Design and synthesis of 4-methoxyphenylacetic acid esters as 15-lipoxygenase inhibitors and SAR comparative studies of them

Article abstract of DOI:10.1016/j.bmc.2009.02.009

A group of 4-methoxyphenylacetic acid esters were designed, synthesized and evaluated as potential inhibitors of soybean 15-lipoxygenase (SLO) on the basis of eugenol and esteragol structures. Compounds 7d-e showed the best IC₅₀ in SLO inhibition (IC₅₀ = 3.8 and 1.9 μM, respectively). All compounds were docked in SLO active site and showed that carbonyl group of compounds is oriented toward the Fe^III-OH moiety in the active site of enzyme and fixed by hydrogen bonding with hydroxyl group. It is assumed that lipophilic interaction of ligand-enzyme would be in charge of inhibiting the enzyme activity. The selectivity of the synthetic esters in inhibiting of 15-HLOb was also compared with 15-HLOa by molecular modeling and multiple alignment techniques.

Full text of DOI:10.1016/j.bmc.2009.02.009

Bioorganic & Medicinal Chemistry 17 (2009) 2327–2335
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of 4-methoxyphenylacetic acid esters as
15-lipoxygenase inhibitors and SAR comparative studies of them
b
b
c,
Hamid Sadeghian a, , Neda Attaran , Zeinab Jafari , Mohammad Reza Saberi
,
*
Mehdi Pordel a, Mohammad Mahdi Riazi b
a Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad 91389-13131, Iran
b Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad 91775-1436, Iran
c School of Pharmacy, Pharmaceutical Research Center, Mashhad University of Medical Sciences, BuAli Square, Mashhad 9196773117, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
A group of 4-methoxyphenylacetic acid esters were designed, synthesized and evaluated as potential
inhibitors of soybean 15-lipoxygenase (SLO) on the basis of eugenol and esteragol structures. Compounds
Received 30 August 2008
Revised 18 January 2009
Accepted 9 February 2009
Available online 13 February 2009
7d–e showed the best IC50 in SLO inhibition (IC50 = 3.8 and 1.9
lM, respectively). All compounds were
docked in SLO active site and showed that carbonyl group of compounds is oriented toward the FeIII
–
OH moiety in the active site of enzyme and fixed by hydrogen bonding with hydroxyl group. It is assumed
that lipophilic interaction of ligand–enzyme would be in charge of inhibiting the enzyme activity. The
selectivity of the synthetic esters in inhibiting of 15-HLOb was also compared with 15-HLOa by molecular
modeling and multiple alignment techniques.
Keywords:
Methylenation
SLO
Protein modeling
Docking
Ó 2009 Elsevier Ltd. All rights reserved.
15-HLO
1. Introduction
within the atherosclerotic lesion.5 In addition, the immediate prod-
ucts of 15-LO oxidation of AA and linoleic acid (LA) have been shown
Our interest in 4-methoxyphenylacetic acid derivatives as
lipoxygenase inhibitors emerges from the early work by our groups,
in which the soybean 15-lipoxygenase inhibition of eugenol and its
esters was reported.1 It is well documented that mammalian lipox-
ygenases (LO’s) are non-heme iron-containing enzymes responsible
for the oxidation of polyunsaturated fatty acids and esters to hydro-
peroxy derivatives.2 There are heterogeneous family of enzymes
distributed widely throughout the plant and animal kingdoms,3
and named according to the position at which a key substrate, ara-
chidonic acid (AA), is oxidized. Among the mammalian lipoxygenas-
es involved in the etiology of human disease, 5-lipoxygenase (5-LO)
is nowwell establishedas a target for reducingthe productionof leu-
kotrienes (important in particular asthma).4 More recently,
15-lipoxygenase (15-LO) has emerged as an attractive target for
therapeuticintervention.5 15-LOhas been implicatedin the progres-
sion of certain cancers6,7 and chronic obstructive pulmonary disease
(COPD).8 Evidence for the inhibitionof 15-LO in the treatmentof vas-
cular disease is, however, most compelling.9 Both transgenic and
knockout studies implicate a role for 15-LO in atherogenesis.10,11
The enzyme is abundantly expressed in macrophages residing
to be pro-inflammatory12 and pro-thrombotic.13
It is also found that 15-LO is linked to cardiovascular complica-
tions due to participation in oxidative modification of low-density
lipoproteins (LDL), leading to the development of atherosclerosis.14
Chanez and colleagues15 explained that in vivo 15-HLOa (hu-
man 15-lipoxygenase) has antitumor effects in human airway car-
cinomas and promote apoptotic pathway. They believed that
neoplastic tissues from human airway carcinomas demonstrated
non-specific staining for human 15-HLOa as compared with nor-
mal tissues. By contrast, in human prostate tumors 15-HLOa was
overexpressed as compared with normal adjacent tissue,7 and
15-HLOb was poorly expressed in prostate tumors.16 In PC3 cells,
13(S)-HODE, one of the 15-HLOa metabolites, upregulated MAP ki-
nase, whereas in contrast 15(S)-HETE, the 15-HLOb metabolite,
downregulated MAP kinase.17 Taken together, these findings
including the upregulation of 15-HLOa within the airway tissue
of smoking patients with chronic bronchitis, provided new evi-
dence of possible acquired abnormalities linked to airway inflam-
mation. The bronchial epithelium is clearly a key player in
inflammation and structural changes in airway diseases. Its rich
content in 15-HLOa- and 15-HLOb-derived products highlight their
potential as new target for therapeutic interventions.
