Synthesis and biological evaluation of thiazolidine-2,4-dione-pyrazole conjugates as antidiabetic, anti-inflammatory and antioxidant agents

Article abstract of DOI:10.1016/j.bioorg.2019.103271

A series of fourteen novel thiazolidine-2,4-dione derivatives clubbed with pyrazole moiety were synthesized via four step reaction procedure. Reactions were monitored by thin layer chromatography and were characterized by physicochemical and spectrophotometric (IR, Mass, ¹HNMR and ¹³CNMR) analysis. The spectral data were in good agreement with their structures. The title compounds were docked against peroxisome proliferated activated receptors (PPAR-γ) and alpha-amylase and further evaluated for in vivo and in vitro antidiabetic, in vitro anti-inflammatory and antioxidant activities. Compound GB14 exhibited significant blood glucose lowering activity and was also found to be active inhibitor of alpha-amylase. Compound GB7 was found to be potent anti-inflammatory agent in terms of reducing inflammatory markers (TNF-α, IL-β, MDA) and also showed antioxidant activity to good extent. Therefore, these compounds may be considered as promising candidates for the development of new antidiabetic agents.

Full text of DOI:10.1016/j.bioorg.2019.103271

Journal Pre-proofs
Synthesis and biological evaluation of thiazolidine-2,4-dione-pyrazole conju-
gates as antidiabetic, anti-inflammatory and antioxidant agents
Garima Bansal, Shamsher Singh, Vikramdeep Monga, Punniyakoti Veeraveedu
Thanikachalam, Pooja Chawla
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S0045-2068(19)30923-X
DOI:
https://doi.org/10.1016/j.bioorg.2019.103271
YBIOO 103271
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Bioorganic Chemistry
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Please cite this article as: G. Bansal, S. Singh, V. Monga, P.V. Thanikachalam, P. Chawla, Synthesis and biological
evaluation of thiazolidine-2,4-dione-pyrazole conjugates as antidiabetic, anti-inflammatory and antioxidant agents,
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Synthesis and biological evaluation of thiazolidine-2,4-dione-pyrazole
conjugates as antidiabetic, anti-inflammatory and antioxidant agents
Garima Bansal, Shamsher Singh, Vikramdeep Monga, Punniyakoti Veeraveedu
Thanikachalam*, Pooja Chawla*
Department of Pharmaceutical Chemistry, ISF College of Pharmacy, Moga-142001, Punjab, India
* Corresponding authors: Dr. Pooja Chawla, Dr. Punniyakoti Veeraveedu Thanikachalam
Department of Pharmaceutical Chemistry,
ISF College of Pharmacy, Moga-142001, Punjab, India.
Voice Contact: +91-1636-324201 (office), +918057952150 (mobile),
Fax: +91-1636-324201
E-mail: pvchawla@gmail.com, nspkoti2001@gmail.com
ABSTRACT
A series of fourteen novel thiazolidine-2,4-dione derivatives clubbed with pyrazole moiety were
synthesized via four step reaction procedure. Reactions were monitored by thin layer
chromatography and were characterized by physicochemical and spectrophotometric (IR, Mass,
1HNMR and 13CNMR) analysis. The spectral data were in good agreement with their structures.
The title compounds were docked against peroxisome proliferated activated receptors (PPAR-γ)
and alpha-amylase and further evaluated for in vivo and in vitro antidiabetic, in vitro anti-
inflammatory and antioxidant activities. Compound GB14 exhibited significant blood glucose
lowering activity and was also found to be active inhibitor of alpha-amylase. Compound GB7 was
found to be potent anti-inflammatory agent in terms of reducing inflammatory markers (TNF-,
IL-β, MDA) and also showed antioxidant activity to good extent. Therefore, these compounds may
be considered as promising candidates for the development of new antidiabetic agents.
Keywords: Thiazolidine-2,4-dione; PPAR-γ, alpha amylase, antidiabetic, anti-inflammatory,
antioxidant
GRAPHICAL ABSTRACT

