Design, synthesis and SAR studies of 4-allyoxyaniline amides as potent 15-lipoxygensae inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.12.065

A group of 4-allyloxyaniline amides 5a-o were designed, synthesized and evaluated as potential inhibitors of soybean 15-lipoxygenase (SLO) on the basis of eugenol and esteragol structures. Compound 5e showed the best IC₅₀ in SLO inhibition (IC₅₀ = 0.67 ± 0.06 μM). All compounds were docked in SLO active site retrieved from RCSB Protein Data Bank (PDB entry: 1IK3) and showed that allyloxy group of compounds is oriented towards the Fe³⁺-OH moiety in the active site of enzyme and fixed by hydrogen bonding with two conserved His⁵¹³ and Gln⁷¹⁶. It is resulted that molecular volume of the amide moiety would be a major factor in inhibitory potency variation of the synthetic amides, where the hydrogen bonding of the amide group could also involve in the activity of the inhibitors.

Full text of DOI:10.1016/j.bmc.2008.12.065

Bioorganic & Medicinal Chemistry 17 (2009) 1614–1622
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and SAR studies of 4-allyoxyaniline amides as potent
15-lipoxygensae inhibitors
a
a
a
Seyed Mohammad Seyedi a, , Zeinab Jafari , Neda Attaran , Hamid Sadeghian ,
*
Mohammad Reza Saberi b, Mohammad Mahdi Riazi a
a Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad 917751436, Islamic Republic of Iran
b School of Pharmacy, Pharmaceutical Research Center, Mashhad University of Medical Sciences, BuAli Square, Mashhad 9196773117, Islamic Republic of 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-allyloxyaniline amides 5a–o were designed, synthesized and evaluated as potential inhibi-
tors of soybean 15-lipoxygenase (SLO) on the basis of eugenol and esteragol structures. Compound 5e
showed the best IC50 in SLO inhibition (IC50 = 0.67 0.06 lM). All compounds were docked in SLO active
site retrieved from RCSB Protein Data Bank (PDB entry: 1IK3) and showed that allyloxy group of com-
pounds is oriented towards the Fe3+-OH moiety in the active site of enzyme and fixed by hydrogen bond-
ing with two conserved His513 and Gln716. It is resulted that molecular volume of the amide moiety would
be a major factor in inhibitory potency variation of the synthetic amides, where the hydrogen bonding of
the amide group could also involve in the activity of the inhibitors.
Received 12 November 2008
Revised 26 December 2008
Accepted 30 December 2008
Available online 6 January 2009
Keywords:
Eugenol
Esteragol
SLO
Ó 2009 Elsevier Ltd. All rights reserved.
Docking
Linoleic acid
1. Introduction
low-density lipoproteins (LDL) leading to the development of
atherosclerosis.13
It is well documented that mammalian lipoxygenases (LOs) are
non-heme iron-containing enzymes responsible for the oxidation
of polyunsaturated fatty acids and esters to hydroperoxy deriva-
tives.1 These are heterogeneous families of enzymes distributed
widely throughout the plant and animal kingdoms,2 and named
according to the position at which a key substrate, arachidonic acid
(AA), is oxidized. Among the mammalian lipoxygenases involved in
the etiology of human disease, 5-lipoxygenase (5-LO) is now well
established as a target for reducing the production of leukotrienes
(important in particular asthma).3,4 More recently, 15-lipoxyge-
nase (15-LO) has emerged as an attractive target for therapeutic
intervention.5 15-LO has been implicated in the progression of cer-
tain cancers6,7 and chronic obstructive pulmonary disease (COPD).6
Evidence for the inhibition of 15-LO in the treatment of vascular
disease is, however, most compelling.8 Both transgenic and knock-
out studies implicate a role for 15-LO in atherogenesis.9,10 The en-
zyme is abundantly expressed in macrophages residing within the
atherosclerotic lesion.5 In addition, the immediate products of
15-LO oxidation of AA and linoleic acid (LA) have been shown to
be pro-inflammatory11 and pro-thrombotic.12
Three different strategies have been developed to inhibit the
LO’s pathway.12 They involve (i) redox inhibitors 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.
There is reasonable homology between the SLO and the human
one.14 This homology becomes more identical (50%) within 8 Å in
the active site pocket. Obviously soybean enzyme is much more
accessible than the human one. Therefore, one can expect that
the results can be extendable to the human LO.
Recently we reported the results of our studies on the soybean
lipoxygenase (SLO) inhibitory activities of some eugenol esters and
on the basis of the SAR (structure activity relationship) studies we
suggested that the inhibitory activity of these molecules largely de-
pends on the orientation of allyl group towards chelated Fe3+-OH
and themolecularvolumeof carboxylatemoietyin active site pocket
of the enzyme withouthydroperoxidation of allylic carbons.14 In this
paper we wish to report the results of a comparative study on the 15-
LO inhibitory activities of benzoate of eugenol, chavicol (4-allylphe-
nol), 4-allyloxyphenol and a group of 4-allyloxyaniline amides.
It is also found that 15-LO is linked to cardiovascular complica-
tions since it is known to participate in oxidative modification of
2. Result and discussion
Considering our previous work on eugenol and esters14 we
tested the inhibitory property of eugenol, eugenyl benzoate, meth-
* Corresponding author. Tel.: +98 05118795162; fax: +98 05118795560.
