2-Prenylated m-dimethoxybenzenes as potent inhibitors of 15-lipo-oxygenase: inhibitory mechanism and SAR studies

Article abstract of DOI:10.1111/cbdd.12779

15-lipo-oxygenases are one of the iron-containing proteins capable of performing peroxidation of unsaturated fatty acids in animals and plants. The critical role of enzymes in the formation of inflammations, sensitivities, and some cancers has been demonstrated in mammals. The importance of enzymes has led to the development of mechanistic studies, product analysis, and synthesis of inhibitors. In this study, a series of allyl and prenyl dimethoxybenzenes were synthesized and their inhibitory potency against soybean 15-Lipo-oxygenase (L1; EC 1,13,11,12) was determined. Among the synthetic compounds, 2,6-dimethoxy-1-isopentenyl-4-methylbenzene, 2,6-dimethoxy-1-geranyl-4-methylbenzene, and 2,6-dimethoxy-1-farnesyl-4-methylbenzene showed the most potent inhibitory activity with IC₅₀ values of 7.6, 5.3, and 0.52?μm, respectively. For some of the compounds, SAR studies showed acceptable relationship between inhibitory potency and enzyme–ligand interactions. Radical scavenging assessment results apart from the SAR studies indicate that electronic properties are the major factors for lipo-oxygenase inhibition potency of the mentioned compounds. Based on the theoretical studies, it was suggested that CH…O intramolecular hydrogen bond between ortho-methoxy oxygen and methine hydrogen atoms is one of the major factors in the stability of 2,6-dimethoxyallyl(or prenyl)benzenes radical via the planarity fixation between phenyl and allyl (or prenyl) pi orbitals.

Full text of DOI:10.1111/cbdd.12779

Received: 14 December 2015
DOI: 10.1111/cbdd.12779
Revised: 6 April 2016
Accepted: 13 April 2016
R E S E A R C H A R T I C L E
2-Prenylated m-dimethoxybenzenes as potent inhibitors of
15-lipo-oxygenase: inhibitory mechanism and SAR studies
Atena Jabbari1
Hamid Sadeghian2,3
Mehdi Bakavoli1
Alireza Salimi1
Mina Mousavian4
Seyed M. Seyedi1
1Department of Chemistry, Faculty of
Science, Ferdowsi University of Mashhad,
Mashhad, Iran
2Neurogenic Inflammation Research
Center, Mashhad University of Medical Sciences,
Mashhad, Iran
3Department of Laboratory Sciences, School of
Paramedical Sciences, Mashhad University of
Medical Sciences, Mashhad, Iran
4Department of Biology, Mashhad Branch, Islamic
Azad University, Mashhad, Iran
15-Lipo-oxygenases are one of the iron-containing proteins capable of performing
peroxidation of unsaturated fatty acids in animals and plants. The critical role of
enzymes in the formation of inflammations, sensitivities, and some cancers has
been demonstrated in mammals. The importance of enzymes has led to the devel-
opment of mechanistic studies, product analysis, and synthesis of inhibitors. In this
study, a series of allyl and prenyl dimethoxybenzenes were synthesized and their
inhibitory potency against soybean 15-lipo-oxygenase (L1; EC 1,13,11,12) was de-
termined. Among the synthetic compounds, 2,6-dimethoxy-1-isopentenyl-4-methylb
enzene, 2,6-dimethoxy-1-geranyl-4-methylbenzene, and 2,6-dimethoxy-1-farnesyl-4-
methylbenzene showed the most potent inhibitory activity with IC50 values of 7.6,
5.3, and 0.52 μm, respectively. For some of the compounds, SAR studies showed
acceptable relationship between inhibitory potency and enzyme–ligand interactions.
Radical scavenging assessment results apart from the SAR studies indicate that
electronic properties are the major factors for lipo-oxygenase inhibition potency of
the mentioned compounds. Based on the theoretical studies, it was suggested that
CH…O intramolecular hydrogen bond between ortho-methoxy oxygen and methine
hydrogen atoms is one of the major factors in the stability of 2,6-dimethoxyallyl(or
prenyl)benzenes radical via the planarity fixation between phenyl and allyl (or
prenyl) pi orbitals.
Correspondence
Hamid Sadeghian, sadeghianh@mums.ac.ir;
Seyed Mohammad Seyedi, smseyedi@um.ac.ir
K E Y W O R D S
2,2-diphenylpicrylhydrazyl, density functional theory, docking, hydrogen bond, rad-
ical scavenging
Lipo-oxygenases form a large class of non-heme iron-
containing enzymes capable of catalyzing the hydroperox-
idation of polyunsaturated fatty acids containing a cis,cis-
1,4-pentadiene moiety, such as arachidonic and linoleic
acids.[1] Nowadays, these enzymes are well known to play
an effective role in many acute and chronic syndromes
such as inflammation, asthma, cancer, stroke, allergy,
and several other inflammation-based diseases.[2] Due to
their importance as therapeutic target, there is currently
much interest in the chemistry and pharmacology for the
discovery and synthesis of novel lipo-oxygenase inhib-
itors. Although several molecules have been synthesized
and biologically evaluated to this regard, they exhibit too
many side-effects, thereby preventing their use as effective
therapeutic drugs.[3]
Allylbenzenes represent a group of secondary metabolites
composed of a single bond junction between the propenyl
group and phenyl ring. These natural compounds (eugenol
and steragol) have been recognized as interesting and valu-
able biologically active phytochemicals.
