Synthesis of novel inhibitors of α-amylase based on the thiazolidine-4-one skeleton containing a pyrazole moiety and their configurational studies

Article abstract of DOI:10.1039/c7md00080d

Postprandial hyperglycemia can be controlled by delaying the absorption of glucose resulting from carbohydrate digestion. α-Amylase is the initiator of the hydrolysis of polysaccharides, and therefore developing α-amylase inhibitors can lead to development of new treatments for metabolic disorders like diabetes mellitus. In the present work, we set out to rationally develop α-amylase inhibitors based on the thiazolidine-4-one scaffold. The structures of all these newly synthesized hybrids were confirmed by spectroscopic analysis (IR, ¹H-NMR, MS). The appearance of two sets of signals for some protons in ¹H NMR revealed the existence of a mixture of 2E,5Z (37.1-42.0%) and 2Z,5Z isomers (58.4-62.8%), which was further supported by DFT studies. All the newly synthesized compounds have potential inhibitory properties as revealed through in vitro α-amylase inhibition activity. Compound 5a at 100 μg mL^-1 concentration showed a remarkable inhibition of 90.04%. In vitro α-amylase inhibition was further supported by docking studies of compound 5a against the active site of human pancreatic α-amylase (PDB ID: 2QV4). The docking studies revealed that the bonding interactions found between 5a and human pancreatic α-amylase are similar to those responsible for α-amylase inhibition by acarbose.

Full text of DOI:10.1039/c7md00080d

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Synthesis of novel inhibitors of αꢀamylase based on thiazolidineꢀ4ꢀone skeleton containing
pyrazole moiety and their configurational studies
Parvin Kumara*, Meenakshi Duhana, Kulbir Kadyana, Jayant Sindhub, Sunil Kumarc,
Hitender Sharmac
aDepartment of Chemistry, Kurukshetra University Kurukshetraꢀ136119
bS D (PG) College, Panipatꢀ132103,
cInstitute of Pharmaceutical Science, Kurukshetra University Kurukshetraꢀ136119
*E.mail: parvinjangra@gmail.com; parvinchem@kuk.ac.in
Abstract
Postprandial hyperglycemia can be controlled by delaying the absorption of glucose resulting
from carbohydrate digestion. αꢀamylase is the initiator of the hydrolysis of polysaccharides,
therefore developing αꢀamylase inhibitors can lead to development of new treatments for
metabolic disorders like diabetes mellitus. In the present work, we set out to rationally develop
αꢀamylase inhibitors based on thiazolidineꢀ4ꢀone scaffold. The structure of all these newly
synthesized hybrids was confirmed by spectroscopic analysis (IR, 1HꢀNMR, MS). Appearance of
1
two sets of signals for some protons in H NMR revealed the existence of a mixture of 2E,5Z
(37.1ꢀ42.0% ) and 2Z,5Z isomer (58.4ꢀ62.8%) which was further supported by DFT studies. All
the newly synthesized compounds have potential inhibitory properties as revealed through in
vitro αꢀamylase inhibition activity. Compound 5a in 100 µg/mL concentration showed
remarkable inhibition of 90.04%. In vitro αꢀamylase inhibition was further supported by docking
studies of compound 5a against active site of human pancreatic αꢀamylase (PDB ID: 2QV4).
Docking studies revealed that the bonding interactions found between 5a and human pancreatic
αꢀamylase are similar to those responsible for αꢀamylase inhibition by acarbose.
Keywords: Thiazolidinꢀ4ꢀone, pyrazole, αꢀamylase, configurational isomer, 1H NMR, docking
Introduction
DM (Diabetes mellitus) is a metabolic disorder characterized by chronic hyperglycemia or
increased blood glucose levels with disturbances in carbohydrate, fat and protein metabolism
resulting from absolute or relative lack of insulin secretion.1 Diabetes, being one of the most
common global diseases, affects approximately 200 million individuals worldwide.2 The