E-mail address: smseyedi@yahoo.com (S.M. Seyedi).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.065

S. M. Seyedi et al. / Bioorg. Med. Chem. 17 (2009) 1614–1622
1615
OH
O
O
O
O
O
O
O
Eugenol
Methyleugenol
Esteragol
Eugenyl benzoate
Figure 1. Molecular structure of some of allylbenzene analogs as lipoxygenase inhibitors.
Table 1
Enzyme inhibitory assessment, Van Der Waals molecular volum (VDW), HOMO energy and docking analysis data of consensus conformers of eugenol, methyleugenol, esteragol,
eugenyl benzoate, compounds 1, 2 and 5a–o
Compound
IC50
Ki
Ed (kcal/mol)
D
Gb (kcal/mol)
RMSD (Å)
EHOMO (eV)
VDW
Eugenol
Methyleugenol
Estragol
Eugenyl benzoat
1
2
38.2 1.9
96.1 3.3
64.1 1.5
7.0 0.4
6.7 0.2
6.2 0.7
6.1 0.2
2.2 0.2
4.1 0.3
6.4 0.7
0.67 0.06
3.3 0.1
3.8 0.1
5.6 0.1
10.1 0.6
6.7 0.4
8.8 0.7
8.1 0.8
13.8 0.6
17.0 0.2
12.9 0.3
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2.09eꢁ6
2.74eꢁ6
4.03eꢁ6
2.65eꢁ5
1.17eꢁ5
1.08eꢁ6
5.49eꢁ7
7.65eꢁ9
1.77eꢁ6
1.21eꢁ6
1.42eꢁ6
1.68eꢁ6
6.33eꢁ7
1.68eꢁ6
1.50eꢁ6
8.82eꢁ7
1.15eꢁ6
1.45eꢁ5
ꢁ9.49
ꢁ8.55
ꢁ8.78
ꢁ8.05
ꢁ8.85
ꢁ9.27
ꢁ9.99
ꢁ12.36
ꢁ9.72
ꢁ9.91
ꢁ9.67
ꢁ9.76
ꢁ10.17
ꢁ9.54
ꢁ9.65
ꢁ9.90
ꢁ9.90
ꢁ8.27
ꢁ7.75
ꢁ7.59
ꢁ7.36
ꢁ6.24
ꢁ6.73
ꢁ8.14
ꢁ8.54
ꢁ11.07
ꢁ7.58
ꢁ8.07
ꢁ7.98
ꢁ7.88
ꢁ8.64
ꢁ7.99
ꢁ7.94
ꢁ8.26
ꢁ8.10
ꢁ6.60
22.89
23.87
23.65
23.79
23.11
23.43
24.07
23.12
23.45
23.47
23.60
23.02
23.42
23.15
23.39
22.91
23.37
23.72
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
ꢁ10.46
ꢁ10.70
ꢁ10.77
ꢁ10.85
ꢁ10.56
ꢁ10.94
ꢁ11.11
ꢁ11.67
ꢁ10.87
ꢁ10.91
ꢁ11.13
ꢁ11.15
ꢁ10.68
ꢁ10.91
ꢁ10.88
55.68
71.02
86.06
101.80
148.82
86.64
89.18
89.18
103.38
103.38
100.95
100.95
111.18
111.18
62.72
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.
yleugenol and esteragol (Fig. 1) on the SLO (substrate: linoleic
acid). The results showed IC50 = 38.2 1.9, 7.0 0.4, 96.1 3.3
and 64.1 1.5 lM for the mentioned compounds, respectively
with optimum conformer (lowest Ki) of eugenyl benzoate was
adopted as the ‘consensus’ structure and used for further analy-
sis.14,16 The results of docking analysis showed that the consensus
structure of eugenyl benzoate, compounds 1 and 2 have the similar
estimated inhibitory constant (Ki) in a rang of 2–4 ꢀ 10ꢁ6 (Table 1).
It seems that the allyl and allyloxy benzene portion of the com-
pounds has hydrophobic interaction with Ile557, Leu565, Leu773 and
Ile572, respectively in such an orientation (Fig. 2A). The most criti-
cal residues that is Ile557, Leu565, Leu773 and Ile572 are close to the
active site. X-ray presentation of LA into SLO17 indicates that
Ile557, Leu565 and Leu773 lay within 4–6 Å of Fe3+-OH and both Leu-
cines are the proximity of the reactive 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 sub-
strate-bonding cavity. Each of these residues provides a large sur-
face to interact with natural substrate, particularly Leu565 and
Leu773. Mutating large residues such as Ile or Leu to an Ala opens
up space within the bonding pocket of SLO, leading to altered HÅ
transfer kinetics.18 The Ile557?Ala and Ile572?Phe mutants de-
creased kcat by twofold from WT (wild type), While Leu565?Ala
and Leu773?Ala decreased kcat by 60- and 1000-fold, respectively,
indicating that these hydrophobic residues (specially Leu565 and
Leu773) contribute significantly to catalysis.18 According to the re-
sult of multiple alignment, three amino acids Ile557, Leu565 and
Leu773 are found to be conserved over all species (Fig. 3).
