Trisubstituted thiophene analogues of 1-thiazolyl-2-pyrazoline, super oxidase inhibitors and free radical scavengers

Article abstract of DOI:10.1016/j.bmc.2012.09.060

Xanthine oxidase (XO) generates superoxide anions and H₂O ₂ for the self-defence system of organism. Abnormal production of this superoxide's (reactive oxygen species) is responsible for a number of complications including inflammation, metabolic disorder, cellular aging, reperfusion damage, atherosclerosis and carcinogenesis. Series of novel trisubstituted thiophenyl-1-thiazolyl-2-pyrazoline libraries are synthesized containing 2,5-dichloro thiophene, 5-chloro-2-(benzylthio) thiophene and 5-chlorothiophene-2-sulphonamide, from chalcones in PEG-400 as green solvent. Superoxide (XO) inhibitory and free radical scavenging activities were also figured out with molecular modeling analysis, bearing in mind their possible future for super oxide inhibitor (Gout) therapeutics, compound 3k shows interesting superoxide inhibitory and free radical scavenger activity with IC₅₀ = 6.2 μM, in comparison with allopurinol.

Full text of DOI:10.1016/j.bmc.2012.09.060

Bioorganic & Medicinal Chemistry 21 (2013) 365–372
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Trisubstituted thiophene analogues of 1-thiazolyl-2-pyrazoline, super
oxidase inhibitors and free radical scavengers
Gajanan G. Mandawad a, Bhaskar S. Dawane a, , Supriya D. Beedkar b, Chandrahas N. Khobragade b,
⇑
Omprakash S. Yemul a
a School of Chemical Sciences, Swami Ramanand Teerth Marathwada University, Nanded 431606, India
b School of Life Sciences, Swami Ramanand Teerth Marathwada University, Nanded 431606, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Xanthine oxidase (XO) generates superoxide anions and H2O2 for the self-defence system of organism.
Abnormal production of this superoxide’s (reactive oxygen species) is responsible for a number of com-
plications including inflammation, metabolic disorder, cellular aging, reperfusion damage, atherosclerosis
and carcinogenesis. Series of novel trisubstituted thiophenyl-1-thiazolyl-2-pyrazoline libraries are syn-
thesized containing 2,5-dichloro thiophene, 5-chloro-2-(benzylthio) thiophene and 5-chlorothiophene-
2-sulphonamide, from chalcones in PEG-400 as green solvent. Superoxide (XO) inhibitory and free radical
scavenging activities were also figured out with molecular modeling analysis, bearing in mind their pos-
sible future for super oxide inhibitor (Gout) therapeutics, compound 3k shows interesting superoxide
Received 5 August 2012
Revised 19 September 2012
Accepted 22 September 2012
Available online 2 November 2012
Keywords:
Thiophenyl thiazolyl pyrazoline
Superoxide inhibitors
Free radical scavengers
Green synthesis
inhibitory and free radical scavenger activity with IC50 = 6.2
lM, in comparison with allopurinol.
Ó 2012 Elsevier Ltd. All rights reserved.
PEG-400
1. Introduction
associated with hyperuricemia for several decades.4 However due
to lack of free radical scavenging activity against superoxide anions
produced, use of allopurinol is associated with some side effects
that include allergy, hypersensitivity reactions, gastrointestinal up-
set, skin rashes and acute interstitial nephritis.5 Therefore there is a
need for the development of novel compounds with better safety
profiles that could be used to relieve associated side effects.
Thiazole, pyrazolo-pyrimidine and its derivatives possess
various biological activities like anticancer,6 antioxidant,7–10 anti-
inflammatory,11 antimicrobial12,13 and XO inhibitory. Similarly
thiophene analogs of compounds exhibit various pharmacological
activities.14–17 As an attempt several purine analogues such as
2-amino-4-hydroxy-6-hydroxymethylpteridine (2),18 6-formylp-
terin,19 6-aminopurine (3), chloromethyl amino purine (4),20 and
3-Hydroxy-1-nitrophenyl-1H-pyrazolo[4,3-c]pyridines (5)21 were
reported as potent XO inhibitors (Fig. 1). However use of purine
analogue leads to development of symptoms for Stevens–Johnson
syndrome and DRESS (Drug Rash with Eosinophylia and Systemic
Symptoms)22,23 therefore extensive research has been done to re-
port nonpurine analogues for XO inhibition. In 1995 Japanese
researchers Masatoshi Chihiro et al. firstly reported, thiazole
derivatives as superoxide inhibitors. (6).24 Recently one of the
nonpurine inhibitor, thiazole derivative, Febuxostat (7) was ap-
proved by the European Medicines Evaluation Agency (EMEA)
and Food and Drug Administration (FDA), has attracted worldwide
attention.25 Scholar review article by Raj Kumar et al extensively
covered all patent survey for xanthine oxidase inhibitors including
Xanthine oxidase (XO) is
a versatile molybdoflavoprotein,
widely distributed, occurring in the milk, kidney, lung, heart, and
vascular endothelium. Liver and intestine are found to possess
highest activity for XO.1 In humans and other uricotelic species,
this enzyme is associated with purine metabolism catalyzing the
conversion of both hypoxanthine and xanthine to uric acid. Excess
level of uric acid (UA) in serum (>7 mg/dl) leads to a hyperuricemic
condition called gout. In a normal person, UA is dissolved in the
blood; however, in a person with hyperuricemia, insoluble UA
forms microscopic crystals in the capillary vessels of joints. These
crystals when deposited at various joints, cause inflammation
and sharp pain which are symptoms of acute gout.2 Moreover dur-
ing the catalytic oxidative hydroxylation of purine substrates, XO
generates superoxide anions and H2O2. These reactive oxygen spe-
cies are responsible for a number of complications including
inflammation, metabolic disorder, cellular aging, reperfusion dam-
age, atherosclerosis and carcinogenesis.3 Therefore selective inhi-
bition of XO may evolve as a promising therapy to treat gout and
other hyperuricemic conditions. Allopurinol (1,5-dihydro-4H-
pyrazolo[3,4-d]pyrimidin-4-one) (1), a purine analogue was the
first XO inhibitor approved by the FDA in 1966 and has been the
cornerstone of the clinical management of gout and conditions
⇑
Corresponding author.