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management of the blood glucose level as close to normal as possible in patients with diabetes
mellitus is the most challenging task. Therefore, enzymes that regulate gluconeogenic or
glycogenolytic pathways are key biological targets for therapeutic interventions. Out of several
enzymes known αꢀamylase is an important key enzyme responsible for carbohydrate digestion.
So inhibitors of αꢀamylase can effectively retard the digestion and assimilation at the early steps
of starch digestion, and thus succeed in a significant delay of postprandial hyperglycemia and
have a beneficial effect on insulin resistance.3 αꢀAmylase are considered to be one of the best
targets for the development of type II diabetes therapeutic agents due to their ability to catalyze
the hydrolysis of αꢀ(1,4)ꢀglycosidic linkages in starch.4,5 Acarbose and voglibose are two known
inhibitors for αꢀamylase which are in clinical use now days. However, they often cause severe
gastrointestinal side effects such as abdominal pain, flatulence, and diarrhea.6 In recent years αꢀ
amylase has been a point of interest for the development of novel antiꢀobesity and antidiabetic
drugs.7
Thiazolidineꢀ4ꢀone represents one of the important class of compounds incorporated in several
antiꢀdiabetic drugs.8 This compound exhibit a wide range of pharmacological activities such as
antiꢀmicrobial,9 antiꢀtubercular activity,10 antiꢀinflammatory,11 antiꢀcancer,12 antiꢀoxidant,13 antiꢀ
hyperglycemic agents14,15 and antiꢀviral.16 There is a wide scope for different pharmacological
properties associated with this molecule due to presence of active methylene position in this
compound. Similarly, pyrazole also represents an important class of compounds not only for
their theoretical interest but also for antiꢀinflammatory, analgesic, antiꢀtumor, antiꢀhypertensive,
antiꢀpyretic, sedatives, antiꢀbacterial and antiꢀdiabetic activities.17ꢀ23
In the light of these facts and in continuation of our interest in the synthesis of heterocycles
containing a multiꢀstructure for biological activity, we, herein report a new class of thiazolidinꢀ4ꢀ
one linked pyrazole to see the additive effect of these rings towards αꢀamylase inhibition.
Result and Discussion
Chemistry
The target compounds 5ꢀ((3ꢀ(aryl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)ꢀ2ꢀ(pꢀtolylimino)
thiazolidinꢀ4ꢀone (5aꢀg) were synthesized by a following synthetic protocol. Firstly, a reaction of
pꢀtolyl thiourea (2) and ethyl bromoacetate was employed to synthesise 2ꢀ(pꢀtolylimino)
thiazolidinꢀ4ꢀone (3) which is a key reactant for the development of novel hybrids. Compound 3
was further subjected to Knoevenagel condensation with 3ꢀ(aryl)ꢀ1ꢀphenylꢀ1Hꢀpyrazoleꢀ4ꢀ

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carbaldehyde24 (4aꢀg) using catalytic amount of piperidine in refluxing ethanol for 10ꢀ12 h. The
progress of the reaction was monitored by TLC using petroleum ether: ethylacetate (60:40, v/v).
After completion of the reaction, the reaction mixture was cooled down at room temperature and
the solid so obtained was filtered to yield 5ꢀ((3ꢀ(aryl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)ꢀ2ꢀ
(pꢀtolylimino) thiazolidinꢀ4ꢀone (5aꢀg) in 72ꢀ85% yield (Scheme 1, Table 1). The formation of
the compounds (5aꢀg) was confirmed by using IR, 1H NMR, Mass and elemental analysis.
Scheme 1: Synthesis of 5ꢀ((3ꢀ(aryl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)ꢀ2ꢀ (pꢀtolylimino)
thiazolidinꢀ4ꢀone 5aꢀg.
Table 1: Synthesis of 5ꢀ((3ꢀ(aryl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)ꢀ2ꢀ(pꢀtolylimino)
thiazolidinꢀ4ꢀone 5aꢀg
S. No.
Compound
ꢀR
Yield (%)
M.pt. °C
298ꢀ300
270ꢀ275
263ꢀ265
292ꢀ295
288ꢀ290
290ꢀ292
288ꢀ290
1
2
3
4
5
6
7
ꢀOCH3
ꢀCH3
ꢀH
85
76
83
78
72
74
85
5a
5b
5c
5d
5e
5f
ꢀCl
ꢀF
ꢀBr
ꢀNO2
5g
Characterization of compounds and their configurational studies
1
The structure of these hybrids was ascertained by IR, H NMR and mass spectral data. The
absorption signals corresponding to N–H stretching appeared at 3,050ꢀ3,127 cmꢀ1 and C=O
stretching appeared in the region of 1,700ꢀ1,720 cmꢀ1 of thiazolidinone ring respectively. The
presence of a band in the range 1,646–1,691 cmꢀ1 reveals the formation of exocyclic C=C bond at
5th position. It is assumed that the compound 5, restricted rotation about (C=C) linkage as well as

