Dicoumarol derivatives: Green synthesis and molecular modelling studies of their anti-LOX activity

Article abstract of DOI:10.1016/j.bioorg.2018.07.021

Dicoumarol derivatives were synthesized in the InCl₃ catalyzed pseudo three-component reactions of 4-hydroxycoumarin with aromatic aldehydes in excellent yields. The reactions were performed in water under microwave irradiation. All synthesized

Full text of DOI:10.1016/j.bioorg.2018.07.021

Accepted Manuscript
Dicoumarol derivatives: green synthesis and molecular modelling studies of
their anti-LOX activity
Dušica Simijonović, Evangelia-Eirini Vlachou, Zorica D. Petrović, Dimitra J.
Hadjipavlou-Litina, Κonstantinos E. Litinas, Nevena Stanković, Nezrina
Mihović, Milan P. Mladenović
PII:
S0045-2068(18)30567-4
DOI:
https://doi.org/10.1016/j.bioorg.2018.07.021
YBIOO 2440
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
11 June 2018
17 July 2018
18 July 2018
Please cite this article as: D. Simijonović, E-E. Vlachou, Z.D. Petrović, D.J. Hadjipavlou-Litina, #.E. Litinas, N.
Stanković, N. Mihović, M.P. Mladenović, Dicoumarol derivatives: green synthesis and molecular modelling studies
of their anti-LOX activity, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.07.021
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Dicoumarol derivatives: green synthesis and molecular modelling studies of
their anti-LOX activity
Dušica Simijonović,*a Evangelia-Eirini Vlachou,b Zorica D. Petrović,a Dimitra J. Hadjipavlou-
Litina,c Κonstantinos E. Litinas,b Nevena Stanković,d, Nezrina Mihović,d Milan P. Mladenovićd
a University of Kragujevac, Faculty of Science, Department of Chemistry, Radoja Domanovića 12, 34000
Kragujevac, Serbia.
bLaboratory of Organic Chemistry, Department of Chemistry, Aristotle University of Thessaloniki,
Thessaloniki 54124, Greece.
cDepartment of Pharmaceutical Chemistry, School of Pharmacy, Faculty of Health Sciences, Aristotle
University of Thessaloniki, Thessaloniki 54124, Greece.
dKragujevac Center for Computational Biochemistry, Faculty of Science, University of Kragujevac,
Radoja Domanovića 12, 34000 Kragujevac, P.O. Box 60, Serbia
Abstract
Dicoumarol derivatives were synthesized in the InCl3 catalyzed pseudo three-component
reactions of 4-hydroxycoumarin with aromatic aldehydes in excellent yields. The reactions were
performed in water under microwave irradiation. All synthesized compounds were characterized
using NMR, IR, and UV-Vis spectroscopy, as well as with TD-DFT. Obtained dicoumarols were
subjected to evaluation of their in vitro lipid peroxidation and soybean lipoxygenase inhibition
activities. It was shown that five of ten examined compounds (3e, 3h, 3b, 3d, 3f) possess
significant potential of antilipid peroxidation (84 tο 97%), and that compounds 3b, 3e, 3h
provided the highest soybean lipoxygenase (LOX-Ib) inhibition (IC50 = 52.5 µM) and 3i
somewhat lower activity (IC50 = 55.5 µM). The bioactive conformations of the best LOX-Ib
inhibitors were obtained by means of molecular docking and molecular dynamics. It was shown
that, within the bioactive conformations interior to LOX-Ib active site, the most active
compounds form the pyramidal structure made of two 4-hydroxycoumarin cores and a central
phenyl substituent. This form serves as a spatial barrier which prevents LOX-Ib Fe2+/Fe3+ ion
activity to generate the coordinative bond with the C13 hydroxyl group of the α-linoleate. It is
worth pointing out that the most active compounds 3b, 3e, 3h and 3i can be candidates for
further examination of their in vitro and in vivo anti-inflammatory activity and that molecular
modeling study results provide possibility to screen bioactive conformations and elucidate the
mechanism of dicoumarols anti-LOX activity.
Keywords: Dicoumarol derivatives, InCl3 catalyzed synthesis, microwave irradiation, anti-
inflammatory activity, molecular docking, molecular dynamics
Corresponding author:
*D. Simijonović: tel.: +381-34-336-223; +381-34-335-039; e-mail: dusicachem@kg.ac.rs