E-mail address: bhaskardawane@rediffmail.com (B.S. Dawane).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.060

366
G. G. Mandawad et al. / Bioorg. Med. Chem. 21 (2013) 365–372
NH2
Cl
NH2
OH
N
N
N
NH2
H
N
N
N
N
N
N
HO
N
N
N
N
H
N
N
N
OH
2-amino-4-hydroxy-6-
Chloromethyl amino
6-aminopurine (3)
Allopurinol (1)
hydroxymethyl pteridin (2)
purin (4)
OH
CN
S
H3C
N
N
N
O
N
O
N
S
N
NO2
OH
3-(4-phenylthiazol-2-yl)
Febuxostat (TEl-6720) (7)
3-Hydroxy-1-nitrophenyl-1H
pyridine (6)
-pyrazolo[4,3]pyridine (5)
O
Cl
R
O
HN
N
O
CH
N
S
X
1-acetyl-3,5-diaryl-4,5-
dihydro 1H Pyrazoles (10)
Iminothiazoles (8)
N-(1,3)-Diaryl-3-Oxopropyl)
amides (9)
H2N
O
CN
NH
N
OH
N
R
N
O
N
N
N
N
NC
N-aryl-5-amino-4-
NC
cynopyrazole (13)
Y-700 (12)
FYX-051 (11)
OH
R3
COOH
O
R1
R2
Flavonide (14)
O
Anacardic acid (15)
O
O
H3CO
OCH3
OH
C
H2
HO
Curcumin (16)
Figure 1. Reported superoxide and XO inhibitors.
nonpurine analogues.26 Kavitha et al. reported substituted imino-
thiazoles as possible antioxidant agents (8).27 Nepali et al. recently
reported synthesis of N-(1,3-diaryl-3-oxopropyl) amides (9) and
1-acetyl-3,5-diaryl-4,5-di-hydro (1H) pyrazoles (10) as a new
template for xanthine oxidase inhibition.28,29 FYX-051 (11) re-
ported by Takahiro Sato et al is currently being evaluated in phase
2 clinical trial.30 Y-700 (12)31 N-aryl-5-amino-4-cyanopyrazole
(13),32 flavonoids (14),33 anacardic acid (15)34 and curcumin
(16)35 also reported to be potent xanthine oxidase inhibitor. As a
continuation of our work on 1-thiazolyl-2-pyrazole deriva-
tives13,36,37 and considering the importance of reported pyrazole,
thiazole derivatives19,20,23,38 as super oxidase inhibitors.
central pyrazole rings that provided a site for hydroxylation near
molybdenum metal. An attempt has been made to insert the thiazol
moiety from febuxostat in pyrazolo derivatives for activity rein-
forcement. Computational tool PASS (Prediction of Activity Spectra
for Substances) predicted antioxidant, free radical scavenger, anti-
inflammatory, oxygen scavenger, gout treatment and xanthine oxi-
dase inhibitor activity for these derivatives. Therefore their XO
inhibitory and free radical scavenging effects were evaluated using
in vitro assay and in silico procedures including docking studies
with inhibitors. The mode of interaction of docked inhibitor was
well described using this approach.
We thought for synthesis of new trisubstituted thiophene-
1-thiazolyl-2-pyrazoline derivatives. In general the known hetero-
cyclic inhibitors of xanthine oxidase consists of unsaturated rings
each containing at least one hetero nitrogen or sulpher atom.
Therefore in an attempt to synthesize nonpurine derivatives, pyr-
azolo ring structures have been incorporated. Nonpurine based
structure excluded the possibility of target compounds being con-
verted, like allopurinol, to unnatural nucleotides. The doubly
bounded N-atom in the ring structure does increase solubility of
compounds.38 Two aromatic or heteroaromatic rings joined by a
2. Materials and methods
2.1. Synthesis
All chemicals and solvents used were laboratory grade and
purified except 2-n-buty-4-chloroformyl imidazole (gifted by
Vijayshree Chemicals, Hyderabad, India) and substituted acetyl
thiophenes II and III were synthesized in lab from
I (2,5-
dichloro-3-acetyl thiophene) as in reported method.49

G. G. Mandawad et al. / Bioorg. Med. Chem. 21 (2013) 365–372
367
2.1.1. General procedure for preparation of 1-thiazolyl-2-
pyrazoline. (3a–l)
