Synthesis and enzyme inhibitory kinetics of some novel 3-(substituted benzoyl)-2-thioxoimidazolidin-4-one derivatives as α-glucosidase/α-amylase inhibitors

Article abstract of DOI:10.1007/s00044-018-2170-4

The present work describes an efficient and convenient synthesis of a library of novel 3-(substituted benzoyl)-2-thioxoimidazolidin-4-ones (3a–j). The benzoyl isothiocyanates were treated with glycine in the presence of pyridine, the reactants got consumed giving a variety of thioxoimidazolidin-4-ones derivatives under mild reaction conditions. The structures of the compounds were determined by elemental analysis, FTIR, ¹H, ¹³C NMR and mass spectral data. The title compounds were tested for their potential to inhibit the activity of enzymes α-glucosidase and α-amylase. It was found that most of the derivatives showed good enzyme inhibitory activity while compound 3j exhibited excellent activity with IC₅₀ values 0.051 and 0.0082 mM for α-glucosidase and α-amylase, respectively. The presence of 3,5-di-NO₂ functional groups at aromatic ring in compound 3j play important role in enzyme inhibitory activity. The enzyme inhibitory kinetic analysis of the most potent derivative 3j revealed that it is a mixed type inhibitor of α-glucosidase with Ki and Ki? values 0.0339 and 0.1562 mM, respectively. It was further investigated that compound 3j formed reversible enzyme inhibitor complex with α-glucosidase. The cytotoxicity of all the synthesized compounds was also evaluated and results showed that none of these compounds displayed toxicity against brine shrimps. Based upon results, it is suggested that compound 3j may act as a lead structure for the development of most potent α-glucosidase inhibitors.

Full text of DOI:10.1007/s00044-018-2170-4

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2170-4
ORIGINAL RESEARCH
Synthesis and enzyme inhibitory kinetics of some novel 3-
(substituted benzoyl)-2-thioxoimidazolidin-4-one derivatives as α-
glucosidase/α-amylase inhibitors
Rabia Qamar1 Aamer Saeed1 Maria Saeed1 Babar Hussain Shah1 Zaman Ashraf2 Qamar Abbas3
Sung Yum Seo4
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Received: 5 January 2018 / Accepted: 19 March 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
The present work describes an efficient and convenient synthesis of a library of novel 3-(substituted benzoyl)-2-
thioxoimidazolidin-4-ones (3a–j). The benzoyl isothiocyanates were treated with glycine in the presence of pyridine, the
reactants got consumed giving a variety of thioxoimidazolidin-4-ones derivatives under mild reaction conditions. The
1
structures of the compounds were determined by elemental analysis, FTIR, H, 13C NMR and mass spectral data. The title
compounds were tested for their potential to inhibit the activity of enzymes α-glucosidase and α-amylase. It was found that
most of the derivatives showed good enzyme inhibitory activity while compound 3j exhibited excellent activity with IC50
values 0.051 and 0.0082 mM for α-glucosidase and α-amylase, respectively. The presence of 3,5-di-NO2 functional groups
at aromatic ring in compound 3j play important role in enzyme inhibitory activity. The enzyme inhibitory kinetic analysis of
the most potent derivative 3j revealed that it is a mixed type inhibitor of α-glucosidase with Ki and Kiʹ values 0.0339 and
0.1562 mM, respectively. It was further investigated that compound 3j formed reversible enzyme inhibitor complex with α-
glucosidase. The cytotoxicity of all the synthesized compounds was also evaluated and results showed that none of these
compounds displayed toxicity against brine shrimps. Based upon results, it is suggested that compound 3j may act as a lead
structure for the development of most potent α-glucosidase inhibitors.
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Keywords 2-thioxoimidazolidin-4-ones Benzoyl isothiocyanates Enzyme inhibitory kinetics Cytotoxicity
Introduction
Diabetes mellitus (DM) is a metabolic syndrome char-
acterized by congenital or acquired insulin secretion defi-
ciency and by declined receptivity of organs to secreted
insulin. Such deficit results in higher blood glucose level
which is damaging for the body’s systems (Matsui et al.
2010). DM is currently one of the most expensive and
* Aamer Saeed
asaeed@qau.edu.pk
aamersaeed@yahoo.com
troublesome chronic diseases that is prevalent along with
* Zaman Ashraf
obesity and ageing all over the world. About 5% of the
global population is affected by diabetes (WHO 2002) and
its treatment without any side effects is still a challenging
task to the medicinal community (Chakrabarti and Raja-
gopalan 2002; Kameswararao et al. 2003).
One therapeutic methodology to treat diabetes is to
decrease the post-prandial hyperglycemia so as to reduce
serious complications and mortality. The inhibitors of
carbohydrate-hydrolyzing enzymes α-glucosidase and α-
amylase retard carbohydrate digestion and delay overall
mzchem@yahoo.com
1
2
3
4
Department of Chemistry, Quaid-i-Azam University,
Islamabad 45320, Pakistan
Department of Chemistry, Allama Iqbal Open University,
Islamabad 44000, Pakistan
Department of Physiology, University of Sindh,
Jamshoro, Pakistan
Department of Biology, College of Natural Sciences, Kongju
National University, Gongju 314-701, Republic of Korea

Medicinal Chemistry Research
time of carbohydrate digestion, causing a decline in glucose
absorption and subsequently suppressing the rise in plasma
glucose concentration (Bhandari et al. 2008; DeFronzo et al.