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the imine (C=N) linkage led to the formation of 4 possible configuration that is 2Z,5E; 2E,5E;
2E,5Z and 2Z,5Z as shown in the (Fig. 1)(1 to 4)
O
O
O
O
H
H
H
H
N
N
N
N
Pyr
Pyr
Ar1
Ar1
S
S
S
N
S
N
Ar1
N
N
Ar1
Pyr
Pyr
2Z, 5E (1)
2E, 5E (2)
2E, 5Z (3)
2Z, 5Z (4)
Figure 1: Four possible isomers for 5.
Literature survey reveals that the 2ꢀaryliminoꢀ4ꢀthiazolidinone 3exists as a mixture of E and Z
1
13
isomers of imino form in dimethylsulphoxideꢀd6 and predominantly in Z form.25 In H and C
NMR of compound 3 also contains two set of signals for NꢀH, ꢀCH2, and aromatic protons as
shown in (Fig. 2).
H
δ = 3.96 (d, 2H, J= 18.8Hz)
H
CH3
2.27 (s, 3H)
S
O
N
N
H
δ = 11.10, 11.66 (2s, NH)
Figure 2: Characteristic peak assigned in1H NMR of compound 3.
In the 1H NMR spectra of compound (5aꢀg) the signals for the olefinic proton (H7) was detected
at δ 7.24ꢀ7.57 ppm, deshielded by the adjacent C=O, a higher chemical shift value than expected
one for E isomer.26 In E isomer, due to lesser deshielding effect of sulphur, such a proton should
resonate at lower delta values.27 Therefore we omit the probability of 2Z,5E and 2E,5E isomers.
The NMR (1H) spectra of compounds 5aꢀg also gave two sets of signals which confirmed the
existence of two configurational isomers in dimethylsulphoxideꢀd6 (2E,5Z and 2Z,5Z) and
according to the literature25ꢀ27 the predominance was assigned to the 2Z,5Z isomer.
Compound 5a was taken as a test sample to study the configurational isomers by means of IR,
1Hꢀ NMR, mass, 1Hꢀ1H COSY (Fig. 3 and Fig.4). Disappearance of signal of methylene proton at
δ 3.80 and appearance of signal in the range of δ 7.24ꢀ7.57 due to C=CꢀH olefinic hydrogen
confirmed the formation of exocyclic C=C bond. The numeration of structure is given
specifically for NMR analysis only. The amide proton (O=CꢀNꢀH) observed at
12.00ꢀ12.53
ppm showed that substitution is on 2nd position of thiazolidinꢀ4ꢀone which is in agreement with a
lactam proton, since an aryl amine proton appears at much higher field.24 Two singlet at 2.30

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(39.94%) and
2.31 (60.55%) were observed due to methyl protons of aryl ring of imine.
Signals at δ 8.52 (61.72%) and δ 8.61 (39.21%) in form of two sharp singlet were observed for
proton of pyrꢀH. Duplication of signals have also been observed for protons showing resonance
in aromatic region associated with the proton of E and Z geometrical isomers of the compound.
Two protons H28/32, exhibits two doublets at δ 7.92 (59.31%) and δ 7.96 (40.74%) with a
coupling constant 7.6 and 8.0 Hz. Two doublets δ 6.97 (J=8.0 Hz) (40.82%) and δ 7.19ꢀ7.21
(59.17%) are due to two protons H10/12. In ESIꢀMS mass spectra of compound 5a, m/z value was
observed at 466.85. In order to understand the effect of solvent on configurational isomer
distribution, the NMR of compound 5a was taken in TFA. Interestingly the % of 2Z,5Z isomer
has been increased over 2E,5Z isomer in a ratio of 95:5 (supplementary file). This may be due to
the solvation and stability of different configuration in different solvent. The chemical shift of
olefinic protons increased from δ 7.43 to δ 8.06. Moreover the protons of OCH3 and CH3were
found downfield. This may be due to the protonation of nitrogen and oxygen.
1
In HꢀNMR of 2ꢀ(pꢀtolylimino)ꢀ5ꢀ((3ꢀaryl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)thiazolidinꢀ4ꢀ
one (5aꢀg), two singlets were observed for ꢀCH3( 2.11ꢀ2.31), pyrꢀH (δ 8.18ꢀ8.64) whereas other
protons of aromatic rings also showed two sets of signals associated with the protons of 2E,5Z
and 2Z,5Z isomers of the compounds. The ratio of 2E,5Z and 2Z,5Z isomers was calculated by
the NMR integration ratio of respective protons in proton NMR analysis. The integration of the
two singlet of methyl (ꢀCH3) and pyrazole proton were used to calculate the relative abundance
of each isomer at 25oC. The percentage of 2E,5Z and 2Z,5Z isomers was found in the range of
37.1ꢀ42.0% and 58.4ꢀ62.8%, respectively as shown in (Table S1).
15
H
O
H
4
26
OCH3
O
3
N
O
H
5
H
25
H
7
27
21
6
N
N
S
2
16
N
H
24
Ar1
1
23
20
8
H17
S
S
N
H
H
13
H
N
Pyr
H
Pyr
N
Ar1
9
N
19
12
H
28
2E,5Z-isomer
δ = 2.30, 8.61, 7.96
2Z,5Z-isomer
δ = 2.31, 8.52, 7.92
22
10
H
11
H
32
CH3
H
29
14
30
31
H
H-H COSY
H
Figure 3: Characteristic peaks for isomers and HꢀH COSY correlation of 5a.

