Design, synthesis and identification of novel substituted 2-amino thiazole analogues as potential anti-inflammatory agents targeting 5-lipoxygenase

Article abstract of DOI:10.1016/j.ejmech.2018.08.098

Human 5-Lipoxygenase (5-LOX) is a key enzyme targeted for asthma and inflammation. Zileuton, the only drug against 5-LOX, was withdrawn from the market due to several problems. In the present study, the performance of rationally designed conjugates of thi

Full text of DOI:10.1016/j.ejmech.2018.08.098

Accepted Manuscript
Design, synthesis and identification of novel substituted 2-amino thiazole analogues
as potential anti-inflammatory agents targeting 5-lipoxygenase
Shweta Sinha, Mukesh Doble, S.L. Manju
PII:
S0223-5234(18)30770-0
10.1016/j.ejmech.2018.08.098
EJMECH 10709
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 December 2017
Revised Date: 28 August 2018
Accepted Date: 31 August 2018
Please cite this article as: S. Sinha, M. Doble, S.L. Manju, Design, synthesis and identification of novel
substituted 2-amino thiazole analogues as potential anti-inflammatory agents targeting 5-lipoxygenase,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.098.
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ACCEPTED MANUSCRIPT

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1
Design, synthesis and identification of novel substituted 2-
amino thiazole analogues as potential anti-inflammatory
agents targeting 5-Lipoxygenase
a,b
b,
a,
Shweta Sinha , Mukesh Doble *, S. L Manju *
a
Department of Chemistry, Vellore Institute of Technology, Vellore, Tamil Nadu, 632014, India.
b
Department of Biotechnology, Indian Institute of Technology, Madras, Tamil Nadu, 600036, India.
*
Corresponding authors
Email: mukeshd@iitm.ac.in, slmanju@vit.ac.in
Keywords: Design, Inflammation, 5-Lipoxygenase, Pharmacophore/Docking, Thiazoles.
Highlights
•
•
•
•
•
Novel thiazoles/thioureas are designed via pharmacophore modeling against 5-LOX.
Most compounds found active against 5-LOX in vitro enzyme inhibition studies.
The best compound, 2m, from thiazole series acts as a competitive 5-LOX inhibitor.
The most potent thiourea, 3f, inhibits 5-LOX via non-competitive (redox) mechanism.
Docking studies demonstrate binding mode of the competitive inhibitors.

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ABSRACT
Human 5-Lipoxygenase (5-LOX) is a key enzyme targeted for asthma and inflammation.
Zileuton, the only drug against 5-LOX, was withdrawn from the market due to several problems.
In the present study, the performance of rationally designed conjugates of thiazole (2) and
thiourea (3) scaffolds from our previously reported 2-amino-4-aryl thiazole (1) is reported. They
are synthesized (total 31 derivatives), characterized, and tested against the 5-LOX enzyme in
vitro and the mode of action of the most active ones are determined. Compound 2m exhibited an
IC of 0.9 ± 0.1 µM acting through competitive (non-redox) mechanism, unlike Zileuton, and
5
0
found to be devoid of radical scavenging properties. Computational studies are in good
agreement with the experimental data supporting its mechanism of action. Another lead molecule
from the thiourea series (3), 3f, exhibited an IC of 1.4 ± 0.1 µM against 5-LOX whose mode of
50
action is redox type (non-competitive). It is promising to note that the activities displayed by
both the lead inhibitors, 2m and 3f, are better than the commercial drug, Zileuton (IC50 = 1.5 ±
0
.3 µM). These inhibitors could be further developed as drugs against inflammation.

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1
. Introduction
Chronic inflammation is implicated in a variety of diseases including asthma, allergy, arthritis,
cancer, cardiovascular and respiratory tract disorders [1-3]. These inflammatory diseases are
directly or indirectly related with the alterations in the production of leukotrienes (LTs) and
prostaglandins (PGs) in the arachidonic acid (AA) pathway [4,5]. Several studies have
demonstrated that 5-lipoxygenase (5-LOX) is an important enzyme which catalyzes AA for the
production of several LTs. It oxygenates AA into 5(S)-hydroperoxyeicosatetraenoic acid (5-
HPETE) and further dehydrates to leukotriene A4 (LTA4) [6,7] Therefore, 5-LOX is regarded as
a valid target against inflammation in the drug discovery. In the past several years, different
classes of low molecular weight 5-LOX inhibitors (i.e. redox, non-redox and iron chelator) have
been developed [8]. Inhibitors including benzoquinones [9], benzimidazoles [10] and triazole
caffeic acid esters [11] have been shown to inhibit this enzyme effectively. The only 5-LOX
inhibitor which could enter the market as an anti-inflammatory drug for the treatment of asthma
was, Zileuton, with an IC50 ~1 µM [12] But its use is restricted, due to drawbacks of liver toxicity
and short half-life, [13] thereby, creating a dire need for the design and development of new
chemical entities against inflammation.
Pharmacophore modeling is one of the widely used strategies in the ligand-based drug design.
Pharmacophore is a set of features comprising of H-bond donor, H-bond acceptor, hydrophobic
centroid and aromatic center, essential for the enzyme-ligand interaction in the active molecule
[14]. Thiazole is a unique heterocycle containing sulphur and nitrogen atoms and is considered
as a building block of many drug candidates in medicinal chemistry. They are known to have
antimicrobial, anticancer, anti-inflammatory and anti-HIV properties [15-17]. Previous reports
have shown that N,4-diaryl-1,3-thiazole-2-amine derivatives containing thiazole as the basic