2004). From this point of view, we dedicated our efforts to
search for more effective and safe inhibitors of α-glucosi-
dase and α-amylase for the treatment of diabetes. In this
regard, 2-thioxoimidazolidin-4-ones attracted our attention
because minor modifications in this class of compounds
made them physiologically active molecules with high
therapeutic efficacy.
2-thioxoimidazolidin-4-ones commonly known as 2-
thiohydantoins, five-membered multiheterocyclic scaffolds
(FMMH) featuring a reactive cyclic thiourea core has been
extensively debated for their pertinence as flexible starting
candidates in drug discovery (Vengurlekar et al. 2012). This
concern arose from the wide range of biological activities
displayed by these scaffolds and their frequent manifesta-
tion in screening hits (Baell and Holloway 2010). Studies
have been conducted to estimate their significance in drug
discovery in terms of their biological activity, chemical
reactivity, aggregation-based promiscuity, and electronic
properties (Mendgen et al. 2012).
A simple change in the substitution array on 2-
thiohydantoin nucleus often leads to incredible diverse
biological activities (Puszyńska-Tuszkanow et al. 2011)
such as anti-bacterial (Salhi et al. 2013), anti-cancer (Khatik
et al. 2011), anti-neoplastic (Carmi et al. 2006), anti-
mutagenic (Takahashi et al. 2005), antimicrobial (Kachha-
dia et al. 2005), as well as potent NADPH oxidase (NOX)
inhibitors (Bae et al. 2016) and as human CB1 cannabinoid
receptor (Muccioli et al. 2005). As part of our ongoing
interest in organo sulfur compounds, herein we reported
the synthesis of new diversely substituted 2-
thioxoimidazolidin-4-ones and their evaluation for α-glu-
cosidase and α-amylase inhibition potential.
5973 mass spectrometer and the elemental analyses were
conducted using a LECO-183 CHNS analyzer.
Synthesis of Acyl isothiocyanates (2), General
procedure
To a 100 ml round bottomed flask was added substituted
aromatic acid (0.2 mmol) in slight excess of thionyl chloride
(1.2 equiv.) and mixture was refluxed for 2 h. To freshly
prepared acid chloride, was added the solution of potassium
thiocyanate (0.2 mmol) in dry acetone and the mixture was
stirred for 1.5 h at room temperature, the precipitated
potassium chloride was removed by filtration to leave a
clear solution of acyl isothiocyanate derivative that was
further used in situ.
Synthesis of 3-(Substituted benzoyl)-2-thioxoimidazolidin-
4-ones (3a-j), General procedure
To a solution of freshly prepared acyl isothiocyanate
(0.2 mmol) in dry acetone, was added glycine (0.2 mmol)
and pyridine (0.2 ml). The reaction mixture was refluxed for
5–8 h with constant stirring until the starting material was
completely consumed as monitored by TLC. Upon com-
pletion, the reaction mixture was cooled to room tempera-
ture and poured onto crushed ice. The crude product was
collected by filtration, washed well with distilled water,
dried and crystallized from n-hexane.
3-(4-Methoxybenzoyl)-2-thioxoimidazolidin-4-one
(3a)
Orange solid; Yield: 80%; Rf: 0.48 (n-Hexane: Ethyl acet-
ate, 1:1); m.p: 124–125 °C; IR (pure, υ cm−1): 3322 (NH),
2930, 2845 (Csp3-H), 1655, 1690 (C=O), 1533, 1425
(Ar–C=C), 1290 (C=S), 1240, 1052 (C–O); 1H-NMR
(300 MHz, (CD3)2CO): δ (ppm) 10.68 (s, 1H, NH),
7.98–7.95 (m, 2H, Ar–H), 7.07–7.03 (m, 2H, Ar–H), 4.09
(s, 2H, CH2), 3.89 (s, 3H, OCH3); 13C-NMR (75.5 MHz,
(CD3)2CO): δ (ppm) 190.7 (C=S), 165.3 (C=O), 163.6
(COAr), 163.5, 130.2, 128.9, 114.2, 60.8 (Ar–OCH3), 57.7
(CH2); GC-MS: m/z (%) = 250.04 (100) [M+]; Anal. Calcd.
for C11H10N2O3S: C, 52.79; H, 4.03; N, 11.19; S, 12.81;
Found: C, 52.72; H, 4.11; N, 11.35; S, 12.88.
Materials and methods
Chemistry
Rf values were determined using aluminum pre-coated silica
gel plates Kiesel 60 F254 from Merck (Darmstadt, Ger-
many). Melting points of the compounds were measured in
open capillaries using Stuart melting point apparatus
(SMP3) and are uncorrected. The IR spectra were recorded
on FTS 3000 MX, Bio-Rad Merlin (Excalibur Model)
3-(4-Methylbenzoyl)-2-thioxoimidazolidin-4-one (3b) Dark
brown; Yield: 81%; Rf: 0.45 (n-Hexane: Ethyl acetate, 1:1);
m.p: 135–136 °C; IR (pure, υ cm−1): 3328 (NH), 2934,
2849 (Csp3–H), 1659, 1687 (C=O), 1537, 1430 (Ar–C=C),
1265 (C=S); 1H-NMR (300 MHz, (CD3)2CO): δ (ppm)
10.60 (s, 1H, NH), 7.91–7.88 (m, 2H, Ar–H), 7.41–7.37 (m,
2H, Ar–H), 4.30 (s, 2H, CH2), 2.40 (s, 3H, Ar–CH3); 13C-
NMR (75.5 MHz, (CD3)2CO): δ (ppm) 188.2 (C=S), 167.0
(C=O), 164.7 (COAr), 159.8, 134.2, 126.4, 114.4, 22.2
1
spectrophotometer as pure compounds. H and 13C NMR
spectra were run on a Bruker 300 MHz NMR spectrometer
in (CD3)2CO solvent using TMS as an internal standard.
