Synthesis, in vitro and in silico screening of 2-amino-4-aryl-6-(phenylthio) pyridine-3,5-dicarbonitriles as novel α-glucosidase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.103879

Inhibition of α-glucosidase enzyme is of prime importance for the treatment of diabetes mellitus (DM). Apart of many organic scaffolds, pyridine based compounds have previously been reported for wide range of bioactivities. The current study reports a series of pyridine based synthetic analogues for their α-glucosidase inhibitory potential assessed by in vitro, kinetics and in silico studies. For this purpose, 2-amino-4-aryl-6-(phenylthio)pyridine-3,5-dicarbonitriles 1–28 were synthesized and subjected to in vitro screening. Several analogs, including 1–3, 7, 9, 11–14, and 16 showed many folds increased inhibitory potential in comparison to the standard acarbose (IC₅₀ = 750 ± 10 μM). Interestingly, compound 7 (IC₅₀ = 55.6 ± 0.3 μM) exhibited thirteen-folds greater inhibition strength than the standard acarbose. Kinetic studies on most potent molecule 7 revealed a competitive type inhibitory mechanism. In silico studies have been performed to examine the binding mode of ligand (compound 7) with the active site residues of α-glucosidase enzyme.

Full text of DOI:10.1016/j.bioorg.2020.103879

Journal Pre-proofs
Synthesis, In vitro and In silico Screening of 2-Amino-4-Aryl-6-(phenylthio)
pyridine-3,5-dicarbonitriles as Novel α-Glucosidase Inhibitors
Muhammad Ali, Khalid Mohammed Khan, Mohammad Mahdavi, Abdul
Jabbar, Shahbaz Shamim, Uzma Salar, Muhammad Taha, Shahnaz Parveen,
Bagher Larijani, Mohammad Ali Faramarzi
PII:
S0045-2068(20)30479-X
DOI:
https://doi.org/10.1016/j.bioorg.2020.103879
YBIOO 103879
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
29 February 2020
16 April 2020
22 April 2020
Please cite this article as: M. Ali, K. Mohammed Khan, M. Mahdavi, A. Jabbar, S. Shamim, U. Salar, M. Taha, S.
Parveen, B. Larijani, M. Ali Faramarzi, Synthesis, In vitro and In silico Screening of 2-Amino-4-Aryl-6-
(phenylthio) pyridine-3,5-dicarbonitriles as Novel α-Glucosidase Inhibitors, Bioorganic Chemistry (2020), doi:
https://doi.org/10.1016/j.bioorg.2020.103879
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Synthesis, In vitro and In silico Screening of 2-Amino-4-Aryl-6-(phenylthio) pyridine-3,5-
dicarbonitriles as Novel α-Glucosidase Inhibitors
Muhammad Ali,1 Khalid Mohammed Khan,1,2,3 Mohammad Mahdavi,4 Abdul Jabbar,5 Shahbaz
Shamim,1 Uzma Salar,6 Muhammad Taha,3 Shahnaz Parveen,7 Bagher Larijani,4 Mohammad Ali
Faramarzi8
1H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
2Pakistan Academy of Sciences, 3-Constitution Avenue G-5/2, Islamabad, Pakistan
3Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam
Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia
4Endocrinology & Metabolism Research Institute (EMRI), Tehran University of Medical
Sciences, Tehran, Iran
5Department of Veterinary Biosciences, Melbourne Veterinary School, Faculty of Veterinary and
Agricultural Sciences, The University of Melbourne, 250 Princes Highway, Werribee, Victoria
3030, Australia
6Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for
Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan
7PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280,
Pakistan
8Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology
Research Center, Tehran University of Medical Sciences, Iran
Abstract: Inhibition of α-glucosidase enzyme is of prime importance for the treatment of diabetes
mellitus (DM). Apart of many organic scaffolds, pyridine based compounds have previously been
reported for wide range of bioactivities. The current study reports a series of pyridine based
synthetic analogues for their α-glucosidase inhibitory potential assessed by in vitro, kinetics and
in silico studies. For this purpose, 2-amino-4-aryl-6-(phenylthio)pyridine-3,5-dicarbonitriles 1-28
were synthesized and subjected to in vitro screening. Several analogs, including 1-3, 7, 9, 11-14,
and 16 showed many folds increased inhibitory potential in comparison to the standard acarbose
(IC50 = 750 ± 10 µM). Interestingly, compound 7 (IC50 = 55.6 ± 0.3 µM) exhibited thirteen-folds
greater inhibition strength than the standard acarbose. Kinetic studies on most potent molecule 7
revealed a competitive type inhibitory mechanism. In silico studies have been performed to
examine the binding mode of ligand (compound 7) with the active site residues of α-glucosidase
enzyme.
Keywords: Synthetic pyridine analogs; α-glucosidase inhibition in vitro, acarbose; structure-
activity relationship; kinetics; in silico study
*Corresponding Author: khalid.khan@iccs.edu; drkhalidhej@gmail.com; Tel. 00922134824910; Fax.
00922134819018
1

