Synthesis, docking, and evaluation of novel thiazoles for potent antidiabetic activity

Article abstract of DOI:10.1007/s00044-017-1851-8

In this study, a series of novel substituted pyrazoles containing indole and thiazole motifs were synthesized and evaluated for their antihyperglycemic activity against α-amylase and α-glucosidase enzymes. Among them, 2-(5-(1H-indol-3-yl)-3-phenyl-1H-pyra

Full text of DOI:10.1007/s00044-017-1851-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1851-8
ORIGINAL RESEARCH
Synthesis, docking, and evaluation of novel thiazoles for potent
antidiabetic activity
1 ●
2 ●
3 ●
4 ●
2 ●
T. V. Sravanthi S. Sajitha Lulu S. Vino M. A. Jayasri A. Mohanapriya
S. L. Manju
5
Received: 17 December 2015 / Accepted: 2 March 2017
©
Springer Science+Business Media New York 2017
Abstract In this study, a series of novel substituted pyr-
azoles containing indole and thiazole motifs were synthe-
sized and evaluated for their antihyperglycemic activity
against α-amylase and α-glucosidase enzymes. Among
them, 2-(5-(1H-indol-3-yl)-3-phenyl-1H-pyrazol-1-yl)-4-(4-
bromo phenyl) thiazole (3f) was identified as the best
antihyperglycemic activity (IC50 = 236.1 µg/mL) in com-
parison with the standard drug (acarbose, IC50 = 171.8
µg/mL). Through reverse screening, glucocorticoid receptor
exploring heteroaryl substituted pyrazoles as potent ther-
apeutic antidiabetic agents.
●
Keywords Heteroaryl substituted pyrazoles Antidiabetic
●
●
activity Molecular docking Molecular dynamics
simulation
(
1NHZ) was identified as the best target and molecular
docking studies was performed to understand the interaction
of the molecules to the active sites of 1NHZ. The docking
study was then conducted against the enzymes (α-amylase
Introduction
Defects in insulin function and secretion are the major
considerations of diabetes mellitus (Matschinsky 1996). α-
Amylase and α-glucosidase are the digestive enzymes
hydrolyzing the larger carbohydrates of food taken into
absorbable glucose (Bhandari et al. 2008). Hence, α-amy-
lase and α-glucosidase inhibitors control the blood glucose
levels and which they can be used as oral anti-diabetic
drugs. Various synthetic antidiabetic agents used for the
sharp reduction of blood glucose levels, however, they have
been limited due to their side effects such as nausea, renal
disorders, gastrointestinal defects, vomiting, and diarrhea
(
4X9Y) and α-glucosidase (2QMJ)) used in the in vitro
studies. A molecular dynamic simulation experiment was
conducted to analyze the behavior of the docked complex.
The analysis confirmed the stability of the docked complex
in terms of energy and hydrogen bonds. In vitro and in
silico evaluations thus generate an interesting insight toward
*
S. L. Manju
(Hanefeld 1998; Israili 2011). Thus, researchers have
slmanju@vit.ac.in
focused on the development of new therapeutic agents for
diabetes with minimal side effects.
1
2
3
4
5
Department of Science & Humanities, Kingston Engineering
College, Vellore, Tamil Nadu, India
Nitrogen and sulfur containing heterocycles play a cri-
tical role in medicinal chemistry and in the development of
potent drug molecules (Valverde and Torroba 2005).
Among them, pyrazoles, indoles, and thiazoles have been
well documented in medicinal chemistry with respect to
their promising biological activities such as anti-inflamma-
tory, anticonvulsant, antiviral, antimalarial antitubercular,
antioxidant, and antitumor activities (Kucukguzelm and
Senkardes 2014; Nagendra Kumar et al. 2013; Shiv Jee
Bioinformatics Division, VIT University, Vellore 632014 Tamil
Nadu, India
Medical Biotechnology Division, VIT University, Vellore 632014
Tamil Nadu, India
Biomolecules and Genetics Division, School of Bio Sciences and
Technology, VIT University, Vellore 632014 Tamil Nadu, India
Organic Chemistry Division, School of Advanced Sciences, VIT
University, Vellore 632014 Tamil Nadu, India

Med Chem Res
et al. 2012). The use of substituted pyrazoles in treating
diabetes mellitus has been reported since 1983 (Soliman
et al. 1983). Pyrazoles are generally synthesized from the
reaction of 1,3-diketones or α,β-unsaturated ketones with
hydrazines (Ryo et al. 2014; Ajay Kumar and Jayaroopa
thiosemicarbazone
(2,
indol-3-yl-(1-phenylallylidene)
hydrazinecarbothioamide). Further, the study demonstrates
here the novel in situ method for the heterocyclization of the
thiosemicarbazone into a thiazole and pyrazole rings upon
treatment with phenacyl bromide in a one pot method. The
generality of this approach has been examined by synthe-
sizing six more targets.
