Novel benzoxazole derivatives featuring rhodanine and analogs as antihypergycemic agents: synthesis, molecular docking, and biological studies

Article abstract of DOI:10.1007/s00044-017-2097-1

A novel series of benzoxazolyl linked benzylidene based rhodanine and their cyclic analogs were synthesized, characterized and evaluated for their α-amyloglucosidase inhibitory activity. Out of eight target compounds, two compounds (4b and 5b) displayed potent inhibitory activity against α-amyloglucosidase with IC₅₀ values in the range of 0.24 ± 0.01–0.94 ± 0.01 μM as compared to standard drug acarbose. Among all the tested compounds, compound 5b containing rhodanine at 3-position of phenyl was found to be the most active inhibitor of α-amyloglucosidase. Docking studies showed the existence of potential H-bonding interactions between synthesized compounds and α-glucosidase which might be responsible for good biological activity.

Full text of DOI:10.1007/s00044-017-2097-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2097-1
ORIGINAL RESEARCH
Novel benzoxazole derivatives featuring rhodanine and analogs as
antihypergycemic agents: synthesis, molecular docking, and
biological studies
1 ●
1 ●
1 ●
1 ●
Varinder Singh Amanjot Singh Gagandeep Singh Raman K. Verma
Rajiv Mall
2
Received: 9 August 2017 / Accepted: 4 October 2017
©
Springer Science+Business Media, LLC 2017
Abstract A novel series of benzoxazolyl linked benzyli-
dene based rhodanine and their cyclic analogs were
synthesized, characterized and evaluated for their α-
amyloglucosidase inhibitory activity. Out of eight target
compounds, two compounds (4b and 5b) displayed potent
inhibitory activity against α-amyloglucosidase with IC50
values in the range of 0.24 ± 0.01–0.94 ± 0.01 µM as
compared to standard drug acarbose. Among all the tested
compounds, compound 5b containing rhodanine at 3-
position of phenyl was found to be the most active inhi-
bitor of α-amyloglucosidase. Docking studies showed the
existence of potential H-bonding interactions between
synthesized compounds and α-glucosidase which might be
responsible for good biological activity.
Introduction
Type I and type II diabetes mellitus develops due to post-
prandial hyperglycemia resulting from insulin resistance or
insulin secretion by β cells (Shaw et al. 2010). Stress on the
β cells to secrete insulin at level higher than they originally
designed for, is because of a high glycaemic loaded diet. In
this condition if blood glucose concentrations near the
normal range are not controlled properly, making diabetes
management and prevention a important health issue. The
number of diabetic patients including adults and teens that
have prediabetes is rapidly rising in United States and
especially in the developing countries such as India, Thai-
land and Indonesia. There is a need to develop new ther-
apeutic strategies for diabetes management. Out of various
therapeutic approaches one is to reduce glucose absorption,
to control postprandial hyperglycemia, to relive stress on β
cells and to manage the both types of diabetes by α-amylase
and α-glycosidase inhibitors. In the digestion and absorp-
tion process of carbohydrates pancreatic α-amylase hydro-
lysis the starch (Gray 1975). Moreover, α-amylase as the
most abundant enzyme in the human saliva, it performs
several distinct functions in the oral cavity. Firstly it breaks
the carbohydrates in oral cavity and secondly it binds to
viridans streptococci in the oral cavity. Bacteria-bound α-
amylase hydrolyses the starch molecules. α-Glycosidase
enzyme play key role in intestinal carbohydrates digestion,
glycogen degradation, maturation and glycoprotein folding
(Ghani 2015). Inhibitors of these enzymes reduce starch
hydrolysis to simple sugars by slowing the action of these
enzymes (Notkins 2002). In the continuous effort to invent
new bioactive compounds (Verma et al. 2012a, b and
2013a, b, c), we reported the synthesis of novel benzoxazole
derivatives containing 2,4-thiazolidinedione (TZD), rhoda-
nine (RhD), hydantoin (HyD), thiohydantoin (THyD)
●
●
●
Keywords Benzoxazole α-amylase α-glucosidase
●
Molecular docking Antihypergycemic
Electronic supplementary material The online version of this article
https://doi.org/10.1007/s00044-017-2097-1) contains supplementary
material, which is available to authorized users.