Introduction
α-Glucosidase enzyme is located in the small intestine and responsible for breaking of
carbohydrates which eventually results in the increased level of α-glucose in the blood [1-3].
Globally diabetes is considered to be one of the most serious threats to humans which can render
severe health complications. Moreover, the most prevalent expressed form of this disease is type
II diabetes [4]. Since 1980 to 2008, the number of individuals suffering from diabetes mellitus
(DM) has expanded from 153 to 347 million around the globe. According to WHO, diabetes is
considered to be the seventh most serious cause of mortality by 2030 [5,6]. Controlling the over-
expression of α-glucosidase, is thought to be a way for the treatment of diabetes and other
associated ailments such as increased body mass [7], gestational diabetes [8], and cardiovascular
diseases [9]. A considerable way to treat the type 2 DM is to inhibit the intestinal α-glucosidase.
Several α-glucosidase inhibitors based drugs such as acarbose, voglibose, and miglitol are
available in the market for the treatment of type 2 DM but their adverse effects do not allow them
to be the appropriate therapeutic agents for the cure of DM [10]. Scientist are still interested in the
identification of antidiabetic agents based on natural and synthetic origins [11-16]. Mollica et. al.
recognized that Turkish hazelnut contains high concentration of phenolic acids and flavonoids
those have the ability to inhibit α-glucosidase enzyme. Similarly, they have also discovered novel
peptides as attractive leads for the treatment of DM by performing computer aided virtual
screening, followed by in vitro, pharmacokinetic evaluation [15,16]. It is essentially important to
design new, safe, and better α-glucosidase inhibitors that could serve as an effective lead candidate
for the better cure of DM.
Pyridine is six-membered aromatic heterocyclic organic compound, synthesized by treating ethyl
acetoacetate, aldehyde, and ammonia which is known as Hantzsch pyridine synthesis. Pyridine
and derivatives have acquired a significant position among the heterocyclic compounds due to
their pharmacological and medicinal importance [17]. Pyridine is used in the synthesis of many
drugs such as tripelennamine, mepyramine (antihistaminic drugs), sulfapyridine used for the
treatment of bacterial and viral infections. Cicletanine is a furopyridine based antihypertensive
drug with diuretic property [18]. Isoniazid is pyridine containing drug used for the treatment of
tuberculosis [19]. In the recent past, we have reported the diversified synthetic pyridine centered
molecules and their medicinal significance found to have α-glucosidase, α-amylase, β-
glucuronidase, antioxidant, and antiglycation potential [20-24] (Figure-1). The encouraging results
motivate us to synthesize a different type of pyridine analogues with several biologically
2

significant substituents to assess their efficacy. Herein, we are reporting the synthesis, in vitro, and
in silico evaluation of 2-amino-4-aryl-6-(phenylthio) pyridine-3,5-dicarbonitriles 1-28.
NO2
O
O
O
O
N
H
-Glucosidase Inhibitors

HO
OH
OH
Cl
Br
N
N
R
Identified
leads for the
treatment of
DM
N
H
N
H
N
N
NC
H2N
CN
S
OH
Antiglycating Agent
-Glucosidase Inhibitors

N
1-28
CH3
N
OCH3
Cl
OH
S
2-Amino-4-aryl-6-(phenylthio)
pyridine-3,5-dicarbonitrile analogs
IC50 = 55.6 ± 0.3 - 474.2 ± 5.0 µM
Acarbose = IC50 = 750 ± 10 µM
H
N
N
Cl
F
S
N
N
HO
N
N
-Glucosidase Inhibitors

