Design, synthesis, biological evaluations and in silico studies of sulfonate ester derivatives of 2-(2-benzylidenehydrazono)thiazolidin-4-one as potential α-glucosidase inhibitors

Article abstract of DOI:10.1016/j.molstruc.2021.131266

A novel series of hydrazolyl linked sulfonate ester analogues of 4-thiazolidinone nucleus has been rationally designed, synthesized and characterized by various spectroscopic techniques including ¹H NMR, ¹³C NMR and mass spectrometry

Full text of DOI:10.1016/j.molstruc.2021.131266

Journal of Molecular Structure 1247 (2022) 131266
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Design, synthesis, biological evaluations and in silico studies of
sulfonate ester derivatives of
2
-(2-benzylidenehydrazono)thiazolidin-4-one as potential
✩
α-glucosidase inhibitors
a
b
c
a,∗
Ramandeep Kaur , Rajnish Kumar , Nilambra Dogra , Ashok Kumar Yadav
a
University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India
b
Department of Neuroscience Care and Society, Division of Neurogeriatrics, Karolinska Institute, Solna 17157, Sweden
c
Centre for Systems Biology & Bioinformatics, Panjab University, Chandigarh 160014, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of hydrazolyl linked sulfonate ester analogues of 4-thiazolidinone nucleus has been ratio-
Received 30 May 2021
Revised 26 July 2021
Accepted 7 August 2021
Available online 9 August 2021
nally designed, synthesized and characterized by various spectroscopic techniques including 1H NMR, 13
C
NMR and mass spectrometry. All of the synthesized derivatives were tested for in vitro α-glucosidase
inhibitory activities and antioxidant potential. The investigated compounds displayed appreciable α-
glucosidase inhibition with IC50 values ranging from 42.80 ± 0.48 to 599.04 ± 1.26 μM in comparison
to acarbose (478.07 ± 1.53 μM) and structure-activity relationship was established. Further, the safety
profile of the most potent derivative (7d) was assessed by MTT assay in normal HEK cells. In vivo disac-
charide loading test endorsed the higher efficacy of (7d) over acarbose (at a dose of 20 mg/kg of body
weight) for reduction of postprandial hyperglycemia after sucrose administration. In silico procedures i.e.
homology modeling, molecular docking, molecular dynamic simulations, binding free energy calculations
and ADME predictive studies further justified the results of in vitro and in vivo biological investigations.
Key Words:
Diabetes
α-glucosidase inhibitors
4
-thiazolidinone
Sulfonate ester
©
2021 Elsevier B.V. All rights reserved.
1
. Introduction
prandial hyperglycemia, which contributes profoundly in the on-
set and development of DM and its associated complications [5–
7]. Such inhibitors retard the intestinal glucose absorption by in-
hibiting the α-glucosidase which catalyzes the cleavage of 1,4-α
glycosidic bonds in complex dietary carbohydrates [8]. Acarbose,
miglitol, and voglibose are the examples of clinically prescribed α-
glucosidase inhibitors for the treatment of T2DM. However, their
extensive use has been limited by the complicated multistep pro-
cedures needed for their synthesis and undesirable side effects
such as gastrointestinal intolerability, diarrhoea and flatulence [9].
Therefore, the search of new small molecules possessing potent
α-glucosidase inhibitory activity and minimal side effects has re-
cently attracted huge attention [10].
Globally, diabetes mellitus (DM) is one of the major health con-
cerns. The number of patients suffering from DM has been in-
creasing sharply, and is expected to reach up to 552 million by
2
030 [1]. The characteristic of this chronic disease is the hyper-
glycemia caused by inadequate production of insulin and/or body’s
inability to fully respond to insulin. An association between DM
and life threatening health complications, including cardiovascu-
lar diseases, nephropathy, and retinopathy has been well estab-
lished. Therefore, effective management of high blood glucose lev-
els can prevent or delay such aligned complication [2,3]. Sulfony-
lureas, biguanides, meglitinides, glitazones, dipeptidyl peptidase
4
inhibitors, thiazolidinediones, sodium-glucose co-transporter in-
4-Thiazolidinone, a 4-carbonyl derivative of tetrahydro form of
thiazole, is an ubiquitous scaffold in drug discovery as it possesses
a range of exceptional pharmacological activities including antibac-
terial [11], antitumor [12], antihistaminic [13], anti-inflammatory
[14] and anticonvulsant activity [15], anti-tubercular [16], antivi-
ral [17]. The keto group present at the 4th position and N-C-S
linkage in thiazolidinones have been accounted for its biological
potential [18]. Apart from these, anti-diabetic potential of thiazo-
lidinone heterocycle has been widely explored, which can be evi-
hibitors, incretin mimetic and α-glucosidase inhibitors are the ma-
jor classes of clinically used antidiabetic drugs acting on differ-
ent biological targets [4]. α-Glucosidase inhibitors have emerged
as one of the effective therapeutic approach to manage the post-
✩
Dedicated to Prof. (Dr.) Manoj Kumar. May His Soul rest in peace.
∗
Corresponding author.
E-mail address: ashoky@pu.ac.in (A.K. Yadav).
https://doi.org/10.1016/j.molstruc.2021.131266
0
022-2860/© 2021 Elsevier B.V. All rights reserved.

R. Kaur, R. Kumar, N. Dogra et al.
Journal of Molecular Structure 1247 (2022) 131266
Fig. 1. α-Glucosidase inhibitors containing aryl sulfonyls, imine and hydrazide scaffolds.
[
19]. These drugs act upon peroxisome proliferator activity receptor
PPARc) to exhibit their hypoglycaemic effect. Also, reports on ap-
(
preciable hypoglycaemic effects produced with thiazolidinones by
acting on other molecular level targets such as aldose reductase
(
ALR2), protein tyrosine phosphatise 1B (PTP1B), α-glucosidase and
α-amylase have been published [18,20–22].
On the other hand, aryl sulfonyls are important synthons in
medicinal chemistry because of their immense bioactivity [23].
They exhibit anti-inflammatory, anti-tumor and anti-fungal activi-
ties, and also inhibit HIV-1 reverse transcriptase, cyclooxygenase-2
Fig. 2. Design strategy for 4-thiazolidinone based hybrids.
(
COX-2) [24]. Our research group has reported the PTB1B inhibitory
potential of aryl sulfonyls for the management of T2DM [25]. Inhi-
denced from the successful clinical use of numerous anti-diabetic
bition of α-glucosidase by aryl sulfonyls has been investigated by
drugs i.e. rosiglitazone, pioglitazone, lobeglitazone and troglitazone
Scheme 1. Reagents and Conditions: (a) Dichloromethane, TEA, stir, rt, 24 h; (b) Ethanol, glacial acetic acid, 30–45 min., 50–60 ˚C, stir; (c) Toluene, DMF, reflux, 4-5 h.
2