(
*
Rajiv Mall
rajivmall@pbi.ac.in
1
Department of Chemistry, Synthetic Organic and Medicinal
Chemistry Laboratory, Punjabi University, Patiala 147002 Punjab,
India
2
Department of Basic and Applied Sciences, Punjabi University,
Patiala 147002 Punjab, India

Med Chem Res
moieties and tested their α-amyloglucosidase inhibitory
activity. Literature revealed that compounds with Benzox-
azole heterocyclic system are promising pharmacophore
exhibited various biological activities including anti-
microbial (Ertan-Bolelli et al. 2016), anticancer and anti-
HIV (Rida et al. 2005), anti-inflammatory (Seth et al. 2014),
antileishmanial (Tipparaju et al. 2008). The docking studies
at the crystal structure of protein (PDB code: 3TOP) using
SYBYL 7.3 (SYBYL 2006, available in our in silico Drug
Design Laboratory) were carried out for prediction of
binding affinities and interactions of the targeted new che-
mical entities as compared to the standard Acarbose (Verma
et al. 2012a, b; Sybyl 7.3 2006).
4-(1,3-Benzoxazol-2-yl)benzaldehyde (4)
M.p. 155–157 °C; yield 62%; FTIR (KBr, cm− ) νmax
1
:
3062, 2843 and 2744 (C–H aldehyde), 1701(C=O), 1602,
1
1575, 1553 (C=C aromatic); H NMR (400 MHz, DMSO-
d6): δ = 10.10 (s, 1H, aldehyde), 8.43 (dd, J = 6.68, 1.64
Hz, 2H, Ar–H), 8.04 (dd, J = 6.76, 1.76 Hz, 2H, Ar–H),
7.83 - 7.80 (m, 1H, Ar–H), 7.63 - 7.61 (m, 1H, Ar–H), 7.43-
1
3
7.38 (m, 1H, Ar–H); C NMR (100 MHz, DMSO-d ): δ =
6
191.46 (H-C=O), 161.62 (N=C-O), 150.96 (Ar-C), 142.02
(Ar–C), 138.01 (Ar–C), 132.32 (Ar–C), 130.13 (Ar–C),
128.11 (Ar–C), 125.99 (Ar–C), 120.51 (Ar–C), 110.85
+
(Ar–C); ESI–MS (m/z): 224.12 [M+1] ; HRMS (m/z):
+
2
24.0682 [M+H] (calcd. for C H NO 224.0712).
1
4
10
2
3-(1,3-Benzoxazol-2-yl)benzaldehyde (5)
Materials and methods
Chemistry
M.p.130–132 °C; yield 65%; FTIR (KBr, cm− ) νmax: 3066,
1
2860 and 2761 (C-H aldehyde), 1692 (C=O), 1601, 1556,
C=C aromatic); H NMR (400 MHz, DMSO-d ): δ =
1
(
6
Chemicals used in the present work were purchased from
Aldrich, Loba Chemie and SD Fine chemicals. Melting/
boiling points was taken by using an open concentrated
10.07 (s, 1H, aldehyde), 8.69 (t, J = 2.84 Hz, 1H, Ar-H),
8.45 (dt, J = 7.76, 2.80 Hz, 1H, Ar-H), 7.99 (dt, J = 7.68,
2.72 Hz, 1H, Ar-H), 7.75 - 7.73(m, 1H, Ar-H), 7.65(t, J =
1
sulphuric acid bath and are uncorrected. The H NMR and
7.72 Hz 1H, Ar-H) 7.56- 7.54 (m, 1H, Ar-H), 7.35 - 7.30
1
3
13
C NMR spectra were measured on Bruker ADVANCE II
00 (400.13 and 100.62 MHz) NMR spectrometer, Bru-
(m, 2H, Ar-H); C NMR (100 MHz, DMSO-d ): δ =
6
4
192.14 (H-C=O),161.12 (N=C-O), 150.27 (Ar-C), 141.36
(Ar-C), 136.77 (Ar-C), 132.42 (Ar-C), 131.98 (Ar-C),
130.03 (Ar-C), 128.07 (Ar-C), 127.39 (Ar-C), 125.67 (Ar-
C), 124.83 (Ar-C), 119.90 (Ar-C), 110.84 (Ar-C); ESI–MS
kerBioSpin, Switzerland, in DMSO-d and CDCl as sol-
6
3
vent using tetramethylsilane (TMS) as an internal standard.
Chemical shifts are expressed as ppm and signals were
designated as: s singlet; d doublet; t triplet; br broad; m
multiplet. Infrared (IR) spectra were recorded on Perkin
Elmer spectrum RX FT-IR spectrometer and values were
+
+
(m/z): 224.12 [M+1] ; HRMS (m/z): 224.0682 [M+H]
(calcd. for C H NO 224.0712).