* Corresponding author. Tel.: +98 0511 7610111; fax: +98 0511 7628088.
E-mail address: hd_sn@yahoo.com (H. Sadeghian).
Tel.: +98 511 7112611/5; fax: +98 511 7112596.
Three different strategies have been developed to inhibit the
LO’s pathway.18 They involve (i) redox inhibitors or antioxidants,
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.009

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H. Sadeghian et al. / Bioorg. Med. Chem. 17 (2009) 2327–2335
Table 1
Enzyme inhibitory assessment and docking analysis data of consensus conformers for SLO
Compound
IC50
(l
M)
Compound
IC50
(
lM)
Ki
Ed (Kcal/mol}
D
Gb (Kcal/mol)
RMSD
Eugenol
Methyleugenol
Esteragol
1
2
3
4
8
38.2 1.9
96.1 3.3
64.1 1.5
43 1.4
137 5.5
33 2.1
145 3.9
>200
7a
7b
7c
7d
7e
7f
7g
7h
71
7J
56.2 1.5
32.0 1.6
11.6 10.2
3.8 0.3
1.13e-4
5.85e-5
3.11e-5
1.07e-5
5.66e-6
6.66e-6
5.72e-5
2.37e-5
2.42e-4
5.73e-5
8.88e-5
1.43e-4
À6.27
À7.05
À7.84
À8.71
À9.50
À9.33
À7.08
À7.49
À6.15
À5.84
À6.44
À5.87
À5.38
À5.77
À6.15
À6.78
À7.16
À7.06
À5.79
À6.31
À4.93
À5.79
À5.53
À5.25
24.86
24.27
24.24
23.98
23.52
22.74
23.72
22.85
23.47
22.93
23.36
23.13
1.9 0.2
35.0 2.1
34.4 0.8
35.7 1.7
88.1 0.6
38.7 0,9
56.5 3.4
64.0 1.1
7k
71
D
Gb: Estimated free energy of bonding, Ed: final docking energy, Ki: estimated inhibition constant and RMSD: root-mean-square deviation from reference structure. The IC50
values are given as mean SD.
which interfere with the redox cycle of 15-LO, (ii) iron-chelator
agents, and (iii) non-redox competitive inhibitors, which compete
with AA to bind the enzyme active site.
In this study, (i) some esters of 4-methoxyphenylacetic acid
were designed, synthesized and their activities were identified as
the mean of IC50 on soybean 15-LO (SLO), then (ii) common bond-
ing model of 4-methoxyphenylacetic acid esters in SLO active site,
(iii) SAR study of inhibitors to propose key features of this class of
inhibitors, (iv) and theoretical potency of these compounds for
inhibiting modeled 15-HLOa and 15-HLOb are reported.
general bonding model of these carbonyl compounds in SLO, a ser-
ies of aliphatic esters of 4-methoxyphenylacetic acid (7b–l) were
synthesized and evaluated for lipoxygenase inhibitory activity.
The synthetic esters 7a–l showed a broad range of inhibition activ-
ity on the enzyme (IC50 = 1.9–88.1 lM; Table 1). Compound 7e
having n-pentyl substituent was the most potent inhibitor at
1.9 0.22 while the isobutyl analogs (7i) presented lowest activity
(IC50 = 88.1 0.64 lM).
The esters 7a–l were docked into the active site of SLO to deter-
mine the inhibitory mechanism. The experimental results matched
with theoretical Ki of docking study for those models in which car-
bonyl group oriented toward iron atom similar to orientation of lin-
oleic acid peroxide in the active site. We generated 100 docked
conformers of 7a–l corresponding in ADT software. A detailed
inspection of each independent inhibitor conformers revealed that
more than 40% of docking results had nearly identical orientations
in which carbonyl group of each inhibitor oriented toward Fe core.
One conformer from each esters cluster whose carbonyl moiety
superposed on linoleic acid peroxy part was adopted as the ‘consen-
sus’ structure and used for further analysis (Fig. 1) (Scheme 1).1,19
2. Results and discussion
Considering our previous work on eugenol and esters1 we
tested the inhibitory property of eugenol, methyleugenol and est-
eragol on the SLO (substrate: linoleic acid). The results showed
IC50 = 38.2 1.9, 96.1 3.3 and 64.1 1.5 lM for the mentioned
enzyme, respectively. It is notable that no other products such as
hydroperoxy are isolated from action of the LO enzyme on methyl-
eugenol and esteragol as substrate (assuming hydroproxy is sup-
posed to be obtained if the redox pathway is blocked and the
inhibitor acts through its allylic group in reaction with the enzyme
active site similar to the oxidation of natural unsaturated fatty
acids).à Considering the IC50 results of the three compounds we
decided to study the effect of some changing in esteragol double
bond on inhibitory potency of this compound. At first 1-methoxy-
4-(2-methylallyl)benzene (2) and 1-methoxy-4-(2-ethylallyl)ben-
zene (4) were synthesized via methylenation of 1-(4-methoxy-
phenyl)acetone (1) and 1-(4-methoxyphenyl)-2-butanone (3). The
results of enzyme assay showed low potency of the mentioned allyl
compounds for inhibition of lipoxygenase activity (IC50 = 137 5.5
and 145 3.9 lM for compounds 2 and 4, respectively). Fixing of
ethyl group of compound 4 in a ring by synthesis of 6-methoxy-2-
methylene-1,2,3,4-tetrahydronaphthalene (6) from 6-methoxy-2-
tetralone (5) lead to no inhibitory potency even at 200
interesting to see that the precursor compounds 1 and 3 showed bet-
ter inhibitory activity towards SLO by 43 1.4 and 33 2.1 M.