2
013). Remogliflozin etabonate containing pyrazole moiety
has been found to reduce the blood glycemic levels, is now
under clinical trials (Fujimori et al. 2008). Pioglitazone,
containing thiazolidinedione moiety is identified as one of
the best antidiabetic drug. Recently, Navarrete-Vazquez
et al. (2014) explored sterically bounded arylsulfonami-
dothiazoles as antidiabetic agents. Also, 1-(4-chlor-
obenzoyl)-5-hydroxy-2-methyl-3-indoleacetic acid derived
from anti-inflammatory drug (indomethacin) has been
identified as potential insulin sensitizing and glucose low-
ering agent (Li et al. 2007). These directed us to explore the
antihyperglycemic activity of substituted pyrazoles con-
taining thiazole and indole nuclei.
Spectral characterization
All the compounds were purified by column chromato-
graphy (petroleum ether and ethyl acetate) and characterized
by spectral analyses such as Fourier transform infrared
spectroscopy (FTIR), nuclear magnetic resonance (NMR),
and mass spectra. The FTIR spectrum of compound (2)
shown characteristic stretching bands for C=N at
−1
−1
1641 cm , C=S at 1220 cm , broad bands of primary
–NH , secondary –NH, and indole NH at 3172.90–3471.84
2
1
−
cm . The appearance of the singlet –NH proton peak at
δ11.194 ppm, singlet indole –NH proton peak at δ11.613
ppm and the olefinic =CH-indole proton peaks as doublet
Results and discussion
Synthesis
1
in the range of δ8.22–8.241 ppm in H NMR spectrum and
the molecular ion peak at 319.8 in the mass spectrum
confirmed the formation of the respective thiosemicarba-
zone. The reaction was exemplified below by describing the
methodology and structural elucidation of compound (3a).
Acetophenone and thiosemicarbazone were dissolved in
The synthesis of the targets has been achieved in three steps
as shown in Scheme 1. Indole-3-carbaldehyde was con-
densed with acetophenone in basic condition to afford the
chalcone (1, 3-(1H-indol-3-yl)-1-phenylprop-2-en-1-one)
methanol,
and
into
this
mixture,
potassium
(
Schmidt 1880; Claisen and Claparede 1881; Kohler and
bromide–bromate mixture (2:1) was added, followed by
drop-wise addition of dilute hydrochloric acid. The addition
of hydrochloric acid to the bromide–bromate mixture
evolves bromine gas (Adimurthy et al. 2008), which
Chadwell 1922). 3-(1H-Indol-3-yl)-1-phenylprop-2-en-1-
one (1) on further treatment with thiosemicarbazide in the
presence of glacial acetic acid yielded the corresponding
Scheme 1 Reagents and
S
NH2
N
conditions: (i) NaOH, ethanol,
RT, Stir, overnight, neutralize
O
H
HN
O
o
O
i
with dil. HCl at 0 C; (ii)
ii
Thiosemicarbazide, ethanol,
glac. AcOH, reflux, 3 h; (iii)
ArCOCH3, KBr-KBrO3, dil.
HCl, RT, 2 h; (iv) Reflux,
overnight
N
H
N
H
N
H
1
2
iii, iV
Ar
N
N
S
N
N
H
(3a-g)
OH
Ar =
3a)
3b)
3c)
3d)
3e)
3f)
3g)
Cl
CH3
OCH3
F
Br

Med Chem Res
underwent electrophilic substitution at the α-methyl carbon
of the acetophenones as well as the addition reaction over
the enone system of the thiosemicarbazone. After the bro-
mine color disappeared, the resultant mixture was refluxed
for 8 h. The resultant phenacyl bromide readily underwent
thiazole cyclization with thioamide part of thiosemicarba-
zone followed by the intramolecular pyrazolization resulted
in the formation of heteroaryl substituted pyrazoles (3a).
