Molecular docking and glucosidase inhibition studies of novel N-arylthiazole-2-amines and Ethyl 2-[aryl(thiazol-2-yl)amino]acetates

Article abstract of DOI:10.1007/s00044-017-2018-3

This study describes an efficient synthesis of a series of novel ethyl 2-[aryl(thiazol-2-yl)amino]acetates (4a–l) from N-arylthiazole-2-amines (3a–l). The reaction conditions were optimized and the best results were obtained when ethyl chloroacetate was used as alkylating agent and NaH as base in THF. α-glucosidase and β-glucosidase inhibition activities of N-arylthiazole-2-amines (3a–l) and ethyl 2-[aryl(thiazol-2-yl)amino]acetates (4a–l) were determined, which revealed that most of the compounds showed high percentage inhibition towards the enzymes. Among the synthesized compounds, 4e appeared to have the highest inhibition towards α-glucosidase having IC₅₀ value of 150.4 ± 1.9 μM which was almost two folds as compared to acarbose (336.9 ± 9.0 μM) taken as standard. Molecular docking of the compounds 3g, 3f, 4a, and 4e was also performed which showed their bonding modes to the enzyme’s active sites via amino and acetate groups, respectively.

Full text of DOI:10.1007/s00044-017-2018-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2018-3
ORIGINAL RESEARCH
Molecular docking and glucosidase inhibition studies of novel
N-arylthiazole-2-amines and Ethyl 2-[aryl(thiazol-2-yl)amino]
acetates
Ayesha Babar1,2 Muhammad Yar1 Hamadeh Tarazi3 Vera Duarte4
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Mohammed B. Alshammari5 Mazhar Amjad Gilani5,6 Haffsah Iqbal1
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Munawwar Ali Munawwar2 Maria J. Alves4 Ather Farooq Khan1
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Received: 15 February 2017 / Accepted: 31 July 2017
© Springer Science+Business Media, LLC 2017
Abstract This study describes an efficient synthesis of a
series of novel ethyl 2-[aryl(thiazol-2-yl)amino]acetates
(4a–l) from N-arylthiazole-2-amines (3a–l). The reaction
conditions were optimized and the best results were
obtained when ethyl chloroacetate was used as alkylating
agent and NaH as base in THF. α-glucosidase and β-glu-
cosidase inhibition activities of N-arylthiazole-2-amines
(3a–l) and ethyl 2-[aryl(thiazol-2-yl)amino]acetates (4a–l)
were determined, which revealed that most of the com-
pounds showed high percentage inhibition towards the
enzymes. Among the synthesized compounds, 4e appeared
to have the highest inhibition towards α-glucosidase having
IC50 value of 150.4 1.9 μM which was almost two folds
as compared to acarbose (336.9 9.0 μM) taken as stan-
dard. Molecular docking of the compounds 3g, 3f, 4a, and
4e was also performed which showed their bonding modes
to the enzyme’s active sites via amino and acetate groups,
respectively.
●
●
keywords α- and β-glucosidase inhibition Thiazoles
Molecular docking
Introduction
Postprandial hyperglycemia is an indication of insulin
deficiency resulting in Diabetes Mellitus (DM) which is the
most common metabolic disorder, causing damages to
many body organs such as heart, kidneys, eyes, ears, and
blood vessels (Xiong et al. 2016) The number of people
suffering from it has increased from 153 million to 347
million worldwide from 1980 to 2008. (Yar et al. 2015)
Glucosidase enzymes are involved in the intestinal digestion
of dietary carbohydrates and release of free mono-
saccharides in blood stream resulting in higher volumes of
glucose causing hyperglycemia (Hirsh et al. 1997). Current
therapeutic approaches to treat Type-II Diabetes include
oral anti-diabetic drugs such as sulfonylureas, thiazolidi-
nediones, metformin based α-glucosidase inhibitors, and
glycosurics. (Ernst et al. 2006) Acarbose, miglitol, and
voglibose etc. are used in management of postprandial
hyperglycemia and are also helpful in circumventing
hyperlipidemia, hyperlipoproteinemia and obesity, however
these are subjected to many side effects including bloating
and headache etc (Hollander 1992). Inhibition of glucosi-
dases can assist in the clinical treatment of Type-II Diabetes
and are well tolerated due to their highly localized action,
non-systemic gastrointestinal side effects and minimal
absorption (Ghani 2015).
* Ather Farooq Khan
atherfarooq@ciitlahore.edu.pk
1
Interdisciplinary Research Centre in Biomedical Materials,
COMSATS Institute of Information Technology, Defence Road,
Off Raiwind Road, Lahore 54590, Pakistan
2
Institute of Chemistry, University of the Punjab, Lahore, Pakistan
3
Department of Medicinal Chemistry, College of Pharmacy,
University of Sharjah, Sharjah, UAE
4
Chemistry Department, College of Sciences and Humanities,
Prince Sattam bin Abdulaziz University, P.O. Box 83, Al-Kharij
11942, Saudi Arabia
5
Department of Chemistry, University of Minho, Campus de
Gualtar, 4710-057 Braga, Portugal
6
Department of Chemical Engineering, COMSATS Institute of
Information Technology, Defence Road, Off Raiwind Road,
Lahore 54590, Pakistan

