Recyclable Amberlite IR-120 Catalyzed domino reaction: Synthesis, anticancer activity and molecular docking studies of biscoumarins

Article abstract of DOI:10.1016/j.molstruc.2021.131093

Under mild conditions, an environmentally friendly method has been developed for the synthesis of bis-coumarins by using a reasonably lesser quantity of Amberlite IR-120 through Domino Knoevenagel–Michael reaction. In which, a series of aryl aldehydes hav

Full text of DOI:10.1016/j.molstruc.2021.131093

Journal of Molecular Structure 1246 (2021) 131093
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Recyclable Amberlite IR-120 Catalyzed domino reaction: Synthesis,
anticancer activity and molecular docking studies of biscoumarins
A.J. Shadaksharia, T.H. Suresha Kumaraa,∗, H.B.V. Sowmyab, Ismailc, B.G. Harishd,
A.J. Yamuna e
a Department of Chemistry, University BDT college of Engineering, Davanagere -577004 (A constituent College Visvesvaraya Technological University,
Belagavi), Karnataka, India
b PG Department of Chemistry, Jain University, 52 Bellary Road, Hebbal, Bangalore, Karnataka 560024, India
c Department of Chemistry, Osmania University, Amberpet, Hyderabad, Telangana, India - 500046, India
d Department of MCA, University BDT college of Engineering, Davanagere -577004 (A constituent College Visvesvaraya Technological University, Belagavi),
Karnataka, India
e Department of Chemistry, School of Chemical Sciences, Kuvempu University, Shankaraghatta-577451, Shimoga, Karnataka, INDIA
a r t i c l e i n f o
a b s t r a c t
Article history:
Under mild conditions, an environmentally friendly method has been developed for the synthesis of
bis-coumarins by using a reasonably lesser quantity of Amberlite IR-120 through Domino Knoevenagel–
Michael reaction. In which, a series of aryl aldehydes have efficiently reacted with 4-hydroxycoumarin
to give respective bis-coumarins through a short reaction time. The acidic Amberlite IR-120 catalyst used
for a reaction was isolated and reused for successive four reactions. As per the observations made, the
activity of the recycled catalyst was preserved. The invitro examination of bis-coumarins against two can-
cer cell lines was proved that, some of these have showed promising anti-proliferative properties. The
molecular docking studies of bis-coumarin derivatives with GlcN-6-P synthase yielded potential confor-
mations with prospective binding energy, docking energy, inhibition constant and electrostatic energy
while comparing with the standard drug chloroquine.
Received 24 November 2020
Revised 8 July 2021
Accepted 10 July 2021
Available online 23 July 2021
Keywords:
Bis-coumarins
Amberlite IR-120 resin
Domino Knoevenagel–Michael reaction
4-Hydroxy coumarin
Aryl aldehydes
© 2021 Published by Elsevier B.V.
Green synthesis
Invitro anticancer activity
Molecular docking studies
1. Introduction
In particular, 4–hydroxy-coumarins have showed important biolog-
ical activities like antitumor, hypotoxicity, enzyme inhibition and
The enzyme, glucosamine-6-phosphatesynthase (EC 2.6.1.16), is
a new target for antimicrobial studies. The enzyme catalytic path-
anticoagulant activities [17, 18]. Above pharmacophores were iso-
lated from natural sources [19,20], exhibited several useful biolog-
ical and medicinal properties like anti-bacterial [21,22], anticancer
[23], antioxidant, anti-inflammatory [24] and antiviral activity [25].
After identifying the considerable importance of bis-coumarins,
several researchers have synthesized through diverse synthetic
methods like Domino Knoevenagel–Michael reaction of 4–hydroxy
coumarin and aromatic aldehydes in the presence of various cat-
alysts viz., piperidine [26], InCl3 [27], cyclic secondary amine
[28], tetrabutylammonium bromide [29], molecular iodine [30],
1–butyl–3-methylimidazolium tetrafluoroborate [31], ionic liq-
uids [32], nanoparticulate silica chloride [33], dodecylbenzenesul-
fonic acid [34], Zn(proline)2 [35], ruthenium(III) chloride hydrate
[36] and sodium dodecyl sulfate (SDS) [37] etc.
ꢀ
way involved produce end product, uridine 5 diphospho-N-acetyl-
d-glucosamine, giving rise to incorporation of N-acetylglucosamine
into several macromolecules such as, chitin in fungi, insects and
crustaceans; teichoic acids, lipopolysaccharides, and peptidoglycan
in bacteria, and glycosaminoglycans mucopolysaccharides and gly-
coproteins in mammals. These molecules are building blocks in the
assembly of the cell wall [1]. Presently, several natural and syn-
thetic origin inhibitors have been reported on GlcN-6-P which ex-
hibited antibacterial and antifungal activity.
Coumarin derivatives are widely distributed in nature [2,3]
and exhibited broad spectrum of biological activities like anti-
inflammatory [4,5], antitumor [6,7], analgesic [8], anticancer [9,11],
antimutagenic [12], antibiotic [13,14] and anti-HIV activity [15,16].
In the current study, we have successfully achieved the one pot
synthesis of biscoumarins (3a-l) using Amberlite-IR 120 resin as an
efficient heterogeneous catalyst. The catalytic activity of amberlite
IR-120 acidic resin has gained importance in green chemistry due
∗
Corresponding author.
E-mail address: suresha.kumara@ubdtce.org (T.H. Suresha Kumara).
https://doi.org/10.1016/j.molstruc.2021.131093
0022-2860/© 2021 Published by Elsevier B.V.

A.J. Shadakshari, T.H. Suresha Kumara, H.B.V. Sowmya et al.
Journal of Molecular Structure 1246 (2021) 131093
170.9,167.9,152.4,139.4,131.1,130.8,128.5,124.9,123.3,119.8,119.0,115.4,
103.1; Mass ESI calcd. C25H15BrO6: ([M + H] +), 490.01; found:
([M + 2] +) 492.6.