The absence of the –C=S band in FTIR spectrum of
Molecular docking
We carried out molecular docking studies of the seven
synthesized compounds (3a–g) to the active sites of gluco-
corticoid receptor (1NHZ) identified as the target through
reverse screening of the compounds using PharmMapper
(Liu et al. 2010). The study has been done to understand the
nature of interactions between the compounds and the active
site amino acids of the targets using the docking program
AutoDock 4.2 (Morris et al. 2009). The PDB structure
1NHZ (resolution 2.3 Å) was used as a receptor for docking
with the molecules. Initially, the bounded water molecules
and hetero atoms of the target were removed checked the
molecule for polar hydrogens and their torsion bonds were
selected. Gasteiger charges were computed and Lamarckian
Genetic Algorithm was employed as the search algorithm in
AutoDock 4.2. The three dimensional grid boxes were cre-
ated by an autogrid algorithm to evaluate the binding ener-
gies on the protein coordinates. Finally, AutoDock was used
to calculate the binding free energy of a compound on
binding with the target. The search was extended over the
whole receptor protein as blind docking. The binding mode
of the best binding compound (3f) to 1NHZ protein and the
interactions are shown in (Fig. 1a). The 2D plot of the best
binding complex of compound (3f) to 1NHZ protein to
identify the active site residues is shown in (Fig. 1b).
1
compound (3a) confirmed the heterocyclization. In the H
NMR spectrum of compound (3a), showed a singlet peak at
δ11.976 ppm corresponding to indole –NH, the singlet peak
at δ7.273 ppm corresponding to the pyrazole –CH proton.
The disappearance of secondary –NH proton peak at
δ11.194 ppm and from the proton count, the formation of
1
3
thiazole ring and the pyrazole ring were confirmed. In
C
NMR spectrum of compound (3a), we have noticed the shift
of the enone carbon peak from δ140.83 to 131.10 ppm and
disappearance of C=S carbon peak. Thus, we have con-
firmed the formation of compound (3a, 2-(5-(1H-indol-3-
yl)-3-phenyl-1H-pyrazol-1-yl)-4-phenylthiazole) from their
spectral analyses. The feasibility of the reaction was
checked by carried out the reaction with other substituted
acetophenones on reaction with thiosemicarbazone, affor-
ded the compounds (3b–g) in good yield.
1
In the H NMR spectra of all the compounds (3b–g), we
observed the same kind of cyclization as evidenced by the
absence of the peak at δ11.194 ppm corresponding to the
aliphatic secondary –NH proton peak of thiosemicarbazone
The indole ring of the compound (3f) was found to be
highly interacting with LEU 732 residue at the bond dis-
tances of 2.4 Å. Table 1 shows the binding energy of the
seven compounds with the active site of 1NHZ. (Fig. 2)
(2). The indole –NH proton peak in the range of
δ11.603–12.112 ppm and pyrazole –CH proton signal at
1
3
δ7.211 to 7.250 ppm were observed. Their C NMR spectra
also displayed the peak at the range of δ130.90–132.07 ppm
corresponding to pyrazole ring carbon attached to indole and
the corresponding aromatic carbon signals.
Molecular dynamic simulation
To understand the conformational variation of the protein
and protein docked complex, the root mean square
Fig. 1 a Interaction of compound (3f) through the active sites of 1NHZ. Red color represents interacting residue and Green dotted lines represents
hydrogen bonds. b 2D plot of the complex shown the interaction of compound (3f) with 1NHZ (color figure online)

Med Chem Res
deviation (RMSD) of backbone after 5 ns simulation with
respect to the structure and structural complex was calcu-
lated (Fig. 3a). The protein and the complex attained stable
conformation in 5 ns. The final RMSD value of protein after
attained stability was about 0.34 nm. Plot of total energy,
hydrogen bonds, root mean square fluctuations (RMSF) in
Fig. 3b indicates that the protein was stable during the
simulation period of 5 ns. It suggests that variation in
RMSD for the optimization of the interaction between the
residues and the overall stability of complex were not
compromised during the simulation. (Fig. 4)
In vitro anti-diabetic study
The synthesized substituted pyrazoles (3a–f) were evaluated
for in vitro antihyperglycemic activity study against α-
amylase and α-glucosidase enzymes (Miller 1959; Matsui
et al. 2007). Acarbose, the most widely used standard drug
for antihyperglycemic activity study (Michael and Fowler
2007). The % inhibition results are represented in (Tables 2
and 3). It was found that all the compounds were shown the
gradual increase of inhibition as the concentration of the
sample increased. From the results, it was found compounds
3e, 3f, and 3g bearing fluoro, bromo, and hydroxyl sub-
stituent on the phenyl ring of the thiazole moiety have
shown maximum inhibition. However, compound (3a)
without any substituent has shown better inhibition than
with methyl and methoxy substitutions. The chloro sub-
stituted compound has shown least inhibition due to its
strong electron-withdrawing character. 2-(5-(1H-Indol-3-
yl)-3-phenyl-1H-pyrazol-1-yl)-4-(4-bromo phenyl) thiazole
(
3f) has shown higher inhibition of 76% at 500 μg/mL,
while compared with the positive control (acarbose) shown
2.08% at 500 μg/mL. It was also observed that all com-
7
pounds were shown significant inhibition above the con-
centration of 300 μg/mL. The IC50 value of compound 3f
was found to be 236.1 μg/mL, whereas it was 171.8 μg/mL
for acarbose.
