Design, synthesis and biological evaluation of novel 5-(imidazolyl-methyl) thiazolidinediones as antidiabetic agents

Article abstract of DOI:10.1016/j.bioorg.2021.105162

A newly designed series of imidazolyl-methyl- l-2,4-thiazolidinediones 9 (a-m) were synthesized and In Silico studies were carried out to rationalize their anti-diabetic activity. Generally, all newly synthesized thiazolidinediones had anti-hyperglycemic activity compared with a diabetic-control group, without toxicity in 3T3 cells (viability ≥ 90%). These studies revealed that the compounds 9e and 9b (11?10^-6mol/kg) lowered blood glucose more effectively when compared to pioglitazone at the same dose. Following the administration of compound 9e, no weight gains or any serious side effects on liver and pancreas were observed. Moreover, the glucose consumption assay results showed a significant glucose-lowering effect (p < 0.001) in HepG2 cells, which were exposed to 11 mM of glucose at concentrations of 1.25–10 mM of compound 9e. Also, the PPAR-γ gene expression study revealed that pioglitazone and 9e showed similar behavior relative to the control group.

Full text of DOI:10.1016/j.bioorg.2021.105162

Bioorganic Chemistry 115 (2021) 105162
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of novel 5-(imidazolyl-methyl)
thiazolidinediones as antidiabetic agents
,
,
d
,
,
,b
Neda Shakoura b, Amirhossein Sahebkarc e, Gholamreza Karimif g, Maryam Pasebanh
,
Aida Tasbandid, Fatemeh Mosaffae, Zahra Tayarani-Najarang, Razieh Ghodsia
,
,
e
Farzin Hadizadehe
,a,*
a Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
b Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
c School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
d Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
e Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran
f Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran
g Department of Pharmacodynamics and Toxicology, School Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
h Department of Physiology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
A newly designed series of imidazolyl-methyl- l-2,4-thiazolidinediones 9 (a-m) were synthesized and In Silico
studies were carried out to rationalize their anti-diabetic activity. Generally, all newly synthesized thiazolidi-
nediones had anti-hyperglycemic activity compared with a diabetic-control group, without toxicity in 3T3 cells
(viability ≥ 90%). These studies revealed that the compounds 9e and 9b (11*10ꢀ 6mol/kg) lowered blood glucose
more effectively when compared to pioglitazone at the same dose. Following the administration of compound 9e,
no weight gains or any serious side effects on liver and pancreas were observed. Moreover, the glucose con-
sumption assay results showed a significant glucose-lowering effect (p < 0.001) in HepG2 cells, which were
exposed to 11 mM of glucose at concentrations of 1.25–10 mM of compound 9e. Also, the PPAR-γ gene
expression study revealed that pioglitazone and 9e showed similar behavior relative to the control group.
Thiazolidinedione
Pioglitazone
Synthetic analogue
Diabetes
Glucose
1. Introduction
cytokines from adipose tissue disrupt insulin signaling pathways in
insulin-sensitive tissues, particularly liver and muscle. The clinically
Diabetes is a multifactorial metabolic disorder and a major health
concern with a high global distribution [1–3]. Therapeutic strategies for
Type 2 diabetes (T2D) include lifestyle modification (mainly through
diet and exercise) and pharmacological agents that either increase in-
sulin sensitivity/secretion, or affect glucose absorption/excretion [4–6]
(Fig. 2). The primary causative mechanism in non-insulin-dependent
diabetes mellitus (NIDDM) is insulin resistance [7,8]. High levels of
circulating fatty acids and certain hormones and inflammatory
used medications which improve insulin sensitivity are thiazolidine-
diones (pioglitazone [9–14] and rosiglitazone [15–17] and biguanides
(phenformin [18–22] and metformin [23–25] (Fig. 2). Thiazolidine-
diones, which activate peroxisome proliferator-activated receptor- γ
(PPAR-γ), are a dominant class of insulin sensitizers that exert remark-
able anti-hyperglycemic effects without causing hypoglycemia. The
mechanism of action for these drugs is to increase peripheral glucose
uptake and insulin sensitivity of target tissue and reduce hepatic
Abbreviations: T2D, Type 2 diabetes; NIDDM, Non-insulin-dependent diabetes mellitus; PPAR-γ, Peroxisome proliferator-activated receptor- γ; TZDs, Thiazoli-
dinediones; MD, Molecular dynamic; RMSF, Root-mean-square fluctuation; RMSD, Root-mean-square derivations; Rg, Radius of gyration; SS, Secondary structures;
TRβ, Thyroid receptor β; AR, Androgen Receptor; GR, Glucocorticoid receptor; TP, Toxic potential; FBG, Fasting blood glucose; AUC, Area under curve; SAR,
Structure-activity relationship; IQR, Interquartile range; RT-PCR, Reverse transcription polymerase chain reaction; TLC, Thin-layer chromatography; ESI, Electro-
spray ionization; DMG, Dimethylglyoxime; PDB, Protein Data Bank; VMD, Visual Molecular Dynamics; PPARc, Peroxisome proliferator-activated receptor c; hERG,
`
Teh human Ether-a-go-go-Related Gene; FBS, Fetal bovine serum; STZ, Streptozocin.
* Corresponding author at: Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran.
E-mail address: hadizadehf@mums.ac.ir (F. Hadizadeh).
https://doi.org/10.1016/j.bioorg.2021.105162
Received 8 May 2021; Received in revised form 5 July 2021; Accepted 8 July 2021
Available online 13 July 2021
0045-2068/© 2021 Published by Elsevier Inc.

N. Shakour et al.
Bioorganic Chemistry 115 (2021) 105162
glycogenesis. PPAR-γ is a glitazone receptor implicated in the expression
of genes involved in glucose metabolism and regulation of insulin
sensitivity and controls insulin secretion, lipid and glucose metabolism,
and is considered the master regulator of adipogenesis [26–33]. It is
mainly expressed in adipose tissue but also found in the liver, heart,
brain, and macrophages. PPAR-γ is linked to diverse pathological con-
ditions, such as diabetes, cardiovascular disorders, Alzheimer’s and
Parkinson’s disease, multiple sclerosis, stroke and cancer [34]. Thiazo-
lidinediones (TZDs) have the highest selectivity toward PPAR-γ (Fig. 1)
formation of TZD derivatives was confirmed by the appearance of a
narrow singlet state for = CH– protons at 8.31 ppm and a broad singlet
state of NH-TZD protons at 12.56 ppm in 1HNMR spectra of 8a-m [52].
The chemical structures of compounds were confirmed by 1H NMR, 13
C
NMR, MS spectroscopy. Finally, compounds 8a-m were converted into
saturated compounds at 0–5 ◦C with a mixture of sodium borohydride,
Dimethylglyoxime and Cobalt(II) nitrate [52]. The addition of acetic
acid revealed the saturated imidazole derivatives of 9a-m precipitates
(Scheme 1). The product was confirmed by three doublets of doublet
state (dd) for CH2 connecting imidazole and thiazolidinedione and CH–
thiazolidinedione protons at 3.06, 3.17 and 4.23 ppm in 1HNMR spectra
of 9a-m. The chemical structures of the final compounds were estab-
lished by 1H NMR, 13C NMR, MS spectroscopy [53].
rather than two other subtypes (PPARs α and β). Activation of PPAR-γ by
TZDs is associated with increased insulin sensitivity in hepatocytes,
adipocytes and skeletal muscle cells, enhanced glucose uptake, oxida-
tion, and transport in insulin-responsive tissues, and simultaneous
reduction of free fatty acids and blood glucose levels [35–39]. Piogli-
tazone improves lipid profile, plasma glucose levels, HbA1c and blood
pressure and significantly increases insulin sensitivity. The main adverse
effects of pioglitazone are weight gain, fluid retention and bone fracture
[40–47]. Therefore, new PPAR-γ ligands with improved activity and less
adverse effects might promise anti-diabetic candidates [48]. In this
study, a novel form of thiazolidinedione structure was designed from
pioglitazone as the lead compound using the principle of bioisosterism
(Fig. 3). To achieve a compound with better biological activity, the
hydrophobic region and the linker were optimized while the main
skeleton structure of pioglitazone was retained. The designed com-
pounds were synthesized at five simple steps with relatively high effi-
ciency as follows: 1) cyclization reaction 2) nucleophilic substitution
reaction 3) alcohol oxidation reaction 4) Knoevenagel and 5) reduction
reaction (Scheme 1).
2.2. HPLC purity analyze of target compound (9e)
Target compound 9e was > 95% pure by HPLC. Purity analysis re-
ports can be found in the Supporting Information (Fig. 37S a, b) [54].
2.3. Molecular docking
In order to rationalize anti-diabetic activity of compounds molecular
docking studies was performed. Pioglitazone is a thiazolidinedione anti-
hyperglycemic agent used in NIDDM [17,43]. In order to validate
docking parameters, the co-crystallized ligand in RCSB with PDB ID:
5y2o was re-docked into the active site of PPAR-γ. The outcome of re-
docking was relatively identical, and the RMSD value (0.02 Å)
confirmed the docking method reliability (Fig. 4). In order to have a real
insight into structural binding mode of PPAR-γ and thirteen synthesized
compounds, the docking process was performed using MOE software.
The result analysis showed that docking scores of target compounds
were almost equal or higher (ꢀ 10.55 to ꢀ 12.69 kcal/mol) compared to
x-ray pioglitazone lead compound (-11.17 kcal/ mol). The docking
scores for compounds 9e, 9i and pioglitazone were almost the same, and
2. Results and discussion
2.1. Chemistry
The target compounds 9a-m were synthesized according to the route
sketched in Scheme 1. Compounds 4a, b were synthesized suitably
through the nucleophilic substitution and cyclization reactions of ben-
zylamine hydrochloride 1a,b with potassium thiocyanate and 1,3-dihy-
droxy acetone dimmer in the presence of the catalytic amount of acetic
acid in 1-butanol [49]. S-alkylation or S-aryl methylation of the com-
pounds 4a, b with alkyl halides or aryl methyl halides was done in
methanol in the presence of sodium hydroxide as base via nucleophilic
substitution reaction. Following purification, the residue was crystal-
lized from ethyl acetate (6a-m) [50]. The manganese dioxide oxidation
of derivatives 6a-m in chloroform at 65–70 ◦C produced compounds 7a-
m via alcohol oxidation reaction [51]. Subsequently, thiazolidinedione
intermediates 8a-m were synthesized via the Knoevenagel condensation
of imidazole aldehyde derivatives with 2, 4-thiazolidinedione in the
presence of piperidine in ethanol and chloroform at 65–70 ◦C. The
they also had one cation-π bond (with Arg288) and one hydrogen bond
(with His449). In this investigation, we have selected compound 9i for
further MD studies. The LigX of MOE depicted the binding mode of the
ligand 9i with PPAR-γ (Fig. 5). An investigation of docking of all syn-
thesized compounds at the receptor active site revealed that the potent
compounds (score > 10 kcal/mol) with the S-benzyl as a substitute on
2,4-thiazolidinedione core had more favorable interactions with PPAR-γ
at a hydrophobic pocket in comparison with the compounds without
benzyl substitute. The outcomes of docking were validated with MD
studies. A map of the docking of the four compounds (9a, 9g, 9i, and 9j)
in the active site of PPAR-γ (PDB code: 5y2o) is shown in Fig. 1S.
2.4. Molecular dynamic (MD) simulation
MD simulation was performed to clarify the performance of PPAR-γ
in ligand binding, stability and ligand-receptor interaction via simula-
tion. The dynamic stability of the secondary structures and conforma-
tional changes in protein alone (apo-form) were compared with
protein–ligand complex through plots of root-mean-square fluctuation
(RMSF), root-mean-square derivations (RMSD) and radius of gyration
(Rg). As shown in Fig. 6, the RMSD of apo-form and protein–ligand
complex attained stability after approximately 20 ns of simulation. The
mean RMSD value for apo-form and protein–ligand complex was 2.2 ±
0.20 Å and 2.22 ± 0.17 Å, respectively. The relatively wider RMSD value
of the complex was probably due to enhanced conformation flexibility of
PPAR-γ following the ligand and protein binding.
The RMSF features macromolecular flexibility and depicts any
regional changes in the protein structure, including dynamic measure-
ments for every atom or residue [55,56]. The RMSF plots of ligand-
bound protein and apo-form are illustrated in Fig. 7 and 2S. The
average RMSF of the protein–ligand complex and apo-form was 0.959 ±
0.52 and 0.958 ± 0.44 Å, respectively. These two systems determine two
Fig. 1. The PPAR-γ protein structure.
2