2.1.1.1. Synthesis of 4,5-dihydropyrazole-1-carbothioamide.
1Ha), 4.0 (dd, J = 9.6 Hz, 1Hb), 5.6 (dd, J = 6.0 Hz 1H, Hx), 7.36 (s,
1H thiazole), 7.38 (dd, J = 6.3, 2H, Ar-H), 7.45 (s, 1H, thiophene),
7.9 (dd, J = 6.3, 2H, Ar-H), 12.3 (s, 1H, imidazole) ppm. Mass (m/
z): 571 (100.0%), Anal. Calcd for C23H19Cl4N5S2; C, 48.35; H, 3.35;
N, 12.26, Found: C, 44.23; H, 3.27; N, 12.17.
(2a–l).
A mixture of heterocyclic chalcones 1a–1 (20 Mmol),
thiosemicarbazide (30 Mmol) and NaOH 5 Mmol was heated at
2–3 h at 55–65 °C with stirring in poly (ethylene) glycol, PEG-
400; progress of reaction was monitored on TLC, after completion
of reaction. Reaction mass cooled to 25–30 °C and slowly poured
in cold distilled water (100) mL under stirring in 20–30 min, inter-
mediate solid (4,5-dihydropyrazole-1-carbothioamide) get sepa-
rated, was filtered and washed by distilled water 20 ꢀ 2 mL, wet
solid dried at 60–65 °C, recrystalized from absolute ethanol to
get pure (4,5-dihydropyrazole-1-carbothioamide) 2a–l.
2.1.3.2.
5-(3-(2-(Benzylthio)-5-chlorothiophen-3-yl)-1-(4-(4-
chlo-rophenyl)thiazol-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)-2-
butyl-4-chloro-1H-imidazole (3b).
Faint green crystalline
,
1H NMR (DMSO-d6, d
solid: IR (KBr): 3220, 3010, 1610, cmꢁ1
ppm) d, 0.91 (t, J = 7.2 Hz, 3H, –CH3), 1.31 (m, 2H, –CH2–), 1.65
(m, 2H, –CH2–), 2.75 (t, J = 7.5 Hz, 2H, –CH2), of n-butyl ring, 3.4
(dd, J = 6.0 Hz 1Ha), 4.0 (dd, J = 9.6 Hz, 1Hb), 4.2 (s, 2H, –CH2 mer-
capto benzyl), 5.6 (dd, J = 6.0 Hz 1H, Hx), 7.36–7.9 (m, 11 H, thia-
zole, thiophene and Ar-H), 12.3 (s, 1H, Imidazole) ppm. Mass (m/
z) 659 (M+ ion), Anal. Calcd for C30H26Cl3N5S3; C, 54.67; H, 3.98;
N, 10.63; Found: C, 54.47; H, 4.02; N, 10.35.
2.1.1.2. Synthesis of 1-thiazolyl-2-pyrazolines (3a–l).
A
mixture of 4,5-dihydropyrazole-1-carbothioamide 2a–l (1 Mmol)
and 2-bromo-1-(4-chlorophenyl) ethanone (1 Mmol) in 5 ml of
poly (ethylene glycol), PEG-400, was stirred at 40–45 °C for 30–
60 min, progress of reaction was monitored by TLC, after comple-
tion of reaction, reaction mass cooled to 25–30 °C and poured in
distilled water 50 mL under stirring, solid gets separated out,
which after filtration dried at 55–60 °C. Dry crude solid was then
recrystalized from absolute ethanol to get pure thiophenyl-
1-thiazolyl-2-pyrazoline 3a–l, All aqueous mother liquors were
combined and distilled under reduced pressure at 65–70 °C to re-
move water, leaving PEG behind, which is reusable up to second
recycle for same reaction.
2.1.3.3. 3-{5-(2-Butyl-5-chloro-3H-imidazol-4-yl)-1-[4-(4-chlor-
ophenyl)-thiazol-2-yl]-4,5-dihydro-1H-pyrazol-3-yl}-5-chloro-
thiophene-2-sulfonamide (3c).
Orange solid: IR (KBr): 3310,
3230, 1610, cmꢁ1 1H NMR (DMSO-d6, d ppm) d, 0.91 (t, J = 7.2 Hz,
,
3H, –CH3), 1.31 (m, 2H, –CH2–), 1.65 (m, 2H, –CH2–), 2.75 (t,
J = 7.5 Hz, 2H, –CH2), 3.52–3.59 (dd, J = 6.3 Hz, 1H, Ha), 4.07–4.14
(dd, J = 9.3 Hz, 1H, Hb), 4.73 (s, br, 2H, –SO2NH2), 5.69–5.74 (dd,
J = 6.3 and 6.9 Hz, 1H, Hx), 7.42–7.44 (dd, J = 6.6 Hz, 2H, Ar-H),
7.51 (s, 1H, thiazole), 7.6 (s, 1H, thiophene), 7.86–7.88 (dd,
J = 6.3, 2H, Ar-H), 13.41 (s,1H –NH of imidazole, D2O exchangeble)
ppm. Mass (m/z) 616 (M+ ion), Anal. Calcd for C23H21Cl3N6O2S3: C,
44.84; H, 3.44; N, 13.64; Found: C, 44.65; H, 3.47; N, 13.47.