3,3ꢀ-((4-Fluorophenyl)methylene)bis
(4–hydroxy-2H-
chromen-2-one) (3c): White solid; mp 223.6–225.1 °C 1H NMR
(400 MHz, CDCl3+DMSO–d6); δ (ppm) 8.96 (s, 1H), 8.03 (d,
j = 8.40 Hz, 2H), 7.46 (m, 2H), 7.35 (m, 5H), 6.88 (m, 2H), 6.22
(s, 1H); 13C NMR (400 MHz, CDCl3); δ (ppm) 168.1, 164.9, 152.9,
138.6, 131.4, 128.8, 128.7, 124.5, 123.3, 120.2, 115.9, 114.8, 114.6,
103.8; Mass ESI calcd. C25H15FO6: ([M + H] +), 430.09; found:
([M + 1] +) 430.8.
Scheme 1. one pot synthesis of biscoumarins (3a-l) through Knoevenagel conden-
sation of 4-hydroxy coumarin (1) and aromatic aldehydes (2a-I) by using Amberlite-
IR 120 resin as an efficient heterogeneous catalyst.
3,3ꢀ-((3-(Benzyloxy)−4-methoxyphenyl)methylene)bis(4–
hydroxy-2H-chromen-2-one) (3d): Light yellow solid; mp
217.8–219.0 °C; 1H NMR (400 MHz, DMSO–d6): δ (ppm) 7.83
(d, J = 7.2 Hz, 2H), 7.52 (m, 2H), 7.27 (m, 6H), 7.13 (m, 3H), 6.78
(m, 2H), 6.76 (s, 1H), 6.17 (s, 1H), 4.84 (s, 2H), 3.70 (s, 3H); 13C
NMR (100 MHz, DMSO–d6); δ (ppm) 168.1, 165.0, 152.9, 147.7,
147.2, 137.7, 135.3, 131.2, 128.5, 128.0, 127.9, 124.5, 123.2, 120.4,
119.7, 115.8, 113.9, 112.2, 104.0, 70.7, 60.2, 56.0; Mass ESI calcd.
C33H24O8: ([M + H] +), 548.15; found: ([M + 1] +), 549.0
to several advantages such as an operational simplicity, non-toxic,
low cost, high yield and efficient heterogeneous catalytic system
for chemical transformations [38], ease of isolation after a reaction
and reusability [39]. We have observed that, highly functionalized
aromatic aldehydes and 4–hydroxy coumarin undergo domino re-
action at room temperature in the presence of Amberlite IR-120
resin, and to give the corresponding symmetric and asymmetric
biscoumarins in good to excellent yield (Scheme 1). Synthesized
compounds were tested invitro for their anti-proliferative proper-
ties against leukemia (K-562) and breast (MD-AMB-231) cancer cell
lines and the possible activity perhaps due to hydrogen bonding
was investigated in detail using molecular docking studies.
3,3ꢀ-((3-Chlorophenyl)
methylene)bis(4–hydroxy-2H-
chromen-2-one) (3e): White solid; mp 249.8–251.0 °C; 1H
NMR (400 MHz, DMSO–d6): δ (ppm) 7.83 (d, J = 6.8 Hz, 2H),
7.54 (m, 2H), 7.28 (m, 3H), 7.26 (m, 2H), 7.16 (s, 1H), 7.07 (m,
2H), 6.26 (s, 1H); 13C NMR (100 MHz, DMSO–d6); δ (ppm) 167.8,
164.9, 152.9, 145.3, 133.0, 131.7, 130.1, 126.7, 126.0, 125.5, 124.5,
123.5, 119.8, 116.0, 103.4; Mass ESI calcd. C25H15ClO6: ([M + H]
+), 446.06; found: ([M + H] +), 446.8.
2. Experimental
2.1. Apparatus and materials
3,3ꢀ-((4-Nitrophenyl)methylene)bis(4–hydroxy-2H-chromen-
2-one) (3f): Yellow solid; mp 295–298.5 °C; 1H NMR (400 MHz,
CDCl3): δ 8.07 (d, J = 9.2 Hz, 2H), 8.02 (d, J = 8.0 Hz, 2H),
7.49–7.48 (m, 4H), 7.23 (m, 4H), 6.43 (s,1H); 13C NMR (100 MHz,
CDCl3): δ 168.3, 164.7, 152.9, 151.9, 145.0, 131.6, 128.2, 124.6, 123.5,
123.5, 120.1, 116.0, 103.1; Mass ESI calcd. C25H15NO8: ([M + H] +),
457.08; found: ([M-H] −), 456.1.
Analytical thin layer chromatography was performed on pre-
coated Aluminum-backed silica gel F254 plates with visualiza-
tion under UV light. Melting points were obtained using a hot-
stage apparatus and are uncorrected. 1H NMR spectra, recorded at
400 MHz are referenced to the residual solvent peak at 7.26 ppm
(CDCl3) and 2.50 ppm (DMSO–d6).13C NMR spectra, recorded at
400 MHz, are referenced to the residual solvent peak at 77.0 ppm
(CDCl3) and 39.52 ppm (DMSO–d6). All reactions were carried un-
der room temperature. All other chemicals and solvents were of
analytical grade.
3,3ꢀ-((3-Nitrophenyl)methylene)bis(4–hydroxy-2H-chromen-
2-one) (3 g): Yellow solid; mp 272–275 °C; 1H NMR (400 MHz,
DMSO–d6); δ 8.46 (s, 2H), 7.76 (d, J = 6.8 Hz, 2H), 7.54–7.49 (m,
4H), 7.35 (m, 2H), 7.35 (m, 4H), 6.46 (s, 1H); 13C NMR (100 MHz,
DMSO–d6); δ 168.3, 163.9, 152.9, 150.0, 135.7, 131.8, 131.4, 130.0,
127.0, 124.5, 124.2, 123.3, 119.9, 115.9, 102.6; Mass ESI calcd.
C25H15NO8: ([M + H] +), 457.08; found: ([M + H] +), 457.9.