1. Introduction
It is known that coumarin derivatives exhibit a lot of pharmacological and biological activities
[1]. 4-Hydroxycoumarin, between them, represent significant structural fragment for many
synthetic and natural products [2]. Therefore, they are important precursors in organic synthesis
and useful pharmacophores in medicinal chemistry. Their derivatives, exert broad spectrum of
biological activities such as anticoagulant [3], antibacterial [4,5], antifungal [5], antiviral [6],
antitumor [7], antioxidant [8,9] and anti-inflammatory [10]. Warfarin (I) (Fig. 1) is an
anticoagulant drug in extended clinical use [11,12]. Derivatives of I [10] as well as the natural
product dicoumarol [13,14] (II) and the synthetic phenprocoumon [11,12] (III) are also
anticoagulants. Moreover, the above compounds present other biological activities such as
anticancer [2a,15], HIV-1 integrase inhibition [16], and urease inhibition [17].
Fig. 1. Some natural and synthetic coumarin derivatives used as anticoagulant drugs
Dicoumarol derivatives are generally synthesized through the reaction of 4-
hydroxycoumarin and aldehydes using different catalysts and media. Recently, several methods
have been reported for the synthesis of these compounds, which includes the use of different
catalyst systems such as p-dodecylbenzenesulfonic acid/piperidine [18], molecular iodine [19],
ionic liquids with Lewis acid and/or Bronsted acid sites [20], SO3H functionalized ionic liquids
[21], Zn(proline)2 [22], heteropoly acid [23], sodium dodecyl sulfate [24], sulfated titania [25],
nano silica catalysts [26], propane-1,2,3triyltris (hydrogen sulfate) [27], Ce2(SO4)×4H2O [28],
Fe3O4 nanoparticles [29], reflux in alcohols [30,31] or heating in aqueous NaCl solution [32].
However, although in the literature there are a lot of studies relating to the synthesis of
dicoumarols under classical conditions, only a few articles can be found related to the synthesis
of these compounds under microwave assisted conditions [33,35]. Microwaves are essential for
the application of green chemistry in science and technology [36]. Water is a characteristic green
solvent, as it is readily available with non-toxicity, non-flammability and high heat capacity.
The lipoxygenases (LOXs) are enzymes which can be found in animals, plants, and fungi.
These enzymes catalyze the oxidative metabolism of polyunsaturated fatty acids. Linoleic acid is
the primary substrate in the reaction of oxygenation of polyunsaturated fatty acids catalyzed by
plant LOX, while the mammalian isozymes mainly catalyze the metabolism of arachidonic acid
[37]. The leukotrienes are products of this metabolism. Leukotrienes, lipid mediators and

bioregulators, have been associated with various pathological processes, such as bronchial
asthma, inflammation, and cancer and that they can regulate tumor progression, apoptosis, and
migration through a lot of signaling [38]. In the literature are described a few types of
lipoxygenases inhibitors: i) redox-active compounds ii) iron-ligand inhibitors with weak redox-
active properties, and iii) non-redox-type inhibitors [39]. Among many investigated compounds,
coumarin derivatives, belong to the group of redox-active lipoxygenase inhibitors. Although, the
mechanism of action of the available LOX inhibitors is not yet fully elucidated, they could
significantly contribute to the suppressing of the negative LOX impact on human health [40].
Hence, the finding of effective LOX inhibitors is challenge for numerous scientific laboratories.
In the course of our interest in the synthesis of coumarin derivatives under MW irradiation
[41], we wish to report here the application of InCl3 catalysis in water, as green solvent, under
MW irradiation for the synthesis of these compounds. To our best knowledge, InCl3 has been
used only for the reactions of dimedone and aldehydes under heating [42], leading to the
synthesis of fused pyran derivatives. In addition, obtained dicoumarol derivatives were
characterized using NMR, IR, and UV-Vis spectroscopy and were tested as inhibitors of lipid
peroxidation and soybean lipoxygenase (LOX-Ib). Since docking study provide possibility to
screen the binding affinity and orientation of bioactive compounds at the active site of the
enzyme, molecular docking and molecular dynamics studies of the most potent LOX-Ib
inhibitors were performed.
2. Results and discussion
2.1. Chemistry
The studied reactions and obtained products are depicted in Scheme 1. The reaction was
performed by treatment of 4-hydroxycoumarin (1) with benzaldehyde (2a) in the presence of 10
mol% InCl3 in water, under MW irradiation and by heating. After cooling and filtration of the
reaction mixture, 3,3’-(phenylmethylene)bis(4-hydroxy-2H-chromene-2-one) (3a) was obtained
[14,19] (90% yield) (Table 1, entry 1). In fact, this reaction is a pseudo three-component reaction
by using twofold equivalents of 4-hydroxycoumarin (1) to benzaldehyde (2).