2.1.2. Chemical analysis
Melting points were determined by open capillary method (Ta-
ble 1) and are uncorrected. 1H NMR spectra were recorded ((in
DMSO-d6, d ppm) on AVANCE-300 MHz spectrometer using TMS
as an internal standard (s = singlet, d = doublet, t = triplet, m = mul-
tiplates and br = broad). Coupling constant (J) are given in (Hz). IR
spectra were recorded (in KBr pallets) on SHIMADZU spectropho-
tometer. The mass spectra were recorded on EI-SHIMDZU-GC–MS
spectrometer. Elemental analyses were performed on a Perkins-
Elmer C, H, N, elemental analyzer. All reactions were monitored
by using thin layer chromatography (TLC) using 0.2 mm silica gel
plates 60 F254 (MERCK). Reaction components were visualized in
UV (255 and 365 nm) or iodine chamber.
2.1.3.4. 5-(4-Chlorophenyl)-1-(4-(4-chlorophenyl)thiazol-2-yl)-
3-(2,5-dichlorothioph-en-3-yl)-4,5-dihydro-1H-pyrazole
(3d).
Reddish colored solid: IR (KBr): 3020, 1605, 1565 cmꢁ1
,
1H NMR (DMSO-d6, d ppm) d, 3.37–3.51 (dd, J = 6, 1H, Ha), 3.93–
4.00 (dd, J = 9.3 Hz, 1H, Hb), 4.24 (s, 2H, –CH2–), 5.56–5.60 (dd,
J = 6 Hz, 1H, Hx), 7.18–7.88 (m, 10 H, thiazole, thiophene and Ar-
H) ppm. Mass (m/z) Mol. Wt.: 524 (M+ ion), Anal. Calcd for
C22H13Cl4N3S2: C, 50.30; H, 2.49; N, 8.00; Found: C, 50.13; H,
2.10; N, 7.93.
2.1.3.5.
3-(2-(Benzylthio)-5-chlorothiophen-3-yl)-5-(4-chlor-
2.1.3. Spectroscopic data of compounds
oph-enyl)-1-(4-(4-chlorophenyl)thiazol-2-yl)-4,5-dihydro-1H-
2.1.3.1. 2-Butyl-4-chloro-5-(1-(4-(4-chlorophenyl)thiazol-2-yl)-
3-(2,5-dichlorothioph-en-3-yl)-4,5-dihydro-1H-pyrazol-5-yl)-
pyrazole (3e).
White solid: IR (KBr): 3150, 1610, 1568,
ppm) d, 3.17–3.24 (dd,
1050 cmꢁ1 1H NMR (DMSO-d6,
,
d
1H-imidazole (3a).
Lemon green solid: IR (KBr): 3233, 2924,
.
J = 5.4 Hz, 1H, Ha), 3.93–4.00 (dd, J = 9.3 and 8.7 Hz, 1H, Hb), 4.24
(s, 2H, –CH2 – mercapto benzyl), 5.56–5.60 (dd, J = 4.5, and
5.4 Hz, 1H, Hx), 7.18–7.88 (m, 15H, thiazole, thiophene and Ar-H)
ppm. Mass (m/z) 613 (M+ ion), Anal. Calcd for C29H20Cl3N3S3; C,
56.82; H, 3.29; N, 6.85; Found: C, 56.54; H, 3.23; N, 6.73.
2854, 1610, 1523, cmꢁ1
1H NMR (DMSO-d6, d ppm) d, 0.91 (t,
J = 7.2 Hz, 3H, –CH3), 1.31 (m, 2H, –CH2–), 1.65 (m, 2H, –CH2–),
2.75 (t, J = 7.5 Hz, 2H, –CH2), of n-butyl ring, 3.4 (dd, J = 6.0 Hz
Table 1
2.1.3.6.
5-Chloro-3-{5-(4-chloro-phenyl)-1-[4-(4-chloro-phe-
Physico-chemical data of synthesized 1-thiazolyl-2-pyrazoline 3(a–l)
nyl)-thiazol-2-yl]-4,5-dihydro-1H-pyrazol-3-yl}-thiophene-2-
Timea
Yield (%)
M.P (°C)
sulfona-mide (3f).
Turmeric yellow powder: IR (KBr): 3010,
Entry
Product
1615, 1350, 1130 cmꢁ1
.
1H NMR (DMSO-d6, d ppm) d, 3.42–3.53
1
2
3
4
5
6
7
8
9
3a
3b
3c
3d
3e
3f
3g
3h
3i
30
40
30
30
50
30
35
45
30
40
35
38
55
68
45
65
42
39
50
65
45
56
70
39
135–137
128–129
157–158
107–108
115–117
130–131
94–95
112–113
125–127
106–109
120–123
146–148
(dd, J = 6.9 Hz, 1H, Ha), 4.03–4.11 (dd, J = 9.3 Hz, 1H, Hb), 4.73 (s,
br, 2H, –SO2NH2), 5.63–5.69 (dd, J = 6 and 6.3 Hz, 1H, Hx), 7.12–
7.84 (m, 10H, thiazole, thiophene and Ar-H). Mass (m/z) 570
(M+ion), Anal. Calcd for C22H15Cl3N4O2S3: C,46.36; H, 2.65; N,
9.83; Found: C, 46.15; H, 2.49; N, 9.65.