Med Chem Res
Synthesis of small heterocyclic molecules to be used as
potential biologically active compounds is an attractive
approach. Azoles and their derivatives have found to pos-
sess wide range of biological activities ranging from anti-
fungal and antibacterial agents to enzyme inhibitions
(Kashtoh et al. 2014). Due to their hetero atomic compo-
sition, they exhibit vast biological properties. Azole drugs
such as fluconazole, itraconazole, and voriconazole are
efficient antimycotics (Sheehan et al. 1999). Thiazole being
present in many naturally occurring biologically active
compounds such as Thiamine, Penicillin, and Vitamin B12,
has found a major role in pharmaceutical industry. N-Phe-
nylthiazol-2-amines have the ability to function as efficient
enzyme inhibitors due to their specific bent geometry
(Babar et al. 2014).
used for the synthesis of various derivatives. Structure
elucidation was done using NMR, HRMS, and X-Ray
crystallography (Babar et al. 2012a; Babar et al. 2012b).
Reaction of 2-anilinothiazole with α-halo acetates in
presence of base is well established. Thus the reaction of N-
phenylthiazol-2-amine with ethyl chloroacetate using strong
base and THF as solvent provided maximum yields of the
desired esters, as per reaction in Scheme 2. Compounds
(4a–l) were synthesized in excellent yields. A variety of
combinations of bases and solvents was attempted and the
reaction conditions were optimized. The best results were
obtained by using NaH as base and chloroethyl acetate as
alkylating agent. The structures of the synthesized products
were established on the basis of NMR and HRMS spec-
troscopic data.
In this study we report a novel series of ethyl 2-[aryl
(thiazol-2-yl)amino]acetates which are prepared by the
reaction of N-phenylthiazol-2-amines and chloro ethylace-
tate (4a–l). α-glucosidase and β-glucosidase assays of N-
phenylthiazol-2-amines (3a–l) and ethyl 2-[aryl(thiazol-2-
yl)amino]acetates (4a–l) are also reported. Most of the
compounds have exhibited potent inhibitory action.
α and β-Glucosidase assays
In order to establish an optimized structure activity rela-
tionship, several aryl thioureas were used for the synthesis
of aryl substituted 2-anilinothiazoles (Table 1) and their
derivatives (Table 2). Electron donating groups such as
methyl and methoxy were introduced on the phenyl ring as
well as derivatives having electron withdrawing groups like
fluoro, chloro and bromo were also prepared. Within these
N-arylthiazole-2-amines, compound 3g having chloro sub-
stituents at 2 and 4 positions at the phenyl ring, appeared to
have the most potent inhibitory action against α-glucosidase
having IC50 value of 173.0 0.3 μM. However, by chan-
ging the position of chloro groups in 3i loss of total activity
was observed. 3e having 4 bromo group also showed high
inhibition with an IC50 value of 189.9 0.7 μM. In general,
presence of methyl and methoxy groups retarded the
activity.
Among the synthesized esters (4a–l), most of the com-
pounds showed high inhibitory action. p-bromo derivative
i.e., 4e showed 99% inhibition with an IC50 value of 150.4
1.9. Compound 4a showed inhibition of both enzymes
having inclination towards β-glucosidase. 4d, 4f, 4j, and 4k
also showed promissing inhibitions. Factors influencing
their activity are dependent upon the nature of substituents
as well as hydrogen bonding ability of side chain.
Results and discussion
Chemistry
The synthesis of N-phenylthiazol-2-amines (3a–l) have
been reported previously by us in good to excellent yields
(70–100 %) (Babar et al. 2014). The reaction was per-
formed using Hantzsch synthetic methodology (Scheme 1)
and the resulting N-phenylthiazol-2-amines were purified
via crystallization. A variety of aryl thioureas containing
chloro, bromo, fluoro, methyl, and methoxy groups were
For further elaboration of the enzyme and inhibitor
relationship and an insight into inhibition mechanism,
molecular modeling studies were performed.
Scheme 1 Synthetic protocol for compounds (3a–l)
Molecular modeling studies
The synthesized compounds were tested towards the
activity against α-glucosidase (EC 3.2.1.20) from baker’s
yeast, as well as against almond β-glucosidase (EC
3.2.1.21). Results of the assays revealed wide margin
of activities against both enzymes. In the case of
Scheme 2 Synthetic protocol for compounds (4a–l)

Med Chem Res
Table 1 α- and β-Glucosidase inhibition activities of compounds (3a–l) and IC50 values (mean SEM, n = 3)
No.
Product
Structure
α-glucosidase %
inhibition (0.5 mM)
IC50 (μM
β-glucosidase %
inhibition (0.5 mM)
IC50 (μM
SEM)
SEM)a
1
3a
28.7
–
41.6
–
2
3
3b
3c
80.6
261.9 16.6
56.2
–
48.2
57.0
–
63.1
57.3
–
4
3d
–
–
5
3e
69.5
189.9 0.7
NI
–
6
3f
90.4
219.9 3.0
65.7
–
7
3g
84.7
173.0 0.3
53.9
–
8
3h
91.2
322.5 5.3
44.2
–