(Table 1). It is notable that no other products such as hydroperoxy
are isolated from action of the LO enzyme on eugenyl benzoate,
methyleugenol and esteragol 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). Considering the IC50 results of the above compounds,
we decided to demethylate esteragol and synthesize the benzoate
ester 1 (Scheme 1) and study its inhibitory potency. Based on the
IC50 of compound 1 (Table 1) comparing to eugenyl benzoate, it is
suggested that the methoxy group of eugenyl benzoate have no
effective role in inhibitory potency. Then the allyl group of com-
pound 1 was replaced with allyloxy (Scheme 1) to study the effect
of HOMO energy of allyl group on inhibitory potency. Interestingly,
the IC50 of allyloxy analog 2 was comparable with compound 1 (Ta-
ble 1). The experimental results matched with theoretical Ki of dock-
ing study for those models in which allylic double bound oriented
towards iron atom similar to orientation of linoleic acid (LA) in the
active site of SLO (Fig. 2A). We generated 100 docked conformers
of desired compounds corresponding in AutoDockTools software.15
One conformer from each esters cluster which had more similarity
As shown in Figure 2A, the molecules in presented orientation
has hydrophilic and hydrophobic interaction with conserved
His513, Gln514 and Gln716. The conserved amino acids Gln514 and
Gln716 play a key role in oxidation potential of Fe3+ via hydrogen
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.

1616
S. M. Seyedi et al. / Bioorg. Med. Chem. 17 (2009) 1614–1622
O
O
BBr3/CHCl3
NaOH (aq)/PhCOCl
NaOH (aq)/PhCOCl
O
Esteragol
1
2
OH
Br
O
K2CO3/water
O
HO
O
Hydroquinone
NO2
NO2
NH2
Br
/EtOH
SnCl2
K2CO3/Acetone
HO
4-Nitrophenol
O
3
O
4
O
H
N
R
Cl
R
4
O
O
K2CO3/Acetone/H2O
5a-o
Compd.
5a
Compd.
5f
R
Compd.
5k
R
R
Cl
Cl
F
5b
5c
5d
5e
5g
5h
5i
5l
5m
5n
5o
O
F
CH3
O
5j
CH3
Scheme 1. General procedures for the synthesis of compounds 1, 2 and 5a–o.
bonding with Asn713 and His518 19
.
This hydrogen bond network is
tion with His513 instead of Gln716. Considering the orientation of
ester moiety of compound 2 towards His513 and Gln716, we decided
to replace the esteric bond by amide to achieve two hydrogen
bonds between the mentioned amino acids and inhibitor (this
can obviously hold the inhibitor more tightly in the active site
pocket). Upon this suggestion, 4-allyloxyphenylbenzamide (5f)
was synthesized (Scheme 1) and its inhibitory potency was deter-
mined. The IC50 value of 5f (3.3 0.1 lM) was nearly two fold less
than 2, which was in accordance with our prediction.
In the next step, other benzoate and cycloalkylate analogs 5a–e
and 5g–n were designed, synthesized and docked into the active
site to obtain the best inhibitor. The synthetic amids 5a–n showed
a broad range of inhibition activity on the enzyme (IC50 = 0.67–
exist in both SLO and 15-RLO (rabbit 15-LO) structures and also
play a steric role in orienting the substrate and inhibitor bound
to LO.19 The C-3–C-8 hydrocarbon tail of LA is flanked by the
hydrophobic portion of the Gln514 and Gln716 (Fig. 2A). Disrupting
this bonding pocket by changing the position of Gln514 and Glu716
may affect the proper positioning of the substrate for C-H bond
cleavage so that abstraction becomes more rate-limiting (as was
observed in the Gln514?Ala, Gln716?Asn and Gln716?Glu mutants
by 4-, 3- and 6-fold decrease in kcat from WT SLO, respectively19).
Proposed inhibitory model of docked molecules have hydrogen
bond with Gln716 via C@O portion except compound 2. The aro-
matic part of benzoate moiety in eugenyl benzoate, compound 1
and 2 is flanked by the hydrophobic portion of the Gln716 side
chain like LA (Fig. 2A). As seen in Figure 2A, the benzoate moiety
of compound 2 is in the far distance of the pocket then the same
moiety of compound 1, causes the hydrogen bonding of C@O por-
17
substituent was the most potent inhibitor at 0.67
and 4-methoxybenzamid analogues (5n and 5m) presented less
activity (IC50 = 17.0 and 13.8 M, respectively).
lM; Table 1). Compound 5e having an adamantanecarboxylate
lM while the 3-
l

S. M. Seyedi et al. / Bioorg. Med. Chem. 17 (2009) 1614–1622
1617
Figure 2. Consensus bonding conformations (stick view) of compounds 1 (red), 2 (blue) and 5f (green) with linoleic peroxide (orange) bonded to Fe in the SLO active site (A).
Consensus bonding conformations (stick view) of compounds 5f–n (B) and 5a–e (C) in the SLO active site. The amino acids bonded to Fe are shown in stick.