Scheme 1. Reaction of formation dicoumarol derivatives. Reagents and conditions: (i) 1 (1
mmol), 2 (0.5 mmol), H2O, InCl3 (0.05mmol), MW, 110℃.
Table 1. Synthesis of dicoumarols 3a-j.
Time
(min)
20
15
15
15
20
20
20
Yield
(%)
90
91
93
96
90
85
87
Entry
R
Product
1
2
3
4
5
6
7
8
9
H
4-Cl
4-F
4-NO2
4-OH
4-CH3
4-CH3O
3a
3b
3c
3d
3e
3f
3g
3h
3i
4-OH, 3-OCH3
4-OH, 3,5-di-OCH3
3,4-di-OH
20
20
20
89
86
85
10
3j
The analogous reactions with aldehydes 2b-j led to the substituted dicoumarol derivatives
3a-j in excellent yields (85-96%) (Table 1). Aldehydes with electron withdrawing group reacted
faster (15 min.) producing the dicoumarols in a little better yield (Table 1, entries 2-4).
Aldehydes with electron donating groups gave slightly lower yields and required longer reaction
time (Table 1, entries 5-10).
2.2. Structural characterization
Synthesized dicoumarols (3a-3j) were experimentally characterized by 1H and 13C NMR, IR and
UV-Vis spectroscopy, as well as with melting points. In addition, the structures of these
compounds were elucidated based on UV-Vis simulated spectral properties. It is worth pointing
out that UV-Vis spectral characterisation of these compounds is reported here for the first time.
1
In the H NMR spectra of all compounds singlets originating from protons of benzylic
carbon, linkage for two 4-hydroxycoumarin moieties and for aromatic ring C appear in the range
of 6.01-6.12 ppm. The aromatic protons of rings B, B’ and C were observed in region of 6.42-
8.19 ppm. Hydroxy groups of 4-hydroxycoumarin part of molecule appear around 11.30 and
11.50 ppm as broad singlets. In the spectra of compounds 3f-3i, singlets originating from protons
of methyl group (3f) and methoxy groups (3g-3i) bonded for aromatic ring at 2.34, 3.79 and 3.75
13
ppm were observed. In C NMR spectra of all dicoumarol derivatives, benzylic carbon atom
appears around 36 ppm. In the range of 104-147 ppm, peaks are assigned to aromatic carbon
atoms. Peaks observed at around 152, 166, and 169 ppm were assigned to C-O, C-OH and C=O
carbon atoms. The spectra of compounds 3f-3i, contain additional peaks at around 21 and 56
ppm for methyl and methoxy group, respectively. The IR spectra of the all compounds contain
weak bands at about 3400, 3060, and 1350 cm-1, which correspond to the vibration of O-H and
C-OH groups. The peaks in region 1615-1670 cm-1 are assigned to the C=O of lactone ring,

while bands present at about 1560 and 1600 cm-1 originate from stretching vibration of double
bond and aromatic rings.
In the UV-Vis spectra of all compounds (experimental and simulated) three major absorption
bands appear around 230 nm, 280 and 310 nm, Figs. 2 and S1. It is worth pointing out, that in all
simulated spectra bands over 300 nm are somewhat redshifted.
Fig. 2. Experimental and simulated UV-Vis spectra of compound 3a
To distinguish the parts of molecules responsible for electronic transitions, Kohn–Sham
orbitals were constructed, Fig. S2-11. Inspection of UV-Vis spectra and constructed orbitals
revealed that bands at lower wavelength (about 230 nm in experimental spectra) are appearing
owing to relatively large energy, but relatively small spatial separation, while the higher
wavelengths are consequence of relatively smaller energy separation of corresponding HOMO
and LUMO, as well as small spatial separation (HOMO, LUMO, and LUMO+1 share same
regions, Fig. S2-11). Detailed UV-Vis characterization is provided in Supplementary material.
A possible mechanistic pathway for the formation of obtained dicoumarols is given in
Scheme 2. 4-Hydroxycoumarin (1) is firstly reacted with an aromatic aldehyde in the presence of
InCl3, as a Lewis acid which increase electrophilicity of carbonyl. After the liberation of water,
intermediate I was formed. The electron-withdrawing groups in aromatic ring of aldehydes
facilitate the addition. This is a Knoevenagel type reaction. Next, second molecule of 1 attacks
the α,β-unsaturated ketone I, via a Michael addition, to generate the intermediate II. Enolization
of the latter intermediate resulted to substituted dicoumarols 3.