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Figure 4: Characteristic peak and comparative 1H NMR of compound 5ain DMSO and TFA.
Computational studies
The 5ꢀ((3ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)ꢀ2ꢀ(pꢀtolylimino) thiazolidinꢀ4ꢀ
one (5a) can exist in 4 possible configuration that is 2Z,5E; 2E,5E; 2E,5Z and 2Z,5Z as shown in the
(Fig. 1)(1 to 4). In this work the 3ꢀdimensional structures of 2ꢀ(pꢀtolylimino)ꢀ5ꢀ((3ꢀaryl)ꢀ1ꢀ
phenylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)thiazolidinꢀ4ꢀone configurational isomer in their neutral state
were obtained by the DFT28 approach utilizing Becke’s three parameter functional29 with the
Vosko et al.30 local and Leeet al.31 nonꢀlocal correlation, abbreviated as B3LYP1(VWN formula
1 RPA in B3LYP). The rotations about the C2ꢀN6 (Fig. 1) (1nd and 2nd), C5ꢀC7 (Fig. 1) (3rd and
4th) bonds respectively were taken into account. We have calculated the rotational energy barrier
by steps of 10° around. All four isomers presented in (Fig. 1) were considered in our study. All
the molecules were geometryꢀoptimized until the rootꢀmean square (RMS) gradient value was
smaller than 10ꢀ6 a.u. To structurally characterize the molecule in detail, a systematic
investigation of its potential energy surface was undertaken at the DFT (B3LYP1)/6ꢀ311G level
of approximation.32 Later, using the surface data generated from firefly software, the distribution
of charge in a molecule was calculated. To obtain a 3D plot of the MEP, the electrostatic
potential cube file was calculated from total SCF density. The contour maps of the electrostatic
potential were then drawn using a distance between grid points of 0.02 Å and the iso value of
0.0004. The firefly software was used to calculate the electrostatic potential maps and surfaces as
the distribution of the potential energy of a unit positive charge in a given molecular space, with
a resolution controlled by the grid density. The vibrational wave numbers were calculated at the
DFT (B3LYP1)/6ꢀ311G level of approximation. It is well known in the quantum chemical

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literature that among the available functional, the B3LYP1 functional yields a good description
of harmonic vibrational wave numbers for small and mediumꢀsized molecules. All calculations
were performed using the fire fly software. The visualizations were prepared by use of the
MASK.33 By considering the calculated relative energy barriers (DFT) for the 2Z,5E; 2E,5E;
2E,5Z and 2Z,5Z configuration, the most stable isomers are 2Z,5Z and 2E,5Z, due to the highest
energy difference (Table S2). However, the minimum difference of energy was observed in 2Z,
5Z and 2E,5Z isomer i.e. (2.8 kcal/mole) which suggest that these two configurations 2Z, 5Z and
2E, 5Z may not be experimentally accessible as isolate species.
Biological studies
In vitro αꢀamylase inhibition
All the synthesized compounds (5aꢀg) and standard drug were explored for their in vitro αꢀ
amylase inhibition studies at different concentrations (50ꢀ200ꢁg/mL) as shown in the Table 2.
All the compounds showed good % inhibition of αꢀamylase when compared with standard drug
acarbose. Compound 5b and 5e were found to be more potent among all the synthesized
compounds when explored at the concentration of 50µg/mL. Compound 5e shows 77.12%
inhibition followed by 5b with 79.24% inhibition. There was a significant rise in % inhibition
when concentration has been changed to 100µg/ml from 50µg/mL. Among all, 5b shows 83.72%
inhibition followed by 5a which showed 90.04% inhibition at 100µg/ml. Apart from this 5a
shows a sudden rise in % inhibition from 75.94 to 90.04 % on increasing concentration to
100µg/mL from 50µg/mL. Inspired by the results obtained at 100µg/mL concentration, all the
synthesized compounds were further screened for there in vitro αꢀamylase inhibition at
200µg/mL. Compounds 5a, 5b, 5c and 5f exhibited a linear rise in % inhibition, but a reduction
in % inhibition has been observed for compounds 5d, 5e and 5g owing to their less solubility at
concentration of 200µg/mL. Among compounds showing enhanced % inhibition, compound 5a
showed % inhibition of 92.5% followed 5f with % inhibition of 97.4% at concentration of
200µg/mL. A comparative graph of % inhibition of compounds 5aꢀg with standard drug
acarbose has been shown in (Fig. 5).

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Table 2: αꢀamylase inhibition activity of compounds 5aꢀg.
Compound
Concentration
(µg/mL)
50
OD at 595
nm
Residual Activity % Inhibition
0.102
0.042
0.032
0.088
0.069
0.068
0.211
0.078
0.066
0.208
0.080
0.106
0.097
0.078
0.093
0.203
0.115
0.011
0.148
0.050
0.178
0.218
0.196
0.154
24.05
9.90
75.94
90.04
92.15
79.24
83.72
83.97
48.08
81.60
84.27
50.94
81.13
75.03
77.12
81.60
78.05
52.12
72.87
97.40
65.09
88.20
58.00
48.58
53.77
63.68
5a
100
200
50
7.54
20.75
16.27
16.04
49.76
18.39
15.73
49.05
18.87
24.97
28.87
18.39
21.95
47.87
27.12
2.59
5b
100
200
50
5c
100
200
50
5d
100
200
50
5e
5f
100
200
50
100
200
50
11.79
34.90
41.98
51.52
46.23
36.32
5g
100
200
50
Acarbose
100
200