2.2. General procedure for preparation of biscoumarin derivatives
3a-l
3,3ꢀ-((2-Hydroxyphenyl)
methylene)bis(4–hydroxy-2H-
chromen-2-one) (3 h): White solid; mp 269–271 °C; 1H NMR
(400 MHz, CDCl3); δ 8.10 (d, J = 8.0 Hz, 2H), 7.53 (m, 3H), 7.30
(m, 4H), 7.07 (t, J = 6.8 Hz, 1H), 6.86 (t, J = 6.2 Hz, 1H), 6.77
(d, J = 6.8 Hz, 1H), 6.48 (s, 1H); 13C NMR (100 MHz, DMSO–d6);
A mixture of 4–hydroxy coumarin (100 mg, 0.616 mmol), alde-
hydes (0.616 mmol), triethylamine (0.08 mL, 0.616 mmol) and Am-
berlite IR-120 (15 wt%) in acetonitrile (10 mL) was stirred at room
temperature for about 1 to 2 h. The course of reaction was mon-
itored by TLC. After completion of reaction, the mixture was fil-
tered to recover the resin. The filtrate was evaporated under re-
duced pressure to remove acetonitrile and the residue obtained
was washed with methyl tertiary butyl ether (MTBE) and dried un-
der vacuum.
δ
164.5, 155.4, 152.7, 130.9, 129.4, 126.4, 124.3, 123.1, 120.5,
118.1, 115.7, 115.0, 110.0, 109.9, 104.2; Mass ESI calcd. C25H16 NO7:
([M + H] +), 428.09; found: ([M-H]−), 427.7.
3,3ꢀ-((2-Fluoro-4-methoxyphenyl)methylene)bis(4–hydroxy-
2H-chromen-2-one (3i): White solid; mp 248–251 °C; 1H NMR
(400 MHz, DMSO–d6); δ 7.84 (d, J = 8.0 Hz, 2H), 7.53 (m, 2H), 7.29
(m, 4H), 6.99 (t, J = 17.6 Hz, 1H), 6.85 (m, 2H), 6.21 (s, 1H), 3.76
(s, 3H); δ 13C NMR (100 MHz, DMSO–d6); δ 166.4, 165.0, 152.7,
145.3, 145.2, 132.1, 124.4, 123.9, 123.0, 118.9, 116.2, 114.8, 114.6,
113.8, 104.2, 56.3; Mass ESI calcd. C26H17 FO7: ([M + H] +), 460.10;
found: ([M + H] +), 460.9.
3,3ꢀ-((4-Chlorophenyl)methylene)bis(4–hydroxy-2H-chromen-
2-one) (3a): White solid; m.p 243.4–245.3 °C; 1H NMR (400 MHz,
CDCl3); δ (ppm) 11.7 (s, 1H), 8.12 (d,
j
=
9.4 Hz, 1H), 7.90
(d, j = 4.0 Hz, 1H), 7.66–7.63 (m, 2H), 7.62 (m, 2H), 7.42–7.40
(m, 4H), 7.30 (m, 2H), 7.16 (m, 2H), 6.04 (s, 1H); 13C NMR
(100 MHz, CDCl3); δ (ppm) 165.9, 164.5, 152.4, 152.1, 133.7, 132.8,
132.5,128.6,127.8,124.8,124.3,116.5,116.2, 105.1, 103.5; Mass ESI
calcd. C25H15ClO6; ([M + H] +), 446.06; found: ([M + H] +),
446.8.
3,3ꢀ-((4-(Dimethylamino)phenyl)methylene)bis(4–hydroxy-
2H-chromen-2-one) (3j): White solid; mp 187–194 °C; 1H NMR
(400 MHz, DMSO–d6); δ 7.81 (d, J = 7.6 Hz, 2H), 7.48 (t, J = 7.6 Hz,
2H), 7.25 (m, 4H), 6.91 (d, J = 7.6 Hz, 2H), 6.57 (d, J = 8.0 Hz, 2H),
6.16 (s, 1H) 2.79 (s, 6H); 13C NMR (100 MHz, DMSO–d6); δ 167.9,
165.0, 152.9, 131.1, 127.6, 123.2, 120.5, 115.8, 112.9, 104.2; Mass
ESI calcd. C27H21NO6: ([M + H] +), 455.14; found: ([M + H]+),
456.0.
3,3ꢀ-((4-Bromophenyl)methylene)bis(4–hydroxy-2H-
chromen-2-one)
279.7 °C 1H NMR (400 MHz, CDCl3);
8.0 Hz, 2H), 7.43 (m, 2H), 7.35 (m, 2H), 7.28 −7.12
(m, 6H), 6.19 (s, 1H); 13C NMR (100 MHz, CDCl3); δ (ppm)
(3b):
Pale
yellow
solid;
mp
277.3–
δ
(ppm) 8.00 (d,
J
=
2

A.J. Shadakshari, T.H. Suresha Kumara, H.B.V. Sowmya et al.
Journal of Molecular Structure 1246 (2021) 131093
3,3ꢀ-((4-(Prop-2-ynyloxy)phenyl)methylene)bis(4–hydroxy-
2H-chromen-2-one) (3k): White solid; mp 215–217 °C; 1H NMR
(400 MHz, DMSO–d6); δ 7.80 (d, j = 1.2 Hz, 2H), 7.49 (t, j = 1.2 Hz,
2H), 7.23 (m, 5H), 7.10 (t, j = 7.2 Hz, 1H), 6.89 (d, j = 7.6 Hz, 1H),
6.81 (t, j = 7.2 Hz, 1H), 6.20 (s, 1H), 4.50 (s, 2H), 3.24 (s, 1H);
13C NMR (100 MHz, DMSO–d6); δ 167.2, 164.4, 155.7, 152.8, 131.0,
129.5, 126.7, 121.2, 120.3, 115.8, 112.7, 104.1, 79.6, 77.8, 60.2, 56.0;
Mass ESI cald. C28H18 O7;:([M + H]+), 466.1; found: ([M + H] +),
467.1.
Table 1
Catalytic performance of solid catalysts on the synthesis of product 3a.
