Synthesis and SAR studies of mono O-prenylated coumarins as potent 15-lipoxygenase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.09.006

All of the mono isopentenyloxy, -geranyloxy and -farnesyloxy derivatives of coumarin were synthesized and their inhibitory potency against soybean 15-lipoxygenase (SLO) and human 15-lipoxygenase-1 (HLO-1) were determined. Amongst the synthetic analogs, 5-farnesyloxycoumarin showed the most potent inhibitory activity against SLO (IC₅₀ = 0.8 μM) while 6-farnesyloxycoumarin was the strongest HLO-1 inhibitor (IC₅₀ = 1.3 μM). The IC₅₀ variations of the farnesyl derivatives for HLO-1 (1.3 to ～75 μM) were much higher than that observed for SLO (0.8-5.8 μM). SAR studies showed that hydrogen bonding, CH/π, anion-π and S-OC interactions with Fe^III-OH, Leu408, Glu357 and Met419 were the distinct intermolecular interactions which can lead to important role of the coumarin substitution site in HLO-1 inhibitory potency, respectively.

Full text of DOI:10.1016/j.ejmech.2012.09.006

European Journal of Medicinal Chemistry 57 (2012) 134e142
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and SAR studies of mono O-prenylated coumarins as potent
15-lipoxygenase inhibitors
Mehrdad Iranshahi a, Atena Jabbari b, Ala Orafaie c, Robabeh Mehri d, Soudabeh Zeraatkar d,
,e,
Taraneh Ahmadi d, Maliheh Alimardani d, Hamid Sadeghian d
*
a Biotechnology Research Center and School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
b Department of Chemistry, School of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran
c Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran
d Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad, Iran
e Microbiology & Virology Research Center, Buali Research Institute, Mashhad University of Medical Sciences, Mashhad 91967-73117, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
All of the mono isopentenyloxy, -geranyloxy and -farnesyloxy derivatives of coumarin were synthesized
and their inhibitory potency against soybean 15-lipoxygenase (SLO) and human 15-lipoxygenase-1 (HLO-
1) were determined. Amongst the synthetic analogs, 5-farnesyloxycoumarin showed the most potent
Received 11 June 2012
Received in revised form
23 July 2012
Accepted 4 September 2012
Available online 11 September 2012
inhibitory activity against SLO (IC50 ¼ 0.8
inhibitor (IC50 ¼ 1.3 M). The IC50 variations of the farnesyl derivatives for HLO-1 (1.3 to w75
were much higher than that observed for SLO (0.8e5.8 M). SAR studies showed that hydrogen bonding,
CH/ , anion-
and SeO]C interactions with FeIIIeOH, Leu408, Glu357 and Met419 were the distinct
m
M) while 6-farnesyloxycoumarin was the strongest HLO-1
m
m
M)
m
p
p
Keywords:
Docking
HLO-1
3-Carboxycoumarin
Farnesyl
intermolecular interactions which can lead to important role of the coumarin substitution site in HLO-1
inhibitory potency, respectively.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Geranyl
1. Introduction
for treatment of inflammatory respiratory disorders such as
asthma, rhinitis, and chronic obstructive pulmonary disease (COPD)
Lipoxygenases (LOs) are a main group of the non heme iron-
containing proteins which can catalyze the hydroperoxidation of
in humans [3]. 15-LO metabolites such as 15-HETE were also found
as important mediators in the development of Th1-allergic
inflammation induced by sensitization with an allergen plus
dsRNA, and the 15-LO pathway was introduced as a novel thera-
peutic target for the treatment of virus-associated asthma charac-
terized by a Th1 immune response to inhaled allergens [4]. The
critical role of the 15-LO-1 metabolite, 13-(S)-hydrox-
yoctadecadienoic acid (13-HODE), in the progression of prostate
cancers and inhibition of 15-LO-1 activity for apoptosis induction in
PC3 cells has been demonstrated [5,6]. It was also found that 15-LO
involved in oxidative modification of low-density lipoproteins
(LDL) leading to the development of atherosclerosis [7e9]. A recent
study on human airway epithelial cells, in cell culture and in human
asthmatic epithelial cells, showed that high levels of 15-LO-1
interact with phosphatidylethanolamine-binding protein-1 to
displace Raf-1 and sustain MAPK/ERK activation [10].
poly unsaturated fatty molecules containing
a
cis,cis-1,4-
pentadiene structure such as arachidonic and linoleic acid [1]. In
mammalians, three classes of signaling compounds leukotrienes,
lipoxines and eoxines, are formed via hydroperoxidation at 5 or 15
carbon position of the arachidonic acid [2,3]. Amongst the
mammalian lipoxygenases involved in the human disease,
15-lipoxygenase-1 (15-LO-1) is recently well-documented as
a target for reducing the biosynthesis of eoxines, one of the known
pro-inflammatory mediators [3]. It has been showed that the
15-LO-1 pathway can generate the pro-inflammatory eoxines in
eosinophils, mast cells, and nasal polyps from allergic subjects,
indicating that inhibition of 15-LO-1 might be an attractive target
* Corresponding author. Microbiology & Virology Research Center, Buali Research
Institute, Mashhad University of Medical Sciences, Mashhad 91967-73117, Iran.
Tel.: þ98 0511 7610111; fax: þ98 0511 7628088.
There is a considerable interest in the development of LO
inhibitors for therapeutic applications. Although a numerous LO
inhibitors has been prepared and biologically studied, due to their
E-mail address: sadeghianh@mums.ac.ir (H. Sadeghian).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.006

M. Iranshahi et al. / European Journal of Medicinal Chemistry 57 (2012) 134e142
135
side effects, consuming of them is usually forbidden or limited [11].