Experimental
Materials and methods
Melting points of the compounds were determined in open
capillary tube method and were uncorrected. Acet-
ophenones and thiosemicarbazide from Sigma-Aldrich,
indole-3-carbaldehyde from AVRA chemicals and solvents
from SD fine chemicals Limited were purchased and used
without further purification. Enzymes, substrate and acar-
bose used in the biological assay were purchased from
Sigma-Aldrich. Mass spectra were recorded on the ESI
THERMO FLEET spectrophotometer and GC-MS

Med Chem Res
Fig. 2 Interaction of compound (3f) through the active sites of 4X9Y and 2QMJ enzymes. Golden color represents interacting residue and blue
dotted lines represents hydrogen bonds (color figure online)
spectrophotometer of GC model Clarus 680 and MS model
Clarus 600 (EI) by used helium as a carrier gas with qua-
litative results. The FTIR spectra were recorded on a Perkin-
Elmer FTIR spectrophotometer using the KBr pellet tech-
nique. NMR spectra were recorded on a Bruker 400 and
129.64, 131.98, 136.77, 140.12, 189.43; GCMS (m/z):
247.1429 [MH +1].
+
Synthesis of indol-3-yl-(1-phenylallylidene)
hydrazinecarbothioamide (2)
1
00 MHz FT-NMR spectrometer. Chemical shifts in NMR
spectra are reported in parts per million (ppm) from tri-
An ethanolic mixture of chalcone (1, 10 mmol) and thio-
semicarbazide (10 mmol) in the presence of 10 drops of
glacial acetic acid was refluxed for 3 h. The reaction mixture
was cooled to room temperature and removed the solvent
using suction pump. The product was washed with 10%
sodium bicarbonate solution and petroleum ether, dried
followed by column purification.
methylsilane (an internal standard) used CDCl /DMSO-d6
3
as a solvent. Splitting patterns are designated as follows: s:
singlet, d: doublet, t: triplet, q: quartet and m: multiplet. The
reaction was monitored by thin layer chromatography on
silica gel protected on aluminium sheets and the compound
spots were detected by exposure to UV-lamp at 256 nm.
The purification of the compounds was done by silica gel
column chromatography using petroleum ether and ethyl
acetate.
−1
Yield: 85%; mp: 168–170 °C; FTIR (KBr, υ cm ):
3415.93 (–NH ), 3172.90 (–NH), 2931.80 (Ar–CH),
2
1641.42 (–C=N), 1631.78 (–CH=CH–), 1577.71
1
(
–C=C–), 1220.94 (–C=S); H NMR (400 MHz, DMSO-
Synthesis of 3-(1H-indol-3-yl)-1-phenylprop-2-en-1-one(1)
d
7
C
6
, δ ppm): 7.120 (m, 3H, Ar–H), 7.356 (m, 3H, Ar–H),
.816 (d, 1H, J = 8 Hz, –CO–CH=), 8.034 (s, 1 H, indole
–H), 8.222 (d, 1H, J = 7.6 Hz,=CH–indole), 8.317 (s,
To an ethanolic mixture of acetophenone (10 mmol) and
indole-3-carbaldehyde (10 mmol), 10 mL of 10% NaOH
was added drop-wise at 0–10 °C and the resultant reaction
mixture was stirred for 10 h. The resultant orange color
reaction mixture was then neutralized with dilute hydro-
chloric acid, obtained the yellow precipitate. The precipitate
was filtered, washed with petroleum ether and dried under
vacuum. The product was recrystallized from ethanol.
