Thiazole-5-carboxylic acid derivatives as potent xanthine oxidase inhibitors: design, synthesis, in vitro evaluation, and molecular modeling studies

Article abstract of DOI:10.1007/s00044-019-02461-y

A series of 22 compounds of thiazole-5-carboxylic acid derivatives was rationally designed and synthesized. All the compounds were characterized by using ¹H and ¹³C NMR and tested against xanthine oxidase enzyme by spectrophotometric assay. Majority of the compounds were found active against the enzyme amongst which GK-20 with an IC₅₀ value of 0.45 μM was found to be most potent. Structure-activity relationship obtained from the biological results revealed that the di-substituted compounds as Ring B were more potent than that of mono-substituted derivatives. Para-substitution on Ring B is crucial for the xanthine oxidase inhibitory potential. Enzyme kinetic studies further revealed their mixed type inhibition behavior. Moreover, the binding pattern of the most potent compound GK-20 within the febuxostat binding site of the enzyme was further analyzed by using docking studies which revealed that it sufficiently block the catalytic active site, which prevents the substrate to bind.

Full text of DOI:10.1007/s00044-019-02461-y

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02461-y
ORIGINAL RESEARCH
Thiazole-5-carboxylic acid derivatives as potent xanthine oxidase
inhibitors: design, synthesis, in vitro evaluation, and molecular
modeling studies
Gurinder Kaur1 Jatinder V. Singh1 Manish K. Gupta2 Kavita Bhagat1 Harmandeep K. Gulati1 Atamjit Singh1
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Preet Mohinder S. Bedi1 Harbinder Singh
Sahil Sharma1
1
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Received: 14 December 2018 / Accepted: 14 October 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
A series of 22 compounds of thiazole-5-carboxylic acid derivatives was rationally designed and synthesized. All the
compounds were characterized by using 1H and 13C NMR and tested against xanthine oxidase enzyme by
spectrophotometric assay. Majority of the compounds were found active against the enzyme amongst which GK-20 with
an IC50 value of 0.45 µM was found to be most potent. Structure-activity relationship obtained from the biological results
revealed that the di-substituted compounds as Ring B were more potent than that of mono-substituted derivatives. Para-
substitution on Ring B is crucial for the xanthine oxidase inhibitory potential. Enzyme kinetic studies further revealed their
mixed type inhibition behavior. Moreover, the binding pattern of the most potent compound GK-20 within the febuxostat
binding site of the enzyme was further analyzed by using docking studies which revealed that it sufficiently block the
catalytic active site, which prevents the substrate to bind.
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Keywords Thiazole derivatives Xanthine oxidase inhibition Enzyme kinetics Molecular modeling
Introduction
hydroxylation of hypoxanthine, and xanthine to produce
uric acid (Stockert et al. 2002; Borges et al. 2002; Hille
2006). It is widely distributed, occurring in milk, kidney,
lungs, heart, and vascular endothelium but extremely active
in liver and intestine in the human body (Dhiman et al.
2012). The functional aberrations of XO lead to many
diseases like gout and oxidative damage to the tissue (White
2018).
Xanthine oxidase (XO) is a form of xanthine oxidor-
eductase, a versatile molybdoflavoprotein that involved in
the metabolism of purines, catalyzing the oxidative
These authors contributed equally: Gurinder Kaur, Jatinder V. Singh
Consequently, XO is an intensified target, inhibition of
which may result in broad-spectrum chemotherapeutic for
gout, cancer, inflammation, and oxidative damage (Stockert
et al. 2002; Borges et al. 2002; Hille 2006; Pacher et al.
2006). Allopurinol (1) (Fig. 1), a purine analog, was
approved by the Food and Drug Administration (1966),
which acts as a suicide inhibitor of XO by its active
metabolite oxypurinol. Oxypurinol can bind only to the
reduced/active form of XO (Borges et al. 2002; Massey
et al. 1970; Krakoff 1966). However, due to some reported
side effects like Steven–Johnson syndrome, worsening of
renal function etc. associated with purine backbone of
allopurinol, limits its clinical use and led to a search for
nonpurine-based XO inhibitors. (Star and Hochberg 1993;
Terkeltaub 1993)
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02461-y) contains supplementary
material, which is available to authorized users.
* Preet Mohinder S. Bedi
bedi_preet@yahoo.com
* Harbinder Singh
singh.harbinder40@gmail.com
* Sahil Sharma
ss.gq2009@gmail.com
1
Department of Pharmaceutical Sciences, Guru Nanak Dev
University, Amritsar, Punjab 143005, India
2
TERI-Deakin Nanobiotechnology Centre, The Energy and
Resources Institute, TERI Gram, Gual Pahari, Gurugram, Haryana
122001, India

Medicinal Chemistry Research
Br
O
O
N
OH
Br
S
O
OH
CH3
HN
O
N
N
N
H
NC
(1)
(3)
O
CH3
(2)
H
N
O
H
N
S
N
O
NO2
N
S
OH
H
N
H
N
O
H3C
S
S
OH
CH3
N
NH2
CH3
O
OH
CN
N
HO
N
O
H3C
(5)
(6)
(7)
H3C
Cl
(4)
Amide Linker
O
Febuxostat architecture
O
C
S
R
Arbitrary Region
HO
H3C
H
N
A
B
N
Target compounds
Fig. 1 Reported xanthine oxidase inhibitors and target compounds
In 1970, a nonpurine-based drug named Benzbromarone
(2) (Fig. 1) was introduced that promotes the excretion of
urate in kidney but not an XO inhibitor itself. Due to its
serious hepatotoxicity, it was rapidly withdrawn from the
market but still marketed in some European countries
(Sinclair and Fox 1975).
After certain efforts, new nonpurine, thiazole containing
drug has been introduced in the market named as Febuxo-
stat (3) (Fig. 1), an oral, potent, and selective XO inhibitor
with lesser side effects as compared with allopurinol/oxy-
purinol. It can bind both oxidized and reduced forms of XO
(Okamoto et al. 2003; Zhao et al. 2003; Horiuchi et al.