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moiety are selective inhibitors of 5-LOX and COX-2 [18]. A series of 4-monosubstituted 2-
aminothiazole pirinixic acid derivatives bearing thiazole as core molecule have been reported as
dual 5-LOX and mPGES1 inhibitors [19]. Recently, 2-amino-4-aryl thiazoles (1) have been
identified by us as a potent competitive type of 5-LOX inhibitors [20]. In continuation of our
earlier work, a series of novel (4-(4-substituted phenyl)-2-(substituted amino-1-yl)thiazol-5-
yl)(phenyl)methanone derivatives (2) have been designed and synthesized by us with better
efficacy and potency (Figure 1). With an aim to increase the potency of basic thiazole
pharmacophore, hydrophobic centroids and H-bond acceptor features were introduced in the
th
0
molecule (2) by incorporating benzoyl group at the 5 and substituted secondary (2 ) amine at
nd
th
the 2 positions in the thiazole ring. Different aryl substitutions at the 4 position of the thiazole
0
ring are incorporated to investigate the effect of benzoyl and 2 amine substituents on the
potency against 5-LOX enzyme.
The known drug, Zileuton (N-(1-benzo[b]thien-2-ylethyl)-N-hydroxyurea) and a compound in
clinical trial, MLN977 (1-(4-(5-((4-fluorophenoxy)methyl)tetrahydrofuran-2-yl)but-3-ynyl)-1-
hydroxyurea) from N-hydroxyurea series have N-hydroxyurea group in common. MLN977 has
also been discontinued in Phase II clinical trials due to side effect, namely elevations in liver
enzyme [21]. Perhaps, N-hydroxyurea group is acting through free radical-mediated lipid
peroxidation of cell membrane and possibly responsible for the hepatotoxicity [22,23]. Based on
these facts as well as to achieve improved safety profile of the drug candidate, the scaffold has
been modified by replacing the urea with a thiourea group as the basic moiety and the designed
another series of novel compounds (3). Further structural modifications are carried out by
substituting both the nitrogens of the thiourea. The first nitrogen is substituted with a secondary
amine and the other with a substituted benzoyl group at N-H position in (3) (Fig. 1).

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Fig. 1: General structures of novel scaffolds A (2) and B (3) including (1) previously reported compound
considered as the starting scaffold for the design of molecule 2.
This paper discusses the ligand based drug design strategy including pharmacophore modeling
and then synthesis of a series of (4-(4-substituted phenyl)-2-(amino-1-yl)thiazol-5-
yl)(phenyl)methanone (2) and 4-substituted-N-(amino-1-carbonothioyl)benzamide (3) scaffolds.
In vitro 5-LOX inhibitory activity as well as their mechanism of action of these synthesized
compounds is also studied.
2
. Results and discussion
2
.1. Design
Thiazole is a vital core scaffold present in several medicinally significant natural and synthetic
compounds. Previous studies indicate that 2-amino-4-aryl thiazole is an effective 5-LOX
inhibitor, among which compound 1 exhibits the highest activity with an IC50 of ~10 µM [20].
To further improve the potency and bring down the IC50 value of molecule 1, hydrophobic
centroids and H-bond acceptor features are introduced (Fig. 2). The reason for introducing these
features are based on pharmacophore studies as described below.

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Fig. 2: Design of novel compound (4-(4-substituted phenyl)-2-(amino-1-yl)thiazol-5-
yl)(phenyl)methanone (2) and 4-substituted-N-(amino-1-carbonothioyl)benzamide (3) from 1 and
Zileuton respectively.
2
.2. Pharmacophore elucidation
A pharmacophore is a set of essential features used to correlate the biological activities for a
group of compounds with their chemical structures [24, 25]. To determine the features essential
for 5-LOX inhibition, a pharmacophore model was developed from the group of known 5-LOX
inhibitors reported in the literature, namely, Zileuton, Atrleuton, MLN977, Licofelone,
Siteleuton, and PF4191834 (Fig. 3).

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Fig. 3: Structures of known 5-LOX inhibitors.
Zileuton is a well-known drug from N-hydroxy urea series available for asthma with restrictions.
Atrleuton and MLN977 are under phase II clinical trials. The latter has been discontinued due to
liver enzyme elevations. All these three inhibitors have a common group, namely N-hydroxy
urea. Licofelone is a multi-targeted inhibitor for FLAP, mPGES1 and 5-LOX with a pyrrolizine
ring. Siteleuton is a coumarin derivative under clinical trials and is terminated due to liver
toxicity. PF4191834, with a pyrazole moeity is a selective 5-LOX inhibitor under clinical trial
and is effective for pain and inflammation [26,27]. These six inhibitors have a four-point
pharmacophoric features (Ph model) containing two hydrophobic centroids (Hyd) and two H-
bond acceptors (Acc2) (Figure 4a). Hence, compound 2 is designed by introducing a
hydrophobic centroid and H-bond acceptor in compound 1, namely a benzoyl group is inserted at
th
0
nd
the 5 position and substituted secondary (2 ) amine at the 2 position in the thiazole ring.
Compound, 3, is designed by incorporating a benzoyl group (H-bond acceptor) at the N-H
position in the thiourea moiety (Fig. 2).

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An in-house conformational database of the designed compounds (listed in Table 1 and 2) are
created and set as a query against Ph model. It is found that all the features of Ph model are
mapped with the best molecule 2m as well as eight other compounds from the (4-(4-substituted
phenyl)-2-(amino-1-yl)thiazol-5-yl)(phenyl)methanone (2) series. Similarly, all the four common
pharmacophore features required for the 5-LOX inhibition (as per Ph model) are mapped on to 3f
as well as fourteen other compounds of the 4-substituted-N-(amino-1-carbonothioyl)benzamide
(3) series (Fig. 4).
Fig. 4: a) Pharmacophore (Ph model) for 5-LOX inhibitors generated by pharmacophore elucidation in
0
MOE 2016.0801 with 3D spatial relationship and geometric distances in A ; b) 3f mapping with Ph model
;
c) 2m mapping with Ph model. Pharmacophore features depicted are Hyd- Hydrophobic (green) and
0
Acc2- H-bond acceptor (orange). Distances are given in A .

