Synthesis and SAR study of 4,5-diaryl-1H-imidazole-2(3H)-thione derivatives, as potent 15-lipoxygenase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.09.050

A series of 4,5-diaryl-1H-imidazole-2(3H)-thione was synthesized and their inhibitory potency against soybean 15-lipoxygenase and free radical scavenging activities were determined. Compound 11 showed the best IC₅₀ for 15-LOX inhibition (IC₅₀ = 4.7 μM) and free radical scavenging activity (IC₅₀ = 14 μM). Methylation of SH at C₂ position of imidazole has dramatically decreased the 15-LOX inhibition and radical scavenging activity as it can be observed in the inactive compound 14 (IC₅₀ >250 μM). Structure activity similarity (SAS) showed that the most important chemical modification in this series was methylation of SH group and Docking studies revealed a proper orientation for SH group towards Fe core of the 15-LOX active site. Therefore it was concluded that iron chelating could be a possible mechanism for enzyme inhibition in this series of compounds.

Full text of DOI:10.1016/j.bmc.2012.09.050

Bioorganic & Medicinal Chemistry 20 (2012) 7160–7166
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and SAR study of 4,5-diaryl-1H-imidazole-2(3H)-thione derivatives,
as potent 15-lipoxygenase inhibitors
Amir Assadieskandar a, Mohsen Amini a, Marjan Salehi a, Hamid Sadeghian b,c, Maliheh Alimardani c,
Amirhossein Sakhteman d, Hamid Nadri d, Abbas Shafiee e,
⇑
a Department of Medicinal Chemistry, Faculty of Pharmacy and Drug Design & Development Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran
b Microbiology & Virology Research Center, Buali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran
c Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad, Iran
d Department of Medicinal Chemistry, Faculty of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
e Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 July 2012
Revised 24 September 2012
Accepted 25 September 2012
Available online 2 October 2012
A series of 4,5-diaryl-1H-imidazole-2(3H)-thione was synthesized and their inhibitory potency against
soybean 15-lipoxygenase and free radical scavenging activities were determined. Compound 11 showed
the best IC50 for 15-LOX inhibition (IC50 = 4.7
Methylation of SH at C2 position of imidazole has dramatically decreased the 15-LOX inhibition and rad-
ical scavenging activity as it can be observed in the inactive compound 14 (IC50 >250 M). Structure
lM) and free radical scavenging activity (IC50 = 14 lM).
l
activity similarity (SAS) showed that the most important chemical modification in this series was meth-
ylation of SH group and Docking studies revealed a proper orientation for SH group towards Fe core of the
15-LOX active site. Therefore it was concluded that iron chelating could be a possible mechanism for
enzyme inhibition in this series of compounds.
Keywords:
Soybean 15-lipoxygenase (SLO)
Radical scavenging activity
Structure–activity relationship studies
4,5-Diarylimidazole
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
such as cancer and chronic obstructive pulmonary diseases
(COPD).12,13 The role of 15-LOX in the progression of atherogenesis
Eicosanoids are a family of lipid mediators derived from the
metabolism of arachidonic acid (AA). Some types of eicosanoids
such as prostanoids and leukotrienes showed to have a wide range
of biological actions including potent effects on inflammation and
immunity. AA is the substrate for various metabolic pathways that
produces biological mediators with cyclooxygenase and lipoxyge-
nase.1,2 Lipoxygenases (LOX) constitute a family of dioxygenases,
that catalyze dioxygenation of 1,4-cis,cis-pentadiene-containing
polyunsaturated fatty acids to produce the corresponding hydro-
peroxy derivatives.3 They are class of non-heme iron containing
enzyme found in both plant and animals. Three main LOXs includ-
ing 5, 12 and 15-lipoxygenases have been distinguished based on
the peroxidation site of AA.4 Comparing the two LOXs of the rabbit
and soybean revealed similarities between the active site of animal
LOX and that of plant in order to its sequence homology and struc-
ture.5–10 The highest level of sequence identity between LOX from
plants and mammals lies in the area of the catalytic domain con-
taining the non-heme iron atom. Since the soybean enzyme is
much more accessible than human, its use as a model for designing
inhibitors could be considered as a tool in structural characteriza-
tion, mechanism elucidation and discovery of new inhibitors.11
15-Lipoxygenase (15-LOX) has been implicated in several diseases
and vascular disease has been also verified.14,15 It was also found
that 15-LOX takes role in the oxidation of low-density protein
(LDL) leading to the progression of atherosclerosis.16 The immedi-
ate products of 15-LOX oxidation were shown to have pro-
inflammatory and pro-thrombotic effects.17,18 Furthermore, 15-
LOX proposed to have
a beneficial role in human airway
carcinomas and promotes apoptotic pathway. Neoplastic tissues
from human airway carcinomas demonstrated non-specific
staining for human 15-LOX as compared with normal tissues.19
In contrast, in human prostate tumors, 15-LOXa was over
expressed as compared with normal adjacent tissue, and 15-LOXb
was poorly expressed in prostate tumors.12,20
It was found that 15-LOXa takes part in minimization of the
brain damage after stroke. An important consequence of stroke is
damage of the mitochondria, leading to the breakdown of the
membrane potential, the cytochrome C release and production of
ROS, which are the major features of neuronal cell death.21
The three different strategies to inhibit the LOX’s pathway are
as follows: (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.22
It has been reported that the iron chelating property of sulfur
atom could be essential for the activity of 15-LOX inhibitors.