1999). Complete XO inhibition was observed with 25 nM
febuxostat, whereas no more than 80% inhibition was seen
with either allopurinol or oxypurinol, even at concentrations
above those tolerated clinically. The success of this prime,
nonpurine XO inhibitor has attracted worldwide attention to
develop a structurally diverse array of molecules without
purine skeleton.
methyl-1,3-thiazole-5-carboxylic acid, (6) (Ali et al. 2016),
2-(7-nitro-5-isopropoxy-indol-2-yl)-4-methylthiazole-5-car-
boxylic acid (7) (Song et al. 2016). (Fig. 1). With these
successive measurements, a new series of thiazole deriva-
tives have been synthesized by keeping intact the thiazole
free acid architecture (Ring A) of febuxostat attached with
the arbitrary region (Ring B) via amide linker (Fig. 1).
Various synthesized analogs were evaluated for their inhi-
bitory potential against XO enzyme using spectro-
photometric assay. The type of inhibition and the
interactions of the most potent inhibitor within the active
site of XO had also been streamlined by using molecular
modeling studies.
Experimental
Materials and measurements
In similar lines, we are also actively involved in this area
for the development of potent nonpurine-based XO inhibi-
tors and have been reported a number of alternate nonpurine
molecules in recent past with great potential against XO
(Dhiman et al. 2012; Shukla et al. 2014; Sharma et al. 2014;
Virdi et al. 2014; Kaur et al. 2015, 2015, 2017; Singh et al.
2014, 2019). Reports on nonpurine thiazole containing XO
inhibitors with promising inhibitory potential are also well
documented, such as pyrazolo[3,4-d] thiazolo[3,2-a]pyr-
imidin-4-one (4) (Khobragade et al. 2010), 2-(3-cyano-2-
isopropyl-1H-indol-5-yl)-4-methylthiazole-5-carboxylic
acid (5) (Song et al. 2015), 2-(2-methylbenzylamino)-4-
The reagents were purchased from Sigma Aldrich, Loba and
CDH, India and used without further purification. All yields
refer to isolated products after purification. Products were
characterized by comparison with authentic samples and by
spectroscopic data (1H and 13C NMR and MASS). 1H NMR
and 13C NMR Spectra were recorded on JEOL AL 300
NMR Spectrometer. The spectra were measured in CDCl3
1
relative to TMS (0.00 ppm). In H NMR chemical shifts
were reported in δ values using tetramethylsilane as an
internal standard with a number of protons, multiplicities (s-
singlet, d-doublet, t-triplet, m-multiplet) and coupling con-
stants (J) in Hz (Hertz) in the solvent indicated. HRMS was

Medicinal Chemistry Research
recorded on micrOTOF-QII Bruker Daltonik LC–MS/MS
High Resolution Mass Spectrometer. Melting points were
determined in open capillaries and were uncorrected.
Bovine Milk XO of grade I ammonium sulfate suspension
was purchased from Sigma Aldrich.
Procedure for synthesis of 2-(benzamido)-4-methylthiazole-
5-carboxylic acid derivatives (GK-1 to GK-22)
Various substituted derivatives of ethyl 2-(benzamido)-4-
methylthiazole-5-carboxylate and potassium carbonate (1
equivalent) were added to a solution of methanol: water
(9:1) and refluxed to break the ester into an acid. On
completion of the reaction, the reaction mixture was cooled
to room temperature and neutralized by adding dropwise
glacial acetic acid. Precipitates of desired product so
obtained were then filtered, washed with cold water, and
recrystallized with ethanol.
Procedure for synthesis of ethyl 2-amino-4-methylthiazole-
5-carboxylate (8)
To a solution of thiourea (0.013 mole) in absolute ethanol
(15 mL), 2-chloroethylacetoacetate (1 equivalent) was
added dropwise with continuous stirring at room tempera-
ture (Ali et al. 2016). After the complete addition of 2-
chloroethylacetoacetate, the reaction mixture was warmed
on a water bath and the completion of the reaction was
monitored by using TLC. After the completion of the
reaction, the reaction mixture was cooled at room tem-
perature, the solid crystalline product so obtained was
washed with 10 mL of ethanol and then treated with satu-
rated sodium hydrogen carbonate to obtain white pre-
cipitates of ethyl 2-amino-4-methylthiazole-5-carboxylate.
The characterization data for ethyl 2-amino-4-methylthia-
zole-5-carboxylate are as follows:
Characterization data of all the synthesized compounds
are given below:
2-(benzamido)-4-methylthiazole-5-carboxylic acid (GK-1)
Yield 80%, mp 133–136 °C. 1H NMR (DMSO-d6,
300 MHz, δ, TMS = 0): 10.77 (1H, bs), 7.91–7.94 (2H, m),
7.60–7.63 (1H, m), 7.49–7.54 (2H, m), 2.44 (3H, s). 13C
NMR (CDCl3, 75 MHz, δ, TMS = 0): 20.19, 131.12,
132.13, 132.58, 133.25, 135.08, 136.14, 152.60, 160.02,
162.70, 168.64. Anal. Calcd. MS: 262.0412; Found m/z:
263.0443 (M+ + 1). Anal. Calcd for C12H10N2O3S: C,
54.95; H, 3.84; N, 10.68; O, 18.30; S, 12.23; Found: C,
54.85; H, 3.92; N, 10.49.
Yield: 90%, mp: 170–174 °C. 1H NMR (CDCl3,
300 MHz, δ, TMS = 0): 4.12 (2H, q, J = 7.2 Hz), 2.05 (2H,
s), 1.69 (2H, bs), 1.23–1.28 (3H, m) (14-09-2016). 13C
NMR (CDCl3, 75 MHz, δ, TMS = 0): 16.81, 19.28, 63.26,
160.04, 165.01, 172.14 (28-10-2016). Anal. Calcd. MS:
186.0463; Found m/z: 187.0495 (M+ + 1). Anal. Calcd. For
C7H10N2O2S: C, 45.15; H, 5.41; N, 15.04; O, 17.18; S,
17.22; Found: C, 45.22; H, 5.35; N, 15.22; S, 17.12.
2-(2-fluorobenzamido)-4-methylthiazole-5-carboxylic acid
1
(GK-2) Yield 77%, mp 170–174 °C. H NMR (DMSO-d6,
300 MHz, δ, TMS = 0): 9.96 (1H, bs), 8.20–8.25 (1H, m),
7.61–7.63 (1H, m), 7.20–7.40 (3H, m), 5.32 (1H. s), 2.63
(3H, s). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 17.52,
125.56, 126.57, 127.64, 158.27, 159.46, 159.94, 163.36,
166.37, 168.54. Anal. Calcd. MS: 280.0318; Found m/z:
281.0349 (M+ + 1). Anal. Calcd for C12H9FN2O3S: C,
51.42; H, 3.24; F, 6.78; N, 9.99; O, 17.13; S, 11.44; Found:
C, 51.54; H, 3.12; N, 10.05.
