Discovery two potent and new inhibitors of 15-lipoxygenase: (E)-3-((3,4-dihydroxybenzylidene) amino)-7-hydroxy-2H-chromen-2-one and (E)-O-(4-(((7-hydroxy-2-oxo-2H-chromen-3-yl) imino)methine) phenyl)dimethylcarbamothioate

Article abstract of DOI:10.1007/s00044-017-1968-9

The mechanisms of action and structural determinants of lipoxygenases inhibitors have been explored on several occasions, but many questions remain unanswered, especially about the differences of the inhibition mechanisms and their effect on the selectivi

Full text of DOI:10.1007/s00044-017-1968-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1968-9
ORIGINAL RESEARCH
Discovery two potent and new inhibitors of 15-lipoxygenase: (E)-3-
((3,4-dihydroxybenzylidene) amino)-7-hydroxy-2H-chromen-2-one
and (E)-O-(4-(((7-hydroxy-2-oxo-2H-chromen-3-yl) imino)methine)
phenyl)dimethylcarbamothioate
Carolina Nuñez1 Nicole Morales2 Olimpo García-Beltran3,4 Carolina Mascayano1
Angelica Fierro2
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Received: 20 February 2017 / Accepted: 19 June 2017
© Springer Science+Business Media, LLC 2017
Abstract The mechanisms of action and structural deter-
minants of lipoxygenases inhibitors have been explored on
several occasions, but many questions remain unanswered,
especially about the differences of the inhibition mechan-
isms and their effect on the selectivity of lipoxygenases
isoenzymes. Thus, REDOX mechanism has been proposed
in this research to clarify the lipoxygenases inhibition by
coumarins derivates on 15-sLOX. A series of fifteen cou-
marin derivatives were synthetized and evaluated as 15-
lipoxygenase inhibitors. The results showed that some
molecules had submicromolar activities and compete with
the substrate as we observed by kinetic studies. The most
relevant and interesting result was found for compound 6
who showed an inhibitory activity comparable to nordihy-
droguaiaretic acid a potent and REDOX inhibitor of
lipoxygenases (0.17 and 0.29 μM, respectively). Finally, the
docking and molecular dynamics studies showed that the
better ligands were accommodated into the binding site
being related with those obtained biological data. In addi-
tion, our findings contribute at the understanding of inhi-
bitor structural requirements and elucidate the inhibition
mechanism of cumarin derivatives on 15-sLOX. Thus, we
point to new parameters for the future design of new ligands
with potential therapeutic utility where are involved the
lipoxygenases enzymes.
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Keywords 15-Lipoxygenase Coumarin derivatives
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Kinetic assay Pseudoperoxidase SAR study
Introduction
The lipoxygenases (LOX) are enzymes that belong to the
non-heme iron-containing dioxygenase family (Ivanov et al.
2010). These enzymes catalyze the stereo- and the regios-
pecific introduction of molecular oxygen into poly-
unsaturated fatty acids such as linoleic acid (LA) and
arachidonic acid (AA) (Brash et al. 2012). The LOX can be
classified according to the position, where the poly-
unsaturated fatty acid was oxygenated, for example, 5, 8,
12, and 15-LOX (Newcomer and Brash 2015).
The AA is an essential precursor of many eicosanoids or
lipid mediators, such as prostaglandins (PG), lipoxins (LX),
leukotrienes (LT), hydroxylated polyunsaturated fatty acids
(e.g., HETE), thromboxane (TX) and other compounds
resulting from AA metabolism. However, deregulation of
these enzymes can trigger serious consequences for human
health due to their involvement in many diseases (Needle-
man et al. 1986), as bronchial asthma (Chauhan and
Ducharme 2012), atherosclerosis (Wuest et al. 2014),
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1968-9) contains supplementary material,
which is available to authorized users.
* Carolina Mascayano
carolina.mascayano@usach.cl
1
Facultad de Química y Biología, Departamento de Ciencias del
Ambiente, Laboratorio de Simulación Molecular y Diseño
Racional de Fármacos, Universidad de Santiago de Chile,
Santiago, Chile
2
Department of Organic Chemistry, Faculty of Chemistry,
Pontificia Universidad Católica de Chile, Casilla 306, Santiago
6094411, Chile
3
Facultad de Ciencias Naturales y Matemáticas, Universidad de
Ibagué, Carrera 22 Calle 67, Ibagué 730001, Colombia
4
Department of Chemistry, Faculty of Sciences, University of
Chile, Santiago 7800024, Chile

Med Chem Res
Table 1 Derivatives of (E)-3-(benzylideneamino)-7-hydroxy-2H-
chromen-2-one
Alzheimer (Giannopoulos et al. 2013), arthritis (Krönke
et al. 2009), and certain types of cancer such as: colorectal,
prostate and lung (Klil-Drori and Ariel 2013).
Some crystal structures of LOX with good resolution
have been elucidated in the last years: i.e., Soybean Lipox-
ygenase-1(PDB ID: 1F8N) (Tomchick et al. 2001), soybean
lipoxygenase-3 (1HU9) (Skrzypczak-Jankun et al. 2003),
rabbit reticulocyte 15-lipoxygenase (1LOX) (Gillmor et al.
1997), coral 8-R-lipoxygenase (2FNQ) (Oldham et al. 2005)
and human 5-lipoxygenase (3O8Y) (Gilbert et al. 2011).
All structures show a hydrophobic zone where the
fatty acid binds to non-heme iron, which has octahedral
coordination geometry; in general, for all LOX’s His and
Ile residues are found in the active site. Mutations of
these residues, for example, in rabbit reticulocyte
15-lipoxygenase (H361, H366, H541, and I663) cause the
complete loss of activity, in particular 15-LOX consists of a
C-terminal catalytic domain and a N-terminal C2-like β-
barrel domain. The catalytic domain contains a non-heme
iron in the active site that acts as an electron acceptor or
donor during catalysis. During enzyme activation, for
example by lipid hydroperoxides, the iron is oxidized from
the ferrous state Fe(II) into the ferric Fe(III) form in the
active enzyme (Kulkarni et al. 2002).