lM. It is
l
Regarding the mechanism of hydroperoxydation and docking
procedure in this study, one might conclude that carbonyl moiety
of compounds 1 and 3 is able to inhibit lipoxygenase activity
through hydrogen bonding interaction with hydroxyl group of iron
atom of the enzyme. We followed the study by testing methyl ester
of 4-methoxyphenylacetic acid (7a) for lipoxygenase inhibitory
activity in which the IC50 was 56.2 1.5 lM. For investigating of
à
Substrate (100 lM) was reacted with soybean LO enzyme (167 U/mL) in 3 mL
borate buffer solution (0.1 M, pH 9) at 20 °C for 15 min. The mixture was then
analyzed by UV at 230–270 nm and no absorption of vinyl benzene formation was
appeared over the blank solution.
Figure 1. Consensus bonding conformations of compounds 7a–l in the SLO active
site (color sticks). Hydrogen bonds are shown by dashed green lines and the Fe
atom bond to hydroxyl group, distinguished by purple color.

H. Sadeghian et al. / Bioorg. Med. Chem. 17 (2009) 2327–2335
2329
HO
O
O
O
O
Eugenol
O
Methyleugenol
Esteragol
O
O
1
3
2
4
O
O
O
O
TiCl4/CH2Cl2
Mg/THF
O
5
6
O
O
a) R = Me
b) R = Et
c) R = n-Pr
d) R = n-Bu
e) R = n-Pan
f) R = n-Hex
g) R = iso-Pr
h) R = sec-Bu
i) R = iso-Bu
j) R = tert-Bu
k) R = Cyclopentyl
l) R = Cyclohexyl
O
R
O
O
O
OH
DBU/benzene
R-Br
7b-l ∗
4-methoxyphenylacetic acid
∗ methyl ester was purchased
Scheme 1. Molecular structures of eugenol, methyleugenol and esteragol. General procedures for the synthesis of compounds 2, 4, 6 and 7b–l.
It seems that the alkyl part of the esters has hydrophobic inter-
action with the cavity formed by side chain of Leu273, Thr274
(Fig. 3). We can also view in Figure 1 that the proposed orientation
of docked molecules has hydrogen bond with conserved His518 and
hydroxyl of iron complex via 4-methoxy and carbonyl group,
respectively.
,
Leu277, Ile557, Leu560, Ile772, Leu773 and Ile857 (pocket A) while ben-
zyl core has the same interaction with Leu515, Trp519, Leu565, Val566
and Ile572 side chains (pocket B) (Fig. 2). Ile857 is one of the high
conserved residues directly bond to the iron atom. The most criti-
cal residues, that is, Ile557, Leu565, Leu773 and Ile572 appeared close
to the active site (Fig. 2). X-ray presentation of LA into SLO20 pre-
sents lipophil interaction of the side chain of Ile857 with C-16–C-
18 of LA. It also indicates that Ile557, Leu565 and Leu773 lay within
4–6 Å of Fe3+–OH and both leucines are near the reactive site, C-
11–C-13, of LA (C-11: hydrogen abstraction site, C-13: oxygenation
site). Although Ile572 is far from Fe3+–OH, (at 9 Å) but still forms
part of the substrate-bonding cavity. Each of these residues pro-
vides a large surface to interact with natural substrate, particularly
Leu565 and Leu773. Mutating of the Ile or Leu to an Ala opens up
space within the bonding pocket of SLO, leading to altered HÅ trans-
fer kinetics.21 The Ile557?Ala and Ile572?Phe mutants decreased
kcat by 2 folds from WT (wild type), While Leu565?Ala and
Leu773?Ala decreased kcat by 60 and 1000-folds, respectively, indi-
cating that these hydrophobic residues (specially Leu565 and
Leu773) contribute significantly to catalysis.21 According to the re-
The Ki of consensus structure of the esters have good non-linear
relationship with IC50 results except for 7f and 7h (Fig. 4). This
relation comes from tendency of the alkyl moiety for filling the
empty lipophilic space of Thr274, Leu277, Ile557, Leu560, Leu773 and
Ile857 side chains (Fig. 2). The exception of 7f and its high increase
in IC50 value versus 7e imply that the lipophilic cavity of pocket A
has a restricted tunnel about 6 Å from oxygenation site of LA. If the
consensus structure of the esters are compared with X-ray presen-
tation of LA into SLO, most conformity between n-pentyl ester (7e)
and LA can be determined. The docking calculation suggests that 7f
would be fairly active. It is notable that the docking performed on
PDB format of X-ray presentation of LA into SLO with no flexibility
of residues in the active site pocket. So it can open a space as large
as n-hexyl portion for suitable occupation and finally results the
best Ki. But in real condition (Section 4) the mentioned space
seems to be smaller which can lead to decrease in IC50 result and
finally causes the observed disconformity between modeling pre-
diction and biological data.
