Discovery of indole-based tetraarylimidazoles as potent inhibitors of urease with low antilipoxygenase activity

Article abstract of DOI:10.1016/j.ejmech.2015.08.011

A series of tetraarylimidazoles (5A-5O) were prepared by one pot four component condensation reactions of 2-arylindole-3-carbaldehydes, substituted anilines, benzil and ammonium acetate in acetic acid. The synthesized compounds exhibited potent antiurease

Full text of DOI:10.1016/j.ejmech.2015.08.011

Accepted Manuscript
Discovery of Indole-based Tetraarylimidazoles as Potent inhibitors of Urease with
Low Antilipoxygenase activity
Sadia Naureen, Faryal Chaudhry, Nadia Asif, Munawar Ali Munawar, Muhammad
Ashraf, Faizul Hassan Nasim, Humera Arshad, Misbahul Ain Khan
PII:
S0223-5234(15)30195-1
10.1016/j.ejmech.2015.08.011
EJMECH 8047
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 April 2015
Revised Date: 20 July 2015
Accepted Date: 5 August 2015
Please cite this article as: S. Naureen, F. Chaudhry, N. Asif, M.A. Munawar, M. Ashraf, F.H. Nasim,
H. Arshad, M.A. Khan, Discovery of Indole-based Tetraarylimidazoles as Potent inhibitors of Urease
with Low Antilipoxygenase activity, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.08.011.
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1
Discovery of Indole-based Tetraarylimidazoles as Potent inhibitors of Urease with Low
Antilipoxygenase activity
Sadia Naureen1*, Faryal Chaudhry1, Nadia Asif1, Munawar Ali Munawar1, Muhammad Ashraf 2,
Faizul Hassan Nasim2, Humera Arshad2 and Misbahul Ain Khan1,2
,
1Institute of Chemistry, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan.
2Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur – 63100, Pakistan.
Abstract: A series of tetraarylimidazoles (5A-5O) were prepared by one pot four component
condensation reactions of 2-arylindole-3-carbaldehydes, substituted anilines, benzil and ammonium
acetate in acetic acid. The synthesized compounds exhibited potent antiurease activity with IC50 values
ranging from 0.12±0.06 µM to 29.12±0.18 µM as compared with thiourea. However, low inhibition
profiles were observed for lipoxygenase. The data show that tetraarylimidazoles containing a substituted
2-penylindole have emerged as a new class of potent inhibitors of urease enzyme.
Graphical Abstract:
1R= H, Br
2 R= H, Cl, CF3, Br 3 R = H, Br, F, Cl, CH3, OCH3
One pot synthesis of tetrasubstituted imidazoles derivatives by four component condensation reaction
Keywords: multicomponent reaction, thiourea, ammonium acetate, urease inhibition, lipoxygenase
inhibition.

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1
Introduction
Indole–imidazole compounds provide an interesting template for synthetic and medicinal chemists. These
compounds have a broad scope of pharmacological activities such as antibacterial, anticancer, anti-
inflammatory, antifungal, antidiabetic, etc. [1, 2]. A series of indole–imidazole compounds [3, 4] has been
reported and is established as modulators of multidrug resistance against tumor cell lines of different
origin e.g. breast, uterine leukemia and colon. N-1-Phenyl-3-(substituted phenyl)-indolo-2, 3-imidazole
derivatives have been reported in the literature. These were prepared by condensation of diverse aromatic
aldehydes with N-1-phenyl isatin in acetic acid in the presence of ammonium acetate. These compounds
displayed anticonvulsant activity [5]. Another series of 1-benzyl-3-(imidazol-1-ylmethyl) indole
derivatives were reported to have antifungal activities [6].
One pot synthesis of 2-[2’,5’-disubstituted-1, H-indole-3’-yl]-benzimidazole by condensation of 2,5-
disubstituted-3-cynoacetylindoles with substituted o-phenylenediamine under microwave and
conventional condition has also been reported using MK-10 Clay as catalyst [7]. Another series of
1,2-disubstituted
4-[(5-substituted2-phenylindol-3-yl)methylene]imidazolin-5(4H)-ones
possessing
antibacterial activity were prepared by the reaction of 5-substituted 2-phenyl indole-3-carbaldehydes and
N-acetylglycine in the presence of acetic anhydride and sodium acetate [8].
1,2,4,5-Tetrasubstituted2-(4’,5’-diphenyl-1’(p-tolyl)-1’H-Imidazole-2’-yl)-1H-indoles
have
been
synthesized in the presence of N-methyl-2-pyrrolidonium hydrogen sulfate as Bronsted acidic ionic liquid
or (P2O5/SiO2) in one pot reaction of indole aldehyde, toluidine, benzil and ammonium acetate [9, 10].
Ureolytic bacteria like a gram negative bacterium Helicobacter pylori produce Ni (II) ions containing
enzyme urease (EC 3.5.1.5) in stomach, the later catalyzes hydrolysis of urea into ammonia and carbonic
acid [11]. This bacterium colonizes gastrointestinal tract because of increased pH around itself which
helps it to survive and grow under hostile conditions in stomach and intestinal tract. It thus causes
development of gastritis and gastroduodenal ulcers and may lead to hepatic coma [12]. Therefore, there is
growing interest in search for inhibitors of urease for the potential treatment of gastric and duodenal
ulcers [13]. Certain rabeprazole [14] (imidazoles) (Fig.1) and indole derivatives [15] (Fig.2) have been
reported as urease inhibitors.
Fig.1 Rabeprazole
Fig.2 5-(1H-Indol-2-yl)-1,3,4-oxadiazole-2(3H)-thione