Scheme 2. The reaction pathway toward the formation of dicoumarols; (i) Knoevenagel type
reaction; (ii) Michael-addition; (iii) enolization.
Preliminary biological tests, which was done by using the AAPH assay [41a,b], revealed
that the examined compounds 3e, 3h, 3b, 3d, 3f presented the high activity, ranged from 84 tο
97%, whereas 3j, 3a, 3i, 3g, 3c showed moderate (39-71%) inhibition of lipid peroxidation, Fig.
3.
% Inhibition of lipid peroxidation induced by AAPH (100 μΜ)
100
90
80
70
60
50
40
30
20
10
0
96
86
97
93
84
87
71
60
56
45
39
Fig. 3. In vitro inhibition of lipid peroxidation (LP%) at 100 μΜ for compounds 3a-3j and
reference compound (Trolox).
Data for the scavenging DPPH radical activity of compounds 3i, 3j can be found in the
literature [9], but not for dicoumarols anti-LOX activity. Since lipoxygenation occurs via a
carbon centered radical, it is to be expected that compounds with potent anti-lipid peroxidation
will exert good LOX inhibition, also. Therefore, all compounds were tested as inhibitors of
soybean lipoxygenase [41a,b], an enzyme implicated in arachidonic acid cascade and different
pathological disorders. The compounds 3b, 3e, 3h provided the highest LOX inhibition (IC50 =

52.5 µM), followed by activity of 3i (IC50 = 55.5 µM) and 3d (IC50 = 60 µM). Compounds 3c, 3f
and 3a exhibit activity in range of 76-92 µM, while compounds 3j and 3g exert the less activity
(39% and 43% at 100 µM), Table 2.
Table 2. In vitro LOX inhibition (IC50)/% at 100µM for compounds 3a-3j and reference
compound (NDGA).
Compounds IC50 (µM) or % at 100µM
92 µM
52.5 µM
76 µM
60 µM
52.5 µM
76 µM
43%
3a
3b
3c
3d
3e
3f
3g
52.5 µM
55.5 µM
39%
3h
3i
3j
NDGA
5.5 µM
2.3. Molecular modeling study
2.3.1. Bioactive conformations of compounds 3b, 3e, 3h, and 3i during the LOX-Ib
inhibition
The lack of the literature data regarding to molecular docking study of coumarin derivatives as
lipoxygenase inhibitors [39a,b] was motivated us to examine the binding affinity of synthesized
dicoumarols and their orientation at the active site of the enzyme. Bioactive conformations of the
best active LOX-Ib dicoumarol inhibitors were revealed by means of flexible ligand-rigid
enzyme (molecular docking, Fig. 4) and induced fit (Molecular Dynamics, MD, Figs. 5-8)
simulations. Both structure-based methodologies supported each other by virtue of accomplished
results, thus confirming that the obtained best docked/fitted conformations are bioactive ones.
Within the enclosed series, the highest potency during the soybean lipoxygenase inhibition was
exerted by four compounds, namely 3b (Fig. 4A), 3e (Fig. 4B), 3h (Fig. 4C), and 3i (Fig. 4D),
respectively. Thus, within the bioactive conformation of 3b, two 4-hydroxycoumarin cores and
p-chlorobenzene are joined into the pyramidal structure, with the chlorine atom, as a top of the
pyramid, positioned inside the hydrophobic pocket compiled of Thr255, Leu534, Leu537,
Leu542, and Leu750. The obtained conformation is snugly situated indoors the active site: the
3b-LOX-Ib reached its stability after 0.6 ns of induced fit simulations and remained stable
onwards (Fig. 5A). The p-chlorobenzene scaffold of 3b is involved in electrostatic interactions
with the side chain hydroxyl group of Thr255 or imidazole ring of His500; particular interactions
are interchanging during the 90% of Molecular Dynamics (MD) run time (1.2 ns) (Figs. 5C, 9A).
Moreover, the benzene moiety is for 30% of MD time involved in π-π hydrophobic interactions
with His500, whereas for the remaining time the scaffold is facing the rest of the LOX-Ib active
site hydrophobic pocked (Figs. 5C, 9A). Being positioned, the p-chlorobenzene scaffold seems to

prevent the LOX-Ib activity to catalyze its own reaction, i.e. the oxygenation of linoleic acid: the
p-chlorobenzene scaffold appears to be the spatial barrier for LOX-Ib Fe2+/Fe3+ ion, where the
HO-Fe3+ cofactor form facilitates the C13 hydroperoxidation of α-linoleate towards
9Z,11E,13S,15Z-13-hydroperoxyoctadeca-9,11,15-trienoate [43]. This can be considered as a key
factor for soybean lipoxygenase inhibition and anti-inflammatory activity of the most active
compounds. In each of the scenarios, the generation of the pyramidal structure is a consequence
of 4-hydroxycoumarin cores interactions. Hence, within the best docked bioactive conformation
of 3b, either the lactone carbonyl oxygen of the first coumarin core or the hydroxyl group at C4
of the second coumarin core establish electrostatic interactions with His495 (Figs. 5C, 9A), a
residue that is involved in the generation of Fe2+/Fe3+ octahedral complex within the enzyme’s
active site. The phenyl part of the first 4-hydroxycoumarin core also establishes weak
hydrophobic interactions with Thr552 and strong steric clash with Trp496 (Figs. 5C, 9A). On the
other hand, the hydroxyl group at C4 of the second 4-hydroxycoumarin core is narrow to a
strong hydrogen bond with the side chain amide carbonyl of Gln419 (dHB = 2.782 Å) (Figs. 5C,
9A). The orientation of the first 4-hydroxycoumarin stays conserved during time, whereas the
second coumarin core in slightly rotated in a manner that the lactone carbonyl oxygen and the
oxygen from the pyrone part of the 4-hydroxycoumarin core are potent hydrogen bond acceptors
to either Gln491 (dHB = 2.696 – 2.804 Å, 83% of MD time) or Gln693 (dHB = 2.904 – 3.206 Å,
10% of MD time) (Figs. 5C, 9A). Particular hydrogen bonds formation is facilitated by the
hydrophobic clash between the phenyl part of the second 4-hydroxycoumarin core His500 (weak
attraction) and Phe553 (strong and stable confrontation, 85% of MD time) (Figs. 5C, 9A).