1. Introduction
Diabetes is an unceasing, metabolic disease characterized by elevated levels of blood glucose (or
blood sugar), which over a period of time leads to serious damage to the heart, blood vessels, eyes,
kidneys, and nerves. India currently represents 49 percent of the world’s diabetes burden, with an
estimated 72 million cases in 2017, a figure expected to almost double to 134 million by 2025 [1].
Being classified as type 1, type 2 and gestational diabetes, Type 2 diabetes mellitus is the common
major form of diabetes which is the consequence of defect(s) in insulin secretion, mainly with a
major contribution from insulin resistance [2].
Thiazolidine-2,4-dione (TZD), a potent agonist of peroxisome proliferated activated receptor
(PPAR-γ) activates it thereby, altering the transcription of several genes involved in glucose and
lipid metabolism and also energy balance [3]. As reported, diabetes is associated with various
complications, among which diabetic nephropathy is one of the most frequent microvascular
complication [4]. Both inflammation and oxidative stress are closely associated with the progress
of diabetic nephropathy (Fig.1) [5-6]. Increase in oxidative stress can increase the production of
inflammatory cytokines and likewise, an increase in inflammatory cytokines can stimulate the
production of free radicals [7]. Therefore, drugs with anti-inflammatory properties such as TZDs
(clubbed with pyrazole) can probably reduce the risk of developing diabetes and diabetes-induced
problems. TZDs inhibit the expression of a variety of proteins with proinflammatory properties,
including cyclooxygenase-2 (COX-2), inducible nitric-oxide synthase (iNOS), and several
cytokines [8]. However, the molecular mechanisms responsible for these activities have not yet
been clarified.
Several studies have shown that diabetes mellitus is accompanied by augmented
development of free radicals (reactive oxygen species) and decreased antioxidant capacity
(reduced free radical scavenging activity and increased plasma oxidisability), leading to oxidative
damage of cell components [9-11]. When the balance between free radicals formation and their

detoxification is disrupted, which is necessary for normal functioning of the cell, a cell enters a
state of oxidative stress and is damaged [12]. As reported, TZDs considerably augmented the
expression of glutathione peroxidase 3 (GPx3), and the antioxidative result of PPAR- was caused
primarily by GPx3 in myotubes [13]. There are many proven studies reporting that TZDs conserve
islet β -cell function in patients with diabetes or high risk for diabetes [14]. In addition, TZDs
progress islet β-cell function and diminish the oxidative stress in islets of db/db mice [15]. Even
though the expression level of PPAR- is pretty low in islets, numerous reports have shown
protective effects of TZDs against glucose- or lipid-induced toxicity in islets [16]. These reports
proposed that TZDs unswervingly provide positive effects on islet β-cells, and the reduction of
oxidative stress may play a vital role in this process. As there are some side effects linked with
TZDs like weight gain, edema, hepatotoxicity, TZDs might be discovered as antidiabetics
including alpha-amylase inhibitors and may reduce the glucose levels that can occur after a meal
by slowing the speed of conversion of starch to monosaccharides [17-18].
Fig. 1. Targets of synthesized derivatives

Derivatization of existing molecules is one of the most resourceful strategies in the designing of
drug and is based on the clubbing of pharmacophoric scaffolds of different bioactive compounds
to fabricate a new fused compound with enhanced affinity and efficiency when compared to the
parent molecules. Guided by the above information and in continuation of our previous work done
on TZD moiety, we have designed pioglitazone/celecoxib hybrids (Fig. 2) containing
thiazolidindione core as central ring (diabetic scaffold) bearing diaryl pyrazole moiety (anti-
inflammatory scaffold) to discover new candidates thereby, possessing anti-diabetic, anti-
inflammatory and antioxidant activities [19-20].
Fig. 2. Designed molecular hybrid
2. Results and discussion
2.1. Chemistry
The proposed compounds (GB1-GB14) were synthesized using a synthetic procedure involving 4
steps and the synthetic strategy is outlined in Scheme 1. Thiourea on refluxing with chloroacetic
acid gives thiazoldine-2,4-dione [21]. Hydrazones were synthesized by treating substituted
acetophenones with phenyl hydrazine followed by Vilsmeier-Haack reaction in the presence of
DMF and POCl3 to give pyrazole carbldehydes [22]. N-allylation and acidification of TZD was
carried out with benzyl bromide and bromoacetic acid, respectively followed by Knoevenagel
condensation with synthesized carbaldehydes to yield TZD clubbed pyrazole adducts (GB1-GB14)
[23-25]. All new compounds were characterized by physico-chemical, IR (Shimadzu 8400 S),
13
1HNMR, CNMR (Bruker Avance II 400 MHz) and mass spectrometry (Shimadzu QP2010
ULTRA GC-MS). The spectral data were in good agreement with their structures. The IR values
are expressed in cm-1 and the chemical shifts are reported in parts per million (δ value) from TMS
(δ 0 ppm for 1H NMR) as an internal standard. Coupling constant are given in Hertz. The structure
of final derivatives is shown in Fig. 3.