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Lipoxygenases (LOXs) oxidize polyunsaturated fatty acids into biologically active eicosanoids which are
involved in maintenance of homeostasis, inflammatory response, asthma, cardiovascular disorders, certain
types of neurodegenerative disorders, tumorigenesis and some types of leukaemia [16]. Recent work
shows that LOX catalysed reactions are also involved in the regulation of expression of redox-sensitive
genes [17]. The catalytic domain contains a non-haem iron in the active site which is oxidized from
ferrous to ferric form during catalysis. The literature also shows that LOX inhibitors are either iron-ligand
inhibitors, or redox active or non-redox type inhibitors. Most of its inhibitors bind to active site and
exhibit competitive type of inhibition. Zileuton is the only approved drug in the market based on this
chemistry but with several side effects [18]. Therefore, there is growing interest in the discovery of
inhibitors for LOX enzyme. Certain derivatives of indoles [19, 20] (Fig.3) and imidazoles [21] (Fig.4)
have also shown high levels of anti-LOX activity.
Fig.3 1-[(4-Chlorophenyl)methyl]-3-[(1,1-dimethylethyl)thio]-
-dimethyl-5-(1-methylethyl)-1H-Indole-2-propanoic acid
Fig.4 Imidazole based sulfonamide
α,α
We have recently reported the synthesis of a number of indole based triarylimidazoles as efficient α-
glucosidase inhibitors [2]. Here, in view of the importance of imidazole and indole ring compounds as
antiurease and anti-LOX agents, we would like to communicate our efforts to prepare some
tetraarylimidazoles containing a 2-indol-3-yl substituents and their screening as urease and LOXs
inhibitors. A multicomponent condensation reaction of substituted 2-phenyl indole-3-carbaldeydes,
benzil, substituted anilines and ammonium acetate was employed to synthesize these tetrasubstituted
imidazoles.

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2.
Results and Discussion
2.1
Chemistry.
Substituted 2-phenylindoles were prepared by the known method of Fischer indole synthesis [22] and
their Vilsmeier-Haack [23] formylation produced the desired 2-arylindole-3-carbaldehydes. A new series
of tetraaryl imidazoles containing substituted 2-phenylindoles substituents (2A-2O) (Table 1) was
prepared by a MCR (multicomponent reaction) route by cyclization reaction of 2-arylindole-3-
carbaldehydes, aromatic amines, 1,2-diketones and ammonium acetate by heating under reflux in acetic
acid (Scheme 1).
The structures of these compounds were established on the basis of spectral data. In their HRMS, the
1
molecular ion peaks were found to correspond to their expected values. The HNMR spectra showed
singlet for –NH indole in the range of
δ
11-12. The compounds substituted with –CH3 and –OCH3 (5C,
2- 4. The splitting patterns of remaining protons of
5D, 5K, 5L) showed upfield singlet in the range of
δ
spectra were as expected, according to the substituent. The 13CNMR spectra and FTIR were also as
expected.
Scheme -1 Synthetic route to compounds (5A-5O).
Table 1 Series of synthesized tetraaryl imidazoles

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2.2
Antiurease Activity
These synthesized compounds were evaluated for their antiurease activity. Thiourea was used as a
standard reference inhibitor having IC50 values of 21.25±0.15µM. Among the twelve synthesized
compounds tested (Table 2), all exhibited potent inhibitory activity as compared to standard. The most
active among these were 5L disubstituted with –CF3 and CH3 (IC50 0.12±0.06 µM) and 5A disubstituted
with Br (IC50 0.15±0.04 µM), 5N disubstituted with –CF3 and F (IC50 0.16±0.02 µM), 5G substituted with
Cl (IC50 0.37±0.01 µM) and 5F disubstituted with Cl and F (IC50 0.44±0.02 µM). Even the least active
compound was comparable with the standard thiourea in its inhibitory potential. The order of activity in
decreasing order is as: 5L > 5A > 5N > 5G > 5F > 5C > 5B > 5O > 5H > 5I > 5J > 5K. These studies
3
show that the placement of –Br and –F groups as R- in 5I and 5J increases IC50 values (11.35±0.08 and
12.13±0.02 µM, respectively) and hence decreases inhibitory activities compared with the most active
compounds. This is also shown by 5K wherein –OCH3 group at this position further increases IC50 value
29.12±0.18 µM. Similar profiles are shown when 3R- is –OCH3, -Cl, -H as shown in compounds 5C, 5B,
5O and 5H, still the IC50 values are < 5.00 µM.
Table 2 Antiurease and anti-LOX activity of indole-imidazole derivatives. Percent inhibition at 0.5mM
is shown in parenthesis. Antiurease activity of 5D, 5E, 5M was not done. Data is mean± sem, n=3.