Procedure for synthesis of ethyl 2-(benzamido)-4-
methylthiazole-5-carboxylate derivatives (9)
To a solution of ethyl 2-amino-4-methylthiazole-5-carbox-
ylate (0.005 mole) and pyridine (4 mL), substituted ben-
zoylchlorides (1 equivalent) were added and kept at room
temperature for 1 h. Completion of the reaction was mon-
itored by TLC. After the completion of reaction, the reac-
tion mixture was poured on crushed ice, solid mass so
obtained was filtered and washed with cold water. The
characterization data for ethyl 2-(3,5-difluorobenzamido)-4-
methylthiazole-5-carboxylate are as follows:
2-(3-fluorobenzamido)-4-methylthiazole-5-carboxylic acid
1
(GK-3) Yield 78%, mp 170–176 °C. H NMR (DMSO-d6,
300 MHz, δ, TMS = 0): 10.56 (1H, bs), 7.53–7.70 (2H, m),
7.46–7.51 (1H, m), 7.30–7.35 (1H, m), 2.53 (3H, s). 13C
NMR (CDCl3, 75 MHz, δ, TMS = 0): 17.41, 125.57,
126.54, 127.63, 158.26, 159.43, 159.96, 163.35, 166.34,
168.55. Anal. Calcd. MS: 280.0318; Found m/z: 281.0350
(M+ + 1). Anal. Calcd for C12H9FN2O3S: C, 51.42; H,
3.24; F, 6.78; N, 9.99; O, 17.13; S, 11.44; Found: C, 51.33;
H, 3.30; N, 9.93.
Yield: 82%, mp: 98–100 °C. 1H NMR (CDCl3,
300 MHz, δ, TMS = 0): 7.50 (2H, d, J = 6.0 Hz), 7.05–7.11
(1H, m), 4.34 (2H, q, J = 7.2 Hz), 2.59 (3H, s), 1.38 (3H, t,
J = 7.2 Hz) (3-jan-2017). 13C NMR (CDCl3, 75 MHz, δ,
TMS = 0): 16.81, 19.28, 63.26, 160.04, 165.01, 172.14 (28-
10-2016). Anal. Calcd. MS: 326.0537; Found m/z:
327.0567 (M+ + 1). Anal. Calcd. For C14H12F2N2O3S: C,
51.53; H, 3.71; F, 11.64; N, 8.58; O, 14.71; S, 9.83; Found:
C, 51.62; H, 3.66; F, 11.75; N, 8.44; S, 9.99.
2-(4-fluorobenzamido)-4-methylthiazole-5-carboxylic acid
1
(GK-4) Yield 91%, mp 155–159 °C. H NMR (DMSO-d6,
300 MHz, δ, TMS = 0): 10.65 (1H, bs), 8.11 (2H, d, J =
9.0 Hz), 7.42 (2H, m), 2.65 (3H, s). 13C NMR (CDCl3,
75 MHz, δ, TMS = 0): 17.54, 125.53, 126.58, 127.67, 158.24,

Medicinal Chemistry Research
159.42, 159.96, 163.34, 166.33, 168.58. Anal. Calcd. MS:
280.0318; Found m/z: 281.0348 (M+ + 1). Anal. Calcd for
C12H9FN2O3S: C, 51.42; H, 3.24; F, 6.78; N, 9.99; O, 17.13;
S, 11.44; Found: C, 51.44; H, 3.18; N, 10.02.
(CDCl3, 75 MHz, δ, TMS = 0): 17.82, 121.55, 125.57,
126.58, 127.63, 133.25, 135.63, 158.24, 159.46, 159.93,
163.35, 168.58. Anal. Calcd. MS: 339.9517; Found m/z:
341.9494 (M+ + 1). Anal. Calcd for C12H9BrN2O3S: C,
42.31; H, 2.66; Br, 23.42; N, 8.21; O, 14.07; S, 9.40;
Found: C, 42.38; H, 2.42; N, 8.244.
2-(3,5-difluorobenzamido)-4-methylthiazole-5-carboxylic
acid (GK-5) Yield 89%, mp 162–168 °C. 1H NMR
(DMSO-d6, 300 MHz, δ, TMS = 0): 10.58 (1H, bs), 7.50
(2H, d, J = 6 Hz), 7.08 (1H, s), 2.58 (3H, s). 13C NMR
(CDCl3, 75 MHz, δ, TMS = 0): 17.78, 125.56, 126.52,
127.63, 129.24, 131.68, 145.63, 153.26, 158.23, 159.44,
159.94, 163.35, 168.52. Anal. Calcd. MS: 298.0224; Found
m/z: 299.0256 (M+ + 1). Anal. Calcd for C12H8F2N2O3S:
C, 48.32; H, 2.70; F, 12.74; N, 9.39; O, 16.09; S, 10.75;
Found: C, 48.44; H, 2.68; N, 9.46.
2-(4-bromobenzamido)-4-methylthiazole-5-carboxylic acid
1
(GK-10) Yield 91%, mp 201–203 °C. H NMR (DMSO-
d6, 300 MHz, δ, TMS = 0): 10.76 (1H, bs), 7.99 (2H, d, J =
8.4 Hz), 7.77–7.82 (2H, m), 2.63 (3H, s). 13C NMR (CDCl3,
75 MHz, δ, TMS = 0): 17.83, 121.57, 125.53, 126.52,
127.65, 133.23, 135.66, 158.27, 159.43, 159.95, 163.32,
168.53. Anal. Calcd. MS: 339.9517; Found m/z: 341.9493
(M+ + 1). Anal. Calcd for C12H9BrN2O3S: C, 42.31; H,
2.66; Br, 23.42; N, 8.21; O, 14.07; S, 9.40; Found: C,
42.23; H, 2.56; N, 8.232.