According to the later study, 4-Methyl-2-(4-methylpiperazinyl)
⇑
Corresponding author. Tel.: +98 21 66406757; fax: +98 21 66461178.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.050

A. Assadieskandar et al. / Bioorg. Med. Chem. 20 (2012) 7160–7166
7161
Cl
Cl
H3CN
S
N
H
N
S
N
N
HN
Ar1
NH
Ar2
a
SCH3
S
N
N
H
N
H
N
H
CH3
11
H3CO
14
H3CO
1-13
MMPB
Scheme 2. Reagents and conditions: (a) CH3I, CH3OH, reflux.
Figure 1. Chemical structure of 4-MMPB and designed compounds.
In one approach for the preparation of unsymmetrical benzoins,
cyanide ion-catalyzed cleavage of benzils derivatives was used to
generate acyl intermediates. The reaction of these intermediates
with various aldehydes furnishes the corresponding esters of
unsymmetrical benzoins. Finally, the products were subjected to
hydrolyze in a basic medium.32 In another approach, various
substituted aromatic hydrocarbons were reacted with appropriate
phenyl acetic acid. The products were then subjected to bromina-
pyrimido[4,5-b]benzothiazine (4-MMPB) was introduced as a po-
tent 15-lipoxygenase23 (Fig. 1).
Additionally, the coordination chemistry of heterocyclic thiones
especially those possessing
a-N-heteroatom is rich due to their
versatility in chelating property.24 Considering this fact that a thio-
urea moiety has constructed an electron rich site, evaluation of
lipoxygenase properties of imidazole-2-thione scaffold has been
of our interest. In continuation of our research work on design
and synthesis of LOX inhibitors,25–28 a new series of 4,5-diaryl-
1H-imidazole-2(3H)-thione derivatives were synthesized and
in vitro 15-LOX inhibitory and free radical scavenging activity of
the synthesized compounds were studied. Structure activity simi-
larity (SAS) and docking experiments were subsequently used to
obtain a pattern of structure activity in this series of compounds.
Furthermore, diarylheterocycle scaffold has been extensively
studied as cyclooxygenase (COX) inhibitors.29
tion using bromine in glacial acetic acid. Subsequently, related a-
bromodesoxybenzoin was refluxed with sodium methoxide in
methanol and the reaction was finally quenched with cold 10%
hydrochloric acid to obtain unsymmetrical benzoin.33 Treatment
of different benzoins with a ten-fold excess of ammonium thiocy-
anate in n-butanol afforded the desired 4,5-diaryl-1H-imidazole-
2(3H)-thiones 1–13 in good yield (Scheme 1).
Finally, alkylation of 11 using methyl iodide in basic media
afforded compounds 14 in good yield (Scheme 2).
2. Results and discussion
2.1. Chemistry
2.2. Inhibition of 15-lipoxygenase
The target compounds 1–14 were evaluated for their inhibitory
activity against Soybean LOX using modified DMAB-MBTH method
reported by Anthon et al.34 In this method the basis of the lipoxy-
genase activity determination is peroxide measurement and the
potent 15-LOX inhibitor 4-methyl-2-(4-methylpiperazinyl)pyrimi-
do[4,5-b]benzothiazine (4-MMPB) was used as the reference com-
pound. The results are summarized in Table 1.
4,5-Diaryl-1H-imidazole-2(3H)-thiones were synthesized by
the reaction of different benzoins and ammonium thiocyanate.30
The cyanide ion-catalyzed condensation of aromatic aldehydes is
a convenient synthetic method for the symmetrical benzoins.
However, synthesis of unsymmetrical benzoins under traditional
conditions imposes to the formation of four different benzoins.31
O
O
a
H
H
+
R1
R2
R2
+
R2
R2
H
O
O
O
O
O
b
c
O
O
OH
R1
R2
R1
R1
R2
g
R2
R2
e
O
HO
O
O
R2
d
f
+
H
N
Br
S
R2
R1
R1
N
H
R1
1-13
R1
Scheme 1. Reagents and conditions: (a) KCN, CH3OH/H2O, reflux; (b) KCN, DMF, rt; (c) NaOH, CH3CN, rt; (d) H3PO4, (CF3CO)2O, 25 °C; (e) Br2, glacial AcOH, rt; (f) CH3ONa,
CH3OH, reflux, 10% HCl; (g) NH4SCN, n-butanol, reflux.

