Comparative α-glucosidase and α-amylase inhibition studies of rhodanine–pyrazole conjugates and their simple rhodanine analogues

Article abstract of DOI:10.1007/s00044-018-2272-z

Novel rhodanine–pyrazole conjugates (6a–i) and their simple rhodanine analogues (8a–e) were prepared and comparatively screened for their antidiabetic activities against enzymatic targets, α-glucosidase and α-amylase. As expected, the molecular hybrids ex

Full text of DOI:10.1007/s00044-018-2272-z

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2272-z
ORIGINAL RESEARCH
Comparative α-glucosidase and α-amylase inhibition studies of
rhodanine–pyrazole conjugates and their simple rhodanine
analogues
Parvesh Singh1 Serisha Mothilal Nagaraju Kerru Ashona Singh-Pillay Lalitha Gummidi
1 ●
1 ●
1 ●
1 ●
●
Ochuko L. Erukainure Md. Shahidul Islam2
2 ●
Received: 10 August 2018 / Accepted: 4 December 2018
©
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Novel rhodanine–pyrazole conjugates (6a–i) and their simple rhodanine analogues (8a–e) were prepared and comparatively
screened for their antidiabetic activities against enzymatic targets, α-glucosidase and α-amylase. As expected, the molecular
−
6
−4
hybrids exhibited significantly greater inhibitory activity against α-glucosidase (IC = 2.259 × 10 –1.160 × 10 mol/L),
5
0
−
4
−4
relative to their simple rhodanine counterparts (IC = 3.056 × 10 –9.494 × 10 mol/L). Amongst the screened derivatives
compounds 6a and 6f displayed a 3-fold and 42-fold greater potency against α-glucosidase (IC = 2.854 × 10 and
.259 × 10 mol/L, respectively) compared to the standard drug, acarbose. The designed molecular conjugates displayed an
50
−
5
5
0
−
6
2
improved binding affinity toward α-glucosidase than α-amylase. Compound 6d was identified as the most potent inhibitor of
−
5
α-amylase (IC = 6.377 × 10 mol/L) with a 1.5-fold greater inhibitory activity than acarbose. Structural assessment of the
5
0
molecules revealed that electron withdrawing (Cl) and electron donating (OCH ) groups at the ortho-position played a
3
significant role in the inhibitory activity. Molecular docking studies of the molecular conjugates and simple rhodanine
analogues in the active site of α-glucosidase were performed to describe and highlight the putative binding interactions
attributing to the selective inhibition. The identification of these novel rhodanine–pyrazole molecular hybrids forms part of a
potential treatment in the management of diabetes.
●
●
●
●
Keywords Pyrazole Rhodanine Molecular hybrids α-glucosidase α-amylase
Introduction
mismanaged has been associated with severe complications,
such as cardiovascular, retinopathic and neuropathic
diseases.
Type II diabetes is a chronic disease which has largely been
characterised by poor lifestyle choices (Browlee 2001;
Sundarram and Murthy 2014; Sales et al. 2012; Taha et al.
Current therapies in the management of diabetes include
oral administration of antidiabetic drugs, intramuscular
insulin injection, as well as dietary (e.g. low glycemic index
foods) and lifestyle changes (Kaku 2014; Mata et al. 2013).
However, these techniques have limited efficacy and suffer
a number of limitations; hence the need to find new and
innovative antidiabetic therapies. Two of the most attractive
targets of antidiabetic chemotherapeutics in the control of
blood glucose levels are α-glucosidase and α-amylase
enzymes.
α-Glucosidase and α-amylase are key enzymes that
degrade carbohydrates into sugars (Fig. 1). The α-glucosi-
dase enzyme is responsible for hydrolysing polysaccharides
into glucose at the terminal non-reducing α-1-4-glycosidic
bond in the small intestine (Taha et al. 2015). Over-
expression of the enzyme can lead to increased levels of
2
017). It is a serious disease that when untreated or
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-018-2272-z) contains supplementary material,
which is available to authorized users.
*
Parvesh Singh
parveshdurban@gmail.com
singhp4@ukzn.ac.za
1
2
School of Chemistry and Physics, University of KwaZulu-Natal,
P/Bag X54001, Westville, Durban 4000, South Africa
Biomedical Research Lab, Department of Biochemistry, School of
Life Sciences, University of KwaZulu-Natal, Westville Campus,
Durban 4000, South Africa

Medicinal Chemistry Research
Glucose
aldose reductase inhibitor which is clinically used for the
treatment of diabetic neuropathy (Terashima et al. 1984). In
a recent study, Andleeb et al. (2016) reported the synthesis
of a series of pyrazole–rhodanine conjugates (compound E,
IC = 1.22 μM) that showed potent aldose reductase inhi-
α-amylase
Polysaccharide
Eg. Starch
5
0
bitory activity.
Nitro-aromatic compounds have demonstrated extensive
target selectivity and are reported to be pharmacologically
active and robust (Patterson and Wyllie 2014; Ju and
Parales 2010; Hall et al. 2011; Hall and Wilkinson 2012).
For example, 2-nitrobenzimidazole has been reported to
possess comparable activity to glibenclamide (an anti-
diabetic drug) in alloxan-induced diabetic rats (Bathini et al.
2013). Amongst different synthetic approaches being used
in drug discovery, molecular hybridization (MH) is gaining
popularity due to its ability to combine two or more bio-
logically active scaffolds with a different or novel
mechanism of action (Berube 2016; Kerru et al. 2017a;
Singh et al. 2017). This supports our research rationale, in
combining the pharmacophore nuclei of pyrazole, rhoda-
nine and nitroaromatic into a single architecture. It was also
envisaged that the structural variations to the 3-phenyl ring
of pyrazole, as well as substituting the acetyl moiety at the
N3-position of the rhodanine may reveal a more potent
antidiabetic inhibitor.
α-glucosidase
Maltose
Fig. 1 Antidiabetic metabolic targets
glucose in the blood. α-Amylase works in a similar way and
is found in the pancreas and saliva. It hydrolyses the α-bond
of large α-linked polysaccharides, such as starch and gly-
cogen, to glucose and maltose (Kazeem et al. 2013). Thus,
the inhibition of the α-glucosidase and α-amylase enzymes
is essential for delaying carbohydrate degradation and glu-
cose absorption, which decreases the amount of overall
glucose in the body, and inevitably reduces the effect of
diabetes (Kazeem et al. 2013).
Current α-glucosidase and α-amylase inhibitors, such as
acarbose, voglibose and miglitol, are stable in the gastro-
intestinal tract and have found clinical application as anti-
diabetic drugs (Moorthy et al. 2012; Shahidpour et al.
2
015). However, they are also allied with various undesir-
Accordingly, in continuation of our interest (Singh et al.
2018; Kerru et al. 2017b) a novel series of rhodanine–
pyrazole conjugates (6a–i) and their simple rhodanine
analogues (8a–e) was developed, and evaluated in vitro
against α-glucosidase and α-amylase enzymes. Further-
more, the structure assessment and molecular docking stu-
dies of these compounds were also performed to describe
the critical pharmacophoric features contributing to inhibi-
tory activity.
able side effects such as diarrhoea, abdominal pain and
flatulence, which leads to some patients discontinuing
therapy. Unfortunately, the various side-effects associated
with the existing α-glucosidase and α-amylase inhibitor
reiterate the need for the development of new therapies, in
the form of potent antidiabetic drugs with no or low-risk
side effects.
In this study, a molecular hybrid approach is employed
which combines the biologically active scaffolds of pyr-
azole, rhodanine and a dinitro-aromatic (Fig. 2), each with
its novel mechanism of action as potential antidiabetic
agents specifically targeting α-glucosidase and α-amylase
enzymes (Kashtoh et al. 2016; Chaudhry et al. 2018).
Pyrazole derivatives have proven to have significant meta-
bolic stability and pharmacological efficiency as anti-
diabetic agents (Viseras et al. 2014; Vazquez et al. 2017).
Teneligliptin is an oral DPP-4 inhibitor containing pyrazole,
and was approved for the treatment of type-II diabetes
Materials and methods
Chemistry
All chemicals (laboratory grade) and solvents used in this
study were purchased from Sigma Aldrich and Merck, and
used as such without any further purification. Dimethyl-
formamide (DMF) was dried for 12 h using molecular
sieves 3 Å, which were regenerated by preheating at 250 °C.
The progress of reactions and the purity of the compounds
were determined using aluminium backed TLC plates
(Kieselgel 60 F254 plates), purchased from Merck. The
spots were visualized using 254 nm wavelength ultraviolet
light. The nuclear magnetic resonance (NMR) analysis was
recorded on a Bruker AVANCE III 400 MHz spectrometer
(
Sharma et al. 2016).
Rhodanine forms an important class of heterocyclic
compounds, which have gained increased attention in
medicinal chemistry and drug discovery over the past few
years (Werner et al. 2015; Ramesh et al. 2014; Reddy et al.
2
014). However, in recent years, there has been an
increased interest in exploiting rhodanine-based compounds
for their antidiabetic activity (Murugan et al. 2009; Agrawal
et al. 2015). An example of this is the drug Epalrestat, an
1
13
(399.995 MHz for H and 100.4296 MHz for C). Chemi-
cal shifts (δ) were reported in parts per million (ppm). The

