Chalcone-Thiazole Hybrids: Rational Design, Synthesis, and Lead Identification against 5-Lipoxygenase

Article abstract of DOI:10.1021/acsmedchemlett.9b00193

A hybrid pharmacophore approach is used to design and synthesize novel chalcone-thiazole hybrid molecules. Herein, thiazole has been hybridized with chalcone to obtain a new class of 5-LOX inhibitors. In vitro biological evaluation showed that most of the compounds were better 5-LOX inhibitors than the positive control, Zileuton (IC₅₀ = 1.05 ± 0.03 μM). The best compounds in the series, namely, 4k, 4n, and 4v (4k: IC₅₀ = 0.07 ± 0.02 μM, 4n: IC₅₀ = 0.08 ± 0.05 μM, 4v: 0.12 ± 0.04 μM) are found to be 10 times more active than previously reported 2-amino thiazole (2m: IC₅₀ = 0.9 ± 0.1 μM) by us. Further, 4k has redox (noncompetitive) while 4n and 4v act through a competitive inhibition mechanism. SAR indicated that the presence of methoxy/methyl either in the vicinity of chalcone or both thiazole and chalcone contributed to the synergistic inhibitory effect.

Full text of DOI:10.1021/acsmedchemlett.9b00193

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Letter
Chalcone-Thiazole Hybrids: Rational Design, Synthesis
and Lead Identification against 5-Lipoxygenase
Shweta Sinha, S.L. Manju, and Mukesh Doble
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00193 • Publication Date (Web): 09 Sep 2019
Downloaded from pubs.acs.org on September 9, 2019
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ACS Medicinal Chemistry Letters
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Chalcone-Thiazole Hybrids: Rational Design, Synthesis and Lead
Identification against 5-Lipoxygenase
Shweta Sinha§#, S. L Manju#*, Mukesh Doble§*
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§Bioengineering and Drug Design Lab, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian
Institute of Technology, Madras, Tamil Nadu, 600036, India.
#Department of Chemistry, School of Advanced Science, Vellore Institute of Technology, Vellore, Tamil Nadu, 632014,
India.
*Corresponding authors Email: mukeshd@iitm.ac.in, slmanju@vit.ac.in
KEYWORDS: Chalcone, thiazole, 5-LOX, pharmacophore, design, hybrid, synthesis, pseudoperoxidase
ABSTRACT: A hybrid pharmacophore approach is used to design and synthesize novel chalcone-thiazole hybrid molecules.
Herein, thiazole has been hybridized with chalcone to obtain a new class of 5-LOX inhibitors. In vitro biological evaluation showed
that most of the compounds were better 5-LOX inhibitor than the positive control, Zileuton (IC50 = 1.05 ± 0.03 µM). The best
compounds in the series namely, 4k, 4n and 4v (4k: IC50 = 0.07 ± 0.02 µM, 4n: IC50 = 0.08 ± 0.05 µM, 4v: 0.12 ± 0.04 µM) are
found to be 10 times more active than previously reported 2-amino thiazole (2m: IC50 = 0.9 ± 0.1 µM) by us. Further, 4k has redox
(non-competitive) while 4n and 4v act through competitive inhibition mechanism. SAR indicated that the presence of
methoxy/methyl either in the vicinity of chalcone or both thiazole and chalcone contributed to the synergistic inhibitory effect.
5-Lipoxygenase (5-LOX) is an important enzyme which
catalyzes arachidonic acid (AA) for the production of several
leukotrienes (LTs) which are well-known mediators of
inflammation related disorders such as rheumatoid arthritis,
allergic rhinitis, atherosclerosis and cardiovascular diseases
among which asthma is the major pathophysiological
implication associated with it.1,2 5-LOX oxygenates essential
polyunsaturated fatty acid (AA) to 5(S)-hydroperoxy-6-trans-
8,11,14-cis-eicosatetraenoic acid (5-HpETE) and further
dehydrates it to the unstable epoxide leukotriene A4 (LTA4).