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The representative plots for the determination of IC50 values are given only for 4 compounds (5L, 5B, 5I,
5K) for urease inhibition.
Fig.5 Representative plot of antiurease activity of compound 5L percent inhibition vs concentration.
Serial dilution from 0.5mM to 0.00125 µM were made and this graph was plotted to calculate IC50 value.
Many dilution points are excluded in this graph for simplicity.
Fig.6 Representative plot of antiurease activity of compound 5B percent inhibition vs concentration.
Serial dilution from 0.5mM to 0.005 µM were made and this graph was plotted to calculate IC50 value.
Many dilution points are excluded in this graph for simplicity.
Fig.7 Representative plot of antiurease activity of compound 5I percent inhibition vs concentration.
Serial dilution from 0.5mM to 0.01 µM were made and this graph was plotted to calculate IC50 value.
Many dilution points are excluded in this graph for simplicity.
Fig.8 Representative plot of antiurease activity of compound 5K percent inhibition vs concentration.
Serial dilution from 0.5mM to 0.03
µM were made and this graph was plotted to calculate IC50 value.
Many dilution points are excluded in this graph for simplicity.

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2.3
Antilipoxygenase activity
This novel series of compounds was also tested for their LOX activities as compared to baicalein as a
standard inhibitor. These compounds exhibited moderate LOX activities as listed (Table 2). Among tested
compounds, compound 5L substituted with CF3 and CH3 showed the highest LOX inhibition of
90.3±1.3% at 0.5 mM and IC50 83±1 µM and compound 5F substituted with -Cl and -F with inhibition of
84.5±1.3% and IC50 89±1 µM. The poorest percent inhibition was exhibited by compound 5C substituted
1
3
with -Br at R and –OCH3 at R as 57.51±1.22 µM with IC50 value of 265±1 µM. This data predict that
such compounds are medium inhibitors of LOX enzyme.

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3.
Conclusions
A new series of novel tetraarylimidazoles containing 2-phenylindoles as substituents (5A-5O) were
synthesized using a multicomponent reaction. The synthesized compounds were evaluated as inhibitors of
urease and LOX and showed potent antiurease activity and lower levels of anti-LOX activity.
4.
Materials and Methods
The chemicals used in this experimental work were commercially available from Merck, Fluka and
Aldrich was used as such. Melting points were determined in open capillary tubes and are uncorrected.
Analytical TLC was performed on DC-Alufolien Silica Gel 60 F254 Merck. UV lamp of short and long
wavelength (model UVGL-25 minor light multiband UV-254/366) was used to visualize TLC plates. IR
1
spectra were recorded on Perkin Elmer Spectrum BX FT-IR. H spectra were recorded at 500 MHZ and
13C NMR spectra were recorded at 126 MHZ on a Brucker Avance AV11500B spectrometer.
4.1 General Experimental Procedure for the Preparation of Tetraubstituted imidazoles containing
a substituted indole (5A-5O)
An equimolar mixture of benzil (1.0 equiv), a substituted 2-phenylindole 3-carbaldehyde (1.0 equiv),),
aromatic amine (1.0 equiv) and ammonium acetate (4.0 equiv) in acetic acid was refluxed for
5-6 hours[2]. After the completion of the reaction (monitored by TLC) and cooling to room temperature,
the reaction mixture was poured into cold water (100 mL). The precipitated product was filtered, washed
with excess of water and recrystallized with EtOH to obtain pure (5A-5O).
4.1.1 5-Bromo-3-(1’-(p-bromophenyl)-4’,5’-diphenyl-1’H-imidazol-2’-yl)-2-phenyl-1H-indole
(5A)
0
Yield: 45% as a white solid. mp: > 250 C; IR (neat): 3077, 1602, 1491, 1443, 1385, 1241, 1071, 1014
1
cm-1; HRMS (ES+) calcd. for C35H24N3Br2 [M+H]+ 644.0377 Found: 644.0353; H NMR (500
MHz, DMSO-d6) δ: 11.85 (s, 1H), 7.79 (s, 1H), 7.55 (d, J = 8.1 Hz, 2H), 7.40 – 7.30 (m, 9H),
13
7.24 – 7.16 (m, 6H), 7.04 (d, J = 8.2 Hz, 2H), 6.37 (d, J = 1.6 Hz, 2H). C NMR (126 MHz,
DMSO-d6) δ: 142.14, 139.47, 137.88, 135.55, 135.19, 135.11, 131.87, 131.65, 131.43, 131.22,