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2
.3. Chemistry
The multi-step synthesis of 4-substituted-N-(amino-1-carbonothioyl)benzamides (3a-q) and (4-
4-substituted phenyl)-2-(amino-1-yl)thiazol-5-yl)(phenyl)methanones (2a-n) is accomplished as
(
shown in schemes 1 and 2 respectively. The intermediates (3a-q) and lead compounds (2a-n)
were synthesized according to the procedure described in the literature with some modification
[28,29]. The first step in the scheme 1 includes the formation of substituted benzoyl
isothiocyanates. This is achieved when substituted benzoyl chloride in toluene is added to the
mixture of aq. solution of potassium thiocyanate and cetrimide. This is followed by the addition
0
of a secondary amine to the aromatic layer at 0-25 C, stirring at room temperature for 2 h to
obtain various benzamides (3a-q) (Scheme1).
Scheme 1: Synthesis of 4-substituted-N-(amino-1-carbonothioyl)benzamide (3a-q) derivatives.
The synthesis of (4-(4-substituted phenyl)-2-(amino-1-yl)thiazol-5-yl)(phenyl)methanone
derivatives (2a-n) is performed by Hantzsch thiazole synthesis procedure [28,29]. Condensation

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of 2-bromoacetophenone with equivalent amount of various benzamides in the presence of
0
catalyst, triethylamine, in acetonitrile at 70 C results in [1+4] thiazole ring formation to give 2a-
n (Scheme 2).
Scheme 2: Synthesis of (4-(4-substituted phenyl)-2-(amino-1-yl)thiazol-5-yl)(phenyl)methanone (2a-n)
derivatives.
2
.4. 5-LOX inhibitory activity
The anti-inflammatory potencies of these synthesized 2-substituted amino-4-substituted phenyl
thiazol-5-yl phenyl methanones (2a-n) and N-(substituted carbonothioyl)-4-substituted
benzamides (3a-q) at a concentration of 10 µM are evaluated in cell free assay using partially
purified human recombinant 5-LOX enzyme (Tables 1 and 2). The activity is determined by
measuring the product, 5-HPETE, produced from AA at λ236 [30,31]. Most of the test compounds
from both the series are found to be significantly active against the enzyme 5-LOX. Zileuton is
used as the positive control while DMSO (2%, v/v) as the negative control. The former inhibited
5
-LOX by 86.9 ± 2.2 % (Table 1) as expected while vehicle control (2% DMSO in assay mixture

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solution) did not show any inhibition. Compounds 2m and 3f exhibited excellent activities,
almost comparable to Zileuton.
Table 1
-yl)(phenyl)methanone derivatives (2a-n). Data are expressed as means ± SD obtained from three
independent experiments.
: 5-LOX inhibition and antioxidant activities of (4-(4-substituted phenyl)-2-(amino-1-yl)thiazol-
5
O
S
R1
N
N
R2
R3
%
5-LOX
% scavenging
Compd
code
S.No.
Structure
Inhibition ± of DPPH ± SD
SD (10 µM)
(20 µM)
2
a
1
2
3
85.2 ± 8.2
8.5 ± 0.2
2
b
85.3 ± 6.6
75.3 ± 3.8
9.5 ± 0.7
N
2
c
N
S
-2.9 ± 0.1
3.6 ± 2.9
O
5
3.1 ± 1.5
2
d
4
5
N
2
e
N
67.8 ± 2.9
S
4.6 ± 3.8
-0.7 ± 1.2
-4.7 ± 0.3
O
2
f
6
7
38.3 ± 2.9
48.6 ± 0.7
2
g

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1
2
2
h
8
9
62.0 ± 9.4
9.6 ± 1.4
2
i
1
7.7 ± 2.9
-1.9 ± 1.8
8.2 ± 4.6
-0.8 ± 0.4
2
j
10
67.8 ± 1.5
50.2 ± 4.4
2k
11
2
l
1
2
3
53.1 ± 2.9
7.8 ± 1.6
2
m
1
92.8 ± 0.7
11.8 ± 2.1
2n
14
79.6 ± 1.5
-2.8 ± 1.0
Zileuton
8
6.9 ± 2.2
17.8 ± 1.2
84.1 ± 0.5
Ascorbic
acid
-
2
.5. Structure activity relationship (SAR) of 5-LOX inhibition
Analogues of 2 and 3 are synthesized to evaluate the roles of the substituents, R , R
1
2
(
hydrophobic) and R (H-bond acceptor) in the parent molecule for the inhibition of 5-LOX.
3
Previous study with thiazole derivatives had indicated, that compound 1 (Fig. 1) was the most
potent 5-LOX inhibitor with an IC50 of ~10 µM [20]. Interestingly, introducing a benzoyl residue
0
and a 2 amine (diphenylamine) in the central thiazole ring (2) yielded a highly potent compound,
2
m, which was 11-folds more active than the former with an IC of 0.9 ± 0.1 µM (Table 3). In
50