Entry∗
Catalyst
Time
Yield (%)∗∗
1
2
3
4
5
6
7
Without Catalyst
24 h
12 h
12 h
12 h
2 h
Trace
45
Silica gel (1mole)
Silica sulfuric acid (1 mole)
Amberlyst-15(1mole)
Amberlite IR-120 (5 wt%)
Amberlite IR-120 (10 wt%)
Amberlite IR-120 (15 wt%)
54
76
86
1 h
90
1 h
94
3,3ꢀ-(Phenylmethylene)bis(4–hydroxy-2H-chromen-2-one)
(3l): White solid; mp 201.1–202.1 °C; 1H NMR (400 MHz, CDCl3);
δ 11.31 (s, 1H), 11.54 (s, 1H), 8.05 (dd, J = 27.92, 7.81 Hz, 2H),
7.68–7.58 (m, 2H), 7.42 (d, J = 8.14 Hz, 3H), 7.37–7.28 (m, 3H), 7.24
(m, 3H+CDCl3) 6.11 (s, 1H); 13C NMR (100 MHz, CDCl3); δ 165.7,
164.5, 164.5, 152.2, 135.1, 132.8, 128.6, 126.8, 126.4, 124.8, 124.3,
116.6, 105.5, 103.8, 103.8; Mass ESI calcd. C25H16 O6; ([M + H] +),
412.09; found: ([M + H] +), 412.9.
∗
Reaction condition: 4-Chlorobenzaldehyde:4-hydroxycoumarin=0.5:1, catalyst,
acetonitrile, rt.
Table 2
Effect of solvents in the synthesis of biscoumarins (3a) with Amberlite IR-120 resin
catalyst.
Entry∗
Solvent
Polarity Index
Time
Yield (%)∗∗
1
2
3
4
5
6
7
Dichloromethane
MTBE
3.4
2.5
2.4
5.1
5.8
9.0
6.6
12 h
12 h
12 h
8 h
64
52
38
73
96
80
78
2.3. Molecular docking studies
Toluene
Ethanol
Acetonitrile
Water
1 h
In the present study, a Graphical User Interface AutoDockTools
is used to prepare the files required for the docking of protein
with ligand. The different conformations of ligand bound to the
active site amino acids of GlcN-6-P synthase were analyzed [39].
The cif format of the compound is converted to 3D structure
(.pdb) using Open able tool. The .pdb file is loaded on to PRO-
DRG server [40] for energy minimization of inhibitor. The coor-
dinate file of GlcN-6-P synthase (PDB ID: 1Jxa) is downloaded
from www.rcsb.org/pdb, was edited by removing the heteroatoms,
adding C-terminal oxygen [41]. For docking calculations, Gasteiger-
Marsili empirical atomic partial charges [42] were assigned to the
inhibitors and non-polar hydrogen atoms were merged. All tor-
sions were allowed to rotate during docking. The Lamarckian ge-
netic algorithm and the pseudo-Solis and Wets methods were ap-
plied for minimization using default parameters. The number of
docking runs was 50, the population in the genetic algorithm was
250, the number of energy evaluations was 100,000, and the max-
imum number of iterations 10,000. The docking results for in-
hibitor against glucosamine-6-phosphate synthase [PDB Id: 1jxa],
showed minimum docking energy, binding energy, inhibition con-
stant, intermolecular energy with RMS as documented. The com-
puter specification used- Operating system: Microsoft Windows XP,
Processor: Intel Pentium 3.40 GHz, RAM: 2 GB, Hard disk: 500 GB,
Python: 2.4.
6 h
Methanol
6 h
∗
Reaction conditions: 4-Chlorobenzaldehyde:4-hydroxycoumarin = 0.5:1, Am-
berlite IR-120 resin (15 wt%), solvents, room temperature.
∗∗
Isolated yields.
hydroxy coumarin (1) and 4–chloro benzaldehyde (2a) was carried
as a probe model reaction with a series of solid catalysts like silica
gel, silica sulfuric acid, Amberlyst-15 and Amberlite IR-120 resins
in acetonitrile solvent at room temperature.
As per the results of current investigations Table 1, negligible
amount of product was observed without the mentioned catalysts.
Amberlyst-15, silica sulfuric acid and silica-gel have not shown the
better catalytic activity as these offered a scope for longer reaction
time, incomplete reaction and poorer yields. Further the study was
continued with Amberlite IR-120 resin. With 15 wt% of Amberlite
IR-120 resin and 1 h reaction time, 96% of the product (3a) was
obtained, with 10 wt% of Amberlite IR-120 resin and 1 h reaction
time, 90% of the product (3a) was obtained, and with 5 wt% of
Amberlite IR-120 resin and 2 h reaction time, 86% of the product
(3a) was obtained. So, 15 wt% of Amberlite IR-120 showed a better
catalytic activity compared with the other selected acidic catalysts.
Further, the study is also focused to know a better solvent
medium, for which a series of solvents like DCM, MTBE, toluene,
ethanol, methanol and water were selected to run above probe
model one pot Domino Knoevenagel–Michael reaction of 1 and 2a
by keeping 15 wt% of Amberlite IR-120 resin as standard. The re-
sults have been summarized in Table 2. Which suggested that, the
reaction time and yield of the product obtained is also dependent
of the nature of the solvent used. Here in general, the solvent with
lower polarity like DCM, MTBE and toluene took longer reaction
time and afforded the product (3a) of lower yield. Moderate yields
were obtained with the polar and protic solvents like ethanol, wa-
ter and methanol. The best result obtained was with acetonitrile
i.e., 96% of biscoumarin (3a).
2.4. Invitro anticancer activity
MTT assay: Cell viability was determined by (3-(4,5-
dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT)
assay. Cells (5 × 103 cells/well) were seeded to 96-well culture
plate and cultured with or without compounds at 1, 10, and 100
mM concentration for 24 h in a final volume of 200 ml. After
treatment, the medium was removed and 20 ml of MTT (5 mg/ml
in PBS) was added to the fresh medium. After 2 h incubation at
37 °C, 100 ml of DMSO was added to each well and plates were
agitated for 1 min. Absorbance was read at 570 nm on a multi-
well plate reader (Synergy Mx, Biotek Inc., USA). Percent inhibition
of proliferation was calculated as a fraction of the control (without
compound).
Trial results of the above procedures were encouraging as the
biscoumarin (3a) obtained was 96% at room temperature. This op-
timized protocol has many advantages viz., highly efficient, cleanly
working, isolation of product is easy and the catalyst is recyclable.
Subsequently, with an optimized protocol, a series of biscoumarins
(3a-l) were synthesized by using 4-hydroxycoumarin and a series
of aldehydes (2a-l). The results have been summarized in Table 3.
The developed procedure worked effectively with all the reactions.