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50 µg/mL
100 µg/mL
200 µg/mL
120
100
80
60
40
20
0
5a
5b
5c
5d
5e
5f
5g
Acarbose
Compound
Figure 5: Comparative analysis of % Inhibition of compounds 5aꢀg.
Molecular docking studies
The aim of specific structural modeling and accurate prediction of activity can be achieved only
by molecular docking studies.34 Interactions between inhibitors and active site of the target
protein can be explored using molecular docking studies. The above results showed that all the
synthesized molecules were stronger inhibitors of alphaꢀamylase as compared to acarbose.
Although compound 5f showed maximum inhibition at 200µg/mL but its % inhibition at
100µg/mL was less than compound 5a which showed a maximum inhibition of 90.04% at
concentration of 100µg/mL. After comparison of their molar mass it has been revealed that lesser
dose of 5a was required for maximum inhibition of αꢀamylase owing to its lower molar mass.
Therefore, for ascertaining the binding conformation and interactions responsible for the activity,
docking simulation of compound 5a was performed against active site of human pancreatic
alphaꢀamylase (PDB ID: 2QV4). The docked pose of 5a with highest binding affinity is shown in
(Fig. 6).

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Figure 6: Interactions (dashed lines) of compound 5a with active site residues of human
pancreatic alphaꢀamylase.
Figure 7: Pose view Image of QV4 in 2QV4.
It can be noticed from the Fig. 6 that mainly hydrophobic interactions are responsible for
anchoring of the compound 5a. Methoxy phenyl ring of the compound was stacked against
hydroxyl phenyl ring of Tyr62 by PiꢀPi interactions. Methyl phenyl ring attached with

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thiazolidinone moiety created Tꢀshaped PiꢀPi interactions with His201. The methyl group was
also involved in Piꢀalkyl interactions with His201. Thiazolidinone, methyl phenyl and phenyl
rings were also involved in Piꢀalkyl interactions with Leu162, Ala188 and Leu165. Gln63 and His201
formed PiꢀHydrogen bonds with phenyl and methyl phenyl rings, respectively by acting as
hydrogen bond donors. Methoxy group made carbon hydrogen bonds with Glu233and Asp300.
These two residues (Glu233 and Asp300) are reported to act as catalytic residues in hydrolytic
reactions of alphaꢀamylase.35 Further; Tyr62 has been shown as an important residue for binding
of some chalcones to active site of alphaꢀamylase.36 Also, Tyr62, Gln63, Glu233 and Asp300 form
hydrogen bonding interactions with the acarbose (Fig. 7). All these facts show that binding of
compound 5a to these active site residues might be the cause of alphaꢀamylase inhibitory
activity. In (Fig. 8), compound 5a is shown along with coꢀcrystalized ligand acarbose in the
active site of alphaꢀamylase.
Figure 8: Compound 5a along with coꢀcrystalized ligand Acarbose in the active site of human
pancreatic alphaꢀamylase (shown as surface)
Experimental
Structures of all the compounds were identified by their spectral data. Silica gel 60 F254
(Precoated aluminium plates) from Merck were used to monitor reaction progress. All the
melting points were determined in open glass capillary tubes and are uncorrected. IR spectra
were taken on a Cary 660 Agilent IR spectrophotometer and the values are expressed as νmax cmꢀ