Medicinal Chemistry Research
Fig. 2 Design of the molecular conjugates (6a–i) as antidiabetic agents
solvent used for NMR analysis was deuterated dimethyl
mL), was added dropwise to sulfuric acid (1.0 mL) while
stirring. The reaction mixture was further stirred for 1 h at
room temperature and then cooled in ice bath to complete
the crystallization. The solid was filtered under suction with
the aid of ethanol.
1
13
sulfoxide-d . The chemical shifts for H and C are refer-
6
enced to DMSO-d at 2.50 ppm and 39.51 ppm, respec-
6
tively. The 2D NMR experiments such as HSQC, HMBC,
COSY and NOESY were conducted with 4 K × 128 data
points (t2 × t1). The spin multiplicities are abbreviated as
follows: singlet (s), doublet (d), doublet of doublets (dd),
triplet (t) and multiplet (m). The NMR data was analysed
using TopSpin 3.1 software (Bruker). Melting points of
compounds were determined in an electro thermal melting
point apparatus (Electro Thermal IA9100) using sealed
capillary tubes, and are uncorrected. Infrared spectra were
recorded on a Perkin Elmer Spectrum 100 FTIR spectro-
meter with universal ATR sampling accessory. High-
resolution mass data were obtained for synthesized com-
pounds using a Bruker Micro ESI-QTOF instrument, which
is operated at ambient temperatures. The concentration of
each sample used in HRMS analysis was approximately 1
ppm. Absorbance readings done for the antidiabetic testing
was obtained using a Synergy/HTX multi-mode reader by
BioTek. The absorbance wavelengths for each enzyme are
specified in the relevant section.
General procedure for the synthesis of substituted
1-(2,4-dinitrophenyl)-3-phenyl-1H-pyrazole-4-
carbaldehydes (4a–i)
To a solution of DMF (6 mL) and POCl (0.5 g, 3.0 mmol),
3
1-phenyletylidene hydrazines (1.0 mmol) (3a–i) was added
and the reaction was allowed to stir at 60 °C for 5 h. The
resulting mixture was poured into ice cold water, neu-
tralized using a saturated solution of sodium hydroxide, and
the solid product was filtered, washed with water, dried and
recrystallized from ethanol.
General procedure for the synthesis of pyrazole–
rhodanine derivatives (6a–i)
Substituted pyrazole-4-carbaldehydes (4a–i) (0.01 mmol),
rhodanine (5) (0.012 mmol) and anhydrous sodium acetate
(0.03 mmol) were dissolved in 15 mL of acetic acid and
refluxed for 3 h. Once the completion of the reaction was
confirmed by TLC, solvent was removed using a high
vacuum. The reaction mixture was then poured on crushed
ice and the obtained solid was filtered and purified using
column chromatography.
General procedure for the synthesis of substituted
(
E)-1-(2,4-dinitrophenyl)-2-(1-phenyl etylidene)
hydrazines (3a–i)
A mixture of acetophenone (1a–i) (0.01 mol) and 2,4-dini-
trophenyl hydrazine (2) (0.02 mol), dissolved in ethanol (10

Medicinal Chemistry Research
1
(
4
Z)-3-Acetyl-5-((1-(2,4-dinitrophenyl)-3-phenyl-1H-pyrazol-
-yl)methylene)-2-thioxo thiazolidin-4-one (6a)
1512.08 (NO ), 1174.07 (C=S), 1083.10 (C–N); H NMR
2
(400 MHz, DMSO-d ): δ 4.26 (s, 3H, CH ), 6.84
6
H
3
(
d, JHH = 8.44 Hz, 2H, ArH), 7.05 (s, 1H, CHolefinic), 7.63
−
1
Orange solid; Yield: 59%; mp 139–140 °C; FTIR νmax (cm ):
(d, JHH = 8.44 Hz, 2H, ArH), 8.17 (d, JHH = 9.0 Hz, 1H,
ArH), 8.56 (d, JHH = 2.76 Hz, 1H, ArH), 8.57–8.60 (dd,
JHH = 2.28, 9.04 Hz, 1H, ArH), 8.84 (s, 1H, CHpyrazole),
3
(
085.92 (C–H), 1703.46 (C=O), 1529.53 (NO ), 1174.34
C=S), 1059.04 (C–N); H NMR (400 MHz, DMSO-d ): δ
.26 (s, 3H, CH ), 6.98 (s, 1H, CHolefinic), 7.50–7.70 (m, 5H,
2
1
6
H
1
3
4
9.73 (s, 1H, OH); C NMR (100 MHz, DMSO-d ): δC
3
6
ArH), 8.41 (d, JHH = 8.92 Hz, 1H, ArH), 8.68–8.71 (dd, JHH
2.52, 8.92 Hz, 1H, ArH), 8.91 (d, J = 2.36 Hz, 1H, ArH),
31.31, 115.59, 115.90, 116.23, 121.11, 122.43, 124.09,
127.18, 127.65, 129.27, 130.01, 132.18, 135.19, 135.61,
141.90, 144.60, 158.45, 169.40, 195.45; HRMS m/z (pos):
534.0612 C H N O S Na (calcd: 534.0614).
=
HH
13
8
2
1
1
5
.98 (s, 1 H, CHpyrazole); C NMR (100 MHz, DMSO-d ): δ
6
C
9.71, 119.72, 121.21, 122.86, 123.33, 126.57, 128.18,
28.57, 128.64, 129.62, 130.30, 135.03, 138.67, 143.01,
46.37, 153.84, 166.45, 176.65, 184.65; HRMS m/z (pos):
18.0590 C H N O S Na (calcd: 518.0591).
2
1
13
5
7 2
(Z)-3-Acetyl-5-((1-(2,4-dinitrophenyl)-3-(3-methoxyphenyl)-
1H-pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one (6e)
21 13 5 6 2
(
1
Z)-3-Acetyl-5-((3-(2-chlorophenyl)-1-(2,4-dinitrophenyl)-
H-pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one (6b)
Yellow solid; Yield: 63%; mp 72.9–77.0 °C; FTIR ν
(cm ): 3068.00 (C–H), 1684.57 (C=O), 1509.07 (NO ),
max
−
1
2
1
1266.68 (C–O–C), 1185.97 (C=S), 1081.50 (C–N); H
Yellow solid; Yield: 78%; mp 190–192 °C; FTIR ν
NMR (400 MHz, DMSO-d ): δ 3.80 (s, 1H, OCH ), 4.25
max
6
H
3
−
1
(
cm ): 3064.49 (C–H), 1700.31 (C=O), 1529.56 (NO ),
(s, 3H, CH ), 7.05 (s, 1H, CHolefinic), 7.11 (d, JHH = 7.92 Hz,
2
3
1
1
172.16 (C=S), 1067.08 (C–N); H NMR (400 MHz,
1H, ArH), 7.38 (s, 1H, ArH), 7.49 (d, JHH = 7.84 Hz, 1H,
DMSO-d ): δ 4.26 (s, 3H, CH ), 7.37 (s, 1H, CHolefinic),
ArH), 8.38 (d, JHH = 8.88 Hz, 1H, ArH), 8.67 (t, JHH
7.40 Hz, 1H, ArH), 8.87 (s, 1H, CHpyrazole), 8.89 (d, JHH
=
=
6
H
3
7
8
.54–7.57 (m, 4H, ArH), 8.40 (d, JHH = 8.88 Hz, 1H, ArH),
.67–8.87 (dd, JHH = 2.44, 8.88 Hz, 1H, ArH), 8.91
1
3
2.04 Hz, 1H, ArH); C NMR (100 MHz, DMSO-d ): δC
6
1
3
(
d, JHH = 2.44 Hz, 1H, ArH), 8.92 (s, 1H, CHpyrazole);
C
31.32, 55.19, 114.11, 114.89, 116.76, 120.41, 120.77,
121.12, 126.27, 126.74, 127.89, 130.28, 131.45, 131.92,
135.06, 142.76, 146.03, 154.77, 159.49, 168.88, 176.63,
194.70; HRMS m/z (pos): 548.9518 C H N O S Na
NMR (100 MHz, DMSO-d ): δC 31.32, 116.73, 120.45,
6
1
1
1
5
21.15, 126.30, 126.90, 127.96, 128.57, 128.74, 128.93,
29.11, 129.56, 130.24, 132.09, 135.14, 142.83, 146.11,
55.07, 168.91, 176.64, 194.93; HRMS m/z (pos):
51.9636 C H N O S ClNa (calcd: 551.9644).
2
2
15
5
7 2
(calcd: 518.9508).
2
1
12
5
6 2
(
Z)-3-Acetyl-5-((1-(2,4-dinitrophenyl)-3-(2-methoxyphenyl)-
(
Z)-3-Acetyl-5-((1-(2,4-dinitrophenyl)-3-(4-fluorophenyl)-1H-
1H-pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one (6f)
pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one (6c)
Orange solid; Yield: 81%; mp 74.8–79.7 °C; FTIR ν
(cm ): 3085.29 (C–H), 1705.29 (C=O), 1503.55 (NO ),
1240.20 (C–O–C), 1175.78 (C=S), 1061.64 (C–N); H
max
−
1
Orange solid; Yield: 74%; mp 142.0–145.8 °C; FTIR ν
max
2
1
−1
(
cm ): 3075.65 (C–H), 1704.53 (C=O), 1541.72 (NO ),
2
1
1
184.29 (C=S), 1080.83 (C–N); H NMR (400 MHz,
NMR (400 MHz, DMSO-d ): δ 3.88 (s, 3H, OCH ),4.26
6
H
3
DMSO-d ): 4.25 (s, 3H, CH ), 7.34 (s, 1H, CHolefinic), 7.44
(s, 3H, CH ), 7.10 (s, 1H, CHolefinic), 7.20–7.28 (m, 3H,
6
3
3
(
8
(
8
t, JHH = 8.72, Hz, 2 H, ArH), 7.58–7.62 (dd, JHH = 5.8,
.72, Hz, 2H, ArH), 8.39 (d, JHH = 8.88 Hz, 1H, ArH), 8.62
d, JHH = 7.48 Hz, 1H, ArH), 8.67–8.69 (dd, J = 2.04,
ArH), 7.55 (t, JHH = 7.64 Hz, 1H, ArH), 8.40 (d, JHH =
8.92 Hz, 1H, ArH), 8.65–8.68 (dd, JHH = 1.8, 8.92 Hz, 1H,
ArH), 8.84 (s, 1H, CHpyrazole), 8.98 (d, JHH = 1.8 Hz, 1H,
HH
13
13
.88 Hz, 1H, ArH), 8.92 (s, 1H, CHpyrazole); C NMR (100
ArH); C NMR (100 MHz, DMSO-d ): δ 31.32, 55.52,
6
C
MHz, DMSO-d ): δ 31.32, 107.11, 115.75, 116.11, 116.33,
112.10, 114.98, 118.12, 118.97, 120.92, 121.14, 124.84,
126.68, 127.92, 131.20, 131.31, 135.20, 140.58, 142.75,
145.94, 153.67, 156.65, 168.96, 176.64, 194.97; HRMS m/z
(pos): 548.1798 C H N O S Na (calcd: 548.1790).
6
C
1
1
1
16.72, 120.33, 121.16, 124.57, 126.45, 126.74, 126.94,
27.72, 127.99, 130.74, 130.82, 132.16, 135.10, 142.82,
45.01, 146.13, 154.08, 168.86, 176.64, 194.90; HRMS m/z
2
2
15
5
7 2
(
pos): 536.0775 C H N O S FNa (calcd: 536.0770).
21 12 5 6 2
(
Z)-3-Acetyl-5-((3-(2,4-dichlorophenyl)-1-(2,4-
(
1
Z)-3-Acetyl-5-((1-(2,4-dinitrophenyl)-3-(4-hydroxyphenyl)-
H-pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one (6d)
dinitrophenyl)-1H-pyrazol-4-yl) methylene)-2-
thioxothiazolidin-4-one (6g)
Orange solid; Yield: 65%; mp 146–147 °C; FTIR ν
Orange solid; Yield: 79%; mp 71.6–75.2 °C; FTIR ν
(cm ): 3084.67 (C–H), 1691.78 (C=O), 1532.18 (NO ),
max
max
−
1
−1
(
cm ): 3405.96 (O–H), 3096.04 (C–H), 1704.81 (C=O),
2