1
4
10
2
−
1
signified in cm . electrospray ionisation mass spectro-
metry (ESI-MS) were determined on (liquid
General procedure for synthesis of targets compounds
(4a–d and 5a–d)
chromatography–mass spectrometry) water, Q-TOF
Micromass UK in ESI mode. HRMS were carried out on
XEVO G2-XS QTOF at IITRPR.
To a stirred solution of the 4-(1,3-benzoxazol-2-yl)benzal-
dehyde (4)/3-(1,3-benzoxazol-2-yl)benzaldehyde (5) (0.448
mmol) and TZD/ RhD / HyD/ ThyD (0.672 mmol) in gla-
cial acetic acid (10 ml) ammonium acetate (0.896 mmol)
was added and the reaction mixture heated under reflux for
General procedure for synthesis of compounds 4 and 5
15 h. The solution was cooled, the precipitate filtered off,
To the solution of 2-chlorobenzoxazole (0.00325 mol) and
washed with water and dried.
4
-formylphenylboronic acid/3-formylphenylboronic acid
(
0.00217 mol), K CO3 (0.00625 mol) was added. The
5-[4-(1,3-Benzoxazol-2-yl)benzylidene]-1,3-thiazolidine-
2
resulting mixture was degassed, stirred at ambient tem-
perature for 20 min and Pd(PPh ) was added in catalytic
amount, then again mixture was degassed and refluxed
under nitrogen gas atmosphere for 12 h. It was allowed to
cool, filtered on celite and extracted using ethyl acetate
2,4-dione (4a)
3
4
M.p. 293–295 °C; yield 75%; FTIR (KBr, cm− ) νmax
3366, 3154 (N-H), 1750 and 1683 (C=O), 1600 (C=C
1
:
1
benzylidene), 1500 (N-H in plane bending); H NMR (400
(
3 × 20 mL). The organic layer was dried using Na SO ,
MHz, DMSO-d ): δ = 12.59 (br s, 1H, N-H), 8.28 (d, J =
2
4
6
concentrated under reduced pressure and cooled to give the
and 5 as solid product.
8.44 Hz, 2H, Ar-H), 7.81 (s, 1H, C=C-H), 7.78-7.69 (m,
4H, Ar-H), 7.42-7.37 (m, 2H, Ar-H); C NMR (100 MHz,
1
3
4

Med Chem Res
DMSO-d ): δ = 167.23 (S-C=O), 167.02 (–CH=C-C=O),
(m, 1H, Ar-H), 7.72-7.70 (m, 1H, Ar-H), 7.44-7.38 (m, 2H,
6
1
3
1
61.30 (N=C-O), 150.28 (S-C=CH), 141.53, 135.92
=CH-Ar), 130.34 (Ar-C), 131.98 (Ar-C), 130.03 (Ar-C),
28.07 (Ar-C), 127.39 (Ar-C), 125.67 (Ar-C), 124.83 (Ar-
C), 119.90 (Ar-C), 110.84 (Ar-C); ESI-MS (m/z): 321.13
Ar-H), 6.52 (s, 1H, C=C-H); C NMR (100 MHz, DMSO-
d6): δ = 179.49 (C=S), 165.63 (C=O), 161.71 (N=C-O),
150.25 (HN-C=CH), 141.60 (Ar-C), 135.62 (=CH-Ar),
130.62 (Ar-C), 128.84 (Ar-C), 127.28 (Ar-C), 126.24 (Ar-
C), 125.34 (Ar-C), 124.61 (Ar-C), 119.66 (Ar-C), 110.60
(
1
+
+
+
[
M-1] , 322.15 [M] ; HRMS (m/z): 323.0459 [M+H]
+
(
calcd. for C H N O S 323.0490).
(Ar-C), 109.60 (Ar-C); ESI-MS (m/z): 320.13 [M-1] ,
1
7 11 2 3
+
+
3
21.17 [M] ; HRMS (m/z): 322.0619 [M+H] (calcd. for
5
-[4-(1,3-Benzoxazol-2-yl)benzylidene]-2-thioxo-1,3-
C H N O S 322.0650).