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A. Assadieskandar et al. / Bioorg. Med. Chem. 20 (2012) 7160–7166
Table 1
As the employment of methoxy (OCH3) and methylthio (SCH3)
substitutes into the para position of phenyl ring is favorable for po-
tency, we substituted such moieties (R1 = OCH3, SCH3 and N(CH3)2)
into p-position of one of the phenyl rings and employed different
substitutes (R2 = Cl, F and OCH3) on the other one. The IC50 value
of resulted derivatives 8–13, defined as disubstituted group, is var-
Enzyme inhibitory assessment and DPPH radical scavenging activity data of the
synthetic compounds in comparison with 4-MMPB and NDGA respectively
R1
N
ied in the range of 4.7–136 lM. Investigation of R1 moiety in disub-
SR
stituted derivatives revealed that the order of 15-LOX inhibitory
potential is OCH3 > SCH3 > N(CH3)2. Comparison of compound 8
with 9 containing N(CH3)2 group, compound 9 (R2 = p-methoxy,
N
H
R2
IC50 = 9.4 lM) is more potent than 8 (R2 = p-Cl, IC50 = 22.3 lM).
Compounds
R
R1
R2
IC50
l
M (DPPH
IC50 lM
Therefore, it seems that one methoxy group in para position was
favorable for potency. Considering the R2 in p-methoxy phenyl
substituted compounds (2, 5, 10 and 11) demonstrated that the
15-LOX inhibitory potency is mainly dependent on the nature of
R2 moiety with the order of Cl ꢀ F > H > CH3O. Thus, it can be de-
duced that the introduction of electron-donating OCH3 in R1 and
electron-withdrawing Cl in R2 were able to improve the activity
of these series. It could be postulated that OCH3 and Cl establish
a balanced resonance in aryl rings in order to increase the iron che-
lating property of sulfur atom. Hofmann et al. reported similar sub-
radical scavenging
activity)
(soybean 15-
LOX)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3O
F
Cl
CH3O
Cl
CH3S
(CH3)2N Cl
(CH3)2N CH3O
CH3O
CH3O
CH3S
CH3S
H
CH3O
F
Cl
H
H
49.2 0.9
15.5 0.7
143.1 2.8
107.4 1.2
87.4 1.1
30.3 0.8
90.0 1.6
15.9 0.3
15.2 0.8
57.2 1.3
14.0 0.4
49.0 0.5
21.2 0.6
>250
15.2 0.8
14.7 0.5
23.4 1.5
62.0 2.5
13.7 0.8
24.9 1.3
5.7 0.4
22.3 0.6
9.4 0.4
11.0 0.9
4.7 0.3
136 4.3
19.7 0.7
>250
H
F
Cl
F
Cl
Cl
stituents in 5-benzylidene-2-phenylthiazolinone scaffold as
a
potent 5-LOX inhibitor.35 Furthermore, comparison of compounds
10–13 showed that replacement of the p-methoxy with a weak
electron-releasing methylthio group (12 vs 10, 13 vs 11) led to de-
crease of 15-LOX activity.
CH3 CH3O
4-MMPBa
NDGAb
—
34.2 1.2
—
6.2 0.4
a
2.3. DPPH radical scavenging activity
4-Methyl-2-(4-methylpiperazinyl)pyrimido[4,5-b]benzothiazine.
Nordihydroguaiaretic acid.
b
The free radical scavenging activity of the compounds 1–14 was
evaluated by DPPH colorimetric method. Several dilutions of the
compounds were prepared for calculation of the IC50 through
regression analysis of the mean values. Among the tested com-
In general, most of compounds demonstrated significant 15-
LOX inhibitory potential. Two of the synthesized compounds, 7
(IC50 = 5.7 M) and 11 (IC50 = 4.7 M), had the best 15-LOX inhibi-
tion compared to reference compound 4-MMPB (IC50 = 34.2 M).
l
l
pounds, compound 11 (IC50 = 14.0 lM) showed the best DPPH rad-
l
ical scavenging activity in comparison with nordihydroguaiaretic
acid (NDGA) as a reference compound. This observation suggests
that the synthetic compounds could possibly inhibit the lipoxyge-
nase activity by their radical scavenging properties and chelating
the iron atom. Finally, S-methylation of compound 11 led to inac-
tive compound 14 (15-LOX inhibition and radical scavenging po-
According to the substitution pattern on the phenyl rings, the syn-
thesized compounds could be categorized in two series: ‘Symmet-
rical derivatives’ which contain the similar group on both phenyl
rings and ‘unsymmetrical derivatives’ with different substituted
moiety on the mentioned phenyl rings. Structure–activity relation-
ship studies showed that the nature of substituted group on the
phenyl ring (R1 and R2) had a profound influence on 15-LOX inhib-
itory activity of compounds in both series.
tency >250 lM).