Explore the alternative strategies to lower the formation of
inflammatory mediators and oncogenic agents with the help of
natural dietary products are necessary. A group of these natural
products are coumarins. Coumarins, consist of fused benzene and
a pyrone rings, comprise a very large class of phenolic derivatives
occurred in plants. More than 1300 derivative of coumarins, pos-
sessing anti-inflammatory and anticancer properties [12], have
been identified as primary or secondary metabolites in green
plants, fungi and bacteria [13]. Several natural and synthetic
derivatives of hydroxycoumarins have been introduced as inhibi-
tors of the lipoxygenase and cyclooxygenase pathways of arach-
idonate metabolism [14,15].
In previous work, umbelliprenin (7-trans,trans-farnesyloxy
coumarin), was examined for in vitro antioxidant activity, in vitro
inhibitory activity against soybean lipoxygenase, and in vivo anti-
inflammatory activity [16]. Lipoxygenase inhibitory activity of
umbelliprenin was determined by using the 1,3-dien formation
method (measuring the decrease in absorbance at 234 nm) where
the linoleic acid was used as the substrate [16].
Umbelliprenin did not show any significant antioxidant activity
in the presence of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) but
exhibited a remarkable and potent inhibition against soybean
15-lipoxygenase (SLO) [16]. This compound, in the in vivo anti-
inflammatory test, could also inhibit the carrageenin induced paw
edema significantly (39%) [16].
In the present study, O-prenylated derivatives involving farne-
syloxy, geranyloxy and isopentenyloxy substituents at positions 3,
4, 5, 6, 7 and 8 of coumarin ring were synthesized and the lip-
oxygenase inhibitory activities of them together with the SAR
studies were evaluated.
5-farnesyloxy derivative (7c) was the best SLO inhibitor by an IC50
value of 0.8 ꢀ 0.1 M. Amongst the farnesyloxy analogs, 7f was the
weakest inhibitor by an IC50 value of 5.8 M. Complementary studies
on inhibitory mechanism showed that the mentioned compounds
inhibit lipoxygenase activity by competitive mechanism (Fig. 1).
In the next step, the inhibitory potential of the farnesyloxy and
geranyloxycoumarins against recombinant human 15-lipoxygenase
m
m
1
(HLO-1) were determined (Table 2). In the experiment,
compounds 7d showed the best inhibitory activity (IC50 ¼ 1.3
m
M)
while the lowest activity was observed for 7a (IC50 > 50
mM).
Considering the data of Table 2, it is found that like SLO, by
decreasing the prenyl length the IC50 value increases. It was inter-
esting that the inhibitory results were different when compared
with SLO. For example, 7a with the second rank of SLO inhibition
was the weakest HLO-1 inhibitor while the ternate SLO inhibitor, 7d,
showed the highest potency for suppressing HLO-1 activity.
In order to give further proof to the action mechanism of
designed inhibitors, molecular models of the complex enzyme-
inhibitor were generated for the synthetic coumarins using X-ray
3D structures of soybean 15-LO (SLO) and rabbit 15-LO-1 (RLO-1).
There is reasonable homology between the HLO-1 and RLO-1
(identities: 81%, positives: 91%, extracted from NCBI-BLAST [22]).
ꢀ
This homology increases (w99%) within 20 A in the active site
pocket. So we can use the 3D structure of RLO-1 for HLO-1 in
docking analysis.
There are significant differences in size, sequence, and substrate
preference between the plant and animal LOs, but the overall fold
and geometry of the non heme iron-binding site are conserved [23].
The structures of SLO and RLO-1 demonstrate a high level of
ꢀ
conservation within 10 A (around the linoleic acid into SLO X-ray
view) in the active site pocket. The structure of RLO-1 was super-
ꢀ
imposed on the SLO with RMS (C-
a
atoms) ¼ 1.24 A in the
2. Result and discussion
mentioned region (Fig. 2). The largest differences between the SLO
and RLO-1 active site pocket were found in residues of initial part of
All of the O-prenylated coumarins were synthesized according
to the previous literature via reaction of each hydroxycoumarin
with the desired prenyl bromide in the presence of potassium
carbonate and acetone (Scheme 1) [17]. 5- and 8-
Hydroxycoumarins were synthesized by starting from related
dimethoxy benzaldehydes and malonic acid. In the synthetic
pathway, 2-hydroxy-6-methoxy benzaldehyde (1c) and 2-hydroxy-
3-methoxy benzaldehyde (1f) reacted with malonic acid in the
mixture of acetic acid and benzene at reflux condition to produce
corresponding methoxy-3-carboxycoumarins (2c and 2f) [18]. After
acidic decarboxylation of the carboxycoumarines [19] and then
demethylation of the resulted methoxycoumarins (3c and 3f) by
melting with anhydrous aluminum chloride [20], the desired
hydroxycoumarins (4c and 4f) were prepared (Scheme 1).
The inhibitory activity of the synthetic compounds against SLO
was determined using modified oxidative coupling of 3-methyl-2-
benzothiazolinone (MBTH) with 3-(dimethylamino) benzoic acid
(DMAB) reported by Anthon et al. [21]. In this method, the basis of
the lipoxygenase activity determination is the peroxide concen-
tration measurement.
a a helix (a2 helix) while the
2 helix. In SLO Leu273eAla280 form
similar residues in RLO-1 (Asp170eLeu177) did not shape any
secondary structure (Fig. 2).
Bonding affinity of the designed molecular structures
(compounds 6aef and 7aef) toward SLO and RLO-1 was studied.