2-(3-chlorobenzamido)-4-methylthiazole-5-carboxylic acid
1
(GK-6) Yield 71%, mp 147–151 °C. H NMR (DMSO-d6,
300 MHz, δ, TMS = 0): 10.56 (1H, bs), 7.50–7.62 (2H, m),
7.46–7.51 (1H, m), 7.28–7.31 (1H, m), 2.51 (3H, s). 13C
NMR (CDCl3, 75 MHz, δ, TMS = 0): 17.41, 125.57,
126.54, 127.63, 158.26, 159.43, 159.96, 163.35, 166.34,
168.55. Anal. Calcd. MS: 296.0022; Found m/z:
297.97.9991 (M+ + 1). Anal. Calcd for C12H9ClN2O3S: C,
48.57; H, 3.06; Cl, 11.95; N, 9.44; O, 16.18; S, 10.81;
Found: C, 48.64; H, 3.01; N, 9.55.
2-(2-iodobenzamido)-4-methylthiazole-5-carboxylic
acid
1
(GK-11) Yield 76%, mp 124–130 °C. H NMR (DMSO-
d6, 300 MHz, δ, TMS = 0): 10.71 (1H, bs), 7.65–7.77 (2H,
m), 7.38-7.47 (2H, m), 4.83 (1H, bs), 2.36 (3H, s). 13C
NMR (CDCl3, 75 MHz, δ, TMS = 0): 17.20, 95.77, 119.03,
131.47, 132.32, 135.75, 142.04, 143.68, 159.12, 163.71,
165.55, 170.13. Anal. Calcd. MS: 387.9379; Found m/z:
388.9410 (M+ + 1). Anal. Calcd for C12H9IN2O3S: C,
37.13; H, 2.34; I, 32.69; N, 7.22; O, 12.36; S, 8.26; Found:
C, 37.24; H, 2.19; N, 7.21.
2-(2,3-dichlorobenzamido)-4-methylthiazole-5-carboxylic
acid (GK-7) Yield 75%, mp 174–178 °C. 1H NMR
(DMSO-d6, 300 MHz, δ, TMS = 0): 10.74 (1H, bs), 8.02
(1H, m), 7.62 (1H, m), 7.44 (1H, m), 2.61 (3H, s). 13C
NMR (CDCl3, 75 MHz, δ, TMS = 0): 17.85, 125.58,
126.59, 127.65, 133.68, 135.24, 153.26, 158.26, 159.48,
159.95, 163.38, 168.58. Anal. Calcd. MS: 331.9633; Found
m/z: 331.9601 (M+ + 1). Calcd for C12H8Cl2N2O3S: C,
43.52; H, 2.43; Cl, 21.41; N, 8.46; O, 14.49; S, 9.68;
Found: C, 43.66; H, 2.33; N, 8.67.
2-(3-nitrobenzamido)-4-methylthiazole-5-carboxylic
acid
1
(GK-12) Yield 84%, mp 180–188 °C. H NMR (DMSO-
d6, 300 MHz, δ, TMS = 0): 10.76 (1H, bs), 9.01 (1H, s),
8.62–7.70 (2H, m), 7.99–8.03 (1H, m), 2.67 (3H, s). 13C
NMR (CDCl3, 75 MHz, δ, TMS = 0): 17.79, 121.53,
125.55, 126.56, 133.27, 135.64, 147.73, 158.26, 159.44,
159.97, 163.36, 168.57. Anal. Calcd. MS: 307.0263; Found
m/z: 308.0294 (M+ + 1). Anal. Calcd for C12H9N3O5S: C,
46.90; H, 2.95; N, 13.67; O, 26.03; S, 10.44; Found: C,
46.99; H, 2.88; N, 13.73.
2-(2-bromobenzamido)-4-methylthiazole-5-carboxylic acid
1
(GK-8) Yield 87%, mp 137–139 °C. H NMR (DMSO-d6,
300 MHz, δ, TMS = 0): 10.75 (1H, bs), 8.05 (1H, d, J =
9.0 Hz), 7.82 (1H, m), 7.65 (1H, m), 7.49 (1H, m), 2.64
(3H, s). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 17.80,
121.54, 125.52, 126.53, 127.64, 133.24, 135.68, 158.28,
159.43, 159.96, 163.33, 168.55. Anal. Calcd. MS:
339.9517; Found m/z: 341.9495 (M+ + 1). Anal. Calcd for
C12H9BrN2O3S: C, 42.3; H, 2.66; Br, 23.42; N, 8.21; O,
14.07; S, 9.40; Found: C, 42.35; H, 2.45; N, 8.235.
2-(4-nitrobenzamido)-4-methylthiazole-5-carboxylic
acid
1
(GK-13) Yield 87%, mp 210–212 °C. H NMR (DMSO-d6,
300 MHz, δ, TMS = 0): 10.73 (1H, bs), 8.55–8.59 (2H, m),
8.32 (2H, d, J = 8.4 Hz), 2.65 (3H, s). 13C NMR (CDCl3,
75 MHz, δ, TMS = 0): 17.80, 121.56, 125.58, 126.55, 133.25,
135.67, 147.78, 158.28, 159.43, 159.96, 163.32, 168.55. Anal.
Calcd. MS: 307.0263; Found m/z: 308.0293 (M+ + 1). Anal.
Calcd for C12H9N3O5S: C, 46.90; H, 2.95; N, 13.67; O, 26.03;
S, 10.44; Found: C, 47.01; H, 2.81; N, 13.78.
2-(3-bromobenzamido)-4-methylthiazole-5-carboxylic acid
1
(GK-9) Yield 81%, mp 180–185 °C. H NMR (DMSO-d6,
2-(3,5-dinitrobenzamido)-4-methylthiazole-5-carboxylic
acid (GK-14) Yield 92%, mp 208–214 °C. 1H NMR
(DMSO-d6, 300 MHz, δ, TMS = 0): 10.77 (1H, bs), 9.48
300 MHz, δ, TMS = 0): 10.71 (1H, bs), 8.23 (1H, s),
7.93–9.04 (2H, m), 7.55 (1H, m), 2.66 (3H, s). 13C NMR

Medicinal Chemistry Research
(1H, s), 9.177 (1H, s), 7.48 (1H, s), 2.66 (3H, s). 13C NMR
(CDCl3, 75 MHz, δ, TMS = 0): 17.20, 95.77, 119.03,
131.47, 132.32, 135.75, 142.04, 143.68, 159.12, 163.71,
165.55, 170.13. Anal. Calcd. MS: 352.0114; Found m/z:
353.0145 (M+ + 1). Anal. Calcd for C12H8N4O7S: C, 40.91;
H, 2.29; N, 15.90; O, 31.79; S, 9.10; Found: C, 40.98; H,
2.34; N, 15.83.