Med Chem Res
Table 1 continued
No.
Product
Structure
α-glucosidase %
inhibition (0.5 mM)
IC50 (μM
β-glucosidase %
inhibition (0.5 mM)
IC50 (μM
SEM)
SEM)a
9
3i
NI
–
NI
–
10
3j
12.4
–
15.7
–
11
12
3k
NI
–
65.0
62.4
–
3l
40.7
–
–
13
Acarbose
65.4
336.9 9.0
NI
–
a All reactions were performed in triplicates and averaged, and SEM is standard mean error of the experiments. NI = No inhibition was detected at
0.5 mM
α-glucosidase, the most active compound (4e) was found to
be more than 2-fold potency of acarbose (IC50 150.4 1.9
μM vs. 336.9 0.9 μM). Furthermore, compounds (3g, 3f,
and 4d) were also found to be highly potent (Table 1 and
Table 2). On the other hand, β-glucosidase assay revealed
that compound (4a) exhibited the highest percent inhibition
among the synthesized series. Accordingly, we decided to
perform molecular modeling studies to identify possible
binding modes and to find plausible reasons of the observed
activities. The previously homology model of α-glucosidase
reported by our group was utilized as a basis for our
docking studies (Yar et al. 2014). Furthermore, we decided
to construct a new homology model for β-glucosidase from
almond. There are some published crystal structures of β-
glucosidase from different sources; however Prunus dulcis
has not been crystallized so far. The cyanogenic β-
glucosidase (EC 3.2.1.21) from white clover (Trifolium
Repens) was found to be the most suitable template because
it shared 64% identity and 76% similarity with our target.
Sequence alignment of both proteins has been presented in
Fig. 1. Based on that alignment and the crystal structure
(PDB code: 1CBG) white clover β-glucosidase, a homology
model of β-glucosidase was built. The model was validated
based on its Discrete Optimized Protein Energy score
compared to that of template (1CBG). Additionally,
Ramachandran plot (Fig. 2) revealed that 97.1% of the
modeled residues were located within the most favored
regions, and 2.7% were located within the allowed region.
Only 0.2% of residues appeared to be outliers.
The α-glucosidase active site contains a number of cat-
alytic residues: two histidines, two aspartates and one glu-
tamate as follow: His111, Asp214, Glu276, His348, and
Asp349. On the other hand, the catalytic residues in the β-
glucosidase are: Glu211, His218, His225, and Glu482.
In the case of N-phenylthiazol-2-amine derivatives, the
most potent compounds (3f and 3g) appeared to share
similar binding mode within the α-glucosidase active site.
Both were able to establish important hydrogen bonding

Med Chem Res
Table 2 α- and β-Glucosidase inhibition activities of compounds (4a–l) and IC50 values (mean SEM, n = 3)
No.
Product
Structure
α-glucosidase %
inhibition (0.5 mM)
IC50
β-glucosidase %
inhibition (0.5 mM)
IC50
(μM SEM)
(μM SEM)a
1
4a
61.2
56.0
37.9
85.2
–
77.8
68.9
39.7
56.7
–
–
–
–
2
3
4
4b
4c
4d
–
–
202.6 1.5
5
4e
99.0
150.4 1.9
NI
–
6
4f
99.0
260.3 4.3
48.8
–

Med Chem Res
Table 2 continued
No.
Product
Structure
α-glucosidase %
inhibition (0.5 mM)
IC50
β-glucosidase %
inhibition (0.5 mM)
IC50
(μM SEM)
(μM SEM)a
7
4g
60.5
–
10.5
–
8
4h
NI
–
NI
–
9
4i
NI
–
NI
–
10
4j
84.1
272.9 5.1
56.8
–
11
4k
99.0
228.3 10.4
NI
–
12
4l
NI
–
NI
–

Med Chem Res
Table 2 continued
No.
Product
Structure
α-glucosidase %
inhibition (0.5 mM)
IC50
β-glucosidase %
inhibition (0.5 mM)
IC50
(μM SEM)
(μM SEM)a
13
Acarbose
65.4
336.9 9.0
NI
–
a All reactions were performed in triplicates and averaged, and SEM is standard mean error of the experiments. NI = No inhibition was detected at
0.5 mM
Fig. 1 Sequence alignment of almond (Prunus dulcis) β-glucosidase and white clover (Trifolium Repens) cyanogenic β-glucosidase (PDB:
1CBG). Both enzymes share 64% identity and 76% similarity. Identical residues are colored in blue, while similar residues are colored in yellow
(color figure online)
with the catalytic residues (Glu276 and Asp349) via their
amino group (Figs. 3a and 4a). An arene-cation interaction
was observed between the thiazole ring of (3f and 3g) and
the corresponding Arg439 residue. On the other hand, both
were seen to establish one hydrogen bond with the Glu211
residue of β-glucosidase (Figs. 3b and 4b), and additional
arene-arene interaction between the phenyl ring of (3f and
3g) and the corresponding Trp396 residue of the β-
glucosidase.
In case of acetate derivatives, the most potent com-
pounds (4a, 4d, and 4e) appeared to share a unique and
consistent binding mode toward the α-glucosidase enzyme
that is different from that of the N-phenylthiazol-2-amine
derivatives. They appeared to interact with different amino
acids other than the catalytic residues. In all of the studied
derivatives, two hydrogen bond interactions were observed;
one with the Asn241 through their acetate carbonyl
and another one with His239 residue through their acetate
oxygen (Figs. 5a, 6a, and Fig. 7a). The N-phenylthiazol-2-
amine derivatives appeared to bind at deeper distance
within the α-glucosidase active site compared to the
acetate derivatives (Fig. 7b). Further arene-cation inter-
action was observed between the thiazole ring of (4d
and 4e) and the corresponding Arg312 residue, which
might explain their enhanced potencies over com-
pound 4a.
On the other hand, investigation of the binding mode for
the compounds (4a and 4d) within β-glucosidase active site
revealed their ability to capture hydrogen bonding with the
catalytic His225 residue (Figs. 5b and 6b), and another