N. Shakour et al.
Bioorganic Chemistry 115 (2021) 105162
O
N
O
S
H
O
H
N
N
N
NH
S
O
N
O
O
O
N
H
Pioglitazone
Glipizide
HN
O
S
N
N
N
O
O
O
H
NH2
NH
NH
S
HN
O
Tolbutamide
O
HN
NH
Rosiglitazone
OH
COOH
Phenformin
OH
O
HO
HO
OH
OH
NH
O
H
OH
HN
OH
OH
N
N
N
NH2
HO
HO
N
OH
NH
NH
Voglibose
Repaglinide
Metformin
Miglitol
Fig. 2. The NIDDM drug’s structures.
O
O
NH
S
NH
S
O
N
N
O
R2
O
S
N
R1
Fig. 3. The chemical structures of the designed compounds, R1: H-, F- and R2: Me-, Et-, Pr-, C6H5-CH2-, p-CH3-C6H4-CH2-, p-FC6H4-CH2-, 2-methylpyridine.
factors regarding protein structure: (a) similar RMSF distribution and (b)
the identical direction of dynamic features. The lower values of RMSF
for residues in the complex could be attributed to the presence of these
residues in the binding site (Arg 288, Ala 292, Ile 326, Leu 330, and
His449). The binding of residues to the ligand does not create a signif-
icant change in protein flexibility.
which increased the ligand-binding affinity for PPAR-γ. The average
intramolecular hydrogen bonds of the protein were 59.03 ± 6.39. The
stability of the PPAR-γ secondary structure is formed by a hydrogen
bond (Fig. 4S).
A simple method to evaluate protein–ligand interaction is the po-
tential energy which was constant in this study (-36822.63 ± 203.94)
after 15.9 ns of simulation and showed the system’s stability during 100
ns of simulation (fig. 5S).
The Rg is an indicator of protein structure compression that portrays
the bound and non-bound systems equilibrium conformation [57]. The
Rg value of apo-form and protein–ligand complex was 18.91 ± 0.068
and 19.07 ± 0.077 Å (mean ± SD), respectively (Fig. 8). The high Rg
value of the protein–ligand complex shows higher compaction of the
complex.
Secondary structures (SS) are folded structures that are crucial for
protein function [58,59]. Calculation of the secondary structure was
performed during MD simulation using VMDSS. Initially, the percentage
of protein secondary structures was studied and then a trajectory of SS
percentage (fig. 7S) and the graphical number of residues were repre-
sented in a line of time (Fig. 6S) [60].
Hydrogen bonds are involved in stabilizing the protein–ligand
complex. The stability of this system created by the bounded ligand at
the protein’s active site is demonstrated in Figure 3S. The outcomes
revealed that ligand created one to two hydrogen bonds with the re-
ceptor (protein).
Secondary structures were determined by distinct colors and found
to be unchanged during simulation [60]. The secondary structure of the
protein–ligand complex was shown in a 3-D column chart (fig. 8S (a))
and a pie chart after 100 ns of the simulation (fig. 8S (b)).
Thiazolidinedione ligand created one hydrogen bond with His 449,
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N. Shakour et al.
Bioorganic Chemistry 115 (2021) 105162
Scheme 1. Reagents and conditions: a)
CH3COOH, 1-Butanol; b) MeOH, NaOH
added as a base; c) MnO2; d) MeOH,
CHCl3, Piperidine; e) NaOH, Co(NO3)2,
Dimethylglyoxime, NaBH4. 9a: R1 = H,
R2 = CH3; 9b: R1 = H, R2 = CH3-CH2–;
9c: R1 = H, R2 = CH3-CH2-CH2–; 9d: R1
= H, R2 = C6H5-CH2–; 9e: R1 = H, R2
=
P-CH3-C6H4-CH2–; 9f: R1 = H, R2 = P-
FC6H4-CH2–; 9g: R1 = H, R2 = 2-Pyr-
idinemethy-; 9h: R1 = F, R2 = CH3; 9i:
R1 = F, R2 = CH3-CH2–; 9j: R1 = F, R2
=
CH3-CH2-CH2–; 9k: R1 = F, R2 = C6H5-
CH2–; 9l: R1 = F, R2 = P-CH3-C6H4-
CH2–; 9m; R1 = F, R2 = P-FC6H4-CH2.
In order to investigate the system during simulation, the ligand-
active site due to a benzyl large substitute and an almost equal score
with the lead compound.
bound protein structure was obtained every ten ns from trajectories.
Images obtained from the simulation of molecular dynamics in Figure 9S
showed that the orientation of the ligand in the active site of PPAR-γ
protein was constant during simulation.
2.5. Toxicity prediction
It was shown that the primary docked structure and the last MD
structure simulation were placed in a matching envelope and pro-
tein–ligand conformation was stable and therefore, docking results were
valid. Fig. 10S illustrates the 3D image of the interaction between
compound 9i and residue (His449) after 100 ns of simulation, which was
unchanged in the primary docked structure and last protein–ligand
complex following MD. The presence of a deep cavity in the hydro-
phobic region of the envelope (Fig. 4) showed that if bulky groups
occupy it, the agonistic effects on the PPAR-γ will be increased. Hence, it
could be concluded that 9e compound acts more stable in the receptor’s
The toxicity of drug-similar candidates can be predicted via potential
“off-target” activities. In this investigation, the toxicity of lead com-
pound and synthesized components was assessed toward binding to 16
target proteins. The binding affinity value for each compound was
defined as 0 (none) or 1 (extreme). The TP values for compounds 9a to
9m were between 0.23 and 0.45, indicating a low binding affinity and a
moderate risk for the receptors (Table 1S). The main target for all
compounds, including pioglitazone and 9a to 9m was the PPAR-γ re-
ceptor and its binding affinity values were 6.3, 12.2, 1.8, 2.4, 2.6, 2.5,
0.7, 10.6, 18.0, 5.0, 15.8, 0.5, 5.0, 8.4 nM, respectively. The constant
4

N. Shakour et al.
Bioorganic Chemistry 115 (2021) 105162
Fig. 4. Map of the re-docking of compound pioglitazone in the active site PPAR-γ with PDB ID: 5y2o. The RMSD value was 0.02 Å.
Fig. 5. Map of the docking of compound 9i in the active site (PDB ID: 5y2o).
Fig. 6. RMSD between PPAR-γ with ligand and without ligand.
binding values for PPAR-γ are lower than the corresponding values for
hERG, meaning that the binding affinity of PPAR-γ for all synthetic
compounds is higher than the hERG receptor and hence, their predicted
cardiotoxicity remains very low. Except for the compounds 9m, 9h and
9f, the synthesized compounds represented no binding to cytochrome
receptors. The predictions also revealed that these compounds might
5