2-(4-trifluoromethylbenzamido)-4-methylthiazole-5-car-
boxylic acid (GK-19) Yield 77%, mp 134–141 °C. 1H NMR
(DMSO-d6, 300 MHz, δ, TMS = 0): 10.74 (1H, bs), 8.21
(2H, d, J = 8.0 Hz), 7.78–7.83 (2H, m), 2.43 (3H, s). 13C
NMR (CDCl3, 75 MHz, δ, TMS = 0): 17.19, 23.45, 120.64,
124.54, 126.65, 132.32, 142.04, 143.68, 159.17, 163.78,
165.54, 170.17. Anal. Calcd. MS: 330.286; Found m/z:
331.318 (M+ + 1). Anal. Calcd for C13H9F3N2O3S: C,
47.27; H, 2.75; F, 17.26; N, 8.48; O, 14.53; S, 9.71; Found:
C, 47.32; H, 2.56; F, 17.45; N, 8.32; S, 9.56.
2-(4-methylbenzamido)-4-methylthiazole-5-carboxylic acid
1
(GK-15) Yield 77%, mp 161–164 °C. H NMR (DMSO-
d6, 300 MHz, δ, TMS = 0): 10.77 (1H, bs), 8.00 (2H, d, J =
8.0 Hz), 7.35–7.40 (2H, m), 2.63 (3H, s), 2.43 (3H, s). 13C
NMR (CDCl3, 75 MHz, δ, TMS = 0): 17.19, 23.45, 120.64,
124.54, 126.65, 132.32, 142.04, 143.68, 159.17, 163.78,
165.54, 170.17. Anal. Calcd. MS: 276.0569; Found m/z:
277.0599 (M+ + 1). Anal. Calcd for C13H12N2O3S: C,
56.51; H, 4.38; N, 10.14; O, 17.37; S, 11.60; Found: C,
56.57; H, 4.24; N, 10.27.
2-(3,5-difluoromethylbenzamido)-4-methylthiazole-5-car-
boxylic acid (GK-20) Yield 91%, mp 182–190 °C. 1H NMR
(DMSO-d6, 300 MHz, δ, TMS = 0): 10.76 (1H, bs),
8.22–8.27 (2H, m), 7.98 (1H, m), 2.74 (3H, s). 13C NMR
(CDCl3, 75 MHz, δ, TMS = 0): 18.85 (thiazole-CH3),
125.56, 126.52, 127.63, 129.24, 131.68, 145.63, 153.26,
158.23, 159.44, 159.94, 163.35 (CONH), 168.52 (C–O).
Anal. Calcd. MS: 398.0160; Found m/z: 399.0190 (M+ +
1). Anal. Calcd for C14H8F6N2O3S: C, 42.22; H, 2.02; F,
28.62; N, 7.03; O, 12.05; S, 8.05; Found: C, 42.27; H, 1.98;
F, 28.74; N, 6.99; S, 8.12.
2-(3,5-dimethylbenzamido)-4-methylthiazole-5-carboxylic
acid (GK-16) Yield 90%, mp 184–189 °C. 1H NMR
(DMSO-d6, 300 MHz, δ, TMS = 0): 10.43 (1H, bs), 8.21
(2H, s), 7.45 (1H, m), 2.68 (3H, s), 2.46 (6H, s). 13C NMR
(CDCl3, 75 MHz, δ, TMS = 0): 20.19, 120.67, 123.83,
124.57, 126.63, 132.36, 142.03, 143.64, 159.13, 163.73,
165.58, 170.11. Anal. Calcd. MS: 290.0725; Found m/z:
291.0757 (M+ + 1). Anal. Calcd for C14H14N2O3S: C, 57.9;
H, 4.86; N, 9.65; O, 16.53; S, 11.04; Found: C, 57.82; H,
4.92; N, 9.61.
2-(4-methoxybenzamido)-4-methylthiazole-5-carboxylic
acid (GK-21) Yield 91%, mp 157–162 °C. 1H NMR
(DMSO-d6, 300 MHz, δ, TMS = 0): 10.71 (1H, bs), 8.17
(2H, d, J = 8.5 Hz), 7.11 (2H, d, J = 8.5 Hz), 3.61 (3H, s),
2.62 (3H, s). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0):
17.34, 60.34, 125.59, 126.56, 127.62, 153.22, 158.21,
159.46, 159.93, 163.37, 165.34, 168.54. Anal. Calcd. MS:
292.0518; Found m/z: 293.0549 (M+ + 1). Anal. Calcd for
C13H12N2O4S: C, 53.42; H, 4.14; N, 9.58; O, 21.89; S,
10.97; Found: C, 53.34; H, 4.19; N, 9.51.
2-(2-trifluoromethylbenzamido)-4-methylthiazole-5-car-
boxylic acid (GK-17) Yield 76%, mp 145–149 °C. 1H NMR
(DMSO-d6, 300 MHz, δ, TMS = 0): 10.71 (1H, bs),
7.65–7.77 (2H, m), 7.38–7.47 (2H, m), 4.83 (1H, bs), 2.36
(3H, s). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 17.20,
95.77, 119.03, 131.47, 132.32, 135.75, 142.04, 143.68,
159.12, 163.71, 165.55, 170.13. Anal. Calcd. MS: 330.286;
Found m/z: 331.319 (M+ + 1). Anal. Calcd for
C13H9F3N2O3S: C, 47.27; H, 2.75; F, 17.26; N, 8.48; O,
14.53; S, 9.71; Found: C, 47.12; H, 2.76; F, 17.23; N, 8.56;
S, 9.65.