2.1.3.7. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-3-(2,5 dichlorothiop-
hen-3-yl)-5-(4-fluoro-phenyl)-4,5-dihydro-1H-pyrazole
10
11
12
3j
3k
3l
(3g).
Off white powder: IR (KBr): 1610, 1569, 1150,
,
1050 cmꢁ1
1H NMR (DMSO-d6, d ppm) d, 3.32 (dd, J = 3.0 and
a
The reaction time is in minutes.
5.1 Hz 1Ha), 4.0 (dd, J = 9.6 Hz, 1Hb), 5.6 (dd, J = 6.0 Hz 1H, Hx),

368
G. G. Mandawad et al. / Bioorg. Med. Chem. 21 (2013) 365–372
7.16 (s, 1H thiazole), 7.38 (dd, J = 6.3, 2H, Ar-H), 7.2–7.85 (m, 9H,
thiophene and Ar-H), ppm. Mass (m/z) Mol. Wt.: 507 (M+ ion), Anal.
Calcd for C22H13Cl3FN3S2; C, 51.93; H, 2.58; N, 8.26, Found: C,
51.88; H, 2.23; N, 8.09.
2.2.2. XO inhibition assay
XO inhibitory activity of synthesized derivatives as test com-
pounds was monitored spectrophotometrically following the
absorbance of uric acid at 292 nm under aerobic condition. The
reaction mixture containing 50 lM potassium dihydrogen phos-
2.1.3.8. 3-(2-(Benzylthio)-5-chlorothiophen-3-yl)-1-(4-(4-chlor-
ophenyl)thiazol-2-yl)-5-(4-fluorophenyl)-4,5-dihydro-1Hpyraz-
ole (3h).
phate buffer (pH 7.4), XO and a solution of test compounds in
DMSO was incubated at room temperature for 15 min. The reaction
Off white: IR (KBr): 3040, 1615, 1587, 1050 cmꢁ1. 1H
was started by addition of Xanthine (100
lM) in the presence of
NMR (DMSO-d6, d ppm), 3.24 (dd, J = 4.5, 5.3 Hz, 1H, Ha), 4.07 (dd,
J = 8.6 Hz, 1H, Hb), 4.24 (s, 2H, –CH2 – mercapto benzyl), 5.56–5.60
(dd, J = 4.5 and 5.7 Hz, 1H, Hx), 7.18–7.80 (m, 15H, thiazole, thio-
phene and Ar-H), ppm. Mass (m/z) 596 (M+ ion), Anal. Calcd for
EDTA (1 M) and uric acid formation was then followed by mea-
l
sure of absorbance at 295 nm. Allopurinol was used as positive
control. The Inhibitory activity of each test compound was indi-
cated by their IC50 values (Table 2) calculated using linear regres-
sion curve. The percent inhibition of XO activity was calculated
using standard formula41
C29H20Cl2FN3S3; C, 58.38; H, 3.38; N, 7.04; Found: C, 58.23; H,
3.13; N, 7.00.
Abs Control ꢁ Abs Sample
2.1.3.9. 5-Chloro-3-[1-[4-(4-chloro-phenyl)-thiazol-2-yl]-5-(4-
fluoro-phenyl)-4,5-dihydro-1H-pyrazol-3-yl]-thiophene-2 sul-
Percent of Inhibition ¼
ꢀ 100
Abs control
where control represents reaction mixture as described above
excluding test compounds instead contain DMSO only whereas
sample represents reaction mixture same as described in method.
fonamide (3i).
White powder: IR (KBr): 3035, 1615, 1570,
1050 cmꢁ1 1H NMR (DMSO-d6, d ppm) d, 3.35 (dd, J = 6.6, and 6.3
.
1H, Ha), 4.1 (dd, J = 9.6, 1H, Hb), 4.7 (s, 2H –SO2NH2), 5.6 (dd,
J = 9.3, 1H Hx), 7.1 (s, 5 H, thiazole), 7.15 (dd, 2H, Ar-H), 7.55
(s,1H, thiophene), 8.0 (dd, 2H, Ar-H), ppm. Mass (m/z) 552 (M+
ion). Anal. Calcd for C22H15Cl2FN4O2S3: C, 47.74; H, 2.73; N,
10.12; Found: C, 47.77; H, 2.40; N, 9.96.
2.2.3. Enzyme inhibition kinetics
Kinetic study was carried out in the absence and presence of the
test compound with varying concentration of xanthine as a sub-
strate i.e., (1–5 lM). Results were analyzed using Lineweaver Burk
plot. Vmax and Km values of inhibition were determined in presence
and absence of inhibitor (Fig. 2).
2.1.3.10. 4-(1-(4-(4-Chlorophenyl)thiazol-2-yl)-3-(2,5-dichlorot-
hiophen-3-yl)-4,5-dihydro-1H-pyrazol-5-yl)-N,N-dimethylbenz-
enamine (3j).