N. Shakour et al.
Bioorganic Chemistry 115 (2021) 105162
9a
9b
9c
9i
9d
9j
9e
9k
9f
9l
9g
9h
50
0
pio
Diab. ctrl
9m
-50
-100
Fig. 7. For every residue, RMSF of PPAR-γ with ligand and without ligand
during 100 ns simulation.
l
j
i
h
9
l
9
f
a
k
e
c
9
o
g
d
b
9
m
9
9
tr
9
9
9
9
9
9
9
pi
c
.
b
Dia
Rg(A°) with ligand
Groups
19.6
19.4
19.2
19
18.8
18.6
18.4
Fig. 9. In vivo anti-diabetic activity of the synthesized compounds vs. per-
centage decrease of FBG level in STZ induced diabetic male rats. The serum
glucose level was checked on day 1 (24 h after the first gavage), day 4, day 7
and day 10. Data is analyzed by one-way ANOVA followed by Dunnett’s test
and expressed as mean ± SEM, (n = 6). *** indicates p < 0.001 vs. diabetic
control. Pio: Pioglitazone; Diab. ctrl: diabetic control; 9a-9m: synthe-
sized compounds.
0
20
40
60
Time(ns)
80
100
ones with S-methyl groups attached to imidazole (9a). Ethyl substitute
(9b) and methyl substitution on the S-benzyl ring (9l, 9e) also produced
a similar increased activity. The p-benzyl ring effect, as substitute
attached to sulfur in the hydrophobic region, on the PPAR-γ agonist
nature was as follows, –CH3 (9e) > no substitute (9d) > -F (9f). The
presence of an alkyl group in the hydrophobic region instead of a benzyl
group reduced the activity of the compounds as follows, CH3CH2– (9b)
> CH3CH2CH2– (9c) > CH3– (9a). The effect of the 2-methyl pyridine
ring attached to sulfur (9g) was like to S-methyl (9a). Interestingly, the
simultaneous presence of fluorine in both positions (para-position of the
S-benzyl ring and para-position of N-benzyl ring) (9m) increased com-
pound activity compared to its presence in either of these positions alone
(9f, 9j, 9h). It has previously been reported that PPAR-γ agonists cause
weight gain [61–63]; in this study, however, oral administration of
compound 9e for ten days in diabetic rats did not induce a significant
change in body weight compared to the pioglitazone group. Body weight
was almost constant on days one and ten like control groups (diabetic-
control, Pioglitazone, healthy) (Fig. 12). For more comprehensive re-
sults, a long term study (at least one month) should be performed.
Fig. 8. Rg of PPAR-γ with ligand and without ligand.
attach to TRβ, AR, and GR receptors. Toxic potential for pioglitazone, 9e
and 9g was low (less than 0.3), whereas the remaining compounds had
moderate toxic potential (0.3 < TP < 0.6).
2.6. The biological assay
2.6.1. Cell survival experiment
The toxicity of compounds 9a to 9m was calculated using statistical
analysis. None of the compounds were toxic and their viability per-
centage was above 82%.
2.6.2. In vivo animal study
2.6.2.1. Blood glucose studies. All synthesized compounds with the
desired results (score ≥ 11.55) in molecular docking were studied in
vivo. As shown in Fig. 9, all compounds 9a ꢀ 9m significantly decreased
fasting blood glucose (FBG) levels (-17.13% to ꢀ 71.60%) compared to
that of diabetic controls (29.39%) (p < 0.001). Besides, compounds 9a,
9g, 9k, 9i, 9d, 9e, 9l, 9c and 9b showed significant statistical differences
in the area under curve (AUC) versus the standard drug in ten days
(Fig. 10). These outcomes showed that treatment of diabetic rats with
thiazolidinedione derivatives could improve blood glucose levels. As
seen in Fig. 10, compounds 9d (-553.8), 9e (-595.4), 9l (-524.6), 9c
(-524.6), 9b (-556.6), 9a (-479.6), 9g (-477.6), 9k (-478.3) and 9i
(-472.9) had higher anti-hyperglycemic activity compared to standard
drug (-416.8). Compounds 9j (-360.1), 9h (-114.8), and 9f (-304.9) had
fewer effects than the standard drug in STZ-induced diabetic rats.
Compound 9m (AUC = -392.8) showed an almost similar effect to pio-
glitazone on lowering blood glucose levels.
2.6.2.2. Histopathological examination and scoring. PPAR-γ agonists
have been reported to cause hepatotoxicity and inflammation [64,65].
In this study, the effects of PPAR-γ agonists on the liver and pancreatic in
four groups (healthy, diabetic-control, pioglitazone and 9e) were
examined. No histopathologic changes, including fibrosis, necrosis,
steatosis, and cholestasis, were seen in liver and pancreas tissues of any
group after 10 days. Compound 9e and pioglitazone caused mild
inflammation that was not significant compared to diabetic control. The
tissue images of the four groups are presented in Figs. 13 and 14. In long
term studies histopathological changes might be observed.
2.6.3. The glucose consumption effect of synthesized compound 9e on
HepG2 cells
Statistical analysis showed that most of the synthesized compounds
in this study had similar or better effects on reducing blood glucose
levels than the standard drug and FBG was significantly decreased on the
7th and 10th days of treatment (Fig. 11).
Different concentrations (1.25, 2.5, 5, and 10 µM) of HepG2 cell lines
with pioglitazone and compound 9e were incubated for 24 h and then
exposed to glucose (5.5, 11, 22 mM) to evaluate glucose uptake
(Fig. 15). Treatment with a concentration of 1.25 µM showed a signifi-
cant glucose-lowering effect in the HepG2 cell line exposed to hyper-
glycemia of 5.5 mM and 11 mM. The same effect was seen in
As mentioned above, the structure–activity relationship (SAR) can be
explained as compounds having an S-benzyl ring without a substitute
(9d) being more active in decreasing blood glucose levels versus the
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N. Shakour et al.
Bioorganic Chemistry 115 (2021) 105162
Fig. 10. AUC of the percentage decrease of FBG level at ten days in STZ induced diabetic male rats was calculated for the glucose test using one-way ANOVA
followed by Dunnett’s test and is revealed as mean ± SEM, (n = 6). ***: p < 0.001 vs. Pio. Diab.ctrl: diabetic control; Pio: Pioglitazone; 9a-9m: synthe-
sized compounds.
concentrations of 2.5 and 10 µM exposed to hyperglycemia of 11 mM.
Moreover, compound 9e in the concentration of 5 µM had the best effect
on HepG2 cell lines exposed to hyperglycemia of 11 and 22 mM,
compared to the control group.
non-toxic to NIH/3T3 normal cells. Ten compounds including 9d, 9k,
9a, 9i, 9l, 9c, 9g, 9m, and particularly 9e and 9b, showed significant in
vivo glucose-lowering effects. Findings from measured toxic potential
(TP) revealed that 9g and 9e had the lowest TP and lacked cardiotox-
icity. Compound 9e was selected as the candidate of choice with no
toxicity to the liver and pancreas, and an oral bioavailability like or
better than the standard drug (pioglitazone). Compound 9e also showed
a significant glucose-lowering effect in the HepG2 cell line exposed to
hyperglycemia. Both compound, 9e and pioglitazone, decreased the
PPAR-γ gene expression by 0.09 and 0.41-fold relative to the control
group, respectively.
2.6.4. PPAR-γ gene expression study
It has been demonstrated that PPAR-γ agonists decrease their mRNA
and protein expression level in 3T3-L1 cells through an auto-regulatory
manner [66–70]. Therefore, mRNA levels of PPAR-γ (as a target gene of
PPAR-γ nuclear receptor activation) in 3T3-L1 cells treated with the
newly synthesized TZD, 9e, or pioglitazone (as a well-known PPAR-γ
agonist) were assessed using quantitative RT-PCR. The results showed
that the expression of the PPAR-γ gene in cultured adipocytes was
significantly reduced in the presence of compound 9e (0.09 fold) and
pioglitazone (0.41 fold) compared to the control group (Fig. 16). Camp
& et al. reported that troglitazone, another PPAR-γ agonist, decreased
PPAR-γ mRNA level in 3T3-L1 adipocytes [70]. They speculated that,
whenever the required level of PPAR-γ activity is achieved, the
continued presence of the ligand results in the receptor downregulation
similar to that seen with other nuclear receptors such as glucocorticoid
and thyroid receptors in the presence of dexamethasone and L-triiodo-
thyronine ligands, respectively [67]. In two other studies, it has been
shown that another TZD, rosiglitazone, also decreased the expression of
mRNA and protein of PPAR-γ2 significantly in the 3T3-L1 cell line
[68,69]. Hence, our results indicated that the 9e compound as a novel
TZD had caused auto-regulation of PPAR-γ the same as other established
PPAR-γ receptor ligands, pioglitazone, troglitazone and rosiglitazone.
4. Methods and materials
Reagents and solvents in this research were purchased from Acros
Organics (Belgium) and Merck & Co, Inc. (Germany) and were used
without further filtration. Halogenated derivatives were purchased from
Kimiaexir Co. (Iran). All reactions were monitored by thin-layer chro-
matography (TLC) using plates with a diameter of 0.25 mm (60 GF-254),
purchased Merck & Co (Germany), and were visualized in UV light.
Melting points were measured by an electric melting point device (UK)
and were not corrected. 1H NMR and 13CNMR spectra were detected
(CHCL3 and DMSO‑d6, TMS) on a Bruker FT-300 MHz appliance (Ger-
many) at 300 MHz and 75 MHz, respectively. The chemical shifts (δ) and
coupling constants (J) were represented in parts per million and Hertz.
Mass spectra were acquired from an LC-MS Q-trap 3200, AB SCIEX
(USA) through an electrospray ionization (ESI) interface. Elemental
analyses were performed on a Cos-Tec model EAS 4010 instrument
(Italy) and the results were within ± 0.4% of the theoretical values. The
purity of the target compound was determined on a Shimadzu LC-SPD-
10A VP system equipped with a UV–Visible detector set to λ = 259 nm
and BRISA LC2 C18 5 µm, 25×0∙46cm column. The analysis was carried
out using the mobile phase containing potassium dihydrogen phosphate
buffer (KH2PO4) at pH 3.4 and acetonitrile (40:60 V/V) in the isocratic
mode, at a flow rate of 0.8 ml/min, for 15 min. The background peaks
between 3.1 and 4.9 min in the mock sample (methanol and mobile
phase with ratio 1:10) were excluded in the quantification of the
compound.
2.7. ADME properties prediction and drug-likeness
The ADME properties of pioglitazone and compound 9e were
calculated utilizing SwissADME online software. This software has been
generated and supported by the molecular modeling Swiss institute
group of bioinformatics (SIB). The colored region in Fig. 11s was re-
ported as a suitable physicochemical region for oral bioavailability. It
showed that properties such as lipophilicity, size, polarity, insolubility,
flexibility, and unsaturation of compound 9e were like standard drug
pioglitazone.
3. Conclusion
4.1. A generic method for preparation of compounds 4a to b
In this study, 13 new compounds were synthesized from a molecular
docking-guided library of compounds. The synthesized compounds were
As previously have been reported, acetic acid (20 ml) and n-butanol
(100 ml) were poured as solvents in a 500 ml round bottom flask using a
7