2-(3,4-dimethoxybenzamido)-4-methylthiazole-5-carboxylic
acid (GK-22) Yield 91%, mp 143–145 °C. 1H NMR
(DMSO-d6, 300 MHz, δ, TMS = 0): 10.71 (1H, bs), 8.19
(1H, s), 7.23–7.31 (2H, m), 3.61 (3H, s), 2.65 (6H, s). 13C
NMR (CDCl3, 75 MHz, δ, TMS = 0): 17.34, 60.34, 125.59,
126.56, 127.62, 153.22, 158.21, 159.46, 159.93, 163.37,
165.34, 168.54. Anal. Calcd. MS: 322.0623; Found m/z:
323.0655 (M+ + 1). Anal. Calcd for C14H14N2O5S: C,
52.17; H, 4.38; N, 8.69; O, 24.82; S, 9.95; Found: C, 52.34;
H, 4.16; N, 8.84; S, 9.76.
2-(3-trifluoromethylbenzamido)-4-methylthiazole-5-car-
boxylic acid (GK-18) Yield 76%, mp 140–142 °C. 1H NMR
(DMSO-d6, 300 MHz, δ, TMS = 0): 10.45 (1H, bs),
7.34–7.39 (2H, m), 7.28–7.31 (1H, m), 7.22 (1H, s), 2.35
(3H, s). 13C NMR (CDCl3, 75 MHz, δ, TMS = 0): 17.20,
95.77, 119.03, 131.47, 132.32, 135.75, 142.04, 143.68,
159.12, 163.71, 165.55, 170.13. Anal. Calcd. MS: 330.286;
Found m/z: 331.317 (M+ + 1). Anal. Calcd for
C13H9F3N2O3S: C, 47.27; H, 2.75; F, 17.26; N, 8.48; O,
14.53; S, 9.71; Found: C, 47.36; H, 2.45; F, 17.34; N, 8.23;
S, 9.76.
In vitro xanthine oxidase assay
All the synthesized compounds were evaluated against XO
enzyme. Bovine milk XO (grade 1, ammonium sulfate
suspension, Sigma-Aldrich) activity was assayed spectro-
photometrically by measuring the uric acid formation at
293 nm using a Hitachi U-3010 UV-visible spectro-
photometer at 25 °C. The reaction mixture contains

Medicinal Chemistry Research
S
C
Scheme 1 Synthesis of thiazole
derivatives. Reagents and
conditions: a Ethanol, reflux,
70 °C; b pyridine, stirring rt, 1 h;
c methanol: water (9:1), K2CO3,
stirring with reflux
H2N
NH2
Thiourea
O
O
a
H3C
CH3
Cl
O
CH3
S
O
H2N
N
CH3
(8)
O
Cl
b
R
O
O
R
R
O
C
O
C
c
S
CH3
S
OH
H
O
H
N
N
N
N
CH3
CH3
(GK-1 to GK-22)
(9)
potassium phosphate buffer (pH 7.6, 50 Mm, 100 µL),
xanthine (75 µM, 20 µL), and XO (0.08 units). Inhibition of
XO activity of synthetics at different concentrations (1, 5,
10, 25, 50 µM) was measured by following the decrease in
the uric acid formation at 293 nm at 25 °C. The enzyme was
preincubated for 5 min with a test compounds, dissolved in
DMSO (1% v/v), and the reaction was started by the
addition of xanthine. The final concentration of DMSO (1%
v/v) did not interfere with the enzyme activity. All the
experiments were performed in triplicate and values were
expressed as the mean of three experiments (Escribano et al.
1988; Takano et al. 2005).
using MGL Tools-1.5.6. Two-dimensional structure of
ligand GK-20 was drawn in ChemDraw and subjected to
energy minimization using the MM2 force field as imple-
mented in Chem 3D Ultra software (Cambridge, USA)
Docking of GK-20 was performed using the AutoDock
Vina software (Trott and Olson 2010). For docking per-
formance, a grid box was generated by fixing the number of
points in x, y, and z directions to 40, each. The spacing was
adjusted to 1.00 Å. The canter of the grid box was fixed to
the point of 96, 52, and 38.
Results and discussion
Enzyme kinetics
Thiazole-5-carboxylic acid derivatives were synthesized via
Scheme 1. To a mixture of thiourea (0.013 moles) and
ethanol (15 mL), 2-chloroethylacetoacetate (1 equivalent)
was added dropwise with continuous stirring at room tem-
perature. After the complete addition of 2-chlor-
oethylacetoacetate, the reaction mixture was warmed on a
water bath and the completion of the reaction was mon-
itored by using TLC. After completion, the reaction mixture
was cooled at room temperature and solid crystalline pro-
duct so obtained was washed with 10 mL of ethanol and
then treated with saturated sodium hydrogen carbonate to
obtain white precipitates of ethyl 2-amino-4-methylthiazole-
5-carboxylate (Ali et al. 2016). Thiazole derivative was
further subjected to benzoylation using various ben-
zoylchlorides in presence of dry pyridine at room tem-
perature. This product was further hydrolyzed by using
Potent XO enzyme inhibitors were further investigated for
the type of inhibition and enzyme kinetics study was carried
out. The Lineweaver–Burk plot was established from which
we could calculate the Km, Vmax of the slope of inhibitor and
the value of α (a constant that defines the degree to which
inhibitor binding affects the affinity of the enzyme for
substrate) (Copeland 2005).
