Synthesis and SAR comparative studies of 2-allyl-4-methoxy-1-alkoxybenzenes as 15-lipoxygenase inhibitors

Article abstract of DOI:10.3109/14756366.2010.495717

A group of 2-alkoxy-5-methoxyallylbenzene were designed, synthesised and evaluated as potential inhibitors of the soybean 15-lipoxygenase (SLO) on the basis of the eugenol and esteragol structures. Compound 4d showed the best half maximal inhibitory concentration (IC₅₀) for SLO inhibition (IC ₅₀=5.9±0.6 μM). All the compounds were docked in the SLO active site retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB entry: 1IK3) and showed that the allyl group of the synthetic compounds similar to the linoleic acid double bond, were oriented toward the Fe³⁺-OH moiety in the active site of the enzyme and this conformation was especially fixed by the hydrophobic interaction of the 2-alkoxy group with Leu⁵¹⁵, Trp⁵¹⁹, Val ⁵⁶⁶ and Ile⁵⁷². It was concluded that the molecular volume and shape of the alkoxy moiety was a major factor in the inhibitory potency variation of the synthetic compounds.

Full text of DOI:10.3109/14756366.2010.495717

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(2): 238–244
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2010.495717
rESEarCH artICLE
Synthesis and SAR comparative studies of 2-allyl-4-methoxy-1-
alkoxybenzenes as 15-lipoxygenase inhibitors
Hamid Sadeghian1, Seyed Mohammad Seyedi2, Neda Attaran2, Atena Jabbari2, and Zeinab Jafari2
1Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad,
IR Iran, and 2Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, IR Iran
abstract
A group of 2-alkoxy-5-methoxyallylbenzene were designed, synthesised and evaluated as potential inhibitors of the
soybean 15-lipoxygenase (SLO) on the basis of the eugenol and esteragol structures. Compound 4d showed the best
half maximal inhibitory concentration (IC50) for SLO inhibition (IC50 =5.9 0.6 µM). All the compounds were docked in
the SLO active site retrieved from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank
(PDB entry: 1IK3) and showed that the allyl group of the synthetic compounds similar to the linoleic acid double
bond, were oriented toward the Fe3+-OH moiety in the active site of the enzyme and this conformation was especially
fixed by the hydrophobic interaction of the 2-alkoxy group with Leu515, Trp519, Val566 and Ile572. It was concluded that
the molecular volume and shape of the alkoxy moiety was a major factor in the inhibitory potency variation of the
synthetic compounds.
Keywords: Eugenol, SLO, docking, linoleic acid
Introduction
shown to be pro-inflammatory [11] and pro-thrombotic
[12]. It has also been found that 15-LO is involved in the
oxidative modification of low-density lipoproteins (LDL)
leading to the development of atherosclerosis [13].
ree different strategies have been developed for
inhibiting LO activity [12]. ey involve (i) redox inhibi-
tors or antioxidants, 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.
ere is reasonable homology between SLO and the
human LO [14]. is homology becomes more identical
(50%) within 8Å of the active site pocket. e soybean
enzyme is much more accessible than the human ver-
sion and therefore, the results could be extendable to
human LO.
Lipoxygenases (LOs) are non-haem iron-containing
enzymes that cause oxidation of polyunsaturated fatty
acids and esters to their hydroperoxy derivatives [1].
ese families of enzymes widely exist in all plant and
animal species [2], and are named according to the oxi-
disedpositionofthecommonsubstrate, arachidonicacid
(AA). Among the mammalian lipoxygenases involved
in human disease, 5-lipoxygenase (5-LO) is now well
documented as a target for reducing the biosynthesis
of leukotrienes [3,4]. e 15-lipoxygenase (15-LO) has
emerged as an attractive target for therapeutic studies
[5], it has been implicated in the progression of certain
cancers [6,7] and chronic obstructive pulmonary dis-
ease (COPD) [6]. Evidence for the inhibition of 15-LO
in the treatment of vascular disease is, however, most
compelling [8] and further studies have shown a role for
15-LO in atherogenesis [9,10]. e enzyme is abundantly
expressed in macrophages residing within the athero-
sclerotic lesion [5]. In addition, the immediate products
of 15-LO oxidation of AA and linoleic acid (LA) have been
Recently we reported the results of our studies on the
SLO inhibitory activities of some eugenol esters and on
the basis of structure activity relationship (SAR) studies.