Yield: 65%; Yellow solid; mp: 178–182 °C; FTIR (KBr,
2
1
3
1H), 11.194 (s, 1H, –NH), 11.613 (s, 1H, Indole–NH);
NMR (100 MHz, DMSO-d , δ ppm): 111.11, 111.74,
120.60, 122.04, 122.61, 123.97, 125.90, 126.55, 128.16,
128.21, 128.40, 130.90, 137.03, 140.83, 157.26 (–C=N),
C
6
+
176.56 (–C=S); LC–MS (m/z): 319.8 [MH ].
Synthesis of 2-(5-(1H-indol-3-yl)-3-phenyl-1H-pyrazol-1-
yl)-4-substituted phenylthiazoles (3a–g)
−
1
υ cm ): 3167.12 (–NH), 3043.67 (aromatic –CH), 1741.72
1
(
–C=O), 1635.64 (–CH=CH–), 1577.77 (–C=C–);
H
To the methanolic mixture of acetophenone (1 mmole) and
thiosemicarbazone (2, 1 mmole), 400 mg of bromide-
bromate mixture (2:1) was added and followed by drop-
wise addition of the dilute hydrochloric acid. The reaction
mixture was continued to stir at room temperature till the
evolved bromine gas color disappears and the brominated
product separated out. Then, the reaction mixture was
NMR (400 MHz, CDCl , δ ppm): 7.203 (m, 7H, Ar–H),
3
7
–
.754 (s, 1H, indole C2–H), 7.952 (1H, d, J = 3.2 Hz,
CO–CH=), 8.174 (d, 3H, J = 8.04 Hz, Ar–H and =CH-
1
3
indole), 11.859 (s, 1H, indole –NH); C NMR (100 MHz,
CDCl , δ ppm): 112.11, 112.50, 113.56, 116.00, 118.86,
3
1
20.33, 121.42, 122.45, 123.05, 123.75, 124.50, 128.69,

Med Chem Res
Fig. 3 a Backbone RMSD of 1NHZ (red) and docked complex 3f-
nhz (black) during 5 ns of molecular dynamic simulation (MDS).
b RMSF of Cα atoms as a function of amino acids for 1NHZ (red) and
3f-1NHZ (black) during 5 ns MDS. c Total energy of 1NHZ (red) and
docked complex (3f–1NHZ, black). d Hydrogen bonds (color figure
online)
1
refluxed for 8 h. Then the reaction mixture was poured into
ice cold water, filtered and dried. The crude product was
column purified (70:30 v/v, petroleum ether and ethyl
acetate) and confirmed the compound (3a) by its spectral
characterization. The same procedure was followed for the
reaction with different substituted acetophenones.
DMSO-d , δ ppm): 111.06, 111.46, 112.51, 114.15, 114.96,
6
116.79, 117.31, 117.85, 119.31, 120.37, 120.47, 121.39,
122.00, 122.70, 123.48, 124.31, 127.66, 128.60, 130.47,
130.90, 135.55, 135.63, 136.25, 136.45, 136.80, 137.17,
+
139.02, 147.82; LCMS (m/z): 492.9 [(MH +K)].
2-(5-(1H-indol-3-yl)-3-phenyl-1H-pyrazol-1-yl)-4-(4-
2
-(5-(1H-indol-3-yl)-3-phenyl-1H-pyrazol-1-yl)-4-phe-
methyl phenyl)thiazole (3c): Yield: 38%; mp: 128–130 °C;
1
nylthiazole (3a): Yield: 42%; mp: 126 °C (132 °C charred);
H NMR (400 MHz, DMSO-d , δ ppm): 7.006 (m, 3H,
Ar–H), 7.229 (m, 5H, Ar–H and pyrazole –CH), 7.517 (d,
H, J = 7.66 Hz, Ar–H), 8.098 (d, 3H, J = 7.49 Hz, Ar–H),
.295 (s, 2H, thiazole C5-H and indole C2-H), 11.976 (s,
H, indole –NH); C NMR (100 MHz, DMSO-d , δ ppm):
12.39, 114.80, 116.09, 116.51, 118.16, 120.79, 121.13,
21.55, 121.70, 122.09, 122.72, 123.43, 124.11, 126.10,
28.08, 128.66, 128.83, 131.10, 135.37, 137.04, 138.37,
H NMR (400 MHz, CDCl , δ ppm): 2.099 (s, 3H, –CH ),
3
3
1
7.206 (m, 5H, Ar–H and pyrazole –CH), 7.418 (d, 1H, J =
Hz, Ar–H), 7.487 (t, 2H, J = 7.10 Hz, Ar–H), 7.660 (d, 1H,
J = 2.73 Hz, Ar–H), 7.829 (d, 1H, J = 2.73 Hz, Ar–H),
6
4
8
1
1
1
1
1
7.978 (m, 3H, Ar–H), 8.358 (d, 1H, J = 7.74 Hz, Ar–H);
1
3
13
C NMR (100 MHz, CDCl , δ ppm): 22.73, 111.16,
6
3
111.80, 111.86, 112.15, 112.30, 117.64, 120.67, 120.99,
121.38, 121.78, 121.82, 122.05, 123.43, 123.52, 124.28,
126.14, 126.58, 128.39, 128.62, 130.87, 132.39, 139.43,
+
+
49.69, 153.85; LCMS (m/z): 440.3 [(MH +Na)].