R. Kaur, R. Kumar, N. Dogra et al.
Journal of Molecular Structure 1247 (2022) 131266
Osella et al., and such molecules exhibited much lower IC50 values
as compared to acarbose (Fig. 1) [26–30].
2. Results and discussion
It was observed from the literature that, imine (-C=N) and hy-
drazide (-NH-NH-) fragments are present in the number of re-
ported α-glucosidase inhibitors (Fig. 1), which contributed consid-
erably towards the inhibition of this enzyme [27,31–42].
Recently, we have reported the α-glucosidase inhibitory po-
tential of thiazolidinedione–isatin [22] and pyrazole-isatin hybrids
2.1. Chemistry
The title 4-thiazolidinone analogues (7a-m) were synthesized
by three steps synthetic pathway as outlined in Scheme 1. Ini-
tially, the O-sulfonylation of 4-hydroxybenzaldehyde (1) was car-
ried out in the presence of triethylamine (basic condition) to yield
aryl/alkylsulfonate esters (3a-m). Further, these sulfonate esters
were condensed with thiosemicarbazide (4) in ethanol contain-
ing catalytic amount of glacial acetic acid to get the Schiff base
intermediates (5a-m). In the final step, Schiff base intermediates
(1.0 mmol) were refluxed with chloro acetic acid (1.0 mmol) (6)
in toluene containing small volume of DMF that resulted in the
formation of 4-thiazolidinone analogues (7a-m). The structures of
the compounds were fully characterized by 1H NMR, 13C NMR
and Mass spectroscopic analysis. In 1H NMR, a signal at δ 11.97–
12.03 ppm due to –NH, and a singlet at δ 3.83–3.91 ppm due
to two –CH2 protons confirmed the formation of 4-thiazolidinone
ring. The signal at δ 173.83–174.42 ppm in 13C NMR appeared be-
[
43]. Therefore, considering the emerging importance of thiazo-
lidinone, aryl sulfonyls, imine and hydrazide fragments, especially
with regard to their α-glucosidase inhibitory properties, and as
continuation of our efforts for the development of new potent α-
glucosidase inhibitors, we have designed a strategy to club these
four pharmacophoric units in a single molecular architecture by
molecular hybridization technique in order to achieve their syn-
ergistic effect (Fig. 2).
Also, the role of oxidative stress has been well established in
the pathophysiology of diabetes and associated complications [44].
Thus, the synthesized compounds were also assessed for their an-
tioxidant properties.
Table 1
In vitro α-glucosidase inhibitory activity and antioxidant potential of synthesized compounds.
(
continued on next page)
3

R. Kaur, R. Kumar, N. Dogra et al.
Journal of Molecular Structure 1247 (2022) 131266
Table 1 (continued)
cause of the carbonyl carbon of 4-thiazolidinone. The mass spectra
further be well explained on the basis of molecular docking and
molecular dynamic simulation studies.
of compounds exhibited molecular ion peaks at [M] and [M+1]+.
+
2
.3. Antioxidant activity
2
.2. In vitro α-glucosidase activity
Prolonged hyperglycemia in T2DM leads to amplified genera-
The newly synthesized analogues (7a-m) showed variable de-
tion of reactive oxygen species (ROS) and impairment of endoge-
nous antioxidants. This oxidative stress have been implicated in the
development of diabetic complications such as diabetes nephropa-
thy, diabetes neuropathy and diabetes retinopathy [45]. Therefore,
it would be beneficial if compounds possess antioxidant activity
along with their α-glucosidase potential. The ferric reducing an-
tioxidant power (PFRAP) assay was used for evaluation of reduc-
ing abilities of compounds through redox reaction with metal ions,
such as iron. All compounds possessed the ability to reduce fer-
ric ions to ferrous ions and exhibited EC50 values ranging from
gree of α-glucosidase inhibitions with IC50 values ranging from
2.80 ± 0.48 to 599.04 ± 1.26 μM (Table 1) in compari-
son with the standard drug acarbose showing an IC value of
4
5
0
4
78.07 ± 1.53 μM. Compound 7d (IC5 = 42.80 ± 0.48 μM) was
0
observed to be the most potent and 7m (IC = 48.19 ± 0.36 μM)
5
0
is as the second most potent derivative of the series. An interesting
structure activity relationship (SAR), depicting the effect of various
substituents at sulfonyl group, can be established from the results
of in vitro enzyme inhibition assay. Different sulfonate esters af-
fected the enzyme inhibitory activity of compounds to different ex-
tents. It is evident from the results that: (i) the arylsulfonate group
appeared crucial for enzyme inhibitory activity, as a sharp de-
cline in inhibitory activity was observed when it was replaced with
alkylsulfonate (7h). (ii) substitution of phenylsulfonate group was
preferred over naphthylsulfonate group when 7c (IC50 = 142.74 ±
6
2.76 ± 0.89 to 223.15 ± 1.45 μM. It can be visualized from
the Table 1 that majority of the compounds were either more or
equipotent to ascorbic acid (EC50 = 196.51 ± 0.48 μM). Compound
7m has been observed to be the most potent among the series
with an EC50 value of 62.76 ± 0.89 μM. It was also observed that
the substituents at phenylsulfonate moiety with +I inductive effect
favored the reducing properties. The reducing ability of the syn-
thesized compounds may serve as an indicator for their potential
antioxidant efficacy that may additionally contribute towards the
management of diabetic complications along with the control of
post prandial hyperglycemia.
0
.51 μM) was compared with 7f (IC50 = 198.71 ± 0.83 μM) (iii)
among the phenylsulfonate derivatives, maximum inhibitory activ-
ity (7d) was achieved when a hydrogen bond acceptor and elec-
tron withdrawing nitro group was present at the ortho position
of phenyl ring, whereas, the electron withdrawing (-I) groups (Cl,
Br, NO2 and CF ) were less influential at the meta and para posi-
3
tions (7b, 7i, 7j and 7l, respectively) (iv) on the other hand, bulky
and electron releasing (+I) groups, in case of compound 7m, fa-
vored the inhibitory activity when present at the ortho and para
positions. Concurrent substitution at the ortho and para positions
appeared to be more beneficial than the mono substitution, as is
in the case of 7g (IC50 = 56.00 ± 0.32 μM) in comparison to 7a
2.4. Cytotoxicity
The most potent compound (7d) was analyzed for its cytotox-
icity in normal HEK cells by MTT assay to demonstrate its safety
profile. Percent viability plot (Fig. 3) of 7d at different assayed con-
centrations revealed that, more than 80% cells were viable even at
(
IC50 = 91.06 ± 0.69 μM). This structure activity relationship will
4