Med Chem Res
arene-arene interaction between their phenyl ring and the
corresponding Trp396 residue.
Furthermore, the ADME profiles for the most potent
compounds were assessed computationally employing the
PreADMET web server (https://preadmet.bmdrc.kr/), so
that to gain insight about their suitability for oral adminis-
tration. According to the data presented in Table 3, the
parameters for our compounds (Caco-2 and %HIA) sug-
gested moderate/high possibility of intestinal absorption,
with enhanced parameters for the acetate derivatives. Our
compounds distributions to human body were assessed by
calculating (%BBB and %PPB). The data suggested middle
distribution to the CNS and weak plasma protein binding.
Additionally, our compounds were predicted to exhibit no
inhibitory activity toward the P-glycoprotein or the Cyto-
chrome P-450 (Table 3).
Conclusion
In conclusion, novel ethyl 2-[aryl(thiazol-2-yl)amino]acet-
ates (4a–l) were derivatized from N-phenylthiazol-2-amines
(3a–l), and both series were subjected to in vitro α-gluco-
sidase and β-glucosidase inhibition studies. The compounds
bearing electron withdrawing groups showed high inhibi-
tion. Among the synthesized compounds, 4e appeared to
have the highest inhibition towards α-glucosidase having
IC50 value of 150.4 1.9 μM which was almost two folds
as compared to acarbose (336.9 9.0 μM) taken as stan-
dard. Molecular docking of the compounds 3g, 3f, 4a, and
4e was also performed which showed their bonding modes
to the enzyme’s active sites via amino and acetate groups
respectively. It is envisaged that this study may lead to the
development of highly effective glucosidase inhibitors.
Fig. 2 Ramachandran plot for the modeled β-glucosidase. Percentage
residues in most favored regions is (97.1%) and residues in allowed
regions (2.7%), while residues in outlier regions is (0.2%)
Fig. 3 Binding mode of compound 3f into the active site of Saccharomyces cerevisiae α-glucosidase a, and the active site of Prunus dulcis β-
glucosidase b. Green dotted lines represent hydrogen bonds. All hydrogen atoms are hidden

Med Chem Res
Fig. 4 Binding mode of compound 3g into the active site of Saccharomyces cerevisiae α-glucosidase a, and the active site of Prunus dulcis
β-glucosidase b. Green dotted lines represent hydrogen bonds. All hydrogen atoms are hidden
Fig. 5 Binding mode of compound 4a into the active site of Saccharomyces cerevisiae α-glucosidase a, and the active site of Prunus dulcis β-
glucosidase b. Green dotted lines represent hydrogen bonds. All hydrogen atoms are hidden
Experimental
spectra were recorded from CDCl3 on a Bruker 300 MHz
NMR spectrometer. The multiplicities were abbreviated as
bs = broad singlet, s = singlet, d = doublet, t = triplet, q =
quartet, dd = double doublet and m = multiplet. Melting
points were determined using a PCSIR Melting point appa-
ratus model no MP-1/02 and are uncorrected. EI Mass
spectrum values were recorded on Varian MAT 312. Ultra-
centrifuge apparatus was purchased from Beckman and SDS
PAGE apparatus was purchased from Bio-Rad. 96-well
General methods
All solvents were dried with Na metal prior to use. Column
chromatography was performed on silica gel 60 from Merck
(40–63 µm). For thin-layer chromatography (TLC) aluminum
backed silica gel was used. The same solvent system used for
TLC was employed in column chromatography. NMR

Med Chem Res
Fig. 6 Binding mode of compound 4d into the active site of Saccharomyces cerevisiae α-glucosidase a, and the active site of Prunus dulcis β-
glucosidase b. Green dotted lines represent hydrogen bonds. All hydrogen atoms are hidden
Fig. 7 Binding mode of compound 4e into the active site of Sac-
charomyces cerevisiae α-glucosidase, where green dotted lines
represent hydrogen bonds a. Relative binding locations of the N-
phenylthiazol-2-amine derivative 3g (CPK, green), and the acetate
derivative 4e (CPK, red) within the α-glucosidase binding site b
microplate reader ELx800 96/384 was purchased from Bio-
tek gen 5. All the chemicals used were of analytical grade.
mixture was refluxed for 2 h. Cold water (50 mL) was added
to it, followed by aqueous NaOH to make the solution
alkaline (pH 8). The resulting precipitates were filtered,
washed with cold water and recrystallization was done
using chloroform and hexane (ration 3:1).
General procedure
Synthesis of N-arylthiazol-2-amine
Synthesis of ethyl 2-[aryl(thiazol-2-yl)amino]acetates
A mixture of N-arylthiourea (0.357 mmol), 2-chloro-1,1-
dimethoxyethane (0.338 mmol) and few drops of con-
centrated HCl were dissolved in water (50 mL), and the
Sodium hydride (60% dispersion in mineral oil 0.353
mmol) was added portion wise to a stirred solution of