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13
2
m, the presence of an electron donating methoxy group (R
3
) in the 4’-position of the phenyl
th
helps to increase the hydrophobicity of the aromatic centroid which is attached at the 4 position
of the thiazole ring (Fig. 4d) and thus might be responsible for the high enzyme inhibition.
Docking studies which is discussed later also suggest that the methoxy group could be
potentially involved in the ionic interactions (H-bond acceptor and donor) with the carbonyl
groups of the amino acids, Ile673, Val175 and Phe177, in the lipophilic side chain of the protein.
The inhibitory activity of 2m is better than that of the standard drug, Zileuton (IC50 = 1.5 ± 0.3
µM) (Table 3). SAR studies indicate that when methoxy is replaced with nitro, 2n (79.6 ± 1.5 %
at 10 µM), (Table 1) the activity decreases by one fold which might be due to the decrease in the
electron density at the p-position of the aromatic ring through a resonance withdrawing effect of
the nitro group. Removal of methoxy or nitro group (R = H, i.e. without any substituent), results
3
0
in a 50 % reduction in the activity (compound 2g: 48.6 ± 0.7 %). Insertion of another bulky 2
nd
amine (N-phenyl piperazine) at the 2 position of the thiazole ring, does not alter the efficiency
against 5-LOX (compound 2f: 38.3 ± 2.9 %). Nevertheless, further variation in the substituents at
the R
3
by –OCH
3
, 2l (53.1 ± 2.9 %) and –NO
2
, 2j (67.8 ± 1.5 %) increases the potency by 1.5 to
1
.8 times when compared to 2f (Table 1). Surprisingly, there is a large increase in the potency
0
nd
when R = H is retained and bulky 2 amine group at the 2 position of the thiazole ring (R &
3
1
0
R ) is exchanged by smaller 2 amines such as piperidine, 2a (IC = 3.1 ± 0.1
µ
M) or
2
50
0
morpholine, 2b (IC = 2.3 ± 0.2 µM) (Table 3). However, these smaller 2 amines are not
5
0
tolerated by any electron donating (-OCH ) or withdrawing group (fluoro) at the R position.
3
3
Thus, a considerable loss in potency is seen in 2h (62.0 ± 9.4 %) and 2k (50.2 ± 4.4 %).
0
Therefore, sustaining R
3
= H and decreasing the bulkiness of 2 amine (R
1
and R
2
) yields
analogues, 2d, 2e and 2c, with high activity. Compared to substituent, N-methyl benzyl amine,

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14
2
d (53.1 ± 1.5 %), the N-methyl aniline analogue, 2e (67.8 ± 2.9 %) is more potent. Insertion of
an electron donating group such as methoxy at R in 2d leads to a considerable decrease in the
3
0
potency (compound 2i: 17.7 ± 2.9 %). 2c (75.3 ± 3.8 %), with a smaller 2 amine (pyrrolidine)
substitution when compared to that in 2d leads to 1.5 times more activity (Table 1).
Table
2:
5-LOX inhibition and antioxidant activities of 4-substituted-N-(amino-1-
carbonothioyl)benzamide derivatives (3a-q). Data are expressed as means ± SD obtained from three
independent experiments.
%
5-LOX
% scavenging
Compd
code
S.No.
Structure
Inhibition ± of DPPH ± SD
SD (10 µM)
(20 µM)
3
a
56.8 ± 2.3
1
1
5
6
23.7 ± 0.7
3
b
79.4 ± 1.7
19.1 ± 1.6
3
c
1
1
7
8
24.5 ± 4.5
80.7 ± 3.8
25.6 ± 2.8
18.4 ± 0.9
3d
O
S
3
e
N
N
1
2
9
0
H
78.7 ± 2.8
13.2 ± 0.4
24.1 ± 0.7
3
f
9
7
1.3 ± 3.8
3.3 ± 6.6
2
1
3
3
g
11.3 ± 1.6
21.8 ± 0.7
h
86.0 ± 1.9
80.7 ± 3.8
2
2
2
3
3i
5
.9 ± 0.9

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15
3
j
68.3 ± 7.2
2
4
3.9 ± 0.1
7.4 ± 0.2
3
k
2
2
2
5
6
7
26.5 ± 1.5
3l
7
6.6 ± 1.5
20.3 ± 3.5
9.1 ± 1.3
3
m
29.5 ± 5.9
3
n
2
2
8
9
23.6 ± 2.9
36.8 ± 2.2
6.3 ± 0.9
3.7 ± 0.7
3
o
3
3
p
q
3
3
0
1
19.2 ± 5.2
8.1 ± 3.5
4.7 ± 5.3
3
0.9 ± 3.7
Another set of analogues with thiourea as the basic moeity (3) with different secondary amines
at R and R positions) are synthesized retaining the presence of benzoyl (H-bond acceptor
(
1
2
property) group which is critical for activity. The highly potent compound in this series, 3f,
0
(
Table 3) with bulkiest 2 amine (phenyl piperazine) substitution replacing R
1
and R
2
while
benzoyl having no substitution (R
3
= H) showed an IC50 of 1.4 ± 0.1 µM (91.3 ± 3.8 % at 10
µM) against 5-LOX. This activity is found to be approximately similar to the standard drug,
Zileuton (IC50 = 1.5 ± 0.3 µM). Replacement of hydrogen (R ) at the para-position of the
3
benzoyl ring with an electron withdrawing groups such as nitro, 3l (76.6 ± 1.5 %) showed a
marginal reduction in the potency whereas considerable loss in inhibitory activity is observed
with fluoro, 3n (23.6 ± 2.9 %) (Table 2). An electron donating methoxy group, 3o (36.8 ± 2.2 %)
0
also could not improve the potency. Bulky 2 amines such as benzyl methyl amine, 3d, or methyl

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16
aniline, 3e, or diphenyl amine, 3g, showed high inhibitory activities (of 80.7 ± 3.8 %, 78.7 ± 2.8
and 73.3 ± 6.6 % respectively). A drastic loss in potency is observed when hydrogen at R in
%
3
3
d is substituted with methoxy group producing 3k (26.5 ± 1.5 %). Similar effects are observed
with methoxy analogue, 3p (19.2 ± 5.2 %), and nitro analogue, 3q (30.9 ± 3.7 %). However,
0
exchanging bulkier amine with smaller 2 amines such as piperidine in 3o led to a significantly
improved compound, 3h, with an IC of 3.3 ± 0.2 µM. There is little change in activity when
5
0
methoxy is replaced with fluoro substituent, 3i (IC50 = 3.8 ± 0.2 µM). But removal of either of
the two substituents, (R = H), led to a drop in the potency (compound 3a).
3
Table 3: IC values of the best six analogues against 5-LOX enzyme in cell free assay, their pseudoperoxidase
50
activity and mechanism of action.
%
Redox absorbance
Type of
mechanism
Compd
Structure
code
5-LOX inhibition,
IC (µM)
(at 10 µM of 13(S)-
HpODE and inhibitor)
50
2
a
3
2
.1 ± 0.1
.3 ± 0.2
+ 0.6
+ 2.1
Non-redox
Non-redox
2
b
Non-redox
(Competitive)
2
m
0.9 ± 0.1
1.4 ± 0.1
+
29.4
3
f
- 46.2
Redox
(
Non-competitive)
3
.3 ± 0.2
3
h
-
35.3
nd
Redox
nd
3
i
3.8 ± 0.2
Zileuton
- 49.6
Redox
1
.5 ± 0.3