1
7 12 3 2
thiazolidin-4-one (4b)
5
-[3-(1,3-Benzoxazol-2-yl)benzylidene]-1,3-thiazolidine-
M.p. 297–299 °C; yield 79%; FTIR (KBr, cm−1) νmax: 3173
2,4-dione (5a)
(
N-H), 1695 (C=O), 1594 (C=C benzylidene), 1500 (N-H
1
M.p. 287–289 °C; yield 81%; FTIR (KBr, cm−1) νmax: 3035
(N-H stretch), 1753 and 1715 (C=O stretch), 1612 (C=C
in plane bending), 1191(C=S); H NMR (400 MHz,
DMSO-d ): δ = 13.71 (br, 1H, N-H) 8.29 (d, J = 8.40 Hz,
2
7
6
1
H, Ar-H), 7.75-7.64 (m, 4H, Ar-H), 7.62 (s, 1H, C=C-H),
stretch of benzylidene), 1550 (N-H in plane bending); H
1
3
.40-7.35 (m, 2H, Ar-H); C NMR (100 MHz, DMSO-d6):
NMR (400 MHz, DMSO-d ): δ = 13.94 (br, 1H, N-H), 8.37
6
δ = 194.69 (C=S), 169.15 (C=O), 161.25 (N=C-O),
(s, 1H, Ar-H), 8.27 (dt, 1H, J = 7.68, 2.68 Hz, Ar-H), 7.84-
7.73 (m, 4H, Ar-H), 7.52 (s,1H, C=C-H) 7.47-7.39 (m, 2H,
1
50.30 (S-C=CH), 141.54 (Ar-C), 135.78 (=CH-Ar),
1
3
1
30.68 (Ar-C), 129.78 (Ar-C), 127.77 (Ar-C), 127.66 (Ar-
Ar-H); C NMR (100 MHz, DMSO-d ): δ = 195.09 (S-
6
C), 127.49 (Ar-C), 125.51 (Ar-C), 124.64 (Ar-C), 119.79
C=O), 169.21 (-CH=C-C=O), 161.26 (N=C-O), 150.31
(S-C=CH), 141.39 (Ar-C), 134 (=CH-Ar), 133.30 (Ar-C),
130.30 (Ar-C), 130.09 (Ar-C), 128.69 (Ar-C), 128.69 (Ar-
C), 128.24 (Ar-C), 127.57 (Ar-C), 127.18 (Ar-C), 125.63
+
(
3
Ar-C), 110.59 (Ar-C); ESI-MS (m/z): 337.14 [M-1] ,
39.33 [M+1] ; HRMS (m/z): 339.0223 [M+H] (calcd.
+
+
for C H N O S 339.0262).
1
7 11 2 2 2
(
Ar-C), 124.82 (Ar-C), 119.93 (Ar-C) 110.85 (Ar-C); ESI-
+
+
5
-[4-(1,3-Benzoxazol-2-yl)benzylidene]imidazolidine-2,4-
MS (m/z): 321.16 [M-1] , 322.17 [M] ; HRMS (m/z):
323.0469 [M+H] (calcd. for C H N O S 323.0490).
+
dione (4c)
1
7 11 2 3
M.p. 308–310 °C; yield 82%; FTIR (KBr, cm−1) νmax: 3168
and 3073 (N-H stretch), 1780 and 1744 (C=O stretch),
5-[3-(1,3-Benzoxazol-2-yl)benzylidene]-2-thioxo-1,3-
thiazolidin-4-one (5b)
1
662 (C=C stretch of benzylidene), 1610 and 1553 (C=C
1
M.p. 292–294 °C; yield 83%; FTIR (KBr, cm−1) νmax: 3008
(N-H), 1724 (C=O), 1599 (C=C benzylidene), 1549 (N-H
in plane bending), 1181 (C=S); H NMR (400 MHz,
stretch aromatic), 1500 (N-H in plane bending); H NMR
(
400 MHz, DMSO-d ): δ = 11.45 (br, 1H, N-H), 10.86 (br,
6
1
1
H, N-H), 8.24 (d, J = 8.44 Hz, 2H, Ar-H), 7.91-7.85 (m,
H, Ar-H), 7.53-7.46 (m, 2H, Ar-H), 6.54 (s, 1H, C=C-H);
4
DMSO-d ): δ = 12.66 (br, 1H, N-H), 8.39 (s, 1H, Ar-H),
6
1
3
C NMR (100 MHz, DMSO-d ): δ = 165.38 (HN-C=O),
8.26 (dt, 1H, J = 7.72, 2.56 Hz, Ar-H), 7.90 (s, 1H, C=C-
6
1
1
61.83 (=C-C=O), 155.69 (N=C-O), 150.23 (HN-C=CH),
41.53 (Ar-C), 136.49 (=CH-Ar), 129.98 (Ar-C), 129.40
H), 7.83-7.80 (m, 2H, Ar-H), 7.78-7.72 (m, 2H, Ar-H),
1
3
7.47-7.40 (m, 2H, Ar-H); C NMR (100 MHz, DMSO-d6):
δ = 167.36 (C=S), 167.09 (C=O), 161.26 (N=C-O),
150.31 (S-C=CH), 141.39 (Ar-C), 134 (=CH-Ar), 133.30
(Ar-C), 130.30 (Ar-C), 130.09 (Ar-C), 128.69 (Ar-C),
128.69 (Ar-C), 128.24 (Ar-C), 127.57 (Ar-C), 127.18 (Ar-
C), 125.63(Ar-C), 124.82 (Ar-C), 119.93 (Ar-C), 110.85
(
Ar-C), 127.42 (Ar-C), 125.67 (Ar-C), 125.48 (Ar-C),
1
24.97 (Ar-C), 119.84 (Ar-C), 110.95(Ar-C), 106.67(Ar-
+
+
C); ESI-MS (m/z): 304.19 [M-1] , 305.21 [M] ; HRMS
+
(
m/z): 306.0840 [M+H]
(calcd. for C H N O
17 12 3 3
3
06.0879).