It was observed that the reaction is more compatible and toler-
able with aromatic aldehydes, whether the nature of the substitu-
3. Result and discussion
Initially, the present study comprises an aim to discover an
efficient heterogeneous solid catalyst for the synthesis of bis
coumarins (3) through Domino Knoevenagel–Michael reaction. Ac-
cordingly, to prepare the compound 3a, one pot reaction of 4–
3

A.J. Shadakshari, T.H. Suresha Kumara, H.B.V. Sowmya et al.
Journal of Molecular Structure 1246 (2021) 131093
Scheme 2. Mechanism of Knoevenagel condensation of 4-hydroxycoumarin (1) with the aldehyde (2) towards biscoumarin (3).
Table 3
Table 4
Recyclability of Amberlite IR-120 resin.
Synthesis of the biscoumarins (3a-l) by one pot condensation of 4-hydroxycoumarin
and different aldehydes (2a-l) by using 15 wt% of Amberlite IR-120 resin.
Cycle
Isolated Amberlite IR-120 (mg)
Isolated Yield (%)
Entry
Product
R
Time (min)
Yield (%)∗
1
2
3
4
15
14
13
12
96
94
92
90
1
3a
3b
3c
3d
3e
3f
4-Cl
60
96
95
93
92
95
91
91
94
90
93
92
95
2
4-Br
60
3
4-F
75
4
5-OMe, 3-OBn
3-Cl
100
60
5
6
4-NO2
120
110
60
7
3 g
3h
3i
3-NO2
15 min to regenerate free -SO3H group, filtered, washed with dis-
tilled water then with acetone and dried and that resin was reused
for next successive reactions. This way it was recycled for four dif-
ferent model reactions. The results are summarized in Table 4. In
the same way, one 15 wt% of Amberlite IR-120 resin was used
for the synthesis of succeeding four different bis coumarins (3)
by using a respective aromatic aldehyde (2). Hence, it is deter-
mined that, the process of synthesis of several biscoumarins (3)
was succeeded efficiently with the recycling of Amberlite IR-120
resin without the significant loss of catalytic activity. During the
successive reactions, the observed weight of the isolated Amber-
lite IR-120 resin was declined by 1 mg (15 mg, 14 mg, 13 mg and
12 mg). And accordingly, the observed yield of the isolated product
was declined slightly by 2%, i.e., from 96%, 94%, 92% and 90%.
8
2-OH
9
2-F, 3-OMe
4-N(Me)2
4-O(propargyl)
H
90
10
11
12
3j
90
3k
3l
110
60
∗
Isolated yield.
tion was electron withdrawing or electron donating which arrive at
good to excellent isolated yield at rt within 60 min to 120 min. the
obtained products were recrystallized from ethanol to obtain pure
biscoumarins (3a-l). Identification of the synthesized compounds
with a new procedure was attained by means of their 1H NMR, 13C
NMR and mass spectral data. The spectral results are summarized
in the experimental section.
The proposed reaction mechanism is shown in Scheme 2.
Which involves a Knoevenagel condensation of 4-hydroxycoumarin
(1) with the aldehyde (2) gives 4, which underwent Michael addi-
tion with another molecule of 1 gives 5, and which upon enoliza-
tion gives 3.
3.1. Biological activity
3.1.1. Molecular docking studies
The newer targets for better antimicrobials acting through novel
mechanisms are the important developments of research in recent
years. One such enzyme present in microbial cells is Glucosamine-
6-Phosphate synthase. The dormant of G-6-P synthase may give
out as a novel approach to find improved antimicrobials. The
molecular docking studies of biscoumarins derivatives and the
standard drug chloroquine with GlcN-6-P synthase yielded all po-
tential conformations with the binding energy, docking energy, in-
Recyclability of the Amberlite IR-120 resin catalyst was also
very important part of this study and was examined cautiously.
So, the catalyst used was recovered from the reaction of 4-
hydroxycoumarin (1) and 4-chlorobenzaldehyde (2a) via filtration.
The filtered triethylammonium salt of resin from the reaction mix-
ture was stirred with sufficient quantity of 0.1 N HCl for about
4

A.J. Shadakshari, T.H. Suresha Kumara, H.B.V. Sowmya et al.
Journal of Molecular Structure 1246 (2021) 131093
Table 5
Molecular docking results of biscoumarins (3a-l) with glucosamine-6-phosphatesynthase protein receptor.
Ligands
Binding Energy (kcal/mol)
Docking Energy(kcal/mol)
Inhibition Constant(μM)
Electrostatic Energy (kcal/Mol)
3a
−6.80
−7.58
−15.83
−15.93
−18.37
−7.92
−7.38
−7.99
−8.94
−17.05
−8.92
−7.92
−6.79
−7.14
−7.97
−16.19
−16.18
−18.73
−8.32
−7.69
−8.79
−9.73
−17.88
−9.12
−8.39
−9.42
1.03 × 10−5
2.78 × 10−6
2.5 × 10−12
2.1 × 10−12
3.4 × 10−14
1.56 × 10−6
3.9 × 10−6
−7.12
−7.89
−16.14
−16.24
−18.68
−8.23
−7.69
−8.93
−9.87
−17.98
−9.30
−8.11
−9.28
3b
3c
3d
3e
3f
3 g
3h
1.39 × 10−6
2.79 × 10−7
3.17 × 10−13
1.58 × 10−6
1.86 × 10−6
1.05 × 10−5
3i
3j
3k
3l
Chloroquine
Table 5a
The% inhibition of growth of cancer cell lines by compounds 3∗.
Harmine was found to be 46 and 52 mM when tested against K-
562 and MDAMB231 cell lines in our MTT assay. The compound
3i therefore appeared to be a promising and potential anticancer
agent of further interest.
K-562 (leukemia)
MDA-MB-231 (breast)
Compounds
100 M
10 mM
1mM
100 mM
10 mM
1 mM
3b
3c
3d
3f
37.6
33.4
55.6
46.9
42.5
43.8
36.2
32.6
50.9
45.7
37.8
34.5
30.9
28.9
46.8
44.6
33.6
32.1
56.5
67.8
65.9
54.8
45.6
51.3
54.8
57.5
61.3
50.8
43.6
45.6
45.8
43.7
48.8
47.8
39.8
41.7
4. Conclusions
In conclusion, the present method demonstrates an opera-
tionally simple and clean procedure for the synthesis of bis-
coumarins using a catalytic amount of Amberlite IR-120 resin.
