research Center, Mazandaran University
Shahr-e Rey Branch, Islamic Azad University, Tehran, Iran
of Medical Sciences, Sari, Iran
Department of Toxicology and Pharmacology, Faculty
of Pharmacy, Mazandaran University of Medical Sciences,
Sari, Iran
Department of Medicinal Chemistry and Pharmaceutical
Sciences Research Center, Faculty of Pharmacy, Mazandaran
University of Medical Sciences, Sari, Iran
1 3