Scheme 1. Synthesis of TZD clubbed pyrazoles (GB1-GB14)

Fig. 3. Structures of final derivatives

2.2. Molecular docking studies
Molecular docking was carried out to investigate some possible structural insights into the
potential binding patterns of the synthesized molecules GB1-GB14 with the active sites of target
PPARγ (PDB ID: 2PRG) and -amylase (PDB ID: 4GQR) using MOE software. The various
possible interactions and orientations were investigated and compared with the binding patterns of
pioglitazone and acarbose for both antidiabetic and -amylase activity. In interaction with PPAR-
γ, compounds GB8, GB11 and GB14 displayed best docking scores with values of -15.13, -16.79
and -17.44 which were better than the reference drug pioglitazone (-12.605). Compound GB8
showed four hydrogen bond interactions, one keto group with water molecule in association to Arg
A288, another keto group with amino acid residues Ser A342 (2.86 Å) and with water molecule in
association with Ser A342 and acidic hydroxyl group interaction with water molecule in
association with Ser A342 (Fig. 4). The hydroxyl group of compound GB11 showed hydrogen
bonding interaction with water in association to Glu A272; whereas other hydrogen bonding
interaction with Glu A272 (1.25 Å) and also with Gln A271 (2.78 Å) (Fig. 5). Compound GB14
showed only one interaction through water molecule in association with Gly A284 as depicted in
Fig. 6. The acidic head of pioglitazone (NH and carbonyl) showed interaction with water molecule
associated with Arg A288 as shown in Fig. 7. In interaction with -amylase, compounds GB11,
GB12 and GB14 displayed best docking scores with values of -16.63, -17.59 and -17.98 whereas
the reference drug acarbose showed docking score with value of -26.33. Compound GB-11 showed
two interactions, one hydroxyl group with water molecule in association to Arg 195, and another
pi-pi interaction with amino acid residues Trp 59 (4.76 Å) (Fig. 8). The acidic carbonyl group of
compound GB-12 showed solvent interaction (H2O) in association with Glu 233, Asn 298, Ala
307 and Asp 300 (Fig. 9). The carbonyl acidic group of compound GB14 showed two hydrogen
bonding interaction with water in association to Ala 307 and Asp 300 and one in direct contact
with Asp 300, compound also showed pi-pi interaction with phenyl ring of Trp 59 (Fig. 10). The
standard acarbose showed many interactions with water molecule associated with Ala 307, Asp
300, Glu 233, Asn 298, Thr 163 and Arg 195. The hydroxyl group also showed interactions with
Ala 307, Thr 163 and Asp 300 as shown in Fig. 11. From the findings, it is evident that the pattern
of binding in the active site of PPAR-γ and -amylase is almost similar to that of pioglitazone and
acarbose which are well-established therapeutic candidates for diabetes.

(a)
(b)

(c)
Fig. 4. (a) 2D interactions of GB8 with PPAR-γ (b) 3D pose of embedded ligand inside the
pocket (c) Possible interactions between ligand and receptor
(a)

(b)
(c)
Fig. 5. (a) 2D interactions of GB11 with PPAR-γ (b) 3D pose of embedded ligand inside the
pocket (c) Possible interactions between ligand and receptor

(a)
(b)

(c)
Fig. 6. (a) 2D interactions of GB14 with PPAR-γ (b) 3D pose of embedded ligand inside the
pocket (c) Possible interactions between ligand and receptor
`
Fig. 7. 2D interactions of pioglitazone with PPAR-γ

(a)
(b)

(c)
Fig. 8. (a) 2D interactions of GB11 with -amylase (b) 3D pose of embedded ligand inside the
pocket (c) Possible interactions between ligand and receptor
(a)

(b)
(c)
Fig. 9. (a) 2D interactions of GB12 with -amylase (b) 3D pose of embedded ligand inside the
pocket (c) Possible interactions between ligand and receptor

(a)
(b)

(c)
Fig. 10. (a) 2D interactions of GB14 with -amylase (b) 3D pose of embedded ligand inside the
pocket (c) Possible interactions between ligand and receptor
Fig. 11. 2D interactions of acarbose with -amylase
2.3. Biological activity
The synthesized derivatives were subjected to pharmacological screening for antidiabetic activity
against STZ-NA induced diabetes in mice, in vitro -amylase activity, in vitro anti-inflammatory
and antioxidant activity using DPPH method.