So far, Zileuton is the only marketed 5-LOX inhibitor against
asthma.2 But it has side-effects of hepatotoxicity and poor
pharmacokinetics. Its hepatotoxicity is associated with its
mechanism involving redox process in which N-hydroxyurea
is involved in electron transfer through free radical-mediated
lipid peroxidation of cell membranes and thiophene forms
chemically reactive metabolites resulting in liver serum
enzyme elevation and toxicity.2 So, there is a need to develop
inhibitors against 5-LOX.
identified as selective 5-LOX whereas aminothiazole pirinixic
acids10 as dual 5‑LOX/mPGES1 inhibitors.
Chalcone (α,β‐unsaturated ketone, collectively defined as an
aromatic ketone and enone) based analogues are found in
many natural compounds including turmeric in the form of
curcumin, liquorice as isoliquiritigenin and synthetic drugs
such as homobutein.11 Chalcones are known to exhibit
biological properties, such as antibacterial, anti-inflammatory,
antioxidant, anticancer, antimalarial and as carbonic anhydrase
inhibitor.11,12 Diarylsulfonylurea‐chalcones12, and di-o-
prenylated chalcones13 have been reported as 5-LOX whereas
chalcone-triazole
Phenylsulphonyl
hybrids14
urenyl
as
15-LOX
inhibitors.
and 3,4-
chalcones15
dihydroxychalcones16 act as dual 5-LOX/COX inhibitors.
Combining two bioactive pharmacophores to design a hybrid
molecule has considerably attracted interest of medicinal
chemists. This molecular hybridization is a useful tool to
generate lead molecules with synergetic and superior
biological activities.17 Considering the anti-inflammatory
potencies of thiazole and chalcone, it is envisaged that
integrating both these moieties in one molecular platform
could possibly produce compounds with synergistic
properties. Therefore, in the present study, as an extension of
our earlier work in the field of development of LT inhibitors,
an attempt has been made to design and synthesize hitherto
novel chalcone-thiazole structural hybrids along with
elaboration of their mechanism to understand their redox and
non-redox behavior to overcome pharmacokinetic problems as
potential 5-LOX inhibitors.
Thiazoles are active constituents of various naturally occurring
biologically significant compounds such as thiamine,
mycothiazole as well as synthetic drugs which act as
antimicrobial, anti-inflammatory, anticancer, antiviral, and
antitubercular.3,4. Thiazol-4(5H)-one derivative, namely
darbufelone and CI-987 are reported as dual COX/LOX
inhibitors.5 Recently, thiazole bearing scaffolds, namely 2-
amino-4-aryl thiazole-5-phenylmethanones6,7, N-aryl thiazole-
2-amines8 and 5-benzylidene-2-phenylthiazolinones9 are
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Rational Design and Pharmacophore Elucidation. The map exactly with the four-point common features of Ph model
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molecular hybridization approach on the concept of combining
two bioactive pharmacophores to achieve superior biological
activities forms the basis of the designed prototype (A) in this
study (Figure 1). Optimization for hybrid molecules (A)
containing both chalcone and thiazole scaffolds have been
pursued via pharmacophore elucidation towards 5-LOX
inhibition. Pharmacophore is a group of essential features
namely, H-bond acceptor, donor, hydrophobic and aromatic
center for a particular biological activity required for enzyme-
ligand interaction.18 To elucidate structure activity relationship
(SAR) of the designed structural hybrid (A), a pharmacophore
model (Ph model) was generated from the set of known and
reported inhibitors of 5-LOX (Figure 2). It included a known
drug: Zileuton, clinical trials inhibitors: Atreleuton,
Siteleuton19 and reported inhibitors: di-o-prenylated and
diarylsulfonylurea chalcones.12,13
such as 4k and 4n (Figure 3c, d). It can be noticed that the
double bond of chalcone is one of the important reasons for
the generation of hydrophobic centroids, contributing
synergistic anti-inflammatory effect. Thus, pharmacophore
elucidation gave a preliminary indication that this designed
hybrid (A) could be a good lead against 5-LOX and hence
these were synthesized and biologically tested.