Docked conformers of 6aef and 7aef were generated in Auto-
DockTools (ADT) software. In docking process, flexible side chain of
the active site pocket residues was allowed to be rotatable (Phe353,
Leu408, Phe415, Ile418, Met419, Ile593, Leu597 in RLO-1 and
Leu277, Leu515, Ile557, Leu560, Leu565, Val566, Ile572, Phe576-
Leu773, Ile857 in SLO). A detailed assessment of the output clusters
ꢀ
(cluster tolerance/A ¼ 2.5) of both enzymes revealed that the most
bonding conformers (17e25%) belong to cluster in which prenyl
portion surrounded by Leu277, Trp519, Ile557, Leu560, Leu565,
Val566, Ile572, Leu773 and Ile857 in SLO and by Leu408, Ala404,
Arg403, Ile400, Asp174 and Ile173 in RLO-1 (Fig. 3). In these clus-
ters, coumarin ring covered by lipophilic side chains of Ser510,
His513, Gln514, Phe576, Gln716, Asp766 and Ile770 in SLO and by
Ile593, Glu548, Leu408, Phe415, Met419, His361, Leu362 and
Glu357 side chains in RLO-1 (Fig. 3). Amongst the mentioned amino
acids Ile557, Leu560, Leu565, Val566, Ile572, Leu773, Ile857, His513,
Gln514, Gln716, Asp766 from SLO and Leu408, Ala404, Ile400,
Glu548, Leu408, His361, Leu362, Glu357 from RLO-1 (as same as
HLO-1) are highly conserved [23]. It is notable that Asp174 and
Ile173 (highly conserved residues in mammalian 15-lipoxygenase-
The enzyme assay was performed according to the end point
protocol while in the previous work it had been down by using the
kinetic procedure (measurement of the enzyme activity within 60e
120 s after addition of the substrate to the enzyme-inhibitor
mixture) [16]. By using the last protocol the lower IC50 values
versus the former is obtained.
1) belong to the
a2 helix have been reported as major residues
Among the three groups of synthetic O-prenylated coumarins,
farnesyloxy derivatives, almost showed the best inhibitory activity
against soybean 15-lipoxygenase (Table 1). The results showed that the
inhibitory potential depends on the prenyl length and it decreases by
8e50foldfromfarnesyltoisopentenylsubstituents.Intheassessment,
which form a distinguished space when compared with SLO [23].
For each farnesyl and geranyl derivatives, it was found that there
is a satisfactory relationship between IC50 values and the lowest
calculated inhibitory constants (Ki) of the docked conformer in the
mentioned clusters (Fig. 4). The described conformer from each

136
M. Iranshahi et al. / European Journal of Medicinal Chemistry 57 (2012) 134e142
1c
2c
3c
4c
O
O
O
OH
COOH
O
OH
O
O
O
O
O
O
malonic acid
NaHSO3
H2SO4
AlCl3/Δ
acetic acid/benzene
COOH
O
OH
1f
O
O
O
O
O
O
O
O
OH
O
2f
3f
4f
R-Br
K2CO3/Acetone/50-60oC
HO
O
R
O
O
O
O
5-7(a-f)
4a-f
*
*
*
R =
R
O
4a
4b
4c
5a
6a
7a
7b
7c
O
O
O
R
5b
6b
O
O
R
O
5c
6c
O
O
O
R
4d
5d
6d
7d
O
O
4e
5e
6e
7e
R
O
O
O
4f
5f
6f
7f
O
O
O
R
O
Farnesyl phenyl ether
Scheme 1. General procedure for the synthesis of O-prenylated coumarins and the chemical structure of farnesyl phenyl ether (FPE). 5c ¼ umbelliprenin; 5b ¼ aurapten.
cluster was named as “consensus structure” and used for further
analysis (Fig. 5).
The higher IC50 variation of HLO-1 as compared with SLO,
implied the important role of the farnesyl substituent situation of
coumarin ring in HLO-1 inhibition. This observation originates from
the different chemical characteristics of the active site pocket in
these enzymes.
Considering the IC50 values of the synthetic compounds, our
founding revealed that the prenyl length played an important role
in the inhibitory activities and when the prenyl moiety composed
of more than 10 carbons (geranyl group), substitution site of
coumarin ring would be substantial.
By considering the docked models of both enzymes it is found
that the coumarin moiety of consensus structures has four

M. Iranshahi et al. / European Journal of Medicinal Chemistry 57 (2012) 134e142
137
Table 1
Table 2
SLO inhibitory assessment data of O-prenylated coumarins, farnesyl phenyl ether
(FPE), caffeic acid and 4-methyl-2-(4-methylpiperazinyl)pyrimido[4,5-b]benzo-
thiazine (4-MMPB: CAS 928853-86-5).
HLO-1 inhibitory assessment data of O-prenylated coumarins (IC50 of FPE > 50
Due to self aggregation the IC50 of FPE, 7a and 6a was not measurable at higher than
50 and 100 M respectively.
mM).
m
Compound IC50
(m
M)
Compound
IC50
(
m
M)
Compound IC50
(m
M)
Compound 7a 7b
7c
7d
7e
7f
5a
5b
5c
5d
5e
5f
4-MMPB
54.4 ꢀ 2.2 6a
43.9 ꢀ 2.4 6b
39.8 ꢀ 2.2 6c
69.4 ꢀ 2.6 6d
60.4 ꢀ 2.7 6e
46.5 ꢀ 2.9 6f
4.7 ꢀ 0.1 7a
5.6 ꢀ 0.2 7b
2.8 ꢀ 0.1 7c
9.6 ꢀ 0.2 7d
6.3 ꢀ 0.2 7e
8.1 ꢀ 0.2 7f
1.7 ꢀ 0.1
3.5 ꢀ 0.2
0.8 ꢀ 0.1
2.1 ꢀ 0.1
3.1 ꢀ 0.1
5.8 ꢀ 0.2
2.7 ꢀ 0.3
IC50 M)
(m
>50 28.8 ꢀ 2.4 3.6 ꢀ 0.3
1.3 ꢀ 0.1 16.7 ꢀ 1.0 15.6 ꢀ 0.8
Compound 6a
6b
6c
6d
6e
6f
IC50 M)
(m
>100 42.6 ꢀ 1.7 12.8 ꢀ 0.3 6.8 ꢀ 0.3 23.9 ꢀ 1.2 39.5 ꢀ 2.2
21.2 ꢀ 1.4 Caffeic acid 39.4 ꢀ 1.3 FPE
of parallel
p-stacking of aromatic compounds on the hydrogen
bonding ability of purine and pyrimidine bases and also pairwise
interaction of aromatic amino acids with anionic amino acids
prominent intermolecular interactions with the RLO-1 (HLO-1)
active site component which is not seen in SLO (Fig. 6):
(anione
Similar to the mentioned intermolecular interactions (CH/
anione interactions by Leu408 and Glu357) are seen for aromatic
p
interactions) has been recently investigated [30e32].
p
and
p
1. Hydrogen bond between FeeOH and phenoxy oxygen of
portion of 2-(1-octynyl)-cinnamate (ligand) in 3D view of RLO-1 co-
crystal structure (PDB entry: 2P0M) [28].
coumarin ring.