2.3.1. In vivo antidiabetic activity
From the docking results, three compounds GB8, GB11 and GB14 showed the best docking scores
and were further evaluated for antidiabetic activity in C57BL/6J mice. After administration of a
single dose of these compounds, the blood glucose levels were monitored as per standard protocols
on 1st, 3rd, 7th and 14th day of the initiation of experiment. As per the analyzed data, compound
GB-14 and GB-8 showed 134.46 ± 0.49 and 136.28 ± 0.79 blood glucose lowering effect as
compared to standard pioglitazone (136.56 ± 0.64) whereas compound GB11 showed moderate
blood glucose lowering effect (139.22 ± 0.66) (Table 1). Therefore compound GB-14 showed the
most potent results which were found to be consistent with the docking results and might occurred
due to the activation of PPAR-γ receptors (Fig. 12).
Table 1
Blood glucose levels of STZ-induced diabetic mice (*P<0.001 when compared with diabetic
control, n=6)
Blood Glucose Level (mg/dL) ± S.D.
Groups
(n = 6)
0 day
3rd day
7th day
109.93 ±
0.51***
14th day
110.79 ±
0.77***
Group I (Control)
112.28 ± 0.77 113.96 ± 0.52***
Group II (Diabetic
control)
113.30 ± 0.45
264.08 ± 0.79
313.14 ± 0.77
387.88 ± 0.76
185.45 ±
0.61***
136.28 ±
0.79***
Group III (GB-8)
Group IV (GB-11)
Group V (GB-14)
115.68 ± 0.53 288.54 ± 0.79***
188.33 ±
0.88***
139.22 ±
0.66***
110.76 ± 0.64
267.48 ± 0.51
189.44 ±
0.52***
134.46 ±
0.49***
115.44 ± 0.73
267.85 ± 0.59
Group VI
(Pioglitazone)
184.90 ±
0.68***
136.56 ±
0.64***
108.42 ± 0.74 250.40 ± 0.69***

Fig. 12. Antidiabetic activity of compounds in STZ-induced diabetic rats. Data are analyzed by
two-way ANOVA followed by Dunnett’s t-test and expressed as mean ± S.D.
2.3.2. In vitro alpha-amylase inhibitory activity assay
All the synthesized compounds were tested for their -amylase activity. It was observed that acidic
derivatives (GB8-GB14) showed more potent -amylase inhibitory action as compared to
benzylated derivatives. Among acidic derivatives, it was observed that compounds GB12 and
GB14 were found to be active inhibitors of -amylase enzyme at different extents but better than
acarbose (Table 2). Log concentrations versus percentage inhibition curves were plotted and IC50
values were calculated for all compounds (Fig. 13). Among these, GB14 has shown the highest
activity against -amylase with IC50 4.08 μg/mL whereas GB-12 was the second most potent -
amylase inhibitor with IC50 7.59 μg/mL. Both of them were found better than acarbose which was
having IC50 8.0 μg/mL.
Table 2
IC50 values obtained by alpha-amylase inhibition
Compound
Code
GB1
% Inhibition (mean ± S.D.)* (µg/mL)
100 150
62.62 ± 0.22 75.56 ± 0.35
IC50
(µg/mL)
41.65
>200
50
200
52.71 ± 0.62
-2.17 ± 0.87
-9.31 ± 0.77
45.89 ± 0.21
7.28 ± 0.98
87.61 ± 0.25
17.88 ± 1.65
13.7 ± 0.84
78.89 ± 0.54
47.24 ± 0.88
GB2
4.81 ± 0.87
-2.21 ± 1.5
11.74 ± 0.87
5.68 ± 1.84
GB3
>200
GB4
57.82 ± 0.56 66.98 ± 0.59
20.21 ± 0.45 33.54 ± 0.52
95.75
>200
GB5