Mass spectra were recorded on Agilent technologies 6890 N
gas chromatograph and an inert mass selective detector

Medicinal Chemistry Research
(Ar–CH3), 58.7 (CH2); GC-MS: m/z (%) = 234.05 (100) [M
+]; Anal. Calcd. for C11H10N2O2S: C, 56.39; H, 4.30; N,
11.96; S, 13.69; Found: C, 56.66; H, 4.39; N, 11.91; S,
13.53.
7.0 Hz, Ar–H), 7.59–7.50 (m, 2H, Ar–H), 4.11 (s, 2H,
CH2); 13C-NMR (75.5 MHz, (CD3)2CO): δ (ppm) 189.4
(C=S), 167.7 (C=O), 165.2 (COAr), 136.8, 134.4, 130.8,
129.7, 127.9, 59.4 (CH2); GC-MS: m/z (%) = 299.94 (100)
[M+]; Anal. Calcd. for C10H7BrN2O2S: C, 40.15; H, 2.36;
N, 9.36; S, 10.72; Found: C, 40.19; H, 2.44; N, 9.39; S,
10.55.
3-(1-Naphthoyl)-2-thioxoimidazolidin-4-one
(3c) Dark
brown solid; Yield: 75%; Rf: 0.42 (n-Hexane: Ethyl acetate,
1:1); m.p: 204–205 °C; IR (pure, υ cm−1): 3325 (NH), 1658,
1685 (C=O), 1537, 1424 (Ar–C=C), 1294 (C=S); 1H-
NMR (300 MHz, (CD3)2CO): δ (ppm) 11.45 (s, 1H, NH),
9.05 (d, 1H, J = 8.4 Hz, Ar–H), 8.91 (d, 1H, J = 8.7 Hz,
Ar–H), 8.51–8.48 (m, 1H, Ar–H), 8.33–8.30 (m, 1H,
Ar–H), 8.22–8.16 (m, 2H, Ar–H), 7.76 (d, 1H, J = 6.3 Hz,
Ar–H), 3.99 (s, 2H, CH2); 13C-NMR (75.5 MHz, (CD3)
2CO): δ (ppm) 170.9 (C=S), 168.0 (C=O), 167.3 (COAr),
134.4, 133.8, 131.5, 131.1, 130.4, 130.2, 130.0, 128.6,
127.5, 126.6, 51.5 (CH2); GC-MS: m/z (%) = 270.05 (100)
[M+]; Anal. Calcd. for C14H10N2O2S: C, 62.21; H, 3.73; N,
10.36; S, 11.86; Found: C, 62.25; H, 3.66; N, 10.41; S,
11.89.
3-(2-Flourobenzoyl)-2-thioxoimidazolidin-4-one
(3g)
Brown sticky oil; Yield: 53%; Rf: 0.40 (n-Hexane: Ethyl
acetate, 1:1); IR (pure, υ cm−1): 3328 (NH), 1667, 1693
(C=O), 1559, 1462 (Ar–C=C), 1297 (C=S); 1H-NMR
(300 MHz, (CD3)2CO): δ (ppm) 10.27 (s, 1H, NH),
8.00–7.95 (m, 2H, Ar–H), 7.42–7.35 (m, 2H, Ar–H), 4.23
(s, 2H, CH2); 13C-NMR (75.5 MHz, (CD3)2CO): δ (ppm)
191.7 (C=S), 166.3 (C=O), 164.7 (COAr), 158.0, 133.8,
125.1, 124.5, 115.6, 55.2 (CH2); GC-MS: m/z (%) = 238.02
(100) [M+]; Anal. Calcd. for C10H7FN2O2S: C, 50.41; H,
2.96; N, 11.76; S, 13.46; Found: C, 50.47; H, 2.99; N,
11.69; S, 13.52.
3-(4-Chlorobenzoyl)-2-thioxoimidazolidin-4-one (3d) Light
brown solid; Yield: 82%; Rf: 0.44 (n-Hexane: Ethyl acetate,
1:1); m.p: 183–184 °C; IR (pure, υ cm−1): 3322 (NH), 1665,
1699 (C=O), 1542, 1435 (Ar–C=C), 1299 (C=S), 774
3-(4-Flourobenzoyl)-2-thioxoimidazolidin-4-one (3h) Off
white powder; Yield: 49%; Rf: 0.46 (n-Hexane: Ethyl
acetate, 1:1); IR (pure, υ cm−1): 3332 (NH), 1654, 1690
(C=O), 1562, 1468 (Ar–C=C), 1283 (C=S); 1H-NMR
(300 MHz, (CD3)2CO): δ (ppm) 10.30 (s, 1H, NH), 8.03 (d,
2H, J = 8.8 Hz, Ar–H), 7.33 (d, 2H, J = 8.8 Hz, Ar–H),
4.10 (s, 2H, CH2); 13C-NMR (75.5 MHz, (CD3)2CO): δ
(ppm) 189.4 (C=S), 167.2 (C=O), 165.7 (COAr), 162.3,
129.8, 129.1, 118.2, 58.5 (CH2); GC-MS: m/z (%) = 238.02
(100) [M+]; Anal. Calcd. for C10H7FN2O2S: C, 50.41; H,
2.96; N, 11.76; S, 13.46; Found: C, 50.47; H, 2.99; N,
11.69; S, 13.52.