The search of selective inhibitors of these enzymes is an
important target for research (Ribeiro et al. 2014; Armstrong
et al. 2014; Sarveswaran et al. 2015). Currently, there is a
commercial inhibitor of 5-hLOX called zileuton, which is
used in the treatment of asthma (Bell et al. 1992), but there
are still not selective inhibitors for 12- and 15-hLOX. For this
reason, the study of inhibitors of these isoforms becomes
important at present. It has been reported that some important
secondary metabolites as flavonoids or isoflavonoids have
the ability to inhibit different isoforms of lipoxygenase
(Ribeiro et al. 2014). Due to similarities in structure and
function, the coumarins are presented as good candidates for
LOX inhibitors. There are not many examples of coumarin
derivatives as LOX inhibitors (Singh and Pathak 2016; Hoult
and Paya 1996; Bansal et al. 2013), but some natural cou-
marin obtained of Haplopappus multiflorus showed to
exhibit activity as 15-LOX inhibitors, with over 50% inhi-
bition of the AA lipoperoxidation (Torres et al. 2013) Also,
there is evidence that compounds bearing a catechol group
such as daphnetin and esculetin block the formation of 5-
LOX products with IC50 values between 1.46 and 10 μM
(Yoshiyuki et al. 1985) However, not only catechol cou-
marins inhibit LOX. The 6-(3-carboxybut-2-enyl)-7-hydro-
xycoumarin, with only one hydroxyl group, was shown to be
a potent inhibitor of 5-LOX (IC50 36.2 μM) (Kotali et al.
2016; Hadjipavlou-Litina et al. 2007). Recently, 7-substituted
coumarins were synthetized and evaluated in-vitro against 5-
LOX obtaining interesting results (Srivastava et al. 2016).
Compounds
R1
R2
R3
R4
1
2
3
4
5
6
H
H
H
OH
OH
H
H
OH
H
H
OH
OH
OCH3
H
H
H
OCH3
OCH3
H
OCH3
H
H
OC(S)N(CH3)2
H
We hypothesized that the new molecules inhibiting LOX
have to affect the non-polar binding cavity through elec-
tronic and steric interactions, combining the structural
properties of the coumarin scaffold and of an extended
aromatic segment linked by an imino group.
Based on these observations, and in search of new
inhibitors toward 12- and 15-LOX, fifteen coumarin deri-
vatives were synthesized and their inhibition determined on
soybean 15-lipoxygenase (15-sLOX). Their structures are
given in Tables 1 and 2.
Materials and methods
Melting points were determined on a Reichert-Jung Galen
III hot-plate apparatus and were not corrected. 1H- and 13C-
nuclear magnetic resonance (NMR) spectra were recorded
on a Bruker Avance 400 spectrometer operating at 400
MHz, using the solvent or the TMS (tetramethylsilane)
signal as an internal standard. The ESIMS of compounds
were recorded on a Waters/Micromass Q-TOF micro high-
resolution hybrid quadrupole orthogonal time-of-flight mass
spectrometer with a constant nebulizer temperature of
100 °C. The experiments were carried out in positive ion
mode, and the cone and extractor potentials were set at 10
and 3.0 V, respectively, with a scan range of m/z 100–600.
The samples were directly infused into the ESI source, via a
syringe pump, at flow rates of 5 μL min–1, through the
instrument’s injection valve. All chemical shifts are reported
in the standard δ notation of parts per million.

Med Chem Res
Table 2 Derivatives of
coumarins with 2-amino-2-
hydroxymethyl-propane-1,3-diol
Compound R1
R2
R3
R4
7
8
CH2C(O)NHC(CH2OH)3
OH
H
CH2C(O)OCH2C[(NH2)(CH2OH)2] OH
H
9
C(O)NHC(CH2OH)3
H
H
H
H
H
H
H
OH
H
10
11
12
13
14
15
C(O)NHC(CH2OH)3
C(O)NHC(CH2OH)3
C(O)NHC(CH2OH)3
C(O)NHC(CH2OH)3
C(O)NHC(CH2OH)3
C(O)NHC(CH2OH)3
N(C2H5)2
H
O(CH2)6Br
H
OCH3
H
H
OH
–CH2CH2NCH2CH2−
OH
OH
13.36 (s, 1H, O–H– –O=C), 10.57 (br, 1H, O–H), 10.37
(br, 1H, O–H), 9.02 (s, 1H, –N=CH–Ar), 7.93 (s, 1H,
=C–H), 7.52 (d, 1H, J = 8.6 Hz), 7.39 (d, 1H, J = 8.6 Hz),
6.81 (dd, 1H, J = 8.0, 2.0 Hz), 6.75 (s, 1H), 6.40 (dd, 1H, J
= 8.0, 2.0 Hz), 6.28 (d, 1H, J = 2.0 Hz). HRMS: [M +
Na]+, C16H11NNaO5, found: 320.0555; calcd.: 320.0535.