2. CH/p interaction between the CH3 of Leu408 side chain and
Non-bonded interactions between a divalent sulfur atom and
carbonyl oxygen atom have recently been characterized in proteins.
Statistical database analyses of various types of SeO]C interac-
tions implied that the S atom tends to approach the O atom either
within the carbonyl plane (in the nO direction) or from the vertical
aromatic portion of coumarin ring.
3. pp stacking interaction between Glu357 carboxylate anion and
coumarin ring.
4. SeO interaction between Met419 sulfur atom and oxygen of
coumarin carboxylate.
ꢀ
direction (in the pO direction) at distance of 3e3.5 A [33]. In the
case of SeO (ester) interactions, the vertical direction is signifi-
cantly preferred [33]. In the mentioned bonding cluster of RLO-1,
carbonyl portion of coumarin ring interacts with the sulfur of
Met419. The steric role of the Met419 in peroxidation site of
arachidonic acid was proved by site direct mutagenesis [34].
Amongst the consensus structures, ones with the suitable
direction of coumarin ring to reach the four mentioned intermo-
lecular interactions would be potent inhibitors. Amongst the
synthetic farnesyl derivatives, compound 7d has the lowest Ki for
this kind of bonding conformation (Fig. 6).
CH/
tions to protein structure and function. Experimental evidence of
CH/ interactions between methyl groups and aromatic amino
acids backbone in proteins has been reported in some literatures
[24]. High-level ab initio analysis of CH/ interactions between
methane and benzene has shown that this interaction is strongest
when a CH bond is pointing directly at the center of the aromatic
ring [25e27]. In the desired bonding cluster of RLO-1, methyl
portion of highly conserved Leu408 [23,28] can axially interact with
p and pp stacking interactions make important contribu-
p
p
p
electrons of benzene portion of coumarin. The pp stacking
interaction between Glu357 carboxylate and coumarin ring can
affect the hydrogen bonding strength between Glu357 and His361
directly bonded to Fe core. The effective role of Glu357eHis361 and
Glu357eGln548eHis545 hydrogen bond network in oxidation
potency of FeIIIeOH has been previously studied [29]. The influence
0.8
5 μM
2.5
1.25
no inhibitor
M
μ
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
M
μ
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0.040
μ
1/[7a] ( M)
Fig. 2. Comparison of the two 15-lipoxygenase 3D structures (in ribbon view). C-a of
Fig. 1. LineweavereBurk plot of SLO inhibition by 7a. Lipoxygenase activity was
measured at 25, 50, 100 and 200 M concentrations of linoleic acid by MBTHeDMAB
method. The Y-intercept average (1/Vmax) of the plotted carves is 0.0574 ꢀ 0.0032
(Km ¼ 23.25 M).
SLO and RLO-1 are superposed and colored in blue and red, respectively. The active site
pockets were mapped by inserting the Fe atom in pink bull view. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)
m
m

138
M. Iranshahi et al. / European Journal of Medicinal Chemistry 57 (2012) 134e142
Fig. 3. Stick and solvent surface view of the SLO (above) and RLO-1 (below) active site residues interacted with consensus structure of 7c and 7d respectively. The residues which
have intermolecular interactions with farnesyl and coumarin moieties are distinguished by green and blue color, respectively. The FeIII bonded to OH is distinguished by pink bull.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
In SLO, important factor of the inhibitor bonding tendency is the
hydrophobic nature of the active site pocket organized by lipophilic
side chain of Leu277, Trp519, Ile557, Leu560, Leu565, Val566, Ile572,
Leu773 and Ile857. This claim could be acceptable when we
consider the low IC50 variation of the prenylated coumarins
particularly isopentenyl analogs. For further proof of the assertion,
inhibitory activity of farnesyl phenyl ether [35] (Scheme 1) against
3. Materials and methods
3.1. Molecular modeling, docking and SAR study
3.1.1. Structure optimization
The structures were designed in chem3D professional; Cam-
bridge software [36]. Output files were minimized under semi-
empirical AM1 method (Convergence limit ¼ 0.01; Iteration
limit ¼ 50; RMS gradient ¼ 0.05 kcal/mol; Fletcher-Reeves opti-
mizer algorithm) in HyperChem7.5 [37,38].
Crystal structure of soybean 15-lipoxygenase (SLO) complex
with 13(S)-hydroproxy-9(Z)-2,11(E)-octadecadienoic acid and
rabbit reticulocyte 15-lipoxygenase (RLO-1) complex with 2-(1-
octynyl)-cinnamic acid was retrieved from RCSB Protein Data
Bank (PDB entry: 1IK3 and 2P0M respectively).
SLO and HLO-1 was determined. The IC50 of SLO was 2.7
mM while it
was higher than 50 M for HLO-1. The results confirm the key role
m
of the prenyl and coumarin moieties in inhibition of SLO and HLO-1
activities respectively.
In summary all of the favorite mono prenyloxycoumarins were
synthesized and their inhibitory potency against herbal and
mammalian 15-lipoxygenase (SLO and HLO-1) were evaluated.
The structure activity relationship (SAR) studies showed the
importance of prenyl length in SLO inhibition and influence of
coumarin substitution site on inhibitory activity for the longest
prenyl chain. It was also found that for farnesyl and geranyl
derivatives the role of coumarin substitution site in HLO-1 inhi-
bition is very predominant. The observed inhibition differences
between the two mentioned enzymes originated from chemical
nature and hydrophobic property of their active side pocket
residues.
3.1.2. Molecular docking
The ligands of the SLO and RLO-1 3D structure were omitted.