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1
13
1. The H and C spectra were recorded on Bruker (AvanceꢀII) at 400 MHz and 100 MHz in
DMSOꢀd6 and trifluoroacetic acid using tetramethylsilane (TMS) as an internal standard.
Chemical shifts (δ) and coupling constants (J) are expressed in ppm and Hz, respectively. All
chemicals used were supplied by Sigma Aldrich and Merck chemical companies. Mass spectrum
was recorded using Waters Micromass QꢀTof Micro. 1ꢀPhenylꢀ3ꢀarylꢀ1Hꢀpyrazoleꢀ4ꢀ
carbaldehydes and 1ꢀpꢀtolylthiourea have been synthesized using reported procedure.37,38
Synthesis of 2ꢀ(pꢀtolylimino)thiazolidinꢀ4ꢀone (3)38
It was prepared by a three component reaction of 1ꢀpꢀtolylthiourea (1) (1.0 mmol), ethyl
bromoacetate (1.0 mmol) and sodium acetate (2.0 mmol) in glacial acetic acid under the
refluxing condition for 2 h. After completion of reaction, reaction was quenched using ice and
the solid so formed was filtered under suction and recrystallized from ethanol. Yield 80%;
M.pt:167ꢀ170°C; 1H NMR (400 MHz, DMSO,d6 ppm): δ 2.27 (s, 3H), 3.96 (d, 2H, J = 18.8 Hz),
6.92 (d, J =7.2, 2H, Ar), 7.18 (d, J = 7.2 Hz, 2H, Ar), 7.59 (d, J =7.6, 2H, Ar), 11.10 & 11.66
(2s, NH); 13C NMR (400 MHz, DMSO, d6, ppm): 20.9, 21.4, 35.6, 38.8, 120.6, 122.1, 129.8,
130.2, 134.3, 134.5, 136.8, 143.2, 177.6, 178.2, 188.5.
Synthesis of 5ꢀ((3ꢀ(aryl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)ꢀ2ꢀ(pꢀtolylimino)thiazoli dinꢀ
4ꢀone(5aꢀg)38
2ꢀAryliminoꢀthiazolidinꢀ4ꢀones 1 (0.5 mmol) and 1ꢀphenylꢀ3ꢀ(pꢀsubstituted phenyl)ꢀ1Hꢀ
pyrazoleꢀ4ꢀcarbaldehydes 2 (0.6 mmol) were dissolved in absolute ethanol. Piperidine (0.5
mmol) was added to the reaction mixture and the reaction mixture was stirred for 8 h at 60°C
until precipitate formed. Then the mixture was cooled to room temperature, and the precipitates
formed were filtered and washed with absolute ethanol to yield the final compound 5aꢀg in good
to excellent yield.
5ꢀ((3ꢀ(4ꢀMethoxyphenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)ꢀ2ꢀ(pꢀtolylimino) thiazolidinꢀ
4ꢀone (5a)
Yield 85%, M.pt. 298ꢀ300°C; IR (vmax cmꢀ1, KBr): 3070 (NꢀH lactam), 3032 (CꢀH str. of
aromatic), 1720 (C=O lactam), 1655 (C=C), 1574 (C=N); 1H NMR (400 MHz, DMSO,d6, ppm):
δ 2.30 & 2.31 (2s, 3H, ꢀCH3), 3.84 (s, 3H, ꢀOCH3), 6.97 (d, J= 8.0 Hz, 2H, H10/H12), 7.11ꢀ7.14
(m, 2H, H24/H26), 7.19ꢀ7.22 (m, 4H, H9/H13, H10/H12), 7.35ꢀ7.43 (m, 2H, H7, H30), 7.49ꢀ7.64 (m,

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6H, H9/H13, H23/H27, H29/H31), 7.92 (d, J=7.6 Hz, 2H, H28/H32), 7.96 (d, J=8.0s Hz, 2H, H28/H32 ),
8.52 & 8.61 (2s, 2H, H 17), 11.95 (broad s, 1H, NH); ESIꢀMS (m/z):466.85;Anal. Calc.
C27H22N4O2S:C, 69.51; H, 4.75; N, 12.01; O, 6.86; S, 6.87; Found: C, 69.45; H, 4.71; N, 11.97;
O, 6.84; S, 6.85.
5ꢀ((3ꢀ(4ꢀMethylphenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)ꢀ2ꢀ(pꢀtolylimino)thiazolidinꢀ4ꢀ
one (5b)
Yield 76%; M.pt. 270ꢀ275°C; IR (vmax cmꢀ1, KBr): 3114 (NꢀH lactam), 3006 (CꢀH str. of
aromatic), 1700 (C=O lactam), 1646 (C=C), 1596 (C=N); 1H NMR (400 MHz, DMSO,d6, ppm):
δ 2.30 & 2.31 (2s, 3H, ꢀCH3), 2.40 (s, 1H, ꢀCH3), 6.96 (d, 2H, J= 7.6 Hz, H10/H12), 7.19ꢀ7.23 (m,
4H, H9/H13, H10/H12), 7.36ꢀ7.43 (m, 4H, H24/H26, H7, H30), 7.50ꢀ7.64 (m, 6H, H9/H13, H23/H27,
H29/H31), 7.93 (d, J=8 Hz, 2H, H28/H32), 7.97 (d, 2H, J=7.6 Hz, H28/H32,), 8.54 & 8.64 (2s, 1H,
H17), 12.08 (broad s, 1H, NH); Calc. For C27H22N4OS: C, 71.98; H, 4.92; N, 12.44; O, 3.55; S,
7.12. Found: C, 71.89; H, 4.82; N, 12.39; O, 3.45; S, 7.09.
5ꢀ((1ꢀphenylꢀ3ꢀpꢀtolylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)ꢀ2ꢀ(pꢀphenylimino)thiazolidinꢀ4ꢀone (5c)
Yield 83%; M.pt: 263ꢀ265°C; IR (vmax cmꢀ1, KBr): 3050 (NꢀH lactam), 3031 (CꢀH str. of
aromatic), 1716 (C=O lactam), 1650 (C=C), 1535 (C=N). 1H NMR (400 MHz, DMSO,d6, ppm):
δ 2.11& 2.13 (2s, 3H, ꢀCH3), 6.74 (d, 2H Hz, J= 8.4 Hz, H10/H12), 6.93ꢀ6.98 (m, 4H, H9/H13,
H10/H12), 7.02ꢀ7.19 (m, 2H, H25,H30), 7.24ꢀ7.35 (m, 5H, H29/H31, H24/H26, H7), 7.38ꢀ7.48 (m, 4H,
H9/H13, H23/H27), 7.66 (d, J=8.0 Hz, 2H, H28/H32), 7.72(d, 2H, J=8.4 Hz, H28/H32), 8.180 & 8.309
(2s, 1H, H17); Anal. Calc. For C26H20N4OS:C, 71.54; H, 4.62; N, 12.83; O, 3.67; S, 7.35. Found:
C, 71.45; H, 4.60; N, 12.79; O, 3.60; S, 7.29.
5ꢀ((3ꢀ(4ꢀChlorophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)ꢀ2ꢀ(pꢀtolylimino)thiazolidineꢀ
4ꢀone (5d)
Yield 78%; M.pt: 295ꢀ298°C; IR (vmax cmꢀ1, KBr): 3072 (NꢀH lactam), 3029 (CꢀH str. of
aromatic), 1718 (C=O lactam), 1656 (C=C), 1598 (C=N); 1H NMR (400 MHz, DMSO, d6 ppm):
δ 2.30 & 2.31 (2s, 3H, ꢀCH3), 6.93ꢀ7.66 (m, 12H, H10/H12 , H9/H13 , H24/H26 , H7, H30 , H23/H27,
H29/H31), 7.88 (d, J=8.0 Hz, 2H, H28/H32), 7.93 (d, 2H, J=7.6 Hz, H28/H32), 8.47 & 8.58 (2s, 1H,
H17); Anal. Calc. For C26H19ClN4OS:C, 66.30; H, 4.07; Cl, 7.53; N, 11.90; O, 3.40; S, 6.81.
Found: C, 66.21; H, 4.01; Cl, 7.45; N, 11.85; O, 3.35; S, 6.75.