2.4. Structure activity similarity
2.2.1. Symmetrical series
In order to obtain a correlation between the activity and struc-
ture of the compounds, structure activity similarity map (SAS) was
generated for this series of compounds.36
Substitution of electron withdrawing halogen groups into the
para position of both phenyls resulted in drastic reduced 15-LOX-
inhibitory potential of symmetrical derivatives; while introduction
of p-methoxy group into the phenyl rings, eventuated in enhanced
inhibitory activity. The symmetrical compound 2 containing p-
dimethoxy phenyl substitutes on the central imidazole core, is
SAS map is a comprehensive tool in developing structure–activ-
ity relationship (SAR) of the synthetic structures. By the aforemen-
tioned method, it is possible to obtain an overview of the chemical
activity space prior to visual inspection of the modifications in each
pair of ligands. In addition, by using SAS maps, it is possible to clus-
ter the data in to four regions of different SAR, which makes it eas-
ier to design more potent compounds based on the similarities of
the active structures.
The plot of the activity and similarity coefficients for the syn-
thesized structures is depicted in Figure 2. According to the pattern
shown in this Figure, most of the points are present in the upper
right part of the plot, which is anticipated for the analogue series
with similarities in the structure and activity. The highest similar-
ity in terms of both activity and structural features was in com-
pounds 5 and 2, with one and two OCH3 substitutions at para
position of the phenyl rings, respectively (Fig. 2a. Tanimoto simi-
larity = 1, Activity similarity = 0.99). On the other hand, the lowest
similarity in the structural features was in compounds 12 and 9
the most potent agent of this series (IC50 = 14.7 lM). However;
the halogen containing derivatives, demonstrated the lowest
inhibitory potential against 15-LOX enzyme that is attributed to
the size of halogen atom along with electron withdrawing property
of substituted halogen. The difluorophenyl containing derivative 3
(IC50 = 23.4
ed compound 4 (IC50 = 62
l
M) is more potent than the dichlorophenyl susbstitut-
M) which could be due to smaller size
l
of fluorine relation to chlorine group.
2.2.2. Unsymmetrical derivatives
Among monosubstituted derivatives of this category (com-
pounds 5–7), p-methylthio containing compound 7 showed signif-
icant LOX inhibitory activity (IC50 = 5.7 lM). Relying on the
para-substituted moiety (R1) of monosubstituted derivatives, the
order of potency is CH3S > CH3O > Cl.

A. Assadieskandar et al. / Bioorg. Med. Chem. 20 (2012) 7160–7166
7163
on the SAS map, reasonable range of structural–activity diversity
(tanimoto similarity 0.5–1, activity similarity 0–1) suggested that
the number of synthesized compounds was suitable for studies
of structure–activity relationship.
2.5. Molecular modeling study
Based on the results, the most important chemical modification
in this series of compounds was methylation of SH at the C2 of imi-
dazol ring, which led to the inactive compound, 14. It was postu-
lated that a possible mechanism for the activity of the SH
containing compounds might be through chelating the iron atom
in the binding site of the lipooxygenase enzyme. In order to inves-
tigate the orientation of the synthesized structures towards the
iron atom in the active site pocket, some docking studies using
autodock vina were performed. After running vina in case of two
active structures (1 and 5), the best docking poses of the ligands
were visualized using VMD. According to Figure 3 (left), a 4.84 Å
distance between the sulfur atoms of the ligands and the iron atom
in the active site was observed. This verifies that the possible
mechanism for the activity of the synthesized compounds could
be through iron chelating activity. Therefore, it looks reasonable
that a decrease in the activity of compound 14 might be caused
by sulfur methylation and reduction of its chelating potency.
As depicted in Figure 3 (right), in the binding pocket formed by
Figure 2. SAS map of the synthesized structures, the color coding represent activity
alterations for each pair of ligands. ‘A’ denounce for Active, ‘M’ for moderate and ‘I’
for the Inactive compounds. (a) Pair comparison between activity similarity and
structural similarity of the compounds 5 and 2. (b) Pair comparison between
the residues Ile 770, Gln 514, Val 566, Leu 773 and Trp 519, a p–p
compound
synthesized structures.
9 and 12. (c) Pair comparisons between compound 14 and other
interaction is seen between Trp 519 and one of the phenyl rings of
compound 1. The other phenyl ring is oriented towards the hydro-
phobic pocket shaped by Ile 770 and Leu 773. This conformation
stabilizes the molecule so that the more hydrophilic mercapto
imidazole moiety orients towards hydrophilic area around the iron
core.
with SCH3 replaced by OCH3 and F replaced by N(CH3)2 (Fig. 2b.
