2-Amino-4-aryl thiazole: A promising scaffold identified as a potent 5-LOX inhibitor

Article abstract of DOI:10.1039/c5ra28187c

Human 5-lipoxygenase (5-LOX) is an important enzyme in the biosynthesis of leukotrienes and is a target for asthma and allergy treatment. Zileuton is the only drug currently marketed that targets this enzyme (IC₅₀ ～ 1 μM). So, the development of novel lead compounds is highly desirable. A series of 2-aryl indole, thiazolopyrazole acid, oxadiazolobenzothiophene, 1,4-disubstituted-1,2,3-triazole, 2-amino-4-aryl thiazole and 4,4′-(1,4-phenylene)bis(1,3-thiazole) derivatives when tested against this enzyme resulted in the identification of a potent compound (1d), p-fluoro substituted 2-amino-4-aryl thiazole, with an IC₅₀ of ～10 μM. Another lead compound identified is (4a), a thiazolopyrazole acid derivative (IC₅₀ ～ 40 μM). All the compounds exhibit poor DPPH radical scavenging activity which suggests that their action occurs not due to the disruption of the redox cycle of iron present in the enzyme (unlike zileuton) but through competitive inhibition, since the V_max remains constant but the K_m increases with an increase in inhibitor concentration. Molecular docking of 1d and 4a to the active site of 5-LOX also supports the experimental data, and suggests that their possible mechanism of action is through competitive inhibition. The current study identifies a promising lead molecule which could be improved further to match the activity of the commercial drug.

Full text of DOI:10.1039/c5ra28187c

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2-Amino-4-aryl thiazole: a promising scaffold
identified as a potent 5-LOX inhibitor†
Cite this: RSC Adv., 2016, 6, 19271
b
b
b
a
Shweta Sinha,ab T. V. Sravanthi, S. Yuvaraj, S. L. Manju and Mukesh Doble
*
*
Human 5-lipoxygenase (5-LOX) is an important enzyme in the biosynthesis of leukotrienes and is a target for
asthma and allergy treatment. Zileuton is the only drug currently marketed that targets this enzyme (IC50 ꢀ 1
mM). So, the development of novel lead compounds is highly desirable. A series of 2-aryl indole,
thiazolopyrazole acid, oxadiazolobenzothiophene, 1,4-disubstituted-1,2,3-triazole, 2-amino-4-aryl
thiazole and 4,40-(1,4-phenylene)bis(1,3-thiazole) derivatives when tested against this enzyme resulted in
the identification of a potent compound (1d), p-fluoro substituted 2-amino-4-aryl thiazole, with an IC50
of ꢀ10 mM. Another lead compound identified is (4a), a thiazolopyrazole acid derivative (IC50 ꢀ 40 mM).
All the compounds exhibit poor DPPH radical scavenging activity which suggests that their action occurs
not due to the disruption of the redox cycle of iron present in the enzyme (unlike zileuton) but through
competitive inhibition, since the Vmax remains constant but the Km increases with an increase in inhibitor
concentration. Molecular docking of 1d and 4a to the active site of 5-LOX also supports the
experimental data, and suggests that their possible mechanism of action is through competitive
inhibition. The current study identifies a promising lead molecule which could be improved further to
match the activity of the commercial drug.
Received 31st December 2015
Accepted 19th January 2016
DOI: 10.1039/c5ra28187c
www.rsc.org/advances
However, weak potency, hepatic toxicity and a short half life are
the therapeutic drawbacks of zileuton.18 So, there is a strong
Introduction
need for the development of safer and efficacious drugs.
Generally, nitrogen and sulphur containing heterocycles
have a signicant role in the discovery of new drugs because of
their physicochemical properties. Various substituted indoles,
pyrazole scaffolds, compounds containing a thiazole ring
moiety and substituted triazoles have been found to have
numerous biological activities including anti-inammatory
activity.19–25 Recently N-1-tosyl indoles with amino acids have
been reported as effective 5-LOX inhibitors. Glycine and tryp-
tophan residues at the end of the C-3 substituent have been
identied as the most active amongst these compounds.26 The
benzothiophene pharmacophore is found to be a potent 5-LOX
inhibitor and the aryl group has been identied to be respon-
sible for inhibition.27 3-Amino-1-phenyl-4,5-dihydro-1H-pyrazol-
5-one has been shown to be a potent human 5-LOX, COX 1 and
COX-2 inhibitor.28 A series of triazole based compounds have
been reported as potent dual mPGES-1 and 5-LOX inhibitors. A
bulky, halogenated phenylether together with a 2-amino group
was found to be very potent for the activity of the compound.29
In addition, 5-benzylidene-2-phenyl-thiazolinone derivatives
have been observed to be effective 5-LOX inhibitors.30 The 2-
amino-5-thiazolyl moiety has been reported to have anti-
inammatory activity in acute carrageenan and chronic
formalin induced rat paw edema models, which indicates that
aliphatic oxime esters attached via a ketone bridge to the 5th
position of thiazole is responsible for the activity.31
Pain, fever, allergy, swelling, etc. are different symptoms of
inammatory diseases suffered by the majority of the human
population.1 Among these, asthma is an inammatory disease
of the respiratory tract affecting the majority of Indians.2–4 The
discovery of the arachidonic acid (AA) metabolic pathway,5 and
the identication of the enzymes involved in it6 have provided
a systematic approach for the development of anti-inamma-
tory drugs.7 Leukotrienes are important mediators of inam-
matory and allergic diseases.8,9 5-LOX is a key enzyme which
catalyzes the two-step conversion of AA to leukotriene A4 (LTA4).