Medicinal Chemistry Research
1
177.34 (C=S), 1075.55 (C–N); 1H NMR (400 MHz,
DMSO-d ): δ 4.26 (s, 3H, CH ), 6.99 (s, 1H, CHolefinic),
(0.03 mmol) were dissolved in 15 mL of acetic acid and
refluxed for 3 h. Once TLC confirmed the completion of the
reaction, solvent was removed using a high vacuum. The
reaction mixture was then poured on crushed ice and the
obtained solid was filtered and purified using column
chromatography.
6
H
3
7
1
8
2
8
.47 (d, JHH = 8.24 Hz, 1H, ArH), 7.61–7.63 (dd, JHH
.8, 8.24 Hz, 1H, ArH), 7.86 (d, JHH = 1.76 Hz, 1H, ArH),
.40 (d, JHH = 8.92 Hz, 1H, ArH), 8.68–8.71 (dd, JHH
.32, 8.92 Hz, 1H, ArH), 8.91 (d, J = 2.44 Hz, 1H, ArH),
=
=
HH
13
.97 (s, 1H, CHpyrazole); C NMR (100 MHz, DMSO-d6):
δC 30.93, 117.90, 119.81, 121.16, 123.61, 126.15, 129.66,
(Z)-3-Acetyl-5-benzylidene-2-thioxothiazolidin-4-one (8a)
1
31.91, 133.00, 133.49, 133.75, 135.08, 135.58, 137.49,
1
43.02, 146.34, 150.72, 152.31, 168.89, 176.63, 194.75;
Yellow solid; Yield: 93%; mp 141.2–145.4 °C; FTIR ν
max
−
1
HRMS m/z (pos): 585.1322 C H N O S Cl Na (calcd:
5
(cm ): 3076.31 (C–H), 1704.57 (C=O), 1185.29 (C=S),
21
11
5
6
2
2
1
85.1327).
1082.27 (C–N); H NMR (400 MHz, DMSO-d ): δ 4.26
6
H
(
s, 3H, CH ), 7.50–7.61 (m, 5H, ArH), 7.64 (s, 1H, CH
3 ole-
1
3
(
1
Z)-3-Acetyl-5-((1-(2,4-dinitrophenyl)-3-(4-methoxyphenyl)-
H-pyrazol-4-yl)methylene)-2-thioxothiazolidin-4-one (6h)
finic); C NMR (100 MHz, DMSO-d ): δ 30.75, 125.54,
6
C
129.42, 130.44, 130.71, 131.58, 132.94, 169.41, 176.66,
1
95.72; HRMS m/z (pos): 288.0928 C H NO S Na (calcd:
12 9 2 2
Orange solid; Yield: 88%; mp 70.4–73.8 °C; FTIR ν
288.0929).
max
−1
(
1
cm ): 3075.83 (C–H), 1708.98 (C=O),1536.98 (NO ),
250.24 (C–O–C), 1173.27 (C=S), 1075.91 (C–N); H NMR
2
1
(Z)-3-Acetyl-5-(4-methoxybenzylidene)-2-thioxothiazolidin-
4-one (8b)
(
400 MHz, DMSO-d ): δ 3.87 (s, 3H, OCH ), 4.26 (s, 3H,
6
H
3
CH ), 7.12 (d, J = 8.60 Hz, 2H, ArH), 7.34 (s, 1H, CHole-
3
HH
finic), 7.47 (d, JHH = 8.56 Hz, 2H, ArH), 8.37 (d, JHH = 8.92
Hz, 1H, ArH), 8.64–8.67 (dd, JHH = 2.12, 8.92 Hz, 1H, ArH),
Yellow solid; Yield: 80%; mp 195–196 °C; FTIR ν
max
−
1
(cm ): 3054.83 (C–H), 1682.73 (C=O), 1257.71 (C–O–
1
8
.85 (s, 1H, CHpyrazole), 8.89 (d, JHH = 2.44 Hz, 1H, ArH);
C), 1169.05 (C=S), 1073.09 (C–N); H NMR (400 MHz,
1
3
C NMR (100 MHz, DMSO-d ): δ 31.31, 55.29, 114.57,
DMSO-d ): δ 3.83 (s, 3H, OCH ), 4.26 (s, 3H, CH ), 7.12
6
C
6
H
3
3
1
1
1
16.62, 121.12, 122.44, 126.05, 126.61, 127.87, 129.90,
31.00, 135.11, 142.70, 145.91, 154.90, 160.29, 160.29,
62.65, 168.92, 176.64, 194.92; HRMS m/z (pos): 548.1510
(d, JHH = 8.70 Hz, 2H, ArH), 7.58 (d, J = 8.70 Hz, 2H,
HH
1
3
ArH), 7.61 (s, 1H, CHolefinic); C NMR (100 MHz, DMSO-
d6): δC 31.63, 55.54, 115.08, 122.18, 125.45, 131.88,
132.67, 161.33, 169.38, 176.65, 195.47; HRMS m/z (pos):
C H N O S Na (calcd: 548.1502).
2
2 15 5 7 2
3
18.0330 C H NO S Na (calcd: 318.0331).
13 11 3 2
(
Z)-3-Acetyl-5-((3-(3,4-dichlorophenyl)-1-(2,4-
dinitrophenyl)-1H-pyrazol-4-yl) methylene)-2-
thioxothiazolidin-4-one (6i)
(Z)-3-Acetyl-5-(2-chlorobenzylidene)-2-thioxothiazolidin-4-
one (8c)
Yellow solid; Yield: 61%; mp 80.2–84.0 °C; FTIR ν
Yellow solid; Yield: 87%; mp 170.0–174.5 °C; FTIR ν
max
max
−1
−1
(
cm ): 3076.56 (C–H), 1705.63 (C=O), 1531.05 (NO ),
(cm ): 3054.53 (C–H), 1693.30 (C=O), 1184.14 (C=S),
2
1
1
1180.64 (C=S), 1069.12 (C–N); H NMR (400 MHz,
1040.24 (C–N); H NMR (400 MHz, DMSO-d ): δ 4.26
6
H
DMSO-d ): δ 4.26 (s, 3H, CH ), 7.35 (s, 1H, CHolefinic), 7.56
(s, 3H, CH ), 7.49–7.54 (m, 3H, ArH), 7.64 (t, J = 5.20
Hz, 1H, ArH), 7.74 (s, 1H, CHolefinic); C NMR (100 MHz,
6
H
3
3 HH
1
3
(d, JHH = 8.64 Hz, 1H, ArH), 7.79 (d, JHH = 15.72 Hz, 1H,
ArH), 7.82 (s, 1H, ArH), 8.87 (d, JHH = 8.32 Hz, 1H, ArH),
DMSO-d ): δ 30.93, 126.07, 128.24, 129.05, 129.27,
6
C
8
8
1
1
1
1
5
.40 (d, JHH = 8.88 Hz, 1H, ArH), 8.68–8.71 (dd, JHH = 2.04,
130.41, 130.81, 132.07, 134.73, 169.10, 176.64, 195.51;
.72 Hz, 1H, ArH), 8.93 (d, J = 2.2 Hz, 1H, ArH), 8.95 (s,
HRMS m/z (pos): 321.0585 C H NO S ClNa (calcd:
HH
12
8
2 2
1
3
H, CHpyrazole); C NMR (100 MHz, DMSO-d ): 28.95,
321.0591)
6
11.38, 113.88, 114.81, 119.54, 119.61, 120.71, 122.37,
23.21, 124.08, 125.71, 126.16, 131.44, 133.89, 136.93,
43.73, 148.40, 166.21, 176.67, 189.28; HRMS m/z (pos):
85.0106 C H N O S Cl Na (calcd: 585.0093).
(Z)-3-Acetyl-5-(3-chlorobenzylidene)-2-thioxothiazolidin-4-
one (8d)
21
11
5
6
2
2
Yellow solid; Yield: 92%; mp 165–167 °C; FTIR ν
max
−
1
General procedure for the synthesis of (Z)-3-acetyl-
-benzylidene-2-thioxothiazolidin-4-ones (8a–e)
(cm ): 3078.09 (C–H), 1704.77 (C=O), 1181.24 (C=S),
1
5
1079.59 (C–N); H NMR (400 MHz, DMSO-d ): δ 4.26
6
H
(
s, 3H, CH ), 7.53–7.57 (m, 3H, ArH), 7.63 (s, 1H, ArH)
3
1
3
Appropriate aromatic aldehydes (7a–e) (0.01 mmol), rho-
danine (5) (0.012 mmol) and anhydrous sodium acetate
7.68 (s, 1H, CHolefinic); C NMR (100 MHz, DMSO-d ): δC
6
29.70, 127.36, 128.10, 129.76, 130.21, 130.26, 131.22,