1
(C–Cl); H-NMR (300 MHz, (CD3)2CO): δ (ppm) 10.37 (s,
1H, NH), 7.89 (d, 2H, J = 6.8 Hz, Ar–H), 7.45 (d, 2H, J =
6.8Hz, Ar–H), 4.01 (s, 2H, CH2); 13C-NMR (75.5 MHz,
(CD3)2CO): δ (ppm) 191.0 (C=S), 166.4 (C=O), 164.0
(COAr), 137.7, 132.3, 130.2, 129.7, 56.3 (CH2); GC-MS:
m/z (%) = 253.99 (100) [M+]; Anal. Calcd. for
C10H7ClN2O2S: C, 47.16; H, 2.77; N, 11.00; S, 12.59;
Found: C, 47.22; H, 2.57; N, 11.36; S, 12.40.
3-(2,4-Dichlorobenzoyl)-2-thioxoimidazolidin-4-one (3e)
Off white powder; Yield: 79%; Rf: 0.40 (n-Hexane: Ethyl
acetate, 1:1); m.p: 174–175 °C; IR (pure, υ cm−1): 3335
(NH), 1669, 1687 (C=O), 1550, 1447 (Ar–C=C), 1295
(C=S), 782 (C–Cl); 1H-NMR (300 MHz, (CD3)2CO): δ
(ppm) 10.52 (s, 1H, NH), 7.83–7.75 (m, 2H, Ar–H), 7.59 (s,
1H, Ar–H), 4.29 (s, 2H, CH2); 13C-NMR (75.5 MHz, (CD3)
2CO): δ (ppm) 185.7 (C=S), 164.4 (C=O), 163.0 (COAr),
139.1, 134.5, 131.8, 130.8, 56.1 (CH2); GC-MS: m/z (%) =
287.95 (100) [M+]; Anal. Calcd. for C10H6Cl2N2O2S: C,
41.54; H, 2.09; N, 9.69; S, 11.09; Found: C, 41.54; H, 2.09;
N, 9.69; S, 11.09.
3-(4-Nitrobenzoyl)-2-thioxoimidazolidin-4-one (3i) Orange
powder; Yield: 84%; Rf: 0.49 (n-Hexane: Ethyl acetate,
1:1); m.p: 193–194 °C; IR (pure, υ cm−1): 3338 (NH), 1672,
1684 (C=O), 1568, 1472 (Ar–C=C), 1542, 1338 (N=O),
1289 (C=S); 1H-NMR (300 MHz, (CD3)2CO): δ (ppm)
10.72 (s, 1H, NH), 8.88 (s, 1H, Ar–H), 8.44–7.78 (m, 3H,
Ar–H), 4.35 (s, 2H, CH2); 13C-NMR (75.5 MHz, (CD3)
2CO): δ (ppm) 182.3 (C=S), 168.4 (C=O), 164.6 (COAr),
148.5, 135.1, 133.6, 129.8, 124.5, 122.4, 54.9 (CH2); GC-
MS: m/z (%) = 265.02 (100) [M+]; Anal. Calcd. for
C10H7N3O4S: C, 45.28; H, 2.66; N, 15.84; S, 12.09; Found:
C, 45.34; H, 2.69; N, 15.90; S, 12.22.
3-(2-Bromobenzoyl)-2-thioxoimidazolidin-4-one (3f) Yel-
low solid; Yield: 83%; Rf: 0.43 (n-Hexane: Ethyl acetate,
1:1); m.p: 144–145 °C; IR (pure, υ cm−1): 3345 (NH), 1649,
1680 (C=O), 1556, 1454 (Ar–C=C), 1293 (C=S), 660 (C-
Br); 1H-NMR (300 MHz, (CD3)2CO): δ (ppm) 10.94 (s, 1H,
NH), 7.98 (d, 1H, J = 6.7 Hz, Ar–H), 7.79 (d, 1H, J =
3-(3,5-Dinitrobenzoyl)-2-thioxoimidazolidin-4-one
(3j)
White solid; Yield: 87%; Rf: 0.51 (n-Hexane: Ethyl acetate,
1:1); m.p: 209–210 °C; IR (pure, υ cm−1): 3342 (NH), 1668,
1681 (C=O), 1565, 1475 (Ar–C=C), 1546, 1343 (N=O),
1292 (C=S); 1H-NMR (300 MHz, (CD3)2CO): δ (ppm)
10.12 (s, 1H, NH), 9.18 (t, 1H, J = 2.1 Hz, Ar–H), 9.07 (d,

Medicinal Chemistry Research
2H, J = 2.4 Hz, Ar–H), 4.20 (s, 2H, CH2); 13C-NMR
(75.5 MHz, (CD3)2CO): δ (ppm) 185.7 (C=S), 165.7
(C=O), 163.1 (COAr), 148.8, 133.4, 128.9, 122.3, 57.6
(CH2); GC-MS: m/z (%) = 310.00 (100) [M+]; Anal. Calcd.
for C10H6N4O6S: C, 38.71; H, 1.95; N, 18.06; S, 10.34;
Found: C, 38.76; H, 1.89; N, 18.10; S, 10.40.
final volume of 100 mL in distilled water). Next, the test tubes
were incubated in a boiling water bath for 5 min and cooled to
room temperature. Then, the reaction mixture was diluted by
adding 400 μL of distilled water, and the absorbance was
measured at 540 nm. For non-enzymatic reactions, the assays
were performed with a blank containing all of the components
except the enzyme. The assay measurements % inhibition and
IC50 calculations were performed using the same procedure as
that for the α-glucosidase assay.