© 2016 John Wiley & Sons A/S.
Chem Biol Drug Des 2016; xx: 1–10
wileyonlinelibrary.com/cbdd
1

2
Jabbari et al.
Allylbenzenes have been recently recognized as lipo-
macromolecule as well as ligands (Gasteiger for the ligands
and Kollman for the protein).
oxygenase inhibitors, they decrease lipo-oxygenase activity
probably by mimicking 1,4-unsaturated bonds by their allyl
moiety.[4–6]
The docking regions of the enzyme were defined by
considering Cartesian chart 18.3, 4.8, and 19.2 as the central
of a grid size with 44, 56, and 62 points in X-, Y-, and Z-
axis.[11] The docking parameter files were generated using
Genetic Algorithm and Local Search Parameters (GALS)
while number of generations and maximum number of
energy evaluations were set to 100 and 2 500 000, respec-
tively. The 100 docked complexes were clustered with a root-
mean-square deviation tolerance (RMSD) of 2.0 Å. Docking
results were submitted to Accelrys DS Visualizer 2.0.1c for
further evaluations.
In the present work, the electronic and steric effects of
methoxy group of dimethoxyallylbenzenes on their lipo-
oxygenase inhibitory activity were studied.
1
|
MATERIALS AND METHODS
1.1 | Molecular modeling, docking, and SAR
study
1.1.1 | Structure optimization
1.2 | Lipo-oxygenase inhibitory assessment
All density functional theory (DFT) calculations were carried
out using GAMESS-US package.[7] Geometry optimizations
of all investigated molecular structures were computed by
DFT using Beck’s three parameter hybrid functional with the
Lee–Yang–Parr correlation functional (B3LYP).[8] The stan-
dard basis set of 6–311++G (d, p) was used for all kinds of
atoms involved in compounds 1–9 in both natural and mono-
radical states. Stationary points were fully optimized and
characterized by vibrational frequency calculations, which
also provided zero-point vibrational energies (ZPE).
Structures 1–11 were simulated in Chem3D profes-
sional (Cambridge software), using the MM2 method (RMS
gradient = 0.1 kcal/mol).a In the second optimization, output
files were minimized under semiempirical AM1 methods
(convergence limit = 0.01; Iteration limit = 50; RMS
gradient = 0.1 kcal/mol; Polak-Ribiere optimizer algorithm
HyperChem 7.5).b
The enzyme solution was prepared by dissolving 1 mg
lyophilized soybean 15-lipo-oxygenase (L1; Type I-B;
EC 1,13,11,12; Sigma Co.) in 5 mL phosphate buffer pH
7.0 (50 mm) and then its activity was determined by diene
formation method at 234 nm.[4] Finally, its activity was
decreased to desired quantity by dilution with the phosphate
buffer. Linoleic acid and two assay solutions (A and B) were
prepared in advance.
Solution A was 50 mm DMAB in an 100 mm phosphate
buffer (pH 7.0). Solution B was a mixture of 10 mm MBTH
(3 mL) and hemoglobin (5 mg/mL, 3 mL) in 50 mm phos-
phate buffer at pH 5.0 (25 mL). A linoleic acid solution
was prepared by mixing 5 mg of linoleic acid with 50 mg
Tween-20 in 3 mL water and then diluted with KOH 100 mm
to a final volume of 5 mL.
In the standard assay, the sample in ethanol (25 μL), SLO
(EC 1,13,11,12; 4000 units/mL; 25 μL) and phosphate buffer,
pH 7.0 (50 mm; 900 μL), were mixed in a test-tube and prein-
cubation was carried out for 5 min at room temperature. A
control test was done with the same volume of ethanol. After
the preincubation, linoleic acid solution (50 μL) was added
to start the peroxidation reaction, and, 7 min later, solution
A (270 μL) and then solution B (130 μL) were added to start
the color formation. Further, 5 min later, 200 μL of a 2% SDS
solution was added to terminate the reaction. The absorbance
at 598 nm was compared with the control test. The enzyme
inhibition results are reported in Table 1.[11,12]
Crystal structure of soybean lipo-oxygenase-3 (arachi-
donic acid 15-lipo-oxygenase) complex with 13(S)-hydro-
proxy-9 (Z)-2,ll(E)-octadecadienoic acid was retrieved from
RCSB Protein Data Bank (PDB entry: 1IK3).