sult of multiple alignment, six amino acids Ile557, Leu565, Ile572
Val566, Leu773 and Ile857 are found to be conserved over all species
,
There are significant differences in size, sequence, and substrate
preference between the plant and animal LOs, but the overall fold

2330
H. Sadeghian et al. / Bioorg. Med. Chem. 17 (2009) 2327–2335
from point of view of C-a situational conformity and lipophilicity,
could be an evidence for selectivity of the large and lipophile esters
toward type b mammalian 15-LO in comparison with type a. To
prove this hypothesis the inhibitory potency of compounds 7b
and 7e, as small and large compound, was determined against
15-lipoxygensae a and b (human recombinant). It was interesting
to see IC50 values of 56.7 1.4 and 63.2 2.1
lM for compound
7e and 30.9 1.3 and 11.7 0.8 M for compound 7e against 15-
l
HLOa and 15-HLOb, respectively. These results confirm that the
large and lipophil ester 7e is selective toward 15-HLOb by ꢀthree-
fold and it cannot be seen for the small analog such as 7b.
In summary the present study introduces that long chain and
lipophile 4-methoxyphenylacetic acid esters such as 7e behave as
the best SLO inhibitors (IC50 = 1.9 lM) and also as a selective inhib-
itor of 15-HLOb when compared with 15-HLOa. The importance of
these compounds could be more highlighted when we consider
their easy synthesis pathway and high yield.
3. Materials and methods
3.1. Chemistry
The methylenation of some carbonyl compounds using
Mg–TiCl4 in THF/CH2Cl2 was reported in last literatures.23 This
Mg–TiCl4-promoted CH2-transfer reaction of CH2Cl2 represents an
extremely simple, practical, and efficient methylenation of a vari-
ety of ketones and aldehydes, especially in enolizable or sterically
hindered ketones. Methylenation of compounds 1, 3 and 5 using
the mentioned method afforded the products 2, 4 and 6 in good
yields (76–88%).
The esters 7b–l were synthesized according to the pervious
work using carboxylic acids and alkyl halides in the presence of
DBU (1,8-diazabicyclo[5,4,0]undec-7-ene) in benzene.24
3.2. Molecular modeling, docking and SAR study
Figure 2. Above: stick view of the consensus bonding conformations of ester 7e
which has lipophilic interaction with amino acids of two pockets A (green symbols)
and B (blue symbols). Below: solvent surface view of pockets A (except for Ile772 and
B with 13(S)-proxy-9(Z)-2,11(E)-octadecadienoic acid (light brown
stick) and 7e (atom colored stick).
3.2.1. Multiple alignment
Conserved amino acids were identified through multiple align-
ment in clustalX 1.81.25 Sequences of lipoxygenase (LO) family
were selected from blasted sequences via ExPASY proteomics ser-
ver.26 Multiple alignment process was then carried out on the se-
lected sequences (protein weight matrix: BLOSUM series,
opening gap penalty = 10).
Leu773) and
and geometry of the non-heme iron-binding site are conserved.22
The structures of SLO and modeled 15-HLOa and 15-HLOb demon-
strate a high level of conservation within 14 Å (around the LA into
SLO X-ray view) in the active site pocket. Thus the structures of
both 15-HLO could be superposed on the SLO with RMS for the
3.2.2. Structure optimization
Structures 7a–l were simulated in CHEM3D professional; Cam-
bridge software; using MM2 method (RMS gradient = 0.05 kcal/
mol).27 Output files were minimized under semi-empirical AM1
method in the second optimization (convergence limit = 0.01; iter-
ation limit = 50; RMS gradient = 0.05 kcal/mol; Fletcher-Reeves
optimizer algorithm) in HYPERCHEM7.5.28,29
Crystal structure of soybean lipoxygenase-3 (arachidonate 15-
lipoxygenase) complex with 13(S)-hydroproxy-9(Z)-2,11(E)-octa-
decadienoic acid and rabbit 15-lipoxygensae (type a) complex with
(E)-3-(2-(oct-1-ynyl)phenyl)acrylic acid was retrieved from RCSB
Protein Data Bank (PDB entry: 1IK3 and 2P0 M, respectively).
In this study, Van der Waals molecular volume (MV) was mea-
sured by QSAR properties tool in HYPERCHEM7.5.28 The lipophilicity of
amino acids was taken from ExPASy26 (ProtScale) by applying
Hphob (Kyte and Doolittle hydropathicity).
C-a atoms of around 1.16–1.24 Å in the mentioned region
(Fig. 5). The largest differences between the SLO and both 15-
HLO active site pockets were found in residues of initial part of
a
2 helix (Fig. 5). In SLO,
a
2 helix consists of 18 residues (Leu273
–
–
Ala291) while the 15-HLOa and 15-HLOb include 16 (Glu176
Leu192) and 18 residues (Glu120–Gln128), respectively (Table 2).