It catalyzes the hydroperoxidation of polyunsaturated fatty
acids.10 Therefore, it is considered as a potential target in the
treatment of asthma, atherosclerosis, prostate cancer, and
allergies.11–15 Different classes of 5-LOX inhibitors have been
reported, which include redox, non redox and iron chelating
agents (for a review see ref. 16). The only 5-LOX inhibitor which
has entered the market is zileuton [N-(1-benzo[b]thien-2-
ylethyl)-N-hydroxyurea],17 an iron chelator which has weak
reducing properties. It is used for the treatment of bronchial
asthma (with an IC50 ¼ 0.5–1 mM in stimulated leukocytes).
aDepartment of Biotechnology, Indian Institute of Technology, Madras, Tamil Nadu
600036, India. E-mail: mukeshd@iitm.ac.in
bDepartment of Chemistry, Vellore Institute of Technology, Vellore, Tamil Nadu
632014, India. E-mail: slmanju@vit.ac.in
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra28187c
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Table 1 The effects of 2-amino-4-aryl thiazole derivatives at 50 mM
concentration on % 5-LOX inhibition and antioxidant activity (* at 40
mM conc.) Data are expressed as the means ꢂ S.D of single determi-
nations obtained in three to four independent experiments
Based on these observations, a variety of scaffolds including
2-aryl indoles, oxadiazolobenzothiophenes, thiazolopyrazole
acid derivatives, 1,4-disubstituted-1,2,3-triazoles and 2-amino-
4-aryl thiazoles bearing indole, benzothiophene, pyrazole, tri-
azole and thiazole as a core structure, respectively, were
synthesized and tested as possible 5-LOX inhibitors. Also the
hitherto unreported oxadiazolobenzothiophenes are syn-
thesised and their activities tested for the rst time. Further-
more, in order to elucidate the mechanism of action of these
compounds, their antioxidant properties were also determined.
The experimental results were rationalized by performing
mathematical modelling and docking of the best inhibitor with
the target enzyme and were compared with those of zileuton.
% inhibition
% scavenging
S. No.
Compounds
R
of 5-LOX ꢂ SD
of DPPH ꢂ SD
Zileuton
MK-886
Ascorbic
acid
1a
1b
1c
1d
1e
97.1 ꢂ 1.3
82.6*
—
45.6 ꢂ 2.2
35.5 ꢂ 1.1
89.8 ꢂ 0.3
1
2
3
4
5
6
H
Cl
CH3
F
NO2
H, o-OH
4.4 ꢂ 3.1
7.3 ꢂ 5.2
23.4 ꢂ 7.2
84.7 ꢂ 1.0
36.5 ꢂ 3.1
47.4 ꢂ 2.1
11.0 ꢂ 1.4
18.3 ꢂ 0.1
12.1 ꢂ 3.1
20.1 ꢂ 0.001
9.6 ꢂ 2.1
1f
20.6 ꢂ 0.1
Results and discussion
In vitro evaluation of 5-LOX inhibition
The inhibitory activity of 2-amino-4-aryl thiazoles (1a–1f), 2-aryl
indoles (2a–2e), oxadiazolobenzothiophenes (3a–3j), thiazolo-
Table 2 Effects of 2-aryl indole derivatives at 50 mM concentration on
% 5-LOX inhibition and antioxidant activity. Data are expressed as
pyrazole acid derivatives (4a–4d), 1,4-disubstituted-1,2,3-tri- means ꢂ S.D of single determinations obtained in three to four inde-
azoles (5a–5c), and 4,40-(1,4-phenylene)bis(1,3-thiazole)s (6a–6f)
pendent experiments
(Fig. 1) at a concentration of 50 mM in 2% DMSO against 5-LOX
% inhibition
% scavenging
(isolated from recombinant plasmid) were evaluated in vitro by
measuring the formation of 5-hydroxy eicosatetraenoic acid
(5-HETE) from AA (Tables 1–5). Zileuton and MK-886 (a 5-LOX
activating protein – FLAP inhibitor which was withdrawn from
clinical trials due to high plasma absorption) (Fig. 2) were used
as positive controls while DMSO (2%, v/v) was used as a vehicle
control. 2%, v/v DMSO (vehicle control) did not show any
inhibition of 5-LOX enzyme activity.