GB6
GB7
10.03 ± 1.5
39.82 ± 0.21
39.11 ± 1.6
50.29 ± 0.14
48.33 ± 1.8
51.21 ± 0.8
58.12 ± 1.9
58.19 ± 0.26
58.19 ± 0.71
57.38 ± 0.25
21.43 ± 1.8
50.21 ± 0.12 58.27 ± 0.21
54.21 ± 1.8 68.89 ± 0.77
59.97 ± 0.65 72.39 ± 0.59
60.39 ± 1.8 73.54 ± 0.48
32.39 ± 0.46
45.41 ± 0.12
69.26 ± 0.77
83.94 ± 1.54
82.56 ± 1.69
85.32 ± 0.44
86.69 ± 0.25
85.32 ± 1.39
87.15 ± 1.65
85.17 ± 1.58
85.78 ± 0.65
>200
102.65
86.44
50.50
57.01
45.10
7.59
GB8
GB9
GB10
GB11
GB12
GB13
GB14
Acarbose
62.71 ± 0.74 75.98 ± 0.54
66.31 ± 1.15 76.33 ± 0.98
62.09 ± 0.47 74.13 ± 1.36
67.32 ± 1.65 77.01 ± 1.24
22.03
4.08
67.99 ± 1.3
77.99 ± 0.87
8.0
* Values are expressed as mean ± standard deviation (n = 3)
(a)

(b)
(c)
Fig. 13. (a) % inhibition of alpha-amylase enzyme by GB1-7 (b) % inhibition of alpha-amylase
enzyme by GB8-14 (c) % inhibition of alpha-amylase enzyme by acarbose at different
concentrations
2.3.3. Anti-inflammatory activity
The in vitro anti-inflammatory studies evaluated the ability of the target derivatives GB1-GB14 to
lower down the expression of inflammatory markers (TNF-, IL-1β and MDA) using an enzyme
immunoassay (EIA). The data (Table 3-5) showed that the target compounds possessed significant

anti-inflammatory activity when compared to control. Benzylated TZDs were shown to give more
potent results than the acidic TZDs and also aming benzylated TZD, compound with nitro
substitution (GB7) came out to be the most potent derivative. Then, comes the more potent
compounds with electron releasing group (Cl or F) (Fig. 14-16). Among all, the best lowering
inflammatory marker was found to be IL-1β.
Table 3.
TNF- activity of synthesized derivatives
Compound
Mean ± S.E.M.
TNF- activity (pg/mL)
i
ii
iii
522
Control
GB1
508
250
210
218
357
197
410
310
467
290
305
183
515
257
215
207
348
190
398
302
458
282
298
189
515 ± 4.04
255.66 ± 2.96
214.33 ± 2.33
212.33 ± 3.17
355 ± 6.24
260
218
212
360
205
412
315
462
275
311
187
GB2
GB3
GB5
GB7
197.33 ± 4.33
406.67 ± 4.37
309 ± 3.78
GB8
GB9
GB10
GB11
GB14
Etoricoxib
462.33 ± 2.60
282.33 ± 4.33
304.67 ± 3.75
186.33 ± 1.76
Table 4.
IL-β activity of synthesized derivatives
Compound
IL-1β activity (pg/mL)
Mean ± S.E.M.
i
680
22
15
28
36
24
62
36
32
ii
iii
Control
GB1
672
18
22
22
28
20
71
29
28
686
26
12
19
31
17
66
32
31
679.33 ± 4.05
22 ± 2.30
GB2
GB3
16.33 ± 2.96
23 ± 2.64
GB5
31.66 ± 2.33
20.33 ± 2.02
66.33 ± 2.60
32.33 ± 2.02
30.33 ± 1.20
GB7
GB8
GB9
GB10