R. Kaur, R. Kumar, N. Dogra et al.
Journal of Molecular Structure 1247 (2022) 131266
tion compared to control at 30 min after sucrose loading. However,
treatment with 7d (10 mg/kg of body weight) reduced the AUC of
plasma glucose curve by 3.03% only in 60 min. It can be clearly de-
duced from the results that 7d suppressed the blood glucose level
in dose-dependent manner. Thus, the results of current study en-
dorse higher efficacy of 7d than standard drug, viz., acarbose, at a
dose of 20 mg/kg of body weight, in suppressing the rise of blood
glucose level after sucrose administration (Fig. 4).
3. In silico studies
3
.1. Homology model building and validation
Fig. 3. Concentration versus percentage cell viability plot of 7d.
Homology model building and validation was performed in
a similar way as reported earlier [22]. Briefly, three-dimensional
structure (a homology model) of α-glucosidase was developed us-
ing RaptorX server [46] taking oligo-1,6-glucosidase (PDB ID: 3A47,
Gene ID: IMA1) of Saccharomyces cerevisiae as template structure.
Validation of the homology model was performed by Ramachan-
dran plot, Qualitative Model Energy ANalysis (QMEAN) server, Pro-
tein Structure Analysis (ProSA) tool and plot of energy as a func-
tion of amino acid sequence position. The homology model was
observed to be free from artefacts and reliable to perform molecu-
lar docking and molecular dynamic simulation studies [22].
the highest tested dose (100 μM). Therefore, compound 7d can be
a candidate for further investigations.
2
.5. In vivo disaccharide loading test
Compound 7d was further selected for in vivo disaccharide
loading test based upon the results of in vitro α-glucosidase in-
hibition and cytotoxicity assay. It was tested for the reduction of
postprandial hyperglycemia after sucrose loading at two different
doses, i.e., 20 mg/kg of body weight (dose equal to standard dose
of acarbose) and 10 mg/kg of body weight (half of standard dose
of acarbose). The control group, administered with 0.5% CMC sus-
pension only, exhibited a rapid increase in blood glucose concen-
tration from 152.4 ± 4.96 to 270.6 ± 10.11 mg/dL after 30 min of
sucrose administration. Subsequently, pretreatment blood glucose
levels were recovered after 120 min.
3.2. Docking studies
The most active inhibitor 7d was selected for detailed molecu-
lar docking simulation studies to predict its interactions in bound
(into the active site of α-glucosidase) conformations. Significant
binding interactions including hydrogen bonding, salt bridges and
hydrophobic interactions were spotted between enzyme and in-
hibitors. The topmost active compound (7d) was found to have a
snug fit (docking score 5.1) inside the active site, 2D and 3D in-
teraction has been shown in Fig. 5. 4-Thiazolidinone ring pene-
trated deep inside the cavity, formed from Ala 278, Arg 212, Tyr 71,
Phe 177, His 348, Arg 439 including catalytic residues i.e. Glu 276,
Asp214 and Asp 349, where it formed two hydrogen bonds with
Arg 212 and Glu 276 through its oxygen and –NH, respectively. Arg
312 established two hydrogen bonds with 7d; one with oxygen of
nitro group and second with oxygen atom of sulfonyl group. Ad-
ditionally, number of hydrophobic interactions along with a salt
bridge between Glu 304 and nitrogen atom of nitro group further
helped to stabilize the enzyme- inhibitor complex. It can be as-
The test group administered with 7d (20 mg/kg of body weight)
and acarbose (20 mg/kg of body weight) showed significant reduc-
tion in blood glucose level (p < 0.001, p < 0.01, respectively) at
3
0 min after sucrose loading vis-à-vis the control. However, 7d ex-
hibited better results than acarbose in regulating the rise of blood
glucose following sucrose administration. Moreover, a decrease of
1
8.33 and 14.88% in area under curve (AUC) for plasma glucose was
observed with 7d and acarbose treatment, respectively from 0 to
0 min.
It was further proposed to evaluate 7d by reducing its dose to
6
half, i.e.10 mg/kg of body weight. There was no statistically signif-
icant difference observed in reduction of blood glucose concentra-
Fig. 4. Effect of 7d and acarbose on blood glucose levels.
5