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Figure 3: (a) Pharmacophoric model (Ph model) generated from
known 5-LOX inhibitors in software MOE 2016.0801; (b) Ph
model with geometric distances (A0) in 3D spatial relationship;
Pharmacophore mapping of (c) 4k and (d) 4n with Ph model.
Pharmacophoric features: Hydrophobic (Hyd - green), H-bond
acceptor (Acc2 - orange) and (blue in (Figure 3a)). Inhibitors
superimposed in (Figure 3a) are: Zileuton- magenta, Atreleuton-
navy blue, Siteleuton- brown, di-o-prenylated- pink and
diarylsulfonylurea chalcone- black.
Figure 1: Design of chalcone-thiazole hybrids via molecular
hybridization.
The synthetic pathways for the novel chalcone-thiazoles are
illustrated in Scheme 1. The key intermediates (2a-d) and (3a-
d) are prepared as per previously reported procedures with
some modifications.20,21 Briefly, benzoyl isothiocyanates (1a-
d) in the 1st step are synthesized by treating substituted
benzoyl chloride with ammonium thiocyanate-acetonitrile.
The product (1) in acetonitrile (ACN) layer in the 2nd step is
o
treated with required amount of ammonium hydroxide at 0 C
to yield various N-carbamothioyl substituted benzamides (2a-
d). In the 3rd step, 3-chloropentane-2,4-dione is added to the
2nd step intermediate, N-carbamothioyl substituted benzamide
(2), followed by reflux to afford the desired intermediates, N-
(5-acetyl-4-methylthiazol-2-yl) substituted benzamides (3a-d)
via Hantzsch thiazole synthesis. The final step for the
synthesis of the target chalcone-thiazole hybrids (4a-w), is
achieved using base catalyzed Claisen–Schmidt condensation
reaction between benzamide thiazoyl ketones (3a-d) and
various substituted aromatic/heteroaromatic aldehydes in
ethanol, and 2.5 N NaOH.14 All the structures were confirmed
using 1H NMR, 13C NMR and HRMS (Supporting
Information).
Figure 2: Structures of reported 5-LOX inhibitors.
A Ph model obtained gave a four-point pharmacophoric
feature consisting of three hydrophobic centroids and one H-
bond acceptors (Figure 3a, b). All the in-house designed
compounds are used as query molecules and subjected to
conformational search to create a library of database. A
pharmacophore search is performed for the in-house database
against Ph model to examine mapping of the designed
molecules to get the hit molecules. Surprisingly, all the 23
compounds designed in the series hit the query and found to
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All the compounds (4a-w) at a concentration of 10 µM are Methylenedioxy group is known to be widely found in natural
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screened for 5-LOX inhibition activity in vitro with Zileuton
as a reference drug in cell free system using human
recombinant 5-LOX enzyme (Table 1,2). The inhibition is
products and drugs. Addition of methylenedioxy bromophenyl
group at R2 yielded 4v (Table 2) which resulted in
significantly promising inhibitory activity (IC50 of 0.12 ± 0.04
µM). But replacement of -H at R1 with fluoro, 4w (50.6 ± 1.4
%) is not tolerated and potency reduced to half. Other
heteroaromatic groups such as furan, thiophene substitutions at
R2 resulted in moderate to good inhibition (60 to 80 %).
measured by determining the conversion of substrate AA to
22
product, 5-HPETE at λ236
.
2% DMSO was taken as negative
control which did not show any inhibition while standard drug,
Zileuton showed an IC50 of 1.05 ± 0.03 µM as expected
according to the literature37 (Table 3). Most of the compounds
exhibited good inhibition. The best active compounds were
tested further at different concentrations (below 10 µM)
against 5-LOX keeping substrate concentration constant to
find out their IC50 (using GraphPad Prism, 5.01). It is
heartening to note that many compounds exhibited better
potency than the reference drug, with IC50s ranging from 0.05
to 0.5 µM (4k, 4n and 4v, being the most potent in the series)
(Table 3).