In the active site pocket of the superposed SLO and both 15-
HLO, the C- of amino acids laying within 7 Å of the LA are well fit-
a
ted except for Leu273, Thr274 and Leu277 of SLO in contrast with
Ile173 and Asp174 of 15-HLOa (Fig. 6). Free space of catalytic region
around the pocket B seems to be larger for SLO and 15-HLOb in
comparison with 15-HLOa (Fig. 6). This lacking comes from steric
occupation of aromatic side chains of Phe353 and Phe552 in the
15-HLOa cavity. The data of Table 2 show that the pocket A of
SLO and 15-HLOb is more lipophile than its equal in 15-HLOa. This
is because of Arg403 and Gln596 in 15-HLOa in contrast with Leu560
and Ile772 in SLO and Leu415 and Leu609 in 15-HLOb. The similarity
between residues of SLO pocket A and the superposed in 15-HLOb,
3.2.3. Molecular docking
Automated docking simulation was implemented to dock 7a–l
into the active site of SLO with AUTODOCKTOOLS version 1.430 using
Lamarckian genetic algorithm.31 This method has been previously

H. Sadeghian et al. / Bioorg. Med. Chem. 17 (2009) 2327–2335
2331
Figure 3. Multiple alignment of SLO (1ik3_A). The conserved residues of two pockets A and B are highlighted in green and blue, respectively.
shown to produce bonding models similar to the experimentally
observed models.29,31,32 The torsion angles of the ligands were
identified, hydrogens were added to the macromolecule, bond dis-
tances were edited and solvent parameters were added to the en-
zyme 3D structure. Partial atomic charges were then assigned to
the macromolecule as well as ligands (Gasteiger for the ligands
and Kollman for the protein).
to the lowest-energy structures. After docking procedure in AD3,
docking results were submitted to Weblab Viewerlite 4.034 and
Swiss-PdbViewer 3.7 (spdbv)35 for further evaluations. The results
of docking processing (DGb: estimated free energy of bonding, Ed:
final docked energy and Ki: estimated inhibition constant) are out-
lined in Table 1.
The regions of interest of the enzyme were defined by consider-
ing Cartesian chart 19.9, 4.3 and 15.5 as the central of a grid size of
30, 30 and 40 points in X, Y and Z-axes. The docking parameter files
were generated using Genetic Algorithm and Local Search Parame-
ters (GALS) while number of generations was set to 100. Com-
pounds 7a–l were each docked into the active site of SLO
enzyme and the simulations were composed of 100 docking runs,
each of 50 cycles containing a maximum of 10,000 accepted and
rejected steps. The simulated annealing procedure was started at
high temperature (RT = 616 kcal/mol, where R is the gas constant
and T is the steady state temperature) and was decreased by a frac-
tion of 0.95 on each cycle33 The 100 docked complexes were clus-
3.2.4. Protein modeling
Three-dimensional models of the 15-HLOa and 15-HLOb se-
quences were constructed by homology modeling. BLAST se-
quence homology searches were performed in order to identify
the template proteins. The rabbit 15-lipoxygensae (type a) com-
plex with (E)-3-(2-(oct-1-ynyl)phenyl)acrylic acid (15-RLOa, PDB
entry: 2P0 M) was chosen as the template for modeling the pro-
teins. The identity of 15-HLOa and 15-HLOb with 15-RLOa is 77%
and 37%, respectively. Model building was performed in the pro-
gram MODELLER9V1 using model-ligand algorithm.1 Several models
at various refinement levels were generated and finally the re-
fined structures involved (E)-3-(2-(oct-1-ynyl)phenyl)acrylic acid
in the active site pocket, were minimized under molecular me-
chanic AMBER method (RMS gradient = 1) in HYPERCHEM7.5.28 All
tered with
a root-mean-square deviation tolerance of 0.2 Å.
Autodock generated 100 docked conformers of 7a–l corresponding

2332
H. Sadeghian et al. / Bioorg. Med. Chem. 17 (2009) 2327–2335
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
after addition of the enzyme and linoleic acid (final concentration:
134 M) as substrate at 20 1 °C. The final enzyme concentration
was 10 g/mL. Synthesized substances were added in DMSO solu-
7i
l
l
tions (final DMSO concentration 1%); whereas DMSO was added in
control experiments with no inhibitor. The mixture of each inhib-
itors and linoleic acid was set as blank sample in testing step. At
least six control test tubes and three tubes for each inhibitor solu-
tion were measured. To ensure constant enzyme activity through-
out the experiment, the enzyme solution was kept in ice, and
controls were measured at regular intervals. Calculation of enzyme
activity was carried out as previously described38 and IC50 values
were determined by linear interpolation between the points
around 50% activity (Table 1).
7l
7k
7a
7j
7h
7f
7g
7b
4. Experimental
4.1. Instruments
7c
R2 = 0.9135
The 1H NMR (100 MHz) spectra were recorded on a Bruker AC
100 spectrometer. Elemental analysis was obtained on a Thermo
Finnigan Flash EA microanalyzer. The IR spectra were obtained
on a 4300 Shimadzu Spectrometer. All measurements of lipoxyge-
nase activities were carried out using an Agilent 8453 spectropho-
tometer. The soybean 15-lipoxygenase and other chemicals were
purchased from Sigma, Aldrich and Merck Co., respectively. Human
recombinant 15-lipoxygenase a and b (I and II, expressed in E. coli)
was prepared from Cyman Chemical Co.