α-Glucosidase inhibition assay
The enzyme inhibition activity for α-glucosidase was per-
formed according to a previously reported procedure briefly
(Watanabe et al. 1997). The substrate p-nitrophenyl-α-D-
glucopyranoside 0.78 mM (Sigma Aldrich, USA) was dis-
solved in a 100 mM phosphate buffer (pH 7.0) containing
0.2 g/L of NaN3, 2 g/L of bovine serum albumin, α-gluco-
sidase (Sigma Aldrich, USA) and an enzyme solution. The
assay solution consisted of 50 µL of an enzyme solution,
and 10 µL of the compound dissolve in less than 1% DMSO
were mixed in a microplate well and incubated at room
temperature for 5 min. Then, a 50 µL solution of the sub-
strate was added and again incubated at 37 °C for exactly
10 min. After incubation, the reaction was terminated by the
addition of 100 µL of (100 mM) of sodium carbonate
solution followed an absorbance measurement at 405 nm
using microplate reader (OPTI Max, Tunable Micro plate
Reader). For non-enzymatic reactions, the assays were
performed with a blank containing all of the components
except the enzyme. Acarbose was used as a reference
inhibitor. Each concentration was analyzed in three inde-
pendent experiments run in triplicate. The percent of inhi-
bition of α-glucosidasewas calculated as following.
Kinetic analysis of the α-glucosidase
A series of experiments were performed to determine the
inhibition kinetics of 3j for the kinetic study. Compound
was selected on the basis of low IC50 values of α-glucosi-
dase. The inhibitor concentrations for 3j are 0.0, 0.025,
0.05, and 0.1 mM. Substrate p-nitrophenyl-α-D-glucopyr-
anoside concentration was between 0.125 to 2 mM in all
kinetic studies. Pre-incubation and measurement time was
the same as discussed in α-glucosidase inhibition assay
protocol. Maximal initial velocity was determined from
initial linear portion of absorbance up to 5 min after addition
of enzyme at a 30 s interval. The inhibition type on the
enzyme was assayed by Lineweaver–Burk plots of inverse
of velocities (1/V) versus inverse of substrate concentration
1/[S] mM−1. The EI dissociation constant Ki was deter-
mined by secondary plot of 1/V versus inhibitors con-
centrations while ESI dissociation constant Kiʹ was
determined by intercept versus inhibitors concentrations.
The reversible kinetics of the enzyme inhibitor complex
was also determined for different concentration of inhibitor
3j versus the enzyme concentration (3, 6, 9, 12, 15, and
20 µg/mL).
Inhibition ð%Þ ¼ ½ðB À SÞ=BŠ  100
Here, the B and S are the absorbances for the blank and
samples.
Cytotoxicity evaluation using brine shrimp assay
The IC50 values were determined via data analysis using
graphing software Origin 8.6 (64-bit).
Culturing and harvesting of Artemiasalina
Artemiasalina cysts were incubated for hatching in a rec-
tangular dish with a plastic divider with several holes
making two uneven compartments. The container was filled
with 3.3% solution of artificial sea water and dry yeast
sprinkled into the larger compartment which was darkened.
The smaller compartment was illuminated with light at 28°
C. After 24 h, hatched A. salinacysts were transferred to
fresh artificial seawater and incubated for a further 24 h
under artificial light and aeriation. The phototropic nauplii
were collected by pipette from the lighted compartment.
α-Amylase inhibition assay
The α-amylase inhibition assay was performed by modifying
a previously reported procedure (Loh and Hadira 2011). 40
μL of the sample solution and 40 μL of a 0.02 M sodium
phosphate buffer (pH 6.9 with 0.006 M sodium chloride)
containing the α-amylase (α-amylase from porcine pancreas,
Sigma Aldrich USA) solution (0.5 mg/mL) were incubated at
25°C for 10 min. After pre-incubation, 40 μL of 1% starch
solution in DMSO was added to each tube. Then, the reaction
mixtures were incubated at 25°C for 10 min, After that
reaction was terminated by the addition of 100 μL of the
DNSA coloring reagent (1 g of 3,5- dinitro salicylic acid, 10 g
of sodium potassium tartrate and 20 mL of 2 N NaOH to a
Brine shrimps assay
A. salinanauplii (20) were counted macroscopically using
Pasteur pipette against a lighted background and transferred

Medicinal Chemistry Research
into each sample vial and the solutions were made to 5 ml
with test compounds using serial dilutions with brine
solution. A drop of dry yeast suspension was added as food
to each vial. All the vials were maintained under light. The
surviving nauplii were counted with the aid of a magnifying
glass after 24 h. The mean mortality at the three dose levels
for compound was determined and repeated thrice. Potas-
sium dichromate was used as reference standard. After 24 h
the LD50 values were calculated.
nucleophile afforded the title compounds 3a-j in good to
excellent yields.