Pell yellow crystals, IR (KBr): 3120, 1611, 1565,
cmꢁ1. 1H NMR (DMSO-d6, d ppm) d, 2.85 (s, 6H, N,N dimethyl), 3.29
(dd, J = 6 Hz, 1H, Ha), 3.95 (dd, J = 9.6 Hz, 1H, Hb), 5.53 (dd, J = 6 Hz,
1H, Hx), 7.09 (s, 1H thiazol), 7.23–7.83 (m, 9H, thiophene and Ar-
H), ppm. Mass (m/z) 533 (M+ ion), Anal. Calcd for C24H19Cl3N4S2:
C, 53.99; H, 3.59; N, 10.49; Found: C, 53.94; H, 3.50; N, 10.53.
2.2.4. DPPH free radical scavenging assay
The stable 1, 1-diphenyl-2-picryl hydrazyl radical (DPPH) was
used for the determination of free radical scavenging activity of
synthesized thiazolyl-pyrazole derivatives 3a–l by following the
method of Koleva et al.42 Different concentrations of test com-
pounds (1
l
M- 50
lM) in methanol were added separately to an
equal volume of 100
l
M methanolic solution of DPPH and the reac-
2.1.3.11. 4-(3-(2-(Benzylthio)-5-chlorothiophen-3-yl)-1-(4-(4-
chlorophenyl)thiazol-2yl)-4,5-dihydro-1H-pyrazol-5-yl)-N,N-
tion mixture was kept at room temperature for 15 min. The absor-
bance of the reaction mixture was recorded at 515 nm using a UV
visible spectrophotometer. Ascorbic acid was used as standard.
Free radical scavenging activity was calculated using the formula
dimethylbenzenamine (3k).
Light yellow crystals; IR (KBr):
3150, 1630, 1579 cmꢁ1 1H NMR (DMSO-d6, d ppm) d, 2.85 (s, 6H,
.
N,N dimethyl), 3.23 (dd J = 6.3, and 5.5 Hz, 1H, Ha), 3.9 (dd, J = 4.5
and 5.7 Hz, 1H, Hb), 4.2 (s, 2H, –CH2–), 5.50 (dd, J = 4.5, 5.5, 1H,
Hx), 7.01–7.83 (m, 15H, thiazole, thiophene and Ar-H), ppm. Mass
(m/z) Mol. Wt.: 621 (M+ ion), Anal. Calcd for C31H26Cl2N4S3: C,
59.89; H, 4.22; N, 9.01; Found: C, 59.83; H, 4.15; N, 9.13.
fðControlODÞꢁðSampleODÞg
%of FreeradicalScavenging¼
ꢀ100
activityðControlODÞ
where control represents reaction mixture containing DPPH and
methanol excluding test compounds whereas sample represents
reaction mixture as described above in the method. The concentra-
tion of test compounds having 50% radical scavenging activity (IC50
was also calculated (Table 2).
2.1.3.12. 5-Chloro-3-[1-[4-(4-chloro-phenyl)-thiazol-2-yl]-5-(4-
dimethylamino-phenyl)-4,5-dihydro-1H-pyrazol-3-yl]-thio-
)
phene-2-sulfonamide (3l).
Faint yellow crystals: IR (KBr):
3035, 1625, 1556, 1128, 1050 cmꢁ1
.
1H NMR (DMSO-d6, d ppm)
d, 2.85 (s, 6H, N,N dimethyl), 3.47 (dd, J = 6.0 and 6.6 Hz 1Ha), 4.0
(dd, J = 9.3 Hz, 1Hb), 5.6 (dd, J = 6.3 Hz 1H, Hx), 7.16 (s, 1H of thia-
zole), 7.23–7.87 (m, 9H, thiophene and Ar-H), ppm. Mass (m/z) 578
(M+ ion), Anal. Calcd for C24H21Cl2N5O2S3; C, 49.82; H, 3.66; N,
12.10; Found: C, 49.73; H, 3.59; N, 12.03.
Table 2
IC50 of XO inhibitory and free radical scavenging activities of 1-thiazolyl-2-pyrazoline
derivatives
Derivatives
XO inhibition IC50
(lM)
Free radical scavenger IC50 (lM)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
26 0.06
20 0.08
23.9 0.76
36 0.76
32 0.66
34.2 0.03
>50
46.6 0.1
>50
9.4 0.7
6.2 0.45
8.3 0.05
31.5 0.77
21.8 0.9
29.3 0.3
44.8 0.23
41.5 0.11
43 0.31
>50
>50
>50
21.09 0.07
15.6 0.08
19.3 0.09
ND
2.2. Bioanalysis
2.2.1. Isolation of XO enzyme from rat liver
XO was isolated and purified from Wister albino rat liver using
standard method.39 In brief, albino rat liver was excised, perfused
and homogenized in ice cold 0.05 M phosphate buffer (pH 7.8) con-
taining 0.1 mM EDTA. The Homogenized solution was then centri-
fuged at 10,000g. The collected supernatant was precipitated using
60% ammonium sulfate precipitation. Precipitate again dialyzed
against equilibrating phosphate buffer (pH 6.8) and used as a
source of enzyme .The protein content was determined by Lowry’s
Method.40
Allopurinol
Ascorbic acid
6
0.7
ND
11.5 0.5
ND, not determined.