N. Shakour et al.
Bioorganic Chemistry 115 (2021) 105162
(C) Day 7
(A) Day 1
20
20
0
***
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0
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-20
-40
-60
-80
-20
-40
-60
-80
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*
*
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***
l
r
j
i
l
f
9
a
9
k
9
e
9
c
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9
h
9
d
9
b
9
9
9
9
i
p
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t
9
l
r
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f
a
k
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9
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9
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9
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9
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i
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9
t
9
9
9
b
9
p
c
a
i
.
b
D
a
i
Groups
D
Groups
(D) Day 10
(B) Day 4
20
50
***
***
0
-20
-40
-60
-80
0
***
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-50
***
***
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**
***
***
***
***
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***
**
-100
l
j
i
l
f
l
r
j
i
l
f
9
a
k
9
e
9
c
9
o
g
h
d
b
a
9
g
9
k
9
h
d
9
e
9
c
9
b
9
r
o
9
9
9
i
m
9
m
9
9
9
9
i
p
t
t
9
9
9
9
9
9
9
p
c
c
.
.
b
b
a
a
i
i
D
D
Groups
Groups
Fig. 11. In vivo anti-diabetic activity of the compounds 9a-9m vs. percentage decrease of FBG level in STZ-induced diabetic male rats: (A) Day 1 (24 h after of first
the gavage) (B) Day 4 (C) Day 7 (D) Day 10. Data was analyzed by one-way ANOVA followed by Dunnett test and expressed as mean ± SEM, (n = 6). ***: p < 0.001;
**: p < 0. 01, *: p < 0.05 and ns: no significance vs. Pio. Diab. ctrl: diabetic control; Pio: Pioglitazone; 9a-9m: synthesized compounds.
magnetic stirrer [71]. Then benzylamine hydrochloride (1a, 3.66 g,
25.5 mmol), 1,3- dihydroxyacetone dimer (3.26 g, 22 mmol) and po-
tassium thiocyanate (6.5 g, 66.6 mmol) were added. The mixture was
stirred for nearly 72 h and then, with filtration, washing and drying,
obtained a crude product of compound 4a as a white precipitate. The
whole method was applied to synthesize all compounds 4a to 4m,
whereas it was white precipitate [72]. (Yield: > 95%)
method was applied to synthesize compounds 6a to 6m, whereas all
were white crystals. (Yield: between 90 and 95%)
4.3. A generic procedure for synthesis of compounds 7a to m
The compounds 7e to 7m were synthesized as previously reported
for compounds 7a to 7d [71]. Compound 6a (1.62 g, 7.0 mmol) was
placed in a 100 ml round bottom flask, and 25 ml of chloroform was used
as a solvent [71]. Then, activated manganese dioxide (2.46 g, 28.3
mmol) was added as an oxidizing agent, and the flask was placed in an
oil bath (≥62 ◦C) using a magnetic stirrer. After adding the reagents, the
reaction was continued for 12–24 h and was monitored by thin-layer
chromatography (TLC). Finally, the mixture was purified and clarified
by filtering through a column (20 × 5 cm) filled with diatomaceous
earth and chloroform. The chloroform was removed and the pure
product of compound 7a was obtained as a sediment. The whole method
was applied to prepare compounds 7a to 7m. (Yield: between 90 and
95%).
4.2. A generic procedure for synthesis of compounds 6a to m
The compounds 6e to 6m were synthesized as previously reported
for compounds 6a to 6d [71], methyl iodide (5a, 0.29 ml, 4.6 mmol)
was added to the stirring suspension of compound 4a (1.06 g, 4.54
mmol) in a combination of methanol (66 ml) as a solvent and sodium
hydroxide (1 N, 4.7 ml) as a base [71]. After stirring overnight, meth-
anol evaporated and the remaining suspension was extracted with
chloroform. After removing the solvent, the remnant was crystallized
from ethyl acetate to obtain compound 6a as a white crystal. The whole
8

N. Shakour et al.
Bioorganic Chemistry 115 (2021) 105162
4.4. A generic procedure for synthesis of compounds 8a to m
Initially, 10 ml of methanol and 70 ml of chloroform were added as
solvents to a 100 ml round bottom flask followed by adding piperidine
(1.2 ml as base) and 2,4-thiazolidinedione (0.43 g, 3.7 mmol). The flask
was placed in an oil bath (65–100 ◦C) using a magnetic stirrer, and then
compound 7a (3.41 g, 14.7 mmol) was added into the flask. After a 24-
hour continuous reaction, the precipitates were washed, filtered and
dried to obtain the crude product compound 8a as a light yellow powder
[52]. The whole method was applied to prepare compounds 8a to 8m,
whereas all were pale yellow powder.
4.4.1. 5-((1-benzyl-2-(methylthio)-1H-imidazol-5-yl) methylene)
thiazolidine-2,4-dione (8a)
C
15H13N3O2S2: MW = 331.04, Light yellow powder, yield 60%, Rf =
0.82 (CHCl3/EtOH (9:1)), m.p. 245–250 ◦C; 1H NMR (300 MHz,
DMSO‑d6) δ 2.66 (s, 3H, CH3), 5.48(s, 2H2, CH2), 7.06–7.50(m, 7H, Ar-
H), 12.49(s, 1H, NH); 13C NMR (75 MHz, DMSO‑d6) δ 15.24, 47.16,
117.36, 120.51, 126.69, 128.26, 129.44, 129.69, 134.48, 136.54,
149.24, 167.31, 167.41; Elem. Anal. Calc. C, 54.36; H, 3.95; N, 12.68.
found: C, 54.24; H, 3.94; N, 12.65; LCMS (ESI, m/z): 332 [M+1]
Fig. 12. Bodyweight curve before (day 0) and after (day 10) treatment for the
H, Diab. ctrl, Pio and 9e groups in male rats. Data were analyzed through
unpaired t-test and reported as mean ± SEM, (n = 6). Diab. ctrl: diabetic
control; Pio: Pioglitazone; H: Health; 9e: synthesized compound.
Fig. 13. The effect of pioglitazone and 9e compound on histological changes in the liver under an optical microscope (Nikon Eclipse E200-LED, Nikon, Tokyo, Japan)
40×. Using one-way ANOVA followed by the Kruskal-Wallis test, (n = 6). The value of P-Value shows p ≥ 0.05 vs. Diab. ctrl group. A) Data on histopathologic scores
were expressed as medians ± IQR B) Section from rat liver with typical histopathologic features C) Sections from the liver of diabetic rats exhibit little inflammation
D) Section from the liver of pioglitazone-treated rats exhibit slight inflammation and little necrosis E) Section from the liver of rats treated with synthesized
compound 9e exhibit slight inflammation without tissue necrosis. Diab. ctrl: diabetic control; Pio: Pioglitazone; H: Health; 9e: synthesized compound.
9