S. No.
Compounds
R
of 5-LOX ꢂ SD
of DPPH ꢂ SD
1
2
3
4
5
2a
2b
2c
2d
2e
H
Cl
F
NO2
H,
10.2 ꢂ 4.5
19.1 ꢂ 6.3
19.7 ꢂ 5.4
8.28 ꢂ 1.8
14.0 ꢂ 8.1
10.9 ꢂ 2.4
11.4 ꢂ 1.0
13.8 ꢂ 3.3
7.38 ꢂ 4.5
9.5 ꢂ 5.3
o-OH
SAR studies. Amongst the 2-amino-4-aryl thiazole derivatives
(1a–1f) tested, compound 1d with a thiazole ring and an elec-
tron withdrawing group (uoro substitution) at the p-position
adjacent to the benzene ring is seen to be the most active with
an IC50 of ꢀ10 mM (Table 1). This activity is found to be closer to
that of the standard drug, zileuton (IC50 ꢀ 1 mM) and FLAP
inhibitor MK-886 (IC50 ꢀ 2 mM) compared to the other
compounds in the series.32 Interestingly, replacing uorine with
chlorine (1b), completely deactivates the compound, which is
a less electronegative atom than uorine. Replacing chlorine
with a methyl group (1c) increases the activity threefold and
with a nitro group (1e) vefold compared to 1b. When there is
no substituent or R ¼ H (1a), the compound is totally inactive
while the addition of a hydroxy group at the ortho position to it
(1f) increases activity 10–11 fold. Structure activity analysis of
compounds (1a–1e) indicates that as log P of the compound
increases, the 5-LOX inhibition decreases (correlation coeffi-
cient, r ¼ ꢁ0.6), indicating that hydrophilic substituents at the
para position enhance the activity. 2-Aryl indole (Table 2)
derivatives (2a–2e), irrespective of substitution at the R position
(halogen, nitro or hydroxyl group) exhibited poor activity. None
of the oxadiazolobenzothiophenes (Table 3) tested showed any
reasonable activity (<35%). 3g with methyl substitution at R2
and phenyl substitution at R3 exhibiting the best activity
(ꢀ32%). The activity was completely lost with hydrogen
substitution at R2 (3a > 3b > 3c). Potency decreased 5–6 fold in
3d and 3f compared to 3g due to the steric hindrance of the p-
methoxy phenyl group at the R3 position. Compound 4a of the
thiazolopyrazole acid derivatives, having a p-uoro phenyl ring
in the R2 position, exhibited good activity of ꢀ70% (Table 4).
Comparable activity was observed for compound 4c in the same
series when R1 attached to the nitrogen of the adjacent benzene
ring was substituted by an electron donating ethyl group.
Replacement of F with Cl reduced the activity by 20 percent
(ꢀ45%). Structure activity analysis of compounds (4a–4d) indi-
cates that as log P of the compound increases, the 5-LOX
Fig. 1 Chemical structures of 2-amino-4-aryl thiazole derivatives (1a–
1f), 2-aryl indole derivatives (2a–2e), oxadiazolobenzothiophene
derivatives (3a–3j), thiazolopyrazole acid derivatives (4a–4d), 1,4-
disubstituted-1,2,3-triazole derivatives (5a–5c), and 4,40-(1,4-phenyl-
ene)bis(1,3-thiazole) derivatives (6a–6f). R, R1, R2, R3 and R4 groups are
described in (Tables 1–6).
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Table 3 Effects of oxadiazolobenzothiophene derivatives at 50 mM concentration on % 5-LOX inhibition and antioxidant activity. Data are
expressed as means ꢂ S.D of single determinations obtained in three to four independent experiments
% inhibition
of 5-LOX ꢂ SD
% scavenging
of DPPH ꢂ SD
S. No.
Compounds
R1
R2
R3
R4
1
2
3
4
5
6
7
8
9
10
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
H
Br
Br
Br
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
OCH3
C6H5
C6H5
C6H5
4-OMeC6H5
4-ClC6H5
4-OMeC6H5
C6H5
4-OMeC6H5
4-ClC6H5
4-OMeC6H5
CH3
C2H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
25.2 ꢂ 1.4
19.4 ꢂ 1.4
16.5 ꢂ 0.9
5.8 ꢂ 0.4
6.0 ꢂ 2.5
3.6 ꢂ 0.3
4.7 ꢂ 2.3
5.2 ꢂ 0.3
5.2 ꢂ 1.2
3.8 ꢂ 1.0
2.6 ꢂ 0.1
3.7 ꢂ 1.9
3.2 ꢂ 1.6
3.6 ꢂ 0.7
15.2 ꢂ 1.8
4.5 ꢂ 1.4
32.0 ꢂ 0.9
13.3 ꢂ 0.9
12.3 ꢂ 1.4
10.4 ꢂ 0.4
3j
Table 4 Effects of thiazolopyrazole acid derivatives at 50 mM concentration on % 5-LOX inhibition and antioxidant activity. Data are expressed as
means ꢂ S.D of single determinations obtained in three to four independent experiments