We suggested that the inhibitory activity of these mole-
cules largely depends on the orientation of the allyl group
Address for Correspondence: Seyed Mohammad Seyedi, Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad,
Mashhad, IR Iran. Tel: +9805118795162; Fax: +9805118795560; E-mail: smseyedi@yahoo.com
(Received 20 March 2010; revised 15 May 2010; accepted 18 May 2010)
238

Synthesis and SAR comparative studies of 2-allyl-4-methoxy-1-alkoxybenzenes 239
toward the chelated Fe3+-OH and the molecular volume
then assigned to the macromolecule as well as ligands
(Gasteiger for the ligand and Kollman for the protein).
e regions of interest of the enzyme were defined by
considering the Cartesian chart 20.6, 7.2 and 20.5 as the
centre of a grid size of 50, 60 and 60 points in the X, Y
and Z axes. e docking parameter files were generated
using Genetic Algorithm and Local Search Parameters
(GALS) while the number of generations was set to 100.
e 100 docked complexes were clustered with a root-
mean-square deviation tolerance of 1 Å. Autodock gen-
erated 100 docked conformers of 4a–j, corresponding to
the lowest-energy structures. After the docking proce-
dures in ADT4, the docking results were submitted to
the Weblab Accelrys DS Visualizer 2.0.1 [31] and Swiss-
PdbViewer 3.7 (spdbv) [32] for further evaluations.
e results of the docking processing (Ki: Estimated
Inhibition Constant) are outlined in Table I.
of the carboxylate moiety in the active site pocket of the
enzyme without hydroperoxidation of allylic carbons
[14]. In this paper we report the results of a compara-
tive study on the 15-LO inhibitory activities of 2-alkoxy
derivatives of 5-methoxyallylbenzene.
materials and methods
Chemistry
Compound 2 was synthesised by the allylation of 4-meth-
oxyphenol (1) in the presence of saturated potassium car-
bonate in water [18]. Compound 3 was synthesised using
the Claisen rearrangement on compound 2[19]. e ethers
4b–j were prepared by the reaction of the desired alkylbro-
mides with phenol 3 in the presence of sodium ethoxide
in ethanol [20]. Compound 4a and methylisoeugenol were
synthesised by the methylation of phenol 3and isoeugenol
[21] using dimethylsulphate [22]. All of the products 4a–j
were purified by column chromatography (Silicagel 230–
400, eluent: hexane/dichloromethane 50:50).
15-LO inhibitory assessment
Lipoxygenase activity was measured in borate buffer
solutions (0.1 M, pH 9) using the method described in the
literature [33,34], by measuring the absorbance at 234 nm
for 60 s after addition of the enzyme (soybean 15-lipoxy-
genase) and linoleic acid (final concentration: 134 µM)
as substrate at 20 1°C. e final enzyme concentration
was 167 U/mL. e synthesised substances were added
as DMSO solutions (final inhibitor concentrations: 200,
100, 50, 25, 12.5, 6.25, 3.12 and 1.5 µM; final DMSO con-
centration 1%); whereas DMSO was added in the control
experiments with no inhibitor. e mixture of each inhib-
itor and linoleic acid were set as the blank sample in the
testing step. At least six control test tubes and three tubes
for each inhibitor solution were measured. To ensure a
constant enzyme activity throughout the experiment, the
enzyme solution was kept on ice, and the controls were
measured at regular intervals. e calculation of enzyme
activity was carried out as previously described [34] and
the IC50 values were determined by using a sigmoidal
dose-response curve in Graphpad Prism 3.02 [35].