153.75; ESI-mass (m/z): 434.47 [MH +2].
2
-(5-(1H-indol-3-yl)-3-phenyl-1H-pyrazol-1-yl)-4-(4-
2-(5-(1H-indol-3-yl)-3-phenyl-1H-pyrazol-1-yl)-4-(4-me
chloro phenyl)thiazole (3b): Yield: 45%; mp: 112–116 °C;
thoxy phenyl)thiazole (3d): Yield: 44%; mp: 140–142 °C;
1
1
H NMR (400 MHz, DMSO-d , δ ppm): 7.029 (t, 1H, J =
H NMR (400 MHz, DMSO-d , δ ppm): 3.846 (s, 3H,
6
6
1
2
4
4.60 Hz, Ar–H), 7.232 (s, 1H, pyrazole –CH), 7.614 (d,
H, J = 7.64 Hz, Ar–H), 7.754 (m, 4H, Ar–H), 7.954 (m,
H, Ar–H), 8.145 (d, 1H, J = 2.8 Hz, Ar–H), 8.344 (d, 1H,
–OCH ), 6.910 (d, 3H, J = 8.8 Hz, Ar–H), 7.220 (m, 4H,
3
Ar–H and pyrazole –CH), 7.462 (d, 2H, J = 6.88 Hz,
Ar–H), 7.877 (m, 5H, Ar–H), 8.223 (d, 2H, J = 7.42,
1
3
J = 2.8 Hz, Ar–H), 8.488 (s, 2H, thiazole C5-H and indole
C2-H), 11.872 (s, 1H, indole –NH); C NMR (100 MHz,
Ar–H), 11.603 (s, 1H, indole –NH); C NMR (100 MHz,
1
3
DMSO-d , δ ppm): 54.45, 111.24, 111.52, 112.46, 112.74,
6

Med Chem Res
Fig. 4 Graphical representation of inhibitory effect of the compounds (3a–g) against amylase and α-glucosidase enzymes
1
1
1
15.22, 117.76, 117.80, 119.28, 120.22, 120.41, 121.38,
21.86, 122.71, 123.49, 124.41, 129.14, 129.38, 129.50,
128.22, 129.20, 131.11, 132.90, 132.95, 133.02, 135.43,
137.08, 138.39, 142.57, 149.71, 153.72, 154.98, 162.87;
ESI-mass (m/z): [MH ].
+
30.48, 130.76, 136.03, 136.36, 136.83, 137.65, 162.44;
+
LCMS (m/z): 469.2 [(MH +Na)].