Moreover, the catalyst used is of low cost, recyclable, less toxic
and moisture compatible. The yields of products (3) were excel-
lent within short reaction time. Therefore, this methodology is one
of the valid contributions to the field of biscoumarin synthesis.
Most of the synthesized biscoumarins (3) have displayed potent
biological activities. The data of molecular docking studies clearly
showed that, most of these docked with the enzyme GlcN-6-P syn-
thase with significant docking energy. The IC50 value of the com-
pounds 3b, 3c, 3d, 3f, 3i and 3k shows that, these have effective
anti-proliferative properties against leukemia (K-562) and breast
(MD-AMB-231) cancer cell lines.
3i
3k
∗Data presented are the average of three experiments.
hibition constant and electrostatic energy (Table 5). The entire set
of molecules docked against the enzyme showed significant dock-
ing energy ranging from −7.14 kcal/mol to −18.73 kcal/mol with an
estimated inhibition constant of 1.03 × 10−5 to 3.4 × 10−14 respec-
tivley. Whereas, docked energy of the standard drug chloroquine
was 9.42 kcal/mol with an inhibition constant of 1.05 × 10−5
.
The molecules 3c, 3d, 3e and 3j showed significant inhibition
against all compounds docked. The compound 3e showed mini-
mum docking energy −18.73 kcal/mol with an inhibition constant
of 3.4 × 10−14 . Theoretically, the activity may be due to inhibi-
tion of enzyme GlcN-6-Psynthase, which catalyzes a complex reac-
tion involving ammonia transfer from l-glutamine to Fru-6-P fol-
lowed by isomerization of the formed fructosamine-6-phosphate
to glucosamine-6-phosphate. In the present study, the derivatives
with chlorine as a halogen have not formed any hydrogen bond
with an active site of amino acids of GlcN-6-P synthase, but
showed excellent docking energy, since the halogens are endowed
with an ability to establish intermolecular bonds in a fashion that
resembles the H-bonds. But the compounds containing halogens
will improve oral absorption, skin penetration and membrane per-
meability [43]. It is generally accepted that halogen atoms are not
capable of significant hydrogen bonding.
Declaration of Competing Interest
“Recyclable Amberlite IR-120 Catalyzed domino reaction: Syn-
thesis, anticancer activity and molecular docking studies of bis-
coumarins” declare that, we don’t have any conflict of interest
among the authors/corresponding author about the manuscript
submission to the Journal of Molecular Structure”
Acknowledgment
THS and SAJ thankful to the Department of Chemistry, UBDTCE,
Davangere (A Constituent College of VTU, Belagavi).
Supplementary materials
3.1.2. Invitro anticancer activity
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.131093.
Some of the compounds (3) synthesized [44] were tested
for their invitro anti-proliferative properties against leukemia (K-
562) and breast (MD-AMB-231) cancer cell lines in a MTT assay.
Harmine, a member of β-carboline family of compounds showed
cytotoxicity against HL60 and K562 cell lines [45] was used as a
reference compound in this assay. The results of active molecules
identified by this assay are presented in Table 5. While 3i and
3k showed good activity against leukemia cells (Table 5a), most
of the compounds, for example, 3b, 3c, 3d, 3f, 3i and 3k were
found to be effective against breast cancer and 3i being the best
among them (Table 5). The compound 3i showed significant anti-
proliferative properties at all the concentrations tested and main-
tained the same at low concentrations. Notably, IC50 value of
References
[1] S. Milewski, Glucosamine-6-phosphate synthase-the multi-facets enzyme,
Biochim. Biophys. Acta 1597 (2002) 173–192.
[2] R.O. Kennedy, R.D. Thornes, Coumarins: Biology, Applications and Mode of Ac-
tion, Wiley: Chichester, U.K, 1997.
[3] W.L. Stanley, L. Jurd, Citrus Coumarins, Agric. Food Chem. 19 (1971) 1106–1110.
[4] C.A. Kontogiorgis, D.J. Hadjipavlou-Litina, Synthesis and antiinflammatory ac-
tivity of coumarin derivatives, J. Med. Chem. 48 (2005) 6400–6408.
[5] G. Melagraki, A. Afantitis, O.I. Markopoulou, A. Detsi, M. Koufaki, C. Konto-
giorgis, D.J. Hadjipavlou-Litina, Synthesis and evaluation of the antioxidant
and anti-inflammatory activity of novel coumarin-3-aminoamides and their al-
pha-lipoic acid adducts, Eur. J. Med. Chem. 44 (2009) 3020–3026.
5

A.J. Shadakshari, T.H. Suresha Kumara, H.B.V. Sowmya et al.
Journal of Molecular Structure 1246 (2021) 131093
[6] M.A. Gouda, M.A. Berghot, E.A. Baz, W.S. Hamama, Synthesis, antitumor and
antioxidant evaluation of some new thiazole and thiophene derivatives incor-
porated coumarin moiety, Med. Chem. Res. 21 (2012) 1062–1070.
[7] L. Raev, E. Voinova, I. Ivanov, D. Popov, Antitumor activity of some coumarin
derivatives, Pharmazie 45 (1990) 696–702.
[29] J.M. Khurana, S. Kumar, Tetrabutylammonium bromide (TBAB): a neutral
and efficient catalyst for the synthesis of biscoumarin and 3,4-dihydropy-
rano[c]chromene derivatives in water and solvent-free conditions, Tetrahedron
Lett. 50 (2009) 4125–4127.
[30] M. Kidwai, V. Bansal, P. Mothsra, S. Saxena, R.K. Somvanshi, S. Day,
T.P. Singh, Molecular iodine: a versatile catalyst for the synthesis of
[8] S. Khode, V. Maddi, P. Aragade, M. Palkar, P.K. Ronad, S. Mamledesai,
A. Thippeswamy, D. Satyanarayana, Synthesis and pharmacological evalu-
bis(4-hydroxycoumarin) methanes in water, J. Mol. Catal. A: Chem 268 (2007)
76–81.
ation of
a novel series of 5-(substituted)aryl-3-(3-coumarinyl)-1-phenyl-2-
pyrazolines as novel anti-inflammatory and analgesic agents, Eur. J. Med.