Med Chem Res
Table 3 Calculated ADME
properties via PreADMET
server for the most potent
compounds
Compound
BBBa
HIAb
Caco-2c
logkpd
PPBe
Pgpf
CYP-450g
3f
3g
4a
4d
4e
a
0.796
0.99
95.255
95.878
98.674
98.685
98.241
29.038
12.801
55.545
54.909
49.958
−2.699
−2.106
−3.237
−3.565
−3.223
72.174
86.834
89.856
88.868
89.696
–
–
–
–
–
–
–
–
–
–
1.62
0.997
1.905
blood-brain barrier penetration (C. brain/C. blood): > 2 high absorption to CNS; between 0.1–2 middle
absorption to CNS, while < 0.1 represents low absorption to CNS
b
percent human intestinal absorption: 0–20% poorly absorbed; 20–70% moderately absorbed, while
70–100% is well-absorbed
c
human colon adenocarcinoma permeability (nm/sec): < 4 low permeability; 4–70 middle permeability,
while > 70 considered of high permeability
d
skin permeability (cm/hour)
e
percent plasma protein binding: > 90% strongly bound; < 90% weakly bound
f
P-glycoprotein inhibitory activity (related to multi-drug resistance)
g
Cytochrome P-450 inhibitory activity
N-aryl-1,3-thiazol-2-amine (0.235 mmol) in THF (10 mL) at
0 °C and the reaction mixture was stirred for 1 h at 0 °C.
Ethyl 2-chloroacetate (0.212 mmol) was added and the
mixture was stirred at room temperature for 2 h under
nitrogen. Methanol (10 mL) was added to it and combined
THF and methanol layers were evaporated under reduced
pressure. Cold water (30 mL) was added and it was
extracted with EtOAc (10 × 3 mL). This layer was dried
over sodium sulfate and evaporated under vacuum. Pure
product was obtained by column chromatography on silica
gel using n-hexane/EtOAc: 4/1 with 2 mL triethyl amine/
liter as eluent.
Ethyl 2-[(2-fluorophenyl)(thiazol-2-yl)amino]acetate (4b)
Yield: 62%. Physical state: Foamy Solid IR (ʋmax/cm−1
)
3400, 2920, 1648.5, 1594, 1565, 1511, 1405, 1315 1H
NMR (300 MHz/CDCl3) δH: 7.73 (m, 2H), 7.43–7.15 (m,
3H), 6.53 (d, J = 3.6 Hz, 1H), 4.60 (s, 2H), 4.33–4.09 (q, J
= 7.5 Hz, 2H), 1.40–1.28 (t, J = 7.5 Hz, 3H) 13C NMR (75
MHz/CDCl3) δC: 170.5 (Cq), 165.7 (Cq), 162 (Cq), 150
(d,1JC,F = 241.5 Hz), 145 (CH), 128 (2JC,F = 11.75 Hz), 124
(3JC,F = 3.75 Hz), 123 (3JC,F = 5 Hz),120 (4JC,F = 0.75 Hz),
116 (2JC,F = 18.75 Hz), 102 (CH), 65.9 (CH2), 64.51 (CH2),
15.9 (CH3) HR-MS (EI) m/z: Calcd. for [C13H13FN2O2S]:
280.0682 Found: 280.0633
Ethyl 2-[phenyl(thiazol-2-yl)amino]acetate (4a)
Ethyl 2-[(2-chlorophenyl)(thiazol-2-yl)amino]acetate (4c)
Yield: 58%. Physical state: Foamy Solid IR (ʋmax/cm−1):
1
3336, 2911, 1648, 1604, 1562, 1512, 1405, 1315 H NMR
(300 MHz/CDCl3) δH: 7.45 (d, J = 3.6 Hz, 1H), 7.38–7.37
(m, 5H), 6.78 (d, J = 3.6 Hz, 1H), 4.3 (q, J = 8.0 Hz, 2H),
4.02 (s, 2H), 1.23 (t, J = 8.0 Hz, 3H) 13C NMR (75 MHz/
CDCl3) δC: 170.02 (Cq), 165.78 (Cq), 150.7 (Cq), 140.1
(CH), 129.5 (CH), 121.98 (CH), 116.05 (CH), 112.12 (CH),
70.35 (CH2), 65.1 (CH2), 15.10 (CH3) HR-MS (EI) m/z:
Calcd. for [C13H14N2O2S]: 262.0776 Found: 262.0701
Yield: 63.8%. Physical state: Foamy Solid IR (ʋmax
/
cm−1): 3306, 2910, 1648, 1605, 1562, 1512, 1405, 1315 1H
NMR (300 MHz/CDCl3) δH: 7.84–7.74 (m, 1H), 7.56–7.45
(m, 1H), 7.38 7.28 (m, 2H), 7.17 (d, J = 3.7 Hz, 1H), 6.47