G. G. Mandawad et al. / Bioorg. Med. Chem. 21 (2013) 365–372
369
with unique properties such as thermal stability, bulk availability,
nonvolatility, miscibility with a number of organic solvents as well
as water and recyclability. Poly (ethylene) glycols, (PEG-
400),13,47,48 are among the one of green solvents to overcome the
toxic solvent lode on environment. Scheme 1 shows synthesis of
thiazolyl pyrazoline incorporating substituted thiophene moiety
3a–l, by reacting different chalcones 1a–l with thiosemicarbazide
fallowed by 2-bromo-1-(4-chlorophenyl) ethanone in Poly (ethyl-
ene) glycol, (PEG-400), all compounds are well conformed on the
basis of spectral data. 1H NMR spectrum of compound displayed
three characteristic signals due to diasterotopic protons (Ha, Hb
and Hx). The Ha proton resonates upfield at d 3.4–3.5 (center) as
doublet of doublets (dd, J = 8.55 Hz), while the Hb proton resonates
downfield at 3.90–4.15 (dd, J = 9.6 Hz). The Hx proton which is vic-
inal to two methylene protons (Ha and Hb) is also observed as dou-
ble doublet at a d value of 5.4–5.6 (dd, J = 9.5 and Hz). The aromatic
protons are observed at the expected chemical shift and integral
values. The 1H proton of imidazole is observed as a singlet at
8.1 ppm apparently due to deshielding caused by the imidazole
ring. The cyclization of chalcone 1a–l into 4,5-dihydropyrazole-
1-carbothioamide derivative 2a–l was further supported by IR
spectral data, picks due to (C@O) straching in (1670–1660)
chalcones get disappeared and new pick observed at 1612, 1580
due to (N@C) straching, moreover the MS (EI) spectrum value of
4,5-dihydropyrazole-1-carbothioamide exhibits an M + 1, M + 2
peak of expected mass. Reaction time, yield and melting point
are summarized in Table 1.
Figure 2. Lineweaver–Burk plots in the absence (Control) and in the presence of 3k
with xanthine as the substrate. V = AA/min; S = Xanthine (lM).
2.2.5. Statistical analysis
All data are expressed as means SD.
3.2. Biological activity
2.2.6. Molecular docking
The coordinates of crystal structure of rat xanthine oxidoreduc-
tase mutant (W335A and F336L) complexed with uric acid were
obtained from Protein Data Bank (PDB entry 2E3T). The structure
was refined using MM3 in the BioMed CaChe (Version 6.1) soft-
ware package. The active site was located in the refined model
using automatic sequence alignment mode in BioMed CaChe work-
space by selecting bound ligand embedded in 5 Å shell of residues
water and HET’s.43
Biological evaluation of all synthesized derivatives was done for
XO inhibitory and free radical scavenging activities. Each derivative
was examined at the concentration range of 1–50 lM. Among 2-
butyl-4-chloro imidazolo 1-thiazolyl-2-pyrazoline derivatives (3a,
3b, 3c) 4-chlorophenyl 1-thiazolyl-2-pyrazoline derivatives (3d,
3e, 3f) 4-flurophenyl 1-thiazolyl-2-pyrazoline derivatives (3g, 3h,
3i) and N,N-dimethyl aniline thiazolyl-2-pyrazoline derivatives
(3j, 3k, 3l) were tested, last group (3j, 3k, 3l) showed satisfactory
XO inhibitory effect. Activity of rat liver XO was reduced to 50%
(IC50) at doses of 9.4 0.7, 6.2 0.45, 8.3 0.05 for 3j, 3k and 3l,
2.2.7. Ligand structure
2D structure of derivatives were drawn in CHEM DRAW (Ver
11.0.) (Fig. 3) and subjected to energy minimization in the MOPAC
module, using the AMI procedure for closed shell systems, imple-
mented in the CS Chem 3D ultra.
respectively, compared to standard inhibitor allopurinol (IC50
=
6
0.7) revealing 3k as most potent among all (Table 2). In order
to evaluate the type of XO inhibition for 3k its inhibitory effect
was tested at different concentrations of the substrate xanthine
(1–5 lM) using Lineweaver–Burk plot (Fig. 3). Competitive type
2.2.8. Docking
of inhibition was shown. Vmax and Km values obtained in presence
of 3k (0.11 and 3.09) were differs from Vmax and Km values in its
absence (0.260 and 2.17), respectively. Although the number of
tested compounds was limited, some structural features ascribed
to XO inhibitory effect can be inferred (Fig. 2). It is clear from the
results that N,N-dimethyl aniline group conferred acceptable XO
inhibitory activity to thiazolyl-2-pyrazoline derivatives (3j, 3k,
3l). 2-butyl-4-chloro imidazole (3a, 3b, 3c) and 4-chlorophenyl
(3d, 3e, 3f) offered XO inhibitory effect at moderately high concen-
Derivatives (ligands) were docked into the active site of XO
using Bio Med CaChe (Version 6.1). Cache automates the docking
of ligands into active site by using a genetic algorithm with a fast,
simplified potential mean force (PMF). The potential of Mean Force
is a knowledge based approach that extracts pair wise atomic
potentials from structure information of known protein ligand
complexes contained in protein data bank. It has been also demon-
strated to show a significant correlation between experimental
binding affinities and its computed score for diverseprotein ligand
complexes.44,45
tration where IC50 ranges (20–36
thiazolyl-2-pyrazoline derivatives decreased XO inhibitory activity
considerably offering IC50 exceed to 50 M. Moreover comparing
lM). Addition of 4-flurophenyl to
Docking parameters involved steric scan, final search for ligand
l
binding site and refinement of the complex.46
among N,N-dimethyl aniline-1-thiazolyl-2-pyrazoline derivatives
(3j, 3k, 3l) with respect to their differing groups, presence of 2-
(benzylthio)-5-chlorothiophene (3k) attributed potent XO inhibi-
tory effect than 5-chlorothiophene-sulfonamide (3l) and 2,5-
dichlorothiophene (3j).