R. Kaur, R. Kumar, N. Dogra et al.
Journal of Molecular Structure 1247 (2022) 131266
Fig. 5. (A) 3D binding pose of 7d, (B) 2D representation of docking interactions.
sumed that the hydrogen bonds might be the attributing reason
3.4. ADME predictive studies
for superior α-glucosidase inhibitory activity of 7d.
Prediction of absorption, distribution, metabolism, excretion
and toxicity (ADMET) of potential drug candidates has become
an imperative part of the drug development process. It has been
considered as an indispensable tool for filtration of compounds
to get the molecules which are less likely to be rejected dur-
ing clinical trials. Therefore, preliminary in silico pharmacokinetic
profiling of the synthesized compounds and acarbose has been
performed using QikProp v5.9 of Schrödinger suite [47]. Various
physico-chemically significant and pharmaceutically relevant de-
scriptors including QPlogPo/w, QPlogS, QPPCaco, QPlogBB, and %
Human oral absorption, etc. for all synthesized compounds and
acarbose have been presented in Table 3. The predictive ability
of used methodology was validated by comparing the calculated
pharmacokinetic property i.e. % Human oral absorption of acarbose
with experimental values. The predicted value of % Human oral ab-
sorption of acarbose was found in good agreement with experi-
3
.3. Binding free energy calculations
The MM-GBSA method was further used to calculate the bind-
ing free energy (ꢀGbind) of the inhibitor enzyme complex. This
method allows decomposing the total free binding energy into in-
dividual components which further facilitated the understanding
of the complex binding pattern in detail i.e. various molecular pa-
rameters contributing in the binding of ligands and their binding
mode with the enzyme. The ꢀGbind and other individual compo-
nents participating to the binding free energy have been given in
Table 2. The free energy of complexation has been observed in
the range from -38.11to -1.15 kcal/mol. The values of the bind-
ing free energy data illustrated that the Coulomb, steric van der
Waals, H-bond and lipophilic interactions contributed favourably
for enzyme-inhibitor complexation.
6

R. Kaur, R. Kumar, N. Dogra et al.
Journal of Molecular Structure 1247 (2022) 131266
Table 2
Binding free energy calculations for the synthesized compounds.
mentally calculated value i.e. 1.2% [48] and its predicted value by
QikProp was 0%. The poor absorption of acarbose from gastroin-
testinal tract means drug stays in intestine and colon for longer
period of time, thus is associated with risk of side effects such as
flatulence, diarrhoea, and hypoglycaemia. Therefore, an inference
may be drawn that the improved % Human oral absorption pre-
dicted for synthesized compounds may benefit in reducing their
poor absorption related side effects. All other predicted parame-
ters for synthesized compounds have also been observed within
the acceptable range defined for human use, illustrating their fa-
vorable ‘druggable’ pharmacokinetic profile.
Deviation (RMSD) plot of 7d with α-glucosidase during 25 ns long
MD simulation has been shown in Fig. 6A.
The RMSD plot indicated that the fluctuation in RMSD value
were not very high, suggesting the stable nature of enzyme-
inhibitor complex. Local changes along the protein chain were
characterized by the Root Mean Square Fluctuation (RMSF) which
gives idea about the areas of the protein that fluctuate the most
over the period of simulation. RMSF trajectories plot (Fig. 6B) of
enzyme indicated that most of the protein residues had RMSF val-
˚
ues lower than 2.5 A. Further, Ligand Root Mean Square Fluctuation
(L-RMSF) was determined to get insights on ligand fluctuations i.e.
changes in the ligand atom positions with respect to the protein.
˚
All ligand atoms exhibited the RMSF values lower than 1.5 A ex-
pect two nitro group oxygen atoms that gave RMSF value equal to
3
.5. Molecular dynamic simulations
˚
1
.5 A (Fig. 6C).
The bar plot (Fig. 7A) showed the interaction fraction of dif-
ferent amino acid residues making different type of contacts with
the ligand. It was observed that the hydrogen bond between Arg
Conventional in silico docking suffer from certain limitations,
majorly related to the static or semi-flexible treatment of ligands
with macromolecules. During the last decade, molecular dynam-
ics (MD) have emerged as an alternative valid tool for simulation
studies. Compared to conventional docking, MD allows deep inside
exploration of drug-macromolecule recognition and binding both
from the mechanistic and energetic aspects. The docked complex
of 7d was subjected to molecular dynamics study to understand its
atomic level interactions with the active site residues and confor-
mation stability of ligand-enzyme complex. The Root Mean Square
312 and sulfonyl oxygen was maintained for 99%, and the hydrogen
bond of carbonyl oxygen with His 348 was retained for 73% of the
simulation period. It was also found that the ligand was majorly
stabilized by the number of hydrophobic interactions and water
bridges. The total number of specific contacts the protein makes
with the 7d over the course of the trajectory and the residue in-
teracting with ligand in each trajectory has been given in Fig. 7B
7

Table 3
Predicted ADME parameters.
1

R. Kaur, R. Kumar, N. Dogra et al.
Journal of Molecular Structure 1247 (2022) 131266
Fig. 6. (A) RMSD trajectories of α-glucosidase and 7d-α-glucosidase complex during simulation of 25 ns, (B) RMSF of α-glucosidase chain over the period of simulation (C)
L-RMSF trajectories of 7d during the simulation.
and C. Lower values of the RMSD and RMSF, and significant num-
ber of contacts deciphered the stability of 7d in the binding cavity
of enzyme.
The plates were examined under UV cabinet at 254 nm. The melt-
ing points were determined on Veego melting point apparatus
and were uncorrected. The structures of the synthesized com-
pounds were confirmed by 1H NMR, 13C NMR and mass spectrom-
etry. 1H and 13C NMR spectra were recorded on Bruker AC-400F
4. Experimental
(
400 MHz) spectrometer in deuterated dimethylsulfoxide (DMSO-
d6). The chemical shifts were reported in parts per million (ppm)
4
.1. Materials and methods
using tetramethylsilane as internal standard. The spin multiplic-
ities were indicated by the symbols: bs (broad singlet), s (sin-
glet), d (doublet), dd (doublet of doublet), t (triplet) and m (multi-
plet). Mass spectrometry was conducted using Waters, Q-TOF Mi-
cromass LCT system in positive mode of electrospray ionisation
The used starting materials and solvents were purchased from
the commercial sources and purified using standard procedures
whenever required. Yields refer to isolated yields of products after
purification. The completion of the reactions and the homogene-
ity of the products were monitored by thin layer chromatography
(
ESI-MS). All of the computational studies were carried out using
Schrödinger Release 2019-1: Schrödinger, LLC, New York, NY, 2019.
(
TLC) using pre-coated TLC-sheets (ALUGRAM® Xtra SIL G / UV254).
9