R1
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O
S
O
step 1
step 2
S
C
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O
ACN
N
N
NH2
NH4OH
NH4SCN
H
65 O
C
Cl
H, OCH3, F, NO2
0o C to room temp.
R1
R1
R1
(1a-d)
=
(2a-d)
O
S
step 3
N
O
O
O
ACN
N
NH2
S
H
N
O
H
In Vitro 5-LOX Inhibition and SAR. To explore the influence
of hydrophobic centroids of chalcones with H-bond acceptor
property of the substituted thiazoles on 5-LOX inhibition, a
series of hybrids (A), (4a-w) have been synthesized.
Substitutions on both thiazole (at R1) and chalcone (at R2, R3,
R4) pharmacophores of prototype (A) are done to analyse their
effect on the inhibition and develop a tentative SAR profile.
The basic chalcone-thiazole core, 4a having no substitutions
exhibited moderate inhibition, 73.5 ± 2.8 % at 10 µM. It is
interesting to note that the introduction of highly
electronegative electron withdrawing halogen, fluoro at para
position (R3) in phenyl ring attached to chalcone, 4b enhanced
the inhibition to 98.0 ± .7 % (Table 1) with IC50 of 0.14 ± 0.06
µM (Table 3). The activities for other para halogen substituted
compounds, 4c (63.7 ± 2.8 %) and 4d (52.3 ± 2.1 %) are found
in the decreasing order of electronegativity of halogens
indicative 4b > 4c > 4d (p-F > p-Cl > p-Br). Again a slight
decrease in the potency is observed when p-Br is replaced with
m-Br, 4e (42.5 ± 3.6 %) as compared to 4d. Insertion of
electron donating substituent, methoxy at R1 in the phenyl ring
of benzamido group attached to thiazole, 4f (IC50 = 0.56 ± 0.15
µM, 88.2 ± 7.8 %) resulted in a fold decrease in the efficiency
with respect to 4b (IC50 = 0.14 ± 0.06 µM). However, even
addition of electron withdrawing group, -NO2 at R1, 4g (48.8 ±
0.7 %) could not improve the potency, instead reduced it by
1.3 fold, as compared to 4c. Surprisingly, replacing bromo by
another electron withdrawing group, -NO2, at the meta
position, 4h (IC50 = 0.24 ± 0.07 µM, 98.0 ± 2.1 %) increased
the inhibitory activity to more than double when compared to
4e (42.5 ± 3.6 %). The inhibition activity seems to increase
with increasing number of electron donating methoxy
substituent (R2, R3, R4) in the phenyl ring adjacent to the
vicinity of chalcone, 4i < 4j < 4k which might be due to
increase in electron density on the substituted phenyl.
Trimethoxy substituted compound, 4k, is a highly active
inhibitor (IC50 of 0.07 ± 0.02 µM). Existence of electron
donating substituents, -OCH3 (R1) and –CH3 (R3) in the
vicinity of both thiazole and chalcone phenyl rings
respectively is well tolerated in 4n which contributed to a
synergistic effect exhibiting an IC50 of 0.08 ± 0.05 µM. A
marginal decrease in potency is observed with the replacement
of -OCH3 by an electron withdrawing fluoro (R1), 4m (0.54 ±
0.10 µM).
