2-Benzamido-4-methylthiazole-5-carboxylic Acid Derivatives as Potential Xanthine Oxidase Inhibitors and Free Radical Scavengers

Article abstract of DOI:10.1002/ardp.201600313

The new chemical entities febuxostat and topiroxostat have been approved by the US Food and Drug Administration, opening new avenues for exploiting different heterocycles other than purines as xanthine oxidase (XO) inhibitors. A different series of substituted 2-benzamido-4-methylthiazole-5-carboxylic acid derivatives (5a–r) was synthesized and characterized by the collective use of IR, ¹H and ¹³C NMR, and mass spectroscopy, for the treatment of gout and hyperuricemia. In vitro studies of the synthesized derivatives revealed that the presence of a fluoro group at the para position in 5b (IC50 = 0.57 μm) and a chloro group in 5c (IC50 = 0.91 μm) signifies excellent XO inhibitory activity among the series, along with their DPPH free radial scavenging activity. In vivo serum uric acid inhibition studies established that 5b and 5c displayed 62 and 53% uric acid inhibition, respectively. Studies on enzyme kinetics indicated that 5b acts as a mixed type inhibitor. In silico prediction by various softwares also helped in the recognition of potent XO inhibitors.

Full text of DOI:10.1002/ardp.201600313

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1600313
Archiv der Pharmazie
Full Paper
2-Benzamido-4-methylthiazole-5-carboxylic Acid Derivatives as
Potential Xanthine Oxidase Inhibitors and Free Radical Scavengers
Md. Rahmat Ali1, Suresh Kumar1, Obaid Afzal1, Nishtha Shalmali1, Wazid Ali2, Manju Sharma3,
and Sandhya Bawa1
1
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Hamdard University, New Delhi, India
Hamdard Institute of Medical Science, Hamdard University, New Delhi, India
Faculty of Pharmacy, Department of Pharmacology, Hamdard University, New Delhi, India
2
3
The new chemical entities febuxostat and topiroxostat have been approved by the US Food and Drug
Administration, opening new avenues for exploiting different heterocycles other than purines as
xanthine oxidase (XO) inhibitors. A different series of substituted 2-benzamido-4-methylthiazole-5-
1
carboxylic acid derivatives (5a–r) was synthesized and characterized by the collective use of IR, H and
13C NMR, and mass spectroscopy, for the treatment of gout and hyperuricemia. In vitro studies of the
synthesized derivatives revealed that the presence of a fluoro group at the para position in 5b
(IC50 ¼ 0.57 mm) and a chloro group in 5c (IC50 ¼ 0.91 mm) signifies excellent XO inhibitory activity
among the series, along with their DPPH free radial scavenging activity. In vivo serum uric acid
inhibition studies established that 5b and 5c displayed 62 and 53% uric acid inhibition, respectively.
Studies on enzyme kinetics indicated that 5b acts as a mixed type inhibitor. In silico prediction by
various softwares also helped in the recognition of potent XO inhibitors.
Keywords: Febuxostat / Kinetic / Reactive oxygen species / Uric acid / Xanthine oxidase
Received: October 25, 2016; Revised: January 1, 2017; Accepted: January 4, 2017
DOI 10.1002/ardp.201600313
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
extent through the gastrointestinal tract. Conclusively, the
excessive production of uric acid or impaired renal excretion
Introduction
Gout is a common metabolic disorder, which is usually
associated with repeated incidences of pain, inflammation,
etc. Such a condition generally originates due to increases in
the deposition of monosodium urate (MSU) crystals in
articular and periarticular tissues, which further generates
characteristic intermittent chronic cartilage injury and acute
inflammation of the gouty joint [1–3]. Uric acid is the final
metabolite of purine degradation pathway in the human
body and is eventually excreted by the kidneys and to a lesser
of uric acid are the main causes of gout and the associated
symptoms [4, 5]. Xanthine oxidase (XO, E.C. 1.1.3.22) is a
complex metalloflavoprotein in the purine scavenging
pathway which activates the transformation of hypoxanthine
to xanthine and xanthine to uric acid [6]. The oxygen
molecules act as electron acceptor through re-oxidation
advancement of OX which produces superoxide radicals
and hydrogen peroxide [7]. These biochemical reactions can
be represented as follows [8]:
Xanthine þ 2O2 þ H2O ! Uric acid þ 2O•2ꢀ þ 2Hþ
Xanthine þ O2 þ H2O ! Uric acid þ H2O2
Correspondence: Dr. Sandhya Bawa, Faculty of Pharmacy,
Department of Pharmaceutical Chemistry, Jamia Hamdard
(Hamdard University), New Delhi 110062, India.
E-mail: drsbawa@rediffmail.com, sbawa@jamiahamdard.ac.in
Fax: þ91 11 26059663
Therefore, xanthine oxidase acts as important biological
source of superoxide radicals, which imparts oxidative stress
effect on human beings. These reactive oxygen species (ROS)
ß 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
(1 of 13) e1600313

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1600313
M. R. Ali et al.
Archiv der Pharmazie
are entangled with several pathological conditions such as
aging, atherosclerosis, inflammation, cancer, etc. [9]. Along
with this, they are also involved in the development of
ischemia-reperfusion injury, various cardiovascular and
inflammatory diseases [10–13]. The nitric oxide signaling
pathway and production of ROS together increase vascular
oxidative stress in patients with chronic heart failure and are
also involved in development of cardiomyopathy in type I
diabetes, which is closely associated with increased activity of
endothelium bound xanthine oxidase [14–16]. Hence the
regulation of xanthine oxidase activity in human body is very
much needed for the anticipation of several diseases and
human health.
Keeping in view the above facts, the use of XO inhibitors
should be regarded as an imperative therapeutic approach
for the treatment of gout and associated complications.
Oxypurinol (2), a metabolite of allopurinol (1), is a renowned
purine analog and XO inhibitor and has been widely used in
treatment of gout for many years [17–19]. However, severe
life-threatening side effects have been reported in some cases
which include a toxicity syndrome executed by vasculitis,
eosinophilia, rash hepatitis, and progressive renal failure due
to purine backbone of allopurinol and its analogs [20, 21].
Therefore, there is an immense need of evolving non-purine
alternativestoallopurinolwithpotentXOinhibitoryactivityand
better safety profile. In 2009, the US Food and Drug Administra-
tion (US FDA) approved febuxostat (3), a thiazole derivative
and non-purine XO inhibitor which attracted worldwide
attention [22, 23]. A recent drug topiroxostat (4) has been
launched in Japan in the year 2013. This derivative is approved
forthe treatment of gout and hyperuricemia[24, 25]. Reduction
inuricacidlevelsmayalsobeachievedbyusinguricosuricagents.
A latest example is lesinurad (11) that increases the urinary
excretion of uric acid by interfering with the urate absorption
from kidney to blood [26].
Higher incidences of toxic effects were observed during
clinical study of above-described drugs and these effects were
also associated with large amount of superoxide anion
generation by XO involved in the various pathological
conditions. Therefore, development of XO inhibitors with
free radical scavenging potential may prove as promising
agents to treat gout. These possibilities have encouraged the
researchers to develop structurally diverse molecules lacking
of purine backbone with promising XO inhibitory activity. The
replacement of thiazole with other heterocyclic rings further
leads to febuxostat analogs having carboxyl ring in their
structures. Some examples are 4-methyl-2-((2-methylbenzyl)
amino)thiazole-5-carboxylic acid (5), Y-700 (6), selenazoles (7),
isoxazoles (8), oxazole (9), pyridines (10), 2-(indol-5-yl)
thiazoles (11), and imidazole’s (13) derivatives (Fig. 1) [27–32].
It was assumed worthy to design structural analogs of
febuxostat rationally as non-purine XO inhibitors with
antioxidant activity and reduced associated toxicities. In
continuation of our earlier efforts on finding novel non-
purine XO inhibitors (5), we have designed, synthesized, and
evaluated 2-benzamido-4-methylthiazole-5-carboxylic acid
derivatives (5a–r) (Fig. 2) as potent XO inhibitors and free
radical scavengers.
Figure 1. Chemical structures of various
inhibitors of xanthine oxidase and its ana-
logs. Allopurinol, febuxostat, and topiroxo-
stat are FDA-approved drugs for the
treatment of gout. Oxypurinol (alloxanthine)
is the hydroxylation product of allopurinol by
XO and inhibits XO via direct coordination of
its N2 atom to Mo of the enzyme active site.
Replaced thiazole with other heterocyclic
rings, including carboxyl moiety containing
inhibitors. Lesinurad a recently approved
drug for the use of urate lowering therapy.
Newly designed compounds (5a–r) as XO
inhibitors.
ß 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
(2 of 13) e1600313