R. Kaur, R. Kumar, N. Dogra et al.
Journal of Molecular Structure 1247 (2022) 131266
Fig. 7. (A) The bar chart of protein-ligand contacts, (B) The timeline representation of interactions and contacts. The top panel shows the total number of specific contacts
the protein makes with the 7d over the course of trajectory and the bottom panel shows the residues interacting with the 7d in each trajectory frame (C) A schematic
representation of detailed 7d atom interactions with the protein residues. Interactions that occurred for more than 30.0% of the simulation time in the selected trajectory
(0.00 through 25.00 ns) have been shown.
4
.2. General procedure for the synthesis of intermediates 3a-m
solution of 4-hydroxybenzaldehyde (1) (10.0 mmol) in
4.4. General procedure for preparation of compounds 7a-m
A
Intermediates 5a-m (3.0 mmol) and chloroacetic acid
(3.0 mmol) were added in toluene (20 mL) containing a small
amount (2.0 mL) of dimethylformamide (DMF). The solution was
refluxed using Dean-Stark apparatus for 4-5 h on oil bath. Progress
of the reaction was monitored by TLC. After the completion of
reaction, toluene was recovered under reduced pressure and the
remaining concentrate was poured into ice cold water. A white
colored solid was separated, which was washed with water, dried
and recrystallized from methanol to obtain pure final products
(7a-m) [49].
dichloromethane (40 mL) was stirred for 10 min. To this so-
lution, triethylamine (11.0 mmol) was added dropwise and fur-
ther stirred for 30 min. Next, various sulfonylchlorides (2a-m)
(
10.0 mmol) were added to the mixture and stirred at room tem-
perature for 24 h. Progress of the reaction was monitored by TLC.
After the completion of reaction, the reaction mixture was repeat-
edly washed with water and brine solution to remove the triethy-
lamine hydrochloride salt. The organic layer was dried over sodium
sulphate and solvent was removed under reduced pressure to yield
intermediates 3a-m.
4
.3. General procedure for the synthesis of intermediates 5a-m
4.5. 4-(((4-Oxothiazolidin-2-ylidene)hydrazono)methyl)phenyl-4-
methylbenzene-sulfonate (7a)
A mixture of intermediates 3a-m (5.0 mmol) and thiosemicar-
bazide (5.0 mmol) were mixed in absolute ethanol (25 mL) along
with few drops of glacial acetic acid. The resulting reaction mix-
ture was stirred at 50–60 °C for 30–45 min. Progress of the reac-
tion was monitored by TLC. After the completion of reaction, the
reaction mixture was allowed to stand for half an hour, and after
which a solid was separated. The solid, thus, obtained was filtered
under vacuum, washed with water and dried to yield pure inter-
mediates 5a-m.
Yield: 79%; mp: 238–240 °C. 1H NMR (400 MHz, DMSO-d6): δ
11.97 (s, 1H, NH), 8.33 (s, 1H, CH=N), 7.72–7.70 (m, 4H, Ar-H), 7.43
(d, 2H, J = 8.16 Hz, Ar-H), 7.04 (d, 2H, J = 8.68 Hz, Ar-H), 3.83
13
(s, 2H, -CH ), 2.45 (s, 3H, CH ) ppm. C NMR (100 MHz, DMSO-
2
3
d6): δ 174.21 (C4-thiazolidinone ring), 164.82 (C2-thiazolidinone
ring), 154.81 (HC=N), 150.28, 145.95, 133.29, 131.18, 130.27, 129.13,
128.23, 122.56 (Aromatic C), 33.00 (C5-thiazolidinone ring), 21.15
+
+
(CH ) ppm. Mass (ES ): 390.1596 (M+1) .
3
10