+
+
(
Ar-C); ESI-MS (m/z): 337.17 [M-1] , 339.35 [M+1] ;
+
5
-[4-(1,3-Benzoxazol-2-yl)benzylidene]-2-
HRMS (m/z): 339.0229 [M+H] (calcd. for C H N O S
17 11 2 2 2
thioxoimidazolidin-4-one (4d)
339.0262).
M.p. 302–304 °C; yield 85%; FTIR (KBr, cm−1) νmax: 3266
and 3059 (N-H), 1731 (C=O stretch), 1656 (C=C benzy-
5-[3-(1,3-Benzoxazol-2-yl)benzylidene]imidazolidine-2,4-
dione (5c)
lidene), 1601 (C=C aromatic), 1552 (N-H in plane bend-
1
M.p. 304–306 °C; yield 82 %; FTIR (KBr, cm−1) νmax
:
ing), 1196 (C=S); H-NMR (400 MHz, DMSO-d ): δ =
6
1
2.39 (br, 1H, N-H), 12.24 (br, 1H, N-H), 8.22 (d, J = 8.40
3189 and 3063 (N-H), 1771 and 1733 (C=O), 1663 (C=C
benzylidene), 1616 (C=C aromatic), 1547 (N-H in plane
Hz, 2H, Ar-H), 7.92 (d, J = 8.44 Hz, 2H, Ar-H), 7.79-7.77

Med Chem Res
1
bending); H-NMR (400 MHz, DMSO-d ): δ = 12.41 (br,
1
synthesized molecules at the respective active sites were
also compared to those of standard inhibitor Acarbose.
6
H, N-H), 12.38 (br, 1H, N-H), 8.42 (s, 1H, Ar-H), 8.18 (d,
J = 7.88 Hz, 1H, Ar-H), 7.94 (d, J = 7.92, 1H, Ar-H), 7.84-
7
7
.78 (m, 2H, Ar-H), 7.64 (t, J = 7.80 Hz, 1H, Ar-H), 7.47-
.40 (m, 2H, Ar-H), 6.60 (s, 1H, C=C-H); C NMR (100
Enzymatic starch digestion assay in vitro
1
3
MHz, DMSO-d ), δ = 179.66 (HN-C=O), 165.57 (=C-
C=O), 161.84 (N=C-O), 150.25 (HN-C=CH), 141.45 (Ar-
C), 133.41 (=CH-Ar), 132.87 (Ar-C), 129.48 (Ar-C),
For in vitro studies starch digestion method was followed
with some modifications (Kasabri et al. 2011). 100 mg corn
starch was taken and gelatinized in 3 mL distilled water
with or without test compounds or Acarbose. To the above
solution 4 μg α-amylase was added vortexed and then
mixture was incubated at 80 °C for 20 min. After incubation
the mixture (3 mL) was diluted to 10 mL with distilled
water and then 1 mL of this mixture was added to 2 mL of
6
1
29.07 (Ar-C), 128.66 (Ar-C), 127.50 (Ar-C), 126.98 (Ar-
C), 125.53 (Ar-C), 124.82 (Ar-C), 119.81 (Ar-C), 110.84
+
(
3
Ar-C), 109.97 (Ar-C); ESI-MS (m/z): 304.19 [M-1] ,
+
+
05.21 [M] ; HRMS (m/z): 306.0836 [M+H] (calcd. for
C H N O 306.0879).