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1600313
Thiazoles as Potential Xanthine Oxidase Inhibitors
Archiv der Pharmazie
Figure 2. Chemical architecture of febuxo-
stat and its analog for development of
2-benzamido-4-methylthiazole-5-carboxylic
acid derivatives (5a–r) as XO inhibitors and
free radical scavengers.
achieved by adding POCl3 dropwise as condensing agent.
An acylating agent was first generated in situ after the
addition of acid which then reacted with the added amine
and finally led to an amide. The acid here acted as an
activating or coupling agent. This one-pot procedure
involved the formation of acylating agents along with
the amide at 0–5°C temperature [36]. The substituted
benzamide esters (4a–r) were hydrolyzed with potassium
carbonate to get substituted 2-benzamido-4-methylthiazole-
5-carboxylic acid derivatives (5a–r) (after acidification) as target
compounds [33]. All synthesized compounds were character-
ized by collective use of IR, 1H and 13C NMR, and mass
Result and discussion
Chemistry
2-Benzamido-4-methylthiazole-5-carboxylic acid derivatives
as XO inhibitors (5a–r) were synthesized as outlined in
Scheme 1. Thiourea and commercially available 2-chloro-
ethylacetoacetate was cyclized to form ethyl 2-amino-
4-methyl-1,3-thiazole-5-carboxylate ester (3) [33–35].
Subsequently, reaction was carried out by stirring equivalent
amounts of substituted aromatic acid and ethyl 2-amino-4-
methyl-1,3-thiazole-5-carboxylate ester in dry pyridine.
In situ formation of amide at 0–5°C temperature was
Scheme 1. Route of synthesis of 2-(benzyla-
mino)-4-methyl-1,3-thiazole-5-carboxylic acid
derivatives (5a–r). Reagents and conditions:
(a) Ethanol, reflux, 70°C; (b) dry pyridine,
POCl3, stirring, RT; (c) K2CO3, stirring with
reflux.
ß 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
(3 of 13) e1600313

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1600313
M. R. Ali et al.
Archiv der Pharmazie
̊
spectroscopy. All spectral values were in accordance with the
assumed structures. IR spectral data characterized the presence
of carbonyl function of carboxylic acid which appeared as
stretching band at 1665 cmꢀ1, and carbonyl function of amide
appeared asstretching band at1742 cmꢀ1. N–H stretching band
appeared at 3243 cmꢀ1, C–N stretching at 1202 cmꢀ1 for
compound 5a, the OH stretching band of carboxyl appeared
at 3063 cmꢀ1. In 1H NMR spectral analysis of compound 4a, the
ester proton resonated as a doublet and quartet at d 1.25 and
4.37 ppm, which disappeared in spectrum of compound 5a,
and a new peak of carboxylic acid was observed at high down-
field values at d 10.52 ppm (bs, 1H) integrating for one proton.
The signal due to –CONH– proton was also observed at high
downfield d 12.22 ppm (D2O exchangeable). The methyl group
of thiazole ring appeared as singlet at d 2.26 ppm and the
aromatic proton appeared as multiplet at d 6.82–7.86ppm of
compound 5a. Furthermore, in 13C NMR spectrum of the
compound 5a, signal due to the carbonyl carbon of amide was
observed at d 163.54 ppm (CONH), and carbonyl carbon of
carboxylic acid was observed at d 167.70 ppm (CO), respectively.
Also a thiazole carbon signal was found in the 13C NMR
spectrum of compounds 5a at 145.86, 156.54, and 158.70, ppm.
Themolecularionpeakofcompound5aappearedatm/z263.01
(M þ H) in mass spectrum. The elucidation of spectral data and
elemental analysis showed successful synthesis of desired
compounds. The specific spectral details of synthesized com-
pounds (5a–r) with their structural characterization data are
mentioned in the Experimental section.
of our compounds were found in the range of 107.50–153.30 A,
which fascinates additional structural optimization for the
development of new molecules. The drug-likeness properties
suggested that compounds having zero or negative values
shouldnotbedeliberatedasdrug-likeapplicant.Inpeculiarityof
allsynthesized compounds impelling drug-likeness scores>0, all
compounds except 5d, 5h, and 5q showed maximum drug-
likeness score in between 1.03 and 5.20 as depicted in Table 1.
The aforesaid online prediction software helped in distinguish-
ing a set of compounds suitable for pharmacological screening.
PASS prediction
PASS (Prediction of Activity Spectra for Substances) identifies
the potential biological activities of a compound on the basis
of comparison of its chemical structure with a database of
existing quantitative structure–activity relationships (2D
QSAR) of over 250000 compounds demonstrating over 4000
types of biological activities together with pharmacologic
effects, mechanism of action, harmful and adverse effects,
interaction with catabolic enzymes and transporters, signifi-
cance on sequence expressions, etc. [43, 44]. Keeping in view
the features of XO inhibitors, PASS prediction of compounds
was performed in order to eliminate the synthesized
compounds with probability of activity values (Pa) less
than 0.400 and the remaining compounds were selected for
gout treatment (Pa – Pi > 0.4) (Table 1). All the synthesized
compounds (5a–r) exhibited the Pa value in the range of
0.466–0.532 and further performed in vitro screening also
concreted the base of potency of the said compounds.
PASS prediction software also helped in recognition of
those compounds which had more reliable results of docking
studies. Low-docked compounds, with moreover lesser dock
outcome or poorly docked poses, were designated as inactive.
In silico computational studies
Physicochemical properties
For the design and development of XO inhibitors with
enriched pharmacological profile, the prediction of physico-
chemical properties is most important. Improved lipophilicity
with poor water solubility is one of these significant
parameters. The drug-likeness characters such as lipophilicity
(clogP), water-solubility (clogS), molecular weight (MW),
number of rotatable bonds (NROTB), and drug-likeness grade
of Lipinski’s rule of five were calculated for the targeted
designed compounds (5a–r) (Table 1) using online Osiris
Property explorer and Molinspiration property calculation
toolkit [37–39]. The solubility (clogS) of synthesized molecules
were found in an acceptable range (<ꢀ4). The lipophilicity-
related clogP enumerating target-oriented drug-likeness
profile, drug potency, pharmacokinetics, and toxicity analysis
has been implicated for many years. Compounds having <5
clogP value was considered as more favorable drug-likeness
profile [40, 41]. Among the series, all the synthesized
compounds were found with <4 clogP value which suggests
their applicability for oral route of administration. Additional
physicochemical parameters such as Topological Polar
Surface Area (TPSA) were also calculated to detect
poorly membrane permeable compounds with less CNS
bioavailability which is beneficial for selecting oral
Molecular docking studies
Molecular docking studies of designed molecules for enzyme–
inhibitor interactions was performed on XO (PDB entry 1N5X)
using Glide XP Docking protocol in Schrodinger 9.4 [45–47].
Molecular docking variations includes van der Waals and
electrostatic synergy between active site and ligand which is
the basis of force field scoring. Chemical structures of the
intended compounds were exposed to the active site of XO
and results were compared with febuxostat. During docking
analysis, validation for the accuracy of Glide docking program
was established. The RMS deviation of Glide XP was calculated
in between the most reasonable binding modes of febuxostat
and co-crystallized structure febuxostat. The result of the
control of Glide docking determined the finest orientation of
the docked inhibitor, febuxostat to be close to that of the
original orientation found in the crystal structure (Fig. 3). The
MS deviation in-between the experimental conformation and
the calculated docked conformation for febuxostat in XDH
̊
(1N5X) was 0.63 A. The carboxylate group, which is the most
tightly bound part in febuxostat was also present in our XO
inhibitors and thus justified the similar binding pattern with
amino acid. The oxygen atom of carboxylate group interacted
̊
drug candidates. Compounds with TPSA values >60 A are
commonly picked for oral drug molecules [42]. The TPSA values
ß 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
(4 of 13) e1600313