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131.08, 130.17, 129.72, 129.24, 128.83, 127.87, 127.07, 126.96, 125.41, 122.15, 121.02, 114.19,
113.25, 102.54.
4.1.2 5-Bromo-3-(1-(4-chlorophenyl)-4,5-diphenyl-1H-imidazole-2-yl)-2-phenyl-1H-indole (5B)
0
Yield: 60% as a yellowish solid. mp: > 250 C; IR(neat):3030 , 1558, 1494, 1444, 1388, 1239, 1086,
1016 cm-1; HRMS (ES+) calcd. for C35H24N3ClBr [M+H]+ 600.0842 found 600.0833. 1H NMR (500 MHz,
DMSO-d6)
δ: 11.84 (s, 1H), 7.79 (bs, 1H), 7.55 (d, J = 7.6 Hz, 2H), 7.35 – 7.27 (m, 9H), 7.24-7.15 (m,
6H), 6.91 (d, J = 7.8 Hz, 2H), 6.43 (d, J = 7.6 Hz, 2H). 13C NMR (126 MHz, DMSO-d6)
δ: 142.18,
139.46, 137.84, 135.19, 135.12, 132.44, 131.86, 131.65, 131.21, 131.10, 130.71, 130.24, 129.43, 129.23,
129.05, 128.95, 128.83, 128.49, 127.86,127.06, 126.94, 125.40, 124.25, 122.14, 114.85, 114.17, 113.23,
102.55.
4.1.3 5-Bromo-3-(1-(4-methoxyphenyl)-4,5-diphenyl-1H-imidazole-2-yl)-2-phenyl-1H-indole (5C)
0
Yield: 51% as a white solid. mp: > 250 C; IR (neat): 3050 ,1510, 1442, 1510, 1243, 1167, 1023 cm-1;
HRMS (ES+) calcd. for C36H27N3OBr [M+H]+ 596.1337 found 596.1335. 1H NMR (500 MHz, DMSO-d6)
δ: 11.81 (s, 1H), 7.73 (bs, 1H), 7.53 (d, J = 7.2 Hz, 2H), 7.40 – 7.34 (m, 3H), 7.31 – 7.14 (m, 12H), 6.44-
6.36 (m, 4H), 3.53 (s, 3H). 13C NMR (126 MHz, DMSO-d6)
δ: 158.50, 142.43, 139.39, 137.51, 135.39,
135.08, 132.01, 131.62, 131.50, 131.46, 130.79, 129.22, 129.09, 128.90, 128.80, 128.76, 127.79, 127.65,
126.90, 125.25, 122.04, 114.08, 113.69, 113.13, 103.08, 56.66.
4.1.4 5-Bromo-3-(4,5-diphenyl-1-(p-tolyl)-1H-imidazole-2-yl)-2-phenyl-1H-indole (5D)
0
Yield: 47% as a white solid. mp: > 250 C; IR (neat): 3120, 2356, 1514, 1443, 1389, 1058 cm-1; HRMS
1
(ES+) calcd. for C36H27N3Br [M+H]+ 580.1388 Found: 580.1381. H NMR (500 MHz, DMSO-d6)
δ:
11.81 (s, 1H), 7.72 (bs, 1H), 7.54 (d, J = 7.9 Hz, 2H), 7.39 – 7.20 (m, 15H), 6.65 (d, J = 7.9 Hz, 2H), 6.39
(d, J = 6.4 Hz, 2H), 2.04 (s, 3H). 13C NMR (126 MHz, DMSO-d6)
: 142.31, 141.83, 139.45, 138.42,
δ
137.66, 137.24, 136.18, 135.38, 135.08, 133.83, 132.22, 131.99, 131.63, 131.48, 130.63, 129.17, 129.09,
129.04, 128.83, 128.76, 127.79, 127.68, 127.54, 126.94, 125.27, 122.71, 122.04, 114.09, 113.17, 104.22,
103.09, 21.00.