Tanimoto similarity = 0.52)
As it can be observed in Figure 2, most of the points are colored
green and are located in the upper right area of the SAS plot. This
means that most compounds are structurally relevant and are
more potent than the control drug. On the other hand, the points
in the lower right side of the plot accounts for the activity cliffs,
compounds with high structural similarity but low similarity in
the activity. Most of the data in this area are related to pair com-
parisons between compounds 14 with other compounds (Fig. 2c).
The inactive compound 14 is showing the lowest similarity in
the activity (<0.1) compared to other structurally similar com-
pounds of high potency in this series. Since SH group is present
in all other structures of this series this dramatic decrease in the
activity can be related to methylation of SH in compound 14. Based
3. Conclusions
A series of 4,5-diaryl-1H-imidazole-2(3H)-thione was synthe-
sized and their inhibitory potency against soybean 15-lipoxyge-
nase and free radical scavenging activities were determined. In
general, most of compounds demonstrated significant 15-LOX
inhibitory potential. Structure–activity relationship studies
showed that the nature of substituent of two arly (R1 and R2) on
the imidazole ring had a profound influence on LOX activity. A pos-
sible mechanism for the activity of the SH containing compounds
Figure 3. (Left) Superposition of the best docked poses for the two active structures 1 and 5 in the active site. (Right) Ball and stick representation of the best docked pose for
compound 1 in the active site of 15-LOX. All visualizations were done using VMD.

7164
A. Assadieskandar et al. / Bioorg. Med. Chem. 20 (2012) 7160–7166
might be through radical scavenging properties and chelating the
iron atom in the binding site of the lipooxygenase enzyme.
(br s, 2H, NH); MS, m/z (%) 288 (M++2, 33), 286 (M+, 100), 252
(20), 227 (15), 193 (15), 168 (18), 104 (10). Anal. Calcd. for
C
15H11ClN2S: C, 62.82; H, 3.87; N, 9.77. Found: C, 62.94; H, 3.68;
4. Experimental
4.1. Chemistry
N, 9.58.
4.1.7. 4-(4-(Methylthio)phenyl)-5-phenyl-1H-imidazole-2(3H)-
thione (7)
Yield, 72%; mp 241–244 °C; IR (KBr, cmꢁ1):
v 3416, 1506, 1209,
1H NMR spectra were recorded on a 500 MHz Bruker spectrom-
eter using CDCl3 or DMSO-d6 as solvent. Chemical shifts (d) are re-
ported in ppm relative to tetramethylsilane (TMS) as internal
standard. Infrared spectra were acquired on a Nicolet Magna
550-FT spectrometer. IR spectra of solids were recorded in KBr
1101, 825, 768, 691; 1H NMR (500 MHz, DMSO-d6): d 2.47 (s, 3H,
SCH3), 7.22 (d, J = 8.5, 2H, H2,6-methylthiophenyl), 7.27 (d, J = 8.5,
2H, H3,5-methylthiophenyl), 7.29–7.39 (m, 5H, phenyl), 12.50 (br
s, 2H, NH); MS, m/z (%) 298 (M+, 100), 283 (25), 250 (9), 193 (7),
165 (6), 77 (4). Anal. Calcd. for C16H14N2S2: C, 64.39; H, 4.73; N,
9.39. Found: C, 64.56; H, 4.85; N, 9.53.
and the absorption band was given in wave numbers
m .
in cmꢁ1
Mass spectra were obtained with a Finnigan Mat TSQ-70 spectrom-
eter. Elemental microanalyses were within 0.4 of the theoretical
values for C, H and N. All measurements of lipoxygenase activities
were carried out using an Spekol 1500 spectrophotometer. The
soybean 15-lipoxygenase and other chemicals were purchased
from Sigma, Aldrich and Merck Co. respectively.
4.1.8. 4-(4-Chlorophenyl)-5-(4-(dimethylamino)phenyl)-1H-
imidazole-2(3H)-thione (8)
Yield, 62%; mp 258–261 °C; IR (KBr, cmꢁ1):
v 3441, 1608, 1531,
1367, 1193, 825; 1H NMR (500 MHz, DMSO-d6): d 2.92 (s, 6H,
NCH3), 6.68 (d, J = 8.5, 2H, H3,5-dimethylaminophenyl), 7.15 (d,
J = 8.5, 2H, H2,6-dimethylaminophenyl), 7.35 (d, J = 8.5, 2H, chloro-
phenyl), 7.40 (d, J = 8.5, 2H, chlorophenyl), 12.36 (br s, 1H, NH),
12.42 (br s, 1H, NH); MS, m/z (%) 331 (M++2, 33), 329 (M+, 100),
270 (7), 164 (8). Anal. Calcd. for C17H16ClN3S: C, 61.90; H, 4.89;
N, 12.74. Found: C, 61.78; H, 4.98; N, 12.86.