N. Shakour et al.
Bioorganic Chemistry 115 (2021) 105162
Fig. 14. The effect of pioglitazone and 9e compound on histological changes in the pancreas under an optical microscope (Nikon Eclipse E200-LED, Nikon, Tokyo,
Japan) 40×. Using one-way ANOVA followed by the Kruskal-Wallis test, (n = 6). The value of P-Value shows p ≥ 0.05 vs. Diab.ctrl group. A) Data on histopathologic
scores were expressed as medians ± IQR B) Section from rat pancreas with typical histopathologic features C) Section from Pancreas of diabetic rats exhibit little
inflammation D) A section of the pancreas of pioglitazone-treated rats exhibits slight inflammation without tissue necrosis E) Section from the liver of rats treated
with synthesized compound 9e exhibits slight inflammation without tissue necrosis. Diab. ctrl: diabetic control; Pio: Pioglitazone; H: Health; 9e: synthe-
sized compound.
4.4.2. 5-((1-benzyl-2-(ethylthio)-1H-imidazol-5-yl) methylene)
thiazolidine-2,4-dione (8b)
DMSO‑d6) δ 4.48(s,2H, CH2), 5.34(s,2H, CH2), 6.92–7.53(m, 12H, Ar-
H), 12.50(s, 1H, NH);13C NMR (75 MHz, DMSO‑d6) δ 37.20, 47.25,
117.29, 120.94, 126.60, 127.94, 128.21, 128.96, 139.35, 129.62,
134.53, 136.50, 137.89, 147.50, 167.31, 167.41; Elem. Anal. Calc. C,
61.89; H, 4.20; N, 10.31. found: C, 61.75; H, 4.18; N, 10.27; LCMS (ESI,
C16H15N3O2S2: MW = 345.06, Light yellow powder, yield 60%, Rf =
0.66 (CHCl3/ EtOH (9:1)), m.p. 207–212 ◦C; 1H NMR (300 MHz,
DMSO‑d6) δ 1.31(t, 3H, J = 7.5, CH3), 3.24 (q, 2H, J = 15, CH2), 5.41(s,
2H, CH2), 7.28–7.50(m, 7H, Ar-H), 12.50(s, 1H, NH);13C NMR (75 MHz,
DMSO‑d6) δ 15.51, 27.38, 47.19, 117.35, 120.63, 126.61, 128.24,
129.43, 129.53, 134.59, 136.63, 148.16, 167.36, 167.45; Elem. Anal.
Calc. C, 55.63; H, 4.38; N,12.16. found: C, 55.51; H, 4.37; N,12.12;
m/z): 408 [M+1] +
.
4.4.5. 5-((1-benzyl-2-((4-methylbenzyl) thio)-1H-imidazol-5-yl)
methylene) thiazolidine-2,4-dione (8e)
LCMS (ESI, m/z): 346 [M+1] +
.
C22H19N3O2S2: MW = 421.09, Light yellow powder, yield 60%, Rf =
0.66 (CHCl3/ EtOH (9:1)), m.p. 238–245 ◦C; 1H NMR (300 MHz,
DMSO‑d6) δ 2.28(s,3H, CH3), 4.45(s, 2H, CH2), 5.35(s, 2H, CH2),
6.92–8.06(m, 11H, Ar-H), 12.47(s, 1H, NH);13C NMR (75 MHz,
DMSO‑d6) δ 21.20, 37.06, 47.24, 117.12, 121.18, 126.61, 127.18,
128.08, 128.20, 129.04, 129.26, 129.32, 129.50, 129.61, 134.47,
134.78, 136.53, 137.16, 147.51, 167.55, 167.63; Elem. Anal. Calc. C,
62.68; H, 4.54; N, 9.97. found: C, 62.57; H, 4.53; N, 9.95; LCMS (ESI, m/
4.4.3. 5-((1-benzyl-2-(propylthio)-1H-imidazol-5-yl) methylene)
thiazolidine-2,4-dione (8c)
C
17H17N3O2S2: MW = 359.08, Light yellow powder, yield 65%, Rf =
0.85 (CHCl3/ EtOH (9:1)), m.p. 240–245 ◦C; 1H NMR (300 MHz,
DMSO‑d6) δ 0.98 (t, 3H, J = 7.5, 5 Hz, CH3), 1.71(m, 2H, CH2), 3.23(t,
2H, J = 6, CH2), 5.46(s, 2H, CH2), 7.08–7.53(m, 7H, Ar-H), 12.54(s, 1H,
NH);13C NMR (75 MHz, DMSO‑d6) δ 13.41, 23.09, 34.08, 47.19, 117.37,
120.55, 126.59, 128.23, 129.42, 129.53, 134.57, 136.63, 148.31,
167.32, 167.43; Elem. Anal. Calc. C, 56.80; H, 4.77; N,11.69. found: C,
z): 422 [M+1] +
.
4.4.6. 5-((1-benzyl-2-((4-fluorobenzyl) thio)-1H-imidazol-5-yl)
methylene) thiazolidine-2,4-dione (8f)
56.68; H, 4.76; N,11.65; LCMS (ESI, m/z): 360 [M+1] +
.
C
21H16FN3O2S2: MW = 425.09, Light yellow powder, yield 55%, Rf
4.4.4. 5-((1-benzyl-2-(benzylthio)-1H-imidazol-5-yl) methylene)
thiazolidine-2,4-dione (8d)
= 0.82 (CHCl3/ EtOH (9:1)), m.p. 265–270 ◦C; 1H NMR (300 MHz,
DMSO‑d6) δ 4.52(s,2H, CH2), 5.37(s,2H, CH2), 7.01–7.58(m, 11H, Ar-
H), 12.54(s, 1H, NH);13C NMR (75 MHz, DMSO‑d6) δ 41.93, 51.99,
122.06, 125.66, 131.35, 132.70, 132.96, 133.71, 133.97, 134.11,
C
21H17N3O2S2: MW = 407.08, Light yellow powder, yield 60%, Rf =
0.82 (CHCl3/ EtOH (9:1)), m.p. 245–250 ◦C; 1H NMR (300 MHz,
10

N. Shakour et al.
Bioorganic Chemistry 115 (2021) 105162
Fig. 15. The lowering effect of glucose in the HepG2 cell line exposed to the hyperglycemia 5.5, 11, and 22 mM for two compound 9e and pioglitazone in various
concentrations A) 1.25, B) 2.5, C) 5, and D) 10 µM was followed by Dunnett test and was expressed as mean ± SEM, (n = 6). A) Investigated effects of glucose
reduction in concentration 1.25 µM; B) Investigation effects of glucose reduction in concentration 2.5 µM; C) Investigated effects of glucose reduction in concen-
tration 5 µM; D) Investigated effects of glucose reduction in concentration 10 µM. Pio: pioglitazone, 9e: synthesized compound 9e, Control: without treatment. ***:
p < 0.001; **: p < 0. 01, *: p < 0.05 and ns: no significance vs. control.
134.36, 139.29, 141.26, 142.5, 152.26, 172.05, 172.16; Elem. Anal.
Calc. C, 59.28; H, 3.79, N, 9.88. found: C, 59.15; H, 3.77, N, 9.84; LCMS
DMSO‑d6) δ 2.55(s, 3H, CH3), 5.44(S, 2H, CH2), 7.14–7.55(m, 6H, Ar-
H), 12.55(s, 1H, NH); 13C NMR (75 MHz, DMSO‑d6) δ 15.22, 46.47,
116.16, 116.45, 117.26, 120.65, 128.82, 128.93, 129.61, 132.74,
132.78, 134.51, 149.18, 167.32, 167.42; Elem. Anal. Calc. C, 51.56; H,
3.46; N, 12.03. found: C, 51.44; H, 3.45; N, 11.98; LCMS (ESI, m/z): 350
(ESI, m/z): 426 [M+1] +
.
4.4.7. 5-((1-benzyl-2-((pyridin-2-ylmethyl) thio)-1H-imidazol-5-yl)
methylene) thiazolidine-2,4-dione (8g)
[M+1] +
.
C
20H16N4O2S2: MW = 408.07, Light yellow powder, yield 60%, Rf =
0.66 (CHCl3/ EtOH (9:1)), m.p. 247–255 ◦C; 1H NMR (300 MHz,
DMSO‑d6) δ 4.60(s, 2H, CH2), 5.38(s, 2H, CH2), 6.98–8.52(m, 11H, Ar-
H), 12.51(s, 1H, NH);13C NMR (75 MHz, DMSO‑d6) δ 47.26, 117.27,
120.98, 123.03, 123.54126.62, 128.23, 129.38, 129.68, 134.51, 136.54,
137.34, 147.54, 149.74, 156.98, 167.37, 167.45; Elem. Anal. Calc. C,
58.80; H, 3.95; N, 13.72. found: C, 58.67; H, 3.93; N, 13.68; LCMS (ESI,
4.4.9. 5-((2-(ethylthio)-1-(4-fluorobenzyl)-1H-imidazol-5-yl) methylene)
thiazolidine-2,4-dione (8i)
C
16H14FN3O2S2: MW = 363.05, Light yellow powder, yield 60%, Rf
= 0.80 (CHCl3/ EtOH (9:1)), m.p. 235–240 ◦C; 1H NMR (300 MHz,
DMSO‑d6) δ 1.36(m, 3H, CH3), 3.21(m, 2H, CH2), 5.42(s, 2H, CH2),
7.11–7.08(m, 6H, Ar-H), 12.43(s, 1H, NH); 13C NMR (75 MHz,
DMSO‑d6) δ 23.08, 34.78, 46.49, 116.44, 116.44, 117.27, 120.68,
128.71, 128.82, 129.45, 132.83, 132.87, 134.61, 148.25, 167.33,
167.44; Elem. Anal. Calc. C, 52.88; H, 3.88; N, 11.56. found: C, 52.74; H,
m/z): 409 [M+1] +
.
4.4.8. 5-((1-(4-fluorobenzyl)-2-(methylthio)-1H-imidazol-5-yl)
methylene) thiazolidine-2,4-dione (8 h)
3.87; N, 11.52; LCMS (ESI, m/z): 264 [M+1] +
.
C
15H12FN3O2S2: MW = 349.04, Light yellow powder, yield 65%, Rf
= 0.78 (CHCl3/ EtOH (9:1)), m.p. 245–247 ◦C; 1H NMR (300 MHz,
11