7e
0
7d
0
25
50
75 100 125 150 175 200 225 250 275
Ki (× 10-6)
Figure 4. Diagram of IC50 value versus Ki for compounds 7a–l.
4.2. General procedure for preparation of compounds 2, 4 and 6
To a 0 °C suspension consisting of Mg (1.92 g, 80 mmol), TiCl4
(3.79 g, 2.1 mL, 20 mmol), and CH2Cl2 (40 mL) was added over a
20 min period a solution of the corresponding ketone (10 mmol)
in CH2Cl2 (30 mL) and THF (20 mL). After being stirred for 30 min
at 0 °C, the resulting green–black mixture was stirred for an addi-
tional 20 min at room temperature. The reaction mixture was re-
cooled to 0 °C. Saturated potassium carbonate solution (100 mL)
was added and the mixture was diluted with ether (200 mL). The
organic layer was separated, dried, evaporated, and purified by
vacuum distillation.
4.2.1. 1-Methoxy-4-(2-methylallyl)benzene 2
Colorless oil; yield 88%; bp 76–78 °C/3 mm; 1H NMR (CDCl3): d
1.68 (s, 3H, –CH3), 3.27 (s, 2H, –CH2–), 3.80 (s, 3H, –OCH3), 4.81 (d,
J = 1.2 Hz, 1H, HC@C), 4.84 (d, J = 1.2 Hz, 1H, HC@C), 6.84 (d,
J = 8.6 Hz, 2H, H-3, H-5), 7.12 (d, J = 8.6 Hz, 2H, H-2, H-6); IR cmÀ1
:
1608 (C@C); Found: C, 81.21; H, 8.83. C11H14O requires: C, 81.44;
H, 8.70.
4.2.2. 1-Methoxy-4-(2-ethylallyl)benzene 4
Figure 5. Comparison of three 15-LO 3D structures (in ribbon view). C-
a of SLO,
Colorless oil; yield 83%; bp 83–84 °C/3 mm; 1H NMR (CDCl3): d
0.90 (t, 3H, J = 7.3 Hz, –CH3), 1.74 (q, 2H, J = 7.3 Hz, –CH2–), 3.27 (s,
2H, –CH2–), 3.80 (s, 3H, –OCH3), 4.81 (d, J = 1.2 Hz, 1H, HC@C), 4.84
(d, J = 1.2 Hz, 1H, HC@C), 6.84 (d, J = 8.6 Hz, 2H, H-3, H-5), 7.12 (d,
J = 8.6 Hz, 2H, H-2, H-6); IR cmÀ1: 1610 (C@C); Found: C, 82.01; H,
9.13. C12H16O requires: C, 81.77; H, 9.15.
modeled 15-HLOa and are superposed and colored in green, blue and red,
b
respectively. The active site pockets were mapped by inserting the consensus
structure of 7e.
models were validated using the program ERRAT at UCLA.36 The
best model had an ERRAT score of 87% and 83% for 15-HLOa and
15-HLOb, respectively.
4.2.3. 1,2,3,4-Tetrahydro-6-methoxy-2-methylenenaphthalen 6
Colorless oil; yield 76%; bp 120–123 °C/3 mm; 1H NMR (CDCl3):
3.3. 15-LO inhibitory assessment
d 2.45 (t, J = 6.5 Hz, 2H, (CH2)2C@C), 2.84 (t, J = 6.3 Hz, 2H,
(CH2)2C@C), 3.46 (s, 2H, –CH2–), 3.77 (s, 3H, –OCH3), 4.81 (d,
J = 1.2 Hz, 1H, HC@C), 4.84 (d, J = 1.2 Hz, 1H, HC@C), 6.65 (s, 1H,
H-5), 6.69 (d, J = 8.6 Hz, 1H, H-7), 6.99 (d, J = 8.6 Hz, 1H, H-8); IR
cmÀ1: 1612 (C@C); Found: C, 82.21; H, 8.08. C12H14O requires: C,
82.72; H, 8.01.
15-Lipoxygenase activity was measured in borate buffer solu-
tions (0.1 M, pH 9) for soybean enzyme and Tris–HCl (50 mM, pH
7.2) for human type enzyme using the method described in litera-
ture,37,38 by measuring the absorbance at 234 nm for 60–120 s

H. Sadeghian et al. / Bioorg. Med. Chem. 17 (2009) 2327–2335
2333
HLOb
Table 2
The Kyte and Doolittle hydropathicity (Hphob) and Van der Waals molecular volume (MV) of superposed amino acids within 7 Å around the LA into SLO X-ray view
SLO
HLOa
HLOb
SLO
HLOa
Leu565
Val566
Ile572
Leu408
Val409
Phe415
Leu420
Ile421
Leu273
Thr274
Leu277
Ile173
Gln119
Glu120
Ala123
Asp174
Val427
Val372
Ala237
Asn238
Arg349
Asp352
Phe353
His356
Glu357
Leu358
His361
Leu362
His366
Ile400
Arg221
Thr222
Arg361
Glu364
Phe365
His368
Glu369
Ala370
His373
Leu374
His378
Ile412
Phe576
His709
Asn713
Glu716
Met419
His541
His545
Glu548
Phe552
Ala558
Pro559
Cys560
Gln590
Ile593
Thr431
His553
Ser557
Gln560
Cys564
Leu570
Pro571
Pro572
Val603
Ala606
Leu607
Leu609
Leu610
Ile676
Asn373
Val506
Asp509
Ser510
His513
Gln514
Leu515
His518
Trp519
His523
Ile557
720
Gly
Arg726
Pro727
Thr728
Asp766
Val769
Ile770
Val594
Gln596
Leu597
Ile663
Ile772
Leu560
Ala561
Arg403
Ala404
Leu415
Ala416
Leu773
Ile857
AA
Hphob
MV
71.21
93.87
126.23
157.91
94.65
AA
Hphob
MV
AA
Hphob
MV
99.58
109.14
120.27
85.01
AA
Hphob
MV
Ala
Cys
His
Met
Thr
1.30
2.50
Arg
Gln
Ile
Phe
Trp
À4.50
À3.50
4.50
2.80
À0.90
151.63
116.24
119.85
144.37
170.47
Asn
Glu
Leu
Pro
Tyr
À3.50
À3.50
3.80
Asp
Gly
Lys
Ser
Val
À3.50
À0.40
À3.90
À0.80
4.20
92.73
54.52
134.37
78.04
À3.20
1.90
À1.60
À0.70
À1.30
149.97
103.62
AA = Amino acid.