GB11
GB14
51
35
16
46
30
24
55
28
23
50.66 ± 2.60
31 ± 2.08
Etoricoxib
21 ± 2.51
Table 5.
MDA activity of synthesized derivatives
Compound
MDA activity (nM/mg)
Mean ± S.E.M.
i)
ii)
iii)
23.6
12
Control
GB1
22.4
10.6
9.2
23.2
11.2
10.5
9.9
10.5
6
23.06 ± 0.35
11.26 ± 0.40
9.83 ± 0.37
10.3 ± 0.26
11.3 ± 0.43
5.36 ± 0.40
18.93 ± 0.98
12.2 ± 0.87
13.26 ± 0.75
11.23 ± 0.64
9.36 ± 0.52
4.73 ± 0.21
GB2
9.8
GB3
10.8
11.4
4.6
10.2
12
GB5
GB7
5.5
GB8
19.6
13.4
14.6
12.2
8.4
17
20.2
12.7
13.2
11.5
9.5
GB9
10.5
12
GB10
GB11
GB14
Etoricoxib
10
10.2
5
4.3
4.9
TNF-
TNF-
600
Compounds
500
400
300
200
Fig. 14. TNF- activity taken in triplicate of the synthesized compounds
100
0

IL-1β
800
600
400
Compounds
Fig. 15. IL-β activity taken in triplicate of the synthesized compounds
200
0
MDA
30
25
Compounds
20
15
10
Fig. 16. MDA activity taken in triplicate of the synthesized compounds
5
0
2.3.4. Antioxidant activity
The synthesized compounds were evaluated for their in vitro antioxidant activity by DPPH method
using ascorbic acid as standard. The results showed that all the synthesized compounds showed
good antioxidant activity in comparison to ascorbic acid, wherein compounds GB4, GB5 and GB8
showed the most potent results with IC50 values of 110.88, 127.18 and 128.55 µg/mL, respectively.
The standard drug ascorbic acid showed the IC50 value 81.12 µg/mL (Table 6 and Fig. 17). Since
all the three compounds (GB4, GB5 and GB8) showed promising results potent analogues and

therefore, can be used as novel lead molecule for future research work and these compounds might
lead to the development of additional treatments directed towards the disorders caused by free
radicals.
Table 6
IC50 values obtained by DPPH assay
Compound
Code
GB1
% Radical scavenging activity (mean ± S.D.)
IC50
(µg/mL)
143.51
159.56
154.27
110.88
127.18
147.56
143.26
128.55
168.11
195.86
146.26
164.36
161.84
210.18
81.12
50 µg/mL
23.52 ± 0.02
19.65 ± 0.12
22.83 ± 0.06
32.97 ± 0.09
34.12 ± 0.13
27.29 ± 0.15
31.79 ± 0.04
32.21 ± 0.12
28.02 ± 0.21
29.22 ± 0.013
27.01 ± 0.09
24.96 ± 0.06
25.88 ± 0.04
25.16 ± 0.01
100 µg/mL
37.69 ± 0.03
33.67 ± 0.15
35.83 ± 0.19
46.96 ± 0.09
44.18 ± 0.08
40.26 ± 0.14
41.29 ± 0.22
43.66 ± 0.21
37.17 ± 0.06
36.14 ± 0.16
40.06 ± 0.23
35.42 ± 0.08
36.86 ± 0.03
36.66 ± 1.4
54.18 ± 0.09
150 µg/mL
52.36 ± 0.03
46.70 ± 0.05
48.38 ± 1.6
61.12 ± 1.7
55.23 ± 2.1
52.33 ± 0.09
51.80 ± 0.04
54.21 ± 0.06
47.33 ± 0.07
42.36 ± 1.2
52.13 ± 0.03
46.78 ± 0.08
46.88 ± 1.3
47.56 ± 2.3
65.13 ± 0.03
200 µg/mL
65.67 ± 2.1
61.69 ± 0.14
62.33 ± 0.19
75.21 ± 2.7
64.81 ± 0.04
60.36 ± 0.09
61.02 ± 0.06
66.96 ± 1.3
55.74 ± 0.04
51.38 ± 0.03
61.36 ± 0.08
58.32 ± 1.3
58.86 ± 1.4
57.36 ± 0.08
78.10 ± 0.04
GB2
GB3
GB4
GB5
GB6
GB7
GB8
GB9
GB10
GB11
GB12
GB13
GB14
Ascorbic acid 43.22 ± 0.19

(a)
(b)