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1600313
Thiazoles as Potential Xanthine Oxidase Inhibitors
Archiv der Pharmazie
Table 1. In silico physicochemical properties for oral bioavailability and bioactivity of the test compounds evaluated
utilizing computational predictive software.
Gout
treatment
Dock
score
Comp. Compound
(kcal/
mol)
XP
score
Rotatable
bonds
Drug
likeliness
Drug
score TPSA
ID
type
Pa
Pa – Pi
cLogP
cLogS
MW
Toxicity
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
H
4-F
4-Cl
0.491
0.476
0.532
0.475
0.529
0.509
0.468
0.458
0.449
0.470
0.459
0.516
0.466
0.510
0.433
0.487
0.475
0.449
0.712
0.487
0.472
0.529
0.471
0.526
0.506
0.464
0.454
0.445
0.466
0.455
0.513
0.462
0.507
0.429
0.483
0.471
0.445
0.716
ꢀ12.202
ꢀ12.249
ꢀ12.424
ꢀ12.158
ꢀ12.123
ꢀ12.017
ꢀ10.609
ꢀ10.011
ꢀ10.014
ꢀ9.504
ꢀ12.202
ꢀ12.249
ꢀ12.424
ꢀ12.158
ꢀ12.123
ꢀ12.017
ꢀ10.609
ꢀ10.011
ꢀ10.014
ꢀ9.504
2.4
2.5
3.0
3.12
2.74
2.05
2.33
1.48
2.5
ꢀ3.35
ꢀ3.67
ꢀ4.09
ꢀ4.19
ꢀ3.70
ꢀ3.06
ꢀ3.37
ꢀ3.81
ꢀ3.67
ꢀ4.09
ꢀ4.19
ꢀ3.70
ꢀ3.37
ꢀ3.06
ꢀ4.40
ꢀ4.09
ꢀ4.19
ꢀ3.67
ꢀ4.27
262
280
296
340
276
278
292
307
280
296
340
276
292
278
314
296
296
340
316
3
3
3
3
3
3
4
4
3
3
3
3
4
3
3
3
3
3
4
3.44
2.97
5.20
0.21
2.36
3.79
3.74
ꢀ8.62
2.01
3.77
ꢀ0.30
3.19
3.74
3.49
1.03
2.12
ꢀ1.09
2.01
ꢀ1.08
0.85
0.81
0.77
0.57
0.79
0.87
0.84
0.42
0.78
0.76
0.53
0.81
0.84
0.87
0.64
0.73
0.46
0.78
0.45
107.50
107.50
107.50
107.50
107.50
127.70
116.70
153.30
107.50
107.50
107.50
107.50
116.70
127.70
107.50
107.50
107.50
107.50
90.46
NM, NT
NM, NT
NM, NT
NM, NT
NM, NT
NM, NT
NM, NT
SM, ST
NM, NT
NM, NT
NM, NT
NM, NT
SM, ST
NM, NT
NM, NT
NM, NT
NM, NT
NM, NT
NM, NT
4-Br
4-CH3
4-OH
4-OCH3
4-NO2
2-F
2-Cl
2-Br
3.0
ꢀ9.981
ꢀ9.981
3.12
2.74
2.33
2.05
3.1
3.0
3.12
2.5
2-CH3
2-NO2
2-OCH3
2-OH
3-Cl, 4-F
3-Cl
ꢀ10.601
ꢀ10.431
ꢀ9.122
ꢀ10.601
ꢀ10.431
ꢀ9.122
ꢀ12.321
ꢀ8.012
ꢀ12.321
ꢀ8.012
ꢀ8.671
ꢀ8.671
3-Br
Febuxostat
ꢀ8.014
ꢀ8.014
ꢀ13.809
ꢀ13.809
4.09
Pa, probability of being active; Pi, probability of being inactive; cLogP, lipophilicity; cLogS, water solubility; TPSA, topological
polar surface area; NM, non-mutagenic; NC, non-carcinogenic; SM, slightly mutagenic; ST; slightely tumorigenic; NP, not
predicted.
with side chain of guanidinium group of Arg880 with
Phe1013, Lys771, and Pro1076. Significant docking score
of most active derivatives 5a–e was found to be ꢀ12.42,
ꢀ12.24, ꢀ12.20, ꢀ12.15, ꢀ12.12 kcal/mol as depicted in
Table 1. According to molecular docking analysis of 5a–e,
we may suggest that the presence of large aromatic
thiazole ring in the structure of 2-benzamido-4-methyl-
thiazole-5-carboxylic acid derivatives gets fits in the active
site of XO and favors their binding and inhibition toward
XO (Figs. 3 and 4).
̊
hydrogen bonds (2.01 A) binding to the side chain, and
̊
hydroxyl with backbone amide of Thr1010 (2.51 A). The N-3 of
thiazole ring led to the formation of hydrogen bond between
the thiazole nitrogen and carboxylate side chain of Glh802
̊
(2.11 A). Whole thiazole ring was rendered between two
̊
̊
phenylalanine residues: Phe1009 (3.73 A) and Phe914 (3.91 A)
with p–p interactions; these interactions may support the
stabilization of the binding patterns and also help in substrate
recognition. A linker (–CONH–) introduced in between the
substituted benzyl and thiazole rings, which confirmed
the structural expansion of our lead molecules through
hydrogen binding with amino acid Ser876. The substituted
benzamide part of designed molecules was observed inter-
leaved in between Leu648, Phe649, Phe1013, Lys771, and
Pro1076 with Ser876-nitrile bond, which provided guidance
for its alignment. Hydrophobic amino acids Leu648, Phe649,
Phe1013, Lys771, Pro1076, Leu873, Leu1014, Val1011,
Thr1010, Ser1008, Arg880, Ala1029, Ala1078, Glh802,
Phe1009, and Phe914 appeared as a birdcage (Fig. 4). The
designed compounds 5a–f mainly embraced hydrophobic
interactions and attained excellent docking score of
>ꢀ12 kcal/mol. The electron donating groups CH3, OH,
OCH3 and electron withdrawing group F, Cl, Br were
appeared encircled by amino acids Leu648, Phe649,
Biological activity
In vitro XO inhibitory activity of compounds 5a–r was carried
out spectrophotometrically by measuring uric acid levels at
295 nm (Table 2) [48–50]. Febuxostat was used as the
reference drug. The synthesized 2-benzamido-4-methylthia-
zole-5-carboxylic acid derivatives demonstrated XO inhibitory
activity, but less than that of febuxostat. The potency of the
aforesaid compounds can be due to the presence of thiazole
ring with free carboxylic acid. Assessment of their inhibitory
effect led to an observation that the presence of electron
withdrawing group leads to good XO inhibitory activity
in comparison to electron donating group. The functional
group located at ortho position slightly diminishes the XO
activity with respect to functional group located at para
position either in case of electron releasing group or electron
ß 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
(5 of 13) e1600313

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1600313
M. R. Ali et al.
Archiv der Pharmazie
Figure 3. Top scored 3-dimensional (3D) diagrams representation showing hydrogen bonding interaction of compound 5b (green)
and febuxostat (yellowish green) with active sites of enzyme xanthine oxidase (PDB ID: 1N5X) for XO inhibitory activity.
withdrawing group. The compounds 5h and 5m were not
evaluated for XO inhibition due to their slight mutagenic
effects as shown by in silico studies. Compounds 5i
(IC50 ¼ 33.19 mm), 5k (IC50 ¼ 59.69 mm), 5n (IC50 ꢁ 100 mm), 5o
(IC50 ¼ 47.25 mm), 5q (IC50 ¼ 27.52 mm), and 5r (IC50 ꢁ 100 mm)
having substitution at ortho or meta position exhibited very
low inhibitory activity. The compounds 5a (IC50 ¼ 14.57 mm),
5e (IC50 ¼ 4.15 mm), 5g (IC50 ¼ 12.67 mm), 5j (IC50 ¼ 6.72 mm), 5l
Figure 4. Top scored 2-dimensional (2D) diagrams showing hydrogen bonding interaction of compound 5b with amino acids of
active sites of enzyme xanthine oxidase (PDB ID: 1N5X).
ß 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
(6 of 13) e1600313