Then the Fe was modified to FeIIIeOH, geometrically optimized by
MMþ method in HyperChem7.5 and outputted in .pdb format for
docking process.
Automated docking simulation was implemented to dock the
minimized structures into the active site of SLO and RLO-1 with

M. Iranshahi et al. / European Journal of Medicinal Chemistry 57 (2012) 134e142
139
hemoglobin (5 mg/mL, 3 mL) in 50 mM phosphate buffer at pH 5.0
(25 mL). A linoleic acid solution was prepared by mixing 5 mg of
linoleic acid with 50 mg Tween 20 in 3 mL water and then diluting
with KOH 100 mM to a final volume of 5 mL.
7d
6.0
5.5
5.0
4.5
4.0
3.5
R2 = 0.932
7c
6d
In the standard assay, the sample in ethanol (25
(4000 units/mL in 50 mM phosphate buffer pH 7.0; 25
m
m
L), SLO
L) and
6c
7f
phosphate buffer pH 7.0 (50 mM; 900 mL) were mixed in a test tube
and preincubation was carried out for 5 min at room temperature.
A control test was done with the same volume of ethanol. After the
6e
7b
6b
7e
preincubation, linoleic acid solution (50
peroxidation reaction, and, 7 min later, solution A (270
then solution B (130 L) was added to start the color formation.
Further 5 min later, 200 L of a 2% SDS solution was added to
mL) was added to start the
7a
6f
mL) and
6a
m
m
terminate the reaction. The absorbance at 598 nm was compared
with control test.
3.5
4.0
4.5
5.0
5.5
6.0
3.2. Experimental section
-log Ki
3.2.1. Instruments
R2 = 0.910
1H NMR (300 & 500 MHz), 13C NMR (75 & 125 MHz) were ob-
tained by using a Bruker Avance DRX-500 Fourier transformer
6.0
5.6
5.2
4.8
7c
7a
spectrometer. Chemical shifts are reported in parts per million (d)
7d
downfield from tetramethylsilane (TMS). Elemental analysis was
obtained on a Thermo Finnigan Flash EA microanalyzer. All
measurements of lipoxygenase activities were carried out using
a Spekol 1500 spectrophotometer. HLO-1 was purchased from
Novus Biologicals Co. SLO and other chemicals were purchased
from Sigma, Aldrich and Merck Co. respectively.
6c
7e
7b
6a
6e
7f
6b
6d
6f
3.2.2. General procedure for preparation of hydroxycoumarins
Desired methoxysalicylaldehyde (0.1 mol; 15.3 g), malonic
acid (0.1 mol; 10.4 g), acetic acid (50 mmol; 3.0 g), aniline
(4 mmol; 0.37 g) and benzene (30 mL) were heated together.
After 8 h the reaction mixture was cooled and filtered. The
produced methoxy-3-carboxycoumarin (2c and 2f) was washed
with benzene and after drying used for next step without further
purification.
5
6
7
8
9
10
-log Ki
Fig. 4. Log IC50 versus ꢁlog Ki for consensus structures of 7aef and 6aef for HLO-1
(above) and SLO (below). The IC50 of 7a and 6a against HLO-1 was predicted as 75
and 150 mM via extrapolation respectively.
8-Methoxy-3-coumarincarboxylic acid (2c): Yellow solid, mp:
203e205 ꢂC; 1H NMR (CDCl3):
d 4.00 (s, 3H, eOCH3), 6.9 (s, 1H, H-8
AutoDock 4.2 [39] using Lamarckian genetic algorithm [40]. This
method has been previously shown to produce bonding models
similar to the experimentally observed models [41,42]. The torsion
angles of the ligands were identified, hydrogens were added to the
macromolecule, bond distances were edited and solvent parame-
ters were added to the enzyme 3D structure. Partial atomic charges
were then assigned to the macromolecule as well as ligands (Gas-
teiger for the ligands and Kollman for the protein).
(coumarin)), 7.1 (s, 1H, H-6 (coumarin)), 7.8 (t, 1H, H-7 (coumarin)),
9.00 (s, 1H, H-4 (coumarin)).
5-Methoxy-3-coumarincarboxylic acid (2f): Yellow solid, mp:
208e210 ꢂC (Lit. [44]: 210 ꢂC.); 1H NMR (CDCl3):
d 4.00 (s, 3H, e
OCH3), 7.32 (s, 3H, H-5, H-6, H-7 (coumarin)), 8.91 (s, 1H, H-4
(coumarin)).
A mixture of methoxy-3-carboxycoumarin (10 g) and sodium
bisulfite (12 g) in water (40 mL) was warmed until no more was
evolved. Then concentrated sulfuric acid was added and refluxing
was continued for 1 h. The mixture was poured on crushed ice and
filtered. The produced methoxycoumarin (3c and 3f) was washed
with water and after drying used for next step without further
purification.
The docking regions of SLO and RLO-1 were defined by
considering Cartesian chart 18.3, 4.8,19.2 and ꢁ50.7, 165.3, 32.7 as
the central of a grid size with 44, 56, 62 and 56, 48, 54 points in X, Y
and Z axes respectively. The docking parameter files were gener-
ated using Genetic Algorithm and Local Search Parameters (GALS)
while number of generations (GA-LS runs) and maximum number
of energy evaluations were set to 200 and 2,500,000 respectively
(number of individuals in population ¼ 300; number of top indi-
viduals to survive to next generation ¼ 1). The 200 docked
complexes were clustered with a root-mean-square deviation
5-Methoxycoumarin (3c): White solid, mp: 87 ꢂC (from water);
Lit. [20]: 85e87 ꢂC.
8-Methoxycoumarin (3f): White solid, mp: 89 ꢂC (from water);
Lit. [44]: 89 ꢂC.
A mixture of desired methoxycoumarin (2 g) and aluminum
chloride (4 g) was heated at 145e150 ꢂC for 3 h. After cooling
a mixture of ice and concentrated hydrochloric acid was added and
the precipitated tiny needles of hydroxycoumarin (4c and 4f) were
separated.