3. Result and discussion
3.1. Chemistry
A docking study could offer more insight into understanding the
enzyme–inhibitor interactions and the structural features of active
site of enzyme.50 Molecular docking on XO (PDB entry 2E3T) was
performed using Biomed Cache V 6.1 software. Docking alterations
The synthetic pathway of the compounds is outlined under the
frame of ‘green chemistry. Liquid polymers or low melting poly-
mers have recently emerged as alternative green solvent systems

370
G. G. Mandawad et al. / Bioorg. Med. Chem. 21 (2013) 365–372
Cl
Cl
Cl
S
S
S
N
N
Cl
N
Cl
Cl
N
N
N
S
N
N
N
N
N
S
N
N
H
N
H
N
H
Cl
Cl
Cl
S
NH2
O
S
S
Cl
O
3c
3a
3b
Cl
Cl
Cl
S
S
N
S
N
N
N
N
N
S
N
N
Cl
N
Cl
Cl
Cl
Cl
Cl
S
NH2
S
S
S
Cl
3f
O
O
3e
3d
Cl
Cl
Cl
S
S
N
N
S
N
N
N
N
N
F
N
F
N
S
F
Cl
Cl
S
S
Cl
S
Cl
NH2
S
3g
3h
3i
O
O
Cl
Cl
Cl
S
S
S
N
N
N
N
N
N
N
N
N
N
N
N
Cl
Cl
Cl
NH2
O
S
S
S
S
3k
S
Cl
3j
3l
O
Figure 3. Synthesized trisubstituted thiophenyl-1-thiazolyl-2-pyrazoline derivatives 3a–l.
The highest scoring orientations for each ligand proposed a feasible
binding mode of the inhibitor in the active site of XO. Docking of 3k
into active site of XO was carried out and results were compared
with allopurinol. Significant dock score ꢁ78 for 3k was obtained
and compared with allopurinol (ꢁ55). Possible explanation for
the fact may be difference in residues involved in binding with
allopurinol and derivative 3k in the active site of enzyme. Residues
Phe 914, Phe 1009, Ala 1078 and Ser 876, Leu873, Phe955, Lys771,
Met770 and Gly647 were observed involved in binding of deriva-
tive 3k (Fig. 4a) that were found absent in binding of allopurinol
in active site (Fig. 4b). The major interactions of derivative with
XO involved arene–arene interactions with Phe1009, Phe914 and
Phe1013. These interactions may promote the stabilization of the
binding positions of the aromatic substrate and may involve in
substrate recognition.51 Hydrophobic amino acids Gly647,
Ser876, Ser1075, Lys771, Leu873, Thr1010, Thr772, Pro1076,
Leu1011 Thr803, Met770, and Ser805 provided a cage for deriva-
tive 3k binding mainly involving hydrophobic interactions. Thiaz-
olo and pyrazolo ring lie parallel to Phe1009 and perpendicular
to Phe 914 providing an energetically favorable arrangement for
derivative.52 N,N-dimethyl aniline group forms hydrophobic inter-
actions with Leu873, Lys771, Ser1075 and Thr 803.
O
S
Cl
N
R
N
NH2
Cl
(i)
S
S
X
R
X
1 a-l
2 a-l
(ii)
S
Cl
Cl
N
N
N
S
3 a-l
R
X
Cl
N
NH
R =
N
(d)
Cl
(b)
F
(a)
(c)
X = -Cl, -S-CH2-Ph, -SO2NH2
Scheme 1. Reagent and conditions: (i) PEG-400, NaOH, NH2CSNHNH2, 75–80 °C 2–
3 h. (ii) p-chloro phenacylbromide, PEG-400, 45–50 °C, 2 h.
From the analysis of binding of 3k we can conclude that regard-
less of presence of larger aromatic rings in the structure, N,N-
dimethyl aniline thiazolyl pyrazoline derivative occupy a position
in the active site of XO that favors its binding and inhibition at
the site.
based on the interaction force field scoring that includes Van der
Waals and electrostatic interactions between active site and ligand.

G. G. Mandawad et al. / Bioorg. Med. Chem. 21 (2013) 365–372
371
the activities. Docking studies were performed that revealed the
binding position of 3k into active site of enzyme and confirmed
the potential of it as a XO inhibitor.
Acknowledgments
One of the authors (B.S.D.) is sincerely thankful to University
Grant Commission, New Delhi for Post Doctoral Research Award
(F. 30-1/2009, SA-II). Authors gratefully acknowledge to Director
NCL Pune for kind analytical support.
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