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1600313
Thiazoles as Potential Xanthine Oxidase Inhibitors
Archiv der Pharmazie
Table 2. In vitro IC50 values of XO inhibitory and free radical scavenging activities of derivatives.
Free radical scavenging
Uric acid inhibition (%)
at 1 h, 10 mg/kg
Comp. ID
Compound
In vitro IC50 (mM)
activity IC50 (mM)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
–
H
4-F
4-Cl
4-Br
4-CH3
4-OH
4-OCH3
4-NO2
2-F
2-Cl
2-Br
2-CH3
2-NO2
2-OCH3
2-OH
3-Cl, 4-F
3-Cl
3-Br
Febuxostat
Ascorbic acid
14.57 ꢂ 0.09
0.57 ꢂ 0.34
0.91 ꢂ 0.79
72.01 ꢂ 0.47
4.15 ꢂ 0.43
28.37 ꢂ 0.60
12.67 ꢂ 0.59
ND
33.19 ꢂ 0.21
6.72 ꢂ 0.37
59.69 ꢂ 0.41
11.7 ꢂ 0.04
ND
29.18 ꢂ 0.51
23.21 ꢂ 0.32
16.37 ꢂ 0.11
63.49 ꢂ 0.17
21.7 ꢂ 0.73
19.71 ꢂ 0.35
28.93 ꢂ 0.76
ND
>100
61.7 ꢂ 0.35
>100
31.21 ꢂ 0.32
ND
44.72 ꢂ 0.47
22.26 ꢂ 0.36
34.79 ꢂ 0.81
58.12 ꢂ 0.93
>100
ND
12.7 ꢂ 0.57
ND
62
53
ND
35
ND
ND
ND
ND
29
ND
ND
ND
ND
ND
41
>100
47.25 ꢂ 0.43
4.97 ꢂ 0.13
27.52 ꢂ 0.07
>100
0.01 ꢂ 0.01
ND
ND
ND
73
–
ND
ND, not determined; % inhibitor, calculated at 10 mM concentration (IC50 value ꢂ SD n ¼ 3).
(IC50 ¼ 11.7 mm), and 5p (IC50 ¼ 4.97 mm) exhibited significant
XO inhibitory activity. Presence of halogen group at para
position such as fluoro in compound 5b (IC50 ¼ 0.57 mm) and
chloro in compound 5c (IC50 ¼ 0.91 mm) signify excellent XO
inhibitory activity among the synthesized derivatives of this
series. IC50 values of all the compounds are depicted in
Table 2. Enzyme kinetics study of most active inhibitor (5b)
was performed. The Lineweaver–Burk plot (Fig. 5) analysis
exhibited that compound 5b is a mixed-type XO inhibitor. The
graph pattern displays that it forms a mixed scenario of
inhibition, the Km, Vmax, and slope all being affected by the
inhibitor. A careful observation of Lineweaver–Burk plot
represented that the intersecting lines on the graph converge
to the left of the y-axis and above the x-axis which stipulated
that the value of a (a constant which defines that the affinity
of the enzyme for substrate is affected by degree of inhibitor
binding) is greater than 1, it was confirmed that the inhibitor
is favorably binding with the free enzyme and not to the
enzyme–substrate complex.
Antioxidant potential of 2-benzamido-4-methylthiazole-
5-carboxylic acid derivatives was also evaluated using DPPH
(1,1-diphenyl-2-picrylhydrazyl) assay [51]. 1,1-diphenyl-2-
picrylhydrazyl (DPPH) is a stable free radical and accepts
electron or hydrogen radical to become a stable diamagnetic
molecule. Ascorbic acid was used as a reference compound.
Derivatives 5d (IC50 ¼ 63.49 mm), 5j (IC50 ¼ 61.7 mm), 5k (IC50
ꢁ 100 mm), 5l (IC50 ¼ 31.21 mm), 5n (IC50 ¼ 44.72 mm), 5p
(IC50 ¼ 34.79 mm), 5q (IC50 ¼ 58.12 mm), and 5r (IC50 ꢁ 100 mm)
specified moderate free radical scavenging activity on the basis
of their IC50 values. Presence of electron or hydrogen radical
releasing groups OCH3, OH, CH3, H in the structures of 5a
(IC50 ¼ 29.12mm), 5e (IC50 ¼ 21.7 mm), 5f (IC50 ¼ 19.71 mm), 5g
(IC50 ¼ 28.93mm), and 5o (IC50 ¼ 22.26 mm) may be responsible
for reduction of DPPH free radical that imparts scavenging
activity to them due to their electron donating activity. The
compounds 5b (IC50 ¼ 23.21mm), 5c (IC50 ¼ 16.37 mm), and 5f
(IC50 ¼ 19.71mm) have significant DPPH free radial scavenging
activity along with potent XO inhibitory activity, which is
compared with standard, ascorbic acid. IC50 values of the
compounds of interest are depicted in Table 2.
Evaluation of the effect of xanthine oxidase inhibitors in
hyperuricemic rat model assay
During evaluation of in vitro XO inhibitory study of
2-benzamido-4-methylthiazole-5-carboxylic acid derivatives
(5a–r), compounds 5b, 5c, 5e, 5j, and 5p were found to be most
active among the series and were subjected to in vivo
evaluation as anti-hyperuricemic agents in potassium
oxonate-induced hyperuricemic rat model [52–54]. Dose of
the compounds and standard was taken as potassium oxonate
300 mg/kg, febuxostat 10 mg/kg and test compounds as 10
and 20 mg/kg. In vivo study demonstrated that the com-
pounds 5b, 5c, 5e, 5j, and 5p exhibited 62, 53, 35, 29, and 41%
of uric acid inhibition at 1 h, at a dose of 10 mg/kg. The
compound 5b is effective almost as the standard drug
febuxostat and showed dose-dependent response graph
(Fig. 6). The percentage uric acid inhibition at 1 h, at a dose
of 10 mg/kg was found to be 62.00% whereas febuxostat
showed 73.00% inhibition at a same dose level (Table 2).
Hence the compound 5b can be used for the management of
gout and hyperuricemia because 5b shows excellent uric acid
inhibition along with free radical scavenging activity which
ß 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
(7 of 13) e1600313

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1600313
M. R. Ali et al.
Archiv der Pharmazie
Figure 6. Serum uric acid concentration (mg/dL). Each value
represents the mean ꢂ SEM (n ¼ 5). Bars with different letters
are significantly different (ꢃꢃp < 0.01, ꢃꢃꢃp < 0.001: ANOVA and
post-Dunnet’s test).
Overall it can be concluded that among the compounds in this
series, compounds 5b and 5c demonstrated significant uric
acid inhibition in both in vitro and in vivo studies. As
compared with febuxostat, these compounds also revealed
their in vitro DPPH free radical scavenging activity which has
not yet been reported for standard. In conclusion, the overall
study confirmed that compound 5b may confer significant
therapeutic benefits and a better safety profile over existing
treatments for gout and hyperuricemia.
Figure 5. Lineweaver–Burk plot in the absence (control) and in
the presence of inhibitor (5b) with xanthine as the substrate.
has not been reported in febuxostat. Therefore, 5b could be
used for further development of lead molecules.
Experimental
Chemistry
General
All the solvents and reagent were obtained from reliable and
authorized sources (S.D. Fi Chemicals, Sigma–Aldrich, Merck
India, CDH Spectrochem, etc). All reactions were monitored by
thin layer chromatography (TLC) using precoated silica gel G
plates at 254 nm under UV lamp/iodine vapors using different
solvent systems. Melting points were determined by the open
capillary method with electric melting point apparatus and
are uncorrected. Mass spectra (ESI-MS) were obtained by
JEOL-AccuTOF JMS-T100LS mass spectrometer (JEOL USA, Inc.,
Peabody, MA, USA), and elemental analysis was performed
on a Vario-EL III CHNOS. IR spectra were recorded on Bruker
FT-IR spectrophotometer and 1H and 13C NMR spectra were
recorded on Bruker DPX 300 MHz spectrophotometer (Bruker
Bio Spin Corporation, Billerica, MA, USA) using DMSO-d6 or
CDCl3 as solvent.
Conclusion
The synthesis of ethyl 2-benzamido-4-methylthiazole-5-car-
boxylate derivatives 5a–r as febuxostat analogs was carried
out successfully as per the scheme outlined in Scheme 1. All
the structures of synthesized compounds were characterized
by collective use of IR, 1H and 13C NMR, and mass spectroscopy.
In vitro studies of these derivatives 5b (IC50 ¼ 0.57 mm) and 5c
(IC50 ¼ 0.91 mm) signify excellent XO inhibitory activity among
all the compounds in this series. Compounds 5b (IC50
¼
23.21 mm), 5c (IC50 ¼ 16.37 mm), and 5f (IC50 ¼ 19.71 mm) also
have a significant DPPH free radial scavenging activity along
with potent XO inhibitory activity. Kinetic studies exhibited
that 5b acts as a mixed-type inhibitor. In vivo serum uric acid
inhibition study on potassium oxonate-induced hyperurice-
mic rat model demonstrated that 5b and 5c displayed 62 and
53% of uric acid inhibition after 1 h at dose of 10 mg/kg.
The NMR spectra and the InChI codes of the investigated
compounds are provided as Supporting Information.
ß 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
(8 of 13) e1600313