ꢀ
tolerance (RMSD) of 2.5 A. Docking results were submitted to
Accelrys DS Visualizer 2.0.1 [43] for further evaluations.
3.1.3. 15-LO inhibitory assessment
Linoleic acid and two assay solutions (A and B) were prepared in
advance.
Solution A was 50 mM DMAB in a 100 mM phosphate buffer
(pH 7.0). Solution B was a mixture of 10 mM MBTH (3 mL),
5eHydroxycoumarin (4c): White solid, mp: 222e224 ꢂC (from
water); Lit. [20]: 223e225 ꢂC.
8-Hydroxycoumarin (4f): White solid, mp: 152 ꢂC (from water);
Lit. [45]: 148e150 ꢂC.

140
M. Iranshahi et al. / European Journal of Medicinal Chemistry 57 (2012) 134e142
Fig. 5. (Above) Consensus structures of compounds 7aef in the SLO (A) and RLO-1 (B) active site pockets with presentation of some of the flexible residues side chain. In both
figures compounds 7c and 7d are distinguished by red color. (Below) Consensus structures of compounds 6c (bleu) & 7c (red) in SLO (C) and 6d (blue) & 7d (red) in RLO-1 (D) active
site pockets. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.2.3. General procedure for preparation of prenyloxycoumarin
A mixture of hydroxycoumarins 4aef (5 mmol), prenyl bromide
(6 mmol) and anhydrous potassium carbonate (0.70 g; 5 mmol) in
dry acetone (3 mL) was refluxed for 12 h and then cooled. The
mixture was diluted with water (10 mL) and then extracted with
ether (2 ꢃ 20 mL). The combined extracts were washed with NaOH
10% (2 ꢃ 10 mL) and dried with anhydrous sodium carbonate. After
removal of the solvent the products were purified by crystallization
from methanol or by column chromatography (silicagel 60; 230e
400; heptane).
3-(3-Methylbut-2-enyloxy)-2H-chromen-2-one (5a): White solid,
mp: 108 ꢂC (from methanol); Lit. [46]: 108e109 ꢂC.
4-(3-Methylbut-2-enyloxy)-2H-chromen-2-one (5b): White solid,
mp: 96e97 ꢂC (from methanol); Lit. [47]: 98 ꢂC.
5-(3-Methylbut-2-enyloxy)-2H-chromen-2-one (5c): White solid,
mp: 64e65 ꢂC (from methanol); Lit. [48]: 62e64 ꢂC.
6-(3-Methylbut-2-enyloxy)-2H-chromen-2-one (5d): White solid,
mp: 128e130 ꢂC (from methanol); Lit. [49]: 129e130 ꢂC.
7-(3-Methylbut-2-enyloxy)-2H-chromen-2-one (5e): White solid,
mp: 76e77 ꢂC (from methanol); Lit. [50]: 74e75 ꢂC.
4-((E)-3,7-dimethylocta-2,6-dienyloxy)-2H-chromen-2-one (6b):
White solid, mp: 47e48 ꢂC (from methanol); Lit. [51]: 48e50 ꢂC.
7-((E)-3,7-dimethylocta-2,6-dienyloxy)-2H-chromen-2-one (6e):
White solid, mp: 64e65 ꢂC (from methanol); Lit. [50]: 62e63 ꢂC.
7-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyloxy)-2H-chro-
men-2-one (7e): white solid, mp: 59e60 ꢂC (from methanol); Lit.
[50]: 57e59 ꢂC.
3-((E)-3,7-dimethylocta-2,6-dienyloxy)-2H-chromen-2-one (6a):
White solid, mp: 70e71 ꢂC; 1H NMR (CDCl3):
d 1.58 (s, 3H, eCH3e,
(geranyl)),1.63 (s, 3H, CH3e, (geranyl)), 1.75 (s, 3H, CH3e, (geranyl)),
2.07e2.12 (m, 4H, e2CH2e, (geranyl)), 4.62 (d, 2H, J ¼ 6.5 Hz (e
OCH2), (geranyl)), 5.044e5.073 (m, 1H, ]CHe (geranyl)), 5.48e
5.51 (m, 1H, ]CHe (geranyl)), 6.82 (s, 1H, H-4 (coumarin)), 7.21e
7.39 (m, 4H, H-5, H-6, H-7, H-8 (coumarin)). 13C NMR (CDCl3)
d
157.73, 149.47, 143.62, 142.46, 131.93, 128.29, 126.39, 124.63,
123.58, 119.78, 117.95, 116.18, 113.69, 110.03, 66.21, 39.47, 26.14,
25.63, 17.69, 16.78. Found: C, 76.48; H, 7.34. C19H22O3 requires: C,
76.51; H, 7.38%.
3-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyloxy)-2H-chro-
men-2-one (7a): White solid, mp: 66e67 ꢂC; 1H NMR (CDCl3):
d 1.58
(s, 6H, 2eCH3e, (farnesyl)), 1.66 (s, 3H, CH3e, (farnesyl)), 1.76 (s, 3H,
CH3e, (farnesyl)), 1.99e2.16 (m, 8H, e4CH2e, (farnesyl)), 4.61 (d,
2H, J ¼ 6.50 Hz, (eOCH2), (farnesyl)), 5.03e5.08 (m, 2H, ]CHe
(farnesyl)), 5.48e5.50 (t, 1H, J ¼ 5.50 Hz, ]CHe (farnesyl)), 6.82 (s,
Fig. 6. Suitable situation of 7d in the RLO-1 active site pocket for formation of
hydrogen bond (hb) with FeeOH,
p p (carboxylate
(coumarin) / s* (H-C of Leu408),
anion of Glu357) / p* (coumarin) and (Met419) SeO]C (coumarin) interaction. The
Van der Waals intermolecular interactions are shown by black arrows.