Cl
Reflux
R1
R1
(2a-d)
(3a-d)
EtOH
NaOH, 2.5 N
step 4
R4
R3
CHO
HO
S
R2-C
R2
O
O
R1
H
H
S
R1
N
N
R2
R2
N
N
O
O
R3
R4
(4
a-
q)
(4r-w)
(4r-w)
(4a-q)
R2
H
H
H
H
Br
H
H
R1
R3
H
F
Cl
Br
H
F
Cl
H
R4
H
H
H
H
H
H
H
H
H
H
R1
H
R2
4a
4b
4c
4d
4e
H
H
H
H
H
O
4r
O
4s
4t
F
H
F
4f -OCH3
4g NO2
S
S
4h
4i
4j
4k
4l
4m
H
H
H
H
H
F
-NO2
H
-OCH3 -OCH3
-OCH3
-OCH3 -OCH3 -OCH3
4u
4v
4w
H
H
H
H
H
-CH3
-CH3
-CH3
-CH3
H
H
H
H
H
O
O
H
F
4n -OCH3
4o NO2
Br
Br
CH3
N
O
O
4p
H
CH3
CH3
N
4q
F
H
H
CH3
Scheme 1: Synthesis of chalcone-thiazole hybrid (4a-w)
derivatives.
5-LOX Kinetic studies. The mode of action of the best active
compounds (4k, 4n and 4v) is estimated by measuring the
initial rate of formation of product for different substrate (AA)
concentrations with three concentrations of these inhibitors (0,
2 and 5 µM).23 Lineweaver-Burke (L-B) plot indicates that the
most active analogue, 4k, acts as a non-competitive inhibitor
against the enzyme (Figure 4a) with constant Km (1.9 ± 0.4,
1.8 ± 0.4, 2.1 ± 0.4 µM) and decreasing Vmax (0.38 ± 0.02,
0.29 ± 0.02, 0.25 ± 0.01 nmol/min) with increase in inhibitor
concentration (0, 2 and 5 µM respectively). So, it can be
concluded that, 4k can bind either enzyme or enzyme-
substrate complex with equal affinity. L-B plots for the other
two compounds, 4n and 4v, showed competitive inhibition
(Figure 4b, c) namely at these three different concentration,
the lines intersect the y-axis indicating constant Vmax (4n: 0.33
± 0.01, 0.32 ± 0.01, 0.31 ± 0.01; 4v: 0.45 ± 0.01, 0.43 ± 0.02,
It was encouraging to substitute targeted hybrid (A) with
hetero-aromatic groups in the vicinity of chalcone.
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0.42 ± 0.02 nmol/min) while Km increases for both compounds
(4n: 0.35 ± 0.10, 0.43 ± 0.10, 0.53 ± 0.13; 4v: 0.31 ± 0.08,
1.41 ± 0.33, 2.91 ± 0.55 µM with increasing concentrations 0,
2 and 5 µM respectively). It suggested that both these
inhibitors probably bind to the enzyme’s active site and thus
hinder the substrate binding site.
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Table 1. 5-LOX inhibition and DPPH radical scavenging activities of N-(5-(3-(substituted phenyl)acryloyl)-4-methylthiazol-2-yl)
benzamide derivatives (4a-q) at 10 µM. Data is expressed as means ± SD from three independent experiments.