Fig. 4. The bioactive conformation of 3b (A); 3e (B); 3h (C); 3i (D). The LOX-Ib amino acids
are presented in white, the LOX-Ib ribbon is colored in pink. For the clarity purpose, only polar
hydrogen atoms are presented.

Fig. 5. RMSDs (A), RMSFs (B) and Interaction diagrams (C) of 3b in complex with LOX-Ib.
Green bars present hydrogen bonds, purple bars hydrophobic interactions, pink bars ionic
interaction, blue bars water bridges

Further on, compound 3e (Figs. 4B, 6A) is of comparable activity and conformation with
3b, with the difference that p-hydroxybenzene is engaged via hydroxyl group in electrostatic
interactions with Thr255 and in hydrophobic and/or induced dipole interactions with the
hydrophobic pocket. Particular electrostatic interaction turns during the induced fit alignment
into the strong and stable hydrogen bond between the hydroxyl group of the p-hydroxybenzene
moiety and Thr255 (dHB = 2.793 – 2.912 Å, 53% of MD time) (Figs. 6C, 9B). In addition, while
the hydroxyl group of the p-hydroxybenzene scaffold is a hydrogen bond donor to the 3e-Ile835-
H2O-Fe hydrogen bond-water bridge network (dHB = 4.125 – 5.563Å, 26% of MD time) between
the anti-inflammatory agent and LOX-Ib iron ion (Figs. 6C, 9B). On this way, 3e is indirectly
associated with Fe ion, interrupting its ability to generate the coordinative bond with the
hydroperoxide derivative of linoleic acid (13-HPOD) as LOX-Ib natural substrate. On the other
hand, the lactone carbonyl of the first 4-hydroxycoumarin core of 3e is bound to hydrogen bonds
with either Gln693 (dHB = 2.517 – 2.716 Å, 73% of MD time) or Gln491 (dHB = 2.314 – 2.552 Å,
78% of MD time) (Figs. 6C, 9B). In addition, particular hydrogen bond network is completed by
the contribution of the hydroxyl group at C4 of the second 4-hydroxycoumarin core which
throughout the entire period of simulation stays in the strong hydrogen bond with the side chain
amide carbonyl of Gln419 (dHB = 2.693 – 2.954 Å, 83% of MD time) (Figs. 5C, 8B). Alike of 3b,
there is no rotation of the second 4-hydroxycoumarin core. Established hydrogen bond network
most certainly contributes to high activity of 3b. Another valuable interconnection is the
hydrogen bod between the lactone carbonyl of the second 4-hydroxycoumarin core and His500
(dHB = 2.216 – 2.454 Å, 83% of MD time) (Figs. 5C, 8B), an amino acid that is coordinatively
bonded to Fe ion. This the second level of 3e’s indirect association with Fe ion.

Fig. 6. RMSDs (A), RMSFs (B) and Interaction diagrams (C) of 3e in complex with LOX-Ib.
Green bars present hydrogen bonds, purple bars hydrophobic interactions, pink bars ionic
interaction, blue bars water bridges