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17
‘
‘
-’ indicates % decrease in absorbance due to consumption of 13(S)-HpODE
+’ indicates % increase in absorbance
nd - not determined
2
.6. Kinetics of 5-LOX inhibition
A steady-state enzyme kinetic analysis of these two most active inhibitors (2m and 3f) was
performed at three different concentrations (0, 5 and 10 µM) in the presence of different
concentrations of the substrate (AA) to determine the initial rate [30,32]. The Lineweaver-Burke
plot for the compound 2m shows that all the lines intersect on the y-axis indicating, it acts as a
competitive inhibitor (Figure 5a). The value of Vmax was found to be constant at these three
concentrations (0.39 ± 0.02, 0.38 ± 0.02, 0.38 ± 0.01 nmol/min) whereas the Km values
increased (0.42 ± 0.15, 0.99 ± 0.26, 1.51 ± 0.20 µM) with increasing concentration of 2m.
Lineweaver–Burk plot for the other compound, 3f, shows that all the three lines are intersecting
in the left hand side of the y-axis (Fig. 5b). Also Vmax (0.48 ± 0.02, 0.40 ± 0.01, 0.34 ± 0.01
nmol/min) decreases while Km values are almost constant (0.7761 ± 0.22, 0.7904 ± 0.13, 0.7674
±
0.09 µM) for increasing concentration of 3f indicating non-competitive (mixed) type of
inhibition where inhibitor may be binding to both the free enzyme and the enzyme-substrate
complex.

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Fig. 5: The Lineweaver-Burk plots for compounds a) 2m tested at 5 and 10 µM concentrations and b) 3f
tested at concentrations 5 and 10 µM. [S] = Substrate (AA) concentration, V = Velocity of reaction.
Control (no inhibitor, 0 µM).
Fig. 6: Pseudoperoxidase activity plot (absorbance measured at 234 nm substracted from control)
showing amount of consumption of 13(S)-HpODE by a) Zileuton, b) 2m c) 3f, d) 2b, e) 3h, f) 2a.

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2
.7. Pseudoperoxidase activity and redox mechanism
Competitive (non-redox), redox and iron chelating are three well-known reported mechanisms
through which compounds inhibit the 5-LOX enzyme [8,33]. Moreover, competitive inhibitors
bind to the active site of the enzyme while iron chelators chelate the metal iron thereby, blocking
the active site and thus exhibit the inhibitory activity. In contratst, redox inhibitors act by
converting the active form of iron to inactive state by redox process and hence inhibit the activity
3
+
of the enzyme [34]. 5-LOX is a non-heme iron containing enzyme having Fe ion in the
2
+
activated form which is reduced to Fe in the presence of a redox inhibitor. Inactivated ferrous
ion can consume lipid peroxide to reactivate the redox cycle of the enzyme. In the
pseudoperoxidase activity assay, the consumption of 13(S)-hydroperoxyoctadecadienoic acid
(13(S) HpODE) in the presence of an inhibitor is determined which is a measure of the redox
potential of the inhibitor [35].
Redox inhibitory activities of 2m, 2a, 2b, 3f and 3h are elucidated here. The decrease in the
amount of peroxide substrate, 13(S) HpODE is determined at 234 nm (Fig. 6). The best 5-LOX
inhibitor, 3f, is found to decrease the absorbance by 46.2 % in 300 s (Table 3, Fig. 6c) indicating
significant degradation of the hydroperoxide product, thus suggesting its redox behaviour. While
Zileuton, a known redox 5-LOX inhibitor shows 49.6 % degradation of the hydroperoxide (Fig.
6
a). Compound 3h also shows considerable degradation of the peroxide (35.3 %) indicating that
it also disrupts the redox cycle of the enzyme. While 2m does not decrease the absorbance
indicating that it does not disturb the redox cycle) (Fig. 6b). However, the graph shows an
(
increase in the absorbance as afunction of time. This increase might be due to an unknown
reaction in the assay mixture leading to UV absorbance at the same wavelength in the absence of
redox activity or compound itself exhibiting UV characterisitcs. Also, when compound 2m alone

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20
is tested without the enzyme at the same wavelength, an increase in the absorbance is observed
as a function of time. Similar increase in absorbance is reported in literature while performing
pseudoperoxidase activity which is attributed to the above mentioned reasons [36,37]. This
compound does not exhibit redox activity and probably binds to the active site of the enzyme as
a competitive inhibitor as seen from the L-B plot (Fig. 5a). Compounds, 2a and 2b did not show
any considerable reduction in the absorbance with time (Fig. 6d, f). So they may be inhibiting the
enzyme through non-redox mechanism.
2
.8. DPPH radical scavenging activity
There is a close relationship between a 5-LOX inhibitor and its radical scavenging property
3
+
because several of them are known to change the oxidation state of the iron from ferric (Fe ) to
2
+
ferrous (Fe ) present in the enzyme [38]. Radical scavenging activity of all the compounds at 20
µM is determined by using free radical, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay method
[39]. Ascorbic acid is used as the standard which exhibited 84.1 ± 0.5 % antioxidant activity at
2
0 µM (Table 1). Most of the compounds from both the series showed poor radical scavenging
property, whereas 3f and 3h showed 24.1 ± 0.7 and 21.8 ± 0.7 % activity respectively (Table 2).
Pseudoperoxidase activity also indicates that these compounds act on the enzyme through the
redox mechanism. While, Zileuton exhibited 17.8 ± 1.2 % free radical scavenging activity
(Table 2), compounds 2m, 2a and 2b showed relatively lower antioxidant activities (11.8 ± 2.1,
8
.5 ± 0.2 and 9.5 ± 0.7 % respectively) indicating that they probably act through a non-redox
mechanism. Pseudoperoxidase activity assay also indicates that these three compounds do not
follow the redox mechanism. L-B plot has also clearly revealed that compound 2m acts through
the competitive mode of inhibition.