4.3. General procedure for preparation of esters 7b–l
4.3.4. Pentyl 2-(4-methoxyphenyl)acetate 7e
Colorless oil; yield 70%; bp 148–149 °C/3 mm; 1H NMR
(CDCl3): 0.91 (t, J = 6.3 Hz, 3H, –CH3), 1.20–1.80 (m, 4H,
A mixture of corresponding alkyl bromide (45 mmol for pri-
mary and 54 mmol for secondary alkyl bromide), 4-methoxyphen-
ylacetic acid (4.98 g, 30 mmol), DBU (4.56 g, 30 mmol) and 60 mL
of benzene was refluxed for 3–6 h regarding the primary or sec-
ondary type of alkyl bromide. The reaction mixture was then
washed with water, dried over anhydrous magnesium sulfate
and distilled. For ester 7b, ethyl iodide was used and the reaction
time reduced to 2 h at room temperature.
d
–OCH2(CH2)2CH3), 3.56 (s, 2H, –CH2–), 3.80 (s, 3H, –OCH3), 4.08
(t, J = 6.4 Hz, 2H, –OCH2(CH2)2CH3), 6.87 (d, J = J = 8.7 Hz, 2H, H-
3, H-5), 7.18 (d, J = 8.7 Hz, 2H, H-2, H-6); IR cmÀ1
: 1735
(C@O); Found: C, 71.27; H, 8.61. C14H20O3 requires: C, 71.15;
H, 8.53.
4.3.5. Hexyl 2-(4-methoxyphenyl)acetate 7f
Ester 7j was synthesized by adding gradually 4-methoxyphenyl
acetyl chloride (1.84 g, 10 mmol) to a mixture of tert-butyl alcohol
(0.72 g, 9.7 mmol), triethylamine (1.2 g, 10 mmol) and dichloro-
methane (1.5 mL) follow refluxing for 1 h. Isolation and purification
of desired product was down according to the mentioned procedure.
Colorless oil; yield 71%; bp 160–161 °C/3 mm; 1H NMR (CDCl3):
d 0.88 (t, J = 6.3 Hz, 3H, –CH3), 1.13–1.81 (m, 8H, –OCH2(CH2)4CH3),
3.54 (s, 2H, –CH2–), 3.79 (s, 3H, –OCH3), 4.10 (t , J = 6.4 Hz, 2H,
–OCH2(CH2)2CH3), 6.86 (d, J = 8.6 Hz, 2H, H-3, H-5), 7.22 (d,
J = 8.6 Hz, 2H, H-2, H-6); IR cmÀ1: 1734 (C@O); Found: C, 72.31;
H, 8.92. C15H22O3 requires: C, 71.97; H, 8.86.
4.3.1. Ethyl 2-(4-methoxyphenyl)acetate 7b
Colorless oil; yield 86%; bp 103–105 °C/3 mm; 1H NMR (CDCl3):
d 1.23 (t, J = 7.1 Hz, 3H, –CH3), 3.52 (s, 2H, –CH2–), 3.77 (s, 3H,
–OCH3), 4.13 (q, J = 7.0 Hz, 1H, –OCH2–), 6.84 (d, J = 8.6 Hz, 2H,
H-3, H-5), 7.19 (d, J = 8.6 Hz, 2H, H-2, H-6); IR cmÀ1: 1733 (C@O);
Found: C, 68.09; H, 7.25. C11H14O3 requires: C, 68.02; H, 7.27.
4.3.6. Isopropyl 2-(4-methoxyphenyl)acetate 7g
Colorless oil; yield 81%; bp 120–122 °C/3 mm; 1H NMR (CDCl3): d
1.20 (d, J = 6.2 Hz, 6H, (H3C)2CHO–), 3.49 (s, 2H, –CH2–), 3.77 (s, 3H,
–OCH3), 4.78–5.15 (m, 1H, –OCH(CH3)2), 6.83 (d, J = 8.6 Hz, 2H, H-3,
H-5), 7.18 (d, J = 8.6 Hz, 2H, H-2, H-6); IR cmÀ1: 1735 (C@O); Found:
C, 69.18; H, 7.70. C12H16O3 requires: C, 69.21; H, 7.74.