M. Iranshahi et al. / European Journal of Medicinal Chemistry 57 (2012) 134e142
141
1H, H-4 (coumarin)), 7.20e7.39 (m, 4H, H-5, H-6, H-7, H-8
(coumarin)). 13C NMR (CDCl3)
157.72, 149.46, 143.62, 142.54,
(coumarin)), 7.65 (d, 1H, J ¼ 9.50 Hz, H-4 (coumarin)). 13C NMR
d
(CDCl3) d 161.04, 155.33, 148.37, 143.29, 141.91, 135.53, 131.34,
135.56, 131.28, 128.29, 126.40, 124.63, 124.25, 123.47, 119.78, 117.92,
116.18, 113.65, 66.20, 39.65, 39.50, 26.68, 26.10, 25.70, 17.68, 16.82,
16.02. Found: C, 78.62; H, 8.10. C24H30O3 requires: C, 78.69; H,
8.20%.
124.26, 123.52, 120.13, 119.13, 118.92, 117.79, 116.96, 111.04, 65.54,
39.68, 39.52, 26.69, 26.14, 25.70,17.69, 16.73, 16.03. Found: C, 78.63;
H, 8.14. C24H30O3 requires: C, 78.69; H, 8.20%.
8-(3-Methylbut-2-enyloxy)-2H-chromen-2-one (5f): White solid,
4-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyloxy)-2H-chro-
mp: 61e62 ꢂC; 1H NMR (CDCl3):
d 1.73 (s, 3H, eCH3e, (prenyl)), 1.76
men-2-one (7b): White liquid, 1H NMR (CDCl3):
d
1.57 (s, 6H, 2e
(s, 3H, CH3e, (prenyl)), 4.64 (d, 2H, J ¼ 7.00 Hz, (eOCH2), (prenyl)),
5.48e5.51 (m, 1H, ]CHe (prenyl)), 6.41 (d, 1H, J ¼ 9.50 Hz, H-3
(coumarin)), 6.93 (d, 1H, J ¼ 3.00 Hz, H-5 (coumarin)), 7.102e7.126
(dd, 1H, J1 ¼ 9.00, J2 ¼ 2.5 Hz, H-7 (coumarin)), 7.24 (d, 1H,
J ¼ 9.00 Hz, H-8 (coumarin)), 7.65 (d, 1H, J ¼ 9.50 Hz, H-4
CH3e, (farnesyl)), 1.67 (s, 3H, CH3e, (farnesyl)), 1.74 (s, 3H, CH3e,
(farnesyl)), 1.91e2.13 (m, 8H, e4CH2e, (farnesyl)), 4.62 (d, 2H,
J ¼ 6.50 Hz, (eOCH2), (farnesyl)), 5.03e5.07 (m, 2H, ]CHe (far-
nesyl)), 5.42e5.45 (t, 1H, J ¼ 5.50 Hz, ]CHe (farnesyl)), 5.77 (s, 1H,
H-3 (coumarin)), 7.33e7.51 (m, 4H, H-5, H-6, H-7, H-8 (coumarin)).
(coumarin)). 13C NMR (CDCl3)
d 160.39, 146.43, 144.10, 143.72,
13C NMR (CDCl3)
d
165.10, 161.22, 155.81, 143.46, 142.25, 135.50,
138.43, 124.22, 119.56, 119.31, 119.28, 116.75, 115.51, 66.17, 25.77,
18.26. Found: C, 72.94; H, 6.00. C14H14O3 requires: C, 73.04; H, 6.08%.
8-((E)-3,7-dimethylocta-2,6-dienyloxy)-2H-chromen-2-one (6f):
131.24, 125.71, 124.28, 116.27, 115.35, 113.12, 112.85, 112.38, 101.52,
65.43, 39.64, 39.49, 26.65, 26.10, 25.70,16.78,16.75,16.02. Found: C,
78.59; H, 8.12. C24H30O3 requires: C, 78.69; H, 8.20%.
white liquid; 1H NMR (CDCl3):
d 1.58 (s, 3H, eCH3e, (geranyl)), 1.64
5-((E)-3,7-dimethylocta-2,6-dienyloxy)-2H-chromen-2-one (6c):
(s, 3H, CH3e, (geranyl)), 1.73 (s, 3H, CH3e, (geranyl)), 2.04e2.12 (m,
4H, e2CH2e, (geranyl)), 4.68 (d, 2H, J ¼ 6.50 Hz, (eOCH2), (ger-
anyl)), 5.04e5.07 (m, 1H, ]CHe (geranyl)), 5.49e5.52 (m, 1H, ]
CHe (geranyl)), 6.40 (d, 1H, J ¼ 9.50 Hz, H-3 (coumarin)), 7.02e7.04
(dd, 1H, J1 ¼ 3.00, J2 ¼ 1.5 Hz, ]CHe H-7 (coumarin)), 7.05e7.65
(dd, 1H, J1 ¼ 8.00, J2 ¼ 1.5 Hz, H-5 (coumarin)), 7.14e7.16 (m, 1H,
H-6 (coumarin)), 7.67 (d, 1H, J ¼ 9.50 Hz, H-4 (coumarin)). 13C NMR
White liquid; 1H NMR (CDCl3):
d 1.61 (s, 3H, eCH3e, (geranyl)), 1.69
(s, 3H, CH3e, (geranyl)), 1.76 (s, 3H, CH3e, (geranyl)), 2.09e2.17 (m,
4H, e2CH2e, (geranyl)), 4.62 (d, 2H, J ¼ 6.00 Hz, (eOCH2), (ger-
anyl)), 5.08e5.11 (m, 1H, ]CHe (geranyl)), 5.48e5.51 (m, 1H, ]
CHe (geranyl)), 6.32 (d, 1H, J ¼ 9.00 Hz, H-3 (coumarin)), 6.72 (d,
1H, J ¼ 7.5 Hz, H-8 (coumarin)), 6.91 (d, 1H, J ¼ 8.00 Hz, H-6
(coumarin)), 7.41e7.44 (t, 1H, J ¼ 8.5 Hz, H-7 (coumarin)), 8.12 (d,
(CDCl3) d 160.40, 146.41, 144.13,143.71,141.71,131.81,124.21,123.72,
1H, J ¼ 7.00 Hz, H-4 (coumarin)). 13C NMR (CDCl3)
d
161.05, 155.51,
120.04, 119.56, 119.32, 119.10, 116.76, 115.61, 66.27, 39.48, 32.46,
26.22, 25.76, 17.69, 16.73. Found: C, 76.40; H, 7.30. C19H22O3
requires: C, 76.51; H, 7.38%.