The introduction of the additional m-OCH3 group causes the comparable activity but
slightly different alignment of compound 3h (Figs. 4C, 7A). The compound is, due to the
contribution of m-OCH3 moiety, still comfortably situated into the hydrophobic chamber. Thus,
the newly introduced m-OCH3 group is facing the pocket where the metal ion is stored, whereas
the p-OH group is turning from electrostatic site to a hydrogen bond acceptor for Thr255. The
established hydrogen bond, or its Thr255-H2O-3h variation, remains stable through time until
reaching the 0.6 ns of MD time. The similar observation is for the contemporaneously formed
3h-Ile835-H2O-Fe hydrogen bond-water bridge network (dHB = 4.125 – 5.563Å, 26% of MD
time) (Figs. 7C, 9C). By being involved in the precise dispersed double hydrogen bond network,
3h exerts high level of anti-inflammatory activity. However, after the 0.6th ns of simulation time,
only the hydrogen bonds with Gln693 (dHB = 2.491 – 2.638 Å, 62% of MD time) or Gln491 (dHB
= 2.443 – 2.784 Å, 55% of MD time) are formed (Figs. 7C, 9C), as previously described for 3b
and 3e, thus maintaining the activity of 3h on remarkable level. Required hydrogen bonds are not
formed in the best docked conformation, only upon the induced fitting.
Similar conformation to 3h is adopted by 3i (Figs. 4D, 8A), mainly due to the existence of
m,m’-dimethoxy-p-hydroxybenzene. The m-methoxy scaffold generates hydrophobic interactions
via methyl group with Leu537, whereas the methyl group of the m’-methoxy moiety is in steric
hindrance with Ile534. There are no hydrogen bonds with Thr255 or Ile835-H2O-Fe network
(Figs. 8C, 9D). The m’-methoxy moiety oxygen is electrostatically attracted by His500, another
coordinative bond donor for iron. Besides the expressive hydrophobic influence of m,m’-
dimethoxy-p-hydroxybenzene, compound 3i owns its high anti-inflamatory potential to already
well acredited hydrogen bonds with Gln693 (dHB = 2.372 – 2.783 Å, 84% of MD time) or
Gln491 (dHB = 2.673 – 2.841 Å, 61% of MD time) (Figs. 8C, 9D). Summarized induced fit
interactions for compounds 3b, 3e, 3h, and 3i are depicted by Fig. 9. The discussion of bioactive
conformation-activity correlation of the remaining less active compounds is reported as
Supplementary material (Bioactive conformations of compounds 3d, 3f, 3c, 3a, 3g and 3j
during the Lox-Ib inhibition section, Fig. S12).

Fig. 7. RMSDs (A), RMSFs (B) and Interaction diagrams (C) of 3h in complex with LOX-Ib.
Green bars present hydrogen bonds, purple bars hydrophobic interactions, pink bars ionic
interaction, blue bars water bridges

Fig. 8. RMSDs (A), RMSFs (B) and Interaction diagrams (C) of 3i in complex with LOX-Ib.
Green bars present hydrogen bonds, purple bars hydrophobic interactions, pink bars ionic
interaction, blue bars water bridges

Fig. 9. Two-dimensional interaction diagram of 3b (A); 3e (B); 3h (C); 3i (D) upon the induced
fit alignment. Hydrogen bonds are presented by purple arrows, hydrophobic interactions are
depicted by green lines.
3. Conclusion
Dicoumarol derivatives were synthesized fast and in excellent yields through the green InCl3
catalyzed reactions of 4-hydroxycoumarin with different aromatic aldehydes, under microwave
irradiation in water. All obtained products were characterized using NMR, IR, and UV-Vis
spectroscopy, as well as with TD-DFT, and then subjected to evaluation of their inhibition
potential of lipid peroxidation and soybean lipoxygenase. The bioactive conformations of the

best LOX-Ib dicoumarol inhibitors were obtained by means of molecular modeling study.
Structural insights based on molecular docking revealed that the most active compounds form
the compact pyramidal structure made of two 4-hydroxycoumarin cores and a central phenyl
substituent. This pyramidal structure is key reason for the formation strong hydrogen bonds and
hydrophobic interactions with the important amino acid side chains in enzyme active site. This
compact skeleton serves as a spatial barrier which is capable to prevent the C13
hydroperoxidation of enzyme substrate and its pro-inflammatory role. On the basis of the
obtained results one can conclude that compounds 3b, 3e, 3h and 3i can be candidates for the
further examination of their in vitro and in vivo anti-inflammatory activity and that may be useful
for the elucidation of the mechanism of the anti-LOX-Ib dicoumarols activity.
4. Experimental
4.1. Material and methods
Melting points were determined on a Kofler hot-stage apparatus and are uncorrected. IR spectra
were obtained with a Perkin-Elmer 1310 spectrophotometer as KBr pellets. The UV-Vis spectra
were measured at room temperature within the 200-500 nm range on the Agilent Technologies,
Cary 300 Series UV-Vis Spectrophotometer. A solution of 2.5×10-5 M of each compound was
prepared in methanol and then 2 mL of the corresponding solution was injected into the 10 mm
quartz cell and recorded spectrum. NMR spectra were recorded on an Agilent (Varian) 500/54
1
13
(500 MHz and 125 MHz) for H and C, respectively using CDCl3 and DMSO as solvent and
TMS as an internal standard. J values are reported in Hz. Mass spectra were determined on a
LCMS-2010 EV Instrument (Shimadzu) under Electrospray Ionization (ESI) conditions. The
MW experiments were performed in a Biotage (Initiator 2.0) scientific MW oven.
4.2. General procedure
The synthesis of dicoumarol derivatives under InCl3 catalysis. In a 5 ml vessel, for reactions in
MW oven, 4-hydroxycoumarin (1) (0.162 g, 1 mmol) and benzaldehyde (2a) (53 mg, 0.5 mmol)
were added in H2O (2 ml) followed by InCl3 (11mg, 0.05 mmol). The mixture was irradiated at
110°C for 20 min. After cooling, the precipitate was filtered, washed thoroughly with H2O (5×2
ml) and dried under vacuum to give 3a (0.185 g, 90% yield). All dicoumarol derivatives 3a-3j
1
were characterized with melting point, H NMR, 13C NMR, IR and UV-Vis spectra, while
compound 3a was characterized additionally with MS (ESI) spectra. The characterizations of
compound 3a is given in main part of the manuscript, while for compounds 3b-3j in
1
Supplementary material, as well as copies of H NMR, 13C NMR and UV-Vis spectra for all
compounds. It is worth pointing out that analytical and spectral data for obtained compounds are
in accordance with literature data [6b,9,19,26b,32].
3,3’-(Phenylmethylene)bis(4-hydroxy-2H-chromene-2-one) (3a) [14,19,32]: (90% yield) white
solid, m.p. 228-230˚C (EtOH-DCM), IR (KBr): 3443, 3068, 2927, 1660, 1604, 1617, 1568,
1496, 1149, 1347, 1304, 1181, 1093, 958, 902, 757, 602, 477, 461 cm-1; UV (λmax) nm: 232.7,