Molecular modelling, docking and SAR study
Multiple alignment
e conserved amino acids were identified through mul-
tiple alignment in clustalX 1.81 [23]. e sequences of
the lipoxygenase (LO) family were selected from blasted
sequences using the ExPASY proteomics server [24]. e
multiple alignment process was then carried out on the
selected sequences (protein weight matrix: BLOSUM
series, opening gap penalty = 10).
Calculations
e structures of the desired compounds were drawn
using the ChemOffice professional (Cambridge software)
[25]. e output files were minimised under the molecular
mechanic MM+ in HyperChem7.5 [26]. enthestructures
were minimised under the semi-empirical AM1 method
in the second optimisation (Convergence limit=1e–5;
Iterationlimit=100;RMSgradient=0.05kcal/mol;Fletcher-
Reeves optimiser algorithm) in HyperChem7.5 [26,27].e
crystal structure of the soybean lipoxygenase-3 (arachido-
nate 15-lipoxygenase) complex with 13(S)-hydroperoxy-
9(Z)-2,11(E)-octadecadienoic acid was retrieved from
the Research Collaboratory for Structural Bioinformatics
(RCSB) Protein Data Bank (PDB entry: 1IK3).
Experimental Section
Instruments
Melting points were recorded on an Electrothermal type
1
9100 melting point apparatus. e H NMR (100 MHz)
spectra were recorded on a Bruker AC-100 spectrometer.
Elemental analysis was obtained on a ermo Finnigan
Flash EA microanalyser. e IR spectra were obtained
on a 4300 Shimadzu Spectrometer. All measurements of
lipoxygenase activities were carried out using an Agilent
8453 photodiode array spectrophotometer. e soybean
15-lipoxygenase, eugenol, methyleugenol and other
chemicals were purchased from Fluka and Merck.
Molecular docking
e automated docking simulation was implemented
to dock 4a–j into the active site of SLO using the
AutoDockTools (ADT) version 1.5.4 [15] with the
Lamarckian genetic algorithm [28]. is method has
been previously shown to produce bonding models
similar to the experimentally observed models [14,
28–30]. e 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 enzyme 3D structure. Partial atomic charges were
1-Allyloxy-4-methoxybenzene (2)
A
mixture of 25.2g (0.2mol) of 4-methoxyphenol,
26.4g (0.22mol) of allylbromide, and 28g of potassium
© 2011 Informa UK, Ltd.

240 Hamid Sadeghian et al.
carbonate (0.2mol) in 40mL water was refluxed for 5
hours and cooled. e mixture was diluted with 100mL of
water and extracted with ether (2×50mL). e combined
extracts were washed with 10% NaOH (2×50mL) and
dried with anhydrous potassium carbonate. After removal
of the solvent, the residual oil was distilled under reduced
pressure.
2-Allyl-1-ethoxy-4-methoxybenzene (4b)
Colourless oil; yield: 79%; 1HNMR (CDCl3): δ 1.42 (t,
J= 6.9 Hz, 3H, -CH3), 3.41 (d, J= 6.2 Hz, 2H, -CH2-), 3.78
(s, 3H, -OCH3), 4.01 (q, J= 6.9 Hz, 2H, -OCH2CH3), 5–5.22
(m, 2H, H2C=), 5.81–6.25 (m, 1H, HC=), 6.62–6.89 (m, 3H,
H-3, H-5, H-6); IR cm−1: 2950 (C=O). Found: C, 74.92; H,
8.33. C12H16O2 requires: C, 74.97; H, 8.39%.
1
Colourless oil; yield: 78%; bp: 118°C/2 mm : HNMR
2-Allyl-4-methoxy-1-propoxybenzene (4c)
(CDCl3): δ 3.78(s, 3H, -OCH3), 4.49 (d, J= 6.2 Hz, 2H,
-CH2-), 5.2–5.52 (m, 2H, H2C=), 5.82–6.25 (m, 1H, HC=),
6.74–6.93 (m, 3H, H-3, H-5, H-6); IR cm−1: 2850 (C=O).