2
-(5-(1H-indol-3-yl)-3-phenyl-1H-pyrazol-1-yl)-4-(4-
Biological assay
fluoro phenyl)thiazole (3e): Yield: 39%; mp: 126–128 °C;
1
H NMR (400 MHz, CDCl , δ ppm): 7.312 (d, 2H, J = 2.82
α-Amylase inhibitory assay
3
Hz, Ar–H), 7.425 (m, 2H, Ar–H and pyrazole –CH), 7.501
(
m, 3H, Ar–H), 7.565 (m, 4H, Ar–H), 8.012 (m, 5H, Ar–H);
% Inhibition of α-amylase was determined by a reporting
method (Miller 1959; McCue and Shetty 2004). Six dif-
ferent concentrations (50, 100, 200, 300, 400, and 500
μg/mL) of the test samples (3a–g) were prepared upto 1 mL
using methanol as a solvent. Starch solution (1%) and α-
amylase enzyme (0.5 mg/mL) were prepared using freshly
prepared phosphate buffer (pH = 6.9). Dinitrosalicyclic acid
(DNSA) was used as a substrate. In test tubes, 100 μL starch
solution was suspended with 100 μL of sample solution and
incubated at 25 °C for 10 min. After incubation, 100 μL of
α-amylase enzyme solution was added and incubated the
reaction mixture at 25 °C for 10 min. Finally, 200 μL of
DNSA color reagent was added to stop the reaction and
incubated in a boiling water bath for 5 min. The reaction
mixtures in the test tubes were cooled and diluted with
distilled water to make the whole mixture upto 1 mL. The
test tube with methanol instead of the sample was used as a
control. The absorbance of the resulting mixture was
recorded at 540 nm using a UV-Vis spectrophotometer. The
% inhibition of α-amylase enzyme was calculated using the
following formulae:
1
3
C NMR (100 MHz, CDCl , δ ppm): 111.70, 112.09,
3
1
1
1
1
13.19, 114.45, 117.85, 120.69, 121.80, 121.90, 123.03,
23.55, 124.38, 125.40, 126.28, 127.11, 128.22, 128.37,
28.59, 130.56, 132.34, 133.09, 135.39, 135.80, 137.37,
+
39.05, 139.21, 191.27; ESI-MS (m/z): 437.07 [MH +1].
2
-(5-(1H-indol-3-yl)-3-phenyl-1H-pyrazol-1-yl)-4-(4-
bromo phenyl)thiazole (3f): Yield: 48%; mp: 98–102 °C
1
(
122 °C charred); H NMR (400 MHz, DMSO-d , δ ppm):
6
7
.055 (d, 2H, J = 18 Hz, Ar–H), 7.222 (m, 10H, Ar–H and
pyrazole –CH), 7.631 (m, 3H, Ar–H), 7.896 (d, 1H, J = 7.6
Hz, Ar–H), 11.137 (s, 1H, indole –NH); C NMR (100
MHz, DMSO-d , δ ppm): 112.48, 113.10, 117.97, 119.34,
1
1
1
1
3
6
20.27, 120.89, 121.47, 123.06, 125.16, 127.23, 128.95,
30.72, 131.48, 132.07, 133.18, 136.54, 137.83, 141.86,
+
42.09; ESI mass (m/z): 497.40 [MH ].
2
-(5-(1H-indol-3-yl)-3-phenyl-1H-pyrazol-1-yl)-4-(2-
1
hydroxy phenyl)thiazole (3g): Yield: 42%; mp: 132 °C; H
NMR (400 MHz, DMSO-d , δ ppm): 5.460 (s, 1H, –OH),
6
6
.430 (t, 1H, J = 14 Hz, Ar–H), 6.757 (d, 1H, J = 7.62 Hz,
Ar–H), 7.029 (m, 2H, Ar–H), 7.215 (m, 6H, Ar–H and
pyrazole –CH), 7.531 (d, 2H, J = 8.20 Hz, Ar–H), 8.024 (m,
ÀÀ
Á
Á
1
3
4
H, Ar–H), 12.112 (s, 1H, indole –NH); C NMR (100
% Amylase inhibition¼ Acontrol ꢀ Asample =Acontrol ꢁ 100
MHz, DMSO-d , δ ppm): 112.41, 114.79, 116.09, 116.52,
6
1
18.16, 120.82, 122.07, 123.40, 124.13, 126.07, 127.00,
where Acontrol was the absorbance of blank at 540 nm;

Med Chem Res
Asample was the absorbance of sample at 540 nm. Triplicates
were done for each sample of every concentration.
α-Glucosidase inhibitory assay
%
Inhibition of α-glucosidase was determined by Matsui
et al. method (Matsui et al. 2007). The assay was carried out
in 96 microwell plate. α-Glucosidase enzyme (1 unit/mL)
and the substrate solution of 5 mM p-nitrophenylglucopyr-
anoside (p-NPG) were prepared using phosphate buffer
(pH = 6.9). 100 μL of α-glucosidase (1.0 U/mL) was pre-
incubated with 50 μL of the different concentrations of the
test samples for 10 min. Then 50 μL of 5 mM p-NPG was
added to start the reaction. The reaction mixture was incu-
bated at 37°C for 30 min. The % inhibition of α-glucosidase
was determined by measuring the amount of yellow-colored
paranitrophenol released from p-NPG at 405 nm using UV-
Vis spectrophotometer. Phosphate buffer was used as a
blank and methanol without sample taken as control. Tri-
plicates were done for each sample of every concentration.