The 2-thiohyantoins 3a-j gave satisfactory C, H, N and S
elemental analyses, which are in agreement with the pro-
posed formula. The infrared spectra of compounds dis-
played characteristic absorptions at 3322–3345, 1649–1699,
1265–1299 cm−1 due to thiohydantoin NH, carbonyl and
thiocarbonyl moieties, respectively, thus supporting the
successful synthesis of target derivatives. In 1H NMR
spectra‚ NH proton resonated at 10.09–10.94 ppm while a
singlet for two protons at 4.01–4.35 ppm corresponds to
CH2. Aromatic protons appeared in their respective
regions. In 13C NMR‚ the thiocarbonyl observed at
182.3–191.7 ppm, carbonyl next to methylene group
appeared in the range of 163.0–167.7 ppm while the car-
bonyl carbon adjacent to the aromatic ring resonated at
slightly shielded chemical shift value of 162.8–165.2.
Free radical scavenging assay
Radical scavenging activity was determined by modifying
method by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay
(Ashraf et al. 2015). The assay solution consisted of 100 mL
of (150 mM) 2,2-diphenyl-1 picrylhydrazyl (DPPH), 20 µL
of increasing concentration of test compounds and the
volume was adjusted to 200 µL in each. This reaction
mixture was then incubated for 30 min at room temperature.
Ascorbic acid (Vitamin C) was used as a reference inhibitor.
The measurements were carried out by using a micro plate
reader (OPTIMax, tunable) at 517 nm. The reaction rates
were compared and the percent inhibition due to the pre-
sence of tested inhibitors was calculated. Each concentra-
tion was analyzed in three independent experiments.
Enzyme inhibition activity
3-(substituted benzoyl)-2-thioxoimidazolidin-4-ones deri-
vatives 3a-j were designed and synthesized to evaluate their
potential to inhibit α-glucosidase and α-amylase activity.
Acarbose is used as standard for comparison purpose and it
was found that most of compounds showed good activity
while compound 3j exhibited excellent α-glucosidase inhi-
bitory activity. On the other hand‚ all the synthesized
compounds displayed better α-amylase inhibitory activity
than the standard acarbose. It has been exposed from
enzyme inhibitory activity (Table 1) that presence of nitro
groups (-NO2) at 3,5-position of the phenyl ring play vital
role in enzyme inhibition. Compound 3j with 3,5-di-NO2
substituted phenyl ring showed excellent α-glucosidase and
α-amylase inhibitory activity with IC50 values 0.051 and
0.0082 mM, respectively. Compound 3f having 4-Cl sub-
stituted phenyl ring while 3d bearing 2-Br substituent
showed better α-glucosidase and α-amylase inhibitory
activity than standard acarbose. Based upon our results‚ it is
proposed that compound 3j may act as a lead structure for
the development of most potent α-glucosidase inhibitors.
Bio-chemical properties and Lipinski’s rule of five
(RO5)
The synthesized ligands (3a–3j) were sketched in ACD/
ChemSketch. The basic biochemical properties and
Lipinski’s rule of five (RO5) of synthesized compounds
(3a–3j) were justified through online computational tools
such as Molsoft (http://www.molsoft.com/) and Molin-
spiration (http://www.molinspiration.com/), respectively.
Furthermore, the pharmacokinetic properties such as
Absorption, Distribution, Metabolism, Excretion and
Toxicity (ADMET) of synthesized ligands were evaluated
through pkCSM online server (Pires et al. 2015).
Results and discussion
Kinetic studies
Chemistry
To understand the inhibitory mechanism of synthetic com-
pounds on α-glucosidase inhibition‚ kinetic studies were
performed. Based upon IC50 results‚ we selected compound
3j to determine their inhibition type and inhibition constant.
The kinetic results of the enzyme by the Lineweaver–Burk
plot of 1/V versus 1/[S] in the presence of different inhibitor
concentration gave a series of straight lines, the results of
compound 3j showed that compound intersected within the
second quadrant. 1/Vmax increased with increasing Km in
the presence of increasing concentration of compound 3j.
The synthesis of target compounds 3-(substituted benzoyl)-
2-thioxoimidazolidin-4-ones was achieved by synthetic
route outlined in Scheme 1. The reaction of substituted
aromatic acids 1 with thionyl chloride furnished the corre-
sponding acid chlorides followed by the addition of thio-
cyanate salt solution in acetone prior to the formation of key
substrate 2. Treatment of in situ generated acyl iso-
thiocyanates with amino acid glycine as nitrogen

Medicinal Chemistry Research
Scheme 1 Synthesis of 3-(substituted benzoyl)-2-thioxoimidazolidin-4-ones 3a-j
Table 1 α-Glucosidase and α-Amylase inhibitory activity of 3-
(substituted benzoyl)-2-thioxoimidazolidin-4-ones 3a-j
noncompetitive manner (Fig. 1a). The secondary plots of
slope versus concentration of compound 3j, showed EI
dissociation constants Ki (Fig. 1b) While ESI dissociation
constants Ki′ were shown by secondary plots of intercept
versus concentration of compound 3j (Fig. 1c). A lower
value of Ki than Ki′ pointed out stronger binding between
enzyme and compound 3j which suggested that it showed
mixed type inhibition kinetics. The kinetic parameters Ki,
Ki′, Km, 1/Vmax and type of inhibition are presented in
Table 2.
The most potent compound 3j was selected to determine
the reversibility of the enzyme-inhibitor complex between
α-glucosidase and inhibitor 3j. The plots on initial rate
versus enzyme concentrations (3, 6, 9, 12, 15, and 20 µg/
mL) for 3j‚ all consisted of straight lines passing through
the origin. Increasing the inhibitor concentration resulted in
a decrease in the slope of the lines, which showed that the
enzyme undergoes a reversible inhibition (Fig. 2).