1
7 12 3 3
0.1 M sodium acetate buffer (pH - 4.75).to this 3 mL of
5
-[3-(1,3-Benzoxazol-2-yl)benzylidene]-2-
mixture, 30 μg α-amyloglucosidase was added and incu-
bated at 60 °C for 30 min. To inhibit the thermophilic
enzymes samples were removed to ice. Control samples
were having neither test compounds nor Acarbose. Using
this assay the inhibitory rate of test compounds were cal-
culated by the following equation.
thioxoimidazolidin-4-one (5d)
M.p. 298–300 °C; yield 87%; FTIR (KBr, cm−1) νmax: 3246
and 3086 (N-H), 1730 (C=O), 1651 (C=C benzylidene),
1
616 (C=C aromatic), 1549 (N-H in plane bending), 1181
1
(
C=S); H-NMR (400 MHz, DMSO-d ): δ = 12.61 (br, 2H,
6
N-H), 8.65 (s, 1H, Ar-H), 8.41 (d, J = 7.92 Hz, 1H, Ar-H),
ð%Þ Inhibition of enzymatic starch digestion
¼ ½A À A =A Š  100
8
7
6
.17 (d, J = 7.84 Hz, 1H, Ar-H), 8.07 - 8.02 (m, 2H, Ar-H),
0
i
0
.87 (t, J = 7.80 Hz,1H, Ar-H), 7.70-7.64 (m, 2H, Ar-H),
1
3
.82 (s, 1H, C=C-H); C NMR (100 MHz, DMSO-d ): δ
Where A = Absorbance of control A = Absorbance of test
6
0
i
=
165.28 (C=S), 161.90 (C=O), 155.76 (N=C-O), 150.25
compound
(
HN-C=CH), 141.47 (Ar-C), 134.04 (=CH-Ar), 132.03
IC50 in μM was determined by plotting graph between %
inhibition of starch digestion verses various concentration
of test compound.
(
Ar-C), 129.43 (Ar-C), 129.28 (Ar-C), 128.00 (Ar-C),
1
27.00 (Ar-C), 126.73 (Ar-C), 125.40 (Ar-C), 124.71 (Ar-
C), 119.74 (Ar-C), 110.74 (Ar-C), 106.85 (Ar-C); ESI-MS
+
+
(
[
m/z): 320.18 [M-1] , 321.19 [M] ; HRMS (m/z): 322.0598
+
M+H] (calcd. for C H N O S 322.0650).
Results and discussion
Chemistry
1
7 12 3 2
Molecular docking methods
The crystal structure of C-terminal domain of human
intestinal α-glycosidase (PDB code: 3TOP) was used as
molecular target. The crystal structure complexed with an
original ligand (Acarbose) was downloaded from RCSB
Protein Data Bank. Water molecules were removed from
enzyme structure whereas, hydrogens added, energy mini-
mized using Gasteiger–Huckel charges and Tripos force
field in the Sybyl software suite (Verma et al. 2015; Singh
et al. 2017). Synthesized compounds were assigned
Gasteiger–Huckel charges and subsequently minimized
using the Powel method by using SYBYL 7.3 software. The
energy-optimized sketched compound were subjected to
docking run at the ligand binding site of α-glucosidase by
Surflex dock in SYBYL7.3. Docking analysis was con-
ducted using the Surflexdock module to predict and
examine the binding modes of the synthesized derivatives at
the active site of α-glucosidase. The hydrogen bonding
interactions (amino acid residues and bond distances) of the
The synthetic process was started from the synthesis of 2-
mercaptobenzoxazole (2) and 2-chlorobenzoxazole (3)
according to the literature protocol (Lazer et al. 1994).
Aldehydes (4 and 5) were synthesized by Suzuki coupling
reaction of 2-chlorobenzoxazole with 3-formylphenylboronic
acid and 4-formylphenylboronic acid respectively under the
catalytic amount of Pd(PPh ) in dioxane:water (4:1). The
3
4
condensation of compounds (4 and 5) with active methylene
containing moieties (TZD, RhD, HyD and THyD) were done
using ammonium acetate in acetic acid under refluxed con-
ditions to afford the targeted compounds (Scheme 1). We
obtained the all targeted compounds as pure solids in good
yields. The structures of all synthesized compounds were
1
13
characterized by H-NMR, C-NMR, FT-IR and ESI-MS
and HRMS techniques. The presence of a 1H singlet for
1
aldehyde proton in H-NMR spectra at δ 10.10 ppm and δ
10.07 ppm along with C-H aldehydic stretch at 2843 and
−1
2860 cm in the IR spectra confirmed the structure assigned

Med Chem Res
NH2
OH
N
O
N
O
a
b
SH
Cl
3
1
2
c
CHO
HO
HO
B
CHO
B
HO
HO
CHO
N
O
N
O
CHO
4
5
X
X
d
d
O
O
N
H
X'
N
H
X'
X'
X
NH
N
O
N
O
O
X
O
4a-d
5a-d
N
H
X'
’
CODE
a and 5a
b and 5b
c and 5c
d and 5d
X
X
O
S
4
S
S
4
4
NH
NH
O
S
4
2 3 5 3 4
Scheme 1 Reagents and conditions: a CS , EtOH, Water, KOH, reflux 3 h b POCl /PCl , DCM stirring 1 h c Pd(PPh ) , Dioxane:Water (4:1),
reflux 12 h d CH
3
COONH
4
, Glacial Acetic Acid, reflux 15 h
1
to 4 and 5, respectively. The H-NMR spectra contained
characteristic signal for benzylidene proton as a singlet in the
synthesized targeted ligands. It was present in the δ 6.52–6.83
ppm range for the compounds (4c, 4d, 5c, and 5d) featuring
HyD and THyD moieties where as a compounds (4a, 4b, 5a,
and 5b) containing TZD and RhD moieties it was found in δ
with protein (PDB code: 3TOP) were given in (Table 1).