1.1.2 | Molecular docking
The fatty acid peroxide bonded to FeIII atom of the SLO 3D
structure was omitted. Then, the Fe was modified to FeIII–
OH, geometrically optimized by MM+ method in Hyper-
Chem 7.5b and outputted in PDB format for docking process.
Automated docking simulation was implemented to dock
the minimized structures into the active site of SLO with
AutoDock 4.2[9] using Lamarckian genetic algorithm.[10]
This method has been previously shown to produce binding
models similar to the experimentally observed models.[4–6]
The torsion angles of the ligands were identified, hydro-
gens were added to the macromolecule, bond distances were
edited, and solvent parameters were added to the enzyme 3D
structure. Partial atomic charges were then assigned to the
1.3 | Determination of DPPH bleaching
In this study, 25 μm solution of DPPH in absolute ethanol
was prepared. This solution was added to an equal volume
of the solution of the test compounds (dissolved in ethanol)
to obtain a desired concentration. Ethanol was used as the
control solution. After 6 h at 40 °C, the absorbance was
recorded at 517 nm (Table 1).[12]

Jabbari et al.
3
O
O
2
Br
O
O
O
O
O
O
4
5
a
O
Br
6
O
O
O
O
O
Br
O
7
8
O
O
O
Br
Br
Br
Br
O
O
b
+
R
O
O
O
O
9
O
O
O
R = H, Me, n-Pr, MeO
10
11
OH
O
O
O
c
3
SCHEME 1 General procedure for the synthesis of compounds 2–11. Reagents and conditions: (a) Mg, I2, & THF and then CuBr, LiBr, & allylbro-
mide; (b) TMED, n-BuLi, and Et2O; and (c) MeI and NaOEt
2
|
EXPERIMENTAL SECTION
Avance DRX 500 Fourier transformer spectrometer (Bruker
Co., Karlsruhe, Germany). Chemical shifts are reported in
parts per million (δ) downfield from tetramethylsilane. The
mass spectra were scanned on a Varian Mat CH-7 instru-
ment at 70 eV. All measurements of DPPH bleaching and
2.1 | Instruments
The IR spectra were obtained on a 4300 Shimadzu Spectrom-
eter. H NMR (500 MHz) was obtained by using a Bruker
1

4
Jabbari et al.
lipo-oxygenase activities were carried out using Spekol 1500
spectrophotometer (Analytikjena Co., Jena, Germany) and
BioTek ELX800 plate reader (Bioteck Co., Winooski, VT,
USA). The soybean 15-lipo-oxygenase (L1; Type I-B; EC
1,13,11,12) was purchased from Sigma Co. Methyl eugenol
and other chemicals were purchased from Aldrich (Sigma-Al-
drich Co., St. Louis, MO, USA) and Merck Co (Merck KGaA,
Darmstadt, Germany).
2.2.3 | 2-Allyl-1,4-dimethoxybenzene (5)
Colorless oil, IR (neat): 1640, 1610, 1590, 1500, 1460, 1040,
1
930 cm−1; H NMR: (CDCl3) δ 3.35 (d, 2H, J = 6.6 Hz),
3.85 (s, 6H), 5.05 (t, 1H, J = 1.4 Hz), 5.13 (m, 1H), 6.10
(m, 1H), 6.61–6.85 (m, 3H);: 34.0, 53.7, 57.4, 110.2, 111.9,
117.1, 119.8, 130.9, 136.0, 152.5, 155.1. MS; m/z: 178
[M+].[17]
2.2.4 | 1-(3,5-dimethoxyphenyl)-propane
2.2 | General procedure for preparation of
Colorless oil, IR (neat): 3080–2840, 1608, 1590, 1449, 1430,
2, 4, 5
1
1340, 1289, 1200 cm−1; H NMR: (CDCl3) δ 0.90 (t, 3H),
1.65 (m, 2H), 2.53 (t, 2H), 3.7 (s, 6H), 6.42 (br s, 3H); 13C
NMR δ 14.2, 24.6, 39.2, 52.8, 59.6, 125.0, 128.1, 128.6,
142.5, 153.3, 157.7. MS; m/z: 180 [M+].[14]
A mixture of 0.25 g (10 mmol) of magnesium turnings and
I2 in 4 mL of dry THF was added into 10 mmol of bromo-
1,3-dimethoxybenzene derivatives, and the mixture was
refluxed for 4 h under Ar. After the mixture was cooled to
room temperature, a catalytic amount of CuBr, codissolved
with LiBr in 1 mL of THF, and 15 mmol of allyl bromide
were added to the reaction mixture with care. The mixture
was stirred overnight in room temperature and then quenched
with 5 mL of saturated aqueous NH4C1. The solvent was
removed under reduced pressure, water was added, the
product was extracted with ether (three times), and the
combined organic layers were washed with 0.5 N aqueous
Na2S2O4 and with water. The solution was dried (MgSO4)
and evaporated to dryness. The resulting oil was purified
by column chromatography (silica, eluent: hexane/ethyl
acetate = 20:1) to give of allyl-1,3-dimethoxybenzene deriv-
atives as a colorless oil.[13] 1-(3,5-dimethoxyphenyl)-propa
ne, one of the precursor of compound 9 (Scheme 1), was also
prepared according to the mentioned synthetic procedure and
its structure was confirmed by comparing with the literature
spectral data.[14]
2.3 | Synthetic procedure of compound 3
A mixture of 0.3 g (4.4 mmol) of sodium ethoxide in 3 mL
ethanol, 0.7 g (4.4 mmol) of 2-ally-6-methoxyphenol, and
5 mmol of the methyl iodide was refluxed for 2 h. After
cooling, the mixture was poured into cold water (10 mL).