General procedure for the preparation of (E)-7-
hydroxy-3-(benzylidenimino)-2H-chromen-2-ones 1–6
3-Amino-7-hydroxy-2H-chromen-2-one (0.56 g, 31 mmol)
and the appropriate benzaldehyde (31 mmol) were dissolved
in absolute EtOH (10 mL) and refluxed for 2 h. The pre-
cipitate formed was collected and washed with hot EtOH. In
this way, the following (E)-7-hydroxy-3-(benzylidenimino)-
2H-chromen-2-ones 1-6 were prepared (Li et al. 2009;
García-Beltrán et al. 2014; García-Beltrán et al. 2012a, b;
García-Beltrán et al. 2015a, b):
(E)-3-((3,4-Dihydroxybenzylidene)amino)-7-hydroxy-2H-
chromen-2-one (3)
Reaction with 3,4-dihydroxybenzaldehyde (31 mmol) gave
3 as a red solid (0.93 g, 92%) mp > 320 °C. 1H NMR
(DMSO-d6): δ 10.46 (br, 1H, O–H), 10.17 (br, 1H, O–H),
9.87 (br, 1H, O–H), 8.80 (s, 1H, –N=CH–Ar), 7.72 (s, 1H,
=C–H), 7.52 (d, 1H, Ar–H, J = 8.0 Hz) 7.49 (s, 1H, Ar–H),
7.30 (d, 1H, Ar–H, J = 8.0 Hz), 6.89 (d, 1H, Ar–H, J = 8.0
Hz), 6.80 (d, 1H, Ar–H, J = 8.0 Hz), 6.74 (s, 1H, OCH3).
HRMS: [M + Na]+, C16H11NNaO5, found: 320.0547;
calcd.: 320.0535.
(E)-7-Hydroxy-3-((2-hydroxybenzylidene)amino)-2H-
chromen-2-one (1)
Reaction with 2-hydroxybenzaldehyde gave 1 (0.85 g, 80
%) as a pale yellow solid, mp 248–250 °C; 1H NMR
(DMSO-d6): δ 12.89 (s, 1H, O–H– –O=C), 10.62 (br, 1H,
O–H), 9.21 (s, 1H, N=CH–Ar), 8.03 (s, 1H,=C–H) 7.60 (d,
1H, Ar–H, J = 8.6Hz), 7.55 (d, 1H, Ar–H, J = 8,6 Hz), 7.40
(dd, 1H, Ar–H, J = 8.6, 2.0 Hz), 6.96 (m, 2H, Ar–H), 6.82
(d, 1H, Ar–H, J = 8.6 Hz), 6.76 (s, 1H, Ar–H); 13C NMR
(DMSO-d6): δ 102.5, 112.1, 114.2, 117.2, 119.7, 119.8,
129.6, 130.4, 132.2, 132.7, 134, 153.3, 158.5, 160.8, 161.6,
164.4. HRMS: [M + Na]+, C16H11NNaO4, found:
304.0592; calcd.: 304.0580.
(E)-3-((2,3-Dimethoxybenzylidene)amino)-7-hydroxy-2H-
chromen-2-one (4)
Reaction with 2,3-dimethoxybenzaldehyde (31 mmol) gave
1
4 as a yellow solid; (0.93 g 92 %). mp 236–238 °C; H
NMR (DMSO-d6): δ 10,63 (br, 1H, O–H), 9.35 (s, 1H,
N=C–Ar), 7.11 (s, 1H,=C–H), 7.66 (m, 2H, Ar–H) 7.30
(m, 3, Ar–H), 6.90 (d, 1H, Ar–H, J = 8.2 Hz), 6.84 (s, 1H,
Ar–H), 6.74 (m, 1H, Ar–H), 3.92 (s, 3H, OCH3), 3.95 (s,
3H, OCH3). HRMS: [M + Na]+, C18H15NO5Na, found:
348.0121; calcd.: 348.0848.
(E)-3-((2,4-Dihydroxybenzylidene)amino)-7-hydroxy-2H-
chromen-2-one (2)
Reaction with 2,4-dihydroxybenzaldehyde (31 mmol) gave
1
2 (0.91 g, 98%) as a red solid, mp > 320 °C. H NMR, δ

Med Chem Res
(E)-3-((2,5-Dimethoxybenzylidene)amino)-7-hydroxy-2H-
chromen-2-one (5)
O–H), 7.63 (d, 1H, Ar–H, J = 8.8 Hz), 6.77 (dd, 1H, Ar–H,
J = 8.8, 2.0 Hz), 6.71 (d, 1H, Ar–H, J = 2.0 Hz), 6.30 (s,
1H,=C–H), 4.79 (br, 2H, –NH2–), 4.10 (s, 2H, –CH2), 3.78
(s, 2H, –CH2–), 3.43–3.30 (m, 4H, –CH2–); 13C NMR
(DMSO-d6): δ 31.2, 64.2, 71.1, 77.2, 102.7, 111.6, 113.4,
127.2, 151.2, 155.4, 160.1, 161.7, 163.1. HRMS: [M +
H]+, C15H17NNaO7, found: 324.0972; calcd: 324.1005.
Reaction with 2,5-dimethoxybenzaldehyde (31 mmol) gave
5 (0.5 g, 89%) as a yellow solid, mp 206–208 °C. 1H NMR
(DMSO-d6): δ 10.53 (br, 1H, O–H), 9.31 (s, 1H, N=C–Ar),
7.81 (s, 1H,=C–H), 7.54 (d, 1H, Ar–H, J = 8.2 Hz) 7.50 (s,
2H, Ar–H), 7.09 (s, 2H, Ar–H), 6.80 (d, 1H, Ar–H, J = 8.2
Hz), 6.74 (s, 1H, Ar–H), 3.92 (s, 3H, OCH3), 3.84 (s, 3H,
OCH3). HRMS: [M + Na]+, C18H15NO5Na, found:
348.1154; calcd.: 348.0848.
N-(1,3-Dihydroxy-2-(hydroxymethyl) propan-2-yl)-7-
hydroxy-2-oxo-2H-chromene-3-carboxamide (9)
It is synthesized from a solution of 7-hydroxy-2-oxo-2H-1-
benzopiran-3-ethyl ester (1.0 g, 4.2 mmol) and 2-amino-2-
(hydroxymethyl) propane-1, 3-diol (tris) (0517 g, 5 mmol)
in 20 ml of EtOH was stirred and refluxed for 20 h. After
that time a precipitate is filtered and washed with hot EtOH,
yielding the compound known as light yellow (9). This was
(E)-O-(4-(((7-hydroxy-2-oxo-2H-chromen-3-yl) imino)
methine) phenyl) dimethylcarbamothioate (6)
3-Amino-7-hydroxy-2H-chromen-2-one (0.56 g, 31 mmol)
and O-(4-formylphenyl) dimethylcarbamothioate (0.65 g,
31 mmol) gave 6 (0.85 g, 80 %) as a yellow solid, m.p.