N. Shakour et al.
Bioorganic Chemistry 115 (2021) 105162
4.5. A generic procedure for synthesis of compounds 9a to m
1.5
1.0
0.5
0.0
Compound (8a, 0.33 g, 1 mmol) was placed in a 25 ml round bottom
flask and 1.5 ml of N, N-dimethyl formamide and 0.3 ml of methanol
were added as solvents using a magnetic stirrer. Then, sodium hydroxide
(2 M, 0.45 ml), cobalt (II) nitrate hexahydrate (0.06 g, 0.2 mmol) and
dimethylglyoxime (DMG) (0.045 g, 0.39 mmol) were added as catalysts.
Sodium borohydride (0.06 g, 1.6 mmol) was also added at a temperature
of 0–5 ◦C as a reducing reagent. After a 4–5-hour continuous reaction,
the suspension was filtered using some active charcoal. The solution pH
was altered to 7 by introducing acetic acid. The produced sediment was
filtered, washed and dried to obtain the crude product compound 9a as a
white powder [52]. The whole method was applied to prepare com-
pounds 9b to 9m, whereas all were white powder.
$$$
***
$$$
***
9e
Control
Pio
Fig. 16. Effects of pioglitazone and synthesize compound 9e on PPAR-γ gene
expression. Quantitative RT-PCR was done in triplicate and was repeated three
times for each sample. Beta-actin was used as a housekeeping gene. Data was
analyzed by one-way ANOVA followed by Dunnett test and expressed as mean
± SEM, (n = 3); ***: p < 0.001; **: p < 0. 01, and *: p < 0.05 vs. control; $$$: p
< 0.001; $$: p < 0. 01, and $: p < 0.05 vs. Pio. Control: without treatment with
0.1% DMSO; Pio: Pioglitazone; 9e: synthesized compound.
4.5.1. 5-((1-benzyl-2-(methylthio)-1H-imidazol-5-yl) methyl)
thiazolidine-2,4-dione (9a)
C
15H15N3O2S2: MW = 333.06, white powder, yield 55%, Rf = 0.43
1
◦
(CHCl3/ EtOH (9:1)), m.p. 147–157 C; H NMR (300 MHz, CDCl3) δ
2.49 (s, 3H, CH3), 3.05(dd, 1H, J = 15, CH), 3.19(dd, 1H, J = 15, CH),
4.18(dd, 1H, J = 9, CH), 5.11(s, 2H, CH2), 6.91–7.30(m, 6H, Ar-H);13
C
NMR (75 MHz, DMSO‑d6) δ 16.21, 27.96, 47.73, 50.69, 126.16, 128.16,
128.26, 128.38, 129.16, 135.62, 144.74, 170.60, 174.48; Elem. Anal.
Calc. C, 54.03; H, 4.53; N, 12.60. found: C, 53.87; H, 4.51; N, 12.54;
LCMS (ESI, m/z): 334 [M+1].
4.4.10. 5-((1-(4-fluorobenzyl)-2-(propylthio)-1H-imidazol-5-yl)
methylene) thiazolidine-2,4-dione (8j)
C17H16FN3O2S2: MW = 377.07, Light yellow powder, yield 65%, Rf
= 0.84 (CHCl3/ EtOH (9:1)), m.p. 215–220 ◦C; 1H NMR (300 MHz,
DMSO‑d6) δ 0.94(t, 3H, J = 15, CH3), 1.66(m, 2H, CH2), 3.19(q, 2H, J =
12, CH2), 5.41(s, 2H, CH2), 7.08–8.03(m, 6H, Ar-H), 12.51(s, 1H,
NH);13C NMR (75 MHz, DMSO‑d6) δ 13.41, 23.08, 34.78, 46.49, 116.44,
117.27, 120.68, 128.71, 128.82, 129.45, 132.83, 132.87, 134.61,
148.25, 167.33, 167.44; Elem. Anal. Calc. C, 54.09; H, 4.27; N, 11.13.
4.5.2. 5-((1-benzyl-2-(ethylthio)-1H-imidazol-5-yl) methyl) thiazolidine-
2,4-dione (9b)
C
16H17N3O2S2: MW = 347.07, white powder, yield 53%, Rf = 0.50
1
◦
(CHCl3/ EtOH (9:1)), m.p. 105–110 C; H NMR (300 MHz, CDCl3) δ
1.30(t, 3H, j = 7.5, CH3), 3.08(m, 2H, CH2), 3.13(m, 1H, CH), 3.31(dd,
1H, j = 15, CH), 4.28(dd, 1H, j = 9, CH), 5.36(s, 2H, CH2), 7–7.36(m, 6H,
Ar-H);13C NMR (75 MHz, DMSO‑d6) δ 14.88; 28.20, 28.82, 47.82, 50.69,
126.01, 128.11, 1287.41, 129.14, 135.82, 143.40, 170.66, 174.54;
Elem. Anal. Calc. C, 55.31; H, 4.93; N,12.09. found: C, 55.31; H, 4.93; N,
found: C, 53.91; H, 4.25; N, 11.09; LCMS (ESI, m/z): 378 [M+1] +
.
4.4.11. 5-((2-(benzylthio)-1-(4-fluorobenzyl)-1H-imidazol-5-yl)
methylene) thiazolidine-2,4-dione (8 k)
12.09; LCMS (ESI, m/z): 348 [M+1] +, 370 [M+23] +
.
C
21H16FN3O2S2: MW = 425.5, Light yellow powder, yield 55%, Rf =
0.79 (CHCl3/ EtOH (9:1)), m.p. 237–242 ◦C; 1H NMR (300 MHz,
DMSO‑d6) δ, 4.54(s, 2H, CH2), 5.40(s, 2H, CH2), 6.98–7.56(m, 11H, Ar-
H), 12.55(s, 1H, NH);13C NMR (75 MHz, DMSO‑d6) δ 37.21, 46.54,
116.06, 116.34, 117.22, 121.04, 127.97, 128.71, 128.82, 128.97,
129.35, 129.52, 132.76, 132.76, 134.58, 137.90, 147.43, 167.30,
167.40; Elem. Anal. Calc. C, 59.28; H, 3.79; N, 9.88. found: C, 59.14; H,
4.5.3. 5-((1-benzyl-2-(propylthio)-1H-imidazol-5-yl) methyl) thiazolidine-
2,4-dione (9c)
C
17H19N3O2S2: MW = 347.07, white powder, yield 52%, Rf = 0.56
(CHCl3/ EtOH (9:1)), m.p. 97–100 ◦C; 1H NMR (300 MHz, CDCl3) δ 0.89
(t, 3H, j = 12, CH3), 1.56(m, 2H, CH2), 2.95(t,2H, j = 6, CH2), 3.01(dd,
1H, j = 15, CH), 3.21(dd, 1H, j = 15, CH), 4.16(dd, 1H, j = 10.5, CH),
5.16(s, 2H, CH2), 6.89–7.29(m, 6H, Ar-H);13C NMR (75 MHz, DMSO‑d6)
δ 13.20, 22.90, 28.22 ,36.46, 47.81, 50.68, 126.10, 126.42, 128.10,
128.23, 128.51, 129.14, 135.81, 143.64, 170.83, 174.71; Elem. Anal.
Calc. C, 56.48; H, 5.30; N, 11.62. found: C, 59.10; H, 4.01; N, 13.72;
3.78; N, 9.84; LCMS (ESI, m/z): 426 [M+1] +
.
4.4.12. 5-((1-(4-fluorobenzyl)-2-((4-methylbenzyl) thio)-1H-imidazol-5-
yl) methylene) thiazolidine-2,4-dione (8 l)
C
22H18FN3O2S2: MW = 439.0, Light yellow powder, yield 60%, Rf =
0.90 (CHCl3/ EtOH (9:1)), m.p. 214–219 ◦C; 1H NMR (300 MHz,
DMSO‑d6) δ 2.32(s, 3H, CH3), 4.49(s, 2H, CH2), 5.40(s, 2H, CH2),
6.98–7.56(m, 10H, Ar-H), 12.55(s, 1H, NH);13C NMR (75 MHz,
DMSO‑d6) δ 21.18, 46.53, 116.02, 116.31, 117.25, 120.98, 128.72,
128.83, 129.26, 129.52, 132.74, 134.59, 134.77, 137.18, 147.54,
167.29, 167.40; Elem. Anal. Calc. C, 60.12; H, 4.13; N, 9.56. found: C,
LCMS (ESI, m/z): 362 [M+1] +
.
4.5.4. 5-((1-benzyl-2-(benzylthio)-1H-imidazol-5-yl) methyl) thiazolidine-
2,4-dione (9d)
C
21H19N3O2S2: MW = 409.09, white powder, yield 52%, Rf = 0.58
(CHCl3/ EtOH (9:1)), m.p. 97–105 ◦C; 1H NMR (300 MHz, CDCl3) δ 2.9
(m, 1H, CH2), 3.03(m, 1H, CH2), 4.09(m, 1H, CH), 4.16(s, 2H, CH2),
4.81(s, 2H, CH2), 6.73–7.31(m, 11H, Ar-H);13C NMR (75 MHz,
DMSO‑d6) δ 28.20, 40.03, 47.60 ,50.62, 126.09, 127.57, 128.04,
128.66, 128.92, 129.01, 129.09, 135.71, 137.45, 142.22, 170.18,
174.01; Elem. Anal. Calc. C, 61.59; H, 4.68; N, 10.26. found: C, 59.10; H,
59.98; H, 4.11; N, 9.52; LCMS (ESI, m/z): 440 [M+1] +
.
4.4.13. 5-((1-(4-fluorobenzyl)-2-((4-fluorobenzyl) thio)-1H-imidazol-5-
yl) methylene) thiazolidine-2,4-dione (8 m)
C
21H15F2N3O2S2: MW = 443.06, Light yellow powder, yield 60%, Rf
= 0.70 (CHCl3/ EtOH (9:1)), m.p. 202–203 ◦C; 1H NMR (300 MHz,
DMSO‑d6)) δ 4.4(s, 2H, CH2), 5.23(s, 2H, CH2), 6.84–7.36(m, 10H, Ar-
H), 12.40(s, 1H, NH);13C NMR (75 MHz, DMSO‑d6) δ 37.21, 46.53,
116.05, 116.34, 117.17, 121.10, 127.97, 128.71, 12128.97, 129.35,
129.53, 132.75, 134.57, 137.89, 147.42, 167.37, 167.44; Elem. Anal.
Calc. C, 56.87; H, 3.41; N, 9.47. found: C, 56.75; H, 3.40; N, 9.43.; LCMS
(ESI, m/z): 443.06 [M]˙+.
4.01; N, 13.72; LCMS (ESI, m/z): 410 [M+1] +
.
4.5.5. 5-((1-benzyl-2-((4-methylbenzyl) thio)-1H-imidazol-5-yl) methyl)
thiazolidine-2,4-dione (9e)
C
22H21N3O2S2: MW = 423.11, white powder, yield 40.8%, Rf = 0.72
1
◦
(CHCl3/ EtOH (9:1)), m.p. 137–142 C; H NMR (300 MHz, CDCl3) δ
2.26(s, 3H, CH3), 2.97(m, 1H, CH), 3.1(m, 1H, CH), 4.11(m, 1H, CH),
4.16(s, 2H, CH2), 4.82(s, 2H, CH2), 6.68–7.19(m, 10H, Ar-H);13C NMR
12