The mixture was acidified with 20% HCl and then extracted
with ether (2 × 10 mL). The combined extracts were washed
with 10% NaOH (2 × 10 mL), dried with anhydrous sodium
sulfate, and evaporated, and then, the residual oil was purified
by column chromatography.[6]
Colorless oil, IR (neat): 1645, 1619, 1590, 1500, 1456,
1
1045, 930 cm−1; H NMR (CDCl3): δ 3.47 (d, J = 6.5 Hz,
2H), 3.82 (s, 3H), 3.86 (s, 3H), 4.96-5.22 (m, 2H), 5.74-
6.25 (m, 1H), 6.60–7.15 (m, 3H); 13C NMR: δ 40.9, 51.0,
99.0, 106.2, 117.4, 130.2, 141.2, 165.7. MS; m/z: 178
[M+].[18]
2.4 | General procedure for preparation of
2.2.1 | 1-Allyl-3,5-dimethoxybenzene (2)
6–11
Colorless oil, IR (neat): 3085–2840, 1600, 1585, 1459, 1425,
1340, 1323, 1289, 1207 cm−1; 1H NMR: (CDCl3) δ 3.30 (d,
2H, J = 6.7 Hz), 3.82 (s, 6H), 5.15 (m, 2H), 6.00 (m, 1H),
6.27 (d, 1H, J = 2.1 Hz), 6.43 (d, 2H, J = 2.1 Hz); 13C NMR:
δ 42.7, 50.6, 100.4, 106.0, 130.4, 140.9, 118.2, 162.3. MS;
m/z: 178 [M+].[15]
To an ice-cold solution of desired compounds (1 mmol)
and 0.25 mL (2.44 mmol) tetramethyl ethylenediamine
(TMED) in 3 mL dry Et2O was added 0.49 mL n-BuLi
(1.22 mmol, 2.50 m in hexane) dropwise under argon. The
solution was stirred at 0 °C for 10 min and at room tempera-
ture for 2 h.[19] So to the prepared lithium anion derivatives
allyl and prenyl bromide (1.2 mmol) dissolved in 2 mL
dry xylene was added dropwise and the reaction mixture
refluxed for 7–24 h. After this time, the reaction was
cooled to room temperature, poured into 5 mL ice water,
and diluted with ether (25 mL). The layers were separated,
and the water layer was extracted with ether (two times).
The ether extracts were combined, washed once with 5%
sodium hydroxide (5 mL), once with water (10 mL), and
once with a saturated sodium chloride solution (10 mL),
2.2.2 | 1-Allyl-2,4-dimethoxybenzene (4)
Colorless oil, IR (neat): 3070–2830, 1630, 1590, 1435, 1340,
1
1330, 1296, 1120 cm−1; H NMR: (CDCl3) δ 3.25 (d, 2H,
J = 6.0 Hz), 3.74 (s, 6H), 5.10 (m, 2H), 6.10 (m, 1H), 6.21
(d, 1H, J = 2.0 Hz), 6.50 (d, 2H, J = 2.1 Hz); 13C NMR: δ
40.5, 51.0, 99.4, 107.0, 129.2, 141.9, 116.4, 166.3. MS; m/z:
178 [M+].[16]

Jabbari et al.
5
TABLE 1 Lipo-oxygenase inhibitory assessment data of the synthetic compounds and DPPH bleaching efficacy (250 μm) of compounds 1–11.