The Ki of consensus structure of amides 5a–n, did not show ra-
tional relation with IC50 results. This may comes from tendency of
the amide moiety for making hydrogen bond with His513 and
Gln716 side chains (Fig. 4). This result can be explained by consid-
ering HOMO energy and Van Der Waals volume (VDW) of amide
moiety of compounds 5a–n (Table 1). A non-linear relation be-
tween IC50 values and VDW of the amides was observed except
for 5e (Fig. 5). The best IC50 belongs to the compounds with molec-
ular volume of 70–80 Å3 (Fig. 5). The proposed relation in Figure 5
between IC50 and molecular volume is only reserved for expansion
of the amide moiety in two dimensions while the adamantanamide
group (compound 5e) which also expand in the third dimension
dose not follow the series. Considering the free space which is
and more strong hydrogen bound of the C@O group. To prove this
observation, isopropylcarboxamide analog (5o) with comparable
molecular volume to cyclopropyl (62.72 vs 55.68 Å3) but having
lower HOMO energy of amide moiety (EHOMO = ꢁ10.88 eV) was
synthesized and its inhibitory potency determined. The IC50 result
of 5o (12.9 0.3 lM) was a good evidence for proving the men-
tioned hypothesis. With the exception of 5a, we see a good non-
linear relationship between IC50 value and EHOMO for compounds
5b–e and 5o (Fig. 6).
It is clear that the inhibitory potency of the aromatic analogs
decrease while their size increases (Fig. 5). We can not see such a
relationship between IC50 and EHOMO for the aromatic analogs. It
might be due to two dimensional expansion of meta and para sub-
stituent of phenyl portion (it is more than the expansions of cyclo-
alkane analog) which cause steric hindrance in filling the amide
pocket. The steric hindrance can disturb the planarity between
phenyl portion and C@O and therefore decreasing in mesomeric
effects.20 The HOMO energy level of amide moiety of 5f–n can
rationalize the IC50 value deference between meta and para isomer.
For example compound 5g with less IC50 value in comparison with
formed by Val372, Ser510, Phe576, Gln716, Gly720, Ile723, Arg726
,
Thr728, Asp766 and Ile770 (amide pocket—Fig. 7), we expected to
see direct relation between molecular volume and inhibitory po-
tency for compounds 5a–e but it did not. As the HOMO energy is
closely related to the electronic density of C@O group and there-
fore support the hydrogen bonding potency,20 it is assumed that
HOMO energy of the amide moiety could be responsible for this
observation.
its isomer 5h (3.8 0.1 vs 5.6 0.1
level and it is the same for compounds 5k and 5l (IC50 = 8.8 0.7
and 8.1 0.8 M, respectively).
lM), has higher HOMO energy
In this regard we found an interesting exception in cyclopropyl
analog. We expected higher IC50 for this analog than the others of
the series (5b–e) regarding its smaller volume but the lower IC50 is
being explained considering higher HOMO (EHOMO = ꢁ10.46 eV)
l
In summary we have carried out a SAR comparative studies on
allyl and allyloxy benzene derivatives as 15-lipoxygenase inhibi-

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S. M. Seyedi et al. / Bioorg. Med. Chem. 17 (2009) 1614–1622
Figure 3. Multiple alignment of SLO (1ik3_A). The conserved residues which have hydrogen bond and lipophilic interactions with docked ligands are highlighted in green and
blue, respectively. The iron-bond residues are highlighted in yellow.
20
18
5n
16
14
12
10
8
5m
5i
5k
5l
5a
5j
5h
6
5d
5c
4
5b
5g
5f
2
R2 = 0.9591
100 110 120
0
50
60
70
80
90
VDW
Figure 4. Stick view of compound 5f interacting with His513 and Gln716 via
hydrogen bonding of amid bond. Hydrogen bonds are revealed by dashed green
lines.
Figure 5. Diagram of IC50 value versus Van Der Waals molecular volume (VDW) for
compounds 5a–d and 5f–n.
tors. It could be suggested that the application of single point prop-
erties like HOMO energy might be applicable to predict the inhib-
itory potency the synthetic amides. This study has also shown the
important role of molecular volume in the inhibitory activity of 4-
allyloxyaniline amides. The importance of these compounds could
be more highlighted when we rank their easy synthesis pathway
and their high yield.
3. Materials and methods
3.1. Chemistry
Compound 1 was synthesized via demethylation of esteragol
using boron tribromide21 and then the followed with benzoyl chlo-

S. M. Seyedi et al. / Bioorg. Med. Chem. 17 (2009) 1614–1622
1619
R2 = 0.8657
16
14
12
10
8
study Van Der Waals molecular volume (VDW) was measured by
QSAR properties tool in HyperChem7.5.
Crystal structure of soybean lipoxygenase-3 (arachidonate 15-
lipoxygenase) complex with 13(S)-hydroproxy-9(Z)-2,11(E)-octa-
decadienoic acid was retrieved from RCSB Protein Data Bank
(PDB entry: 1IK3).
5o
5d
5a
6
5c
4
3.2.3. Molecular docking
5b
2
Automated docking simulation was implemented to dock euge-
nylbenzoate, 1, 2 and 5a–o into theactive site of SLO with AutoDock-
Tools(ADT) version 1.415 using Lamarckiangenetic algorithm.31 This
method has been previously shown to produce bonding models sim-
ilarto the experimentally observedmodels.14,31,32 Thetorsionangles
of the ligands were identified, hydrogens were added to the macro-
molecule, bond distances were edited and solvent parameters were
added to the enzyme 3D structure. Partial atomic charges were then
assigned to the macromolecule as well as ligands (Gasteiger for the
ligands and Kollman for the protein).