(c)
Fig. 17. (a) % of radical scavenging by GB1-7 (b) % of radical scavenging by GB8-14 (c) % of
radical scavenging by ascorbic acid at different concentrations
2.4. Structure activity relationship (SAR)
As regards the relationship between the structures of the various synthesized scaffolds and the
observed antidiabetic, anti-inflammatory and antioxidant activity, it showed varied biological
activity. The SAR of the synthesized compounds are analysed on the basis of substitution on aryl
ring attached to pyrazole core along with nature of N-substitution (TZD head). In case of
antidiabetic activity, the acidic head derivatives were found to be most potent in comparison to
benzylated TZD along with electron withdrawing substitution on aryl ring of pyrazole (GB14).
Compounds containing electron releasing groups further showed decreased activity (GB2 and
GB3). In case of anti-inflammatory activity, benzylated TZDs exhibited significant results and
along with electron withdrawing group substitution on pyrazole ring (GB7). In case of antioxidant
activity, the benzylated TZDs showed more potent results (GB4 and GB5) rather than acidic
TZDs.
3. Conclusion
A total of fourteen novel diaryl pyrazolyl thiazolidinediones were synthesized via Knoevenagel
condensation of substituted pyrazole carboxaldehdyes with TZDs and along with N-substitution
using benzyl bromide and bromoacetic acid, respectively. The resulting compounds were docked
in order to study the ligand protein interactions of designed compounds with PPAR-γ (PDB ID-
2PRG) and alpha-amylase (PDB ID-4GQR) using MOE software version 2008.10. Compounds
GB8, GB11 and GB14 were well docked in the active site of PPAR-γ and compounds GB11,
GB12 and GB14 were well docked in the active site of alpha-amylase with the best docking scores.

The compounds with best docking scores were further screened for antidiabetic activity in STZ-
induced diabetic mice. Compound GB14 equipped with 4-nitro phenyl group along with acidic
head showed significant blood glucose lowering effect (134.46 mg/dL) quite comparable to
pioglitazone (136.56 mg/dL). Compounds GB12 (IC50: 7.59 μg/mL) and GB14 (IC50: 4.08 μg/mL)
were found to be active inhibitors of alpha-amylase enzyme at different extents and also better
than acarbose (IC50: 8.0 μg/mL) on in vitro evaluation for alpha-amylase activity. The in vitro anti-
inflammatory screening results showed that the benzylated TZDs along with NO2 (GB7) exhibited
the most potent results. Additionally, compounds were also evaluated for antioxidant activity by
DPPH method. As a result, compounds (GB4, GB5 and GB8) exhibited promising results and
showed IC50 values of 110.88, 127.18 and 128.55 µg/mL, respectively and the results were
compared to standard drug acarbose (81.12 µg/mL). The compounds with good activity profiling
may be used as novel lead for future research work.
4. Experimental section
All the chemicals were purchased from CDH, Hi-Media, Loba Chemie and Sigma-Aldrich.
Reactions were monitored by thin layer chromatography (TLC) on precoated silica-gel plates and
visualized using ultraviolet light and iodine. Recrystallization of compounds was performed with
chloroform-methanol and ethylacetate mixture. The melting points were determined by open
capillary method and are uncorrected. IR spectra were recorded on Shimadzu 8400 S FT-IR
spectrophotometer using KBr pellets from 400-4000 cm-1. Proton (1H) Nuclear Magnetic
Resonance (1HNMR) and 13CNMR spectra were obtained using Bruker Avance II 400 MHz and
400 MHz respectively using DMSO as a solvent. Mass spectra was recorded on Shimadzu QP2010
ULTRA GC-MS. Chemical shifts were obtained in parts per million (ppm) with tetramethylsilane
(TMS) as an internal standard, and coupling constant (J) values are stated in Hertz (Hz). The
following notations expressed the peak types in the spectra: singlet (s), doublet (d), triplet (t) and
multiplet (m).
4.1. Chemistry
4.1.1. Synthesis of thiazolidine-2,4-dione (3)
A solution of chloroacetic acid (0.6 M) in 60 mL of water and thiourea (0.6 M) in 60 mL of water
was stirred for 15-20 min to obtain white precipitates. To the content, 60 mL of concentrated
hydrochloric acid was added. Then, the reaction mixture was refluxed for 8-10 h at 100-110 oC.
On cooling, the contents were solidified into white needles as TZD (3) and was filtered and washed
with water. The compound was then recrystallized from ethyl alcohol. The purity of synthesized
compound was ascertained by TLC using ethyl acetate: hexane (80:20) as the mobile phase. Yield:
64%; IR (KBr, cm-1): 1736.74 and 1671.24 (two C=O), 3482.62 (N-H), 1338.53 (C-H, bend),
3052.17 (C-H, strech), 627.85 (C-S); 1H NMR (400MHz, DMSO, δ ppm): 3.9 (s, 2H, CH2), 9.8
(s, 1H, NH).
4.1.2. Synthesis of pyrazole-4-carbaldehydes (7a-g)