4.1.1. 4,5-Diphenyl-1H-imidazole-2(3H)-thione (1)
Yield, 78%; mp 226–229 °C; IR (KBr, cmꢁ1):
v 3441, 1598, 1495,
1214, 758, 691, 558; 1H NMR (500 MHz, DMSO-d6): d 7.29–737 (m,
10H, phenyl), 12.50 (br s, 2H, NH); MS, m/z (%) 252 (M+, 100), 218
(14), 193 (20), 165 (21), 104 (10), 77 (8). Anal. Calcd. for C15H12N2S:
C, 71.40; H, 4.79; N, 11.10. Found: C, 71.28; H, 4.61; N, 11.23.
4.1.9. 4-(4-(Dimethylamino)phenyl)-5-(4-methoxyphenyl)-1H-
imidazole-2(3H)-thione (9)
Yield, 51%; mp 231–233 °C; IR (KBr, cmꢁ1):
v 3416, 1618, 1516,
4.1.2. 4,5-bis(4-Methoxyphenyl)-1H-imidazole-2(3H)-thione (2)
Yield, 71%; mp 261–264 °C; IR (KBr, cmꢁ1):
v 3426, 1613, 1506,
1362, 1244, 1173, 819; 1H NMR (500 MHz, DMSO-d6): d 2.89 (s, 6H,
NCH3), 3.75 (s, 3H, OCH3), 6.68 (d, J = 8.5 Hz, 2H, H3,5-dimethylam-
inophenyl), 6.91 (d, J = 8.5 Hz, 2H, H3,5-methoxyphenyl), 7.16 (d,
1250, 1168, 1029, 840; 1H NMR (500 MHz, DMSO-d6): d 3.75 (s, 6H,
OCH3), 6.91 (d, J = 8.5, 4H, H3,5-methoxyphenyl), 7.27 (d, J = 8.5, 4H,
H
2,6-methoxyphenyl), 12.34 (br s, 2H, NH); MS, m/z (%) 312 (M+,
J = 8.5, 2H,
2,6-methoxyphenyl), 12.24 (br s, 2H, NH); MS, m/z (%) 325 (M+,
100), 315 (21), 266 (10), 251 (8), 162 (8). Anal. Calcd. for
18H19N3OS: C, 66.43; H, 5.88; N, 12.91. Found: C, 66.56; H, 5.67;
H2,6-dimethylaminophenyl), 7.28 (d, J = 8.5, 2H,
100), 297(30), 238 (11), 156 (5), 134 (4). Anal. Calcd. forC17H16N2O2S:
C, 65.36; H, 5.16; N, 8.97. Found: C, 65.52; H, 5.27; N, 8.81.
H
C
4.1.3. 4,5-bis(4-Fluorophenyl)-1H-imidazole-2(3H)-thione (3)
N, 12.75.
Yield, 64%; mp 244–247 °C; IR (KBr, cmꢁ1):
v
3436, 1603, 1511,
1229, 1157, 840; 1H NMR (500 MHz, DMSO-d6): d 7.20 (t, J = 8.5, 4H,
4.1.10. 4-(4-Fluorophenyl)-5-(4-methoxyphenyl)-1H-imidazole-
2(3H)-thione (10)
H
3,5-fluorophenyl), 7.37 (dd, J = 8.5, J = 5.5, 4H, H2,6-fluorophenyl),
12.56 (br s, 2H, NH); MS, m/z (%) 288 (M+, 100), 254 (10), 229
(21), 201 (20), 122 (15), 95 (9). Anal. Calcd. for C15H10F2N2S: C,
62.49; H, 3.50; N, 9.72. Found: C, 62.24; H, 3.36; N, 9.58.
Yield, 72%; mp 253–256 °C; IR (KBr, cmꢁ1):
v 3426, 1608, 1511,
1250, 1029, 835; 1H NMR (500 MHz, DMSO-d6): d 3.76 (s, 3H,
OCH3), 6.93 (d, J = 8.5 Hz, 2H, H3,5-methoxyphenyl), 7.20 (t,
J = 8.5, 2H, H3,5-fluorophenyl), 7.26 (d, J = 8.5, 2H, H2,6-methoxy-
phenyl), 7.36 (dd, J = 8.5, J = 5.5, 2H, H2,6-fluorophenyl), 12.44 (br
s, 1H, NH), 12.46 (br s, 1H, NH); MS, m/z (%) 300 (M+, 100), 286
(70), 241 (25), 226 (22), 198 (20), 171 (10), 150 (11), 122(8),
95(6). Anal. Calcd. for C16H13FN2OS: C, 63.98; H, 4.36; N, 9.33.