Chem. 44 (2009) 1682–1688.
[9] C. Zwergel, S. Valente, A. Salvato, Z. Xu, O. Talhi, A. Mai, A. Silva, L. Altucci,
G. Kirsch, Novel benzofuran–chromone and –coumarin derivatives: synthesis
and biological activity in K562 human leukemia cells, Med. Chem. Commun. 4
(2013) 1571–1578.
[31] J.M. Khurana, S. Kumar, Ionic liquid: an efficient and recyclable medium for the
synthesis of octahydroquinazolinone and biscoumarin derivatives, Monatsh.
Chem. 141 (5) (2010) 561–564.
[32] [a] A. Tzani, A. Douka, A. Papadopoulos, E.A. Pavlatou, E. Voutsas, A. Detsi, Syn-
thesis of biscoumarins using recyclable and biodegradable task-specific ionic
liquids, ACS Sustain. Chem. Eng.
1 (2013) 1180–1185; [b] W. Li, Y. Wang,
[10] F. Belluti, G. Fontana, L.D. Bo, N. Carenini, C. Giommarelli, F. Zunino, Design,
synthesis and anticancer activities of stilbene-coumarin hybrid compounds:
identification of novel proapoptotic agents, Bioorg. Med. Chem. 18 (2010)
3543–3550.
Z. Wang, L. Dai, Y. Wang, Novel SO3H-functionalized ionic liquids based on
benzimidazolium cation: efficient and recyclable catalysts for one-pot synthe-
sis of biscoumarin derivatives, Catal. Lett. 141 (2011) 1651–1658.
[33] R. Karimian, F. Piri, A.A. Safari, S.J. Davarpanah, One-pot and chemoselective
synthesis of bis(4-hydroxycoumarin) derivatives catalyzed by nano silica chlo-
ride, J. Nanostruct. Chem 3 (2013) 52–57.
[11] D.M. Manidhar, R.K. Kesharwani, N.B. Reddy, C.S. Reddy, K. Misra, Designing,
synthesis, and characterization of some novel coumarin derivatives as probable
anticancer drugs, Med. Chem. Res. 22 (2013) 4146–4157.
[12] C.A. Kontogiorgis, D.J. Hadjipavlou-Litina, Synthesis and antiinflammatory ac-
tivity of coumarin derivatives, J. Med. Chem. 48 (2005) 6400–6408.
[13] N. Hamdi, M. Saoud, A. Romerosa, 4-Hydroxy coumarine: a versatile reagent
for the synthesis of heterocyclic and vanillin ether coumarins with biological
activities, Top. Heterocycl. Chem. 11 (2007) 283–301.
[14] K.M. Khan, Z.S. Saify, M.Z. Khan, Zia-Ullah, M.I. Choudhary, A.U. Rahman,
S. Parveen, Z.H. Chohan, C.T. Supuran, Synthesis of coumarin derivatives with
cytotoxic, antibacterial and antifungal activity, J. Enzyme Inhib. Med. Chem. 9
(2004) 373–379.
[34] B. Pawar, V. Shinde, A. Chaskar, n-Dodecylbenzene Sulfonic Acid (DBSA) as
a novel brønsted acid catalyst for the synthesis of Bis(indolyl)methanes and
Bis(4-hydroxycoumarin-3-yl)methanes in water, Green Sustain. Chem 3 (2013)
56–60.
[35] Z.N. Siddiqui, T.N. Mohammed Musthafa, S. Praveen, F. Farooq,
Zn(Proline)2-catalyzed Knoevenagel condensation under solvent-free/aqueous
conditions and biological evaluation of products, Med. Chem. Res. 20 (2011)
1438–1444.
[36] K. Tabatabaeian, H. Heidari, A. Khorshidi, M. Mamaghani, N.O. Mahmoodi, Syn-
thesis of biscoumarin derivatives by the reaction of aldehydes and 4-hydrox-
ycoumarin using ruthenium (III) chloride hydrate as a versatile homogeneous
catalyst, J. Serb. Chem. Soc. 77 (4) (2012) 407–413.
[15] D. Bhavsar, J. Trivedi, S. Parekh, M. Savant, S. Thakrar, A. Bavishi, A. Radadiya,
H. Vala, J. Lunagariya, M. Parmar, Synthesis and in vitro anti-HIV activity
of N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chromen-4-yl)acetamide derivatives
using MTT method, Bioorg. Med. Chem. Lett. 21 (2011) 3443–3446.
[16] C. Spino, M. Dodier, S. Sotheeswaran, Anti-HIV coumarins from Calophyllum
seed oil, Bioorg. Med. Chem. Lett 8 (24) (1998) 3475–3478.
[37] H. Mehrabi, H. Abusaidi, Synthesis of biscoumarin and 3,4-dihydropy-
rano[c]chromene derivatives catalysed by sodium dodecyl sulfate (SDS) in neat
water, J. Iran. Chem. Soc. 7 (2010) 890-594.
[38] [a] N. Tewari, D. Katiyar, V.K. Tiwari, R.P. Tripathi, Amberlite IR-120 catalysed
efficient synthesis of glycosyl enamines and their application, Tetrahedron Lett.
44 (2003) 6639–6642; [b] K. Akagawa, S. Sakamoto, K. Kudo, Resin-supported
acid- and base-catalyzed one-pot sequential reaction including an enantiose-
lective step, Tetrahedron Lett. 48 (2007) 985–987.
[17] A. Dolmella, S. Gatto, E. Girardi, G. Bandoli, X-ray structures of the anticoagu-
lants coumatetralyl and chlorophacinone. Theoretical calculations and SAR in-
vestigations on thirteen anticoagulant rodenticides, J. Mol. Struct. 513 (1999)
177–199.
[18] F. Rosskopf, J. Kraus, G. Franz, Immunological and antitumor effects of
coumarin and some derivatives, Die Pharmazie 47 (2) (1992) 139–142.