N. Shakour et al.
Bioorganic Chemistry 115 (2021) 105162
1
◦
(75 MHz, DMSO‑d6) δ 21.19, 28.18, 39.67, 47.00, 50.64, 115.87,
116.16, 127.84, 127.95, 128.47, 128.83, 119.34, 131.40, 131.44,
134.21, 137.34, 142.41, 170.32, 174.27; Elem. Anal. Calc. C, 62.39; H,
5.00; N, 9.92. found: C, 59.10; H, 4.01; N, 13.72; LCMS (ESI, m/z): 424
(CHCl3/ EtOH (9:1)), m.p. 123–133 C; H NMR (300 MHz, CDCl3) δ
2.88(m, 1H, CH2), 3.03(m, 1H, CH2), 4.13(m, 1H, CH), 4.25(s,2H, CH2),
4.81(s, 2H, CH2), 6.66–7.47(m, 10H, Ar-H);13C NMR (75 MHz,
DMSO‑d6) δ 28.19,40.04, 46.93, 50.70,115.91, 116.19, 127.62, 127.82,
127.93, 128.69, 128.91, 131.37, 131.41, 137.42, 142.07,170.70,
174.60; Elem. Anal. Calc. C, 59.00; H, 4.24; N, 9.83. Found: C, 59.10; H,
[M+1] +
.
4.5.6. 5-((1-benzyl-2-((4-fluorobenzyl) thio)-1H-imidazol-5-yl) methyl)
thiazolidine-2,4-dione (9f)
4.01; N, 13.72; LCMS (ESI, m/z): 428 [M+1] +, 450 [M+23] +
.
C
21H18FN3O2S2: MW = 427.08, white powder, yield 70%, Rf = 0.59
4.5.12. 5-((1-(4-fluorobenzyl)-2-((4-methylbenzyl) thio)-1H-imidazol-5-
yl) methyl) thiazolidine-2,4-dione (9l)
1
◦
(CHCl3/ EtOH (9:1)), m.p. 155–160 C; H NMR (300 MHz, CDCl3) δ
2.96(dd, 1H, j = 18, CH2), 3.1(dd, 1H, j = 15, CH2), 4.09(m, 1H, CH),
4.17(s, 2H, CH2), 4.81(s, 2H, CH2); 6.72–7.21(m, 10H, Ar-H);13C NMR
(75 MHz, DMSO‑d6) δ 28.21, 40.11, 47.61 ,50.68, 126.9, 127.58,
128.04, 128.67, 128.85, 128.93, 129.09, 135.68, 137.40, 142.10,
170.72, 174.56; Elem. Anal. Calc. C, 59.00; H, 4.24; N, 9.83. found: C,
59.10; H, 4.01; N, 13.72; LCMS (ESI, m/z): 427 [M]+.
C
22H20FN3O2S2: MW = 441.10, white powder, yield 80%, Rf = 0.69
1
◦
(CHCl3/ EtOH (9:1)), m.p. 123–133 C; H NMR (300 MHz, CDCl3) δ
2.37(s, 3H, CH3), 3.07(m, 1H, CH), 3.23(dd, 1H, j = 15, CH), 4.19(m,
1H, CH), 4.48(s, 2H, CH2), 4.97(s, 2H, CH2), 6.84–7.30(m, 9H, Ar-
H);13C NMR (75 MHz, DMSO‑d6) δ 21.20, 28.22, 39.72, 47.68, 50.67,
126.13, 128.03, 128.64, 128.85, 129.06, 129.32, 134.31, 135.73,
137.28, 142.46, 170.43, 174.29; Elem. Anal. Calc. C, 59.84; H, 4.57; N,
9.52. found: C, 59.10; H, 4.01; N, 13.72; LCMS (ESI, m/z): 442 [M+1] +.
4.5.7. 5-((1-benzyl-2-((pyridin-2-ylmethyl) thio)-1H-imidazol-5-yl)
methyl) thiazolidine-2,4-dione (9g)
C
20H18N4O2S2: MW = 410.09, white powder, yield 77%, Rf = 0.40
4.5.13. 5-((1-(4-fluorobenzyl)-2-((4-fluorobenzyl) thio)-1H-imidazol-5-
yl) methyl) thiazolidine-2,4-dione (9m)
1
◦
(CHCl3/ EtOH (9:1)), m.p. 160–164 C; H NMR (300 MHz, CDCl3) δ
2.94(m, 1H, CH), 3.12(dd, 1H, j = 15, CH), 4.12(dd, 1H, j = 9, CH), 4.33
(s, 2H, CH2), 4.95(s, 2H, CH2), 6.17–8.48(m, 10H, Ar-H);13C NMR (75
MHz, DMSO‑d6) δ 28.12, 40.92, 47.75, 50.65, 122.51, 123.46, 126.07,
126.40, 128.04, 128.25, 128.81, 128.90, 129.08, 135.66, 137.04,
142.03, 149.38. 157.08, 170.59, 174.43; Elem. Anal. Calc. C, 58.52; H,
4.42; N, 13.65. found: C, 59.10; H, 4.01; N, 13.72; LCMS (ESI, m/z): 411
C
21H17F2N3O2S2: MW = 445.07, white powder, yield 70%, Rf = 0.53
1
◦
(CHCl3/ EtOH (9:1)), m.p. 125–130 C; H NMR (300 MHz, CDCl3) δ
2.95(dd, 1H, j = 15, CH), 3.09(dd, 1H, j = 15, CH), 4.12(m, 1H, CH),
4.19(s, 2H, CH2), 4.76(s,3H, CH3), 6.66–7.20(m, 9H, Ar-H);13C NMR
(75 MHz, DMSO‑d6) δ14.16, 28.21, 46.92, 50.67, 115.91, 116.20,
127.63, 127.83, 127.94, 128.70, 128.76, 128.84, 128.92, 131.35,
137.41, 141.99, 170.83, 174.73; Elem. Anal. Calc. C, 56.62; H, 3.85; N,
9.43. Found: C, 59.10; H, 4.01; N, 13.72; LCMS (ESI, m/z): 445 [M]+.
Spectra data (1H NMR and 13C NMR) for compounds 8a-m and 9a-
m were shown in Figures 12S-36S in the supplementary.
[M+1] +, 433 [M+23] +
.
4.5.8. 5-((1-(4-fluorobenzyl)-2-(methylthio)-1H-imidazol-5-yl) methyl)
thiazolidine-2,4-dione (9 h)
C
15H14FN3O2S2: MW = 351.05, white powder, yield 60%, Rf = 0.80
1
◦
(CHCl3/ EtOH (9:1)), m.p. 145–150 C; H NMR (300 MHz, CDCl3) δ
2.51(s, 3H, CH3), 3.06(m, 1H, CH2), 3.17(m, 1H, CH2), 4.23(dd, 1H, j =
9, CH); 5.07(S, 2H, CH2), 6.86–6.99(m, 5H, Ar-H); 13C NMR (75 MHz,
DMSO‑d6) δ 16.13, 27.89, 47.07, 50.73, 116.02, 116.31, 127.91,
128.02, 128.25, 131.30, 131.34, 144.69, 170.81, 174.80; Elem. Anal.
Calc. C, 51.27; H, 4.02; 11.96. found: C, 59.10; H, 4.01; N, 13.72; LCMS
4.6. Molecular docking section
Molecular docking, one of the most common techniques in drug
design, was used to investigate the interaction of small molecules with
suitable target binding sites. The docking was performed using Moe
2019 (www.chemcomp.com) to predict the best conformations of
selected compounds in the active site of a PPAR-γ protein. The crystal
structure of the x-ray of PPAR-γ with PDB ID: 5y2o and resolution 1.801
Å was obtained from the Protein Data Bank (PDB). Polar hydrogen atoms
(corresponding to pH 7) were connected to the receptor considering the
suitable ionization states, water molecules were eliminated and the
protein was saved in PDB format. The two-dimensional structure of
these synthesized compounds was provided through Chem Draw Ultra
(v.12.0 Cambridge Soft, UK, 2010, http://www. cambridgesoft.com).
The 3D format was generated by Hyper Chem7 (Hypercube, Inc, USA)
using the PM3 semi-empirical method. The docking was done through
the triangle matcher method and London DG scoring with 100 poses.
Refinements were done using a rigid receptor method and GBWI/WSA
DG was scored with five poses. Also, every generated conformation of
compounds was analyzed by the LigX module of MOE. Docking was
done for 13 compounds [73]. In the last step, the best conformation was
evaluated in terms of the number of hydrogen bonds and the Gibbs free
energy. Finally, the best docking state of compound 9i was selected for
molecular dynamic (MD) simulation. A docking map of the four com-
pounds (9a, 9g, 9i, and 9j) in the PPAR-γ active site is shown.
(ESI, m/z): 352 [M+1] +
.
4.5.9. 5-((2-(ethylthio)-1-(4-fluorobenzyl)-1H-imidazol-5-yl) methyl)
thiazolidine-2,4-dione (9i)
C
16H16FN3O2S2: MW = 365.07, white powder, yield 76%, Rf = 0.65
1
◦
(CHCl3/ EtOH (9:1)), m.p. 120–125 C; H NMR (300 MHz, CDCl3) δ
1.29(t, 3H, j = 7.5, CH3), 3.08(m, 2H, CH2), 3.11(m, 1H, CH), 3.22(m,
1H, CH), 4.32(m, 1H, CH), 5.22(s, 2H, CH2), 6.91–7.30(m, 5H, Ar-
H);13C NMR (75 MHz, DMSO‑d6) δ 14.84, 28.18, 28.84, 47.15, 50.73,
115.98, 116.27, 127.85, 127.96, 128.30, 128.49, 131.50, 131.54,
143.29, 160.73, 164.00; Elem. Anal. Calc. C, 52.59; H, 4.41; N, 11.50.
found: C, 59.10; H, 4.01; N, 13.72; LCMS (ESI, m/z): 366 [M+1] +
.
4.5.10. 5-((1-(4-fluorobenzyl)-2-(propylthio)-1H-imidazol-5-yl) methyl)
thiazolidine-2,4-dione (9j)
C
17H18FN3O2S2: MW = 379.08, white powder, yield 50%, Rf = 0.70
1
◦
(CHCl3/ EtOH (9:1)), m.p. 145–150 C; H NMR (300 MHz, CDCl3) δ
0.89(t, 3H, j = 7.5, CH3), 1.53(m, 2H, CH2), 2.98(m, 2H, CH2), 3.05(m,
1H, CH), 3.18(dd, 1H, j = 15, CH), 4.21(dd, 1H, j = 9, CH), 5.12(s, 2H,
CH2), 6.87–7.19(m, 5H, Ar-H);13C NMR (75 MHz, DMSO‑d6) δ 13.21,
22.92, 28.10, 36.28, 47.11, 50.64, 116.00, 116.29, 127.83, 127.93,
128.04, 128.52, 131.57, 143.79, 170.07, 174.06; Elem. Anal. Calc. C,
53.81; H, 4.78; N, 11.07. found: C, 59.10; H, 4.01; N, 13.72; LCMS (ESI,
4.7. Molecular dynamic (MD) simulation
MD simulation studies were done to probe the protein–ligand
interaction in atomic details based on the equation of motion and
Newton’s second law (as an aqueous solution with the actual physio-
logical state at P = 1 atm, T = 37 ◦C). NAMD Git-2018–04-24 did
calculate for Linux-x86_64-multicore-CUDA (www.ksuiuc.edu). MOE
was used to generate input files for namd2. Viewing and analyzing the
m/z): 380 [M+1] +
.
4.5.11. 5-((2-(benzylthio)-1-(4-fluorobenzyl)-1H-imidazol-5-yl) methyl)
thiazolidine-2,4-dione (9k)
C21H18FN3O2S2: MW = 427.08, white powder, yield 80%, Rf = 0.61
13