The regions of interest of the enzyme were defined by consider-
ing Cartesian chart 19, 2 and 19 as the central of a grid size of 50,
50 and 50 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. The men-
tioned compounds 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-
0
5e
-11.0 -10.9 -10.8 -10.7 -10.6 -10.5 -10.4 -10.3
EHOMO (eV)
Figure 6. Diagram of IC50 value versus HOMO energy (EHOMO) of the amide moiety
for compounds 5a–e and 5o.
ride in aqueous solution of sodium hydroxide.14 Compound 2 was
synthesized by allylation of hydroquinone in the presence of satu-
rated potassium carbonate in water22 and immediate reaction with
benzoyl chloride in aqueous solution of sodium hydroxide. The
amides 5a–o were prepared by reaction of desired acid chlorides
with 4-allyloxyaniline (4) in the presence of potassium carbonate
in acetone–water.23 4-Allyloxyaniline was synthesized via reduc-
tion of 4-allyloxynitrobenzene (3)24 using stannous chloride.25
3.2. Molecular modeling, docking and SAR study
3.2.1. Multiple alignment
Conserved amino acids were identified through multiple align-
ment in clustalX 1.81.26 Sequences of lipoxygenase (LO) family
were selected from blasted sequences via ExPASY proteomics ser-
ver.27 Multiple alignment process was then carried out on the se-
lected sequences (protein weight matrix: BLOSUM series,
opening gap penalty = 10).
tered with
a root-mean-square deviation tolerance of 0.2 Å.
Autodock generated 100 docked conformers of 1, 2 and 5a–o-cor-
responding to the lowest-energy structures. After docking proce-
dure in ADT4, 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 inhi-
bition constant) are outlined in Table 1.
3.2.2. Calculations
Structures of desired compounds were simulated in chem3D
professional; Cambridge software; using MM2 method (RMS gradi-
ent = 0.05 kcal/mol).28 Output files were minimized under semi-
empirical AM1 method in the second optimization (Convergence
limit = 1eꢁ5; Iteration limit = 100; RMS gradient = 0.05 kcal/mol;
Fletcher-Reeves optimizer algorithm) in HyperChem7.5.29,30 Single
point properties of molecules such as energy of HOMO and LUMO
were calculated using ab initio RHF/6-311G* methods (Conver-
gence limit = 1eꢁ5; Iteration limit = 100) in HyperChem7.5. In this
3.3. 15-LO inhibitory assessment
Lipoxygenase activity was measured in borate buffer solutions
(0.1 M, pH 9) using the method described in literature,36,37 by mea-
suring the absorbance at 234 nm for 60 s after addition of the en-
zyme (soybean 15-lipoxygenase), and linoleic acid (final
Figure 7. The cavity for amide moiety of consensus structure of compounds 5a–o is shown in stick (right) and solvent surface (left) view.

1620
S. M. Seyedi et al. / Bioorg. Med. Chem. 17 (2009) 1614–1622
concentration: 134
l
M) as substrate at 20 1 °C. The final enzyme
cmꢁ1: 1730 (OC@O). C16H14O3 requires: C, 75.57; H, 5.55. Found:
C, 75.37; H, 5.53.
concentration was 167 U/mL. Synthesized substances were added
in DMSO solutions (final DMSO concentration 1%); whereas DMSO
was added in control experiments with no inhibitor. The mixture
of each inhibitors and linoleic acid was set as blank sample in test-
ing step. At least six control test tubes and three tubes for each
inhibitor solution were measured. To ensure constant enzyme
activity throughout 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 described37 and
IC50 values were determined by linear interpolation between the
points around 50% activity.
4.4. General procedure for preparation of compounds 5a–o
A
mixture of 69.5 g (0.50 mol) of 4-nitrophenol, 66.0 g
(0.55 mmol) of allyl bromide and 70.0 g of anhydrous potassium
carbonate (0.50 mol) in dry acetone (150 mL) was refluxed for
8 h and cooled. The mixture was diluted with water (250 mL)
and then extracted with ether (2 ꢀ 150 mL). The combined ex-
tracts were washed with NaOH 10% (2 ꢀ 100 mL) and dried with
anhydrous sodium carbonate. After removal of the solvent the
residual oil of 3 was distilled under reduced pressure (74.4 g,
83% yield).
4. Experimental
4.1. Instruments
A mixture of 64.4 g (0.36 mol) of 3 and 406.0 g (1.80 mol) of
SnCl2ꢂ2H2O in 600 mL of absolute ethanol was refluxed under
nitrogen for 20 min. After cooling; the mixture was poured into
cold water (1500 mL). The pH was made basic by adding sodium
bicarbonate. The 4-(allyloxy)aniline (4) was extracted by ethyl ace-
tate (3 ꢀ 300 mL). The combined organic phase dried with anhy-
drous sodium sulfate. After removal of the solvent the residual
oil of 4 was distilled under reduced pressure (40.3 g, 75% yield).
To a stirred mixture of 4 (1.1 g, 7.3 mmol), potassium carbonate
(1.5 g, 10.8 mmol), water (10 mL), and acetone (5 mL), was added
desired acid chlorides (9.0 mmol) at 0 °C dropwise. The mixture
was stirred at room temperature for 30 min and then diluted with
water (20 mL). The resulting precipitate of 5a–o was separated and
recrystallized from ethanol.
Melting points were recorded on an Electrothermal type 9100
melting point apparatus. The 1H NMR (500 MHz) spectra were re-
corded on a Bruker Avance DRX-500 spectrometer. Elemental anal-
ysis was obtained on a Thermo Finnigan Flash EA microanalyzer.