Med Chem Res
(d, J = 3.7 Hz, 1H), 4.56 (s, 2H), 4.27–4.09 (q, J = 8.0 Hz,
2H), 1.32–1.17 (t, J = 8.0 Hz, 3H) 13C NMR (75 MHz/
CDCl3) δC: 170.5 (Cq), 164.7 (Cq), 149 (Cq), 140.2 (CH),
130.71 (CH), 128.29 (CH), 125.8 (CH), 124.12 (Cq), 115.9
(CH), 112.10 (CH), 65.5 (CH2), 64.29 (CH2), 15.3 (CH3)
HR-MS (EI) m/z: Calcd. for [C13H13ClN2O2S] 296.0386
found 296.0389
Ethyl 2-[(2,4-difluorophenyl)(thiazol-2-yl)amino]acetate (4f)
Ethyl 2-[(4-fluorophenyl)(thiazol-2-yl)amino]acetate (4d)
Yield: 72%. Physical state: Foamy Solid IR (ʋmax/cm−1):
3340, 2910, 1647, 1602, 1561, 1511, 1403, 1314 H NMR
1
(300 MHz/CDCl3) δH: 7.53–7.22 (m, 4H), 6.67 (d, J = 3.6,
1H), 4.59 (s, 2H), 4.22 (m, 2H), 1.28 (m, 3H) 13C NMR (75
MHz / CDCl3) δC: 169.65 (Cq), 165.17 (Cq), 158.9 (Cq),
155.2 (Cq), 135.90 (CH), 126.9 (Cq), 116.9 (CH), 114.10
(CH), 112.1 (CH), 111.1 (CH), 63.9 (CH2), 61.51 (CH2),
12.9 (CH3) HR-MS (EI) m/z: Calcd. for [C13H13F2N2O2S]
298.05888 found 298.0592
Yield: 54%. Physical state:Foamy Solid IR (ʋmax/cm−1
)
Ethyl 2-[(2,6-dichlorophenyl)(thiazol-2-yl)amino]acetate (4g)
1
3335, 2900, 1648, 1596, 1565, 1515, 1400, 1314 H NMR
(300 MHz/CDCl3) δH: 7.56–7.45 (m, 2H), 7.20–7.19 (d, J
= 3.6 Hz, 1H), 7.16–7.10 (m, 2H), 6.49 (d, J = 3.6 Hz, 1H),
4.59 (s, 2H), 4.20 (m, 2H), 1.28 (t, J = 7.6H, 3 H) 13C NMR
1
(75 MHz/CDCl3) δC: 169.15 (Cq), 167 (Cq), 159 (d, JC,F
= 249 Hz), 140 (CH), 136 (4JC,F = 2.25 Hz), 120 (3JC,F
=
7.5 Hz), 116 (2JC,F = 22.5 Hz), 108 (CH), 64.9 (CH2), 63.51
(CH2), 14.9 (CH3) HR-MS (EI) m/z: Calcd. for
[C13H13FN2O2S] 280.0682 Found: 280.0610
Yield: 63%. Physical state: Foamy Solid IR (ʋmax/cm−1):
1
3495, 2910, 1646, 1600, 1568, 1514, 1406, 1317 H NMR
(300 MHz/CDCl3) δH:mδ 7.54–7.38 (m, 2H), 7.42–7.19 (m,
2H), 6.60 (d, J = 3.7 Hz, 1H), 4.54 (2H, s), 4.18 (m, 2H),
1.26 (m, 3H) 13C NMR (75 MHz/CDCl3) δC: 169.5 (Cq),
165.9 (Cq),149.01 (Cq), 140.1 (CH), 129.2 (CH), 125.5
(Cq), 119.2 (CH), 112.9 (CH), 63.9 (CH2), 60.9 (CH2), 14.9
(CH3) HR-MS (EI) m/z: Calcd. for [C13H12Cl2N2O2S]:
331.9997 Found: 331.9923
Ethyl 2-[(4-bromophenyl)(thiazol-2-yl)amino]acetate (4e)
Ethyl 2-[(2,4-dichlorophenyl)(thiazol-2-yl)amino]acetate (4h)
Yield: 50.11 mg, 87%. Physical state: Foamy Solid IR
(ʋmax/cm−1): 3266, 2910, 1649, 1600, 1565, 1511, 1405,
1
1315 H NMR (300 MHz/CDCl3) δH: 7.63–7.51 (m, 1H),
7.47–7.35 (m, 1H), 7.32–7.19 (m, 3H), 6.54 (d, J = 3.6 Hz,
1H), 4.62 (s, 2H), 4.32–4.12 (q, J = 8.0 Hz, 2H), 1.38–1.23
(t, J = 8.0 Hz, 3H) 13C NMR (75 MHz/CDCl3) δC: 169.10
(Cq), 165.01 (Cq), 149.9 (Cq), 140.3 (CH), 135.1 (CH),
116.2 (CH), 115.8 (Cq), 112.9 (CH), 64.9 (CH2), 63.01
(CH2), 14.9 (CH3) HR-MS (EI) m/z: Calcd. for
[C13H13BrN2O2S]: 339.9881 Found: 339.9832
Yield: 78%. Physical state: Foamy Solid IR (ʋmax/cm−1):
3306, 2910, 1648, 1600, 1562.9, 1514, 1401, 1313 1H
NMR (300 MHz/CDCl3) δH: 7.79 (dd, J = 8.6, 1.2 Hz, 1H),
7.57 (d, J = 2.3 Hz, 1H), 7.36 (d, J = 3.7 Hz, 1H), 7.26 (m,
1H), 6.55 (d, J = 3.7 Hz, 1H), 4.57 (s, 2H), 4.33–4.10 (q, J