% inhibition
of 5-LOX ꢂ SD
% scavenging
of DPPH ꢂ SD
S. No.
Compounds
R1
R2
1
2
3
4
4a
4b
4c
4d
CH3
CH3
CH2CH3
CH2CH3
4-F-C6H5
4-Cl-C6H5
H
69.9 ꢂ 3.8
45.2 ꢂ 3.8
65.7 ꢂ 0.1
41.8 ꢂ 4.8
8.2 ꢂ 5.7
6.1 ꢂ 0.3
14.1 ꢂ 8.5
12.1 ꢂ 12.1
C6H5
Table 5 Effects of 1,4-disubstituted-1,2,3-triazole derivatives at 50 mM
concentration on % 5-LOX inhibition and antioxidant activity. Data are
expressed as means ꢂ S.D of single determinations obtained in three
to four independent experiments
position and N-methylaniline is replaced with N-ethylaniline at
the NHR1R2 position (6e), compared to 6d and 6f. The activity is
reduced by half when nitro is replaced with chloro at the X
position and aniline is added at the NHR1R2 position (6b). So it
appears that an electron withdrawing group (such as NO2) is
essential for good activity.
% inhibition
of 5-LOX ꢂ SD
% scavenging
of DPPH ꢂ SD
S. No.
Compounds
R1
R2
The Vmax and Km values for the control are 0.99 ꢂ 0.01 nmol
minꢁ1 and 0.25 ꢂ 0.04 mM, respectively. In the presence of 10 and
50 mM of compound 1d, Vmax almost remained the same at 0.90 ꢂ
0.03 and 0.85 ꢂ 0.11 nmol minꢁ1 and the Km of the enzyme
increased to 0.38 ꢂ 0.013 and 2.03 ꢂ 0.12 mM, respectively.
Similarly, the Vmax and Km values for the control are 1.21 ꢂ 0.03
nmol minꢁ1 and 0.10 ꢂ 0.05 mM, respectively. In the presence of
10 and 50 mM of compound 4a, Vmax almost remained the same at
1.05 ꢂ 0.03 and 1.33 ꢂ 0.3 nmol minꢁ1 and the Km of the enzyme
increased to 0.40 ꢂ 0.09 and 3.44 ꢂ 0.35 mM, respectively.
1
2
3
5a
5b
5c
Br
Cl
F
H
H
H
25.4 ꢂ 0.9
24.1 ꢂ 0.9
48.9 ꢂ 1.3
12.0 ꢂ 0.001
11.5 ꢂ 4.8
13.3 ꢂ 2.9
In vitro evaluation of antioxidant activity
Fig. 2 Chemical structures of zileuton, MK-886 and ascorbic acid.
Studies have suggested that most LOX inhibitors are also anti-
oxidants or free radical scavengers.33 Compounds which inhibit
LOX and have the ability to reduce Fe3+ at the active site to the
catalytically inactive Fe2+ in the enzyme are closely related.34,35
LOXs contain a “non-heme” iron in the enzyme active site as
Fe2+ in the native form and Fe3+ in the activated state. Redox
inhibitors reduce Fe3+ to inactive Fe2+. Iron chelating agents
bind to the ferric iron and block the binding ability of AA
without changing the iron state.36
inhibition decreases (correlation coefficient, r ¼ ꢁ0.7). A 1,4-
disubstituted-1,2,3-triazole with a uoro group, 5c, exhibited
49% activity (Table 5). Replacement of F with Br or Cl reduced
the activity by half (ꢀ25%). Of the compounds in the bisthiazole
series (Table 6), the best compounds 6d and 6f share the same
activity of around 59% with a nitro group at the X position
irrespective of substitution at the NHR1R2 position. There is
a reduction in activity when the nitro is replaced with H at the X
The results show that all the compounds show poor antioxi-
dant activity as measured by DPPH assay. Ascorbic acid exhibited
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Table 6 Effects of 4,40-(1,4-phenylene)bis(1,3-thiazole) derivatives at 50 mM concentration on % 5-LOX inhibition and antioxidant activity. Data
are expressed as means ꢂ S.D of single determinations obtained in three to four independent experiments
% inhibition
of 5-LOX ꢂ SD
% scavenging
of DPPH ꢂ SD
S. No.
Compounds
NHR1R2
X
1
2
3
4
5
6
6a
6b
6c
6d
6e
6f
N,N-Dimethylamine
Aniline
n-Butylamine
N-Methylaniline
N-Ethylaniline
Pyrrolidine
Cl
Cl
Cl
NO2
H
35.4 ꢂ 2.6
29.1 ꢂ 1.7
42.6 ꢂ 1.3
58.6 ꢂ 1.7
52.0 ꢂ 4.8
58.6 ꢂ 5.3
2.8 ꢂ 1.0
2.2 ꢂ 0.5
2.8 ꢂ 0.5
3.6 ꢂ 1.4
2.5 ꢂ 1.8
2.8 ꢂ 0.2
NO2
89.8% activity while zileuton and MK-886 showed 45.6% and
35.5% radical scavenging activity, respectively.