Found: C, 63.64; H, 4.54; N, 9.56.
4.1.4. 4,5-bis(4-Chlorophenyl)-1H-imidazole-2(3H)-thione (4)
Yield, 68%; mp 287–290 °C; IR (KBr, cmꢁ1):
v 3395, 1501, 1091,
830; 1H NMR (500 MHz, DMSO-d6): d 7.35 (d, J = 8.5, 4H, chloro-
phenyl), 7.44 (d, J = 8.5, 4H, chlorophenyl), 12.64 (br s, 2H, NH);
MS, m/z (%) 322 (M++2, 67), 420 (M+, 100), 300 (15), 227 (14),
138 (16), 111 (15), 69 (25). Anal. Calcd. for C15H10Cl2N2S: C,
56.09; H, 3.14; N, 8.72. Found: C, 56.27; H, 3.32; N, 8.63.
4.1.11. 4-(4-Chlorophenyl)-5-(4-methoxyphenyl)-1H-imidazole-
2(3H)-thione (11)
Yield, 74%; mp 247–250 °C; IR (KBr, cmꢁ1):
v 3426, 1598, 1516,
4.1.5. 4-(4-Methoxyphenyl)-5-phenyl-1H-imidazole-2(3H)-
1250, 758, 830; 1H NMR (500 MHz, DMSO-d6): d 3.77 (s, 3H, OCH3),
6.95 (d, J = 8.5, 2H, H3,5-methoxyphenyl), 7.27 (d, J = 8.5, 2H,
thione (5)
Yield, 74%; mp 255–257 °C; IR (KBr, cmꢁ1):
v 3400, 1608, 1506,
1255, 1024, 830, 763, 691; 1H NMR (500 MHz, DMSO-d6): d 3.76
(s, 3H, OCH3), 6.93 (d, J = 8.5, 2H, H3,5-methoxyphenyl), 7.28 (d,
J = 8.5, 2H, H2,6-methoxyphenyl), 7.25–7.38 (m, 5H, phenyl), 12.43
(br s, 1H, NH), 12.44 (br s, 1H, NH). MS, m/z (%) 282 (M+, 100), 267
(23), 223 (10), 180 (11), 152 (6), 77 (5). Anal. Calcd. for C16H14N2OS:
C, 68.06; H, 5.00; N, 9.92. Found: C, 68.22; H, 5.17; N, 9.84.
H
2,6-methoxyphenyl), 7.35 (d, J = 8.5, 2H, chlorophenyl), 7.41 (d,
J = 8.5, 2H, chlorophenyl), 12.47 (br s, 1H, NH), 12.50 (br s, 1H,
NH); 13C NMR (DMSO-d6):
55.65, 114.74, 121.07, 123.05,
d
123.75, 128.12, 129.19, 129.41, 129.73, 132.75, 159.71, 161.50;
MS, m/z (%) 318 (M++2, 33), 316 (M+, 100), 302 (22), 252 (15),
180 (10), 152 (8), 69 (7). Anal. Calcd. for C16H13ClN2OS: C, 60.66;
H, 4.14; N, 8.84. Found: C, 60.86; H, 4.25; N, 8.67.
4.1.6. 4-(4-Chlorophenyl)-5-phenyl-1H-imidazole-2(3H)-thione
(6)
4.1.12. 4-(4-Fluorophenyl)-5-(4-(methylthio)phenyl)-1H-
imidazole-2(3H)-thione (12)
Yield, 76%; mp 264–267 °C; IR (KBr, cmꢁ1):
v 3452, 1603, 1506,
1219, 1091, 830, 763, 697; 1H NMR (500 MHz, DMSO-d6): d 7.31–
7.39 (m, 7H, aromatic), 7.43 (d, J = 8.5, 2H, chlorophenyl), 12.59
Yield, 74%; mp 242–245 °C; IR (KBr, cmꢁ1):
v
3436, 1629, 1506,
1209, 1157, 845; 1H NMR (500 MHz, DMSO-d6): d 2.48 (s, 3H,

A. Assadieskandar et al. / Bioorg. Med. Chem. 20 (2012) 7160–7166
7165
SCH3), 7.20–7.24 (m, 4H, aromatic), 7.26 (d, J = 8.5, 2H, H3,5-meth-
ylthiophenyl), 7.39 (dd, J = 8.5, J = 5.5, 2H, H2,6-fluorophenyl), 12.52
(br s, 2H, NH); MS, m/z (%) 316 (M+, 100), 301 (23), 268 (6), 242 (5),
211 (5), 183 (4), 158 (4). Anal. Calcd. for C16H13FN2S2: C, 60.73; H,
4.14; F, N, 8.85. Found: C, 60.89; H, 4.26; N, 8.63.