-Amylase Inhibitor

Figure-1: Rationale of the current study.
Results and Discussion
Chemistry
2-Amino-4-aryl-6-(phenylthio) pyridine-3,5-dicarbonitrile derivatives 1-28 were prepared by
following reaction scheme. First, the reaction of different aryl aldehydes with malononitrile,
catalyzed by triethyl amine to form the 2-arylidenemalononitrile which was monitored by TLC
analysis. Then, 2-benzylidenemalononitrile intermediates were treated with thiophenol and
malononitrile, in the presence of triethyl amine to afford products in good yields (Scheme-1).
Intermediates as well as the final product formation were examined by thin layer chromatography
(TLC system = Hexane:Ethyl acetate (8:2)).
SH
O
R
R
H
R
Et3N, EtOH
Reflux, 1 h
Et3N, EtOH
Reflux, 4 h
NC
CN
S
CN
H2N
N
CN
Intermediate
N
N
N
N
1-28
Scheme-1: Synthesis of 2-amino-4-aryl-6-(phenylthio) pyridine-3,5-dicarbonitriles 1-28.
3

1
Chemical structures of all compounds were deduced by EI-MS and H-NMR spectroscopic
techniques. To the best of our knowledge eleven compounds 3, 4, 9-11, 14, 18, 20, 21, 25, and 26
are structurally new derivatives, so they were also subjected to HREI-MS and 13C-NMR for further
structural confirmation.
In vitro α-glucosidase inhibitory screening
All synthetic 2-amino-4-aryl-6-(phenylthio) pyridine-3,5-dicarbonitriles were subjected for in
vitro α-glucosidase inhibitory screening. It is worth mentioning that all molecules except 8, 22, 24,
25, and 28 were found to possess many folds increased inhibitory potential ranging from IC50 =
55.6 ± 0.3 to 474.2 ± 5.0 µM as compared to the standard acarbose (IC50 = 750.0 ± 10.0 µM)
(Table-1).
Table-1: In vitro α-glucosidase inhibitory activities of pyridine derivatives 1-28.
R
NC
CN
S
H2N
N
α-Glucosidase
Inhibitory
α-Glucosidase
Inhibitory
Comp.
No.
R
Comp.
15
R
Activity
Activity
(µM ± SEMa)
(µM ± SEMa)
Br
1
2
3
162.7 ± 1.7
103.7 ± 0.9
184.3 ± 2.1
205.0 ± 2.5
315.4 ± 3.6
OMe
OMe
OMe
OMe
16
17
OMe
OMe
NO2
750 <
OMe
OMe
OMe
Me
4
5
273.6 ± 3.1
304.5 ± 3.5
18
19
332.3 ± 3.9
OMe
OMe
750 <
N
OMe
4

OMe
OEt
F
6
7
450.6 ± 4.7
55.6 ± 0.3
20
21
750 <
OMe
OMe
448.7 ± 5.5
OH
OH
8
9
750 <
22
23
125.0 ± 1.0
156.7 ± 1.6
474.2 ± 5.0
OH
OMe
10
11
750 <
24
25
130.8 ± 1.1
O
OMe
OH
OMe
OMe
F
O
227.4 ± 2.8
175.1 ± 1.9<
OMe
OH
Cl
160.5 ± 1.7
324.7 ± 3.8
12
13
240.1 ± 2.9
92.7 ± 0.7
26
27
I
OMe
Br
Cl
OMe
Br
Cl
326.5 ± 3.8
OMe
14
191.6 ± 2.3
28
OMe
OMe
Standard (Acarbose)b
750.0 ± 10.0
SEMa (standard error mean); Acarboseb (Standard for α-glucosidase inhibitory activity).
5