N. Shakour et al.
Bioorganic Chemistry 115 (2021) 105162
results were carried out using the Visual Molecular Dynamics (VMD)
1.9.2 program [74]. The compound 9i was employed as a template
molecule to illustrate the MD simulations. Minimization of energy in the
protein structure and docked ligand was done in the Amber force field.
At the height of 2 nm from the protein surface, the water solvent was
added to the box center with dimensions 4.5 * 5 * 5 nm. Also, the MOE
program adding charge was done for all atoms. Then, MD simulations
were done in 0 to 100 ns, during which the first five nanoseconds were
for minimization. The system trajectory was collected every 1 ps and
was checked out by VMD analyzer tools. The system was complicated
and needed notable computation. In this work, we used systems with
AMD Rayzen 1950X processors (32 core). The 3D structure, number of
hydrogen bonds, and hydrophobic interactions between compound 9i
and active site of PPAR-γ were taken from PLIP (https://projects.biotec.
tu-dresden.de/plip-web/plip).
SP.1396.196. Rats were purchased from animal facilities at Mashhad
University of Medical Sciences, Iran. All rats were ensconced in non-
opaque nest boxes and were kept under regulated temperature (18
ꢀ 23 ◦C), illumination (12-hour dark and 12-hour light) with access to
tap water and feed (Behparvar animal feed company, Tehran, Iran).
After a two-week adaptation, the rats were randomly divided into four
groups (6 rats per group): 1) Diabetic rats that were orally fed with a
solution containing 0.25% CMC (carboxymethyl cellulose Sigma, USA),
2) healthy rats that received no treatment and were orally fed with
0.25% CMC solution, 3) Diabetic rats orally fed with pioglitazone
(Sigma, USA) (as 0.25% CMC suspension at a dose of 3.8 mg/kg (0.011
mmol/kg) and 4) Diabetic rats that were fed oral synthesized com-
pounds (as 0.25% CMC suspension) with an equal dose of a standard
drug. After 12–14 h of fasting, diabetes was induced in rats by intra-
peritoneal injection (45 mg/ kg) of STZ (Keocyt, France) in a standard
saline solution. The rats were considered diabetic if their blood glucose
values were above 250 mg/dL on the 3rd day of injection. The oral
gavage of compounds 9a- 9m and pioglitazone was started on the third
day of STZ injection and continued for ten days. Then the serum glucose
level was checked on day 0 (3rd day after STZ injects), day 1 (24 h after
the first gavage), day 4, day 7 and day 10 [85]. Experimental methods on
animals were performed under moral guidelines.
4.8. Predict toxicity
The number of aromatic interactions within the residues existing in
the PPAR-γ active site is a possible cause for the notably cytotoxic ac-
tivity of compound 9a to 9 m. Advances in bioinformatics and computer
sciences have made it possible to achieve a measured toxic potential
(TP). An available powerful program for predicting TP in terms of
metabolic or endocrine disruption, carcinogenicity and cardiotoxicity is
the Virtual ToxLabTM program [75]. This program calculates the
binding affinity (binding constant) and the toxic potential (TP) of each
molecule for 16 proteins, including four members of the cytochrome
P450 enzyme family (3A4, 2D6, 2C9, and 1A2), ten receptors (miner-
alocorticoid, Peroxisome proliferator-activated receptor c (PPARc),
4.9.2.2. Histopathological examination and scoring. After completing of
the treatment period (i.e., on the 10th day), four groups (healthy,
diabetic-control, pioglitazone, and 9e) were decapitated. Sections of
liver and pancreas tissues were placed in 10% formalin solution for
24–48 h. Paraffin-embedded 5 mm-thick specimens were stained using
eosin and hematoxylin and were then observed under an optical mi-
croscope (Nikon Eclipse E200-LED, Nikon, Japan) by a pathologist who
was blind to the study. The specimens were scored from 0 to 3 based on
the following histopathologic changes: inflammation, fibrosis, necrosis,
steatosis and cholestasis (0: Normal, 1: mild, 2: Moderate, 3: Severe)
[86].
thyroid β, thyroid
α, liver X, progesterone, estrogen β, estrogen α,
androgen, and glucocorticoid), one potassium ion channel (hERG) and
one transcription factor (aryl hydrocarbon receptor) [75–79].
4.9. The biological assay
4.10. Glucose consumption
4.9.1. Cell survival experiment
NIH/3T3 cells, mouse embryonic fibroblast cells (Pasteur Institute,
Iran) were kept in RPMI-1640 medium with 10% fetal bovine serum
(FBS) (USA), streptomycin- penicillin 100 U/ml (USA) at 37 ◦C in a
humidified atmosphere of 5% CO2. All other reagents were purchased
from commercially available sources. The final concentrations of test
compounds at the wells were 1.25, 2.5, 5, and 10 mM. AlamarBlue®
(resazurin) is a non-fluorescent active blue ingredient reduced in live
cells to a red color with a high fluorescence [80]. About 104 cells were
seeded in 96-well plates and were exposed to different concentrations of
each synthesized analog. For each study regarding time and concen-
tration, there was a control sample that was kept untreated and received
an equal volume of DMSO as a blank control group. Also, there was a
positive control sample at each the test that received doxorubicin (1.25,
2.5, 5 µg/ml). Pioglitazone was employed as a control group throughout
the experiment. After 24 h, and the addition of AlamarBlue® to each
well according to the manufacturer’s instructions, an ELISA microplate
reader measured the absorbance value (A) of each well at the wave-
lengths of 570 nm, and 600 nm and ultimately cell survival was deter-
mined [81]. Each experiment was repeated in triplicate and was
repeated three times.
The HepG2 cells liver hepatocellular carcinoma cells, (Institut Pas-
teur, Iran) were grown in a low glucose medium (i.e., DMEM, Institut
Pasteur, Iran) containing 2∙7*10ꢀ 8 M of insulin and 10% fetal bovine
serum. Initially, cells were placed in 96-well plates and by the time they
reached the junction, the medium was removed. Then, the medium was
completed using 0.2% bovine serum albumin or BSA (Sigma, USA) and
the addition of glucose of different concentrations (5.5, 11, and 22 mM).
After 12 h, the medium of all wells was replaced with the same BSA and
DMEM containing pioglitazone and 9e. Finally, the medium was
removed and glucose concentrations were calculated by the glucose
oxidase method [87–89]. Each experiment was repeated in triplicate
and was repeated twice.
4.11. Predicting ADME properties and drug-likeness
The profile of ADME will have a significant influence on the chances
of success of a drug. ADME properties are most commonly based on
experiential strategies such as QSAR. These methods are highly efficient
and applicable for many molecules; they require high-quality data to
elicit a relationship between structure and activity. In this study, pre-
diction of ADME properties and Drug likeness was calculated using a
free online program, SwissADME (http://www.swissadme.ch/index.
php) for the standard drug (pioglitazone) and thiazolidinedione deriv-
ative (9e). This prediction included water solubility, drug-likeness,
pharmacokinetics, physicochemical properties, lipophilicity, and me-
dicinal chemistry [90–93].
4.9.2. In vivo animal study
4.9.2.1. Blood glucose investigation studies. According to the reported
method, a diabetic model induced by streptozocin (STZ) was used to
create a state of diabetes [82–84]. The empirical study was carried out in
adult male Wistar rats aged six weeks with 190 to 240 (g). All procedures
on animals were approved by the Ethics Committee of Mashhad Uni-
versity of Medical Sciences on Animal Use, with number IR.MUMS.
14

N. Shakour et al.
Bioorganic Chemistry 115 (2021) 105162
[2] Z. Huiying, C. Guangying, Z. Shiyang, Design, synthesis and biological activity
evaluation of a new class of 2, 4-thiazolidinedione compounds as insulin
enhancers, J. Enzyme Inhib. Med. Chem. 34 (1) (2019) 981–989.
4.12. PPAR-γ gene expression study
4.12.1. Cell culture
[3] C. Kharbanda, M.S. Alam, H. Hamid, K. Javed, A. Dhulap, S. Bano, Y. Ali,
Antidiabetic effect of novel benzenesulfonylureas as PPAR-γ agonists and their
anticancer effect, Bioorg. Med. Chem. Lett. 25 (20) (2015) 4601–4605.
[4] M. Spanou, K. Tziomalos, Bariatric surgery as a treatment option in patients with
type 2 diabetes mellitus, World J. Diabet. 4 (2) (2013) 14.
Preadipocytes (3T3-L1) cells were allowed to grow in DMEM
comprising 10% calf serum (USA) until approximate full confluence
during which preadipocytes differentiated to adipocytes spontaneously
with large triglyceride droplets visible under microscope [94]. 3T3-L1
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centrifuge tubes with a 1.5 ml volume. Total RNA separation was done
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Declaration of Competing Interest
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The authors declare that they have no known competing financial
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This work was supported by grant no 960816 from Mashhad Uni-
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