4-MMPB (CAS 928853-86-5) was used as control for lipo-oxygenase assaying
Compound
IC50 (μm)
% DPPH bleaching
Compound
IC50 (μm)
% DPPH bleaching
1
2
33.3 1.8
43.2 3.1
49.5 3.1
39.1 1.8
7
8
83.2 2.7
30.5 1.9
25.1 1.7
38.3 1.4
3
128.5 6.1
16.0 1.5
9
7.6 0.7
52.0 1.9
4
31.4 2.8
34.3 1.7
10
5.3 0.3
53.1 1.4
5
6
102.9 3.6
53.2 2.6
24.6 1.4
36.2 1.7
11
4-MMPB
0.52 0.1
22.6 2.8
51.2 1.1
95<
and dried with MgSO4 and evaporated to dryness oil. The
resulting product was purified by column chromatography
(silica, eluent: hexane/ether = 20:1) to give corresponding
derivatives as a colorless oil.[20]
2.4.1 | 2-Allyl-1,3-dimethoxybenzene (6)
Colorless oil, IR (neat): 3080–2840, 1625, 1600, 1480, 1470,
1
1290, 1070, 995 cm−1; H NMR: (CDCl3) δ 3.43 (d, 2H,
J = 6.6 Hz), 3.75 (s, 6H), 4.95 (t, 1H, J = 1.5 Hz), 5.05 (m,
1H), 6.08 (m, 1H), 6.18–6.93 (m, 3H); 13C NMR δ: 29.9,
52.5, 59.4, 111.3, 112.9, 119.0, 120.0, 131.4, 132.8, 153.2,
157.2. MS; m/z: 178 [M+].[21]
FIGURE 1 DPPH bleaching potency of the synthetic compounds
against their SLO inhibition activity. Due to high deviation, the data of
compounds 1, 10, and 11 were omitted
2.4.2 | 2-Allyl-1,3,5-trimethoxybenzene (7)
Colorless oil, IR (neat): 3080–2850, 1639, 1605, 1500,
1
1460, 1070, 960, 925 cm−1; H NMR: (CDCl3) δ 3.30 (d,
2H, J = 6.2 Hz), 3.73 (s, 6H), 4.86–5.05 (2H, m), 5.60–
6.18 (1H, m), 6.40 (s, 2H); 13C NMR δ 32.1, 54.2, 56.1,
110.1, 112.6, 117.8, 136.2, 154.4, 160.3. MS; m/z: 208
[M+].[22]
2.4.5 | (E)-2-(3,7-Dimethylocta-2,6-dien-1-
yl)-1,3-dimethoxy-5-methylbenzene (10)
Colorless oil, IR (neat): 3095–2900, 1605, 1590, 1080,
980 cm−1; 1H NMR (CDCl3) δ 1.54 (s, 3H), 1.63 (s, 3H), 1.75
(s, 3H), 2.00 (m, 4H), 2.44 (s, 3H), 3.36 (d, 2H, J = 7 Hz),
3.83 (s, 6H), 5.09 (t, 1H, J = 7 Hz), 5.35 (t, 1H, J = 7H), 6.88
(d, 1H, J = 8 Hz), 7.21 (d, 1H, J = 8 Hz); 13C NMR δ 16.7,
17.7, 21.1, 25.7, 26.4, 33.5, 39.7, 51.7, 52.2, 117.0, 122.5,
124.2, 130.0, 131.46, 136.4, 139.3, 149.0, 155.8. MS; m/z:
288 [M+].[24]
2.4.3 | 2-Allyl-1,3-dimethoxy-5-
methylbenzene (8)
Colorless oil, IR (neat): 3095–2840, 1600, 1595, 1465,
1
1425, 1100, 970 cm−1; H NMR (CDCl3) δ 2.40 (s, 3H),
3.47 (m, 2H), 3.96 (s, 6H), 5.15 (m, 2H), 5.75–6.28 (m,
1H), 6.62 (s, 2H); 13C NMR δ 21.4, 38.3, 52.1, 56.3, 115.8,
127.9, 128.5, 135.7, 140.1, 142.6, 155.7, 159.6. MS; m/z:
192 [M+].[20]
2.4.6 | 1,3-Dimethoxy-5-methyl-2-((2E,6E)-
3,7,11-trimethyldodeca-2,6,10-trien-1-yl)
benzene (11)
2.4.4 | 1,3-Dimethoxy-5-methyl-2-(3-
methylbut-2-en-1-yl)benzene (9)
Colorless oil, IR (neat): 3090–2900, 1605, 1580, 1117,
1
980 cm−1; H NMR (CDCl3) δ 1.4 (m, 12H), 2.1 (m, 8H),
Colorless oil, IR (neat): 3095–2910, 1610, 1587, 1110,
1
990 cm−1; H NMR (CDCl3) δ 1.79 (s, 3H), 1.81 (s, 3H),
2.2 (s, 3H), 3.38 (br d, 2H), 3.82 (s, 6H), 5.06–5.39 (br m,
3H), 6.38 (s, 2H); 13C NMR δ 15.9, 17.5, 21.7, 21.9, 23.3,
25.6, 26.66, 31.85, 39.6, 39.8, 55.5, 104.7, 115.4, 123.4,
123.8, 124.7, 129.00, 134.00, 136.43, 157.82. S; m/z: 365
[M+].[25]
2.42 (s, 3H), 2.50 (d, 2H, J = 6.6 Hz), 3.9 (s, 6H), 5.54 (m,
1H), 6.7 (m, 2H); 13C NMR δ 21.2, 25.1, 25.4, 39.8, 51.9,
52.5, 108.9, 112.3, 120.0, 137.6, 141.6, 143.2, 154.2, 156.3.
MS; m/z: 220 [M+].[23]

6
Jabbari et al.