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21
2
.9. Molecular docking studies
The experimental studies indicated that compounds 2m, 2a and 2b act as competitive inhibitors
so these ligands were docked into the active site of the enzyme, 5-LOX to understand their mode
of binding and interaction they made with the active site amino acid residues. The docking was
not performed for the compound 3f which was found to be acting through non-competitive
mode. The crystal structure of the protein (pdb ID 3o8y) was obtained from Brookhaven Protein
Data Bank (PDB) and docking was performed with Molecular Operating Environment (MOE
2
016.0801) software. Compound 2m showed several interactions with the active site residues
with a binding score of -7.0 kcal/mol (Fig. 7). The –OCH group of the phenyl linker attached to
3
th
the 4 position of the thiazole ring interacts with Ile673 (3.74 Å), Val175 (4.11 Å) and Phe177
0
(
3.69 Å) in the hydrophobic pocket of the protein through H-bond. 2 amine containing phenyl
groups are also seen to make H-bonds with Asn187 (3.75 Å), Lys183 (4.21 Å) and Phe610 (4.09
Å). Polar amino acid residues such as Gln609 and Asn180 are observed to possess pi-H
0
interactions with one of the phenyl ring of 2 amine and benzoyl ring respectively (Fig. 7).

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22
Fig. 7: Binding of compound 2m with 5-LOX. (a) Ligplot showing residues involved in the interaction
between the amino acids of the enzyme with compound 2m. (b) Docked pose of compound 2m in a stick
model. Interactions are shown by dotted lines. Colours: carbon: grey, hydrogen: cyan, nitrogen: blue,
sulphur: yellow, fluorine: green and oxygen: red.
Molecular docking of 2a also showed appreciable interactions in the active site of 5-LOX with
binding score of -5.1 kcal/mol. Side chain H-bond interactions between piperidine ring and
Ile673 (3.99 Å), Val175 (3.91 Å) and Asn180 (3.63 Å) and between hydrogen of phenyl ring at
th
the 4 position of thiazole moiety and carbonyl of Asn187 (3.86 Å) are observed. This side chain
phenyl ring also exhibits arene pi-H interaction with Lys183. Similar type of pi-H interaction is
also seen with benzoyl group and Asn180 (Fig. 8).
Fig. 8: Binding of compound 2a with 5-LOX. (a) Ligplot showing residues involved in the interaction
between the amino acids of the enzyme with compound 2a. (b) Docked pose of compound 2a in a stick
model. Interactions are shown by dotted lines. Colours: carbon: grey, hydrogen: cyan, nitrogen: blue,
sulphur: yellow, fluorine: green and oxygen: red.

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23
Compound 2b with a docking score of -5.3 kcal/mol is found to possess side chain H-bond
interactions with morpholine and Ile673 (3.96, 3.70 Å) and Val175 (3.99, 3.48 Å). Also, a back
bone H-bond donor is detected between sulphur of thiazole ring and Gly174 (3.49 Å) (Fig. 9).
These computational studies indicate that these compounds (competitive and non-redox mode)
bind effectively through H-bond and pi-H interactions with the amino acid residues in the active
site of the enzyme.
Fig. 9: Binding of compound 2b with 5-LOX. (a) Ligplot showing residues involved in the interaction
between the amino acids of the enzyme with compound 2b. (b) Docked pose of compound 2b in a stick
model. Interactions are shown by dotted lines. Colours: carbon: grey, hydrogen: cyan, nitrogen: blue,
sulphur: yellow, fluorine: green and oxygen: red.
2
.10. Pan-Assay Interference Compounds (PAINS)
Chemical compounds which react non-specifically with multiple biological targets and often
depict false positives in high throughput screenings and they are interpreted as PAINS. On the
contrary, a selective inhibitor or drug-like molecule will interact specifically with the target

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24
protein. But These PAINS show activity in many assays because of probably due to covalent
modifications, chelation, autofluorescence, agglomeration or degradation of the target protein
[40]. Many classes of compounds have been categorized as PAINS including rhodanines,
isothiazolones, enones, and quinones [41,42]. So it is important to determine if compounds that
are being studied as drug candidates posses these structural features which may lead to them to
be classified as PAINS. The currently synthesized thiazoles and thioureas were tested using
ZINC15 database and found to be PAINS negative.
3
. Conclusion
5
-LOX is an important target in the leukotriene pathway which is implicated in asthma and other
inflammatory disorders. The drug, Zileuton, is withdrawn from the market due to its non-
selective nature. In the present study, novel inhibitors of the enzyme 5-LOX, were designed
using pharmacophore modeling technique, and then chemically synthesized (31 compounds) and
tested against this enzyme. Out of 31 compounds synthesized, 19 compounds (2d, 2f, 2g, 2i, 2j,
2
k, 2l, 2m, 2n, 3d, 3f, 3h, 3i, 3k, 3l, 3m, 3n, 3o, 3p) are completely novel as verified by
Scifinder and for other 12 compounds (2a, 2b, 2c, 2e, 2h, 3a, 3b, 3c, 3e, 3g, 3j, 3q), no reports
are available for their biological activity against 5-LOX in literature. Our previous studies
identified a thiazole inhibitor (1, IC50 ~10 µM). Introduction of hydrophobic and H-bond
th
acceptor properties in the molecule (1) by incorporating a benzoyl group at the 5 and
0
nd
substituted secondary (2 ) amine at the 2 positions respectively in the basic thiazole moiety
provided a potent analogue (2m) with an IC of 0.9 ± 0.1 µM. This compound is found more
5
0
potent than the drug, Zileuton (IC50 = 1.5 ± 0.3 µM). Another compound (3f) exhibited an IC50 of
.4 ± 0.1 µM. Structure activity relationship studies revealed that the presence of electron
1