It is observed that in compound 1d, the sulfur of the thiazole
ring and the nitrogen of the amino group attached to the thia-
zole ring both form hydrogen bonds with the backbone oxygen
of amino acid Ala606 in the active site of 5-LOX with a binding
score of ꢁ4.78 kcal molꢁ1 (Fig. 3). However, the second best
compound, 4a, with a better binding score of ꢁ6.92 kcal molꢁ1
exhibits one H-bond between the nitrogen of its pyrazole ring
Molecular docking studies
To support the in vitro anti-inammatory results exerted by 1d
and 4a, they are docked to the active site of 5-LOX (Fig. 3 & 4)
and compared with the results for zileuton (Fig. 5).
Fig. 3 Binding of compound 1d with 5-LOX. (a) Ligplot showing residues involved in the interaction between the amino acids of the enzyme with
compound 1d. (b) Docked pose of compound 1d in a stick model. Interactions are shown by dotted lines. Colours: carbon: grey, hydrogen: cyan,
nitrogen: blue, sulphur: yellow, fluorine: green and oxygen: red.
Fig. 4 Binding of compound 4a with 5-LOX. (a) Ligplot showing residues involved in the interaction between the amino acids of the enzyme with
4a. (b) Docked pose of 4a 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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Fig. 5 Binding of zileuton with 5-LOX. (a) Ligplot showing residues involved in the interaction between the amino acids of the enzyme with
zileuton. (b) Docked pose of zileuton in a stick model. Interactions are shown by dotted lines. Colours: carbon: grey, hydrogen: cyan, nitrogen:
blue, sulphur: yellow, fluorine: green and oxygen: red.
and the amino acid Glu612, and is found to be oriented towards
Nevertheless, zileuton is the only approved drug to emerge
the residues in the binding pocket (Fig. 4). In comparison, the against the 5-LOX enzyme.17 It is known to be a metal ion chelator
binding score of zileuton is ꢁ4.93 kcal molꢁ1 (Fig. 5). It is found and it also inhibits 5-LOX via the reduction of ferric iron (active
that the nitrogen in the amino group of the ligand forms state) to ferrous (inactive state) and is classed as a redox inhib-
a H-bond with the backbone oxygen of residue Asn613. Another itor.29 However, the transfer of an electron from zileuton to the
H-bond is seen between the oxygen atom of the ligand hydroxyl ferric iron of the enzyme does not occur in isolation, and electron
group and the sidechain of amino acid Asp170. One more H-bond transfer in its redox cycle is carried out through the N-hydroxy-
is formed between the sulfur of the thiophene ring of zileuton urea group present, which ultimately generates unstable intra-
and the side chain of amino acid Ser171. Also of importance is the cellular lipid alkoxide and thiol radicals.42
metal contact between the nitrogen of the N-hydroxy urea group
Here from experimental data, it is visible that compounds 1d
present in zileuton and iron in the enzyme (Fig. 5) which is (ꢀ85%) and 4a (ꢀ70%) show very good 5-LOX inhibition while
responsible for electron transfer and thus the inhibition of the they exhibit poor DPPH radical scavenging activities (20% and
enzyme. The molecular docking results indicate that the mech- 8% respectively). At the same time, docking studies indicate that
anism of action of 1d and 4a is not similar to that of zileuton.
they bind and interact with active site amino acids, thus blocking
the substrate binding site. Poor antioxidant activity indicates that
these compounds are not acting by reducing ferric iron. In
contrast, zileuton has marvellous 5-LOX inhibition (ꢀ97%) and
also possesses radical scavenging activity (ꢀ45%) which indicates
that it acts by reducing the ferric iron and thus it is classied as
a redox inhibitor. In addition, this study is also supported by
docking studies which highlight that the N-hydroxy urea group is
involved in the reduction of ferric iron.
Simultaneously, Lineweaver–Burke plots for both the
compounds show that Vmax remains almost the same and Km
value increases when the concentrations of the compounds are
increased, suggesting them to be competitive inhibitors towards
the 5-LOX activity of the protein.
Insight into the potential mechanism of action
5-LOX inhibitors can be generally classied into three main
categories: (1) redox (reductive or electron transfer type), (2) non
redox (competitive or mixed inhibitors) and (3) iron chelators
(iron ligands).37,38 Several inhibitors have been reported in these
classes such as zileuton, nordihydroguaiaretic acid (NDGA), and
atreleuton as chelative and reductive; and setileuton (MK-0633)
and PF-4191834 as competitive and non reductive inhibitors
(Fig. 6).39–41
From the experiments, binding energies in terms of Gibbs
free energy for compounds 1d and 4a are calculated, where the
value for the control is 3419.0 ꢂ 0.03 J molꢁ1 which decreased to
2395.8 ꢂ 0.02 J molꢁ1 in the presence of 10 mM and further to
a negative value of ꢁ1754.12 ꢂ 0.65 J molꢁ1 in the presence of
50 mM of inhibitor 1d. For compound 4a the value for the
control is 5723.2 ꢂ 0.04 J molꢁ1 which decreased to 2254.6 ꢂ
0.06 J molꢁ1 in the presence of 10 mM and further to a negative
value of ꢁ3047.4 ꢂ 0.32 J molꢁ1 in the presence of 50 mM of
inhibitor 4a. The decreased and negative values of Gibbs free
Fig. 6 Classification of 5-LOX inhibitors based on their mechanism.