The docking interactions for all the novel targeted ligands
(4a-d and 5a–d) in comparison to Acarbose (Standard
Drug) were illustrated in (Fig. 1). All the final synthesized
compounds were docked (with Surflex dock module of
Sybyl 7.3, Tripose Inc. software available at our in silico
drug design Laboratory) at the binding site of C-terminal
domain of human intestinal α-glucosidase (PDB Code:
3TOP) for the prediction of binding affinities in reference to
standard molecule (Acarbose). It was found from the ana-
lysis of docking results that the binding affinity of Acarbose
in term of G-score is −160.51 and it binds through
hydrogen bonds formation with ASP1526, ASP1279,
ASP1157, HIS1584, ARG1510, and LYS1460 amino acid
residues. Out of two sets of benzoxazolyl linked meta/para
substituted phenyl containing H-bonding parts, para
7
.52–7.90 ppm range. In FT–IR spectra (=C–H) benzylidenic
−1
13
13
stretch ranged from 2736–2857 cm . C peaks in the C-
NMR spectra corresponding to all the concerned inter-
mediates and targeted compounds were found at appropriate
chemical shift positions.
Molecular docking
The docking results in terms of Gold score energies (G
score) and H-bond interactions of the studied compounds

Med Chem Res
Table 1 Docking results of the
synthesized targeted compounds
as compared to acarbose
Compound
Acarbose
G score
H-bonding interactions between the ligands and the active site amino acid (AA)
residues
*
(
standard drug) at the active site
Atoms of ligands
AA residues
Bond distance (Å)
No. of H-bonds
11
of α-glucosidase
−160.51
OH
ASP1279
HIS1584
ASP1526
ASP1526
ARG1510
ARG1510
ARG1510
ASP1157
ASP1157
ASP1157
LYS1460
THR1586
ARG1510
ASP1157
ASP1157
ASP1157
ARG1510
THR1586
ASP1157
LYS1460
LYS1460
LYS1460
LYS1460
LYS1460
ASP1157
ASP1526
1.99
1.95
2.65
1.85
2.41
2.63
2.08
1.91
1.85
2.75
2.59
2.40
2.19
2.11
1.74
2.34
2.32
2.06
2.24
2.78
2.36
2.28
2.04
2.13
1.80
2.14
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
4
4
a
−160.25
−180.87
N– of Benzoxazole
3
3
3
-CO of TZD
-NH of TZD
4
b
4-NH of RhD
4
5
-NH of RhD
-CO of RhD
4
4
c
−141.50
−145.76
O– of Benzoxazole
4-NH of THyD
1
3
d
5
-CO of THyD
-CO of THyD
5
5a
5b
5c
−123.10
−194.22
−89.88
N– of Benzoxazole
5-CO of RhD
1
1
2
N– of Benzoxazole
4
-NH of HyD
5
d
−113.53
2-NH of THyD
1
*
The particular atom of the ligands involved in H-bonding has been indicated by bold face
substituted compounds (4a, 4c and 4d) have better binding
scores than their corresponding meta substituted com-
pounds (5a, 5c, and 5d) except in case of RhD based
compounds (4b and 5b). The comparison of the predicted
binding affinities in terms of G score of the synthesized
ligands with that of acarbose showed that both RhD based
biological activity studies. The hydrogen bond interactions
between the synthesized ligands and the AA residues in the
active sites of 3TOP were explored and compared with
those of Acarbose to explain the binding modes and binding
affinities of the ligands. The hydrogen bonds for each of the
novel ligands are less in number compared to Acarbose
(eleven hydrogen bonds) at the active site. The interacting
AA residues were all similar to that of Acarbose, as the new
ligands occupy a same region in the binding cavity. The H-
bond distances (Å) between the ligands and the interacting
AA residues are in range and in most of the cases are shorter
than those of Acarbose. Generally, the acidic units of the
ligands are involved in hydrogen bond formation in all the
cases. The TZD based compound 4a and 5a interacts with
important AA residues (THR 1586, ARG1510 and ASP
1157, LYS1460 respectively) through N of benzoxazole,