Medicinal Chemistry Research
control sample without inhibitors by the formula:
%
inhibition¼
ꢂꢀ
ꢁ
ꢃ
absorbance of blankÀ
=
absorbance of blank à 100
absorbance of compound
Determination of α-amylase inhibitory activity
The α-amylase inhibitory activity of the synthesised com-
pounds were determined using a modified method described
by Shai et al. (2010), in which 250 µL of each synthesised
compound, or acarbose, dissolved in ethanol to obtain dif-
ferent concentrations (15, 30, 60, 120 and 240 µg/mL), was
mixed with 500 µL of porcine pancreatic amylase (2 U/mL)
in phosphate buffer (100 mM, pH 6.8) and incubated for 10
min at 37 °C. Thereafter, 50 µL of 1% starch, dissolved in
the same buffer, was added to the reaction mixture and the
samples were incubated again at 37 °C. After 30 min,
dinitrosalicylate (DNS) colour reagent (1 mL) was and the
samples were allowed to boil for 10 min. Absorbance was
then measured at 540 nm. The results were expressed as a
percentage of the control by the formula below:
Fig. 3 Ramachandran plot
1
34.01, 135.12, 169.27, 176.66, 195.38; HRMS m/z (pos):
3
19.0590 C H NO S ClNa (calcd: 319.0591).
1
2
8
2 2
%
inhibition¼
ꢂꢀ
ꢁ
ꢃ
(
Z)-3-Acetyl-5-(2,5-dimethoxybenzylidene)-2-
absorbance of blankÀ
thioxothiazolidin-4-one (8e)
=absorbance of blank à 100
absorbance of compound
Yellow solid; Yield: 96%; mp 140–142 °C; FTIR ν
max
−
1
(
cm ): 3067.11 (C–H), 1704.37 (C=O), 1184.97 (C=S),
Homology modelling
1
1
184.97 (C–O), 1080.99 (C–N); H NMR (400 MHz,
DMSO-d ): δH 3.83 (s, 6H, OCH ), 4.26 (s, 3H, CH ),
The protein sequence of Saccharomyces cerevisiae α-glu-
cosidase was obtained from UniProt (http://www.uniprot.
org) with accession number P53341. X-ray crystal structure
of S. cerevisiae isomaltase (PDB ID code 3AJ7 of 1.30 Å
resolution) having a 72.4% sequence identity with S. cere-
visiae α-glucosidase was selected as a template for the
homology modelling as employed in previous studies
(Guerreiro et al. 2013; Khan et al. 2014). The query and
template sequence were aligned using the pairwise sequence
alignment tool of ClustalOmega. The three-dimensional
6
3
3
13
7
.12–7.21 (m, 3H, ArH), 7.61 (s, 1H, CHolefinic); C NMR
(
100 MHz, DMSO-d ): δC 30.92, 55.52, 56.03, 113.12,
6
1
1
13.81, 118.47, 121.72, 125.62, 126.62, 152.52, 153.15,
69.32, 184.62, 195.81; HRMS m/z (pos): 346.1361
C H NO S Na (calcd: 346.1372).
1
4
13
4 2
Antidiabetic activity testing methods
Determination of α-glucosidase inhibitory activity
(3D) structural model of α-glucosidase for S. cerevisiae was
The α-glucosidase inhibitory activities for the synthesised
compounds were determined using a modified method
described by Ademiluyi and Oboh (2013). Briefly, 100 μL
of 1.0 U/mL of α-glucosidase, in a phosphate buffer (100
mM, pH 6.8), with 50 μL of each synthesised compounds,
or acarbose, dissolved in ethanol to obtain different con-
centrations (15, 30, 60, 120 and 240 µg/mL), was incubated
in a 96-well plate at 37 °C for 20 min. Thereafter 50 μL of 5
mM of p-nitrophenyl-α-D-glucopyrnoside (pNPG) solution
was added to the reaction mixture and was further incubated
at 37 °C for 30 min. The absorbance was then measured at
obtained by homology modelling using MODELER 9.19
integrated in Chimera (Sievers and Higgins 2014; Webb
and Sali 2014; Pettersen et al. 2004). The model with the
lowest discrete optimized protein energy (DOPE) score was
selected for validation.
The modelled S. cerevisiae α-glucosidase structure was
validated by generating the phi/psi Ramachandran plot (Fig.
3) distributions obtained by using protein preparation
wizard of Schrodinger modelling suite and RAMPAGE
Ramachandran plot analysis server. In addition, the sig-
nificance consistency between the modelled structure and
the selected template was evaluated using ProSA server
4
05 nm and the results expressed as a percentage of the