4.3.2. Propyl 2-(4-methoxyphenyl)acetate 7c
Colorless oil; yield 82%; bp 122–124 °C/2 mm; 1H NMR (CDCl3):
d 0.90 (t, J = 7.2 Hz, 3H, –CH3), 1.45–1.85 (m, 2H, –OCH2CH2CH3),
3.54 (s, 2H, –CH2–), 3.78 (s, 3H, –OCH3), 4.03 (t, J = 6.8 Hz, 2H,
–OCH2CH2CH3), 6.84 (d, J = 8.6 Hz, 2H, H-3, H-5), 7.19 (d,
J = 8.6 Hz, 2H, H-2, H-6); IR cmÀ1: 1735 (C@O); Found: C, 69.29;
H, 7.80. C12H16O3 requires: C, 69.21; H, 7.74.
4.3.7. sec-Butyl 2-(4-methoxyphenyl)acetate 7h
Colorless oil; yield 83%; bp 127–128 °C/3 mm; 1H NMR (CDCl3):
d 0.86 (t, J = 7.1 Hz, 3H, –OCH(CH3)CH2CH3), 1.20 (d, J = 6.06 Hz, 3H,
–OCH(CH3)CH2CH3), 1.41–1.75 (m, 2H, –OCH(CH3)CH2CH3), 3.54 (s,
2H, –CH2–), 3.79 (s, 3H, –OCH3), 4.68–5.03 (m, 1H,
–OCH(CH3)CH2CH3), 6.86 (d, J = 8.6 Hz, 2H, H-3, H-5), 7.22 (d,
J = 8.6 Hz, 2H, H-2, H-6); IR cmÀ1: 1735 (OC@O); Found: C, 70.39;
H, 8.13. C13H18O3 requires: C, 70.24; H, 8.16.
4.3.3. Butyl 2-(4-methoxyphenyl)acetate 7d
Colorless oil; yield 80%; bp 136–138 °C/3 mm; 1H NMR (CDCl3):
d 0.92 (t, J = 6.3 Hz, 3H, –CH3), 1.18–1.85 (m, 4H, –OCH2(CH2)2CH3),
3.58 (s, 2H, –CH2–), 3.93 (s, 3H, –OCH3), 4.07 (t, J = 6.4 Hz, 2H,
–OCH2(CH2)2CH3), 6.85 (d, J = 8.7 Hz, 2H, H-3, H-5), 7.21 (d,
J = 8.7 Hz, 2H, H-2, H-6); IR cmÀ1: 1735 (C@O); Found: C, 70.21;
H, 8.06. C13H18O3 requires: C, 70.24; H, 8.16.
4.3.8. Isobutyl 2-(4-methoxyphenyl)acetate 7i
Colorless oil; yield 79%; bp 129–131 °C/3 mm; 1H NMR (CDCl3):
d 0.87 (d, J = 6.6 Hz, 6H, (H3C)2CHCH2O–), 1.71–2.14 (m, 1H,
–OCH2CH(CH3)2), 3.54 (s, 2H, –CH2–), 3.76 (s, 3H, –OCH3), 3.85
(d, J = 6.6 Hz, 2H, –OCH2CH(CH3)2), 6.83 (d, J = 8.6 Hz, 2H, H-3,

2334
H. Sadeghian et al. / Bioorg. Med. Chem. 17 (2009) 2327–2335
Figure 6. Stick view of LA bond to Fe and C-
a
superposed amino acids of 15-HLOa (blue), 15-HLOb (red) and SLO (green) which have high differences in molecular volume
and lipophilicity.
H-5), 7.20 (d, J = 8.6 Hz, 2H, H-2, H-6); IR cmÀ1: 1735 (C@O);
Found: C, 70.17; H, 8.19. C13H18O3 requires: C, 70.24; H, 8.16.
4.3.11. Cyclohexyl 2-(4-methoxyphenyl)acetate 7l
Colorless oil; yield 74%; bp 155–156 °C/2 mm; 1H NMR (CDCl3):
d 1.18–2.00 (m, 10H, –CH2– (cyclohexyl)), 3.54 (s, 2H, –CH2–), 3.80
(s, 3H, –OCH3), 4.61–4.93 (m, 1H, –CH– (cyclohexyl)), 6.86
(d, J = 8.6 Hz, 2H, H-3, H-5), 7.22 (d, J = 8.6 Hz, 2H, H-2, H-6);
IR cmÀ1: 1735 (C@O); Found: C, 72.31; H, 8.05. C15H20O3 requires:
C, 72.55; H, 8.12.
4.3.9. tert-Butyl 2-(4-methoxyphenyl)acetate 7j
Colorless oil; yield 48%; bp 89–92 °C/3 mm; 1H NMR (CDCl3): d
1.44 (s, 9H, (H3C)3CO–), 3.46 (s, 2H, –CH2–), 3.78 (s, 3H, –OCH3),
6.85 (d, J = 8.6 Hz, 2H, H-3, H-5), 7.22 (d, J = 8.6 Hz, 2H, H-2, H-
6); IR cmÀ1: 1738 (OC@O); Found: C, 70.33; H, 8.10. C13H18O3 re-
quires: C, 70.24; H, 8.16.
References and notes
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