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5ꢀ((3ꢀ(4ꢀFluorophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)ꢀ2ꢀ(pꢀtolylimino)thiazolidineꢀ4ꢀ
one (5e)
Yield 72%; M.pt: 288ꢀ290°C; IR (vmax cmꢀ1, KBr): 3127 (NꢀH lactam), 3006 (CꢀH str. of
aromatic), 1706 (C=O lactam), 1664 (C=C), 1598 (C=N); 1H NMR (400 MHz, DMSO,d6, ppm):
δ 2.30 & 2.31 (2s, 3H, ꢀCH3), 6.93ꢀ7.88 (m, 14H, H10/H12 , H9/H13 , H24/H26 , H7, H30 , H28/H32,
H23/H27, H29/H31), 8.42 & 8.54 (2s, 1H, H17); Anal. Calcd. For C26H19FN4OS: C, 68.71; H, 4.21;
F, 4.18; N, 12.33; O, 3.52; S, 7.05. Found: C, 68.69; H, 4.14; F, 4.08; N, 12.30; O, 3.45; S, 6.98.
5ꢀ((3ꢀ(4ꢀBromophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)ꢀ2ꢀ(pꢀtolylimino)thiazolidineꢀ
4ꢀone (5f)
Yield 74%; M.pt: 290ꢀ292°C; IR (vmax cmꢀ1, KBr): 3064 (NꢀH lactam), 3035 (CꢀH str. of
aromatic), 1720 (C=O lactam), 1654 (C=C), 1596 (C=N); 1H NMR (400 MHz, DMSO,d6, ppm):
δ 2.30 & 2.31 (2s, 3H, ꢀCH3), 6.93 (d, J= 8.0 Hz, 2H, H10/H12), 7.11ꢀ7.18 (m, 4H, H9/H13,
H10/H12), 7.32ꢀ7.43 (m, 2H, H30, H7), 7.46ꢀ7.72 (m, 8H, H24/H26,H9/H13, H23/H27, H29/H31), 7.87
(d, J=8.0 Hz, 2H, H28/H32), 7.92 (d, J=8.0 Hz, 2H, H28/H32), 8.44 & 8.56 (2s, 1H, H17); Anal.
Calc. For C26H19BrN4OS:C, 60.59; H, 3.72; Br, 15.50; N, 10.87; O, 3.10; S, 6.22. Found: C,
60.55; H, 3.69; Br, 15.45; N, 10.81; O, 3.01; S, 6.19.
5ꢀ((3ꢀ(4ꢀNitrophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ4ꢀyl)methylene)ꢀ2ꢀ(pꢀtolylimino)thiazolidineꢀ4ꢀ
one (5g)
Yield 85%; M.pt: 288ꢀ290°C; IR (vmax cmꢀ1, KBr): 3072 (NꢀH lactam), 3037 (CꢀH str. of
aromatic), 1716 (C=O lactam), 1691 (C=C), 1600 (C=N); 1H NMR (400 MHz, DMSO, d6, ppm):
δ 2.30 & 2.31 (s, 3H, ꢀCH3), 6.91 (d, J= 8.0 Hz, 2H, H10/H12), 7.16 (d, J= 8.0 Hz, 2H, H9/H13,
H10/H12), 7.34ꢀ7.41 (m, 1H, H30), 7.48ꢀ7.57 (m, 3H, H29/H31, H7), 7.62ꢀ7.64 (d, 2H, H9/H13, J=8.4
Hz), 7.87ꢀ7.91 (m, 2H, H27/H31), 7.93ꢀ7.96 (m, 2H, H28/H32), 8.35ꢀ8.39 (m, 2H, H24/H26); 8.49 &
8.61 (2s, 1H, H17); Anal. Calc. For C26H19N5O3S:C, 64.85; H, 3.98; N, 14.54; O, 9.97; S, 6.66.
Found: C, 64.81; H, 3.87; N, 14.43; O, 9.91; S, 6.60.
Inhibition assay for αꢀAmylase activity
A stock solution of 10 mg/10 mL concentration was prepared by using DMSO solvent. Activity
of amylase was assayed with different concentrations (50, 100, 200 µg/mL) of sample with
control and reagent solution without test sample was used as control. Starch solution (1% w/v) or
(0.5% w/v) was prepared by stirring and boiling 0.5 g of soluble potato starch in 50 mL of