%
Glucosidase inhibition was calculated as follows:
%
Glucosidase inhibition ¼
ððControl A405 ꢀ Corrected sample A405Þ=Control A405Þꢁ100
where, control A405 was the absorbance of the control at
405 nm; corrected sample A405 was the corrected absor-
bance of the sample at 405 nm.
To eliminate the background readings, sum of the
absorbance of the samples with enzyme and substrate
mixture were subtracted from the absorbance of samples
alone as follows:
Corrected A405 of sample ¼
A405 of the sample; enzyme and substrate mixture
ꢀ
A405 of sample alone
Molecular docking
Molecular docking is widely used to predict the binding
affinities of ligands with targets. In our current work,
molecular structures of the synthesized compounds were
built by using Avogadro (Marcus Hanwell et al. 2012). The
targets were identified through reverse screening approach
through PharmMapper server (Liu et al. 2010). The Glu-
cocorticoid receptor (1NHZ) regulating gene expression in a
ligand-dependent fashion and also reveals the highest hit
among the other transcription factors. Glucocorticoid
receptor (1NHZ) was docked with the synthesized com-
pounds (3a–g) to understand the mode of interaction of the
small molecules against the macromolecular target. Blind
docking was performed using Autodock 4.2 (Morris et al.

Med Chem Res
Table 3 % of α-glucosidase inhibitory assay
Compound
50 μg/mL
100 μg/mL
200 μg/mL
300 μg/mL
400 μg/mL
500 μg/mL
IC50 (μg/mL)
3
3
3
3
3
3
3
a
b
c
d
e
f
34.78 ± 0.01
28.62 ± 0.03
21.38 ± 0.02
14.49 ± 0.01
32.25 ± 0.03
11.60 ± 0.03
9.06 ± 0.05
36.96 ± 0.02
32.25 ± 0.04
34.78 ± 0.02
–
46.74 ± 0.01
40.95 ± 0.09
36.96 ± 0.04
22.10 ± 0.02
48.19 ± 0.04
50.36 ± 0.03
42.39 ± 0.05
48.56 ± 0.02
–
53.62 ± 0.11
59.78 ± 0.03
46.01 ± 0.05
75.73 ± 0.02
68.84 ± 0.04
63.04 ± 0.15
–
56.16 ± 0.03
61.01 ± 0.06
59.78 ± 0.04
76.09 ± 0.02
83.70 ± 0.05
89.13 ± 0.03
76.09 ± 0.05
–
–
37.69 ± 0.10
44.57 ± 0.01
48.55 ± 0.05
54.35 ± 0.05
60.51 ± 0.06
–
318.5
308.2
190.9
240.8
36.23 ± 0.01
47.10 ± 0.06
23.19 ± 0.03
g
–
not found
2
009). To examine the best conformation space of the
2.4 Å through hydrogen bonding with lowest binding
energy of −11.27 kcal/mol. The docking results have
shown the strong correlation with the in vitro anti-
hyperglycaemic assay results. Compounds (3e, 3f, and 3g)
could be considered as promising scaffolds towards the
development of novel antidiabetic agents.
ligand, the Lamarckian Genetic Algorithm was employed
Lydia and Sudarsanam 2014) and the other parameters
(
were kept as default. The result was quantified based on
lowest binding energy conformation.
Molecular dynamic simulations (MDS)
Acknowledgements T V Sravanthi thanks VIT University, Vellore,
Tamil Nadu, India, for providing Research Associateship. The VIT
DST-FIST for NMR and GC-MS facilities and VIT-TBI for FT-IR at
VIT University are greatly acknowledged.
The ligand coordinates of the best conformation were
prepared using PRODRG server. Simultaneously, the
protein complex was prepared using Gromacs 4.5.5
package (Hess et al. 1997). To validate the stability of
the protein trajectory and protein-ligand complex trajec-
tories were compared. MD simulation was carried out in
cubic water box using single point charge water mole-
cules. The simulation was carried out for 5000 ps. The
stability was validated by comparing the RMSD, RMSF
and total energy of the protein and protein–ligand com-
plexes. The hydrogen bonds formed between the protein
and the ligand was monitored using hydrogen bond
parameter available using Gromacs 4.5.5 package (Scott
et al. 1999).
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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