1
Colourless oil; yield: 75% : HNMR (CDCl3): δ 1.05 (t,
J = 7.3 Hz, 3H, -CH3), 1.62–2 (m, 2H, -OCH2CH2CH3),
3.39 (d, J = 6.6 Hz, 2H, -CH2-), 3.76 (s, 3H, -OCH3), 3.88
(t, J = 6.3 Hz, 2H, -OCH2CH2CH3), 5–5.23 (m, 2H, H2C=),
5.8–6.23 (m, 1H, HC=), 6.6–6.84 (m, 3H, H-3, H-5, H-6);
IR cm−1: 2950 (C=O). Found: C, 75.89; H, 8.83. C13H18O2
requires: C, 75.69; H, 8.80%.
2-Allyl-4-methoxyphenol (3)
e allyl ether 3 (35 g) was refluxed for 3 hours under
a nitrogen atmosphere, then cooled. e oil was dis-
solved in 100 mL of ether and the solution was extracted
with 10% NaOH (3 × 50 mL). e combined alkaline
extracts were then acidified with 50 mL of concentrated
HCl diluted with 50 mL of water, and the mixture was
extracted with ether (3 × 50 mL). e combined ether
extracts were dried with anhydrous sodium sulphate,
evaporated and the residual oil was distilled under
reduced pressure.
2-Allyl-1-isopropoxy-4-methoxybenzene (4d)
Colourlessoil;yield:50%;1HNMR(CDCl3):δ1.32(d, J= 6.1
Hz, 6H, (H3C)2CHO-), 3.38 (d, J= 6.6 Hz, 2H, -CH2-), 3.75
(s, 3H, -OCH3), 4.25–4.58 (m, 1H, -OCH(CH3)2), 4.97–5.21
(m, 2H, H2C=), 5.78–6.19 (m, 1H, HC=), 6.59–6.87 (m, 3H,
H-3, H-5, H-6); IR cm−1: 2950 (C=O). Found: C, 75.77; H,
8.74. C13H18O2 requires: C, 75.69; H, 8.8%.
1
Colourless oil; yield: 70%; bp: 120°C/2 mm : HNMR
(CDCl3): δ 3.38 (d, J= 6.2 Hz, 2H, -CH2-), 3.76 (s, 3H,
-OCH3), 4.64 (s, 1H, -OH), 5.04–5.25 (m, 2H, H2C=), 5.8–
6.26 (m, 1H, HC=), 6.59–6.97 (m, 3H, H-3, H-5, H-6); IR
cm−1: 2850 (C=O), 3400 (O-H).
2-Allyl-1-butoxy-4-methoxybenzene (4e)
Colourless oil; yield: 53%; 1HNMR (CDCl3): δ 0.95 (t,
J= 6.8 Hz, 3H, -CH3), 1.23–1.94 (m, 4H, -OCH2(CH2)2CH3),
3.35 (d, J= 6.6 Hz, 2H, -CH2-), 3.74 (s, 3H, -OCH3), 3.89 (t,
J= 6.2 Hz, 2H, -OCH2(CH2)2CH3), 4.94–5.18 (m, 2H, H2C=),
5.78–6.21 (m, 1H, HC=), 6.57–6.81 (m, 3H, H-3, H-5, H-6);
IR cm−1: 2950 (C=O). Found: C, 76.47; H, 9.14. C14H20O2
requires: C, 76.33; H, 9.15%.
General procedure for preparation of compounds (4a–j)
A mixture of 0.6g (8.8 mmol) of sodium ethoxide in 5mL
ethanol, 1.4g (8.8 mmol) of ally ether 3 and 11 mmol of
the desired alkyl bromide (for compounds 4a and 4b dim-
ethyl sulphate and ethyl iodide were used, respectively)
was refluxed for 1hour (2 hours for compound 4a). After
cooling, the mixture was poured into cold water (10mL).
e mixture was acidified with 20% HCl and then extracted
with ether (2×10mL). e combined extracts were washed
with 10% NaOH (2×10mL) dried with anhydrous sodium
sulphate and evaporated then the residual oil was purified
by column chromatography.