ACCEPTED MANUSCRIPT
25
0
donating groups such as methoxy (2m) at 4’- position along with nitrogen containing groups, 2
amine as bulky side chain in scaffold (2) increases the hydrophobicity in the molecule and thus
0
leads to high inhibitory potency. In contrast, a bulky 2 amine side chain in moiety (3) does not
require any substitution at R position (3f) to possess high potency. Pharmacophore studies also
3
indicated that the presence of hydrophobic and H-bond acceptor is essential for the enzyme
inhibition (Figure 4). Additional experiments such as pseudoperoxidase, kinetic and antioxidant
assays gave a clear picture that the best compound, 2m, acts through a competitive mode of
inhibition (non-redox process). 3f, on the other hand acts by non-competitive (redox)
mechanism. This compound also has high antioxidant activity. Computational analysis was in
good agreement with the experimental data. The non-redox inhibitors, 2a, 2b and 2m, interact
with the protein through H-bond and pi-H interactions in the active site (Fig. 7). Highly potent
both redox and non-redox type of 5-LOX inhibitors are reported here which could act as novel
chemical entities for further development as an anti-inflammatory drugs.
4
. Experimental
4
.1. General
All solvents and reagents were purchased from chemical suppliers Sigma-Aldrich Chemie
GmbH (Steinheim, Germany), Alfa-Aesar GmbH & Co. (Heysham, England), Acros Organics
(Geel, Belgium) and Thermo Fisher Scientific (Waltham, United States) and were used without
1
13
further purification. H and C NMR spectra were obtained on Bruker AVANCE III 500 MHz
(AV-500) NMR Spectrometer using CDCl and DMSO-d as solvents with tetramethylsilane
3 6
(TMS) as an internal standard. Chemical shifts (δ) are expressed in parts per million (ppm) and
coupling constants (J) values are expressed in hertz (Hz) unit. High resolution mass spectra

ACCEPTED MANUSCRIPT
26
(HRMS) were recorded on Thermo Scientific Orbitrap Elite Mass spectrometer equipped with
dual pressure linear ion trap coupled with high-field orbitrap Mass analyzer. IR spectra were
-1
recorded on Perkin Elmer Spectrum1 FT-IR instrument with a resolution of 1.0 cm and MIR
-1
4
50-4000 cm . Melting points were recorded in capillaries on GUNA melting point apparatus
and are uncorrected. Synthetic reactions were monitored by thin layer chromatography (TLC) on
TLC Silica gel 60G F₂₅₄ plates from Merck KGaA (Darmstadt, Germany), Millipore. Column
chromatography was performed using 60−120 mesh silica. Visualization of compounds was done
using UV light or iodine chamber. Purities of all title compounds synthesized here were 95 % or
higher.
4
.1.1. General procedure for the synthesis of compounds 3a-q. Substituted benzoyl chloride (1.5
mmol) in 10 mL toluene was added to a mixture of aqueous solution of potassium thiocyanate
1.9 mmol) and cetrimide (Scheme 1). The reaction mixture was stirred for 2 h at room
(
temperature. Aromatic yellow layer was separated from aqueous layer. N,N-dialkylamines (1.5
0
mmol) was added to the aromatic layer at 0 C and stirred at room temperature for 3-4 h. The
solid obtained was filtered and the crude product was washed with saturated aqueous sodium
bicarbonate solution, dried in vacuo and further recrystallized with ethanol/ethyl acetate to obtain
pure products.
4
.1.1.1. N-(piperidine-1-carbonothioyl)benzamide (3a). Cream solid, yield 74 %, mp 114-115
0
-1
C. IR (KBr, cm ) 3439 (N-H str), 3168 (aromatic C-H str), 1650 (amide C=O str), 1580 (N-H
1
bend), 1530 (aromatic C=C str), 1250 (aromatic amine C-N str). H NMR (500 MHz, CDCl
3
)
δ:
8
.46 (s, 1H, NH), 7.86 (d, J = 7.2 Hz, 2H, Ar-H), 7.60 (t, J = 7.4 Hz, 1H, Ar-H), 7.50 (t, J = 7.7
Hz, 2H, Ar-H), 4.19 (s, 2H, piperidin-H), 3.62 (s, 2H, piperidin-H), 1.84 – 1.73 (m, 6H,
1
3
piperidin-H). C NMR (126 MHz, CDCl ) δ: 178.25, 163.19, 132.94, 132.62, 128.82, 127.82,
3