R. Kaur, R. Kumar, N. Dogra et al.
Journal of Molecular Structure 1247 (2022) 131266
4
.6. 4-(((4-Oxothiazolidin-2-ylidene)hydrazono)methyl)phenyl-4-
4.11. 4-(((4-Oxothiazolidin-2-ylidene)hydrazono)methyl)phenyl-2,4,6-
trimethyl benzenesulfonate (7g)
chlorobenzenesulfonate (7b)
Yield: 78%; mp: 243–245 °C. 1H NMR (400 MHz, DMSO-d6):
δ 11.99 (s, 1H, NH), 8.35 (s, 1H, CH=N), 7.87–7.84 (m, 2H, Ar-
H), 7.76–7.73 (m, 2H, Ar-H), 7.71–7.68 (m, 2H, Ar-H), 7.12–7.08 (m,
Yield: 77%; mp: 242–244 °C. 1H NMR (400 MHz, DMSO-d6):
δ 12.00 (s, 1H, NH), 8.37 (s, 1H, CH=N), 7.76–7.72 (m, 2H, Ar-H),
7.15 (s, 2H, Ar-H), 7.10–7.07 (m, 2H, Ar-H), 3.90 (s, 2H, CH ), 2.48
2
2
H, Ar-H), 3.85 (s, 2H, CH ) ppm. 13C NMR (100 MHz, DMSO-
(s, 6H, CH ), 2.30 (s, 3H, CH ) ppm. 13C NMR (400 MHz, DMSO-
2
3
3
d6): δ 174.42 (C4-thiazolidinone ring), 165.01 (C2-thiazolidinone
ring), 154.78 (HC=N), 150.07, 140.22, 133.53, 132.86, 130.17, 130.06,
d6): δ 174.05 (C4-thiazolidinone ring), 164.83 (C2-thiazolidinone
ring), 154.85 (HC=N), 150.10, 144.39, 139.75, 133.21, 131.87, 129.38,
129.11, 122.34 (Aromatic C), 32.99 (C5-thiazolidinone ring), 22.11
1
29.24, 122.63 (Aromatic C), 33.00 (C5-thiazolidinone ring) ppm.
+
+
+
+
Mass (ES ): 411.0873 (M+1) .
(CH ), 20.57 (CH ) ppm. Mass (ES ): 417.9610 (M) , 418.9575
3
3
(
M+1)+.
4
.7. 4-(((4-Oxothiazolidin-2-ylidene)hydrazono)methyl)phenyl
4
.12. 4-(((4-Oxothiazolidin-2-ylidene)hydrazono)methyl)phenyl
benzenesulfonate (7c)
methanesulfonate (7h)
Yield: 76%; mp: 247–248 °C. 1H NMR (400 MHz, DMSO-d6):
δ 12.02 (s, 1H, NH), 8.38 (s, 1H, CH=N), 7.89–7.87 (m, 2H, Ar-H),
Yield: 80%; mp: 225–227 °C. 1H NMR (400 MHz, DMSO-d6):
δ 12.01 (s, 1H, NH), 8.39 (s, 1H, CH=N), 7.77–7.73 (m, 2H, Ar-H),
7
.85–7.82 (m, 1H, Ar-H), 7.75 (d, J = 8.76 Hz, 2H, Ar-H), 7.70–7.67
7
.16–7.12 (m, 2H, Ar-H), 3.90 (s, 2H, CH ), 3.61 (s, 3H, CH ) ppm.
(
m, 2H, Ar-H), 7.27 (d, J = 8.72 Hz, 2H, Ar-H), 3.91 (s, 2H, CH ) ppm.
2
3
2
13C NMR (100 MHz, DMSO-d6): δ 174.06 (C4-thiazolidinone ring),
64.84 (C2-thiazolidinone ring), 154.78 (HC=N), 151.11, 130.11,
29.42, 122.54 (Aromatic C), 38.32 (CH ), 32.98 (C5-thiazolidinone
1
3
C NMR (100 MHz, DMSO-d6): δ 174.17 (C4-thiazolidinone ring),
64.81 (C2-thiazolidinone ring), 154.80 (HC=N), 150.21, 135.16,
34.08, 133.37, 129.86, 129.15, 128.20, 122.56 (Aromatic C), 32.99
1
1
1
1
3
+
+
+
+
+
ring) ppm. Mass (ES ): 313.7012 (M) , 314.7013 (M+1) .
(
C5-thiazolidinone ring) ppm. Mass (ES ): 376.1496 (M+1) .
4
.13. 4-(((4-Oxothiazolidin-2-ylidene)hydrazono)methyl)phenyl-4-
4
.8. 4-(((4-Oxothiazolidin-2-ylidene)hydrazono)methyl)phenyl-2-
bromo benzenesulfonate (7i)
nitrobenzenesulfonate (7d)
Yield: 84%; mp: 232–233 °C. 1H NMR (400 MHz, DMSO-d6): δ
Yield: 80%; mp: 255–257 °C. 1H NMR (400 MHz, DMSO-d6):
δ 12.03 (s, 1H, NH), 8.41 (s, 1H, CH=N), 8.22 (dd, J = 8.0 Hz,
1
2.01 (s, 1H, NH), 8.48–8.44 (m, 2H, Ar-H), 8.39 (s, 1H, CH=N),
8
2
.18–8.15 (m, 2H, Ar-H), 7.79–7.75 (m, 2H, Ar-H), 7.19–7.16 (m,
1
.0 Hz, 1H, Ar-H), 8.10–8.06 (m, 1H, Ar-H), 8.02–7.99 (m, 1H, Ar-
H, Ar-H), 3.90 (s, 2H, CH ) ppm. 13C NMR (100 MHz, DMSO-
2
H), 7.91–7.87 (m, 1H, Ar-H), 7.82–7.79 (m, 2H, Ar-H), 7.28–7.25 (m,
H, Ar-H), 3.90 (s, 2H, CH ) ppm. 13C NMR (100 MHz, DMSO-d6):
d6): δ 174.09 (C4-thiazolidinone ring), 164.87 (C2-thiazolidinone
ring), 154.72 (HC=N), 151.10, 149.89, 139.25, 133.74, 130.04, 129.32,