3-(4-substituted-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde was prepared by the reaction of
acetophenone and its derivatives with phenyl hydrazine in presence of ethanol as a solvent and
acetic acid as catalyst, then the resulted hydrazone derivative was treated with the Vilsmeier-Haack
reagent (DMF–POCl3) leading to the corresponding 4-carboxaldehyde functionalized pyrazole
ring (7a-g). The progress of the reaction was ascertained by TLC using ethyl acetate: hexane
(70:30) as the mobile phase. Yield: 80%; IR (KBr, cm-1): 3060.82 (aromatic C-H), 1600.98 and
1450.37 (aromatic C=C), 697.81 (aromatic C=C-H bending), 1656.63 (C=O).
4.1.3. Synthesis of N-substituted TZD (10 and 11)
The synthesis of N-benzylated TZD was carried out on stirring at 50-55 oC by taking 1.7 mmol of
TZD, benzyl bromide (1.1 equivalents) and potassium carbonate (2 equivalents) in solvent DMF
(4 mL). The reaction gets completed in 2-3 h and was monitored by TLC. After stirring, the
reaction mixture was poured into ice cold water and the content was precipitated out and filtered
and washed with cold water. Similarly, for N-acidic TZD, the reaction was carried out between
TZD and bromoacetic acid at same reaction conditions. The products (10 and 11) were then
recrystallised from ethanol. Yield: 85% (benzylated) and 65% (acidic); IR (KBr, cm-1): 1174 (C-
N), 1432 (CH2), 1648 and 1771 (2 ring C=O), 1541 (aromatic C=C), 3058.75 (aromatic C-H),
1720 (acidic C=O), 3240 (OH stretch), 946 (OH bend).
4.1.3. Synthesis of final derivatives (Knoevenagel condensation) (GB1-14)
The final step involves the Knoevenagel condensation of various pyrazole carbaldehydes with N-
substituted TZDs (both benzylated and acidic). The perfect spot was observed in TLC the product
was obtained in good yield. The solvent system used for TLC is toluene: ethyl acetate: formic acid
(8.0:1.5:0.5). All the title compounds GB (1-14) were obtained in satisfactory yield after
purification by recrystallization.
4.1.3.1. 3-benzyl-5-((1,3-diphenyl-1H-pyrazol-4-yl)methylene)thiazolidine-2,4-dione (GB1)
Yield: 85%; IR (KBr, cm-1): 1739 and 1686 (two C=O), 1614 (C=N), 3067 (aromatic C-H), 1597
and 1456 (aromatic C=C), 2924 (aliphatic C-H); 1H NMR (400MHz, DMSO, δ ppm): 8.51-8.49
(1H, s, pyrazolyl H), 7.45-7.43 (1H, s, =CH-), 4.85-4.83 (2H, s, N-CH2), 7.82-7.23 (15H, m,
aromatic H); 13C NMR (400MHz, DMSO, δ ppm): 168.04-165.37 (2C=O), 139.82-139.03 (-CH=),
155.37-154.23 (C-3 and 5, pyrazolyl ring), 44.63-44.46 (N-CH2), 119-137 (aromatic C); Mass
(m/z): 437.51 [M+H]+.
4.1.3.2. 3-benzyl-5-((3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)thiazolidine-2, 4-
dione (GB2)
Yield: 82%; IR (KBr, cm-1): 1740 and 1686 (two C=O), 1613 (C=N), 3056 (aromatic C-H), 1598
1
and 1452 (aromatic C=C), 1598 (aliphatic C=C), 2946 (aliphatic C-H), 1243 (C-F stretch); H
NMR (400MHz, DMSO, δ ppm): 8.81-8.79 (1H, s, pyrazolyl H), 7.73-7.71 (1H, s, =CH-), 4.36-