240–242 °C; 1H NMR (DMSO-d6): δ 8.59 (s, 1H,
N=C–Ar), 7.94–7.92 (d, 1H, Ar–H, J = 8.6 Hz), 7.78 (s,
1H,=C–H), 7.56–7.54 (d, 1H, Ar–H, J = 8.0 Hz),
7.22–7.20 (d, 1H, Ar–H, J = 8.0 Hz), 6.81 (dd, 1H, Ar–H,
J = 8.0, 2.0 Hz), 6.75(d, 1H, Ar–H, J = 2.0 Hz), 3.36 (s,
3H, N(CH3)2), 3.32 (s, 3H, N(CH3)2); 13 C NMR (DMSO-
d6): δ 39.1, 43.3, 102.3, 112.4, 114.0, 123.9, 130.0, 130.1,
132.3, 132.4, 133.8, 154.0, 156.7, 158.5, 161.1, 161.8,
186.3. HRMS: [M+H]+, C19H16N2O4S, found: 369.0895;
calcd.: 369.4143.
1
a return of (0.98 g, 75.5%). mp 252–254; H NMR (400
MHz, DMSO-d6): 8.98 (s, 1H), 8.74 (s, 1H), 7.72 (d, 1H, J
= 8.6), 6.79 (dd, 1H, J = 8.6, 2,0), 6.684 (d, 1H, J = 2,0),
7.435 (t, 1H), 4.78 (br, 3H), 3.63 (s, 6H); 13C NMR (100
MHz, DMSO-d6): δ 60.8, 62.7, 102.4, 110.6, 112.5, 115.9,
132.3, 148.35, 157.3, 161.88, 162.6, 166.8. HRMS: [M +
Na]+, C14H15NNaO7, found: 332.0746; calcd: 332.0746.
7-(Diethylamino)-N-(1,3-dihydroxy-2-(hydroxymethyl)
propan-2-yl)-2-oxo-2H-chromene-3-carboxamide (10)
A solution of ethyl 7-(diethylamino)-2-oxo-2H-chromene-
3-carboxylate (3.0 g, 10.37 mmol) and 2-amino-2-(hydro-
xymethyl)propane-1,3-diol (TRIS) (1.33 g, 11.0 mmol) in
30 mL of EtOH was stirred and refluxed for 20 h. The
precipitate formed was filtered and washed with hot EtOH,
yielding the compound (10) as light yellow crystals (2.83 g,
Synthesis of derivatives of coumarins with 2-amino-2-
hydroxymethyl-propane-1,3-diol (7 and 8)
1.0 g of 2-(7-hydroxy-2-oxo-2H-chromen-4-yl) acetate (4
mmol) and 0.6 g of 2-amino-2- (hydroxymethyl) propane-
1,3-diol (“tris”) (5 mmol) were dissolved in 30 mL of etha-
nol and the mixture was heated at reflux for 20 h (Huff et al.
1999; García-Beltrán et al. 2012a; García-Beltrán et al.
2013; Aliaga et al. 2014; Kostova and Saso 2013). The
product was purified by column chromatography using as
mobile phase DCM / MeOH 98:2 to obtain compound 7:
1
74.9%); mp 159–161 °C; H NMR (400 MHz, DMSO-d6):
δ 9.02 (s, 1H), 8.66 (s, 1H), 7.66 (d, J = 8.0, 1H), 6.79 (dd,
J = 8.6, 1.0, 1H), 6.62 (d, J = 1.0, 1H), 3.63 (s, 6H), 3.47
(q, J = 8.0, 4H), 1.13 (t, J = 8.0, 6H); 13C NMR (100 MHz,
DMSO-d6): δ 12.7, 44.7, 57.0, 63.0, 96.2, 108.0, 109.9,
110.5, 131.9, 148.0, 152.8, 157.7, 162.1, 162.9. HRMS:
[M]+, C18H28N2NaO6, found: 364.9670; calcd: 364.3930.
1
0.9 g, white solid, 70%, mp 145–147 °C; H NMR (400
MHz, DMSO-d6): δ 7.65 (s, 1H), 7.62 (d, 1H, J = 8.6), 6.75
(dd, 1H, J = 8.6, 2.0), 6.69 (d, 1H, J = 2.0), 6.17 (s, 1H),
4.70 (br, 3H), 3.70 (s, 2H), 3.55 (s, 6H); 13C NMR (100
MHz, DMSO-d6): δ 60.8, 62.9, 102.7, 111.7, 111.8, 113.5,
127.3, 152.1, 155.5, 160.8, 162.0, 169.2. HRMS: [M +
Na]+, C15H17NNaO7, found: 346.0897; calcd: 346.0903.