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4.1.5 5-Bromo-2-phenyl-3-(1,4,5-triphenyl-1H-imidazole-2-yl)-1H-indole (5E)
Yield: 75% as a yellow solid. mp: > 250 0C; IR (neat): 3062, 2357, 1598, 1386, 1260, 1026 cm-1; HRMS
1
(ES+) calcd. for C35H25N3Br [M+H]+ 566.1232 Found: 566.1211; H NMR (500 MHz, DMSO-d6)
δ:
11.79 (s, 1H), 7.70 (bs, 1H), 7.52 (d, J = 8.3 Hz, 2H), 7.37 – 7.17 (m, 15H), 6.98 – 6.79 (m, 3H), 6.47 (bs,
2H). 13C NMR (126 MHz, DMSO-d6)
: 142.19, 139.48, 137.65, 136.31, 135.26, 135.06, 131.89, 131.63,
δ
131.35, 131.33, 130.52, 129.25, 129.10, 128.79, 128.50, 127.89, 127.78, 126.91, 125.27, 121.99, 114.10,
113.12, 102.92.
4.1.6 2-(4-Chlorophenyl)-3-(1-(4-fluorophenyl)-4,5-diphenyl-1H-imidazole -2-yl)-1H-indole (5F)
Yield: 88% as a white solid. mp: > 250 0C;
IR (neat): 3083, 1509, 1388, 1223, 1094, 1017 cm-1 HRMS (ES+) calcd. for C35H24N3FCl [M+H]+
540.1643 found 540.1656. 1H NMR (500 MHz, DMSO-d6)
(d, J = 8.0 Hz, 2H), 7.46-7.38 (m, 3H), 7.36 – 7.04 (m, 12H), 6.78-6.72 (m, 2H), 6.57 (bs, 2H). 13C NMR
(126 MHz, DMSO-d6) : 160.37, 142.70, 137.71, 136.61, 136.36, 128.78, 127.71, 126.83, 123.10, 120.80,
δ: 11.69 (s, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.54
δ
120.71, 120.22, 115.53, 115.30, 112.12, 103.57.
4.1.7 2-(4-Chlorophenyl)-3-(1,4,5-triphenyl-1H-imidazole-2-yl)-1H-indole (5G)
Yield: 75% as a white solid. mp: > 250 0C; IR (neat): 3065 , 1597, 1496, 1329, 1095, 1025 cm-1; HRMS
1
(ES+) calcd. for C35H25N3Cl [M+H]+ 522.1737 found 522.1730. H NMR (500 MHz, DMSO-d6)
δ: 11.65
(s, 1H), 7.61 – 7.51 (m, 3H), 7.45 – 7.37 (m, 3H), 7.32 – 6.85 (m, 15H), 6.55 (d, J = 6.6 Hz, 2H). 13C
NMR (126 MHz, DMSO-d6) : 142.60, 139.69, 137.70, 136.63, 136.40, 135.30, 133.57, 133.11, 131.68,
δ
131.39, 131.25, 130.42, 130.32, 129.56, 129.23, 129.18, 129.11, 129.08, 128.88, 128.76, 128.73, 128.55,
127.79, 127.66, 126.97, 123.03, 120.67, 120.11, 112.08, 111.31, 103.82.
4.1.8 2-(4-Bromophenyl)-3-(1,4,5-triphenyl-1H-imidazole-2-yl)-1H-indole (5H)
Yield: 52% as a white solid. mp: > 250 0C; IR (neat): 3079 , 1597, 1496, 1421, 1389, 1245, 1072, 1014
1
cm-1 ; HRMS (ES+) calcd. for C35H25N3Br [M+H]+ 566.1232 found 566.1227. H NMR (500 MHz,
DMSO-d6)
δ: 11.66 (s, 1H), 7.61 – 7.51 (m, 5H), 7.39 (d, J = 8.1 Hz, 1H), 7.33 – 6.85 (m, 15H), 6.56 (d,
J = 6.5 Hz, 2H). 13C NMR (126 MHz, DMSO-d6)
δ: 142.59, 137.71, 136.66, 136.42, 135.30, 132.18,
131.68, 131.60, 131.39, 130.43, 129.58, 129.50, 129.11, 128.88, 128.76, 128.55, 127.96, 127.81, 126.94,
123.05, 121.74, 120.67, 120.12, 112.09, 103.79.