our application implemented in .NET together with their corre-
sponding activity vector. Tanimoto similarity index for each pair
of compounds was calculated based on FP2 fingerprint of the two
compounds using Open Babel.37 In the SAS maps, pair wise
Tanimoto similarity coefficients on horizontal axis represent the
structural similarities between the two ligands, meanwhile exper-
imental activity similarity (vertical axis) for each ligand pair (i,j) is
calculated as follows:
4.1.13. 4-(4-Chlorophenyl)-5-(4-(methylthio)phenyl)-1H-
imidazole-2(3H)-thione (13)
Yield, 71%; mp 259–262 °C; IR (KBr, cmꢁ1):
v 3390, 1634, 1501,
SimActivityði; jÞ ¼ 1 ꢁ ½ðjActivityi ꢁ ActivityjjÞ=ðActivitymax
ꢁ ActivityminÞꢂ
1219, 1086, 819; 1H NMR (500 MHz, DMSO-d6): d 2.48 (s, 3H,
SCH3), 7.24 (d, J = 8.5, 2H, H2,6-methylthiophenyl), 7.28 (d, J = 8.5,
2H, H3,5-methylthiophenyl), 7.35 (d, J = 8.5, 2H, chlorophenyl),
7.43 (d, J = 8.5, 2H, chlorophenyl), 12.56 (br s, 2H, NH); 13C NMR
The two metrics Activitymax and Activitymin denounce the IC50
s
of the most and the least potent compounds, respectively. A new
color coding system was implemented in our application to show
all possible activity alterations in each pair of ligands. In this study
for color coding system, the two cutoff values 34.2 and 64 were
used to show the range of activity for moderate structures. The va-
lue 34.2 was selected based on the activity of the positive control
compound (IC50 = 34.2).
(DMSO-d6):
d 14.56, 122.12, 126.24, 127.55, 128.50, 129.05,
129.27, 129.66, 133.86, 141.02, 141.40, 177.40; MS, m/z (%) 334
(M++2, 33), 332 (M+, 100), 317 (19), 284 (5), 227 (4), 166 (4). Anal.
Calcd. for C16H13ClN2S2: C, 57.73; H, 3.94; N, 8.42. Found: C, 57.92;
H, 3.75; N, 8.53.
4.1.14. 5-(4-Chlorophenyl)-4-(4-methoxyphenyl)-2-
(methylthio)-1H-imidazole (14)
4.4. Molecular modeling studies
Yield, 85%; mp 179–181 °C; IR (KBr, cmꢁ1):
v 3446, 1613, 1506,
1250, 1168, 1091, 830; 1H NMR (500 MHz, CDCl3): d 2.67 (s, 3H,
SCH3), 3.83 (s, 3H, OCH3), 6.89 (d, J = 8.5, 2H, H3,5-methoxyphenyl),
7.26 (d, J = 8.5, 2H, H2,6-methoxyphenyl), 7.37 (d, J = 8.5, 2H, chlo-
rophenyl), 7.47 (d, J = 8.5, 2H, chlorophenyl); MS, m/z (%) 332
(M++2, 33), 330 (M+, 100), 297 (61), 257 (38), 243 (19), 133 (7).
Anal. Calcd. for C17H15ClN2OS: C, 61.72; H, 4.57; N, 8.47. Found:
C, 61.58; H, 4.36; N, 8.71.
Docking simulation studies were performed using Autodock
Vina.38 For this purpose, two active structures (1 and 5) were con-
verted to pdbqt using open babel 2.3.1. The PDB of the soybean
lipoxygenase was also retrieved from protein data bank (1IK3)
and converted to into pdbqt via autodock tools. The exhaustiveness
parameter was set to 100 and the box center was set to the dimen-
sions of iron atom in the protein file (x = 23.944, y = 2.975,
z = 15.444). The box size in all directions was set to 50 Å, num of
modes was set to 9 and energy range was set to 3 in autodock vina
conf.txt.
4.2. Biological assays
4.2.1. 15-Lipoxygenase inhibition assay
For 15-LO Inhibitory assessment, Linoleic acid and two assay
solutions (A and B) were prepared in advance. Solution A was
50 mM DMAB (3-dimethylaminobenzoic acid) in a l00 mM phos-
phate buffer (pH 7.0) while solution B was a mixture of l0 mM
MBTH (3-methyl-2-benzothiazolonhydrazone) (3 mL), hemoglobin
(5 mg/mL, 3 mL) in 50 mM phosphate buffer at pH 5.0 (25 ml). A
linoleic acid solution was prepared by mixing 5 mg of linoleic acid
with 0.5 mL ethanol and then diluting with KOH (100 mM) to a fi-
nal volume of 5 mL. In the standard assay, the sample in ethanol
Acknowledgment
This project was supported by Grants from Research Council of
Tehran University of Medical Sciences.
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