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27
+
5
3.05, 52.77, 26.04, 25.25, 23.66. HRMS (ESI) m/z for C13
16 2
H N OS [M + Na] calcd 271.0876,
found 271.0880.
4
.1.1.2. N-(morpholine-4-carbonothioyl)benzamide (3b). White solid, yield 67 %, mp 124-126
0
-1
C. IR (KBr, cm ) 3452, 3243 (N-H str), 3021 (aromatic C-H str), 1665 (amide C=O str), 1580
(N-H bend), 1523 (aromatic C=C str), 1269 (aromatic amine C-N str), 1114 (morpholine C-O-C
1
str). H NMR (500 MHz, CDCl
3
)
δ: 8.61 (s, 1H, NH), 7.86 (d, J = 7.7 Hz, 2H, Ar-H), 7.61 (t, J =
7
.4 Hz, 1H, Ar-H), 7.50 (t, J = 7.6 Hz, 2H, Ar-H), 4.25 (s, 2H, morpholin-H), 3.85 (s, 4H,
1
3
morpholin-H), 3.68 (s, 2H, morpholin-H). C NMR (126 MHz, CDCl3)
33.18, 132.32, 128.97, 127.83, 66.23, 52.51, 51.60. HRMS (ESI) m/z for C H N O S [M +
δ: 179.34, 163.34,
1
12
14
2
2
+
Na] calcd 273.0668, found 273.0670.
4
.1.1.3. N-(pyrrolidine-1-carbonothioyl)benzamide (3c). White solid, yield 64 %, mp 120-123
0
-1
C. IR (KBr, cm ) 3442 (N-H str), 3145 (aromatic C-H str), 1644 (amide C=O str), 1579 (N-H
1
bend), 1537 (aromatic C=C str), 1253 (aromatic amine C-N str). H NMR (500 MHz, CDCl
3
)
δ:
8
.56 (s, 1H, NH), 7.81 (d, J = 7.7 Hz, 2H, Ar-H), 7.55 (t, J = 7.4 Hz, 1H, Ar-H), 7.44 (t, J = 7.6
Hz, 2H, Ar-H), 3.85 (t, J = 7.0 Hz, 2H, pyrrolidin-H), 3.67 (t, J = 6.7 Hz, 2H, pyrrolidin-H), 2.05
1
3
–
1
1.99 (m, 2H, pyrrolidin-H), 1.98 – 1.92 (m, 2H, pyrrolidin-H). C NMR (126 MHz, CDCl ) δ:
3
76.59, 163.51, 133.13, 132.76, 129.03, 128.11, 54.72, 53.10, 26.43, 24.84. HRMS (ESI) m/z for
+
C H N OS [M + H] calcd 235.0900, found 235.0900.
12
14
2
4
.1.1.4. N-(benzyl(methyl)carbamothioyl)benzamide (3d). Cream solid, yield 93 %, mp 112-115
0
-1
C. IR (KBr, cm ) 3450, 3186 (N-H str), 3071 (aromatic C-H str), 2926 (methyl C-H str), 2892
3
(
CH C-H str) 1682 (amide C=O str), 1580 (N-H bend), 1539 (aromatic C=C str), 1483 (C-H sp
2
1
bend), 1260 (amine C-N str). H NMR (500 MHz, CDCl )
δ: 8.60 (s, 1H, NH), 7.90 (d, J = 7.5
3

ACCEPTED MANUSCRIPT
28
Hz, 2H, Ar-H), 7.61 (t, J = 6.9 Hz, 1H, Ar-H), 7.56 – 7.44 (m, 4H, Ar-H), 7.41 (t, J = 7.3 Hz,
2
H, Ar-H), 7.34 (t, J = 7.2 Hz, 1H, Ar-H), 5.31 (s, 1H, CH ), 4.82 (s, 1H, CH ), 3.35 (s, 1H, N-
2
2
1
3
CH ), 3.19 (s, 2H, N-CH ). C NMR (126 MHz, CDCl3)
δ: 181.54, 163.52, 134.86, 133.08,
3
3
1
32.51, 128.93, 128.83, 127.94, 127.88, 59.65, 40.37. HRMS (ESI) m/z for C H N OS [M +
16 16 2
+
Na] calcd 307.0876, found 307.0880.
4
.1.1.5. N-(methyl(phenyl)carbamothioyl)benzamide (3e). Light yellow solid, yield 84 %, mp
0
-1
1
17-119 C. IR (KBr, cm ) 3455, 3195 (N-H str), 3029 (aromatic C-H str), 2932 (methyl C-H
3
str), 1695 (amide C=O str), 1597 (N-H bend), 1515 (aromatic C=C str), 1436, 1382 (C-H sp
1
bend), 1261 (amine C-N str). H NMR (500 MHz, CDCl )
δ: 8.45 (s, 1H, NH), 7.52 (dd, J =
3
1
3
2
4.9, 17.9 Hz, 3H, Ar-H), 7.42 – 7.34 (m, 7H, Ar-H), 3.78 (s, 3H, N-CH ). C NMR (126 MHz,
3
CDCl3)
δ: 180.55, 162.84, 145.29, 132.92, 132.67, 129.46, 128.73, 127.90, 127.55, 125.54,
+
4
5.59. HRMS (ESI) m/z for C H N OS [M + H] calcd 271.0900, found 271.0900.
1
5
14
2
4
.1.1.6. N-(4-phenylpiperazine-1-carbonothioyl)benzamide (3f). Cream solid, yield 75 %, mp
0
-1
1
22-125 C. IR (KBr, cm ) 3434, 3259 (N-H str), 3058 (aromatic C-H str), 1668 (amide C=O
1
str), 1599 (N-H bend), 1532 (aromatic C=C str), 1273, 1221, 1161 (amine C-N str). H NMR
(
500 MHz, CDCl3)
Ar-H), 7.52 (t, J = 7.5 Hz, 2H, Ar-H), 7.32 (t, J = 7.6 Hz, 2H, Ar-H), 6.95 (dd, J = 12.5, 7.8 Hz,
H, Ar-H), 4.40 (s, 2H, piperazine-H), 3.86 (s, 2H, piperazine-H), 3.40 (d, J = 22.6 Hz, 4H,
δ: 8.58 (s, 1H, NH), 7.89 (d, J = 8.0 Hz, 2H, Ar-H), 7.63 (t, J = 7.2 Hz, 1H,
3
1
3
piperazine-H). C NMR (126 MHz, CDCl
3
)
δ: 179.18, 163.34, 150.35, 133.17, 132.41, 129.32,
1
29.00, 127.86, 120.56, 116.46, 51.65, 51.20, 48.96, 48.62. HRMS (ESI) m/z for C18H N
19 3
OS
-
-
[
M + H] calcd 326.1322, found 326.1321.