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deionized water for 15 minutes. The enzyme solution (1 unit/mL) was prepared by mixing 100
mg in 100 mL of 20 mM sodium phosphate buffer (pH 6.9).The color reagent was a solution
containing 96 mM 3,5ꢀdinitrosalicylic acid (DNSA) (20 mL), 5.31 M sodium potassium tartrate
in 2 M NaOH (8 mL) and deionized water (12 mL). Acarbose was used as a standard at the
concentration of 1mg/mL. 100 µl of test solution and 100 µL of enzyme solution were mixed in
viols and incubated at 25°C for 30 min. To this mixture 100 µL of color reagent was added and
the mixture was heated on water bath at 85°C for 15 min. After then, the reaction mixture was
removed from water bath, cooled and absorbance value determined at 595 nm. Individual blanks
were prepared for correcting the background absorbance. Control experiment was conducted in
the same manner by replacing the drug sample with 1 mL DMSO. Inhibition percentage of αꢀ
amylase was calculated by the formula.39 Enzyme activity was calculated and percentage of
inhibition shown in the table.
%Inhibition = ꢀꢁꢂꢃꢄꢁꢅꢆꢇꢈꢉꢃ×100
ꢀꢁꢂꢃꢄꢁꢅ
Docking simulations
The 3D structure of the compound 5a was made and optimized with Marvinsketch.40 The
enzyme alphaꢀamylase (PDB ID:2QV4) was downloaded from Brookhaven Protein Data Bank
(http://www.rcsb.org/pdb). The preparation of protein was performed with UCSF Chimera41 and
Auto Dock Tools42 using reported procedure.43 The docking simulation was performed using
well established AutoDock Vina program.44 The validation of the docking protocols was done
following the reported technique45 and these protocols were adopted for carrying out docking
studies. The search space for docking was centered at x = 11.0976587428, y = 47.4125222511,
and z = 26.0545925088 with size x = 25.0, y = 25.0 and z = 25.0. The exhaustiveness was set to
be 100. The results were visualized with Discovery Studio46 and PyMol47.
Conclusion
In conclusion, the present study describe the synthesis of seven novel 5ꢀ((3ꢀ(aryl)ꢀ1ꢀphenylꢀ1Hꢀ
pyrazolꢀ4ꢀyl)methylene)ꢀ2ꢀ(pꢀtolylimino)thiazolidinꢀ4ꢀone (5aꢀg) compounds by condensation of
1ꢀphenylꢀ3ꢀ(aryl)ꢀ1Hꢀpyrazoleꢀ4ꢀcarbaldehyde (4aꢀg) with 2ꢀ(pꢀtolylimino)thiazolidinꢀ4ꢀone (3)
and evaluated for their αꢀamylase inhibition. The structures of all newly synthesized compounds
were confirmed by elemental and spectroscopic analysis (IR, 1HꢀNMR, MS).1H NMR shows that
2Z,5Zꢀisomer is present in 58.4ꢀ62.8% which was further supported by DFT studies. The

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biological potential of newly synthesized compounds was investigated through in vitro αꢀ
amylase inhibition activity. The results showed that some of the synthesized compounds
exhibited significant inhibitory activities. Compound 5a in 100 µg/mL concentration showed
remarkable inhibition of 90.04%. Docking studies of compound 5a was performed against active
site of human pancreatic alphaꢀamylase (PDB ID: 2QV4). It has been revealed from docking
studies that the bonding interactions found between 5a and human pancreatic αꢀamylase are
similar to those responsible for αꢀamylase inhibition by acarbose.
Acknowledgement
Meenakshi Duhan thanks Haryana State Council of Science and Technology (HSCST) for the
award of research fellowship.
Conflict of Interest
The authors declare no competing interests.
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