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1600313
Thiazoles as Potential Xanthine Oxidase Inhibitors
Archiv der Pharmazie
Synthesis of ethyl 2-amino-4-methyl-1,3-thiazole-5-
carboxylate (3) [33–35]
2-Benzamido-4-methylthiazole-5-carboxylic acid (5a)
Yield: 54%; white solid; m.p.: 133–135°C; IR (KBr v max):
3219, 3057, 1696, 1665, 1488, 1300, 1200, 813 cmꢀ1; 1H NMR
(300 MHz, CDCl3); d 2.26 (s, 3H, CH3), 6.82–7.86 (m, 5H, Ar-H),
12.22 (bs, 1H, D2O exchangeable CONH); 13C NMR (75 MHz,
CDCl3); 17.86 (thiazole-CH3), 128.31, 128.91, 129.13, 129.26,
To a mixture of anhydrous ethanol (100 mL) and thiourea
(0.098 mol), ethyl-2-chloro acetoacetate (0.097 mol) was
added drop-wise under constant stirring at room tempera-
ture. Once addition of ethyl-2-chloro acetoacetate is com-
pleted, reaction mixture was heated at 70–80°C for 15 min.
Then reaction mass was cooled to room temperature and the
solid precipitate was isolated, washed with 100 mL of cold
ethanol and further washed with saturated sodium bicarbon-
ate solution to obtain white solid which was finally dried
under vacuum at 50°C for 8 h. Yield: 91%; white crystalline
solid; m.p.: 170–172°C; IR (KBr v max): 3222, 3051, 1598, 1415,
–
145.86, 156.54, 158.70, 163.54 (CONH), 167.70 (C O); ESI-
–
MS: m/z 263.01 (MþH). Anal. calcd. for C12H10N2O3S: C,
54.95; H, 3.84; N, 10.68%. Found: C, 54.93; H, 3.85; N,
10.70%.
2-(4-Fluorobenzamido)-4-methylthiazole-5-carboxylic
acid (5b)
1299, 698 cmꢀ1
;
1H NMR (300 MHz, DMSO-d6); d 1.36 (s, 3H,
Yield: 49%; light pink solid; m.p.: 156–58°C; IR (KBr v max):
CH3), 2.62 (s, 3H, CH3), 4.32 (q, 2H, CH2CH3), 5.09 (bs, 2H,
D2O exchangeable NH2); ESI-MS: m/z 187.07 (MþH); Anal.
calcd. for C7H10N2O2S: C, 45.15; H, 5.41; N,15.04%. Found: C,
45.13; H, 5.42; N, 15.09%.
3257, 3071, 1743, 1670, 1499, 1323, 1045, 772 cmꢀ1 1H NMR
;
(300 MHz, CDCl3); d 3.42 (s, 3H, CH3), 8.10–8.15 (t, 2H, Ar-H),
8.67–8.72 (m, 2H, Ar-H), 10.52 (bs, 1H, COOH), 12.13 (bs, 1H,
D2O exchangeable CONH); 13C NMR (75 MHz, CDCl3); 17.79
(thiazole-CH3), 128.20, 128.36, 128.94, 135.53, 147.38, 149.09,
–
Synthesis of substituted ethyl 2-benzamido-4-
152.80, 165.03 (CONH), 169.05 (C O); ESI-MS: m/z 282.01
–
methylthiazole-5-carboxylate (4a–r)
(Mþ2). Anal. calcd. for C12H9FN2O3S: C, 51.42; H, 3.24; N,
To a solution of substituted aromatic acids (0.001 mol) in
10 mL of dry pyridine, ethyl 2-amino-4-methyl-1,3-thiazole-5-
carboxylate (3) (0.001 mol) was added and stirred at room
temperature in flat bottom flask. The external temperature
was maintained at 0–5°C. To the stirring saturated reaction
mixture, 0.5 mL of POCl3 was added dropwise and it was
further stirred at room temperature till the completion of
reaction. A solid precipitate was transferred into beaker
containing crushed ice, and neutralized with glacial acetic
acid, filtered and washed with water and dried, it was
recrystallized with ethanol to give white solid products [34]
(4a–r). The progress of the reaction and purity of the
compound were checked by TLC, using toluene/ethyl ace-
tate/formic acid (5:4:1) as mobile phase.
9.99%. Found: C, 51.43; H, 3.25; N, 10.01%.
2-(4-Chlorobenzamido)-4-methylthiazole-5-carboxylic
acid (5c)
Yield: 49%; light pink solid; m.p.: 187–189°C; IR (KBr v max):
3263, 3039, 1729, 1656, 1554, 1423, 1179, 1023, 812 cmꢀ1
;
1H NMR (300 MHz, CDCl3); d 2.60 (s, 3H, CH3), 7.28 (d, 2H,
J ¼ 7.80 Hz, Ar-H), 8.01 (d, 2H, J ¼ 8.10 Hz, Ar-H), 12.11 (bs, 1H,
D2O exchangeable CONH); 13C NMR (75 MHz, CDCl3); 17.07
(thiazole-CH3), 127.33, 128.78, 129.66, 144.13, 156.79, 160.34,
–
163.95 (CONH), 165.88 (C O); ESI-MS: m/z 297.13 (Mþ1). Anal.
–
calcd. for C12H9ClN2O3S: C, 48.57; H, 3.06; N, 9.44%. Found: C,
48.58; H, 3.07; N, 9.42%.
2-(4-Bromobenzamido)-4-methylthiazole-5-carboxylic
acid (5d)
Ethyl 2-benzamido-4-methylthiazole-5-carboxylate (4a)
Yield: 63%; white crystalline solid; m.p.: 187–189°C; IR (KBr v
Yield: 53%; light yellow solid; m.p.: 201–203°C; IR (KBr v max):
max): 3273, 3083, 1733, 1665, 1489, 1376, 12009, 998 cmꢀ1
;
3294, 3071, 1734, 1650, 1558, 1473, 1041, 679 cmꢀ1 1H NMR
;
1H NMR (300 MHz, DMSO-d6); d 1.25 (d, 3H, CH3), 2.54 (s, 3H,
CH3), 4.37 (q, 2H, CH2CH3), 7.28–8.01 (m, 5H, Ar-H), 12.10 (bs,
1H, D2O exchangeable CONH). Anal. calcd. for C14H14N2O3S: C,
57.92; H, 4.86; N, 9.65%. Found: C, 57.94; H, 4.85; N, 9.66%.
(300 MHz, CDCl3); d 3.71 (s, 3H, CH3), 6.76 (d, 2H, J ¼ 8.10 Hz,
Ar-H), 7.85 (d, 2H, J ¼ 8.10 Hz, Ar-H), 12.14 (bs, 1H,
D2O exchangeable CONH); 13C NMR (75 MHz, CDCl3); 16.79
(thiazole-CH3), 127.98, 128.53, 129.73, 143.34, 156.75, 160.59,
–
163.82 (CONH), 167.31 (C O); ESI-MS: m/z 339.43 (MꢀH). Anal.
–
Synthesis of substituted ethyl 2-benzamido-4-
methylthiazole-5-carboxylate (5a–r)
calcd. for C12H9BrN2O3S: C, 42.24; H, 2.66; N, 8.21%. Found: C,
42.26; H, 2.67; N, 8.23%.
Substituted ethyl 2-benzamido-4-methylthiazole-5-carboxyl-
ate (4a–r) (0.075 mol) and potassium carbonate (0.3 mol) were
added to a mixture of methanol/water (9:1) (20 mL). The
solution was refluxed with stirring until the reaction was
completed. The clear solution was cooled and neutralized
with glacial acetic acid and stirred further for 25–30 min.
Precipitate obtained was filtered, washed with water, dried
and recrystallized with ethanol [33]. The progress of the
reaction and purity of the compounds were checked by TLC,
using benzene/acetone (8:2) as mobile phase.
4-Methyl-2-(4-methylbenzamido)thiazole-5-carboxylic
acid (5e)
Yield: 42%; nature: white solid; m.p.: 153–154°C; IR (KBr v
max): 3367, 3031, 1753, 1649, 1563, 1421, 1029 cmꢀ1; 1H NMR
(300 MHz, CDCl3); d 2.59 (s, 3H, CH3), 2.65 (s, 3H, Ar-CH3), 7.28
(d, 2H, J ¼ 7.80 Hz, Ar-H), 7.91 (d, 2H, J ¼ 8.10 Hz, Ar-H), 12.03
(bs, 1H, D2O exchangeable CONH); 13C NMR (75 MHz, CDCl3);
17.13 (thiazole-CH3), 21.50, 128.19, 128.94, 129.22, 143.38,
–
156.37, 160.80, 162.91 (CONH), 165.65 C O); ESI-MS: m/z
–
ß 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
(9 of 13) e1600313