Medicinal Chemistry Research
Scheme 1 Synthetic pathway of
pyrazole–rhodanine hybrids
(6a–i)
2 4 3
Reagents and conditions: (a) EtOH, H SO , RT, 1 h; (b) DMF, POCl , 60 °C, 5 h;
(
c) AcONa, AcOH, EtOH, reflux, 3 h.
(
Sastry et al. 2013; Lovell et al. 2003; Wiederstein and
Sippl 2007).
Molecular docking method
Scheme 2 Synthesis of substituted phenyl–rhodanine hybrids (8a–e)
Prior to molecular docking, a total of 5000 steps of energy
minimization were performed for the modelled structure
using Amber14 force field of AMBER 14 molecular
dynamic simulation package (Case et al. 2014; Trott and
Olson 2010). Molecular docking was performed using
Autodock Vina software package integrated in Chimera
software. The software’s default settings were used for the
docking procedures. A Chimera graphical user interface tool
was used to assign Geister partial charges during the docking
process. The binding site pocket of S. cerevisiae α-glucosi-
dase for molecular docking was defined by a grid box
around key active site residues Glu276, Asp214, Asp349,
Arg439, Arg312, Asp408, Gln350, Ser438, Val108, Phe157,
Phe177 and Phe300 of the minimized α-glucosidase mod-
elled structure. The X, Y and Z dimensions were defined as
and 2. The MH reported herein consists of two steps, the
synthesis of the pyrazole aldehyde derivatives which is a
key intermediate for further transformation, followed by
coupling of these derivatives with commercially available
rhodanine. The pyrazole aldehydes (4a–i) were achieved
by cyclisation of the synthesized hydrazone intermediates
(3a–i). Specifically, the reaction of appropriate substituted
acetophenones (1a–i) with 2,4-dinitrophenylhydrazine (2)
was conducted in the presence of sulfuric acid in absolute
ethanol and water to afford the hydrazone intermediates
(3a–i). The synthesized hydrazone intermediates then
undergo a Vilsmeier–Hack reaction (DMF-POCl ) at 80 °C
3
for 5 h to afford pyrazole aldehydes (4a–i) (Andleeb et al.
2016). The pyrazole–rhodanine hybrid compounds (6a–i)
were prepared via a Knoevenagel condensation of rhoda-
nine (5) with the synthesised pyrazole aldehydes (4a–i),
by refluxing both in acetic acid, in the presence of a
catalytic amount of sodium acetate for 3 h. The same
mechanistic pathway and reaction conditions were used to
synthesize (Z)-3-acetyl-5-benzylidene-2-thioxothiazolidin-
2
6.0448, 24.4198, and 27.4387, respectively whereas the X,
Y and Z centres were defined by 34.4671, 46.5256 and
9.559, respectively. Polar hydrogens were assigned for all
4
compounds. The docked complex structures were visualized
by the UCSF chimera graphical user interface and molecular
interactions were also visualized using Molegro Molecular
Viewer (http://www.clcbio.com).
4-ones (8a–e) via the reaction of various substituted aro-
matic aldehydes (7a–e) with rhodanine.
Results and discussion
After purification, the structure of all the synthesized
compounds were characterized using spectroscopic techni-
ques (IR, HRMS, H and C NMR). For example, the H
NMR of compound 6b exhibits three singlets. The most
characteristic singlet appeared at δ 7.37 ppm which corre-
sponds to the one vinylic proton (=CH) between the
1
13
1
Chemistry
Synthesis of the molecular hybrids (6a–i) and simple rho-
danine analogues (8a–e) has been outlined in Schemes 1

Medicinal Chemistry Research
Additionally, these assignments were supported by het-
eronuclear multiple bond correlation (HMBC) correlations,
selected HMBC correlations used in the structural elucida-
tion of 6b are provided in Fig. 4. The structure of 6b was
1
3
further confirmed by the C NMR in which the most
characteristic peak at δ 31.32 ppm corresponding to the
methyl carbon, and the peak at δ 121.15 ppm which cor-
responds to vinylic carbon. The most deshielded peaks at δ
194.93 and 168.91 ppm correspond to the thiocarbonyl
(which is correlation with acetyl group protons) and car-
bonyl (which is correlation with vinylic proton) in the
rhodanine ring, respectively. Whereas, the acetyl carbonyl
functional group resonated in the region 176.64 ppm, these
1
3
peaks, along with the other 16 peaks seen in the C NMR
were confirmed using HMBC and HSQC spectra.
These results suggested Knoevenagel condensation
obtained between pyrazole aldehyde and rhodanine, as well
as the presence of the acetyl carbon peak and the methyl
1
13
Fig. 4 HBMC ( H→ C) correlation of compound 6b
1
3
1
carbon peak in the C NMR and acetyl protons peak in H
NMR spectra confirms that acetylation occurred in the
synthesis of the pyrazole–rhodanine derivatives. Further-
more, the IR spectrum of compound 6b showed character-
istic absorption peak exhibited at 1700.31 and 1172.16
pyrazole and rhodanine moieties. The other two singlets are
seen at δ 8.92 ppm and δ 4.26 ppm which correspond to the
proton in the pyrazole ring, and three protons of N-acetyl
group attached to the rhodanine respectively. J coupling
was then used to assign the protons present in the 2,4-
dinitrophenyl ring, which were seen to be highly deshiel-
ded, and therefore downfield, due to the presence of the two
nitro groups. The doublet at δ 8.40 ppm corresponds to H-6
and is ortho coupled to H-5 (JHH = 8.88 Hz). On the other
hand, H-5 is a doublet of doublets at δ 8.67 ppm and is
ortho coupled to H-6 and meta coupled to H-3 (JHH = 2.44,
−
1
cm due to the (C=O) and (C=S) groups, respectively.
The successful synthesis of 6b was further supported
by HRMS in which the molecular-ion peak was found at
m/z = 551.9636.
In vitro α-glucosidase and α-amylase inhibitory
activity
8
.88 Hz). H-3 appeared as a doublet at δ 8.91 ppm (JHH
=
Two different molecular hybrid scaffolds were synthesized,
namely the pyrazole–rhodanine scaffolds (6a–i) and the
phenyl–rhodanine scaffolds (8a–e). This was done to allow
the comparison of the two different scaffolds, in terms of
their antidiabetic activity, with the hope that the pyrazole–
rhodanine scaffold would have a significantly better activity
as it possesses two pharmacophores. The antidiabetic
activities of the synthesized compounds were determined
via in vitro testing against the α-glucosidase and α-amylase
enzymes, from which the IC50 values were determined and
compared to the standard reference drug, acarbose. The IC50
values of each compound obtained from both inhibitory
studies summarized in Table 1.
2
.44 Hz) and is the most downfield proton due to it being in-
between two nitro groups. A multiplet is also seen in the
region of δ 7.54–7.57 ppm, which corresponds to the four
protons in the chloro substituted phenyl ring. Along with
coupling constants, COSY was also used to confirm the
assignment of the proton peaks, especially those of the 2,4-
dinitrophenyl ring.
Although, hypothetically, two geometric isomers, E and
Z, are probable for the Knoevenagel condensed products, all
the compounds displayed only the Z-configuration, as
anticipated from reported studies, which is confirmed by the
peak for =CH appeared between 6.98 and 7.74 ppm in the
1
H NMR spectra, as the vinylic proton is more deshielded in
For the inhibition of the α-glucosidase enzyme, the IC50
values of the synthesized compounds (6a–i and 8a–e) were
the Z-isomer than the E-isomer due to the magnetic aniso-
tropy effects of the carbonyl group on the vinylic proton.
Hence, Z-configuration due to the high degree of thermo-
dynamic stability attributed to intramolecular hydrogen
bond that can be formed between the rhodanine carbonyl
oxygen atom and the =CH group of hydrogen atom
−5
compared to that of acarbose (IC = 9.667 × 10 mol/L).
5
0
Compounds 6a–i exhibited better α-glucosidase inhibitory
activity compared to acarbose, with IC values ranging
5
0
from 2.259 × 10− to 1.160 × 10 mol/L. Although, the IC
6
−4
50
−4
values of compounds 8a–e ranged between 3.086 × 10 and
> 9.494 × 10− mol/L. For the α-amylase inhibitory activity,
the synthesized compounds exhibited good inhibition with a
4
(
Mendoza et al. 2014; Kumar and Nanjan 2010; Mandal
et al. 2016).