The IR spectra were obtained on a 4300 Shimadzu Spectrometer.
All measurements of lipoxygenase activities were carried out using
an Agilent 8453 spectrophotometer. The soybean 15-lipoxygenase
and other chemicals were purchased from Sigma, Aldrich and
Merck Co., respectively.
4.2. 4-Allylphenyl benzoate (1)
4.4.1. 1-(Allyloxy)-4-nitrobenzene (3)
Colourless oil; pb (3 mm): 126–129 °C; 1H NMR (CDCl3); d 3.47
(s, 2H, –NH2), 4.49 (d, J = 5.20 Hz, 2H, –CH2–), 5.18–5.53 (m, 2H,
H2C@), 5.89–6.29 (m, 1H, HC@), 6.88 (d, J = 8.90 Hz, 2H, H-3, H-
5), 7.48 (d, J = 8.90 Hz, 2H, H-2, H-6).
A solution of 0.30 g (2 mmol) of esteragol in 5 mL of CHC13 was
added during 2 min to a well-stirred solution of 3.0 g (12 mmol) of
BBr3 in 35 mL of CHC13 maintained in the range 23–26 °C. Stirring
was continued for 15 min at 23–26 °C. The reaction mixture was
then poured into a well-stirred mixture of 30 g of ice-water. After
15 min the organic phase was separated, washed with water
(2 ꢀ 20 mL) and then extracted by NaOH 5% (2 ꢀ 10 mL). The ex-
tract was washed with CHCl3 (2 ꢀ 20 mL) and then acidified by
HCl 10% to appearance of milky emulsion of 4-allylphenol. The
product was extracted by ether and after removal of the solvent,
dissolved in stirred NaOH 5% (5 mL) and then benzoyl chloride
(0.3 mL) was added. After 30 min stirring, the precipitate of com-
pound 1 was separated and recrystallized from methanol 90%
(0.32 g, 67% yield).
4.4.2. 4-(Allyloxy)aniline (4)
Colourless oil; bp (3 mm): 108–110 °C; 1H NMR (CDCl3); d 3.47
(s, 2H, –NH2), 4.49 (d, J = 5.20 Hz, 2H, –CH2–), 5.18–5.53 (m, 2H,
H2C@), 5.89–6.29 (m, 1H, HC@), 6.7 (AB, qarter, 4H, H-2, H-3, H-
5, H-6).
4.4.3. N1-(4-(Allyloxy) phenyl)-1-cyclopropanecarboxamide
(5a)
White solid; mp: 147–148 °C; 1H NMR (CDCl3): d 0.20–1.20 (m,
4H, –CH2– (cyclopropyl)), 1.24–1.73 (m, 1H, –CH– (cycloropyl)),
4.51 (d, J = 5.1 Hz, 2H, –CH2–), 5.17–5.54 (m, 2H, H2C@), 5.85–
6.27 (m, 1H, HC@), 6.83 (d, J = 8.94 Hz, 2H, H-3, H-5), 7.42 (d,
J = 8.94 Hz, 2H, H-2, H-6), 7.53 (s, 2H, –NH–); IR cmꢁ1: 1653
(NC@O). C13H15NO2 requires: C, 71.87; H, 6.96; N, 6.45. Found: C,
72.02; H, 6.93; N, 6.41.
White solid; mp: 61–62 °C; 1H NMR (CDCl3):
d 3.42
(d, J = 6.6 Hz, 2H, –CH2–), 5.79–6.23 (m, 2H, H2C@), 5.94–6.33 (m,
1H, HC@), 7.00–7.36 (m, 5H, H-2, H-4, H-30, H-40, H-50), 7.56
(d, J = 9.5 Hz, 2H, H-3, H-5), 8.24 (d, J = 7.9 Hz, 2H, H-20, H-60); IR
cmꢁ1: 1735 (OC@O). C16H14O2 requires: C, 80.65; H, 5.92. Found:
C, 80.39; H, 5.83.
4.3. 4-(Allyloxy)phenyl benzoate (2)
4.4.4. N1-(4-(Allyloxy) phenyl)-1-cyclobutanecarboxamide (5b)
White solid; mp: 129–130 °C; 1H NMR (CDCl3): d 1.21–2.12
(m, 8H, –CH2– (cyclobutyl)), 2.96–3.32 (m, 1H, –CH– (cyclobu-
tyl)), 4.53 (d, J = 5.1 Hz, 2H, –CH2–), 5.20–5.52 (m, 2H, H2C@),
5.88–6.29 (m, 1H, HC@), 6.84 (d, J = 9 Hz, 2H, H-3, H-5), 7.12
(s, 2H, –NH–), 7.42 (d, J = 9 Hz, 2H, H-2, H-6); IR cmꢁ1: 1652
(NC@O). C14H17NO2 requires: C, 72.70; H, 7.41; N, 6.06. Found:
C, 72.89; H, 7.33; N, 6.05.
A mixture of 5.5 g (50 mmol) of hydroquinone, 6.6 g (55 mmol)
of allyl bromide and 7.0 g of anhydrous potassium carbonate
(50 mmol) in water (12 mL) was refluxed for 5 h and cooled. The
mixture was extracted with ether (2 ꢀ 20 mL) and washed with
water (3 ꢀ 20 mL). After removal of the solvent, the residual oil
was dissolved in stirred NaOH 10% (50 mL) and then benzoyl chlo-
ride (7.0 mL) was added. After 30 min stirring, the precipitate of
compound
(5.5 g, 43% yield).