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1600313
M. R. Ali et al.
Archiv der Pharmazie
277.04 (MþH). Anal. calcd. for C13H12N2O3S: C, 56.51; H, 4.38;
2-(2-Bromobenzamido)-4-methylthiazole-5-carboxylic
acid (5k)
N, 10.14%. Found: C, 56.52; H, 4.39; N, 10.15%.
Yield: 44%; light pink solid; m.p.: 137–139°C; IR (KBr v max):
2-(4-Hydroxybenzamido)-4-methylthiazole-5-carboxylic
acid (5f)
3342, 3071, 1751, 1654, 1587, 1029, 667 cmꢀ1 1H NMR
;
(300 MHz, CDCl3); d 2.29 (s, 3H, CH3), 7.67–7.89 (m, 4H,
Ar-H), 12.10 (bs, 1H, D2O exchangeable CONH); ESI-MS: m/z
341.17 (Mþ2). Anal. calcd. for C12H9BrN2O3S: C, 42.24; H, 2.66;
N, 8.21%. Found: C, 42.25; H, 2.67; N, 8.23%.
Yield: 29%; creamy white solid; m.p.: 185–187°C; IR (KBr v
max): 3322, 3143, 3056, 1731, 1653, 1578, 1454, 1029,
809 cmꢀ1; 1H NMR (300 MHz, CDCl3); d 2.43 (s, 3H, CH3), 6.67
(d, 2H, J ¼ 7.80 Hz, Ar-H), 7.53 (d, 2H, J ¼ 8.10 Hz, Ar-H), 9.47
(bs, 1H, Ar-OH), 11.79 (bs, 1H, D2O exchangeable NH);
ESI-MS: m/z 279.09 (MþH). Anal. calcd. for C12H10N2O4S: C,
51.79; H, 3.62; N, 10.07%. Found: C, 51.80; H, 3.63; N,
10.09%.
4-Methyl-2-(2-methylbenzamido)thiazole-5-carboxylic
acid (5l)
Yield: 39%; light orange solid; m.p.: 174–176°C; IR (KBr v
max): 3274, 3051, 1771, 1679, 1551, 1422, 1027 cmꢀ1
;
1H NMR (300 MHz, CDCl3); d 2.32 (s, 3H, CH3), 2.58 (s, 3H,
CH3), 7.15 (m, 3H, Ar-H), 7.66 (s, 1H, J ¼ 8.40 Hz, Ar-H), 9.92
(bs, 1H, COOH), 11.92 (bs, 1H, D2O exchangeable CONH);
13C NMR (75 MHz, CDCl3); 18.53 (thiazole-CH3), 20.58,
128.18, 128.32, 128.91, 129.12, 146.88, 150.76, 158.66,
2-(4-Methoxybenzamido)-4-methylthiazole-5-carboxylic
acid (5g)
Yield: 47%; white solid; m.p.: 159–161°C; IR (KBr v max):
3329, 3097, 1701, 1678, 1573, 1447, 1029, 973 cmꢀ1; 1H NMR
(300 MHz, CDCl3); d 2.51 (s, 3H, CH3), 3.88 (s, 3H, Ar-OCH3),
6.97 (d, 2H, J ¼ 6.90 Hz, Ar-H), 7.87 (d, 2H, J ¼ 7.20 Hz, Ar-H);
10.30 (bs, 1H, COOH), 11.80 (bs, 1H, D2O exchangeable
CONH); 13C NMR (75 MHz, CDCl3); 18.36 (thiazole-CH3),
60.78 (Ar-OCH3), 128.36, 128.93, 129.12, 129.35, 130.03,
–
162.76 (CONH), 168.66 (C O); ESI-MS: m/z 277.19 (Mþ2).
–
Anal. calcd. for C13H12N2O3S: C, 56.51; H, 4.38; N, 10.14%.
Found: C, 56.53; H, 4.39; N, 10.13%.
4-Methyl-2-(2-nitrobenzamido)thiazole-5-carboxylic acid
(5m)
–
142.12, 152.80, 158.69, 162.62 (CONH), 169.07 (C O);
–
ESI-MS: m/z 293.13 (MþH). Anal. calcd. for C13H12N2O4S:
C, 53.42; H, 4.14; N, 9.58%. Found: C, 53.43; H, 4.15; N,
9.57%.
Yield: 43%; pale yellow solid; m.p.: 163–165°C; IR (KBr v max):
3312, 3047, 1732, 1661, 1582, 1426, 1376, 1029 cmꢀ1; 1H NMR
(300 MHz, CDCl3); d 3.43 (s, 3H, CH3), 8.15–8.65 (m, 4H, Ar-H),
12.52 (bs, 1H, D2O exchangeable CONH); ESI-MS: m/z 308.82
(Mþ2). Anal. calcd. for C12H9N3O5S: CC, 46.90; H, 2.95; N,
13.67%. Found: C, 46.91; H, 2.96; N, 13.69%.
4-Methyl-2-(4-nitrobenzamido)thiazole-5-carboxylic acid
(5h)
Yield: 41%; dark yellow solid; m.p.:210–212°C; IR (KBr v max):
3387, 3101, 1751, 1654, 1532, 1477, 1029 cmꢀ1
;
1H NMR
2-(2-Methoxybenzamido)-4-methylthiazole-5-carboxylic
acid (5n)
(300 MHz, CDCl3); d 2.77 (s, 3H, CH3), 7.97 (d, 2H, J ¼ 6.90 Hz,
Ar-H), 8.32 (d, 2H, J ¼ 7.20 Hz, Ar-H); 10.51 (bs, 1H, COOH),
12.15 (bs, 1H, D2O exchangeable CONH); ESI-MS: m/z 308.35
(MþH). Anal. calcd. for C12H9N3O5S: C, 46.90; H, 2.95; N,
13.67%. Found: C, 46.92; H, 2.94; N, 13.68%.
Yield: 33%; nature: white solid; m.p.: 141–143°C; IR (KBr v
max): 3376, 3067, 1752, 1672, 1581, 1437, 1284, 1061 cmꢀ1
;
1H NMR (300 MHz, CDCl3); d 2.43 (s, 3H, CH3), 3.69 (s, 3H, CH3),
6.90 (s, 1H, J ¼ 7.20 Hz, Ar-H), 7.03 (s, 1H, Ar-H), 7.47 (d, 2H,
Ar-H), 11.87 (bs, 1H, D2O exchangeable NH); ESI-MS: m/z
293.13 (Mþ2). Anal. calcd. for C13H12N2O4S: C, 53.42; H, 4.14;
N, 9.58%. Found: C, 53.43; H, 4.13; N, 9.56%.
2-(2-Fluorobenzamido)-4-methylthiazole-5-carboxylic
acid (5i)
Yield: 49%; light orange solid; m.p.: 171–173°C; IR (KBr v max):
3373, 3031, 1773, 1644, 1563, 1462, 1029, 912 cmꢀ1
;
1H NMR
2-(2-Hydroxybenzamido)-4-methylthiazole-5-carboxylic
acid (5o)
Yield: 37%; dark brown solid; m.p.: 196–198°C; IR (KBr v
(400 MHz, CDCl3); d 2.31 (s, 3H, CH3), 7.68–8.03 (m, 4H, Hz,
Ar-H), 11.93 (bs, 1H, D2O exchangeable CONH); ESI-MS: m/z
282.07 (MþH). Anal. calcd. for C12H9FN2O3S: C, 51.42; H, 3.24;
N, 9.99%. Found: C, 51.43; H, 3.25; N, 10.02%.
max): 3335, 3137, 3052, 1753, 1653, 1540, 1410, 1242,
1029 cmꢀ1 1H NMR (300 MHz, CDCl3); d 2.41 (s, 3H, CH3),
;
7.01–7.56 (m, 4H, Ar-H), 8.97 (bs, 1H, OH), 11.87 (bs, 1H,
D2O exchangeable NH); ESI-MS: m/z 279.21 (Mþ2). Anal.
calcd. for C12H10N2O4S: C, 51.79; H, 3.62; N, 10.07%. Found: C,
51.77; H, 3.63; N, 10.06%.
2-(2-Chlorobenzamido)-4-methylthiazole-5-carboxylic
acid (5j)
Yield: 36%; creamy white solid; m.p.: 149–151°C; IR (KBr v
max): 3362, 3037, 1763, 1673, 1591, 1413, 1029, 783 cmꢀ1
;
1H NMR (300 MHz, CDCl3); d 2.32 (s, 3H, CH3), 7.44 (m, 3H,
Ar-H), 7.86 (s, 1H, J ¼ 7.80 Hz, Ar-H), 12.16 (bs, 1H,
D2O exchangeable CONH), ESI-MS: m/z 298.05 (Mþ2). Anal.
calcd. for C12H9ClN2O3S: C, 48.57; H, 3.06; N, 9.44%. Found: C,
48.55; H, 3.07; N, 9.46%.
2-(3-Chloro-4-fluorobenzamido)-4-methylthiazole-5-
carboxylic acid (5p)
Yield: 47%; light pink solid; m.p.: 163–165°C; IR (KBr v max):
3389, 3278, 3007, 1742, 1649, 1561, 1432, 989, 734 cmꢀ1
;
1H NMR (300 MHz, CDCl3); d 2.27 (s, 3H, CH3), 7.07 (s, 1H, Ar-H),
ß 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
(10 of 13) e1600313