carbonyl group and NH of TZD. The RhD based com-
pounds 4b and 5b interacts with AA residues (ASP 1157
(
(
meta/para linked) compounds 4b (−180.47) and 5b
−194.22) have higher binding affinities than that of
Acarbose (−160.51) which is expected as RhD derivatives
are known to possess antidiabetic activities. The TZD based
compounds 4a (−160.25) showed comparable binding
affinity to that of Acarbose (−160.51) whereas 5a
(
−123.10) have lesser binding affinity. The HyD and THyD
based meta/para compounds 4c (−141.50), 4d (145.76), 5c
−89.88) and 5d (−113.53) have lesser binding affinity to
(
that of Acarbose (−160.51). Furthermore out of these TZD
and RhD based compounds, RhD linked compounds have
higher binding affinities which is also in accordance with

Med Chem Res
Fig. 1 Molecular interactions of
standard drug Acarbose and
benzoxazole derivatives in the
active site of α-glucosidase
amino acid residues. (a)
Acarbose (b) compound 4a (c)
compound 4b (d) compound 4c
(e) compound 4d (f) compound
5
a (g) compound 5b (h)
compound 5c (i) compound 5d
and ARG 1510, LYS1460 respectively) through carbonyl
group and NH of RhD. The HyD and THyD based com-
pounds 4c and 5c, 4d and 5d interacts with AA residues
Table 2 α-Amyloglucosidase inhibitory activity of the synthesized
compounds
Compounds
IC50 (µM)
(
THR 1586, ASP 1157, ASP 1526 and LYS1460) through
4
4
4
a
b
c
15.03 ± 1.04
0.94 ± 0.01
19.93 ± 0.79
18.43 ± 0.70
22.00 ± 1.21
0.24 ± 0.01
29.03 ± 1.11
25.47 ± 1.33
15.01 ± 1.09
O of benzoxazole, NH of THyD, carbonyl group of THyD,
N of benzoxazole and NH of HyD. The results of molecular
docking studies are found to be in accordance with in vitro
studies and hence, supported the biological activity.
4d
5
5
5
5
a
b
c
In vitro studies
d
These eight newly synthesized compounds (4a–d and 5a–d)
were evaluated for their in vitro α-amylase and α-glucosi-
dase inhibitory activity using Acarbose as positive control
Acarbose
(
(
IC50 value of 15.01 ± 1.09 µM) (Table 2). The compounds
4b and 5b) were more inhibitive to these enzymes than the
22.00 ± 1.21 µM) showed much lesser inhibition potential
than standard drug (IC50 15.01 ± 1.09 µM). The HyD and
THyD based compounds 4c (IC50 19.93 ± 0.79 µM), 4d
(IC50 18.43 ± 0.70 µM), 5c (IC50 29.03 ± 1.11 µM) and 5d
(IC50 25.47 ± 1.33 µM) have also lesser inhibition potential
than standard drug. Molecular docking studies in terms of G
score values and binding interaction of novel targeted
compounds in comparison to standard drug (acarbose) in
rest of the tested compounds with IC50 values of much lower
than that of the positive control (Standard drug Acarbose). It
has also been concluded from biological results that RhD
based compounds 4b (IC50 0.94 ± 0.01 µM) and 5b
(
IC50 0.24 ± 0.01 µM) have better inhibition than standard
drug (IC50 15.01 ± 1.09 µM). The TZD based compound 4a
IC50 15.03 ± 1.04 µM) showed comparable while 5a (IC50
(

Med Chem Res
Acknowledgements Authors are thankful to the Punjabi University,
Patiala authorities for providing the necessary research facilities. We
are also grateful to Director and Mr. Avtar Singh of Sophisticated
Analytical Instrumentation Facility (SAIF), Panjab University, Chan-
digarh, respectively, for extending the facilities for spectral analysis of
the compounds reported in this paper. One of the authors, Mr. Var-
inder Singh, is thankful to UGC for providing Maulana Azad National
fellowship.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interest.
Fig. 2 The correlation graph between the predicted G score data and
IC50 value
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