Commercial drugs and those in clinical trials are marked in black and
our best inhibitors marked in red.
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energy indicate that the compound at higher concentrations spectrophotometer. The solvents used were DMSO-d6, CDCl3
has better binding affinity and stability with the protein 5-LOX. and CD3OD, and tetramethylsilane (TMS) was used as an
internal standard. Mass spectra were recorded using an LC-MS
instrument. All new compounds characterized gave satisfactory
spectral data.
Conclusions
The present study shows that among six different scaffolds
tested,
a 2-amino-4-aryl thiazole derivative with uorine
Chemistry
General procedure for the synthesis of 2-amino-4-aryl thia-
substitution (1d) possesses the highest anti-inammatory
activity (IC50 ¼ 10 mM) followed by a thiazolopyrazole acid
derivative (4a with IC50 ¼ 40 mM). Both the compounds have
electron withdrawing p-uoro substitution on the phenyl ring.
Moreover, they do not posses hydroxyl radical scavenging
activity and hence exhibit poor antioxidant activity. So, their
possible mechanism of action against this enzyme may be
through competitive inhibition and non redox type (unlike
zileuton). Indeed, the hepatotoxicity of zileuton is perhaps
related to its mechanism of action, particularly to the N-
hydroxyurea moiety which is a known toxicant, and it acts via
the free radical-mediated lipid peroxidation of cell
membranes.43 It takes part in electron transfer in the redox cycle
resulting in the elevation of serum liver enzyme.44 In addition,
the thiophene moiety which forms chemically reactive metab-
olites in liver is an alternative reason for its hepatotoxicity.45,46
So, efforts are made to focus on the development of non redox
type inhibitors for 5-LOX. Here, in the present work, we have
identied a scaffold exhibiting a non redox type of inhibition
which could be a signicant contribution to the development of
5-LOX inhibitors.
zoles. The 2-amino-4-aryl thiazole derivatives (1a–1f) were
synthesized as described briey here (Scheme 1).47 Ethanolic
solution of a-bromoarylethanones (1 mmol) and thiourea
(1 mmol) were taken in boiling tubes and ultrasonicated at
45 ꢃC in an ultrasonic bath. The reaction was monitored by TLC
every 5 min. It was found that heterocyclisation was completed
within 20–35 min. The reaction mixture was allowed to cool,
and solvent was evaporated to obtain the products. Recrystal-
lization of the crude products was carried out using ethanol and
petroleum ether to obtain pure 2-amino-4-substituted phenyl-
1,3-thiazoles.
Scheme 1 General synthesis of 2-amino-4-aryl thiazole derivatives
(1a–1f).
So, it can be concluded that the poor antioxidant activity but
good anti-inammatory activity of compounds 1d and 4a indi-
cates that their possible mechanism of action is non redox type.
Constant Vmax and increasing Km with an increase in inhibitor
concentration indicates that they probably act by blocking the
substrate binding site thereby possibly acting as a competitive
inhibitor. In contrast, zileuton acts by non competitive (redox
and iron chelation) inhibition. The docking studies also
support these experimental observations. By further optimizing
the structure and SAR of compound 1d, it may be possible to
achieve activity comparable or better than that of the
commercial drug zileuton (which exhibits an IC50 of ꢀ 1 mM).
Also, based on our studies on the other four scaffolds which
exhibit poor 5-LOX inhibition, we wish to conclude that further
studies and SAR in this direction may not bear useful leads.
General procedure for the synthesis of 2-aryl indoles. The
procedure for the synthesis of 2-aryl indoles (2a–2e) is briey
described here (Scheme 2).48 Equimolar amounts of acetophe-
none (0.5 mmol) and phenylhydrazine (0.5 mmol) were dis-
solved in methanol. The mixture was then warmed in a water
bath and glacial acetic acid was added until the solution
became clear. Clayzic catalyst (200 mg) was added to the above
reaction mixture, which was reuxed. The reaction was moni-
tored by TLC using petroleum ether and ethyl acetate (4 : 1).
Upon completion of the reaction (in 20 min), it was cooled to
room temperature, ltered and poured over crushed ice. The
product formed was then extracted with dichloromethane,
rotary evaporated and further puried by column chromatog-
raphy using petroleum ether and ethyl acetate (90 : 10).