Medicinal Chemistry Research
Table 1 In vitro IC50 values of α-glucosidase and α-amylase inhibition activities of synthesised compounds 6a–i and 8a–e
compounds 6a-i and 8a-e.
IC50 (mol/L)
Comp.
Structures
α-Glucosidase α-Amylase
6
a
2
.854 x 10-5
3.227 x 10-4
7.729 x 10 -4
3.491 x 10-5
1.065 x 10-4
2.099 x 10-5
6
b
3.093 x 10-4
6.377 x 10-5
6
c
6
d
4.116 x 10-5
3.680 x 10-4
6
e

Medicinal Chemistry Research
2.259 x 10-6
6.183 x 10-5
3.080 x 10-4
6
f
1.693 x 10-4
1.325 x 10-4
2.231 x 10-4
6
g
1.160 x 10-4
6.383 x 10-5
6
h
6
i
>9.494 x 10-4
3.086 x 10-4
3.809 x 10-4
6.251 x 10-4
3.439 x 10-4
>8.395 x 10-4
8
a
8
b
8
c
>8.395 x 10-4 >8.395 x 10-4
8
d
7.623 x 10-4
9.667 x 10-5
3.114 x 10-4
1.038 x 10-4
8
e
Standard
Acarbose

Medicinal Chemistry Research
range of IC50 values from 6.377 × 10 to 7.730 × 10−4 mol/
−
5
the substituted phenyl moiety, the resulting compounds, 8a–e,
displayed less inhibitory activity against the α-glucosidase
−4
−4
L and 3.114 × 10 to 6.251 × 10 mol/L, for 6a–i and 8a–
e, respectively, with acarbose having an IC value of
.038 × 10 mol/L. Unlike the α-glucosidase inhibitory
enzyme. However, in the case of compound 8b (IC =
50
50
−4
−4
1
3.086 × 10 mol/L), where the 4-methoxy group was present,
activity, only a slight difference is observed between the α-
amylase inhibitory activity of the pyrazole–rhodanine deri-
vatives (6a–i) and the substituted phenyl–rhodanine deri-
vatives (8a–e). However, there were certain pyrazole–
rhodanine derivatives that showed good inhibitory activity.
This proves that the presence of the pyrazole ring, and
possibly the nitro groups present on the phenyl ring adds to
the inhibitory activity of the pyrazole–rhodanine series.
Although all the structural features, for instance, the
pyrazole ring and N-acetyl rhodanine moieties, are plea-
santly playing their part in signifying the inhibitory poten-
tial, the variation of the substituents in the phenyl ring on
the pyrazole moiety was seen to result in a large range of
IC50 values. A structural assessment was conducted to
examine the effects of the different substitution at the aryl
moiety (R) of the pyrazole–rhodanine derivatives.
the derivative showed the highest inhibition of the α-gluco-
sidase enzyme. The difference in activity displayed between
the two series of derivatives suggests that the pyrazole moiety
might be helpful in the binding of the pyrazole–rhodanine
derivatives with the active site of the α-glucosidase enzyme.
In the case of the α-amylase inhibition activity, com-
pound 6d, which is the para-hydroxy substituted pyrazole–
rhodanine derivative, exhibited the highest inhibitory
activity, with an IC50 value of 6.377 × 10− mol/L, which is
approximately 1.5-fold higher than acarbose (IC =
5
50
1.038 × 10− mol/L). The 4-methoxy substituted derivative
4
(6h) had the second highest inhibitory activity, with an IC
50
value of 1.325 × 10− mol/L. When comparing the position
of the electron donating methoxy group attached to the
phenyl ring on the pyrazole moiety, the 4-methoxy sub-
stituted derivative (6h) was found to be more active than the
2-methoxy (6f) and 3-methoxy (6e) derivatives which had
4
In terms of the α-glucosidase inhibitory activity, com-
−
6
−4
−4
pounds 6f (IC = 2.259 × 10 mol/L), 6e (IC = 4.116 × 10
IC50 values of 3.080 × 10
and 3.680 × 10 mol/L,
5
0
50
−5
−4
mol/L) and 6h (IC = 1.160 × 10 mol/L), which all
respectively. In contrast, when looking at the electron
withdrawing chloro group on the phenyl ring on the pyr-
50
contain a methoxy group at phenyl ring, showed potent
activity, with 6f having a 42-fold stronger potency than the
azole moiety, the 2,4-dichloro substituted derivative 6g had
−5
−4
standard acarbose (IC = 9.667 × 10 mol/L). The enhanced
a higher activity (IC = 1.693 × 10 mol/L) than the 3,4-
50
50
activity of compound 6f may be due to the presence of an
electron donating methoxy group at the ortho position, which
may be responsible for its binding with the α-glucosidase
enzyme. In comparison, the relatively decreased activity of
compounds 6e and 6h may be due to the change in the
position of methoxy group from ortho to meta and ortho to
para, respectively, which were approximately 18-fold and 51-
fold less potent than that of compound 6f, respectively. The
replacement of the methoxy group with an electron with-
drawing chloro group at ortho position (6b) reduced the
inhibitory efficacy, but still showed an inhibitory activity 2-
fold higher than the standard inhibitor. The ortho position was
again favoured for the chloro substituents, with the 2-chloro
substituted phenyl of pyrazole derivative (6b) displaying good
inhibitory activity, with an IC50 value of 3.491 × 10− mol/L,
while the 2,4-dichloro (6g) and 3,4-dichloro (6i) substituted
dichloro (6i) and 2-chloro (6b) derivatives (IC50 value of
2.231 × 10 and 7.730 mol/L, respectively). Compounds 6a,
which is an unsubstituted derivative, and 6c, the 4-fluoro
substituted derivative, were found to be moderately active
(IC50 values of 3.227 × 10− and 3.093 mol/L respectively).
Furthermore, all the substituted phenyl–rhodanine deriva-
tives (8a–e) displayed moderate to weak activity with IC
4
50
values ranging from 3.114 × 10− to 6.251 × 10 mol/L.
Thus, it can be seen from the results that the presence of the
pyrazole moiety in the pyrazole–rhodanine derivatives is
essential for the potent inhibitory activity compared to the
substituted phenyl–rhodanine derivatives for both α-gluco-
sidase and α-amylase inhibition. In general, the present
study explored a new class of α-glucosidase inhibitors
based on pyrazole–rhodanine compounds. Figure 5 pro-
vides a concise summary of the structural analysis poten-
tially related to the activity.
4
−4
5
−5
derivatives displayed IC values of 6.183 × 10 mol/L and
5
0
−5
6
.383 × 10 mol/L, respectively. This also shows that the
mono chloro-substituted derivative was favoured over a di-
chloro substituted derivative. It is also interesting to note that
the unsubstituted derivative (6a) showed the second highest
Molecular docking
The homology model of glucosidase was validated using
reported methods. Docking calculations of most active
inhibitors were carried out against the homology built
enzyme. For comparison, molecular docking of standard
inhibitor acarbose was also carried out.
−4
activity, with an IC50 value of 2.854 × 10 mol/L, which is
more than 3-fold better than that of acarbose. Compounds
−
5
containing para hydroxy (6d; IC = 2.099 × 10 mol/L) and
50
−4
para fluoro (6c; IC = 1.065 × 10 mol/L) substituted phe-
50
nyl rings on the pyrazole moiety were also found to be active.
On the other hand, when the pyrazole moiety was changed to
Analysis of the binding profile of acarbose in the active
site of α-glucosidase (PDB ID: 5NN8) highlights significant