2-Allyl-1-sec-butoxy-4-methoxybenzene (4f)
Colourlessoil;yield:73%;1HNMR(CDCl3):δ0.86(t,J=7.1Hz,
3H, -OCH(CH3)CH2CH3), 1.2 (d, J=6.1 Hz, 3H, -OCH(CH3)
CH2CH3), 1.41–1.75 (m, 2H, -OCH(CH3)CH2CH3), 3.74 (s,
3H, -OCH3), 3.89 (m, 1H, -OCH (CH3)CH2CH3), 4.94–5.18
(m, 2H, H2C=), 5.78–6.21 (m, 1H, HC=), 6.57–6.81 (m, 3H,
H-3, H-5, H-6); IR cm−1: 2950 (C=O). Found: C, 76.37; H,
9.19. C14H20O2 requires: C, 76.33; H, 9.15%.
2-Allyl-1-isobutoxy-4-methoxybenzene (4g)
2-Allyl-1,4-dimethoxybezene (4a)
Colourless oil; yield: 70%; 1HNMR (CDCl3): δ 1.32
(d, J = 6.1 Hz, 6H, (H3C)2CHO-), 3.38 (d, J = 6.6 Hz,
2H, -CH2-), 3.75 (s, 3H, -OCH3), 4.25–4.58 (m, 1H,
-OCH(CH3)2), 4.97–5.21 (m, 2H, H2C=), 5.78–6.19 (m,
1H, HC=), 6.59–6.87 (m, 3H, H-3, H-5, H-6); IR cm−1:
Colourless oil; yield: 74%; 1HNMR (CDCl3): δ 3.35 (d,
J= 6.2 Hz, 2H, -CH2-), 3.74 (s, 3H, -OCH3), 3.76 (s, 3H,
-OCH3), 4.94–5.2 (m, 2H, H2C=), 5.76–6.2 (m, 1H, HC=),
6.62–6.9 (m, 3H, H-3, H-5, H-6); IR cm−1: 2850 (C=O).
O
O
O
O
OH
O
O
O
Eugenol
Methyl eugonol
Methyl isoeugenol
2,5-Dimethoxyallylbenzene
Figure 1. Molecular structure of some of allylbenzene analogues as lipoxygenase inhibitors.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and SAR comparative studies of 2-allyl-4-methoxy-1-alkoxybenzenes 241
OH
O
OH
K2CO3 (aq)
Allylbromide
Reflux
O
O
O
1
2
3
EtONa/EtOH
R-Br
Compound
Compound
R
R
4a
4b
4c
4d
4e
Methyl
4f
4g
4h
4i
sec-Butyl
iso-Butyl
R
O
Ethyl
n-Propyl
iso-Propyl
n-Butyl
tert-Butyl
Cyclopantyl
Cyclohexyl
O
4j
4a-j
Scheme 1. General procedures for the synthesis of compounds 4a–j.
2950 (C=O). Found: C, 76.27; H, 9.1. C14H20O2 requires:
C, 76.33; H, 9.15%.
Table 1. Enzyme inhibitory assessment and docking analysis data
of consensus conformers of eugenol, methyl eugenol, methyl
isoeugenol and compounds 4a–j. Ki (M): Estimate d Inhibition
Constant. e IC50 (µM) values are given as mean SD.
2-Allyl-1- tert-butoxy-4-methoxybenzene (4h)
Compound
IC50 (μM)
34.1 2.1
96.1 3.3
138.9 3.1
93.1 5.9
91.1 4.2
34.3 2.3
5.9 0.6
Ki (μM)
-
Colourless oil; yield: 18%; 1HNMR (CDCl3): δ 1.37 (s, 9H,
-CH3), 3.39 (d, J= 6.1 Hz, 2H, -CH2-), 3.74 (s, 3H, -OCH3),
4.97–5.27 (m, 2H, H2C=), 5.62–6.21 (m, 1H, HC=), 6.30–
7.02 (m, 3H, H-3, H-5, H-6); IR cm−1: 2950 (C=O). Found:
C, 76.19; H, 9.09. C14H20O2 requires: C, 76.33; H, 9.15%.