FIGURE 2 (Above) Stick view of the consensus
bonding conformation of 11 in the active site pocket
of the SLO in the presence of 13 (S)-hydroperoxy-
9(Z),11(E)-octadecadienoic acid (green stick)
bonded to the iron core (PDB code: 1IK3). (Below)
Solvent surface view of the aforementioned
compounds
Among
the
synthetic
dimethoxyallylbenzenes,
3
|
RESULTS AND DISCUSSION
2,4-dimethoxyallylbenzene (4) and methyl eugenol (1)
showed the highest lipo-oxygenase inhibitory potency and
2,3-dimethoxyallylbenzene (3) was the weakest inactivator
(IC50: 33.3 1.8 μm versus 128.5 6.1 μm; Table 1). The
SLO inhibition activity of the para-methyl and para-methoxy
derivatives of 2,6-dimethoxyallybenze was evaluated to study
the electronic effects of the mentioned groups on the lipo-
oxygenase inhibition activity. It is interesting to note that the
introduction of methyl (8) and methoxy groups (7) increased
and decreased inhibitory potency, respectively. The radical
scavenging potency of all the synthetic compounds was
measured by the DPPH bleaching test in thermodynamic
conditions (concentration of sample: 250 μm, operating
temperature: 40 °C, duration: 6 h).[26] As a result of the low
radical trapping potency of allylbenzenes, the aforemen-
tioned conditions made a suitable condition for comparing
the radical scavenging potency of the synthetic compounds.
Compound 3 was synthesized through the methylation of
O-eugenol.[6] Compounds 2, 4, and 5 were synthesized
through reaction of allyl bromide with the desired dime-
thoxybenzyl magnesium bromide.[13] Compounds 6–11 were
synthesized through coupling of ally or prenyl bromides with
the desired 1,3-dimethoxybenzyl lithium (Scheme 1).[20]
Compound 1 (methyl eugenol) was commercially available.
The lipo-oxygenase inhibitory activity of all the synthetic
compounds against SLO (soybean 15-lipo-oxygenase) was
determined using peroxide formation method reported in
the previous literatures.[11,12] In this method, the basis of
the lipo-oxygenase assay is the measurement of fatty acid
peroxide concentration. All the measurements were down
in comparison with one of the well-known lipo-oxygenase
inhibitors: 4-methyl-2-(4-methylpiperazinyl)pyrimido[4,5-b]
benzothiazine (4-MMPB; CAS 928853-86-5).

Jabbari et al.
7
FIGURE 3 The diagrams show the variations
of three expressions of the mean estimated inhibitory
constants (−Log KiAC, −Log KiMPC, and −Log KiLEC
versus −Log IC50 (SLO inhibition) of compounds
1–11. The data are presented as SD
)
Among the synthetic dimethoxyallylbenzenes, compound
bleaching potency of compounds 2–9 is in accordance with
their lipo-oxygenase inhibition activity (Figure 1).
Changing the allyl moiety of 8 to isopentenyl (9) led to
increase in both DPPH bleaching and LOX inhibition (four-
fold) ability. It is interesting to note that by increasing the
1 showed the best radical scavenging activity, while the lowest
activitywasobservedfor3(Table1). Compound8withmethyl
substitution at the para position of 2,6-dimethoxyallybenze
showed more activity than 6. It is notable that the DPPH

8
Jabbari et al.
TABLE 2 The stabilization energies of natural (En) and radical (Er) states, bond dissociation enthalpy (BDE), number, and distance of intermolecular
hydrogen bond (HB) are related to compounds 1–9 (energies in Hartree and distances in Angstrom)
Compound
En
Er
BDE
HB (no.)
HB (distance)
1
2
−577.918314
−577.924937
−577.302953
−577.307899
0.115464
0.117141
0
0
–
–
3
−577.916256
−577.296924
0.119435
1
2.244
4
−577.923691
−577.305948
0.117846
1
2.220
5
6
−577.921392
−577.922098
−577.302509
−577.303418
0.118986
0.118783
1
2
2.205
2.211/2.184
7
–
–
0.118192
2
2.221/2.195
8
–
–
0.117447
2
2.217/2.190
9
–
–
0.117051
2
2.198/2.166
prenyl chain length, via insertion of geranyl (10) and farnesyl
(11), the inhibition potency was increased by 6- to 60-folds.
In spite of lipo-oxygenase inhibition enhancement of the
geranyl and farnesyl derivatives, in comparison with 9, the
DPPH bleaching potency did not change.
To rationalize the observed variation in the radical scav-
enging potency and also explain the contradiction seen for
compounds 1, 10–11, computer-assisted modeling studies
were performed on the synthetic compounds. The modeling
involved two calculative procedures: SLO inhibitor docking
analysis and single point calculations, both were performed
on the energy-minimized 3D structures of the synthetic
compounds.