Compounds α-Glucosidase
IC50 SEM
α-Amylase
IC50 SEM
(mM)
DPPH
% inhibition (100
µg/mL)
(mM)
3a
0.290 0.02
0.440 0.02
0.498 0.02
0.245 0.01
0.870 0.03
0.127 0.01
0.496 0.02
0.781 0.05
0.842 0.04
0.051 0.02
0.250 0.02
–
0.034 0.02
0.041 0.02
0.038 0.01
0.037 0.04
0.038 0.01
0.030 0.02
0.028 0.01
0.039 0.01
0.026 0.01
28.33 2.1
33.91 7.2
13.02 4.2
28.73 2.8
2.77 0.05
50.89 4.6
17.89 3.7
8.97 1.0
3b
3c
3d
3e
3f
3g
3h
3i
47.98 5.1
3j
0.0082 0.02 3.086 0.1
Acarbose
Vitamin C
1.800 0.06
–
–
95.63 1
Cytotoxicity evaluation
This behavior indicated that synthetic compound
inhibits the α-glucosidase by two different pathways,
competitively forming enzyme inhibitor (EI) complex and
interrupting enzyme-substrate-inhibitor (ESI) complex in
The synthesized compounds were screened for their cyto-
toxicity using brine shrimp assay. The brine shrimp test has
been frequently used as primary screen for cytotoxicity

Medicinal Chemistry Research
Fig. 1 Lineweaver–Burk plots for inhibition of α-glucosidase in the
presence of 3j. a Concentrations of 3j were 0.0, 0.025, 0.05 and
0.1 mM, Substrate p-nitrophenyl-α-Dglucopyranoside concentrations
were 0.0625, 0.12, 0.5, 1, and 2mM. b The insets represent the plot of
the slope (c) the vertical intercepts versus inhibitors concentrations to
determine inhibition constants
Table 2 Enzyme inhibitory kinetic analysis of compound 3j
Compound
Dose (mM)
1/Vmax (ΔA /Sec)
Km (mM)
Inhibition type
Mixed
Inhibition
Reversible
Ki (mM)
Kiʹ (mM)
3j
0.0
24615.384
30000.462
33846.153
40769.230
0.0881
0.1254
0.1545
0.2091
0.0339
0.1562
0.025
0.05
0.1
evaluation. The cytotoxicity data of synthetic compounds is
presented in Table 3. From results, it is evident that all the
synthesized compounds are non-toxic to the brine shrimps.
The LD50 values calculated for these compounds ranging
from 190.33 µM to 270.35 µM while LD50 value of the
potassium dichromate is 0.89 µM.
mass values should be less than 5 and 500 (g/mol),
respectively. Moreover, compounds should possess no more
than 10 HBA and 5 HBD. Literature data exposed that the
exceed values of HBA and HBD results in poor permeation.
The hydrogen-bonding capacity has been considered as
significant parameter for drug permeability. Our results
justified that all synthesized compounds possess <10 HBA
and <5 HBD values which were comparable with standard
values. However, molecular weight and logPof all com-
pounds are exceeded from standard values (>500 g/mol;
>5), respectively. Whereas, NA4 and NA10 have 3.29 and
4.10 logP values which are less than standard values. The
predicted score values favor the synthetic compounds. All
compounds exhibited positive value which means the
ligands have some therapeutic potential.
Bio-chemical properties and Lipinski’s rule of five
(RO5) validations
The biochemical properties of all the synthesized com-
pounds (3a–3j) were predicted by using computational
tools. The basic predicted values are mentioned in Table 4.
Furthermore, all the synthesized compounds were validated
through RO5. It has been observed that logP and molecular

Medicinal Chemistry Research
ADMET assessment of synthesized compounds
The pharmacokinetics properties (ADMET) of newly syn-
thesized compounds were considered as major hallmark to
predict the lead structures. Table 5 showed the pharmaco-
kinetic properties of synthesized compounds 3a–3j. The
absorption properties like water absorption (log mol/L),
intestinal solubility (% absorbed) and skin permeability
(logKp) values justified the strong therapeutic potential of
synthetic compounds. Literature report depicted that ligands
having good absorption value have more chance to cross
gut barrier by passive penetration (Pires et al. 2015). The
water solubility results justified that compounds 3a–3j
showed good absorption prediction. Furthermore, all com-
pounds 3a–3j exhibited good intestinal solubility results
which are comparable to standard value (>30 %abs). The
skin permeability values of all compounds were also com-
parable with standard value (−2.5 logKp) which justified
their drug likeness behavior. Moreover, Blood Brain Barrier
(BBB) and Central Nervous System (CNS) permeability
values of all screened compounds were also comparable
with the standard values (>0.3 to <−1 log BB and >−2 to <
−3 logPS), respectively. It has been observed that com-
pounds with greater than 0.3 log BB value have potential to
cross BBB, while with less than −1 value are poor dis-
tributed to brain. Similarly, the compounds have >−2 logPS
value are considered to penetrate the CNS, while with <−3
are difficult to move in the CNS. Results showed that