Eugenol
Methyleugenol
-
Methylisoeugenol
-
4a
4b
4c
4d
4e
4f
31.88
27.18
3.88
1.14
17.16
7.91
3.43
33.15
14.12
13.81
2-Allyl-1-cyclopentyloxy-4-methoxybenzene (4i)
1
Colourless oil; yield: 65%; HNMR (CDCl3): δ 1.57–1.63
58.6 3.7
20.8 1.4
35.8 3.4
11.4 0.6
40.2 3.7
44.1 2.6
(m, 8H, -CH2- (cyclopentyl)), 3.33 (d, J=6.6 Hz, 2H, -CH2-),
3.75 (s, 3H, -OCH3), 4.82–4.6 (m, 1H, -CH- (cyclopentyl)),
4.97–5.2 (m, 2H, H2C=), 5.77–6.22 (m, 1H, HC=), 6.54–6.74
(m, 3H, H-3, H-5, H-6); IR cm−1: 2950 (C=O). Found: C,
77.45; H, 8.64. C15H20O2 requires: C, 77.55; H, 8.68%.
4g
4h
4i
4j
2-Allyl-1-cyclohexyloxy-4-methoxybenzene (4j)
Colourless oil; yield: 63% : HNMR (CDCl3): δ 1.24–2.13
(m, 10H, -CH2-(cyclohexyl)), 3.37(d, J= 6.6Hz, 2H, -CH2-),
3.74 (s, 3H, -OCH3), 3.95–4.22 (m, 1H, -CH- (cyclohexyl)),
4.92–5.2 (m, 2H, H2C=), 5.76–6.15 (m, 1H, HC=), 6.54–6.88
(m, 3H, H-3, H-5, H-6); IR cm−1: 2950 (C=O). Found: C,
78.13; H, 9.04. C16H22O2 requires: C, 78.01; H, 9%.
1
among the dimethoxy compounds, methyl isoeugenol
showed a reduced inhibitory activity. It is notable that no
other products such as hydroperoxy were isolated from
the action of the LO enzyme on the methylated com-
pounds as substrate (assuming hydroproxy is supposed
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).†
results and discussion
Considering the IC50 results of the above compounds,
and approved site direction of the allyl group in the active
site pocket of SLO, we decided to synthesis other 2-alkyl
analogues of 2,5-dimethoxyallylbenzene to investigate the
interactive effect of the 2-alkyl moiety with the lipophilic
pocket formed by the conserved residues Leu515, Val566,
Leu565 Ile572 and Leu773 on the inhibitory potency. To
investigate this, some 2-alkyl and cycloalkyl analogues
of 2,5-dimethoxyallylbenzene (compounds 4a–j) were
synthesised) and their inhibitory potency was deter-
mined. e synthetic ethers 4a–j showed a broad range of
Considering our previous work on eugenol and esters
[14], we tested the inhibitory properties of eugenol,
methyl eugenol, methyl isoeugenol and 2,5-dimethoxy-
allylbenzene on SLO (substrate: linoleic acid) to study
the effect of the methoxy group position on the inhibi-
tory activity. e results showed half maximal inhibitory
concentration (IC50) of 34.1 2.1, 96.1 3.3, 138.9 3.1
and 93.1 2.9 µM for the above mentioned compounds,
respectively (Table I). It was found that the protection
of the hydroxyl group of eugenol decreased its IC50 and
© 2011 Informa UK, Ltd.

242 Hamid Sadeghian et al.
inhibition activity on the enzyme (IC50=5.9 to 93.1 µM,
see Table I). Compound 4d, with an isopropyl substitu-
ent, was the most potent inhibitor at 5.9 0.6 µM while the
methoxy and ethoxy analogues (4a and 4b) showed less
activity (IC50 =93.1 2.9 and 91.1 3.2µM, respectively).
e secondary ethers (4d, 4f, 4i and 4j) showed the best
inhibitory activity in comparison with the primary types.