Experimental section
Dimethyl sulfoxide (DMSO) was purchased from Merck, USA.
Luria–Bertani (LB) medium was purchased from BD, Biosci-
ences USA. AA, Zileuton and MK-886 were obtained from
Cayman Chemical (Inalco, Milan, Italy). All other chemicals
reported here were purchased from Sigma, St. Louis, USA.
Reagents were of analytical grade and were purchased from
HiMedia Biosciences, Mumbai, India. Fourier transform
infrared (FT-IR) spectra were recorded using a Shimadzu IR
Affinity-1 CE model with resolution 4 cmꢁ1 and a Perkin-Elmer
FT-IR (Spectrum 1000). 1H and 13C nuclear magnetic resonance
(NMR) spectra were recorded using a Bruker AMX (400 MHz)
Scheme 2 General synthesis of 2-aryl indole derivatives (2a–2e).
General procedure for the synthesis of 1,4-disubstituted-
1,2,3-triazoles. Various 1,4-disubstituted-1,2,3-triazoles (5a–5c)
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were synthesized by click chemistry reactions involving Huisgen THF : water (2 ml : 1 ml), sodium hydroxide (0.370 mmol) was
[1,3] dipolar cycloaddition between azides and acetylenes to added and stirred at room temperature overnight. The reaction
form triazoles with some modications to the procedure mixture aer reaction completion was neutralized with citric
(Scheme 3).49
acid and extracted with ethyl acetate (10 ml ꢄ 2). This ethyl
acetate layer was washed with water (10 ml) and brine (10 ml).
Then, the organic layer was separated, dried over sodium
sulfate and concentrated to get the compounds.
Scheme
3 General synthesis of 1,4-disubstituted-1,2,3-triazole
derivatives (5a–5c).
General procedure for the synthesis of oxadiazolobenzo
thiophenes. The procedure for the synthesis of 2-(5-substituted-
[1,2,4]-oxadiazol-5-yl)-benzo[b]thiophen-3-yl bis sulfonamide
derivatives (3a–3j) is briey described here (Scheme 4). 3-
Amino-substituted benzo[b]thiophene-2-carboxylic acid methyl
esters were prepared from the reaction between o-halonitriles
(1.0 eq.) and methyl 2-mercaptoacetate (1.0 eq.) in the presence
of potassium t-butoxide (1.1 eq.) as a base in N,N-dime-
Scheme
(4a–4d).
5 General synthesis of thiazolopyrazole acid derivatives
General procedure for the synthesis of 4,40-(1,4-phenylene)
bis(1,3-thiazole)s. The 4,40-(1,4-phenylene)bis(1,3-thiazole)
derivatives (6a–6f) were synthesized as described briey here
(Scheme 6) as per our previously reported method.51 The bis-
thiazoles were obtained by treating substituted N,N0-bis(amino-
carbonothioyl)terephthalamides, synthesized by the reaction of
terephthaloyl chloride and potassium thiocyanate, with the
appropriate N,N0-diaryl/alkyl amines and further heterocyclisa-
tion with the corresponding phenacyl bromides. Thus, the 4,40-
(1,4-phenylene)bis(1,3-thiazole)s52 were synthesized by the
ꢃ
thylformamide at 80 C for 3 h. The (E)-N0-hydroxy-benzimida-
mides were prepared from the reaction of the nitriles and
hydroxylamine in ethanol at reux for 2 h. The rst reaction step
product (1.0 eq.) was treated with different (E)-N0-hydroxy-ben-
zimidamide (1.1 eq.) in ethanol with sodiumethoxide as base at
80 ꢃC to get substituted-2-(3-substituted-1,2,4-oxadiazol-5-yl)
benzo[b]thiophen-3-amines. These compounds were further
treated with sulfonyl chloride (2.5 eq.) in dichloromethane and
triethylamine as a base to get the 2-(5-substituted-[1,2,4]-oxa-
diazol-5-yl)-benzo[b]thiophen-3-yl
bis
sulfonamide
title
condensation
of
N,N0-bis(amino-carbonothioyl)tereph-
compounds.
thalamides with the appropriate phenacylbromides in DMF.
Scheme 4 General synthesis of oxadiazolobenzothiophene deriva-
tives (3a–3j). These are novel compounds reported for the first time.
Methodology and the spectral data are given in the ESI.†
Scheme 6 General synthesis of 4,40-(1,4-phenylene)bis(1,3-thiazole)
derivatives (6a–6f).