Medicinal Chemistry Research
Fig. 5 Structural analysis of
substituted phenyl of pyrazole–
rhodanine derivatives as potent
antidiabetic agents
(interaction distance of 3.60/3.99, 3.90, 3.99 and 3.97 Å,
respectively). The second subunit of acarbose positioned in
the binding pocket of α-glucosidase forms a water bridge
between the surrounding solvent and donor residue Asp616
at an angle of 117.3° and 83.5° and donor–water distance of
2.85 Å.
The binding of acarbose to α-amylase (PDB ID: 2QV4)
is more comprehensive and stabilized by predominantly
hydrogen bond interactions (Fig. 7). The residues involved
include hydrogen bond donors: Gln63 and Arg195 (bond
distance = 1.69 and 2.11 Å; bond angle = 126.2° and
1
42.3°, respectively); as well as the hydrogen bond accep-
tors: Gln63, Ala106, Gly164, Glu233, Asp300 and His305
bond distance = 2.90, 1.87, 2.53, 2.68/2.47, 2.23/2.40 and
(
3
1
.09 Å, respectively; bond angle = 103.0°, 153.3°, 139.9°,
37.7/138.4°, 140.9/145.8° and 151.5°, respectively).
Similar to the binding of acarbose to α-glucosidase, the α-
amylase complex forms three water bridges with the sur-
rounding solvent and amino acid residues Asn105, Ile235
and Asn298 (distance between donor and water = 3.22,
Fig. 6 Acarbose interacting with active site residues of α-glucosidase
(
PDB ID: 5NN8)
3.66 and 2.90 Å, respectively; water angle = 101.4°, 83.56°
and 75.89°, respectively).
contribution by hydrogen bonds with two substrate subunits
subunit 1—represented as X , and subunit 2—X , where X
is the residue identifier) (Fig. 6). These interactions are
From elucidation of the binding profile of acarbose to the
two enzyme binding pockets, it is evident that to ensure
selective inhibition and maintain reversibility of ligand–
protein complexation there needs to be a significant con-
tribution from hydrogen bond interactions. The molecular
docking of synthesized ligands was performed on a
homology model of α-glucosidase built from S. cerevisiae
α-glucosidase (Fig. 8). The model was validated according
to the experimental method outlined in homolgy modelling
section.
(
a
b
observed between residues Asp91 , Gly123 , Trp126 ,
a
a
a
Cys127 , Asp282 , Asp518 , Arg600 , Asp616 and
a
b
b
b
b
His674b. The bond distance between the proton and
acceptor measured 2.55, 2.42, 2.87, 1.81/2.29, 2.27, 3.14/
3
.24, 1.89/2.97 and 3.14 Å, respectively; with a bond angle
of 113.5°, 161.3°, 105.6°, 142.9/110.0°, 145.7°, 136.3/
15.2°, 179.1/129.7°, 125.0° and 171.9°, respectively.
1
Acarbose is a pseudo tetrasaccharide comprising of four six-
membered rings linked in a single chain which facilitates a
hydrophobic interaction within the binding pocket between
residues Trp126a, Trp376b, Trp481b and Phe649b
Compounds 8a–e displayed no measurable activity
against α-glucosidase. The measured total polar surface area
(TPSA) of compounds 8a–e is significantly lower than that
for 6a–i which relates to the poor solubility of the 8a–e

Medicinal Chemistry Research
derivatives (Table 2). This reduces their bioavailability and
subsequent activity against α-glucosidase. As mentioned
earlier, an extensive hydrogen bond interaction network
facilitates potent inhibition of the enzyme, due to the lack of
polarity of the ligands there is poor interaction between the
ligand and protein.
Compounds 6a and 6f displayed the most potent inhi-
bitory activity against α-glucosidase. The binding interac-
tion profile of 6a (Fig. 9) comprises a significant hydrogen
bond contribution from the donor side chain of residues:
Arg212 (amine group), Thr215 (hydroxyl group), Asn241
(amine group), Asp349 (carboxyl group) and Arg439
(amine group) (bond distance = 3.70/2.59, 2.28, 2.20, 3.35
and 3.58 Å, respectively; bond angle = 104.6/104.2°,
130.8°, 132.2°, 115.2° and 109.8°, respectively) with the
oxygen of the nitro groups of the ligand. Three distinct
hydrophobic interactions were observed between the con-
jugated rings and residues Phe157 and Arg312 at a distance
of 3.64, 3.60 and 3.74 Å, respectively. A T-shaped π-
stacking interaction is measured between Phe177 at 5.05 Å
and angle of 74.43°. Compound 6f (Fig. 9) displays a
similar interaction profile; however, the binding of the
ligand is supported by a greater contribution from hydro-
phobic interactions. These include residue binding with
Phe157, Phe300, Pro309 and Arg312 (bond distance =
3.30/3.19/3.65/3.87, 4.00, 3.94 and 3.84 Å). Three hydro-
gen bonds exist between residues Arg212 (amine group),
Asn241 (amine group) and Asp349 (carboxyl group) and
Table 2 Docking scores of synthesised compounds 6a–i and 8a–e in
the homology model of α-glucosidase as well as their clogP and total
polar surface area
Entry Compound ID Docking scores (kcal/mol) clogP TPSA (Å2)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
6a
6b
6c
6d
6e
6f
−10.8
−11.1
−11.2
−10.9
−11.1
−11.0
−10.9
−10.7
−10.9
−8.00
−8.00
−8.30
−8.60
−8.20
4.176 158.300
4.829 158.300
4.315 158.300
3.294 180.220
4.185 167.530
5.483 158.300
5.483 158.300
4.185 167.530
5.483 158.300
2.443 46.610
2.434 37.380
3.088 37.380
3.088 37.380
2.451 55.840
6g
6h
6i
0
1
2
3
4
8a
8b
8c
8d
8e
Fig. 7 Active site residues of α-amylase (PDB ID: 2QV4) interacting
with acarbose
Fig. 8 3D representation of the
homology model of α-
glucosidase highlighting the
catalytic site (A)

Medicinal Chemistry Research
Fig. 9 Structure of 6a and 6f highlighting critical interactions in the enzyme active site
Fig. 10 Structure of 6b, 6d, 6e, 6g and 6i interacting with α-glucosidase binding pocket residues
oxygen of the nitro groups of the ligand (bond distance =
secondary amines and other alkoxide groups may improve
efficacy, i.e. inhibitory activity.
3
1
.19, 3.26 and 3.20 Å, respectively; bond angle = 152.8°,
05.2° and 115.3°, respectively). A T-shaped π-stacking is
Compounds 6b, 6d, 6e, 6g and 6i (Fig. 10) have
demonstrated improved inhibitory activity compared to the
drug standard acarbose. The binding landscape of each of
these compounds is similar to one another. An extensive
hydrophobic interaction system exists in each ligand–protein
complex, along with hydrogen bond interactions and a
exerted by the conjugate rings and residue Phe177 at 4.82 Å
and angle of 69.6°. The presence of the strongly activating
electron-donating group at position 2 of the conjugated ring
in 6f improves the potency of the antidiabetic drug candi-
date, thus substitution at this position with primary and

Medicinal Chemistry Research
Fig. 11 2D representation of 6c and 6h in the binding pocket of α-glucosidase
Table 3 Binding interactions of 6c and 6h
distinct T-shaped π-stack. The binding structure of 6b
incorporated: hydrophobic interactions with residues
Phe157, Arg312 and Tyr313 at a bond distance of 3.73/3.87/
Compound Residue
Interaction
type
Bond distance Bond
(Å) angle
identifier
3
.57, 3.70 and 3.85 Å, respectively; hydrogen bonds
6
c
Phe157
Hydrophobic 3.69, 3.23, 3.79, –
.90
between Arg212 (amine group), Thr215 (hydroxyl group),
Asn241 (amine group) and Asp349 (carboxyl group) with
the ligands oxygen nitro groups (bond distance = 3.34/2.48,
3
Phe177
Pro309
Arg312
Arg212
Hydrophobic 3.49, 3.87
Hydrophobic 3.85
–
–
–
2
.23, 1.92 and 3.64 Å, respectively; bond angle = 113.4/
Hydrophobic 3.71
1
16.2°, 129.8°, 166.7° and 110.7°, respectively); as well as a
Hydrogen
bond
3.22, 2.65
113.6°,
141.3°
π-stack with amino acid Phe177 at 4.96 Å and angle of
1.76°. Compound 6d has five hydrophobic interactions
with Phe157, Phe300 and Arg312 at a bond distance of 3.32/
.76/3.80, 3.83 and 3.90 Å, respectively. The ligand acts as a
7
Asn241
Asp349
Hydrogen
bond
2.40
119.6°
3
Hydrogen
bond
2.98
123.7°
hydrogen bond acceptor facilitating four distinct hydrogen
bonds with Arg212 (amine group), Asn241 (amine group)
and
6h
Phe157
Phe177
Arg312
Arg212
Hydrophobic 3.22, 3.49, 3.68 –
Asp349
(carboxyl
group)
Hydrophobic 3.61
Hydrophobic 3.85
–
(
bond distance = 3.20/3.51, 2.12 and 3.19 Å, respectively;
–
bond angle = 152.7/112.6°, 130.4° and 117.3°, respec-
tively); as well as π-stacks with Phe177 at 4.81 Å and angle
of 67.3°. The binding of 6e in the binding pocket of α-
glucosidase is stabilized by six hydrophobic interactions, six
hydrogen bonds and a π-stack. The hydrophobic interactions
involve residues Phe157, Pro309 and Arg312 (bond dis-
tance = 3.78/3.68/3.79, 3.80 and 3.37/3.79 Å, respectively)
with a π-stacking with amino acid Phe177 (bond distance =
Hydrogen
bond
2.40
2.37
2.52
121.4°
Asn241
Asp349
Hydrogen
bond
118.4°
132.3°
Hydrogen
bond
5
.18 Å and bond angle = 77.08°). Hydrogens bonds origi-
hydrogen bonding is exerted through donor residues Arg212
(amine group), Asn241 (amine group) and Asp (carboxyl
group) at a bond distance of 3.17, 3.53 and 3.20 Å and bond
angle of 153.0°, 117.9° and 115.0°, respectively. Residues
Phe157, Pro309 and Arg312 contribute to the hydrophobic
network at a distance of 3.30/3.19/3.65/3.88, 3.91 and 3.84
Å, respectively. In addition, compound 6i has a unique
binding profile driven by a distinct electrostatic interaction
with chloride of the ligand and the carboxyl group of
Asp408 at distance 3.71 Å, donor angle = 137.3° and
nate from the proton donors Thr215 (side chain hydroxyl
group), Asn241 (side chain amine), Glu276 (side chain
carboxyl group), Arg312, Asp349 (side chain carboxyl
group) and Arg439 (side chain amine) with the nitro group
oxygen’s of the ligand (bond distance = 2.37, 1.89, 3.17,
2
1
.11, 3.16 and 3.49 Å, respectively; bond angle = 135.3°,
57.0°, 104.5°, 147.8°, 124.1° and 121.7°, respectively).
The compound 6g shares a π-stack with Phe177, bond dis-
tance of 4.82 Å and bond angle of 69.60°. Significant