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4.1.9 2-(4-Bromophenyl)-3-(1-(4-bromophenyl)-4,5-diphenyl-1H-imidazole-2-yl)-1H-indole (5I)
Yield : 56% as a white solid. mp: > 250 0C; IR (neat): 3079 , 1549, 1492, 1387, 1329, 1222, 1069, 1016
1
cm-1 ; HRMS (ES+) calcd. for C35H24N3Br2 [M+H]+ 644.0337 found 644.0322. H NMR (500 MHz,
DMSO-d6)
δ: 11.71 (s, 1H), 7.64 – 7.51 (m, 5H), 7.41 (d, J = 8.1 Hz, 1H), 7.36 – 7.05 (m, 14H), 6.49 (d,
J = 6.5 Hz, 2H). 13C NMR (126 MHz, DMSO-d6)
δ: 142.51, 139.47, 137.84, 136.65, 136.42, 135.75,
135.17, 132.21, 131.72, 131.59, 130.12, 129.77, 129.53, 129.30, 128.71, 127.25, 127.07, 126.94, 126.55,
123.19, 122.74, 121.87, 121.61, 120.94, 120.15, 119.81, 118.90, 118.82, 118.05, 115.63, 114.41, 112.57,
112.24, 103.02.
4.1.10 2-(4-Bromophenyl)-3-(1-(4-fluorophenyl)-4,5-diphenyl-1H-imidazole-2-yl)-1H-indole (5J)
0
Yield: 62% as a white solid. mp: >250 C; IR(neat):3070, 1508, 1422 (C=C), 1219, 1152, 1073(C-N)
1
cm-1; HRMS (ES+) calcd. for C35H24N3BrF [M+H]+ 584.1138 found 584.1128. H NMR (500 MHz,
DMSO-d6)
δ: 11.69 (s, 1H), 7.65 – 7.51 (m, 5H), 7.40 (d, J = 8.1 Hz, 1H), 7.35 – 7.04 (m, 12H), 6.78 –
13
6.71 (m, 2H), 6.58 (bs, 2H). C NMR (126 MHz, DMSO-d6)
δ: 159.91, 142.68, 137.70, 136.63, 136.48,
135.63, 135.22, 132.74, 132.39, 132.21, 131.71, 131.58, 131.23,130.41, 129.89, 129.82, 129.56, 129.47,
129.19, 128.99, 128.78, 127.93, 126.92, 123.55, 123.13, 122.63, 121.85, 121.06, 120.72, 120.58, 120.20,
117.77, 115.53, 115.35, 114.60, 112.13, 103.50.
4.1.11 2-(4-Bromophenyl)-3-(1-(4-methoxyphenyl)-4,5-diphenyl-1H-imidazole-2-yl)-1H-indole
(5K)
0
Yield: 60% as a white solid. mp: >250 C; IR (neat): 3050, 1513, 1301, 1250, 1035 cm-1; HRMS (ES+)
1
calcd. for C36H27N3OBr [M+H]+ 596.1337 found 596.1330. H NMR (500 MHz, DMSO-d6)
δ: 11.66 (s,
1H), 7.61 – 7.50 (m, 5H), 7.39 (d, J = 7.9 Hz, 1H), 7.34 – 7.15 (m, 11H), 7.08 – 7.03 (m, 1H), 6.56 – 6.39
(m,4H), 3.55 (s, 3H). 13C NMR (126 MHz, Acetone-d6)
: 158.79, 149.89, 147.70, 142.61, 136.60,
δ
135.56, 131.71, 131.42,129.33, 129.24, 128.71, 128.50, 128.27, 128.11, 128.07, 128.01, 126.87, 126.54,
126.19, 122.76, 122.68, 120.33, 120.14, 113.13, 54.77.
4.1.12 3-(4,5-Diphenyl-1-(p-tolyl)-1H-imidazole-2-yl)-2-(4-(trifluoromethyl)phenyl)-1H-indole (5L)
0
Yield: 68% as a yellow solid. mp: >250 C; IR (neat): 3078, 1618, 1601, 1516, 1390, 1160, 1114, 1065,
1
1019 cm-1 ; HRMS (ES+) calcd. for C37H27N3F3 [M+H]+ 570.2157 found 570.2134. H NMR (500 MHz,
DMSO-d6)
(d, J = 8.3 Hz, 1H), 7.33 – 7.17 (m, 9H), 7.11 – 7.06 (m, 1H), 6.65 (d, J = 7.9 Hz, 2H), 6.45 (d, J = 6.9
Hz, 2H), 2.04 (s, 3H). 13C NMR (126 MHz, DMSO-d6)
: 142.41, 137.70, 137.34, 136.64, 136.31,
δ: 11.78 (s, 1H), 7.73 (d, J = 8.2 Hz, 2H), 7.63 (d, J = 7.9 Hz, 1H), 7.55 – 7.45 (m, 4H), 7.42
δ