Med Chem Res
= 7.5 Hz, 2H), 1.88 (t, J = 7.5 Hz, 3H) 13C NMR (75 MHz/
CDCl3) δC: 171 (Cq), 163 (Cq), 142.9 (Cq), 138 (CH), 131.2
(CH), 129.7 (CH), 124.1 (Cq), 123.8 (Cq), 116.3 (CH),
112.8 (CH), 64.3 (CH2), 61.92 (CH2), 14.8 (CH3) HR-MS
(EI) m/z: Calcd. for [C13H12Cl2N2O2S]: 331.9997 Found:
331.9946.
Ethyl 2-[(2-fluorophenyl)(thiazol-2-yl)amino]acetate (4k)
Ethyl 2-[(3,4-dichlorophenyl)(thiazol-2-yl)amino]acetate (4i)
Yield: 67%. Physical state: Foamy Solid IR (ʋmax/cm−1):
1
3338, 2910, 1648, 1594, 1562, 1512, 1405, 1315 H NMR
(300 MHz / CDCl3) δH: 7.21 (d, J = 3.6 Hz, 1H), 7.01–6.97
(m, 3H), 6.44 (d, J = 3.6 Hz, 1H), 4.38 (s, 2H), 4.30–4.10
(q, J = 7.3 Hz, 2H), 2.31 (s, 3H), 2.27 (s, 3H), 1.27 (t, J =
7.3 Hz, 3H) 13C NMR (75 MHz/CDCl3) δC:170.1 (Cq),
166.7 (Cq), 151.1 (Cq), 141.01 (CH), 139.7 (Cq), 127.9
(CH), 119.9 (Cq), 118.7 (CH), 114.5 (CH), 112.9 (CH),
64.5 (CH2),61.9 (CH2), 19.8 (CH3), 14.9 (CH3) HR-MS
(EI) m/z: Calcd for [C15H18N2O2S]: 290.1089 Found:
290.1051
Yield: 69%. Physical state: Foamy Solid IR (ʋmax/cm−1):
3336, 2910, 1640, 1601, 1562.9, 1512.8, 1405, 1315 H
1
NMR (300 MHz/CDCl3) δH: 7.62 (d, J = 2.6 Hz, 1H), 7.50
(d, J = 8.6 Hz, 1H), 7.37 (dd, J = 8.6, 2.5 Hz, 1H), 7.24 (d,
J = 3.6 Hz, 1H), 6.57 (d, J = 3.7 Hz, 1H,), 4.60 (s, 2H), 4.19
(m, 2H), 1.28 (t, J = 7.2 Hz, 3H) 13C NMR (75 MHz/
CDCl3) δC: 168.5 (Cq), 164.9 (Cq), 150.9 (Cq), 140.12
(CH), 135.89 (Cq), 128.1 (CH), 125.8 (Cq), 116.3 (CH),
114.5 (CH), 112.8 (CH), 65.9 (CH2), 60.1 (CH2), 15.9
(CH3). HR-MS (EI) m/z: Calcd. for [C13H12Cl2N2O2S]:
331.9997 Found: 331.9966
Ethyl 2-[mesityl(thiazol-2-yl)amino]acetate (4l)
Yield: 65%. Physical state: Foamy Solid IR (ʋmax/cm−1):
3339, 2920, 1648, 1595, 1569, 1517, 1405, 1315 H NMR
1
Ethyl 2-[(2-chloro-5-methoxyphenyl)(thiazol-2-yl)amino]
acetate (4j)
(300 MHz/CDCl3) δH: 7.20 (d, J = 3.6 Hz, 1H), 6.97 (s,
2H), 6.43 (d, J = 3.6 Hz, 1H), 4.38 (s, 2H), 4.30–4.10 (q, J
= 7.3 Hz, 2H), 2.31 (s, 3H), 2.27 (s, 6H), 1.27 (t, J = 7.3,
3H) 13C NMR (75 MHz/CDCl3) δC: 169.9 (Cq), 167.8 (Cq),
142.9 (Cq), 141.02 (CH), 130.9 (Cq), 128.9 (CH), 125.9
(Cq), 112.9 (CH), 64.9 (CH2), 62.9 (CH2), 22.9 (CH3), 19.9
(CH3), 15.9 (CH3) HR-MS (EI) m/z: Calcd. for
[C16H20N2O2S]: 304.1245 Found: 304.1278
Yield: 56%. Physical state: Foamy Solid IR (ʋmax/cm−1):
3296, 2910, 1650, 1601, 1563, 1512, 1406, 1315 H NMR
Biological assays
1
(300 MHz/CDCl3) δH: 7.34 (d, J = 3.6 Hz, 1H,), 7.21–6.97
(m, 3H), 6.92 (d, J = 3.6 Hz, 1H,), 4.60 (s, 2H), 4.21–4.10
(m, 2H,), 3.80 (s, 3H,), 1.29 (t, J = 7.3, 3H) 13C NMR (75
MHz/CDCl3) δC: 170.1 (Cq), 169.5 (Cq), 152.3 (Cq), 140.9
(CH), 136.9 (Cq), 129.4 (Cq), 118.9 (CH), 114.6 (CH),
112.9 (CH), 63.9 (CH2), 61.9 (CH2), 58.5 (CH3), 15.1
(CH3) HR-MS (EI) m/z: Calcd. for [C13H15ClN2O3S]:
326.0492 Found: 326.0446
α-Glucosidase assay procedure
Total volume of 100 µL reaction mixture contained, 70 µL
50 mM phosphate buffer, pH 6.8, 10 µL (0.5 mM) test
compound, followed by the addition of 10 µL (0.020 units,
Sigma G-5003) enzyme. The contents were mixed, pre-
incubated for 10 min at 37 °C and pre-read at 400 nm. The
reaction was initiated by the addition of 10 µL of 0.5 mM