Medicinal Chemistry Research
acceptor angle = 104.1°. Hydrophobic interactions with
Phe157, Phe177 and Pro309 stabilize the ligand–protein
complex (bond distance = 3.65/3.21/3.84/3.65/3.96, 3.50/
glucosidase) and hypertension (angiotensin I converting enzyme)
in vitro. Exp Toxicol Pathol 65:305–309
Agrawal YP, Agrawal MY, Gupta AK (2015) Design, synthesis and
evaluation of rhodanine derivatives as aldose reductase inhibitors.
Chem Biol Drug Des 85:172–180
3
.87 and 3.87, respectively). Hydrogen bonds between
Arg212, Asn241 and Asp349 enhance the selective binding
profile of 6i (bond distance = 3.23/2.73, 2.45 and 3.05 Å,
respectively; bond angle = 135.5/142.3°, 118.0° and 121.4°,
respectively).
Andleeb H, Tehseen Y, Shah SJA, Khan I, Iqbal J, Hameed S (2016)
Identification of novel pyrazole–rhodanine hybrid scaffolds as
potent inhibitors of aldose reductase: design, synthesis, biological
evaluation and molecular docking analysis. RSC Adv 6:77688–
7
7700
Compounds 6c and 6h have the least inhibitory activity
against α-glucosidase. The binding landscape of both
ligands lacks the presence of a π-stack (Fig. 11). Table 3
summarizes the highlights of the integral residue interac-
tions between the active site and ligand.
Bathini P, Kameshwari L, Vijaya N (2013) Antidiabetic effect of 2
nitro benzimidazole in alloxan induced diabetic rats. Int J Basic
Clin Pharma 2:814–818
Berube G (2016) An overview of molecular hybrids in drug discovery.
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Browlee M (2001) Biochemistry and molecular cell biology of diabetic
complications. Nature 14:813–820
Case DA, Babin V, Berryman J, Betz RM, Cai Q, Cerutti DS,
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Gusarov S, Homeyer N, Janowski P, Kaus J, Kolossvary I,
Kovalenko A, Lee TS, LeGrand S, Luchko T, Luo R, Madej B,
Merz KM, Paesani F, Roe DR, Roitberg A, Sagui C, Salomon-
Ferrer R, Seabra G, Simmerling CL, Smith W, Swails J, Walker
RC, Wang J, Wolf RM, Wu X, Kollman PA (2014) AMBER 14.
University of California, San Francisco, p 29–31
Chaudhry F, Naureen S, Choudhry S, Huma R, Ashraf M, Al-Rashida
M, Jahan B, Khan MH, Iqbal F, Munawar MA, Khan MA (2018)
Evaluation of α-glucosidase inhibiting potentials with docking
calculations of synthesized arylidene-pyrazolones. Bioorg Chem
77:507–514
Conclusions
Two series of rhodanine containing compounds were suc-
cessfully synthesized using the MH approach. Pyrazole–rho-
danine conjugates (6a–i) exhibited superior α-glucosidase
inhibitory activity to their simple rhodanine analogues (8a–e).
The most potent conjugate 6f (IC = 2.259 × 10 mol/L)
displayed 42-fold better than that of acarbose. Interestingly,
−
6
5
0
−5
the unsubstituted derivative 6a (IC = 2.854 × 10 mol/L)
50
showed the second highest α-glucosidase inhibitory activity,
Guerreiro LR, Carreiro EP, Fernandes L, Cardote TAF, Moreira R,
Caldeira AT, Guedes RC, Burke AJ (2013) Five-membered
iminocyclitol α-glucosidase inhibitors: synthetic, biological
screening and in silico studies. Biorg Med Chem 21:1911–1917
Hall BS, Bot C, Wilkinson SR (2011) Nifurtimox activation by try-
panosomal type I nitroreductases generates cytotoxic nitrile
metabolites. J Biol Chem 286:13088–13095
Hall BS, Wilkinson SR (2012) Activation of benznidazole by trypa-
nosomal type I nitroreductases results in glyoxal formation.
Antimicrob Agents Chemother 5:115–123
Ju KS, Parales RE (2010) Nitroaromatic compounds, from synthesis to
biodegradation. Microbiol Mol Biol Rev 74:250–272
Kaku K (2014) Efficacy of voglibose in type-2 diabetes. Expert Opin
Pharmacother 15:1181–1190
Kashtoh H, Muhammad MT, Khan JJ, Rasheed S, Khan A, Perveen S,
Javaid K, Atia-tul W, Khan KM, Choudhary MI (2016) Dihy-
dropyrano [2,3-c] pyrazole: novel in vitro inhibitors of yeast α-
glucosidase. Bioorg Chem 65:61–72
Kazeem MI, Adamson JO, Ogunwande IA (2013) Modes of inhibition
of α-amylase and α-glucosidase by aqueous extract of Morinda
lucida Benth Leaf. BioMed Res Int 2013:527570
which is also 3-fold better than that of acarbose. On the other
−
5
hand, only two derivatives, 6d (IC = 6.377 × 10 mol/L)
50
−4
and 6h (IC = 1.325 × 10 mol/L) displayed strong inhibi-
50
tory potential against α-amylase when compared to the stan-
dard inhibitor. Molecular docking studies further revealed
three essential pharmacophoric features that are critical to
exert α-glucosidase inhibition. This includes the nitro sub-
stituents on the phenyl ring; the hydrophobic bulk introduced
by the pyrazole and conjugated ring structures, and; the 2-
position of the unsubstituted phenyl ring (6a), in which the
introduction of a strongly activating electron donating sub-
stituent (6f) at this position has been noted to improve inhi-
bitory activity. Thus, only an electron donating substitution at
position 2 would be tolerated to maintain or improve the drug
candidate’s antidiabetic activity.
Acknowledgements The authors would like to acknowledge the sup-
port from the NRF South Africa (Grant UID: 99563) as well as the
CHPC in Cape Town for access to computational resources.
Kerru N, Bhaskaruni SVHS, Maddila S, Singh P, Jonnalagadda SB
(2017a) Synthesis and antioxidant evaluation of a new class of
thienopyrimidine–rhodanine hybrids. Lett Drug Des Discov
1
5:118–126
Kerru N, Singh P, Koorbanally N, Raj R, Kumar V (2017b) Recent
Compliance with ethical standards
advances (2015–2016) in anticancer hybrids. Eur J Med Chem
1
42:179–212
Conflict of interest The authors declare that they have no conflict of
Khan KM, Rahim F, Wadood A, Kosar N, Taha M, Lalani S, Khan A,
Fakhri MI, Junaid M, Rehman W (2014) Synthesis and molecular
docking studies of potent α-glucosidase inhibitors based on bis-
coumarin skeleton. Eur J Med Chem 81:245–252
Kumar BRP, Nanjan MJ (2010) Novel glitazones: design, synthesis,
glucose uptake and structure–activity relationships. Bioorg Med
Chem Lett 20:1953–1956
interest.
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