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O
R2
R3
S
HN
O
R1
N
R4
Compd
code
% DPPH radical
scavenging ± SD
% 5-LOX inhibition
± SD
R1
R2
R3
R4
H
H
H
H
H
H
H
H
H
H
4a
4b
4c
H
H
H
H
73.5 ± 2.8
98.0 ± .7
2.5 ± 1.5
3.0 ± 1.7
H
F
Cl
Br
H
H
H
63.7 ± 2.8
52.3 ± 2.1
8.8 ± 1.2
2.4 ± 1.2
4d
4e
H
H
H
Br
42.5 ± 3.6
88.2 ± 7.8
5.7 ± 1.1
2.1 ± 1.6
4f
4g
4h
4i
-OCH3
NO2
H
H
F
H
Cl
H
48.8 ± 0.7
2.4 ± 1.9
-NO2
H
98.0 ± 2.1
58.8 ± 2.1
8.7 ± 3.5
7.9 ± 1.0
6.4 ± 1.8
5.7 ± 2.9
H
-OCH3
-OCH3
-OCH3
-CH3
4j
H
-OCH3
-OCH3
H
70.2 ± 8.5
4k
4l
H
-OCH3
~ 100 ± 1.4
H
H
H
H
75.1 ± 2.1
91.5 ± .7
3.0 ± 0.2
15.0 ± 2.6
5.5 ± 0.2
4m
4n
F
H
-CH3
-OCH3
H
-CH3
~ 100 ± 2.8
CH3
N
4p
H
F
H
H
H
H
CH3
88.2 ± 3.6
70.2 ± 2.8
~ 0 ± 0.9
3.9 ± 0.8
CH3
N
4q
CH3
Zileuton
86.6 ± 2.8
10.3 ± 0.5
60.5 ± 2.3
-
-
-
-
-
-
-
-
Ascorbic
acid
-
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Table 2. 5-LOX inhibition and DPPH radical scavenging
show significant fall (Table 3) in the absorbance and the lines
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2
3
4
5
6
activities of N-(5-(3-substituted acryloyl)-4-methylthiazol-2-yl)
benzamide derivatives (4r-w) at 10 µM. Data is expressed as
means ± SD from three independent experiments.
are almost paralell to the x-axis suggesting that they act by
non-redox process (Figure 5c, d, e, h, i). Therefore, these
compounds probably interact in the active site. These
pseudoperoxidase activity results are supported by L-B plots
also.
O
S
R2
HN
O
R1
N
7
8
9
Compd
code
% DPPH
radical
scavenging
± SD
% 5-LOX
Inhibition
± SD
R1
R2
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O
O
4r
4s
H
F
57.2 ± 5.7
3.4 ± 1.6
8.2 ± 0.8
65.3 ± 3.6
71.9 ± 6.4
81.7 ± 0.7
~ 100 ± 2.8
S
S
4t
H
F
2.2 ± 0.7
4u
4v
~ 0 ± 1.8
5.4 ± 2.3
O
O
H
F
Br
Br
Figure 5: Pseudoperoxidase activity plots for the consumption of
13(S)-HpODE (at 234 nm; control substracted) by (a) Zileuton,
(b) 4k (c) 4n, (d) 4v, (e) 4b, (f) 4h, (g) 4m, (h) 4f, (i) 4p as a
function of time (in sec).
O
O
4w
50.6 ± 1.4
86.6 ± 2.8
5.1 ± 0.8
Zileuton
Ascorbic
acid
10.3 ± 0.5
-
-
-
-
-
60.5 ± 2.3
DPPH Radical Scavenging Activity. 5-LOX contains Fe3+ in
the activated state and most of the 5-LOX inhibitors are found
to be good scavenging radicals. Therefore, scavenging
property of all the derivatives is determined by 2, 2-diphenyl-
1-picrylhydrazyl (DPPH) methodology as per previously
reported procedure.24 Positive control (ascorbic acid) showed
60.5 ± 2.3 % antioxidant activity (Table 1). Results showed
that almost all compounds are poor radical scavengers,
showing below ~10 % activity and Zileuton exhibited 10.3 ±
0.5 % at 10 µM. Whereas 4m posseses about 15 % of
antioxidant activity for which pseudoperoxidase assay also
suggested that it acts by disrupting the redox cycle of iron. The
other best inhibitors namely 4n and 4v, showed antioxidant
activities in the range of ~5 %. In support, pseudoperoxidase
assay also indicated that both compounds act through non-
redox mechanism.
Figure 4: Lineweaver-Burk plots for inhibitors at 0, 2 and 5 µM
concentrations (a) 4k (b) 4n (c) 4v. [S] = concentration of the
substrate (AA, in µM), V = reaction rate.