Molecular modeling studies
Three-dimensional structure of bovine milk xanthine
dehydrogenase complexed with fabuxostat (a potent XO
inhibitor) was retrieved from RCSB protein data bank (PDB
code 1N5X) (Okamoto et al. 2003). For docking simula-
tions, hydrogens were added onto the protein structure

Medicinal Chemistry Research
Table 1 Various substituted thiazole-5-carboxylic acid derivatives
Table 2 Percentage inhibition and IC50 values of thiazole-5-carboxylic
acid derivatives against XO
O
Compound Percent inhibition
1 μM 5 μM 10 μM 25 μM 50 μM
IC50 (μM SD)
R4
R5
R1
S
OH
CH3
HN
O
N
R3
GK-1
15
13
28
51
53
18
52
5
28
24
33
58
58
24
59
9
38
36
43
64
69
39
67
14
14
23
8
57
48
54
71
72
55
72
18
17
29
11
33
36
41
69
72
63
49
69
87
56
60
69
61
68
83
86
67
89
24
19
32
15
40
43
55
75
78
69
55
71
98
62
69
14.70 1.03
29.14 2.44
13.88 2.09
0.75 0.09
0.53 0.08
23.45 2.33
0.71 0.05
ND
R2
GK-2
GK-3
Code
R1
R2
R3
R4
R5
mp (°C)
GK-4
GK-1
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
132–136
170–174
170–176
155–159
162–168
147–151
174–178
137–139
180–185
201–203
124–130
180–188
210–212
208–214
161–164
184–189
145–149
140–142
134–141
182–190
157–162
143–145
GK-5
GK-2
H
H
H
GK-6
GK-3
H
H
H
H
Cl
Br
H
H
I
F
H
H
GK-7
GK-4
H
F
H
GK-8
GK-5
F
H
F
GK-9
7
11
16
5
ND
GK-6
Cl
H
H
GK-10
GK-11
GK-12
GK-13
GK-14
GK-15
GK-16
GK-17
GK-18
GK-19
GK-20
GK-21
GK-22
Febuxostat
Allopurinol
11
4
ND
GK-7
Cl
H
H
ND
GK-8
H
H
H
15
18
10
44
47
41
22
43
55
31
40
21
21
22
53
57
48
31
51
63
38
48
29
30
32
60
66
56
38
59
76
45
54
ND
GK-9
Br
H
H
H
ND
GK-10
GK-11
GK-12
GK-13
GK-14
GK-15
GK-16
GK-17
GK-18
GK-19
GK-20
GK-21
GK-22
Br
H
H
44.45 3.56
4.01 0.46
2.44 0.32
8.11 0.86
ND
H
H
H
H
H
H
H
CF3
H
H
H
H
H
NO2
H
H
H
NO2
H
H
NO2
H
NO2
H
CH3
H
5.05 0.87
0.45 0.03
12.55 0.81
8.40 0.97
0.03
CH3
H
CH3
H
H
CF3
H
H
H
CF3
H
H
CF3
H
CF3
H
8.69
OCH3
OCH3
The bold values provided of standard drugs, provided to compare the
biological results with synthesized compounds
OCH3
H
potassium carbonate in the presence of methanol: water
(9:1) to obtain the target compounds. All the reactions
proceeded smoothly with diverse benzoylchlorides and
products were obtained in good yields (Table 1). The
structures of the synthesized compounds were elucidated by
1H and 13C NMR. All spectral data were in accordance with
assumed structures.
showed 98% inhibition at 50 µM concentration. The con-
centration to inhibit the 50% of the enzyme (IC50) was only
calculated for those compounds, which displayed more than
60% inhibition at 50 µM concentration. The obtained IC50
values were ranging from 0.45–44.45 µM. Nine compounds
(GK-4, GK-5, GK-7, GK-15 to GK-17, GK-19, GK-20,
and GK-22) were found more potent than standard drug
allopurinol (IC50 = 8.69 µM). The most potent compound
(GK-20) was endowed with the most potent inhibitory
activity with the IC50 value of 0.45 µM, which was found
almost 20 times more potent than allopurinol. GK-4, GK-5,
and GK-7 are other potent compounds of the series with the
IC50 values of 0.75, 0.53, and 0.71 µM, respectively. The in-
vitro screening of the thiazole-5-carboxylic acid derivatives
revealed some interesting aspects of the structure-activity
relationship. Unsubstituted Ring B showed moderate inhi-
bitory activity against the enzyme. Careful examination of
the biological data (Table 2) indicates that mono-substituted
compounds (as Ring B) are less active than di-substituted
compounds (compare GK-4 with GK-5, GK-6 with GK-7,
In vitro biological evaluation
For in vitro screening of the thiazole-5-carboxylic acid
derivatives, enzymatic assay using bovine milk XO (grade
1, ammonium sulfate suspension) was performed as
described in the literature (Escribano et al. 1988; Takano
et al. 2005). Febuxostat and allopurinol were employed as
reference inhibitors (Escribano et al. 1988; Takano et al.
2005). The results of the in vitro assay (Table 2) indicated
that the derivatives exhibited significant XO inhibitory
activity amongst which the most potent compound (GK-20)

Medicinal Chemistry Research
GK-13 with GK-14, GK-15 with GK-16, GK-19 with GK-
20, and GK-21 with GK-22). It has also been cleared from
the evaluation results that para position of Ring B for
halogen atoms is more sensitive towards the XO inhibitory
activity. It means that compounds with halogen atoms on
para position of Ring B were found more active than ortho
and meta halogenated compounds (compare GK-4 and GK-
20 with GK-2, GK-3, GK-6, and GK-11).
GK-20 were generated and ranked according to their in
silico binding affinity. The conformation with a highest
binding affinity (−8.7 kcal/mol) was selected for discussion
(Fig. 3a, b).
The Two-dimensional depiction of various residues
surrounding GK-20 is presented in Fig. 3b. The binding site
residues and overall binding mode of GK-20 is similar to
those observed with febuxostat (Okamoto et al. 2003),
salicylic acid (Enroth et al. 2000), and curcumin (Shen and
Ji 2009). GK-20 gets stabilizes at the binding cavity by
various electrostatic interactions. The major interactions
include two π–π stacking and three H-bond interactions
(Fig. 3c). The thiazole ring gets sandwiched between
Phe914 and Phe1009 and involved in face-to-face and edge-
to-face π–π stacking interactions, respectively (d = 4.387 Å
and 3.430 Å). This arrangement of energetically favorable
π–π stacking interactions has also seen in the co-crystal
structure of XO with febuxostat and salicylate (Okamoto
et al. 2003; Enroth et al. 2000). Its conservation argues for
an important role in stabilizing the binding positions of
aromatic substrates and it might well represent one of the
key features of substrate recognition. The carboxylate
function at thiazole ring (H-bond acceptor) has shown two
H-bond interactions with strongly basic guanidine group of
Arg880 (H-bond donor; d = 2.225 Å and 2.537 Å). H-bond
interaction observed between carbonyl function of amide
linker chain (H-acceptor) and the hydroxyl group of Ser876
(H-bond donor; d = 2.051 Å) makes it a suitable con-
firmation similarly as seen in previously designed lead
molecules (Ali et al. 2016). The 3,5-di-substitutes phenyl
ring gets positioned in a cavity formed by Leu648, Phe649,
Leu873, Val1011, Leu1014, and Lys771 and stabilized by
van der Waals and/or dispersion interactions. The study
suggests that GK-20 may block the activity of the XO
sufficiently enough to prevent the substrate from binding to
its active site.