compounds displayed a significant potential to cross the
barriers and may be target directly to receptor molecule. The
excretion and toxicity predicted values were also justified
the drug likeness behavior of these compounds on the basis
of total clearance (log ml/min/kg), AMES toxicity, max-
imum tolerated dose (MTD) and LD50 values. The non-
mutagenic and non-toxic behavior was also observed from
AMES toxicity prediction. The positive and negative
hepatotoxicity behavior shows toxic and less sensitive
effects. The ligands 3a-b and 3i-j showed hepatotoxic
behavior, while no one with skin sensitive behavior. The
Fig. 2 Effects of concentrations of α-glucosidase on its activity for the
catalysis of pitrophenyl-α-D-glucopyranoside at different concentra-
tions of 3j
Table 3 Cytotoxicity evaluation of 3-(substituted benzoyl)-2-
thioxoimidazolidin-4-ones 3a-j
Compounds
LD50 (µM))
3a
248.46 15.3
270.35 20.8
215.31 19.4
240.69 12.1
210.54 9.22
203.48 10.11
263.23 22.3
265.71 18.2
228.62 10.5
195.33 10.32
0.89 0.01
3b
3c
3d
3e
3f
3g
3h
3i
3j
Potassium dichromate
Table 4 Biological properties of synthesized thiohydantoins (3a-j)
Properties
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
Mol.Wt
250
4
234
3
270
3
253
3
287
3
297
3
238
3
238
3
265
5
310
7
No. HBA
No. HBD
Mol. LogP
PSA (A2)
SC
1
1
1
1
1
1
1
1
1
1
0.4
48
0.7
40
0
1.6
40
0
1.0
40
0
1.6
40
0
1.0
40
0
0.4
40
0
0.6
40
0
0.06
79
0.2
117
0
Mol.Vol (A3)
248
−0.89
237
−0.7
266
−0.5
233
−0.5
249
−0.1
237
−0.6
221
−0.5
222
−0.7
242
268
Drug score
−0.7
−0.5
MR molar refractivity, SC stereo centers, HBA hydrogen bond acceptor, HBD hydrogen bond donors

Medicinal Chemistry Research
Table 5 Pharmacokinetic assessment of synthesized ligands (3a-j)
ADMET properties
Absorption
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
WS
−2.66
79.08
−3.43
−0.32
−0.14
−2.96
No
−2.62
78.48
−3.30
−0.18
−0.09
−2.88
No
−3.05
95.03
−2.89
0.13
0.04
−2.19
Yes
−3.07
82.25
−3.28
−0.25
−0.11
−2.91
No
−3.77
91.52
−3.27
−0.28
0.093
−2.91
No
−3.09
82.08
−3.3
−0.28
−0.15
−2.90
No
−2.47
82.14
−3.32
−0.38
−0.10
−2.92
No
−2.65
82.31
−3.36
−0.36
−0.11
−2.93
No
−2.94
77.53
−2.81
−0.36
−0.53
−2.68
No
−3.50
74.61
−2.75
−0.39
−0.88
−2.89
No
IS(%abs)
SP(log Kp)
VDss
Distribution
BBBP
CNSP
Metabolism (Inhibitors)
CYP1A2
CYP2C19
CYP2C9
CYP3A4
TC
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Excretion
Toxicity
0.03
No
−0.06
No
−0.03
Yes
−0.13
No
0.06
No
−0.10
No
0.054
No
−0.15
No
0.011
Yes
0.308
Yes
AMES
MTD
0.62
2.37
Yes
0.302
2.485
Yes
−0.25
2.50
No
0.64
2.52
No
0.54
2.66
No
0.386
2.59
No
0.544
2.528
No
0.609
2.302
No
−0.14
1.96
Yes
−0.90
1.91
Yes
ORAT(LD50)
HT
SS
No
No
No
No
No
No
No
No
No
No
WS water solubility(log mol/L), IS intestinal solubility, SP skin permeability, VDss volume of distribution at steady state (log L/kg) BBBP blood
brain barrier permeability(Log BB), CNSP CNS permeability(LogPS), TC total clearance(log ml/min/kg), ORAT oral rat acute toxicity, HT
hepatotoxicity, SS skin sensitization, MTD maximum tolerated dose
ADMET properties results justified that these novel syn-
thesized compounds showed good lead like potential with
little hepatotoxic and no skin sensitive effects.
structure for the development of most potent α-glucosidase
inhibitors.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Conclusion
A convenient synthesis of 3-(substituted benzoyl)-2-thiox-
oimidazolidin-4-one 3a-j was described. The aroyl iso-
thiocyanates were reacted with glycine catalyzed by
pyridine in a one pot progression to afford title compounds
by tailoring with various substituents on benzene ring in
satisfactory yields. The α-glucosidase and α-amylase inhi-
bitory activity of the synthesized compounds was deter-
mined and it was found that most of the compounds showed
good enzyme inhibitory activity while compound 3j
exhibited excellent activity with IC50 values 0.051 and
0.0082 mM for α-glucosidase and α-amylase, respectively.
Compound 3j bearing 3,5-di-NO2 functional groups at
phenyl ring is the most potent one among the synthesized
compounds. The enzyme inhibitory kinetics of 3j showed
that it is a mixed type inhibitor of α-glucosidase with Ki and
Kiʹ values 0.0339 and 0.1562 mM, respectively. It was
further investigated that compound 3j formed reversible
enzyme inhibitor complex with α-glucosidase. The cyto-
toxicity of all the synthesized compounds was also eval-
uated and results showed that none of these compounds
displayed toxicity against brine shrimps. Based upon our
results‚ it is proposed that compound 3j may act as a lead
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