2
2
δ 174.09 (C4-thiazolidinone ring), 164.82 (C2-thiazolidinone ring),
54.71 (HC=N), 149.83, 147.91, 137.16, 133.89, 133.11, 131.81, 129.41,
25.91, 125.44, 122.48 (Aromatic C), 33.01 (C5-thiazolidinone ring)
ppm
1
25.05, 122.63 (Aromatic C), 33.00 (C5-thiazolidinone ring) ppm.
1
+
+
+
Mass (ES ): 455.8911 (M+1) , 456.9318 (M+2) .
1
4
.14. 4-(((4-Oxothiazolidin-2-ylidene)hydrazono)methyl)phenyl-4-
+
+
+
Mass (ES ): 420.9240 (M) , 421.9440 (M+1) .
nitrobenzenesulfonate (7j)
4
.9. 4-(((4-Oxothiazolidin-2-ylidene)hydrazono)methyl)phenyl-4-
Yield: 80%; mp: 263–265 °C. 1H NMR (400 MHz, DMSO-d6):
δ 12.02 (s, 1H, NH), 8.39 (s, 1H, CH=N), 7.91–7.88 (m, 2H, Ar-H),
methoxybenzenesulfonate (7e)
7
.81–7.79 (m, 2H, Ar-H), 7.78–7.75 (m, 2H, Ar-H), 7.17–7.14 (m, 2H,
Yield: 76%; mp: 260–261 °C. 1H NMR (400 MHz, DMSO-d6):
δ 12.01 (s, 1H, NH), 8.38 (s, 1H, CH=N), 7.81–7.78 (m, 2H, Ar-
H), 7.77–7.73 (m, 2H, Ar-H), 7.18–7.15 (m, 2H, Ar-H), 7.13–7.09 (m,
Ar-H), 3.90 (s, 2H, CH ) ppm. 13C NMR (100 MHz, DMSO-d6): δ
2
174.08 (C4-thiazolidinone ring), 165.97 (Aromatic C), 164.84 (C2-
thiazolidinone ring), 154.77 (HC=N), 150.07, 133.53, 133.30, 130.13,
2
H, Ar-H), 3.90 (s, 2H, CH ), 3.86 (s, 3H, OCH ) ppm. 13C NMR
2
3
129.41, 129.24, 122.62 (Aromatic C), 33.01 (C5-thiazolidinone ring)
(
(
100 MHz, DMSO-d6): δ 174.07 (C4-thiazolidinone ring), 164.82
C2-thiazolidinone ring), 154.86 (HC=N), 164.08, 150.35, 133.23,
+
+
+.
ppm. Mass (ES ): 420.9640 (M) , 421.9740 (M+1)
1
30.65, 129.12, 125.24, 122.62, 114.98 (Aromatic C), 55.91 (OCH ),
3
4.15. 4-(((4-Oxothiazolidin-2-ylidene)hydrazono)methyl)phenyl-4-
+
+
3
3.00 (C5-thiazolidinone ring) ppm. Mass (ES ): 405.9191 (M) ,
06.9012 (M+1)+.
(
tert-butyl) benzenesulfonate (7k)
4
Yield: 73%; mp: 281–283 °C. 1H NMR (400 MHz, DMSO-d6):
4
.10. 4-(((4-Oxothiazolidin-2-ylidene)hydrazono)methyl)phenyl
δ 12.00 (s, 1H, NH), 8.39 (s, 1H, CH=N), 7.83–7.80 (m, 2H, Ar-
naphthalene-2-sulfonate (7f)
H), 7.78–7.74 (m, 2H, Ar-H), 7.71–7.68 (m, 2H, Ar-H), 7.16–7.13 (m,
2
H, Ar-H), 3.90 (s, 2H, CH ), 1.31 (s, 9H, CH ) ppm. 13C NMR
2 3
Yield: 80%; mp: 248–250 °C. 1H NMR (400 MHz, DMSO-d6):
δ 12.00 (s, 1H, NH), 8.62–8.61 (m, 1H, Ar-H), 8.35 (s, 1H, CH=N),
(100 MHz, DMSO-d6): δ 174.11 (C4-thiazolidinone ring), 164.87
(C2-thiazolidinone ring), 158.39 (Aromatic C), 154.82 (HC=N),
150.27, 133.29, 131.43, 129.14, 128.09, 126.70, 122.50 (Aromatic C),
35.13 (C(CH ) ), 33.00 (C5-thiazolidinone ring), 30.59 (CH ) ppm.
8
.23 (d, 2H, J = 9.0 Hz, Ar-H), 8.12 (d, 1H, J = 8.0 Hz, Ar-
H), 7.89 (dd, 1H, J = 8.5 Hz, J = 2.0 Hz, Ar-H), 7.81–7.78
1
2
3
3
3
+
+
+
(
m, 1H, Ar-H), 7.73–7.70 (m, 3H, Ar-H), 7.16–7.14 (m, 2H, Ar-H),
.89 (s, 2H, CH ) ppm. 13C NMR (100 MHz, DMSO-d6): δ 173.99
Mass (ES ): 431.9892 (M) , 432.9820 (M+1) .
3
2
(
(
C4-thiazolidinone ring), 164.84 (C2-thiazolidinone ring), 154.72
HC=N), 150.19, 135.01, 133.28, 131.34, 131.06, 130.25, 129.99,
4.16. 4-(((4-Oxothiazolidin-2-ylidene)hydrazono)methyl)phenyl-3’-
(trifluoromethyl) benzenesulfonate (7l)
1
3
4
29.96, 129.57, 129.08, 128.02, 127.91, 122.52, 122.36 (Aromatic C),
+
+
Yield: 76%; mp: 236–237 °C. 1H NMR (500 MHz, DMSO-d6):
δ 12.01 (s, 1H, NH), 8.39 (s, 1H, CH=N), 8.25 (d, 1H, J = 8.0 Hz,
2.89 (C5-thiazolidinone ring) ppm. Mass (ES ): 425.9560 (M) ,
26.9821 (M+1)+.
11