To approach this aim, semiempirical energy-minimized
3D structure of 1–11 was docked into the SLO (PDB entry:
1IK3) active site pocket, while the Fe core was modified to
FeIII–OH and side chain of Leu277, Leu515, Leu557, Leu560,
Leu565, Val566, Ile572, Phe576, Leu773, and Ile857 was
made flexible. The bonding affinity of the designed molecular
structures toward SLO was studied. One hundred (100) docked
conformers of the compounds were generated in Auto Dock
Tools (ADT) software.d A detailed assessment of the output
clusters (cluster tolerance/Å = 2.0) of the enzyme revealed
that for each of the docked molecules, the most popular cluster
exists. For compounds 1–8, the number of conformations
(22–41%) belongs to a cluster in which the phenyl moiety
is surrounded by His513, Gln514, His518, Ile572, Phe576,
FIGURE 4 Linear regression between C–HBDE and DPPH bleaching
activity of compounds 1–5, 7, and 8
Gln716, Asp766, Ile770, and Lue773. For compounds 9–11,
the popular cluster involves conformations in which the phenyl
moiety is surrounded by Leu277, His523, Ile557, Leu560,
Ala561, Leu565, Val769, Leu773, and Ile857 and prenyl chain
is covered by His513, Ser510, Gln514, His518, Trp519, Ile572,
Phe576, Gln716, and Arg726 (Figure 2). Except Leu277,
Phe576, Arg726, and Trp519, all the mentioned amino acids
are conserved residues.[5] It was noted that the bonding
conformers with the least estimated inhibitory constant (Ki)
FIGURE 5 Ball and stick view of the geometry-
optimized structures of compound 6 in the natural
(left) and radical (right) forms

Jabbari et al.
9
D2O
SLO-FeIII-OD
SLO-Fe-OH
H
O
O
H
O
O
O
H
SLO-FeIII-OD
SLO-FeII-OHD
D2O
+
+
+
+
O
H
O
H
O
D
SLO-FeIII-OD
SLO-FeII-OD2
FIGURE 6 Suggested mechanism for deuteri-
um exchange of allyl hydrogens of compound 6
belonged to the mentioned clusters. Among all the conformers
ineachclusterofthedockedinhibitors, onewiththeleastKiwas
named ‘consensus structure’ and used for further analysis.[3]
In all the consensus structures, the allyl moiety was situated
near the Fe–OH core (Figure 2). The mentioned orientation
was suitable for interfering with Fe–OH hydrogen abstraction
during the peroxidation process. To perform better analysis on
docking results, the average Ki of all the conformers from the
most populated cluster (KiMPC), average of all the lowest Ki
from each cluster (KiLEC), and average Ki of all the conformers
(KiAC) were calculated. Acceptable conformity between the
KiMPC and IC50 variations was observed for compounds 2–5
and 9–11 (Figure 3A–C, respectively). For compounds 1, 6–8,
observing the disconformities between IC50 variations and Ki
results apart from the convergence between DPPH bleaching
and IC50 values indicates that electronic properties are one of
the major factors for the lipo-oxygenase inhibition potency of
the aforementioned compounds.
The stabilization energies related to the optimized struc-
tures of compounds 1–9 in both natural and mono-radical
states were computed using the B3LYP/6-311++G (d, p)
level of theory (Table 2). No acceptable relationship was
found between stabilization energy and radical scavenging
activity. Considering the hydrogen donating mechanism
of allylbenzene in the radical scavenging process,[27] C–H
bond dissociation enthalpy (C–HBDE) of the methylene
portion of the mentioned compounds was also calculated.
The obtained data were responsible for DPPH bleaching
variations of compounds 1–5, 7, and 8 with good regression
(Figure 4). The acquired results persuaded us to do further
studying for finding rational factors affecting the stability of
the radical state of compounds 6 and 9. Among the effective
factors, interamolecular hydrogen bonds could explain the
radical scavenging potency exception of compounds 6 and
9. It was suggested that the hydrogen bonding between the
methoxy groups and methine hydrogen (CH…O) fixing the
planarity between phenyl and allyl pi orbitals (Figure 5) plays
an important role in radical stability, so compounds 6 and 9
with two methoxy groups at the ortho position and shortest
intramolecular hydrogen bond distance showed the DPPH
bleaching efficacy similar to compounds with low C–HBDE
(e.g., compound 9 versus 1 and compound 6 versus 2 & 4).
Similarly, the role of interamolecular hydrogen bonds on the
stability of phenoxy radical has been reported by Foti et al.[28]
and Praveena et al.[29]
In compounds 1 and 2 with no situation for intramolecular
hydrogen bond formation between methoxy and methylene
hydrogen in radical state, inductive and mesomeric effects
are the major factors in radical scavenging. The experiment
showed the best DPPH bleaching for 1 as compared to 2. C–
HBDE calculation showed lower energy for radical formation
of 1 which arises from both inductive and mesomeric effects
of meta- and para-methoxy substitution, respectively.