Structure-activity relationship (SAR)
Albeit, all structural characteristics of synthetic analogs seem to have an indispensable role in the
inhibitory potential but structure-activity relationship (SAR) has been established on the basis of
varying substituents on the phenyl ring “R”.
Compound 1 (IC50 = 162.7 ± 1.7 µM) with unsubstituted phenyl ring found to be four-folds more
active than the standard acarbose (IC50 = 750 ± 10 µM). Incorporation of substituents significantly
affected the inhibitory potential. Such as compound 2 (IC50 = 103.7 ± 0.9 µM) with m-nitro
substitution at phenyl ring, showed significant increased inhibitory potential as compared to
compound 1 and the standard acarbose. The enhanced inhibition by compound 2 might be due to
the electron-withdrawing effect of the nitro group which makes the ring more polarizable to
interact with the active binding site of an enzyme. However, in some cases substitution leads to
the decline in the inhibitory potential as observed in case of o-methyl and o-fluoro substituted
compounds 4 (IC50 = 273.6 ± 3.1 µM) and 6 (IC50 = 450.6 ± 4.7 µM), respectively. The decreased
activity might be due to the occupied ortho position which may create some steric hindrance during
binding. However, it is still worth-mentioning that compound 4 and 6 are much better active than
the standard acarbose. One exceptional derivative 5 (IC50 = 304.5 ± 3.5 µM) having pyridine ring
instead of phenyl as “R”, revealed two-fold more potent inhibitory potential than the standard
acarbose (Figure-2) which gave us an idea to explore the effect of substituted pyridine as “R” on
the inhibitory potential in the near future.
NO2
Me
NC
CN
NC
CN
S
H2N
N
S
H2N
N
N
5
4
2
IC50 = 103.7 ± 0.9 µM
IC50 = 273.6 ± 3.1 µM
NC
F
CN
NC
CN
S
NC
CN
S
H2N
N
S
H2N
N
H2N
N
6
1
IC50 = 450.6 ± 4.7 µM
IC50 = 304.5 ± 3.5 µM
IC50 = 162.7 ± 1.7 µM
Figure-2: Structure-activity relationship of compounds 1, 2, 4-6.
6

p-Hydroxy substituted compound 7 (IC50 = 55.6 ± 0.3 µM), the most potent member of this
synthetic library, identified as thirteen-fold more potent in comparison to the standard acarbose
(IC50 = 750 ± 10 µM). This promising inhibition potential could be attributed to the hydroxy group,
specifically at the para position, might be the most appropriate position to bind with the active site
of an enzyme. m-Hydroxy substituted residue 8 (IC50 = 750 ± 10 µM) with the same structural
feature but the different position of hydroxy group, resulted in several folds decreased inhibitory
potential suggests that position of a substituent at a specific position is very important to bind with
the active site of an enzyme. Comparison of analog 9 (IC50 = 474.2 ± 5.0 µM) with compound 7,
having almost identical structure but only incorporation of methoxy group adjacent to hydroxy,
resulted in decrease of inhibitory strength might be due to factor of steric hindrance by the methoxy
substituent adjacent to hydroxy. Furthermore, compound 10 (IC50 = 750< µM) distinctly similar
to compound 9 but with reverted positions of hydroxy and methoxy substituents, displayed
significantly lower activity as compared to compound 9 but similar inhibitory strength to the
standard acarbose. Activity of derivative 9 can be compared with another derivative 11 (IC50 =
227.4 ± 2.8 µM) having fluoro group instead of hydroxy, revealed enhanced inhibitory strength
which suggests that being an electronegative group, fluoro is playing a critical role in the efficacy
of this analogue. Similarly, compound 12 (IC50 = 240.1 ± 2.9 µM) with an additional iodo group
at meta position also showed almost two-folds enhanced inhibitory strength, compared to
compound 9 which reveals the significance and active participation of halogen group in the activity
(Figure-3).
OH
OMe
OMe
HO
OH
NC
CN
S
H2N
N
NC
CN
S
NC
CN
S
9
IC50 = 474.2 ± 5.0 µM
H2N
N
H2N
N
OH
F
8
10
IC50 = 750<
I
OMe
OMe
OH
IC50 = 750 ± 10 µM
NC
CN
S
NC
CN
S
NC
CN
S
H2N
N
H2N
N
11
IC50 = 227.4 ± 2.8 µM
12
IC50 = 240.1 ± 2.9 µM
H2N
N
7
IC50 = 55.6 ± 0.3 µM
7