R. Kaur, R. Kumar, N. Dogra et al.
Journal of Molecular Structure 1247 (2022) 131266
Ar-H), 8.21 (d, 1H, J = 8.0 Hz, Ar-H), 8.12 (s, 1H, Ar-H), 7.95
4.20. In vivo oral disaccharide loading test
(
t, 1H, J = 8 Hz, Ar-H), 7.79–7.76 (m, 2H, Ar-H), 7.20–7.17 (m,
1
3
2
H, Ar-H), 3.90 (s, 2H, CH ) ppm. C NMR (125 MHz, DMSO-
In vivo biological efficacy of the most potent compound (7d) has
been evaluated by earlier reported methods [22,43]. Male albino
mice were used to perform the sucrose loading test for 7d. An-
imals (30–40 g) were fasted overnight and divided into different
groups with five animals in each. The control group and reference
group was orally administered with vehicle (0.5% carboxymethyl
cellulose [CMC] solution) and standard drug acarbose (20 mg/kg
body weight) dispersed in 0.5% CMC, respectively. Test groups were
administered with homogenized suspension of 7d (in 0.5% CMC
and distilled water) at doses of 20 and 10 mg/kg body weight. Af-
ter 20 min, the mice were given sucrose solution (2.5 g/kg body
weight). Blood glucose level was estimated at 0 (before), 30, 60
and 120 min after sucrose solution loading using the Dr. Morepen
GlucoOne (Morepen Laboratories Limited, Delhi, India) blood glu-
cose monitoring system. The results were expressed as mean ±
SEM. Area under blood glucose-time curve (AUC) over a period
of 30 and 60 min after sucrose administration was calculated by
GraphPad Prism version 5.01 (GraphPad, CA, USA). Statistical analy-
sis was performed by two-way analysis of difference (ANOVA), fol-
lowed by Bonferroni tests.
2
d6): δ 173.83 (C4-thiazolidinone ring), 164.82 (C2-thiazolidinone
ring), 154.65 (HC=N), 149.82, 135.13, 133.58, 132.30, 131.84, 131.51,
1
29.13, 124.61, 122.53, 121.78 (Aromatic C), 124.58 (CF ), 32.88 (C5-
3
+
+
thiazolidinone ring) ppm. Mass (ES ): 443.9059 (M) , 444.8917
(
M+1)+.
4
.17. 4-(((4-Oxothiazolidin-2-ylidene)hydrazono)methyl)phenyl-2,4,6-
triisopropylbenzenesulfonate (7m)
Yield: 76%; mp: 290–292 °C. 1H NMR (500 MHz, DMSO-d6): δ
1
2.00 (s, 1H, NH), 8.39 (s, 1H, CH=N), 7.77–7.72 (m, 2H, Ar-H), 7.30
(
(
s, 2H, Ar-H), 7.09–7.06 (m, 2H, Ar-H), 3.90 (s, 2H, CH ), 3.82–3.89
2
m, 3H, CH), 1.11 (d, J = 7.2 Hz, 6H, CH ), 1.02 (d, J = 6.8 Hz,
3
13
12H, CH ) ppm. C NMR (125 MHz, DMSO-d6): δ 174.12 (C4-
3
thiazolidinone ring), 164.84
C2-thiazolidinone ring), 154.51 (HC=N), 150.36, 150.04, 133.18,
29.14, 128.67, 124.10, 122.58 (Aromatic C), 34.18 (CH), 33.00 (CH),
2.86 (C5-thiazolidinone ring), 24.56 (CH ), 23.51 (CH ) ppm. Mass
(
1
3
3
3
+
+
+
(ES ): 501.8402 (M) , 502.8513 (M+1) .
4
.18. In vitro α-glucosidase inhibition assay
4.21. MTT assay
The α-glucosidase inhibitory activities of compounds (7a-m)
Cell viability was measured by standard colorimetric assay
(MTT assay) following the procedure reported in our recent pub-
lication [22].
were tested as per earlier described procedures [22,43]. In a 96-
well plate, 60 μL of sample solution, prepared by dissolving test
compounds in 5% DMSO and phosphate buffer [pH 6.8]), and 50 μL
of 0.1 M phosphate buffer (pH 6.8) containing α-glucosidase solu-
tion (Saccharomyces cerevisiae) (0.2 U/ml) was incubated at 37 °C
for 10 min. To this solution, 50 μL of 5 mM p-nitrophenyl-α-D-
glucopyranoside (PNPG) solution in 0.1 M phosphate buffer (pH
5. In silico studies
5.1. Homology model building and validation
6
.8) was added and incubated at 37 °C for another 20 min. To stop
The sequence of α-glucosidase was retrieved from UniProt (ac-
cess code P53341) in FASTA format which was used for building
of homology model through RaptorX server [50]. 3D crystal struc-
ture of oligo-1,6-glucosidase (PDB ID: 3A47, Gene ID: IMA1) of Sac-
charomyces cerevisiae was selected as best template for homology
modeling. It has a high sequence similarity of 72.1 % and a p-value
of 5.44e-20. Further, PROCHECK server (https://servicesn.mbi.ucla.
edu/PROCHECK/) was used for obtaining Ramachandran plot. The
the reaction, 40 μL of 0.8 M sodium carbonate was added to each
well and the absorbance was recorded at 405 nm by microplate
reader. For control, 60 μL of 5% DMSO was taken in place of the
sample solution and acarbose was used as standard. The tests were
performed in triplicate and percentage inhibition was calculated as
follows:
Inhibition(%) = Absorbance of control − Absorbance of sample/
Absorbance of control × 100
3
D model structure and its quality has been validated by using
QMEAN server and ProSA web server [51,52].
The concentration of inhibitors corresponding to 50% of the α-
glucosidase inhibition under the assay conditions was defined as
the IC50 value
5
.2. Molecular docking
The docking studies were performed using standard Glide
(
Glide, 2019) molecular docking protocol implemented in Mae-
stro molecular modeling suite of Schrӧdinger, LLC,New York, NY,
019. Modeled protein was treated using protein preparation wiz-
4
.19. In vitro antioxidant activity (PFRAP assay)
2
The procedures for the determination of reducing potential of
ard of Schrӧdinger suit where bond orders were assigned, hy-
drogens were added, bonds to metals were deleted, the formal
charges on the metal and the neighbouring atoms were adjusted
and finally water molecules were deleted. The protein structure
was refined with the help of restrained minimization. The dock-
ing was performed using Glide extra precision mode (XP) [53–55].
The 3D crystal structure of oligo-1,6-glucosidase (isomaltase, PDB
ID: 3A4A) of Saccharomyces cerevisiae was aligned on the homol-
ogy model of alpha-glucosidase. Since, the binding site between of
these two proteins is very similar, we used the co-ordinates of co-
crystallised alpha-D-glucose from the isomaltase structure to de-
fine the docking site. The receptor grid was generated using the
centroid of the co-crystallized alpha-D-glucose. During the dock-
ing studies, receptor was kept rigid and ligands were kept flexible.
The final assessment was made on the basis of glide score (docking
score).
compounds (7a-m) were similarly followed as reported in our re-
cent publications [22,43]. In a test tube, 2.5 ml of sample solution
was mixed with 2.5 ml of phosphate buffer (0.2 M; pH 6.6) and
2
.5 ml of 1% potassium ferricyanide solution. The mixture was in-
cubated at 50 °C for 20 min, then rapidly cooled and mixed with
.5 ml of 10% trichloroacetic acid. Then ferric chloride solution
0.5 ml, 0.1%) was added to an aliquot (2.5 ml) of the mixture di-
2
(
luted with 2.5 ml of distilled water and the solution was allowed
to stand for 10 min. An aliquot of 5% DMSO (control) was treated
similarly. Finally, absorbance was measured at 700 nm using UV
spectrophotometer. The test samples were analyzed in triplicate
and the data presented as mean ± standard error of the mean
(SEM). The percent reducing power was calculated using the fol-
lowing formula:
Reducing power(%) = Atest − Acontrol/Atest × 100
12

R. Kaur, R. Kumar, N. Dogra et al.
Journal of Molecular Structure 1247 (2022) 131266
The molecular simulation studies were carried out to calcu-
late the binding free energies (ꢀGbind) of the synthesized hybrids
with α-glucosidase. The Prime MM-GBSA panel was used to de-
termine the ligand-binding energies and ligand strain energies for
a set of ligands and α-glucosidase [56]. The Molecular Mechan-
ics/Generalized Born Surface Area (MM-GBSA) procedure was used
to calculate the binding free energy for all ligand- α-glucosidase
complexes. The MM-GBSA method can be conceptually summa-
rized as:
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inhibitor among the studied compounds. The rise of blood glu-
cose level, after sucrose administration during in vivo studies, was
more effectively controlled by 7d (at a dose of 20 mg/kg of body
weight) than acarbose. The results of MTT cytotoxicity assay estab-
lished the safety profile of 7d. Molecular docking studies identi-
fied the key binding interactions responsible for the snag fit of 7d
within the binding cavity of enzyme. The confirmation stability of
enzyme-7d complex over 25 ns long MD simulation further sup-
ported the higher enzyme inhibitory potential of 7d. Additionally,
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Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
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