When the reaction is extended for an additional 48 h
allowing the almost complete evaporation of the solvent, a
mixture of compounds 7 and 8 was obtained and fractio-
nated by column chromatography using as mobile phase
DCM/MeOH 98:2, compound 8. 0.35 g, white solid, 27%,
7-((6-bromohexyl) oxy)-N-(1,3-dihydroxy-2-
(hydroxymethyl)propan-2-yl)-2-oxo-2H-chromene-3-
carboxamide (11)
A mixture of 9 (1.5 g, 4.8 mmol), K2CO3 (2.07 g, 15 mmol),
and 1,6-dibromohexane (10.98 g, 45 mmol), was dissolved
in DMF (100 mL). The solution was stirred for 4 h at 60 °C
and then diluted with water (100 mL). An organic layer that
formed was extracted with methylene chloride, and the
extract was washed with water, dried, and concentrated to
dryness under reduced pressure. The residue was purified by
1
m.p. 240–242 °C; H NMR (DMSO-d6): δ 10.58 (br, 1H,

Med Chem Res
1
CC using as mobile phase Hex:AcOEt, affording crystals
(1.97 g, 78.8 %): mp 176–178. 1H NMR (200 MHz,
CDCl3); δ 8.97 (s, 1H), 8.86 (s, 1H, H-4), 7.89 (d, 1H, J =
8.0 Hz), 7.12 (d, 1H, J = 2.0 Hz), 7,03 (dd, 1H, J = 8.0, 1.0
Hz), 4.85 (t, 2H, J = 8.0 Hz), 3.66 (m, 2H), 3.54 (t, 2H, J =
8.0), 3.35 (s, 6H), 1.78 (m, 2H), 1.45 (m, 2H). HRMS: [M
+ Na]+, C20H26BrNNaO7, found: 494.1125; calcd:
494.0790.
(14) as yellow solid (1.1 g, 78.1 %); mp 172–174 °C; H
NMR (400 MHz, DMSO-d6): δ 7.98 (s, 1H), 6.56 (s, 1H),
4.79 (s, 3H), 3.55 (s, 6H), 3.14 (s, 4H), 2.46 (s, 6H), 1.08 (s,
4H); 13C NMR (100 MHz, DMSO-d6): δ 20.6, 21.4, 22.4,
27.3, 49.5, 49.8, 61.8, 64.8, 106.5, 107.8, 111.3, 129.7,
147.2, 160.3, 167.9. HRMS: [M + Na]+, C20H24N2NaO6,
found: 411.1552; calcd: 411.1532.
N-(1,3-dihydroxy-2-(hydroxymethyl) propan-2-yl)-7-
N-(1,3-dihydroxy-2-(hydroxymethyl) propan-2-yl)-7,8-
methoxy-2-oxo-2H-chromene-3-carboxamide (12)
dihydroxy-2-oxo-2H-chromene-3-carboxamide (15)
A solution of ethyl 7-methoxy-2-oxo-2H-chromene-3-car-
boxylate (1.0 g, 4.0 mmol) and 2-amino-2-(hydro-
xymethyl)-propane-1,3-diol (TRIS) (0.517 g, 5 mmol)
in 20 mL of EtOH was stirred and refluxed for 20 h. The
precipitate formed was filtered and washed with hot
EtOH, yielding the compound (12) as white solid (0.95 g,
A solution of ethyl 7,8-dihydroxy-2-oxo-2H-chromene-3-
carboxylate (0.75 g, 3.0 mmol) and 2-amino-2-(hydro-
xymethyl)-propane-1,3-diol (TRIS) (0.4 g, 4.0 mmol) in 20
mL of EtOH was stirred and refluxed for 20 h. The pre-
cipitate formed was filtered and washed with hot EtOH,
yielding the compound (15) as light yellow solid (0.92 g,
94.4 %); mp 274–276 °C; 1H NMR (400 MHz, DMSO-d6):
δ 9.14 (s, 1H), 8.50 (s, 1H), 7.09 (d, J = 8.0, 1H), 6.55 (d, J
= 8.0, 1H), 3.62 (s, 6H); 13C NMR (100 MHz, DMSO-d6):
δ 60.2, 60.9, 61.1, 62.6, 106.8, 108.4, 116.2, 123.4, 133.7,
142.7, 148.2, 162.7, 164.2, 164.6. HRMS: [M + Na]+,
C14H15NNaO8, found: 411.1552; calcd: 411.1532.
1
70 %); mp 139–141 °C; H NMR (400 MHz, DMSO-d6):
δ 8.98 (s, 1H), 8.84 (s, 1H), 7.88 (d, J = 8.0, 1H), 7.11
(d, J = 1.0, 1H), 7.03 (dd, J = 8.0, 1.0, 1H), 4.85 (t, J = 8.0,
3H), 3.89 (s, 3H), 3.68 (m, J = 8.0, 6H); 13C NMR (100
MHz, DMSO-d6): δ 56.6, 60.6, 62.7, 100.6, 112.4, 114.1,
115.3, 131.9, 148.2, 156.6, 161.4, 161.8, 164.8. HRMS:
[M + Na]+, C15H17NNaO7, found: 346.1005; calcd:
346.0903.
Kinetics and IC50 assay
N-(1,3-dihydroxy-2-(hydroxymethyl) propan-2-yl)-8-
hydroxy-2-oxo-2H-chromene-3-carboxamide (13)
All compounds were screened for activity as soybean 15-
lipoxygenase (15-sLOX) inhibitors. In all cases the pre-
liminary evaluation was done at 1 μmol/mL. The inhibition
of enzyme activity was determined following the formation
of the conjugated diene product at 234 nm (ε = 25,000
M−1cm−1) using a Perkin-Elmer Lambda 25 UV/Vis
spectrophotometer, relative to control rates measured for the
carrier solvent DMSO, as previously published. All reac-
tions were done in a volume of 2 mL constantly stirred
using a magnetic stir bar at room temperature (23 °C). The
15-sLOX specific activity was 100,000 U/mg, (Cayman
Chemical Catalog Number 60712). Reactions were carried
out in 25 mM HEPES (pH 7.4), 0.01% Triton X-100 and 10
µM AA. The concentration of AA was quantitatively
determined by allowing the enzymatic reaction to reach
completion. IC50 values were obtained by determining the
enzymatic rate at various inhibitor concentrations and
plotted against inhibitor concentration, followed by a
hyperbolic saturation curve fit. The data used for the
saturation curves were obtained in duplicate or triplicate,
depending on their quality. Additionally, the Ki and inhi-
bitor behavior of the most potent compound (6) was
determined against 15-sLOX (Holman et al. 1998;
Deschamps et al. 2007; Carroll et al. 2001; Wecksler et al.