Itisinterestingtonotethatbyintroducingmethylandmethoxy
substituents at the para position of 2,6-dimethoxyallylbenzene,
the DPPH bleaching activity changes according to both C–
HBDE and CH…O length results. It shows consonant role of
both electronic and steric effects in the radical scavenging
tendency of compounds 7 and 8. The opposite result is seen
for compounds 4 and 5. In these compounds, relocation of
the methoxy group from para (4) to meta (5) position leads to
decrease in CH…O length but finally increase in both DPPH
and lipo-oxygenase deactivation ability is seen according to
C–HBDE results. For further proof, compounds 5 and 6, with
similar C–HBDE, were treated with SLO in D2O at pH 8.0
(phosphate buffer) for 12 h. Then, the mixtures were extracted
by diethyl ether and after solvent removal were analyzed by
H NMR. The H NMR spectra showed 30% exchange of allyl
hydrogens (–CH2–) of compound 6 compared to compound 5
and controls (controls: each of the compounds in D2O at pH
8.0 without enzyme). It demonstrates the deuterium exchange
of allyl hydrogen during hydrogen abstraction of FeIII–OH[30]
in the active site of the enzyme (Figure 6) and also it empha-
sizes on the role of CH…O length in stability of radical form
of compound 6. It was notable that no oxidized form of the
allylbenzene was detected after the experiment. Deuterium
exchange of allyl hydrogens shows that the allylbenzenes mimic
the 1,3-diene moiety of unsaturated fatty acids and compete

10
Jabbari et al.
[3] C. Charlier, C. Michaux, Eur. J. Med. Chem. 2003, 38, 645.
with them for occupying the enzyme active site pocket, but
they are only involved until the hydrogen abstraction step with
no further reaction with oxygen.
[4] H. Sadeghian, S. M. Seyedi, M. R. Saberi, Z. Arghiani, M. Riazi, Bioorg.
Med. Chem. 2008, 16, 890.
[5] H. Sadeghian, N. Attaran, Z. Jafari, M. R. Saberi, M. Pordel, M. M.
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[6] H. Sadeghian, S. M. Seyedi, N. Attaran, A. Jabbari, Z. Jafari, J. Enzyme
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In summary, it was shown that allylbenzenes can inhibit
lipo-oxygenase activity via radical scavenging mechanism
arising from C–H dissociation of the allylmethylene moiety
with no more reaction with oxygen. On the other hand, it was
suggested that the CH…O hydrogen bond strength between
ortho-methoxy oxygen and methine hydrogen atoms is one
of the major factors in the stability of dimethoxyallylben-
zenes radical via the planarity fixation between phenyl and
allyl pi orbitals. In some cases, the mentioned phenomenon
is more efficient than the inductive and mesomeric effect of
the electron-withdrawing substituents. Now, based on this
study, other ortho-methoxyallylbenzenes can be designed
and synthesized as more potent lipo-oxygenase inhibitors.
[9] G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S.
Goodsell, A. J. Olson, J. Comput. Chem. 2009, 30, 2785.
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[13] H. K. A. C. Coolen, J. A. M. van Meeuwis, P. W. N. N. Leeuwen, R. J.
M. Nolte, J. Am. Chem. Soc. 1982, 117, 11906.
FUNDING
[14] D. R. Compton, W. R. Prescott, B. R. Martin, C. Siegel, P. M. Gordon,
R. K. Razdan, J. Med. Chem. 1991, 34, 3310.
This study was supported by grant nos. 910502 and 3/25470
from Mashhad University of Medical Sciences and Ferdowsi
University, respectively.
[15] G. C. Dol, P. C. J. van Kamer, P. W. N. M. Leeuwen, Eur. J. Org. Chem.
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[16] E. Wenkert, J. B. Fernandes, E. L. Michelotti, C. S. Swindell, Synthesis
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DECLARATION OF INTEREST
[18] G. Dayaker, F. Chevallier, P. C. Gros, F. Mongin, Tetrahedron 2010,
66, 8904.
The authors state no conflict of interest and have received no
payment in preparation of this manuscript.
[19] A. Takeuchi, Y. G. Wang, Y. Kobayashi, Org. Lett. 2006, 8, 2699.
[20] A. E. Guthrie, J. E. Semple, M. M. Joullie, J. Org. Chem. 1982, 47,
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[21] V. D. Kaliakoudasl, C. H. Eugster, P. Riiedi, Helv. Chim. Acta 1990,
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NOTES
a ChemDraw® Ultra, Chemical Structure Drawing Standard, CambridgeSoft
Corporation, 100 Cambridge Park Drive, Cambridge, MA 02140 USA, http://
www.cambrigesoft.com.
b HyperChem® Release 7, Hypercube Inc., http://www.hyper.com/.
c http://accelrys.com/products/discovery-studio.
d Auto Dock Tools (ADT). The Scripps Research Institute, 10550 North Torrey
Pines Road, La Jolla, CA 92037-1000, USA, http://www.scripps.edu/pub/
olson-web/doc/autodock/.
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