5-LOX Pseudoperoxidase Activity and Mechanism. Known
mechanisms for 5-LOX inhibition are redox, non-redox and
iron chelating.10 The inhibitors acting via redox mechanism
disrupt the redox cycle by affecting the iron’s ionic state from
Fe3+ to Fe2+. Deactivated ferrous ion has a tendency to
consume lipid peroxide for activating the redox cycle of 5-
LOX. Here, the substrate, 13(S)-hydroperoxyoctadecadienoic
acid (13(S) HpODE) in the pseudoperoxidase assay is
consumed in the presence of a redox inhibitor. The decrease in
absorbance (at 234 nm) with respect to time is the measure of
consumption of the substrate.7 Pseudoperoxidase activities of
4k, 4n, 4v, 4b, 4h, 4m, 4f and 4p are performed and reported
here (Figure 5). The most active inhibitor, 4k, decreases 56.6
% of absorbance in 300 s (Table 3, Figure 5b) depicting
significant consumption of the hydroperoxide, exhibiting
redox nature. Zileuton is a well known reported redox
inhibitor. When analysed experimentally it showed 63.4 %
hydroperoxide consumption (Figure 5a). 4h and 4m also show
significant decrease in the peroxide by 40.2 and 56.3 %
respectively suggesting they also interrupt the redox cycle of
the enzyme. While compounds 4n, 4v, 4b, 4f and 4p do not
Docking Studies. The binding interactions of competitive
inhibitors, 4n and 4b, were evaluated by docking them into the
active site of 5-LOX. The Molecular Operating Environment
(MOE 2017.0801) software was used and 5-LOX protein (pdb
ID 3O8Y) was obtained from Protein Data Bank (PDB). 4n
showed a binding score of -9.09 kcal/mol along with H-bond
and pi-H interactions (Figure 6a,b). The carbon in the double
bond of chalcone interacted with the polar amino acids,
Gln363, forming a H-bond donor with a distance of 2.62 Å.
Thiazole ring forms a pi-H interaction with the Leu414 (4.7 Å)
in the hydrophobic pocket of the enzyme. 4v with a binding
score of -8.03 kcal/mol showed side chain H-bond interaction
with the polar Gln557 (3.25 Å) and bromo group of benzo
ethylenedioxide substituent. Other two pi-H interactions are
seen with the polar residues of thiazole (Leu414) and benzene
rings of the benzoamide group attached to thiazole (Tyr181)
(Figure 6c,d).
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Table 3. 5-LOX inhibition IC50, Lipinski parameters, pseudoperoxidase activity and type of mechanism for the best compounds of the
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series.
Pseudoperoxidased
5-LOX
Total polar
surface area
(TPSA)c
Å2
activity in terms of %
change in absorbance (at
10 µM of 13(S)-HpODE
and inhibitor)
Compd code
4k
Structure
inhibition,
Lipophilicity
Type of mechanism
IC50 (µM)a
C log Pb
O
OCH3
OCH3
S
N
HN
O
Redox
(Non competitive)
0.07 ± 0.02
0.08 ± 0.05
3.56
4.84
114.99
96.53
- 56.6
+ 5.6
OCH3
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O
S
HN
Non-redox
(Competitive)
O
4n
4v
N
O
CH3
O
S
O
O
HN
O
5.16
4.40
105.76
87.30
+ 1.2
Non-redox
(Competitive)
0.12 ± 0.04
0.14 ± 0.06
N
Br
O
S
- 12.5
HN
O
Non-redox
4b
4h
4m
4f
N
F
O
NO2
S
HN
O
0.24 ± 0.07
0.54 ± 0.10
0.56 ± 0.15
4.00
4.91
133.12
87.30
- 40.2
- 56.3
Redox
Redox
N
F
O
H
S
N
N
O
CH3
O
H3CO
H
N
S
4.49
96.53
90.54
+ 0.3
Non-redox
Non-redox
N
O
F
O
S
HN
O
- 12.2
4p
N
0.64 ± 0.08
N
4.42
2.48
HO
O
N
1.05 ± 0.03
94.80
- 63.4
Redox
Zileuton
NH2
S
CH3
adetermined experimentally
ddetermined experimentally. Negative values indicate % decrease in absorbance due to consumption of 13(S)-HpODE and positive values indicate %
increase in absorbance.