Enzyme kinetics
The most potent XO inhibitor among the series (GK-20)
was further investigated for the type of inhibition by
enzyme kinetic studies. The reactions were performed at
different concentrations of inhibitor GK-20 (at 1, 10 and
50 µM). The pattern of the Lineweaver–Burk plot indicates
that it inhibited the enzyme by mixed type inhibition sce-
nario, where Km, Vmax, and slope are all affected by the
inhibitor. (Fig. 2) The inhibitor has increased the Km and
slope (Km/Vmax) while decreasing the Vmax. Moreover, on
careful observation, it was found that intersecting lines on
the graph converge to the left of the y-axis and above the x-
axis, which indicates that the value of α (affinity constant) is
>1 (Copeland 2005). This confirms that the inhibitor pre-
ferentially binds to the free enzyme and not the
enzyme–substrate complex.
Molecular modeling studies
In order to understand the binding conformation of inhibi-
tors, the most potent XO inhibitor GK-20 was docked into
the febuxostat binding site using the AutoDock Vina soft-
ware (Fig. 3). Docking procedure was validated by repro-
ducing co-crystallized conformation of febuxostat. The
AutoDock Vina was able to reproduce the co-crystallized
conformation of febuxostat with the RMSD value of
0.792 Å (Fig. 3a), which ensures the acceptability of the
selected docking protocol. Several docked conformations of
In silico study
Furthermore, to confirm that whether the compounds can be
used for animal studies, in silico physicochemical properties
of active compounds were determined by using the web-
based applications MarvinSketch (http://www.chemaxon.
com/) and PreADMET (http://preadmet.bmdrc.org/). Table
3 indicated that the compounds are predicted to have lower
blood–brain barrier permeation which suggests that they are
found less likely to cause neurotoxicity. Parameters of the
Lipinski rule of five of all the synthesized compounds were
determined with ChemAxon software MarvinSketch (Table
4). Tabular values indicate that all the molecules have a
molecular weight in the range of 262–398 which lies in the
limit of 180–500. Compounds have no H-bond donating
Fig. 2 Lineweaver–Burk plot of compound GK-20

Medicinal Chemistry Research
Fig. 3 a Superimposition of co-
crystalized (green) and docked
conformation (magenta) of
febuxostat (RMSD: 0.792 Å); b.
Two-dimensional representation
of GK-20 at febuxostat binding
site; c. Three-dimensional
Docking conformation of GK-
20 (Hydrogens which are
involved in H-bond interactions
are shown)
Table 3 In silico ADME properties of active compounds
Absorption
Distribution
Compound Human intestinal
absorption (HIA)%
In vitro Caco-2 cell
permeability (nm/s)
In vitro MDCK cell
permeability (nm/s)
In vitro skin
permeability
(logKp) cm/h
In vitro plasma In vivo
protein
blood–brain barrier
binding (%)
penetration (C.
brain/C.blood)
GK-1
89.13
89.24
89.24
89.24
89.34
94.49
97.11
13.28
90.52
91.75
90.80
90.80
92.23
86.61
83.68
1.02
0.91
0.91
10.05
0.84
4.39
4.28
1.10
0.87
3.16
1.19
0.89
30.00
3.65
2.81
40.10
43.06
37.45
42.20
34.10
20.06
3.84
−3.79
−4.06
−4.06
−4.04
−4.21
−3.87
−3.82
−3.79
−3.71
−3.70
−2.68
−2.71
−2.10
−3.99
−4.15
62.56
72.61
71.65
70.66
78.04
73.66
83.55
87.32
70.81
80.46
81.82
75.94
96.73
67.70
66.62
0.30
0.35
0.35
0.03
0.40
0.49
0.82
0.04
0.43
0.16
0.66
0.18
0.01
0.09
0.11
GK-2
GK-3
GK-4
GK-5
GK-6
GK-7
GK-14
GK-15
GK-16
GK-17
GK-19
GK-20
GK-21
GK-22
9.89
28.13
27.45
20.70
0.06
0.04
23.66
2.87
and accepting properties within the described limits. Molar
refractivities are consistent and lie well in the range of
40–130. From the log P values, it has been cleared that the
compounds are not very lipophilic in nature. These results
make the compounds pharmacologically efficient for use in
the animal model.

Medicinal Chemistry Research
Table 4 Physicochemical
parameters of active compounds
Compound Molecular weight No. of H-
bond donors
No. of H-bond Molar
acceptors
Log P No. of
refractivity
Lipinski
violation
GK-1
262
280
280
280
298
296
329
352
276
290
330
330
398
292
322
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
4
4
4
4
4
4
4
8
4
4
4
4
4
5
6
67.91
68.12
68.12
68.12
68.34
72.71
77.51
82.55
72.95
77.99
73.88
73.88
79.85
74.37
80.83
2.33
2.47
2.47
2.47
2.61
2.93
3.54
2.21
2.84
3.36
3.21
3.21
4.09
2.17
2.01
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
GK-2
GK-3
GK-4
GK-5
GK-6
GK-7
GK-14
GK-15
GK-16
GK-17
GK-19
GK-20
GK-21
GK-22
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Conclusion
A novel series of thiazole-5-carboxylic acid derivatives was
synthesized and in vitro evaluated against XO enzyme.
Compounds were found significantly active against the
enzyme and exhibited the IC50 values ranging from 0.45 to
44.45 µM. The compound GK-20 was found to be the most
potent compound with 3,5-di(trifluoromethyl) substitution
on Ring B, which exhibited almost 20 folds potent inhibi-
tion (IC50 = 0.45 µM) against the enzyme than allopurinol.
The established structure-activity relationship revealed that
di-substituted compounds as Ring B were more potent than
that of mono-substituted compounds. The para-substitution
was found to be the most preferable one for the inhibitory
potential, other than ortho- and meta-substitution. Mixed
type inhibition scenario was observed for the compound
GK-20 through enzyme kinetic studies. Various types of the
binding interactions within the active site of XO enzyme
were also streamlined by using docking studies. Thus the
compounds could act as hit lead for the further development
of potent XO inhibitors.
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Acknowledgements Authors are grateful to the University Grants
Commission for providing funds under Rajiv Gandhi National Fel-
lowship (RGNF), National Fellowship for Other Backward Classes
(NFOBC) and are thankful to Council of Scientific & Industrial
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Compliance with ethical standards
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