[19] P. Sengupta, M. Sen, P. Karuri, Structure of gerberinol novel
dimethyldicoumarol from Gerbera lanuginose benth, J. Indian Chem.Soc.
62 (1985) 916–919.
[39] N. Mulakayala, K.M.K. Ismail, R.K. Rapolu, B. Kandagatla, P. Rao, S. Oruganti,
M. Pal, Catalysis by Amberlite IR-120 resin: a rapid and green method for the
synthesis of phenols from arylboronic acids under metal, ligand, and base-free
conditions, Tetrahedron Lett. 53 (2012) 6004–6007.
[40] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, Autodock4 and AutoDockTools4: automated docking with selective
receptor flexibility, J. Computational Chem. 16 (2009) 2785–2791.
[41] A.K. Ghose, G.M. Crippen, Atomic physicochemical parameters for three-di-
mensional-structure-directed quantitative structure-activity relationships. 2.
Modeling dispersive and hydrophobic interactions, J. Chem. Inf. Comput. Sci.
27 (1987) 21–35.
[20] K.P. Link, The anticoagulant 3,3-methyl-bis-4-hydroxycoumarin, Fed. Proc
(1945) 176–182.
4
[21] M. Gersch, J. Kreuzer, S.A. Sieber, Electrophilic natural products and their bio-
logical targets, Nat. Prod. Rep. 29 (2012) 659–682.
[22] K. Petnapapun, W. Chavasiri, P. Sompornpisut, Structure-activity relation-
ꢀ
ships of 3,3 -Phenylmethylene-bis-4-hydroxycoumarins: selective and potent
inhibitors of gram-positive bacteria, The Sci. World J. 2 (2013) 178649.
[42] T.A. Binkowski, S. Naghibzadeg, J. Liang, Computed atlas of surface topography
of proteins, Nucleic Acids Res. 319 (2003) 3352–3355.
[23] [a] N.H. Kim, S.N. Kim, J.S. Oh, Y.K.Kim S.Lee, Anti-mitotic potential of
ꢀ
7-diethylamino-3(2 -benzoxazolyl)-coumarin in 5-fluorouracil-resistant human
[43] J. Gasteiger, M. Marsili, Iterative partial equalization of orbital electronegativ-
ity, Tetrahedron 36 (1980) 3219–3228.
gastric cancer cell line SNU620/5-FU, Biochem. Biophys. Res. Commun. 418
(2012) 616–621; [b] S.N. Kim, N.H. Kim, Y.S. Park, H. Kim, S. Lee, Q. Wang,
[44] For identification of novel anticancer agents, see:
ꢀ
Y.K. Kim, 7-Diethylamino-3(2 -benzoxazolyl)-coumarin is a novel microtubule
[a] N. Mulakayala, B. Kandagatla, Ismail, R.K. Rapolu, P. Rao, C. Mulakayala,
C.S. Kumar, J. Iqbal, S. Oruganti, InCl3-catalysed synthesis of 2-aryl quina-
zolin-4(3H)-ones and 5-aryl pyrazolo[4,3-d]pyrimidin-7(6H)-ones and their
evaluation as potential anticancer agents, Bioorg. Med. Chem. Lett. 22 (2012)
5063–5066.
inhibitor with antimitotic activity in multidrug resistant cancer cells, Biochem.
Pharmacol 77 (2009) 1773–1779.
[24] N. Au, A.E. Rettie, Pharmacogenomics of 4-hydroxycoumarin anticoagulants
drug, Metab. Rev. 40 (2008) 355–375.
[25] D.K. Mayank, N. Kaur, R. Giri, N. Singh, A biscoumarin scaffold as an efficient
anti-Zika virus lead with NS3-helicase inhibitory potential: in vitro and in silico
investigations, New J. Chem. 44 (2020) 1872–1880.
[b] N. Mulakayala, D. Rambabu, M.R. Raja, M. Chaitanya, C.S. Kumar, A.M. Kalle,
G.R. Krishna, C.M. Reddy, M.V.B. Rao, M. Pal, Ultrasound mediated catalyst free
synthesis of 6H-1-benzopyrano[4,3-b]quinolin-6-ones leading to novel quino-
line derivatives: their evaluation as potential anti-cancer agents, Bioorg. Med.
Chem. 20 (2012) 759–768.
[26] [a] J.J. Xin, J. Li, Z. Zhang, X. Hu, M. Li, Biscoumarin derivatives: synthe-
sis, crystal structure, theoretical studies and induced apoptosis activity on
bladder urothelial cancer cell, J. Mole. Str. 1084 (2015) 200–206; [b] Fen Li,
Chang-wei Lv, Zi-dan Zhang, Jing Li, Zheng Hou, Xiao-hui Yang, Jiang-tao Li, Xi-
ao-xing Luo, Ming-kai Li, Crystal structure and activities of three biscoumarin
derivatives against Staphylococcus aureus, J. Mole. Stru 1097 (2015) 117–123.
[27] S. Kolita, L. Dutta, P. Das, P.J. Bhuyan, One-pot synthesis of unsymmetrical
Bis(4-Hydroxycoumarin-3-yl)methanes, Synlett 28 (2017) 2291–2294.
[28] F.A. El-Samahy, H.A. Abd El Salam, N.F. El-Sayed, E.M. Shalaby, M.F. Dondeti,
Synthesis of unexpected novel bis-coumarin derivatives via three component
reactions of 4-hydroxycoumarin, aldehydes and cyclic secondary amines. Con-
formation in the solid state and pharmacological evaluation, Naturforsch 72
(10) (2017) 705–716 b.
[c] R.M. Rao, C.H.U. Reddy, A. Nakhi, N. Mulakayala, M. Alvala,
M.K. Arunasree, R.R. Poondra, J. Iqbal, M. Pal, Sequential cou-
pling/desilylation–coupling/cyclization in a single pot under Pd/C–Cu catalysis:
synthesis of 2-(hetero)aryl indoles, Org. Biomol. Chem. 9 (2011) 3808–3816.
[45] F. Jahaniani, S.A. Ebrahimi, N. Rahbar-Roshandel, M. Mahmoudian, Xanthomi-
crol is the main cytotoxic component of Dracocephalum kotschyii and a po-
tential anti-cancer agent, Phytochemistry 66 (2005) 1581–1592.
6