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1600313
Thiazoles as Potential Xanthine Oxidase Inhibitors
Archiv der Pharmazie
7.23 (s, 1H, J ¼ 6.40 Hz, Ar-H), 7.48 (s, 1H, Ar-H), 12.11 (bs, 1H,
D2O exchangeable CONH); 13C NMR (75 MHz, CDCl3); 17.23
(thiazole-CH3), 124.03, 127.19, 128.23, 128.72, 129.89, 148.59,
157.56, 158.63, 162.13 (CONH), 166.93 (CO); ESI-MS: m/z
317.09 (Mþ2); Anal. calcd. for C12H8ClFN2O3S: 45.80; H, 2.56;
N, 8.90%. Found: 45.82; H, 2.57; N, 8.91%.
strategies were based on rapid and perfect computational
methods for the approximation of receptor-ligand binding
affinities. Using Glide XP module, the docking of molecules
was done with Epik state penalties for different ionizations
and tautomeric states. XP docking works as more stringent
scoring functions include terms for hydrophobic enclosure
and desolvation penalties. Different docking poses of ligands
were generated and analyzed for interpretation of final
results.
2-(3-Chlorobenzamido)-4-methylthiazole-5-carboxylic
acid (5q)
Yield: 33%; light orange solid; m.p.: 169–171°C; IR (KBr v max):
3343, 3103, 1787, 1653, 1549, 1487, 1263, 1082, 771 cmꢀ1
;
In silico study for bioactivity and physicochemical
properties
1H NMR (300 MHz, CDCl3); d 2.28 (s, 3H, CH3), 6.20 (bs, 1H,
D2O exchangeable NH), 7.08 (d, 1H, Ar-H), 7.21 (d, 1H, Ar-H),
7.49 (s, 1H, Ar-H); ESI-MS: m/z 298.08 (Mþ2). Anal. calcd. for
For development of XO inhibitors with enhanced pharmaco-
logical profile, the prediction of physicochemical properties
are most emphasizing parameters. All the designed com-
pounds were evaluated for their oral bioavailability, physico-
chemical properties, toxicity, and online pharmacological
activity by utilizing these online softwares Osiris Property
Explorer, Molinspiration, and PASS computational study.
clogP, clogS, MW, NROTB, Lipinski’s rule of five, drug likeness,
and toxicity were calculated using online Molinspiration
property calculation toolkit and online OSIRIS Property
explorer. With the help of Osiris Property Explorer software,
toxicities were predicted which indicated that the synthesized
compounds would be free of mutagenicity, tumorigenicity,
reproductive side effects and irritation. Potential biological
activities of a compound based on 2D QSAR including drugs,
drug-candidates, leads, and toxic compounds exhibiting
biological activities including pharmacological effects, mech-
anism of action, toxic and adverse effects, interaction with
metabolic enzymes and transporters, influence on gene
expressions, etc. can be predicted by PASS online software
[43, 44]. The PASS prediction software helped in fast
recognition of a set of probable compounds having much
more reliable results of docking. This above online prediction
software helped in quickly identifying a set of probable
compounds and is also useful for screening of compounds for
pharmacological activities.
C
12H9ClN2O3S: C, 48.57; H, 3.06; N, 9.44%. Found: C, 48.54; H,
3.07; N, 9.46%.
2-(3-Bromobenzamido)-4-methylthiazole-5-carboxylic
acid (5r)
Yield: 41%; light brown solid; m.p.: 182–184°C; IR (KBr v max):
3307, 2997, 1761, 1642, 1569, 1453, 1281, 1041, 673 cmꢀ1
;
1H NMR (300 MHz, CDCl3); d 2.48 (s, 3H, CH3), 7.08 (bs, 1H,
D2O exchangeable NH), 8.13 (s, 1H, Ar-H), 8.16 (s, 1H, Ar-H),
8.78 (s, 1H, Ar-H); ESI-MS: m/z 341.27 (Mþ2). Anal. calcd. for
C
12H9BrN2O3S: C, 42.24; H, 2.66; N, 8.21%. Found: C, 42.23; H,
2.65; N, 8.23%.
In silico computational studies
Molecular docking studies
Molecular docking study was carried out using Glide XP
Docking protocol in Schrodinger 9.4 [45–47]. Co-crystal
structure of xanthine dehydrogenase (XDH) with febuxostat
̊
(PDB entry 1N5X; resolution 2.8 A) was selected based on
superior crystal structure parameters and compared with
other XO or XDH structures. There was no difference seen
in the binding sites and co-crystal structures of XO and XDH.
The schematic ligand database is designed on the basis of
previous literature and synthetic schemes utilized for the
structure-based drug design (SBDD). Using LIGPREP module
within Maestro BUILD, ligand was prepared by default
setting. The tautomeric forms of ligands were generated at
physiological pH (7.0 ꢂ 2.0 pH) which includes keto and enol
forms of ligands. Thirty conformations for each ligand were
generated and lowest energy conformers were used for the
docking analysis. Protein was prepared, optimized, and
minimized by Protein Preparation Wizard using OPLS-2005
Biology
Xanthine oxidase inhibitory activity
All the synthesized compounds were evaluated using bovine
milk XO (grade 1, ammonium sulfate suspension, purchased
by Sigma–Aldrich) for XO inhibition assay. For measuring uric
acid formation at 293 nm at 25°C under aerobic condition, the
UV-visible spectrophotometer (EI 2371) was used. The
reaction mixture containing 1 mL xanthine (0.15 mM),
2.5 mL potassium phosphate buffer (50 mM, pH 7.4), and
0.5 mL of XO solution (0.405 U/mL) was incubated for 5 min at
25°C. Inhibition of XO activity by different inhibitors was
calculated by following the decrease in the uric acid creation
at 293 nm at 25°C. The blank was prepared without enzyme
solution. Febuxostat was used as positive control. The enzyme
was pre-incubated for 5 min with test compounds (dissolved
in DMSO, 1% v/v), and the reaction started by the addition of
xanthine. DMSO (1% v/v) did not interfere with the enzyme
̊
force field and the RMSD limit from the structure of 0.3 A
using the Impref module of impact 5.9. Bond orders were
assigned, hydrogen atoms were added, formal charges were
treated, and water molecules were deleted. Active site for
docking was defined as a grid box of dimensions 25 ꢄ 25 ꢄ
3
̊
25 A around the centroid of the ligand assuming that the
ligands to be docked are of similar size as the co-crystallized
ligand. Availability of crystal structure of human XO
facilitated the use of structure-based drug designing strate-
gies for the search of novel bioactive molecules. SBDD
ß 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
(11 of 13) e1600313

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1600313
M. R. Ali et al.
Archiv der Pharmazie
activity. All the experiments were performed in triplicate and
values were stated as mean of three experiments [48–50].
XO activity was expressed as percentage inhibition of XO as
follows:
administration, rats were treated with selected test com-
pounds dissolved in a solution of polyethylene glycol 400
and ethanol (2:1) which were orally administered as 10 mg
and 20 mg/kg, po, and standard (febuxostat 10 mg/kg, po) and
after the time period of 1 h, blood was collected. Serum was
obtained by allowing the blood sample to settle for 1 h at
room temperature and after centrifugation (Sigma 3K-30) at
4000 rpm for 20 min. Serum uric acid levels were measured
spectrophotometrically at 550 nm (at 25°C) using an auto
analyzer (AUTOSPAN Liquid Gold Uric Acid Kit, Span
diagnostic Ltd., Surat, India) in HAH Centenary Hospital,
Jamia Hamdard New Delhi.
XO Inhibition rate ð%Þ ¼ ðAC ꢀ AS=ACÞ ꢄ 100
where AS and AC represent the absorbances of the test
compound and control, respectively. The IC50 values, i.e., the
mg concentration of inhibitor necessary for 50% inhibition,
were determined by using GraphPad (Prism) software.
Four different concentrations of inhibitor 5b were used
for kinetic studies with varying concentration of xanthine
(0.2–1.0 mg/mL)andfixedconcentrationofenzyme(0.0405 U/mL)
in potassium phosphate buffer (50 mM, pH 7.4) [51].
Statistical analysis
GraphPad Prism ver. 5.0 (GraphPad Software, San Diego, CA)
was used for statistical analysis and results are expressed as
mean ꢂ SEM. Groups of data were equated with the analysis
of variance (ANOVA) followed by Dunnett’s t-test. The values
p < 0.001 and p < 0.01 were considered for statistically
significant.
DPPH free radical scavenging assay
The stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) was used
for the determination of free radical scavenging activity
of synthesized compounds (5a–r) by Koleva et al.
method [52]. Dissimilar concentrations of test compounds
(6.25–100 mM) in methanol were added independently to an
equal volume of 100 mM methanolic solution of DPPH and
the reaction mixture was kept at room temperature for
15 min. The absorbance of the reaction mixture was recorded
at 515 nm using a UV visible spectrophotometer. The control
sample contained DPPH and methanol excluding test
compounds. Ascorbic acid was used as standard. % Free
radical scavenging activity was calculated using the follow-
ing formula:
Authors are thankful to Department of Pharmaceutical
Chemistry, Jamia Hamdard, New Delhi for providing necessary
facilities. Thanks is also due to Jamia Hamdard, IIT Delhi and
JNU, New Delhi for recording spectral data. One of the
authors (Md. Rahmat Ali) is grateful to the UGC (Moulana
Azad National Fellowship), New Delhi, India, for providing
financial assistance.
The authors have declared no conflicts of interest.
Free radical scavenging ð%Þ ¼ ðAC ꢀ AS=ACÞ ꢄ 100
where C represents control and S represents sample reaction
mixture as described above in the method. The concentration
of test compounds having 50% radical scavenging activity
(IC50) was calculated by using GraphPad (Prism) software.
References
[1] E. Suresh, Postgrad. Med. J. 2005, 81, 572–579.
[2] A. Shoji, H. Yamanaka, N. Kamatani, Arthritis Rheum.
2004, 51, 321–325.
In vivo efficacy: Animals
Animals were obtained from Central Animal House Facility,
Jamia Hamdard (Hamdard University), New Delhi-110062.
Registration number and date of registration is 173/CPCSEA,
28 Jan, 2000. All the experimental protocols were carried out
with the permission from Institutional Animal Ethics Commit-
tee (IAEC) and Committee for the Purpose of Control and
Supervision on Experiments on Animals (CPCSEA), Govern-
ment of India, application no. 1136.
[3] J. U. Adams, Nat. Biotech. 2009, 27, 309.
[4] P. Richette, T. Bardin, Lancet 2010, 375, 318–328.
[5] Y. Shi, J. Evans, K. Rock, Nature 2003, 425, 516–522.
[6] P. Pacher, A. Nivorozhkin, C. Szabo, Pharmacol. Rev.
2006, 58, 87–114.
[7] I. Fridovich, J. Biol. Chem. 1970, 245, 4053–4057.
[8] C. Paul, L. Ying, C. Mario, P. H. Jia, C. Kanyanga, V. P. Bart,
P. Luc, J. V. Arnold, V. B. Dirk, J. Nat. Prod. 1998, 61,
71–76.
In vivo efficacy: Potassium oxonate-induced hyperuricemic
rat-model assay
[9] B. Halliwell, J. M. C. Gutteridge, C. E. Cross, J. Lab. Clin.
Med. 1992, 119, 598–620.
The compounds showing significant in vitro XO inhibitory
activity in comparison to the standard drug febuxostat were
further evaluated by in vivo animal activity using potassium
oxonate-induced hyperuricemic rat-model assay [52–54].
Overnight fasted Wistar rats of body weight 180–200 g, were
grouped according to serum uric acid levels and then treated
with potassium oxonate (300 mg/kg) intraperitoneally to
induce hyperuricemia. One hour after potassium oxonate
[10] A. Meneshian, G. B. Bulkley, Microcirculation 2002, 9,
161–175.
[11] C. E. Berry, J. M. Hare, J. Physiol. 2004, 555, 589–606.
[12] A. Boueiz, M. Damarla, P. M. Hassoun, Am. J. Physiol.
Lung. Cell. Mol. Physiol. 2008, 294, L830–L840.
[13] M. Houston, A. Estevez, P. Chumley, M. Aslan,
S. Marklund, D. A. Parks, B. A. Freeman, J. Biol. Chem.
1999, 274, 4985–4994.
ß 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
(12 of 13) e1600313