2 was separated and recrystallized from ethanol
4.4.5. N1-(4-(Allyloxy) phenyl)-1-cyclopantanecarboxamide (5c)
White solid; mp: 138–140 °C; 1H NMR (CDCl3): d 1.64–1.91 (m,
8H, –CH2– (cyclopentyl)), 2.50–2.85 (m, 1H, –CH– (cyclopentyl)),
4.52 (d, J = 5.1 Hz, 2H, –CH2–), 5.20–5.54 (m, 2H, H2C@), 5.85–
6.26 (m, 1H, HC@), 6.85 (d, J = 8.94 Hz, 2H, H-3, H-5), 7.23 (s, 1H,
–NH–), 7.42 (d, J = 8.94 Hz, 2H, H-2, H-6); IR cmꢁ1: 1652 (NC@O).
White solid; mp: 102–103 °C; 1H NMR (CDCl3): d 3.40 (d,
J = 6.6 Hz, 2H, –CH2–), 4.94–5.19 (m, 2H, H2C@), 5.74–6.19 (m,
1H, HC@), 7.56 (d, J = 9.5 Hz, 2H, H-3, H-5), 7.20–7.38 (m, 5H, H-
2, H-4, H-30, H-40, H-50), 8.22 (d, J = 7.9 Hz, 2H, H-20, H-60); IR

S. M. Seyedi et al. / Bioorg. Med. Chem. 17 (2009) 1614–1622
1621
C15H19NO2 requires: C, 73.44; H, 7.81; N, 5.71. Found: C, 73.59; H,
7.83; N, 5.75.
cmꢁ1: 1650 (NC@O). C16H14ClNO2 requires: C, 66.79; H, 4.90; N,
4.87. Found: C, 66.86; H, 4.83; N, 4.92.
4.4.6. N1-(4-(Allyloxy) phenyl)-1-cyclohexanecarboxamide (5d)
White solid; mp: 148–149 °C; 1H NMR (CDCl3): d 1.10–2.36 (m,
6H, cyclohexyl), 4.50 (d, J = 5.1 Hz, 2H, –CH2–), 5.19–5.53 (m, 2H,
H2C@), 5.84–6.26 (m, 1H, HC@), 6.85 (d, J = 8.9 Hz, 2H, H-3, H-5),
4.4.14. N1-(4-(Allyloxy) phenyl)-3-chlorobenzamide (5l)
White solid; mp: 157–158 °C; 1H NMR (CDCl3):
d 4.55
(d, J = 5.1 Hz, 2H, –CH2–), 5.22–5.54 (m, 2H, H2C@), 5.88–6.44 (m,
1H, HC@), 6.82 (d, J = 9.8 Hz, 2H, H-3, H-5), 7.16–7.88 (m, 5H, H-
20, H-30, H-50, H-60, –NH–), 7.55 (d, J = 9.8 Hz, 2H, H-2, H-4); IR
cmꢁ1: 1648 (NC@O). C16H14ClNO2 requires: C, 66.79; H, 4.90; N,
4.87. Found: C, 66.71; H, 4.92; N, 4.79.
7.12 (s, 1H, –NH–), 7.40 (d, J = 8.9 Hz, 2H, H-2, H-6); IR cmꢁ1
:
1645 (NC@O). C16H21NO2 requires: C, 74.10; H, 8.16; N, 5.40.
Found: C, 74.21; H, 8.13; N, 5.48.
4.4.7. N1-(4-(Allyloxy) phenyl)-1-admantancarboxamide (5e)
White solid; mp: 180–181 °C; 1H NMR (CDCl3): d 1.76 (m, 6H,
–CH2– (adamantylyl)), 1.91–2.22 (m, 9H, –CH–, –CH 2– (adamantyl)),
4.52 (d, J = 5 Hz, 2H, –CH2–), 5.19–5.52 (m, 2H, H2C@), 5.88–6.28 (m,
1H, HC@), 6.88 (d, J = 8.90 Hz, 2H, H-3, H-5), 7.21 (s, 1H,
–NH–), 7.48 (d, J = 8.90 Hz, 2H, H-2, H-6); IR cmꢁ1: 1650 (NC@O).
C20H25NO2 requires: C, 77.14; H, 8.09; N, 4.50. Found: C, 76.99; H,
8.15; N, 4.46.
4.4.15. N1-(4-(Allyloxy) phenyl)-4-methoxybenzamide (5m)
White solid; mp: 162–163 °C; 1H NMR (CDCl3): d 3.83 (s, 3H,
–OCH3), 4.52 (d, J = 5.5 Hz, 2H, –CH2–), 5.23–5.54 (m, 2H, H2C@),
5.86–6.32 (m, 1H, HC@), 6.88 (d, J = 10 Hz, 2H, H-3, H-5), 6.88
(d, J = 10 Hz, 2H, H-30, H-50),7.50 (d, J = 10 Hz, 2H, H-2, H-6), 7.80
(d, J = 10 Hz, 2H, H-20, H-40), 8.02 (s, 1H, –NH–); IR cmꢁ1: 1645
(NC@O). C17H17NO3 requires: C, 72.07; H, 6.05; N, 4.94. Found: C,
72.19; H, 6.13; N, 5.01.