155.18, 142.05, 138.84, 132.24, 131.96, 123.59, 118.66, 114.39, 109.89,
108.98, 106.28, 65.75, 39.49, 26.23, 25.69, 17.72, 16.73. Found: C,
76.45; H, 7.34. C19H22O3 requires: C, 76.51; H, 7.38%.
8-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyloxy)-2H-chro-
5-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyloxy)-2H-chro-
men-2-one (7f): white liquid; 1H NMR (CDCl3):
d 1.59 (s, 3H, eCH3e,
men-2-one (7c): White liquid; 1H NMR (CDCl3):
d
1.61 (s, 3H, eCH3e
(farnesyl)), 1.60 (s, 3H, CH3e, (farnesyl)), 1.67 (s, 3H, CH3e, (farne-
syl)), 1.75 (s, 3H, CH3e, (farnesyl)), 1.95e2.15 (m, 8H, e4CH2e,
(farnesyl)), 4.71 (d, 2H, J ¼ 6.50 Hz, (eOCH2), (farnesyl)), 5.06e
5.11 (m, 2H, ]CHe (farnesyl)), 5.52e5.55 (m, 1H, ]CHe (farne-
syl)), 6.42 (s, 1H, J ¼ 9.50 Hz, H-3 (coumarin)), 7.04e7.05 (dd, 1H,
J1 ¼ 8.00, J2 ¼ 1.5 Hz, H-7 (coumarin)), 7.07e7.09 (dd, 1H, J1 ¼ 8.50,
J2 ¼ 15.00 Hz, H-5 (coumarin)), 7.16e7.19 (m, 1H, H-6 (coumarin)),
, (farnesyl)), 1.61 (s, 3H, CH3e, (farnesyl)), 1.68 (s, 3H, CH3e, (far-
nesyl)), 1.76 (s, 3H, CH3e, (farnesyl)), 1.96e2.17 (m, 8H, e4CH2e,
(farnesyl)), 4.65 (d, 2H, J ¼ 6.50 Hz, (eOCH2), (farnesyl)), 5.07e
5.13 (m, 2H, ]CHe (farnesyl)), 5.49e5.51 (t, 1H, J ¼ 5.50 Hz, ]
CHe (farnesyl)), 6.32 (s, 1H, J ¼ 9.50 Hz, H-3 (coumarin)), 6.72 (d,
1H, J ¼ 7.5 Hz, H-8 (coumarin)), 6.90 (d, 1H, J ¼ 8.50 Hz, H-6
(coumarin)), 7.40e7.43 (t, 1H, J ¼ 8.00 Hz, H-7 (coumarin)), 8.12 (d,
7.68 (d, 1H, J ¼ 9.50 Hz, H-4 (coumarin)). 13C NMR (CDCl3)
d 160.38,
1H, J ¼ 7.50 Hz, H-4 (coumarin)). 13C NMR (CDCl3)
d
161.03, 155.51,
146.47, 144.19, 143.65, 141.61, 135.47, 131.29, 124.32, 124.19, 123.61,
119.58, 119.58, 119.29, 119.08, 116.83, 115.62, 66.29, 39.67, 39.51,
26.70, 26.19, 25.70, 17.69, 16.77, 16.03. Found: C, 78.59; H, 8.10.
155.17, 142.03, 138.83, 135.58, 132.25, 131.37, 124.24, 123.47, 118.69,
114.37, 109.86, 108.96, 106.25, 65.74, 39.69, 39.49, 26.70, 26.12,
25.70, 25.51, 17.69, 16.74, 16.06. Found: C, 78.60; H, 8.12. C24H30O3
requires: C, 78.69; H, 8.20%.
C24H30O3 requires: C, 78.69; H, 8.20%.
6-((E)-3,7-dimethylocta-2,6-dienyloxy)-2H-chromen-2-one (6d):
Acknowledgments
White solid, mp: 96e97 ꢂC; 1H NMR (CDCl3):
d 1.60 (s, 3H, eCH3e,
(geranyl)), 1.66 (s, 3H, CH3e, (geranyl)), 1.74 (s, 3H, CH3e, (geranyl)),
2.07e2.15 (m, 4H, e2CH2e, (geranyl)), 4.56 (d, 2H, J ¼ 6.50 Hz, (e
OCH2), (geranyl)), 5.06e5.09 (m, 1H, ]CHe (geranyl)), 5.46e5.49
We are grateful to Mashhad University of Medical Sciences for
financial support of this work (project 88820).
(m, 1H, ]CHe (geranyl)), 6.41 (d, 1H,
J
¼
9.50 Hz, H-3
Appendix A. Supplementary material
(coumarin)), 6.93 (d, 1H, J ¼ 3.00 Hz, H-5 (coumarin)), 7.1e7.12 (dd,
1H, J1 ¼ 9.00, J2 ¼ 2.5 Hz, H-7 (coumarin)), 7.24 (d, 1H, J ¼ 9.00 Hz,
H-8 (coumarin)), 7.65 (d, 1H, J ¼ 9.50 Hz, H-4 (coumarin)). 13C NMR
Supplementary material associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.ejmech.
2012.09.006.
(CDCl3)
d 161.05, 155.32, 148.36, 143.31, 141.89, 131.89, 123.65,
120.15, 119.18, 118.90, 117.77, 116.94, 111.07, 65.55, 39.50, 32.43,
26.24, 25.68, 17.70, 16.71. Found: C, 76.44; H, 7.32. C19H22O3
requires: C, 76.51; H, 7.38%.
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