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12
135.96, 135.32, 133.79, 131.69, 131.42, 130.57, 129.56, 129.11, 129.04, 128.89, 128.74, 128.12, 127.58,
126.92, 126.10, 126.07, 123.44, 120.82, 120.42, 112.23, 104.87, 21.14.
4.1.13 2-(4-(Trifluoromethyl)phenyl)-3-(1,4,5-triphenyl-1H-imidazol-2-yl)-1H-indole (5M)
0
Yield: 52% as a yellow solid. mp: >250 C; IR (neat): 1324, 1113, 778 cm-1 ; HRMS (ES+) calcd. for
C36H25N3F3 [M+H]+ 556.2001, found 556.2002;1H NMR (500 MHz,DMSO-d6) δ: 11.79(s,1H), 7.72(d,
J=8.0,2H), 7.64(d, J=9.0, 1H), 7.55-7.40(m, 5H), 7.35-7.0(m,10H), 7.0-6.8(m, 3H), 6.54(d, J=7.0, 2H).
13C NMR (126 MHz, DMSO-d6)
δ:142.35, 137.82, 136.66, 136.31, 136.05, 135.27, 131.67, 131.33,
130.47, 129.48, 129.01, 128.88, 128.75, 128.50, 128.29, 128.14, 127.92, 127.78, 126.97, 126.12, 125.91,
123.75, 123.46, 120.83, 120.43, 112.24, 104.74.
4.1.14 3-(1-(4-Fluorophenyl)-4,5-diphenyl-1H-imidazole-2-yl)-2-(4-(trifluoromethyl)phenyl)-1H-
indole (5N)
0
Yield: 50% as a yellow solid. mp: 220 C; IR (neat): 3070, 1617, 1510, 1324, 1234, 1066 cm-1 ; HRMS
1
(ES+) calcd. for C36H24N3F4 [M+H]+ 574.1906 found 574.1912. H NMR (500 MHz, DMSO-d6)
δ: 12.38
(s, 1H), 7.81 – 7.70 (m, 3H), 7.60 – 7.45 (m, 6H), 7.40 – 7.13 (m, 10H), 6.76 (bs, 3H). 13C NMR (126
MHz, DMSO-d6) : 142.20, 136.83, 136.42, 131.79, 130.85, 129.97, 129.61, 129.24, 128.89, 128.44,
δ
127.26, 126.26, 125.85, 125.68, 123.74, 122.32, 121.09, 120.52, 115.64, 115.53, 112.51.
4.1.15 3-(1-(4-Chlorophenyl)-4,5-diphenyl-1H-imidazole-2-yl)-2-(4-(trifluoromethyl)phenyl)-1H-
indole (5O)
0
Yield: 45% as a yellow solid. mp: > 250 C; IR (neat): 2340, 1618, 1494, 1319, 1112, 1065, 1017 cm-1;
1
HRMS (ES+) calcd. for C36H24N3FCl [M+H]+ 590.1611 Found: 590.1605; H NMR (500 MHz, DMSO-
d6)
δ: 11.85 (s, 1H), 7.76 – 7.64 (m, 3H), 7.57 – 7.42 (m, 5H), 7.36 – 7.06 (m, 10H), 6.91 (d, J = 8.1 Hz,
2H), 6.56 (d, J = 6.7 Hz, 2H). 13C NMR (126 MHz, DMSO-d6)
δ: 142.41, 137.96, 136.80, 136.30, 136.01,
132.51, 131.77, 131.05, 130.27, 129.55, 129.34, 129.18, 128.79, 128.55, 128.26, 127.08, 126.92, 126.13,
125.85, 123.69, 123.52, 120.82, 120.51, 112.35, 104.32.
5
Enzyme assays
Urease assay
5.1
This assay was modified from Berthelot assay and was employed for the determination of urease activity
[24]. The assay is based on the hydrolysis of urea into ammonia which reacts with phenol-hypochlorite to

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13
form light blue colored complex measured at 625 nm. A total volume of 85 µl assay mixture contained 10
µl of 50 mM phosphate buffer, pH 7.0, 10 µl of sample solution and 25 µl of jack beans urease (Sigma)
solution (0.015 units). The contents were pre-incubated at 37ºC for 10 min. Then, 40 µl of urea stock
solution (20 mM) was added to each well and incubation continued at 37ºC for further 10 min and pre-
read at 625nm using the 96-well plate reader Synergy HT (Biotek Inc.). Phenol hypochlorite (115 µl)
reagent was added in each well (freshly prepared by mixing 45 µl phenol reagent with 70 µl of alkali
reagent). For color development, incubation was done at 37oC for another 10 min. Absorbance was again
measured at 625 nm. The percentage enzyme inhibition was calculated by the following formula:
Inhibition (%) = 100 – [(Abs. of test sample / Abs. of control) × 100]
IC50 values (concentration at which enzyme inhibition is 50%) of active compounds were determined by
measuring activities at further dilutions and the data was computed by using EZ-Fit Enzyme software
(Perrella Inc, USA). The absorbance units of uninhibited enzyme were between 1.0-1.2.
5.2
LOX assay
LOX activity was assayed according to the reported method, but with slight modifications [25]. A total
volume of 200 µl assay mixture contained 140 µl sodium phosphate buffer (100 mM, pH 8.0), 20 µl test
compound and 15µl (600 unit) purified soybean lipoxygenase enzyme (Sigma, USA). The contents were
mixed, preincubated for 10 minutes at 25°C and preread at 234 nm using Synergy HT (BioTek, USA) 96-
well plate reader. The reaction was initiated by the addition of 25 µl substrate solution (linoleic acid). The
change in absorbance was observed after 6 min at 234 nm. All reactions were performed in triplicates.
The positive and negative controls were included in the assay. Baicalein was used as a positive control.
The percentage inhibition was calculated by the formula given above for urease assay. The absorbance
units of uninhibited enzyme were between 1.0-1.2.