2009a, b).
A solution of ethyl 8-hydroxy-2-oxo-2H-chromene-3-car-
boxylate (1.0 g 4.2 mmol) and 2-amino-2-(hydroxymethyl)
propane-1,3-diol (TRIS) (0.517 g, 5 mmol) in 20 mL of
EtOH was stirred and refluxed for 20 h. The precipitate
formed was filtered and washed with hot EtOH, yielding the
compound (13) as whitish solid (0.95 g, 70 %); mp
245–247 °C; 1H NMR (400 MHz, DMSO-d6): δ 9.07 (s,
1H), 8.67 (s, 1H), 7.30 (m, 1H), 7.20 (m, 2H), 3.63 (s, 6H);
13C NMR (100 MHz, DMSO-d6): δ 63.9, 69.2, 112.1,
117.5, 118.2, 121.2, 122.9, 127.5, 143.9, 144.7, 160.1,
162.7. HRMS: [M + Na]+, C14H15NNaO7, found:
332.0755; calcd: 332.0746.
N-(1,3-dihydroxy-2-(hydroxymethyl) propan-2-yl)-11-oxo-
2,3,5,6,7,11-hexahydro-1H-pyrano[2,3-f]pyrido[3,2,1-ij]
quinoline-10-carboxamide (14)
A solution of ethyl 11-oxo-2,3,5,6,7,11-hexahydro-1H-
pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxylate (1.0
g, 3.2 mmol) and 2-amino-2-(hydroxymethyl)propane-1,3-
diol (TRIS) (0.48 g, 4.0 mmol) in 20 mL of EtOH was
stirred and refluxed for 20 h. The precipitate formed was
filtered and washed with hot EtOH, yielding the compound

Med Chem Res
Pseudoperoxidase activity assay
Computational studies
The reductive properties of the compounds were determined
by monitoring the pseudo-peroxidase activity of 15-sLOX
in the presence of the inhibitor and 13-HPODE. Activity
was characterized by direct measurement of the product
degradation following the decrease of absorbance at 234 nm
using a Perkin-Elmer Lambda 25 UV/Vis spectrometer (25
mM HEPES (pH 7.4), 0.01% Triton X-100, and 10 μM
13-HPODE). All reactions were performed in 2 mL of
buffer with constant magnetic stirring (22 °C) (Riendeau
et al. 1991; Hoobler et al. 2013).
Reaction was initiated by addition of 10 μM inhibitor
(1:1 ratio to product), and a positive result for activity
reflected a loss of more than 40% of product absorption
using nordihydroguaiaretic acid (NDGA), a known reduc-
tive inhibitor. Individual controls were conducted with
inhibitor alone with product and enzyme alone with pro-
duct. These negative controls formed the baseline for the
assay, reflecting non-pseudoperoxidase dependent hydro-
peroxide product decomposition. To rule out the auto-
inactivation of the enzyme from pseudo-peroxidase cycling,
15-sLOX residual activities were measured after the assay
was complete, 20 μM AA was added to the reaction mixture
and the residual activity was determined by comparing the
initial rates with inhibitor and 13-(S)-HPODE vs. inhibitor
alone, since the inhibitor by itself inherently lowers the rate
of the oxygenation. Activity is characterized by direct
measurement of the product formation with the increase of
absorbance at 234 nm.
The structures of ligands were built using GaussView
5.0 software. The Chelpg charges were obtained at the
B3LYP/6-31G** level of theory, employing the Gaussian
09 package. Docking studies of 3, 5, 6, 7 and NDGA into
the active site of the crystal structures of 15-sLOX (PDB
ID: 1N8Q) (Borbulevych et al. 2004), human 5-LOX (PDB
ID: 3O8Y) (Gilbert et al. 2011), human 12-S-LOX (PDB
ID:3D3L) (Tresaugues et al. 2008), rabbit 15-LOX-1 (PDB
ID: 2P0M) (Choi et al. 2008) and human 15-LOX-2 (PDB
ID:4NRE) (Kobe et al. 2014) were done using the
Lamarckian algorithm in AutoDock 4.2 (Morris et al. 1996)
and assuming total flexibility of the inhibitors and partial
flexibility of the His residues coordinated to Fe(III) inside
the binding site. The grid maps were built with 60 × 60 ×
60 points, with a grid-point spacing of 0.375 Å. The
AutoTors option was used to define the ligand torsions, and
the docking results were then analyzed by a ranked cluster
analysis, resulting in conformations with the highest overall
binding energy (lowest ΔGbinding value). When the com-
plexes for 6 and NDGA had been obtained, the whole
systems were inserted into a water box (TIP3) and ions were
added creating an overall neutral system using NaCl. The
ions were equally distributed in the water box. The final
systems contained approximately 35,000 atoms and were
submitted to a molecular dynamics (MD) simulation for 10
ns using NAMD 2.6 (Phillips et al. 2005).
The NPT ensemble was used to perform MD calcula-
tions. Periodic boundary conditions were applied to the
system in the three coordinate directions. A pressure of 1
atm. and a temperature of 310 K were maintained.
Evaluation of radical-trapping activity using DPPH
The DPPH scavenging capacity assay was performed using
20 µL of different compound concentrations in methanol,
which were added to 1 mL of 0.1 mM DPPH in methanol.