4.4.8. N1-(4-(Allyloxy) phenyl)benzamide (5f)
4.4.16. N1-(4-(Allyloxy) phenyl)-3-methoxybenzamide (5n)
White solid; mp: 109–110 °C; 1H NMR (CDCl3): d 3.88 (s, 3H, –
OCH3), 4.52 (d, J = 5.5 Hz, 2H, –CH2–), 5.19–5.52 (m, 2H, H2C@),
5.88–6.28 (m, 1H, HC@), 6.92 (d, J = 9.8 Hz, 2H, H-3, H-5), 6.88
(d, J = 10 Hz, 2H, H-20, H-60), 7.50 (d, J = 9.8 Hz, 2H, H- 2, H-6),
White solid; mp: 150–151 °C; 1H NMR (CDCl3): d 4.55 (d, 2H,
–CH2–), 5.22–5.58 (m, 2H, H2C@), 5.91–6.31 (m, 1H, HC@), 6.92
(d, J = 9.5 Hz, 2H, H-3, H-5), 7.29–7.65 (m, 5H, H-2, H-4, H-30, H-
40, H-50), 7.86 (d, J = 7.9 Hz, 2H, H-20, H-60), 8.02 (s, 1H, –NH–); IR
cmꢁ1: 1648 (NC@O). C16H15NO2 requires: C, 75.87; H, 5.97; N,
5.53. Found: C, 75.79; H, 6.03; N, 5.59.
7.55 (d, J = 10 Hz, 2H, H-30, H-50), 7.74 (s, 1H, –NH–); IR cmꢁ1
:
1648 (NC@O). C17H17NO3 requires: C, 72.07; H, 6.05; N, 4.94.
Found: C, 71.94; H, 6.00; N, 4.88.
4.4.9. N1-(4-(Allyloxy) phenyl)-4-fluorobenzamide (5g)
White solid; mp: 169–170 °C; 1H NMR (CDCl3): d 4.55 (d,
J = 5 Hz, 2H, –CH2–), 5.20–5.57 (m, 2H, H2C@), 5.86–6.28 (m, 1H,
HC@), 6.95 (m, 2H, H-3, H-5), 7.18 (d, J = 9.50 Hz, 2H, H-30, H-50),
7.50 (m, 2H, H-2, H-6), 7.71 (s, 1H, –NH–), 7.88 (d, J = 9.50 Hz,
2H, H-20, H-60); IR cmꢁ1: 1649 (NC@O). C16H14FNO2 requires: C,
70.84; H, 5.20; N, 5.16. Found: C, 70.92; H, 5.14; N, 5.11.
4.4.17. N1-(4-(Allyloxy) phenyl)-2-methylpropanamide (5o)
White solid; mp: 121–122 °C; 1H NMR (CDCl3): d 1.22 (d, J = 7 Hz,
6H, –CH3 (propan)), 2.26–2.64 (m, 1H, –CH– (propan)) 4.52 (d, J =
5.1 Hz, 2H, –CH2–), 5.18–5.52 (m, 2H, H2C@), 5.85–6.28 (m, 1H,
HC@), 6.85 (d, J = 9.8 Hz, 2H, H-3, H-5), 7.14 (s, 1H, –NH–), 7.42 (d,
J = 9.8 Hz, 2H, H-2, H-4); IR cmꢁ1: 1648 (NC@O). C13H17NO2 requires:
C, 71.21; H, 7.81; N, 6.39. Found: C, 71.37; H, 7.83; N, 6.45.
4.4.10. N1-(4-(Allyloxy) phenyl)-3-fluorobenzamide (5h)
White solid; mp: 155–156 °C; 1H NMR (CDCl3):
d 4.71
References and notes
(d, J = 5.1 Hz, 2H, –CH2–), 5.35–5.70 (m, 2H, H2C@), 6.02–6.44 (m,
1H, HC@), 7.09 (d, J = 9.8 Hz, 2H, H-3, H-5), 7.29–7.85 (m, 4H, H-
20, H-30, H-50, H-60), 7.62 (d, J = 9.8 Hz, 2H, H-2, H-4), 7.94 (s, 1H,
–NH–); IR cmꢁ1: 1650 (NC@O). C16H14FNO2 requires: C, 70.84; H,
5.20; N, 5.16. Found: C, 70.69; H, 5.18; N, 5.20.
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4.4.11. N1-(4-(Allyloxy) phenyl)-4-methylbenzamide (5i)
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4.4.12. N1-(4-(Allyloxy) phenyl)-3-methylbenzamide (5j)
White solid; mp: 108–109 °C; 1H NMR (CDCl3): d 2.45 (s, 3H,
–CH3), 4.52 (d, J = 5 Hz, 2H, –CH2–), 5.20–5.50 (m, 2H, H2C@),
5.88–6.27 (m, 1H, HC@), 6.92 (d, J = 9.8 Hz, 2H, H-3, H-5), 7.28–
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6.41; N, 5.24. Found: C, 76.29; H, 6.37; N, 5.20.
4.4.13. N1-(4-(Allyloxy) phenyl)-4-chlorobenzamide (5k)
White solid; mp: 179–180 °C; 1H NMR (CDCl3):
d 4.53
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1622
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