bcalculated using ChemBioDraw Ultra 11.0
ccalculated using Swiss ADME software
group along with thiazole for 5-LOX inhibition with a
synergistic effect. As a result, molecules (4k: IC50 = 0.07 ±
The active inhibitors 4k, 4n and 4v have higher total polar
surface area (TPSA) (Table 3) than Zileuton indicating
chances
app.com/technical-info/core-properties/logp.htm). The Swiss
ADME prediction indicated that these inhibitors satisfy
Lipinski rule of five as well as Ghose, Veber, Egan, Muegge
rules signifying that they have good druglikeness properties
along with C log P value more than that of Zileuton indicating
their higher lipophilic nature.25
0.02 µM, 4n: IC50 = 0.08 ± 0.05 µM) synthesized here are
found to be 10-100 times better potent than previously
reported thiazole molecules (2m: IC50 = 0.9 ± 0.1 µM, 1d: IC50
= 10 µM) by us and also better than Zileuton (IC50 = 1.05 ±
0.03 µM). 6,7 SAR indicated that the presence of more number
of electron donating groups namely, methoxy and methyl
either in the vicinity of chalcone or both thiazole and chalcone
contributed synergistic effect and yielded highly active
molecules. Pharmacophore elucidation revealed that the
double bond of chalcone is one of the important reasons for
hydrophobic centroid generation for inhibition. Further,
pseudoperoxidase assay and kinetic studies revealed the
mechanism of 4k has a redox type (non-competitive) while 4n
and 4v act through competitive inhibition. Docking studies of
4n and 4v gave molecular binding interactions with the active
site amino acids. Therefore, the results has laid a foundation
of
lesser
adverse
effects
(www.asteris-
In conclusion, a series of novel chalcone-thiazole molecules
were
pharmacophore hybridization approach and evaluated against
enzyme 5-LOX. All the 23 compounds synthesized are
completely new molecules, verified by Scifinder. In analogy to
our previous study7, it was possible to incorporate chalcone
rationally
designed
and
synthesized
using
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AA, Arachidonic acid; LT, Leukotriene; 5-LOX, 5-Lipoxygenase;
for the discovery of novel hybrids which will help further to
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LTA4, Leukotriene A4; DPPH, 2,2 -diphenyl-1-picrylhydrazyl; 5-
HPETE, 5(S) hydroperoxyeicosatetraenoic acid; 13(S) HpODE,
13(S)-hydroperoxyoctadecadienoic acid; LB, Lineweaver–Burk;
PMSF, Phenylmethyl sulphonyl fluoride; Ph, Pharmacophore;
TPSA, Total polar surface area.
develop a new class of next generation 5-LOX inhibitors
targeting inflammation.
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8
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ASSOCIATED CONTENT
Supporting Information
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AUTHOR INFORMATION
Corresponding Authors
*Phone: +91 9884691871. E-mail: mukeshd@iitm.ac.in
*Phone: +91 9443449594. E-mail: slmanju@vit.ac.in
Author Contributions
SS performed the experiments and written the manuscript. MD
and SLM designed the experiments and corrected the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
Shweta Sinha acknowledges DST, New Delhi, WOS-A
(SR/WOS-A/CS-126/2013), GoI for funding and fellowship for
the research. The authors thank SAIF, IIT-Madras for NMR and
IR spectral analysis and Department of Biotechnology, IIT-
Madras for HRMS analysis. The authors thank VIT, Vellore for
providing ‘VIT Seed Grant’ for the research.
13. Reddy, N. P.; Aparoy, P.; Reddy, T. C. M.; Achari, C.;
Sridhar, P. R.; Reddanna, P. Design, synthesis, and
biological evaluation of prenylated chalcones as 5-LOX
inhibitors. Bioorganic & medicinal chemistry 2010, 18,
5807-5815.
ABBREVIATIONS
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