Med Chem Res
substrate (p-nitrophenyl α-D-glucopyranoside, Sigma Inc.).
After 30 min of incubation at 37 °C, absorbance of yellow
color produced due to the formation of p-nitrophenol was
measured at 400 nm using Thermo Scientific Varioskan
Flash 96-well microplate reader. Acarbose was used as
positive control. All experiments were carried out in tri-
plicate. The percent inhibition was calculated by the fol-
lowing equation:
Sequence alignment of target and template was performed
with ClustalW2, using BLOSUM62 matrix, gap opening
penalty 10 and gap extension penalty 0.2 values (Larkin
et al. 2007) Automodel from Modeler 9.15 was applied to
generate the homology model for the enzyme (Eswar et al.
2006) Further model refinement was accomplished by
conjugate gradients optimization and molecular dynamics
simulations. The model was evaluated based on its Discrete
Optimized Protein Energy score (DOPE-score) and Rama-
chandran plot assessment (Lovell et al. 2003).
Inhibition ð%Þ ¼ ðabs of control
À abs of test=abs of controlÞ Â 100
IC50 values (concentration at which there is 50% inhi-
bition in enzyme catalyzed reaction) were calculated vary-
ing the concentration of the test compound.
Ligand docking
The generated homology models for α and β-glucosidases
were used as the basis for our docking study. Docking
experiments were performed using the program Autodock
vina (Trott and Olson 2010). Autodock Tools (MGL Tools
1.5.6rc2) were used for preparing the homology models for
docking by adding polar hydrogen atoms and generating
Gasteiger–Marsili charges. The investigated compounds
were drawn using ChemDraw Ultra 8.0 software ((http://
www.cambridgesoft.com) and USA.), and were optimized
for energy and geometry using MMFF94 force field.
Investigated compounds were treated employing the pre-
vious preparation steps mentioned earlier. A grid boxes of
the size 22 cubic Å were established to cover the active site
of the macromolecules, with a spacing of 1.0 between the
grid points, centered towards the coordinates of 21.89 (x),
−7.10 (y), 23.5 (z) for α-glucosidase and 37.27 (x), 32.62
(y), 86.01 (z) for the β-glucosidase. The exhaustiveness and
the number of poses were set to 12 and 10, respectively.
The results were viewed in Discovery studio 3.1 client
(http://accelrys.com/).
β-Glucosidase assay procedure
Total volume of 100 µL reaction mixture contained, 70 µL
50 mM phosphate buffer, pH 6.8, 10 µL (0.5 mM) test
compound, followed by the addition of 10 µL (0.020 units,
Sigma G-0395) enzyme. The contents were mixed, pre-
incubated for 10 min at 37 °C and pre-read at 400 nm. The
reaction was initiated by the addition of 10 µL of 0.5 mM
substrate (p-nitrophenyl β-D-glucopyranoside, Sigma Inc.).
After 30 min of incubation at 37 °C, absorbance of the
yellow color produced due to the formation of p-nitrophenol
was measured at 400 nm using Thermo Scientific Varioskan
Flash 96-well microplate reader. All experiments were
carried out in triplicate. The percent inhibition was calcu-
lated by the following equation:
Inhibition ð%Þ ¼ ðabs of control
À abs of test=abs of controlÞ Â 100
Molecular modeling studies
Evaluation of ADME properties
Homology modeling
The pharmacokinetic (ADME) parameters for the most
potent compounds including; blood-brain barrier penetra-
tion (BBB), percent human intestinal absorption (%HIA),
human colon adenocarcinoma permeability (Caco-2), skin
permeability (logkp), percent plasma protein binding (%
PPB), P-glycoprotein inhibition activity (Pgp), and the
Cytochrome P-450 inhibitory activity (CYP-450) were
calculated employing PreADMET web server (https://pre-
admet.bmdrc.kr/).
The baker’s yeast α-glucosidase homology model pre-
viously reported by our group was used as the basis for our
docking approach. On the other hand, a new homology
model for almond β-glucosidase was developed according
to the following procedure; sequence of almond β-glucosi-
dase was obtained from Uniprot (No. H9ZGD8) (Uniprot
and http://www.uniprot.org/), then Blast program (Blast
2.2.25+ and https://blast.ncbi.nlm.nih.gov/Blast.cgi/) was
utilized to search for homologous proteins. Cyanogenic β-
glucosidase from white clover was found to be the most
identical and similar to our target. Full sequence of template
and its 3D structure were downloaded from Uniprot (No.
P26205) and Protein Data Bank (1CBG), respectively.
Acknowledgements A.F. Khan is thankful to Higher Education
Commission, Pakistan for providing funding under NRPU project No.
1690 for this research.

Med Chem Res
Compliance with ethical standards
Hirsh AJ, Yao S, Young JD, Cheeseman CI (1997) Inhibition of
glucose absorption in the rat jejunum: a novel action of
alpha-D-glucosidase inhibitors. Gastroenterology 113:205–211
Hollander P (1992) Safety profile of acarbose, an α-Glucosidase
Inhibitor. Drugs 44:47–53. doi:10.2165/00003495-199200443-
00007
Conflict of interest The authors declare that they have no competing
interests.
Kashtoh H et al. (2014) Oxadiazoles and thiadiazoles: novel α-
glucosidase inhibitors. Bioorgan Med Chem 22:5454–5465
Larkin MA et al. (2007) Clustal W and Clustal X version 2.0.
Bioinformatics 23:2947–2948
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