277.1, 303.2 nm; 1H-NMR (CDCl3, 500 MHz) δ: 6.11 (s, 1H), 7.22-7.34 (m, 5H), 7.36-7.44 (m,
4H), 7.60-7.66 (m, 2H), 8.00 (d, 1H, J= 7.5 Hz), 8.07 (d, 1H, J= 7.5 Hz), 11.29 (s, 1H,
13
exchangeable with D2O), 11.52 (s, 1H, exchangeable with D2O); C-NMR (DMSO, 125 MHz)
δ: 36.2, 116.6, 124.4, 124.9, 126.5, 126.9, 128.7, 132.8, 132.9, 135.2, 141.2, 152.5, 163.1, 164.7;
MS (ESI): m/z 413 [M+H]+.
4.3. Density functional theory calculations
The Gaussian 09 program package was used to perform all calculations [44]. The equilibrium
geometries of all compounds were calculated using the B3LYP functional in conjunction with
the 6-311+G(d,p) basis set [45]. Simulations of UV-Vis spectra were performed using TD-DFT
and structures optimized in methanol, since experimental spectra were acquired using this
solvent.
4.4. Biological experiments
4.4.1. In vitro assays
The compounds were dissolved in DMSO. All tests were undertaken on three replicates, the
results were averaged and standard deviation was considered to be less than 10%.
1) Antilipid peroxidation. Production of conjugated diene hydroperoxide by oxidation of linoleic
acid in an aqueous dispersion is monitored at 234 nm. AAPH is used as a free radical initiator.
Linoleic acid sodium salt solution (10 ml of 16 mM) was added to the UV cuvette containing
0.93 ml of 0.05 M phosphate buffer, pH 7.4 pre-thermostated at 37˚C. The oxidation reaction
was initiated at 37˚C under air by the addition of 50 ml of 40 mM AAPH solution. Oxidation
was carried out in the presence of aliquots (10 ml) in the assay without antioxidant. Lipid
oxidation was measured in the presence of the same level of DMSO. The rate of oxidation at
37˚C was monitored by recording the increase in absorption at 234 nm caused by conjugated
diene hydroperoxides and compared with the appropriate standard Trolox [40 a,b].
2) Lipoxygenase inhibition. The tested compounds were incubated at room temperature with
sodium linoleate (100 μl) and 200 μl of enzyme LOX type Ib solution (1/9 x 10-4w/v in saline)
and buffer tris pH 9. The conversion of sodium linoleate to 13- hydroperoxylinoleic acid at 234
nm was recorded and compared with the appropriate standard inhibitor NDGA [40 a,b]. Several
concentrations were used in order to determine IC50 values of the more potent compounds.
4.3. Molecular modeling experiments
4.3.1. Soybean lipoxygenase crystal structure preparation
To the best of our knowledge, there are no crystal structures containing co-crystallized structures
of compounds investigated in this study with soybean lipoxygenase. Therefore, molecular
docking was performed on soybean lipoxygenase-Ib (PDB ID: 5T5V). Initially, protein was