Biology
Expression and purication of human 5-LOX from Escher-
ichia coli (E. coli). Recombinant human 5-LOX in a pT3 plasmid
General procedure for the synthesis of thiazolopyrazole
acids. The procedure for the synthesis of pyrazol-3-yl thiazole 4-
carboxylic acid derivatives (4a–4d) is described as per our
previously reported methodology50 with some modications
(Scheme 5). Pyrazole carboxylates (0.246 mmol), (synthesized as
per the previously reported method) were dissolved in
˚
(a kind gi from Prof. Olof Radmark, Karolinska Institute,
Stockholm, Sweden) was expressed in E. coli BL21 bacteria.53
A
ꢃ
bacterial culture was grown at 37 C overnight with shaking in
LB medium (150 mg mlꢁ1 ampicillin) to an OD600 above 0.5. The
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expression of 5-LOX was induced with 0.5 mM of isopropyl-D- absorbance was measured at 517 nm using a Multimode Plate
thiogalactopyranoside and the culture was cooled to 18 ꢃC with reader (Enspire Perkin Elmer, version 4.10.3005.1440). Ascorbic
shaking for overnight growth. Cells were then pelleted out by acid was used as a reference compound. DPPH scavenging
centrifugation at 5000 rpm and 4 ꢃC for 15 minutes. Cell pellets percentage was calculated using the formula:
were resuspended in a chilled buffer at pH 8.0, containing 50
ðA0 ꢁ AsÞ
mM triethanolamine HCl, 5 mM EDTA, 60 mg mlꢁ1 trypsin
inhibitor, 1 mM PMSF and 500 mg mlꢁ1 lysozyme, with 30
minutes incubation in ice and then sonicated for 42 seconds.
The cell lysate was centrifuged at 19 000 ꢄ g and 4 ꢃC for 15
minutes. Ammonium sulfate was added to the supernatant
(50% w/v) which was stirred in ice for 45 minutes, centrifuged at
16 000 ꢄ g and 4 ꢃC for 30 minutes and aliquoted. Semi-puried
5-LOX was immediately used for activity assays.
% inhibition ¼
ꢄ 100
A0
where A0 ¼ absorbance of the control (blank, without sample)
and As ¼ absorbance of the test samples. All tests were per-
formed in triplicate.
Molecular modeling studies. Docking experiments were per-
formed using Molecular Operating Environment (MOE; version
2011.10, Chemical Computing Group, Suite 910, Canada) to
determine the binding free energies of ligands with different
poses. The X-ray crystal structure of 5-LOX was obtained from the
Brookhaven Protein Data Bank (http://www.rcsb.org/pdb). The
protein target (PDB Code 3O8Y) was selected from the PDB
database and all the heteroatoms (i.e., nonreceptor atoms such as
water, ions, etc.) were removed. Polar hydrogens were added and
Kollmann charges were assigned using forceeld AMBER99; also
the energy of the protein was minimized by using AMBER99 force
eld. The structures of the ligands were built in MOE and their
energies were minimized using MMFF94x force eld. The ligands
were then docked into the protein for the best docking pose and
its binding free energy. LondondG was used as the rst and GBVI/
WSAdG as the second rescoring methodology. The binding
energies of the top 100 conformations were computed. The more
negative the binding score, the more favorable the binding of the
ligand to the protein was.
5-LOX enzyme activity assay. Aliquots of semi-puried 5-LOX
prepared as described were added to a mixture containing 25
mM of HEPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-
sulfonic acid (pH 7.3), 0.4 M of ethylenediaminetetraacetic acid
ꢃ
(EDTA), 10 mM of CaCl2, and 4 mM of ATP at 4 C. The total
activity of the enzyme was measured by lipoxygenase reaction
using UV absorbance at a lmax of 236 nm with 100 ml of nal
reaction mixture containing 96.75 ml of assay mixture, 2 ml of
DMSO (2% in nal mixture), and 1.25 ml of the substrate (30 mM
of AA). The test compounds dissolved in DMSO were added to
the reaction mixture to a nal concentration of 50 mM and the 5-
LOX reaction was initiated by the addition of the substrate AA
(30 mM). The activity was determined by measuring the
conversion of AA to the product 5-HETE in terms of OD at l236
.
Percentage inhibition was calculated by comparing 5-LOX
activity in the presence and absence of the inhibitor.
Commercial drug zileuton and FLAP inhibitor MK-886 were
used as positive controls. Each assay was repeated thrice. We
compared the IC50 values of our two highly potent 5-LOX
inhibitors to the IC50 of zileuton experimentally and with the
literature. Zileuton displayed an IC50 value of ꢀ1 mM, which is
in good agreement with the literature value of 0.5 mM.54,55
Assay for mechanism of action. The 5-LOX activity of the
protein in terms of 5-HETE produced was measured for between
1 and 30 mM of AA in the presence of 10 and 50 mM of compounds
1d or 4a in the same way as the activity assay with UV absorbance
at a lmax of 236 nm. The mechanism of inhibition was deter-
mined from a Lineweaver–Burke plot. Further, the binding
energy of the substrate to the protein in terms of Gibbs free
energy was estimated from the equilibrium constants for the
control and in the presence of inhibitor using the formula:56,57
Acknowledgements
The authors acknowledge the gi of a pT3 5-LOX plasmid from
Prof. Olof Radmark, Karolinska Institute, Stockholm, Sweden.
Shweta Sinha is grateful to the DST-WOS-A scheme for the
nancial support and fellowship.
˚
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