Among the butoxy analogues, the tertiary type (4h) pos-
sessed the greatest inhibitory potential with an IC50 value
of 11.4 0.6 µM. e results indicated that an increase in
the C-O substituent number led to an improvement in
inhibitory potency for this series of compounds.
e binding affinity of compounds 4a–j towards
SLO was studied. 100 docked conformers of the desired
compounds were generated using the AutoDockTools
software in which the side chain of Leu515, Val566, Leu565
Ile572 and Leu773 was flexibilised [15]. e experimental
results matched with the theoretical Ki for the docking
study for those models in which the allylic double bond
oriented toward the iron atom in a similar orientation to
that found in linoleic acid (LA) in the active site of SLO.
One conformer from each ether cluster which had more
similarity with the optimum conformer (lowest Ki) of 4d
was adopted as the “consensus” structure and used for
further analysis [14,16].
Leu 515
Vel 566
Ile 572
e results of the docking analysis showed that the
consensus structure of 4a–j had an estimated inhibitory
constant (Ki) in the range of 1 to 33 × 10−6 M (Table I). It
appeared that the allyl benzene portion of the consensus
structure has a hydrophobic interaction with the back-
bone of the Trp519, Ile557, Leu565, Ile572, and Leu773 in such
an orientation. e Ile557 → Ala and Ile572 → Phe mutants
decreased the kcat by two fold from the WT (wild type),
While Leu565 → Ala and Leu773 → Ala decreased the kcat by
60 and 1000 fold respectively, indicating that these hydro-
phobic residues (specially Leu565 and Leu773) contributed
significantly to catalysis [17]. Mutating the residues Ile or
Leu to Ala, shape an empty space in the binding pocket
of SLO, led to a decreased H• transfer kinetics [17].
Leu 565
Fe
Leu 773
Figure 2. Stick (right) and solvent surface (left) view of flexible
residues, surrounding the consensus bonding conformations of
compounds 4a–j in the SLO active site.
Figure 3. Multiple alignment of SLO (1ik3_A). e residues which have lipophilic interactions with docked ligands are highlighted in green respectively.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and SAR comparative studies of 2-allyl-4-methoxy-1-alkoxybenzenes 243
100
80
60
40
20
0
4a
4b
4e
Primary ether
Secondary ether
Tertiary ether
Leu 565
4j
4i
4g
4c
4f
4h
4d
0
10
20
30
40
Leu 773
Ki (µM)
Figure 4. Diagram of IC50 value versus Ki for consensus structure of
compounds 4a–j.
Ile 557
Fe
According to the multiple alignment results, the amino
acids Ile557, Leu565 and Leu773 were found to be conserved
over all species (Figure 3).
Figure 5. Stick view of the consensus bonding conformation of
compound 4d which has lipophilic interaction through its isopropyl
moiety with hydrophobic pockets (surface view) formed by Leu515
,
Trp519,Val566 andIle572 sidechain.Inthisfigurewecanseetheorientation
of linoleic acid (green stick) bonded to Fe via proxy bridge.
e Ki variations of the consensus structure of the pri-
mary and secondary ethers were distinct, in contrast with
the IC50 changes (Figure 4). is relation comes from the
tendency of the alkyl moiety to fill the empty lipophilic
space formed by the Leu515, Trp519, Val566 and Ile572 side
chains. e docking results and IC50 values of the syn-
thetic ethers, indicated that the aforementioned lipo-
philic cavity of SLO has an organised space for accepting
the isopropyl group in comparison with the other struc-
tural isomer.
In summary, we carried out SAR comparative studies
on some methoxyallylbenzene derivatives as 15-lipox-
ygenase inhibitors. We suggest that the application of
docking results such as Ki can be applied to predict the
inhibitory potency of the synthetic ethers. is study has
also shown the important role of molecular structure in
the inhibitory activity of 2-alkoxy-5-methoxyallylben-
zene. e importance of these compounds is highlighted
for having a straightforward synthesis pathway with a
high yield.
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declaration of interest
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e authors report no conflicts of interest. e authors
alone are responsible for the content and writing of the
paper.
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BN,
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