prepared for molecular modelling after being loaded into the UCSF Chimera v1.10.1 software
[46] for Linux 64 bit architecture and visually inspected. Protein was than energy minimized as
follows: through the leap module of the Amber suite [47] they were solvated with water
molecules (TIP3 model, SOLVATEOCT Leap command) in a box extending 10 Å in all
directions, neutralized with either Na+ or Cl- ions, and refined by a single point minimization
using the Sander module with maximum 1000 steps of the steepest-descent energy minimization
followed by maximum 4000 steps of conjugate-gradient energy minimization, with a non-bonded
cut off of 5 Å.
4.3.2. Generation of dicoumarol derivatives structures
All of the dicoumarol derivatives structures were modeled by applying the Chemaxon's msketch
module [48] by means of molecular mechanics’ optimization upon which the hydrogen atoms,
appropriate for pH 7.4, were assigned.
4.3.3. Molecular docking protocol
The active site definition was performed upon the 5T5V crystal had been aligned to soybean
lipoxygenase-3 co-crystallized with 13(s)-hydroperoxy-9(Z),11(E)-octadecadienoic acid (PDB
ID: 1IK3) [49], by means of UCSF Chimera MatchMaker module. The protein part of 1IK3
crystal was prepared similarly to the 5T5V one. Thus, an 1IK3 inhibitor was extracted from the
complex after which either protein or ligand were improved by assigning the hydrogen atoms at
pH of 7.4 and Amber parameters [47] using Antechamber semi-empirical QM method. Upon the
thorough examination, the A chain of each complex was retained for further manipulation and
aligned to obtain common relative coordinates.
The prepared structures of 5T5V and 1IK3 were imported into AutoDockTools graphical
user interface. Nonpolar hydrogen atoms were removed while Gasteiger charges and solvent
parameters were added. All of the tested compounds were used as ligands for docking. The rigid
root and rotatable bonds were defined using AutoDockTools. The docking on 5T5V was
performed with AutoDock 4.2 [50] by applying the cuboid docking grid coordinates provided
from 1IK3 complex as follows: the xyz coordinates (in Ångströms) for the computation were
Xmin/Xmax = 8.328/31.096, Ymin/Ymax = -15.291/15.029, Zmin/Zmax = 3.579/31.555; the
coordinates setup was performed in a manner to embrace the minimized inhibitor spanning 10 Å
in all three dimensions. The Lamarckian Genetic Algorithm was used to generate orientations or
conformations of ligands within the binding site. The procedure of the global optimization
started with a sample of 200 randomly positioned individuals, a maximum of 1.0 x 106 energy
evaluations and a maximum of 27 000 generations. A total of 100 runs were performed with
RMS Cluster Tolerance of 0.5 Å.
4.3.4. Molecular dynamics protocol

The merged ligand-Lox-Ib complexes were used to perform explicit solvent molecular dynamics
simulations. The parallelized Desmond Molecular Dynamics System and associated analysis
tools, available within the Schrödinger suite 2009 [51], were used for this purpose. The models
were prepared using the ‘system builder’ utility. The OPLS_2005 force field parameters were
assigned for all the simulation systems. Each inhibitors-enzyme complex was placed in the
centre of a orthorhombic box ensuring 10 Å solvent buffers between the solute and the boundary
of simulation box in each direction. The TIP3 model was used to describe the water molecules.
More, Na+ and Cl- ions were added at physiological concentration of 0.15 M to neutralize. The
model systems were relaxed using to 0.5 Å using the minimization protocol and subjected to a
production phase, using the NPT ensemble and periodic boundary conditions for a period of 1.2
ns. The pressure control was used to maintain the pressure of the cell at 1.013 bar using the
isotropic coupling method. The Nose-Hoover thermostat method was used to control the
temperature at 310.15 K. Heavy atom-hydrogen covalent bonds were constrained using the
SHAKE algorithm which allowed a 2-fs integration step to be used. The integration of the
equation of motion as implemented according to the RESPA multiple time step scheme was used
in the simulations. Long-range electrostatic forces were computed using the smooth Particle-
Mesh Ewald (PME) method. The cutoff distance for calculating short-range electrostatics and
Lennard-Jones interaction was set to 9.0 Å. The trajectories and the energies were recorded at
every 4.8 ps, respectively. The simulation quality analysis tool was used to analyze the
trajectories obtaining RMSD and RMSF values, hydrogen bond distances, angles, and van der
Waals interactions over the simulation trajectories.
Acknowledgments
This work was supported by the Ministry of Education, Science and Technological Development
of the Republic of Serbia (Projects No. 172016 and III43004).
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Graphical abstract

Highlights
• Dicoumarol derivatives were synthetized in green InCl3 catalyzed reaction.
• Structures of all obtained products were elucidated by spectroscopic techniques.
• Dicoumarols were subjected to evaluation of their in vitro lipid peroxidation and
soybean lipoxygenase inhibition activities.
• The bioactive conformations of the best LOX inhibitors were obtained by means
of molecular docking and molecular dynamics.