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1600313
Thiazoles as Potential Xanthine Oxidase Inhibitors
Archiv der Pharmazie
[14] U. Landmesser, S. Spiekermann, S. Dikalov, H. Tatge,
R. Wilke, C. Kohler, D. G. Harrison, B. Hornig, H. Drexler,
Circulation 2002, 10, 3073–3078.
[15] Z. Ungvari, S. A. Gupte, S. Rkai, F. A. Recchia, S. Batkai,
P. Pacher, Curr. Vasc. Pharmacol. 2005, 3, 221–229.
[16] M. C. Desco, M. Asensi, R. Marquez, J. Martinez-Valls,
M. Vento, F. V. Pallardo, J. Sastre, J. Vina, Diabetes 2002,
51, 1118–1124.
[17] R. Hille, Eur. J. Inorg. Chem. 2006, 10, 1913–1926.
[18] F. Arellano, J. A. Sacristan, Ann. Pharmacother. 1993, 27,
337–343.
[19] K. Okamoto, B. T. Eger, T. Nishino, S. Kondo, E. F. Pai,
T. Nishino, J. Biol. Chem. 2003, 278, 1848–1855.
[20] G. W. Smith, V. Wright, Br. J. Clin. Pract. 1987, 41,
710–711.
[35] P. C. Mhaske, K. S. Vadgaonkar, R. P. Jadhav,
V. D. Bobade, J. Kor. Chem. Soc. 2011, 55, 882–886.
[36] A. G. N. Christian, C. A. G. N. Montalbetti, V. Falque,
Tetrahedron 2005, 61, 10827–10852.
[37] T. Hou, J. Wang, W. Zhang, X. Xu, J. Chem. Inf. Model.
2007, 47, 460–463.
[38] Molinspiration software or free molecular property
calculation services (2 screens). Available from URL:
www.molinspiration.com/cgi-bin/properties (last accessed
11.02.16).
[39] T. Sander, Osiris Property Explorer, 2001. Available from
URL: http://www.organic-chemistry.org/prog/peo/ (last
accessed 18.02.16).
[40] N. A. Meanwell, Chem. Res. Toxicol. 2001, 24, 1420–1456.
ꢀ
[41] A. Tarcsay, K. Nyıri, G. M. Keseru, J. Med. Chem. 2012, 55,
[21] A. M. Youssef, E. G. Neel, E. B. Villanueva, M. S. White,
I. M. El-Ashmawy, B. Patrick, A. Klegeris, A. S. Abd-El-Aziz,
Bioorg. Med. Chem. 2010, 18, 5685–5696.
[22] M. K. Reinders, T. L. Jansen, Clin. Interv. Ageing 2010, 5,
7–18.
[23] S. Ishibuchi, H. Morimoto, T. Oe, T. Ikebe, H. Inoue,
A. Fukunari, M. Kamezawa, I. Yamada, Y. Naka, Bioorg.
Med. Chem. Lett. 2001, 11, 879–882.
1252–1260.
€
[42] A. L. Hopkins, G. M. Keseru, P. D. Leeson, D. C. Rees,
C. H. Reynolds, Nat. Rev. Drug Discov. 2014, 13,
105–121.
[43] A. Lagunin, A. Stepanchikova, D. Filimonov, V. Poroikov,
Bioinformatics 2000, 16, 747–748.
[44] PASS software free molecular property calculation
services.
Available
from
URL:
http://www.
[24] “New Drugs FY”. (2013), Pharmaceuticals and Medical
Devices Agency, Japan.
pharmaexpert.ru/passonline/predict.php (last accessed
21.02.16).
€
[25] T. Sato, N. Ashizawa, K. Matsumoto, T. Iwanaga,
H. Nakamura, T. Inoue, O. Nagata, Bioorg. Med. Chem.
Lett. 2009, 19, 6225–6229.
[26] Synthesis of Lesinurad, AstraZeneca’s potential block-
buster drug for gout. Published 24 February (2014).
[27] M. R. Ali, S. Kumar, O. Afzal, N. Shalmali, M. Sharma,
S. Bawa, Chem. Biol. Drug Des. 2016, 87, 508–516.
[28] Q. Guan, Z. Cheng, X. Ma, L. Wang, D. Feng, Y. Cui,
K. Bao, L. Wu, W. Zhang, Eur. J. Med. Chem. 2014, 85,
508–516.
[45] Maestro. (2013) Version 9.4, Schrodinger, LLC,
New York, NY.
[46] J. P. Hughes, S. Rees, S. B. Kalindjian, K. L. Philpott, Br. J.
Pharmacol. 2011, 162, 1239–1249.
[47] C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney,
Adv. Drug Deliv. Rev. 2001, 46, 3–26.
[48] P. Valentao, E. Fernandes, F. Carvalho, P. B. Andrade,
R. M. Seabra, M. L. Bastos, J. Agric. Food Chem. 2001, 49,
3476–3479.
[49] S. D. Beedkar, C. N. Khobragade, S. S. Chobe,
B. S. Dawane, O. S. Yemul, Int. J. Biol. Macromol.
2012, 50, 947–956.
[29] R. Kumar, K. Darpan, S. Sharma, R. Singh, Expert. Opin.
Ther. Patents 2011, 21, 1071–1108.
[30] S. Wang, J. Yan, J. Wang, J. Chen, T. Zhang, Y. Zhao,
M. Xue, Eur. J. Med. Chem. 2010, 4, 2663–2670.
[31] J. U. Song, S. P. Choi, T. H. Kim, C. K. Jung, J. Y. Lee,
S. H. Jung, G. T. Kim, Bioorg. Med. Chem. Lett. 2015, 25,
1254–1258.
[32] S. Chen, T. Zhang, J. Wang, F. Wang, H. Niu, C. Wu,
S. Wang, Eur. J. Med. Chem. 2015, 103, 343–353.
[33] Process for the preparation of febuxostat and salts
thereof, Patent No-WO2011/073617A1.
[50] L. D. Kong, X. Pan, R. X. Tan, C. H. Cheng, Cell. Mol. Life.
Sci. 2000, 57, 500–505.
[51] L. Costantino, G. Rastrelli, A. Albasini, Int. J. Pharm. 1992,
86, 17–23.
[52] I. I. Koleva, T. A. Van-Beek, J. P. Linssen, A. de-Groot,
L. N. Evstatieva, Phytochem. Anal. 2002, 13, 8–17.
[53] Y. Osada, M. Tsuchimoto, H. Fukushima, K. Takahashi,
S. Kondo, M. Hasegawa, K. Komoriya, Eur. J. Pharmacol.
1993, 241, 183–188.
[34] Aminothiazole derivatives as human stearoylcoa desa-
turase inhibitors, Patent No-WO2007130075A1.
[54] L. Kuraka, T. Kalnovicova, B. Lia, P. Turanil, Clin. Chem.
1996, 42, 756–760.
ß 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
(13 of 13) e1600313