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14
6.
Acknowledgements
The authors are thankful to Higher Education Commission of Pakistan (HEC) for the award of IRSIP
fellowship. M. A. Khan puts on record his thanks for the generous support of HEC through their
Analytical support provisions. Prof. Gary S. Molander of Department of Chemistry, University of
Pennsylvania, USA, was gracious enough to provide necessary laboratory and other analytical facilities to
S. N. and deserves our thanks. Thanks are also due to anonymous referee for useful comments in the
improvement of this manuscript.

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15
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Tables
Table 1 Series of synthesized tetraarylimidazoles.
Sr. No.
Sample code
1R
Br
Br
Br
Br
Br
H
2R
H
3R
Br
1
2
5A
5B
5C
5D
5E
5F
H
Cl
3
H
OCH3
CH3
H
4
H
5
H
6
Cl
F
7
5G
5H
5I
H
Cl
H
8
H
Br
Br
Br
Br
CF3
CF3
CF3
CF3
H
9
H
Br
10
11
12
13
14
15
5J
H
F
5K
5L
H
OCH3
CH3
H
H
5M
5N
5O
H
H
F
H
Cl

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Table 2 Antiurease and anti-LOX activity of indole-imidazole derivatives.Percent inhibition at 0.5mM is
shown in parenthesis. Antiurease activity of 5D, 5E, 5M was not done. Data is mean± sem, n=3.
Sr.No.
1
Sample code
Antiurease activity
IC50 (µM)
Anti-LOX activity
IC50 (µM)
178±1
5A
5B
0.15±0.04
(93.2±0.4)
4.66±0.04
(87.8±0.3)
4.62±0.06
(88.7±0.8)
-
(78.5±1.3)
101±1
(83.2±1.2)
265±1
(57.5±1.2)
143±1
2
3
5C
4
5D
(81.4±1.2)
134±1
5
5E
-
(97.2±1.3)
89±1
(84.5±1.3)
276±1
(58.3±1.3)
197±1
(59.3±1.2)
173±1
(61.1±1.2)
162±1
(87.8±1.3)
206±1
(61.7±1.3)
83±1
(90.3±1.3)
117±1
6
5F
0.44±0.02
(85.6±0.2)
0.37±0.01
(80.3±0.7)
4.81±0.01
(89.4±0.4)
11.35±0.08
(87.8±0.7)
12.13±0.02
(86.7±0.3)
29.12±0.18
(85.2±0.5)
0.12±0.06
(79.7±0.8)
-
7
5G
8
5H
9
5I
10
11
12
13
14
15
16
17
5J
5K
5L
5M
5N
(86.2±1.2)
106±1
(80.2±1.3)
215±1
(62.15±1.3)
-
0.16±0.02
(85.2±0.5)
4.75±0.05
(70.3±0.7)
21.25±0.15
(88.7±0.8)
-
5O
Thiourea
Baicalein
22.4±1.3
(93.7±1.3)

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FIGURES
tBu
S
iPr
CO2H
N
Cl
Fig.1 1-[(4-Chlorophenyl)methyl]-3-[(1,1-dimethylethyl)thio]- α,α-dimethyl-5-(1-methylethyl)-1H-Indole-
2-propanoic acid
O
HN S
2R
O
n
N
OCH3
N
H
1R
Fig.2 Imidazole based sulfonamide
Fig.3 Rabeprazole
H
S
NH
N
O
N
Fig.4 5-(1H-Indol-2-yl)-1,3,4-oxadiazole-2(3H)-thion

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Fig.5 Representative plot of antiurease activity of compound 5Lpercent inhibition vs concentration.
Serial dilutions from 0.5mM to 0.00125µM were made and this graph was plotted to calculate IC50 value.
Many dilution points are excluded in this graph for simplicity.
Fig.6 Representative plot of antiurease activity of compound 5B percent inhibition vs concentration.
Serial dilutions from 0.5mM to 0.005 µM were made and this graph was plotted to calculate IC50 value.
Many dilution points are excluded in this graph for simplicity.

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Fig.7Representative plot of antiurease activity of compound 5I percent inhibition vs concentration. Serial
dilutions from 0.5mM to 0.01 µM were made and this graph was plotted to calculate IC50 value. Many
dilution points are excluded in this graph for simplicity.
Fig.8Representative plot of antiurease activity of compound 5K percent inhibition vs concentration.
Serial dilutions from 0.5mM to 0.03 µM were made and this graph was plotted to calculate IC50 value.
Many dilution points are excluded in this graph for simplicity.

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Scheme
(1)
(2)
(3)
(4)
1R= H, Br
2 R= H, Cl, CF3 ,Br 3 R= H, Br, F, Cl, CH3, OCH3
(5A-5O)
Scheme -1
Synthetic route to compounds (5A-5O).

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Highlights
•
•
•
Synthesis of a series of novel indole based tetraarylimidazoles.
A new class of potent inhibitors of urease enzyme.
Lower inhibition profiles for lipooxygenase.