Each mixture was vortexed for a few seconds and stirred in
the dark for 30 min at room temperature. The absorbance
(A) of each reaction mixture was measured at 517 nm
against a blank of methanol using a UV–visible spectro-
meter (Perkin-Elmer 25). The scavenging capacity of each
sample was calculated on the basis of an ascorbic acid
standard curve. Duplicate reactions were carried out with
each sample. The correlation among the IC50 values for
enzyme inhibition and the scavenger properties of the
compounds was evaluated using a correlation matrix
including the IC50 for enzyme inhibition of twelve com-
pounds and the percentage of remaining DPPH radical for
three independent experiments with each enzyme. The
Pearson correlation for the IC50 and % remaining DPPH
was done using GraphPad Prism v.5 DEMO for windows,
considering an alpha value of 0.05 (Miliauskas et al. 2004;
GraphPad Software).
Results and discussion
Compounds 1–6 were synthetized starting from 2, 4-dihy-
droxybenzaldehyde, which was subsequently condensed
with acetylglycine (Knoevenagel) and hydrolyzed in one
step to afford 3-amino-7-hydroxycoumarin (SI). The cou-
marins were condensed with appropriately substituted
benzaldehydes to obtain compounds 1–6 (Li et al. 2009;
García-Beltrán et al. 2014; García-Beltrán et al. 2012a, b,
2015a, b), in yields higher than 90%. Finally, compounds
7–15 were synthetized according to literature protocols
(Scheme 1) (Huff et al. 1999; García-Beltrán et al. 2012a;
García-Beltrán et al. 2013; Aliaga et al. 2014; Kostova and
Saso 2013).
A preliminary screen was carried out (Holman et al.
1998; Deschamps et al. 2007; Carroll et al. 2001; Wecksler
et al. 2009a, b), for all prepared compounds against 15-
sLOX. The soybean (15-sLOX) enzyme was taken as a
study model because, although the human enzyme is also

Med Chem Res
Scheme 1 Synthesis of (E)-3-(benzylideneamino)-7-hydroxy-2H-chromen-2-one and 2-amino-2-hydroxymethyl-propane-1,3-diol derivatives
commercially available, it is less stable than that of plant
origin, besides exhibiting low biological activity in in vitro
studies. An alignment of the sequences of human and
soybean LOX yielded a 28% identity. Although this could
be taken as an indication of little identity between the two
isoforms, a comparative study of their active sites showed
that all residues that are part of the binding site, and that
interact with the AA and iron (Wecksler et al. 2009a), are
highly conserved in both systems. Thus, we can consider
15-sLOX as a first good model for studying the mechanisms
of inhibition of 12- or 15-LOX, an assumption that was
supported by docking studies.
This preliminary screen was expected to reveal poten-
tially active inhibitors of the human enzyme. The percen-
tages of inhibition assayed at 1 µM concentration of the
coumarin derivatives are displayed in Table 3.
This preliminary screening singled out coumarins 3, 5, 6,
and 7 as the most promising derivatives, with percentage
inhibition near or higher than 50%. IC50 values for these
molecules were then determined at different inhibitor con-
centrations. Two of them (3 and 6) proved effective at a
submicromolar, and the other two (5 and 7) at a micromolar
concentration. With the aid of the Cheng-Prusoff Eq. (1)
(Cheng and Prusoff 1973) the corresponding inhibition
constants Ki for these derivatives were calculated.
IC50
Ki ¼
ð1Þ
½Sꢀ
1 þ Km
Table 3 lists the IC50 and the Ki values for coumarin
derivatives 3, 5, 6, and 7. For the sake of comparison, the
corresponding values for 7-hydroxy-8-methyl-3-phenyl-4H-
chromen-4-one (IR-204) and for the potent competitive
inhibitor nordihydroguaiaretic acid (NDGA) were also
included.
To determine the type of inhibition of these compounds,
kinetic studies were carried out the Fig. 1 reproduces
Lineweaver-Burk plots for the most promising inhibitor 6,
in an indication of a competitive behavior for this molecule.

Med Chem Res
% DPPH
Table 3 Biological results of
the most promising inhibitors
against 15-sLOX
Compound
Percentage of
Inhibition ( ) SD
IC50 (µM)
( ) SD
Ki (µM)
Pseudoperoxidase
activity
1
33 0.20
10 1.10
60 0.30
2
3
0.37 0.00
0.18
+
93.7
4
4
0.10
5
48 2.80
65 4.70
36 1.70
19 1.10
16 1.20
13 2.20
19 6.00
11 3.70
12 1.40
24 2.50
18 1.50
1.49 0.06
0.34 0.02
5.13 0.37
0.76
0.17
2.63
+
+
+
71.3
81.1
21.9
6
7
8
9
10
11
12
13
14
15
NDGAa
IR204b
a
1.13 0.02
0.29
+
N.D
N.D
>100
>100
–
Nordihydroguaiaretic acid
b
7-hydroxy-8-methyl-3-phenyl-4H-chromen-4-one
effectiveness of these 15-sLOX inhibitors could be ascribed
to their antioxidant activities.
An important result of Table 3, is that two of the new
coumarin derivatives (compounds 3 and 6) showed com-
parable and even better activities than the potent REDOX
inhibitor NDGA with a Ki = 0.29 μM (Somvanshi et al.
2008). Our kinetic studies revealed that 6 compete with the
substrate for the binding site of the enzyme, in the same
way as NDGA.
On the other hand, steric effects apparently play an
important role in the interaction with the binding site as we
observed in compounds 5 and 7 which showed the worst
results of IC50 (5.13 and 1.49 μM respectively), particular
the compound 7, had the lowest percentage of DPPH
bleaching seen in this series, despite that above, this results
explain the different chemical interaction responsible for
LOX inhibition and that the presences of catechol or
hydroxyl group not guaranteed the potency of drugs.
In order to gain some insight into the structural factors
responsible for the inhibitory activity of the coumarin
derivatives, docking studies of these molecules and NDGA
into the active site of 15-sLOX were carried out (Gauss-
View, Version 5; Frisch et al. 2009; Berman et al. 2000;
Morris et al. 1996; Phillips et al. 2005). Starting from the
known structure of crystalline 15-sLOX (PDB ID: 1N8Q)
(Borbulevych et al. 2004), docking studies revealed a
variety of interactions of this molecule with polar and non-
polar amino acids.
Fig. 1 Biological activity of compound 6 and Lineweaver−Burk plot
of compound 6 in front of 15-sLOX
A possible correlation between the inhibitory activities of
3, 5, 6, and 7 and their pseudo-peroxidase and antioxidant
activities was next investigated (Table 3). It is seen that all
tested inhibitors exhibited pseudoperoxidase activity
(Riendeau et al. 1991; Hoobler et al. 2013), suggesting that
the coumarin derivatives affect the oxidation state of iron in
the active site, reducing it from ferric (active) to ferrous
(inactive). Additionally, the four derivatives were capable
of bleaching DPPH (Miliauskas et al. 2004; GraphPad
Software), and the percentage bleaching varied inversely
with their inhibitory constants Ki, suggesting that the

Med Chem Res
Fig. 2 a Representation of LOX b and c shows the main interactions of NDGA and 6 respectively inside the binding cavity. The hydrogen bonds
generated during the simulation are displayed in (d) NDGA and e compound 6
Studies with the best LOX inhibitor revealed a relevant
π-cation interaction between the chromene ring and the iron
cation located in the binding site. The presence of the
catechol group in compound 3 also favored the inhibition, a
result that can be related with the antioxidant and pseudo-
peroxidase activities shown in Table 3, common to other
good inhibitors assayed in our group (Mascayano et al.
2013, 2015).
Compounds 3, 5, and 7 (IC50 0.37, 1.49, and 5.13 μM
respectively) were next docked into the binding site of 15-
sLOX. The results showed that for compounds 6, 5, and 3,
the imine group was located close to the iron ion. This was
not observed for compound 7, which had the 2-amino-2-
hydroxymethyl-propane-1,3-diol substituent positioned
near the metal. A good relationship was found between the
binding energies and the activities of compounds 6 and 7
(ΔGbinding = −5.85 and −1.89 Kcal/mol), the most and
least active, respectively. Docking thus provided a pre-
liminary approximation and insight into the protein–ligand
interactions in the enzyme cavity.
The complex LOX-6 was studied using MD simulations
in order to rationalize the system from a dynamic and
energetic point of view. (Fig. 2c). Is important to mention
that Q514, which has been described an important residue
that aid to the fatty acid to binding into the catalytic site is in
coordination with H518 along the MD helping to maintain
the central ring of 6 close to the metal due to the electro-
static interaction formed with imine group.
The docking results with compound 6 showed that the
dimethylcarbamothioate substitutent is located near the
basic residue R726, favoring a strong interaction between
ligand and protein through the sulphur atom (Fig. 2c).
Compound 6 is located close (≈4 Å) to Fe(III) and ΔGbinding
of −5.85 Kcal/mol and exhibits strong interactions with
Q514, H518, W519, H523 and R726. Among these resi-
dues, Q514 plays a decisive role in promoting the binding
of fatty acids into the catalytic site. By coordinating with
H518, it draws the central ring of 6 close to the metal,
through interactions with its imine group. Docking of 6
against other isoforms of LOX reveals a lower affinity in the
human crystal structure of 5-LOX (−0.38 Kcal/mol), while
for human 12-S-LOX and 15-LOX-2, affinities (−11.09 and
−5.47 Kcal/mol respectively) were comparable or better
than for 15-sLOX. Comparison of binding energies from
docking studies with human 15-LOX-1 shows similar
values to those with 15-sLOX (used in this study). It is
important to mention that in all cases the ligands interact in
a similar way in the LOX cavity, positioning the imine
group near the iron cation (≈3 Å).
As was mentioned above, NDGA is a REDOX inhibitor
of LOX. In order to evaluate the specific interactions
between NDGA and the protein computational studies were
carried out. The docking results (ΔGbinding −6.42 Kcal/mol)
showed that the presence of the catechol group is funda-
mental for their interaction with iron (1.7 Å), but the
hydrophobicity of pocket is also relevant, residues as F, V, I
and W allow the correct positioning of ligand into the

Med Chem Res
Brash AR, Schneider C, Hamberg
M
(2012) Applications of
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enzyme.
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The analysis of LOX-NDGA complex by MD showed a
strong interaction between the catechol group and the iron
cation. This would validate the suggestion from kinetic
studies that inhibition by NDGA occurs by hydrogen
transfer to iron (Somvanshi et al. 2008) in the similar way
that coumarin 6, in both cases the REDOX mechanisms
were the responsible. In addition, NDGA established more
hydrogen bonds with surrounding residues than 6 (Fig. 2d, e).
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Deschamps JD, Gautschi JT, Whitman S, Johnson TA, Gassner NC,
Crews P, Holman TR (2007) Discovery of platelet-type 12-human
lipoxygenase selective inhibitors by high-throughput screening of
structurally diverse libraries. Bioorg Med Chem 15:6900–6908
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In conclusion, among the newly studied coumarins, 3, 5, 6,
and 7 all of them showed promising inhibitory activities
against 15-LOX. The most powerful inhibitor, 6 (IC50 0.34
µM), proved even more potent than NDGA (IC50 1.13 µM).
Kinetic measurements suggested a competitive mechanism
(Ki 0.17 µM) for its action, in agreement with its excellent
REDOX and pseudoperoxidase activities. Molecular simu-
lation studies supported these observations and revealed that
binding inside the active site occurs mainly through van der
Waals and electrostatic interactions.
Acknowledgements Financial support from DICYT 021641MC;
Fondecyt 1120379, Fondecyt 1161375 and the Millennium Scientific
Initiative (Grant P05-001-F).
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
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García-Beltran O, Mena N, Friedrich LC, Netto-Ferreira JC, Vargas V,
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