Figure-3: Structure-activity relationship of compounds 7-12.
Among the di- and tri-alkoxy substituted analogs, compound 13 (IC50 = 92.7 ± 0.7 µM) with di-
methoxy substitutions at two meta positions and a bromo at para, found to be the second most
potent molecule of this series as well as seven-times more active than the standard acarbose. Its
good inhibitory potential might be due to symmetrical substitution pattern which makes it
favorable to bind with the enzyme. Replacement of bromo with another methoxy as in case of
compound 17 (IC50 = 750< µM) leads to a decline in the inhibitory potential which revealed a
crucial role of bromo group in inhibition of α-glucosidase. However, lack of bromo group in
compound 20 (IC50 = 750< µM) also leads to decreased inhibition potential. Similarly, replacement
of bromo and one of the methoxy as in cases of compounds 14 (IC50 = 191.6 ± 2.3 µM) and 15
(IC50 = 205.0 ± 2.5 µM) brings out the decline in the inhibitory strength as compared to compound
13 which gives the perception that switching of bromo position actually drives away the inhibition
potential to a considerable extent and bromo group at para position is suitable to interact with the
binding pocket of enzyme. Tri-methoxy substituted compound 16 (IC50 = 315.4 ± 3.6 µM) also
showed more than two-fold increased inhibitory potential. Regretfully, tri-methoxy substituted
compound 17 didn’t show activity comparable to compound 16 might be due to not satisfying the
conformational requirement to bind with the active site of α-glucosidase. Activity of analog 16 can
be compared with residue 18 (IC50 = 332.3 ± 3.9µM) having almost similar structural feature but
lack of one methoxy at meta position, displayed a slight decreased in inhibitory strength which
shows that m-methoxy is not worth important for the inhibitory potential. Similarly, lack of o-
methoxy in case of compound 19 (IC50 = 750< µM) displayed a sharp decline in the inhibitory
strength as compared to compound 16 which indicates that the ortho position has worth importance
in this activity. Activity comparison of compound 19 (IC50 = 750< µM) with 21 (IC50 = 448.7 ±
5.5 µM) revealed that insertion of ethoxy instead of methoxy at para position lifted up the
inhibitory potential up to a good extent (Figure-4).
8

OMe
MeO
Br
OMe
OMe
MeO
OMe
NC
CN
S
Br
OMe
CN
NC
CN
NC
H2N
N
15
H2N
N
S
H2N
N
S
IC50 = 205.0 ± 2.5 µM
14
16
IC50 = 315.4 ± 3.6 µM
IC50 = 191.6 ± 2.3 µM
Br
OMe
OEt
MeO
OMe
MeO
OMe
MeO
NC
CN
S
NC
CN
S
NC
CN
S
H2N
N
H2N
N
H2N
N
13
17
21
OMe
IC50 = 92.7 ± 0.7 µM
IC50 = 750 <
IC50 = 448.7 ± 5.5 µM
MeO
OMe
OMe
OMe
OMe
NC
H2N
CN
NC
CN
S
N
S
H2N
N
NC
CN
S
18
20
IC50 = 332.3 ± 3.9 µM
IC50 = 750 <
H2N
N
19
IC50 = 750 <
Figure-4: Structure-activity relationship of compounds 13-21.
Compounds 3 and 22-25 with extended conjugation showed multifold enhanced inhibitory
strength in the range of IC50 = 125.0 ± 1.0 - 184.3± 2.1 µM as compared to the standard acarbose
(IC50 = 750 ± 10µM). Amongst them, compound 22 (IC50 = 125.0 ± 1.0µM) having the 1-naphthyl
substitution was found to be six-fold more potent than standard and interestingly the fourth most
potent molecule of this library. The considerable inhibition strength of compound 22 possibly due
to the interaction of the extended π-electronic system with the active site of enzyme which resulted
in the significant inhibition. Similarly, analog 23 (IC50 = 156.7 ± 1.6µM) with 2-naphthyl
substitution also showed many folds enhanced inhibition potential. The inhibition strength of
compound 23 is slightly lower than the compound 22 that could occur only due to some
conformational differences. Furthermore, 3- benzyloxy-4-methoxy substituted compound 24 (IC50
9

= 130.8 ± 1.1µM) is amongst the most potent member of this library. Its potent inhibitory strength
might be due to combined effects of methoxy and benzyloxy group on inhibition. However,
switching of benzyloxy to para position and absence of methoxy group as in case of compound
25 (IC50 = 175.1 ± 1.9 µM) leads to decrease in the inhibition potential which suggests that the
position of benzyloxy and presence of methoxy group plays an important role in the inhibitory
potential. Exceptionally, compound 3 (IC50 = 184.3± 2.1µM) with a biphenyl ring has also
displayed four-folds more inhibition potential as compared to the standard, most probably due to
extended conjugation which makes it better interact with the active site of an enzyme (Figure-5).
NC
CN
S
NC
CN
S
O
N
OMe
H2N
N
O
H2N
N
23
3
IC50 = 156.7 ± 1.6 µM
IC50 = 184.3 ± 2.1 µM
NC
NC
CN
S
NC
CN
S
CN
S
H2N
N
H2N
H2N
N
22
IC50 = 125.0 ± 1.0 µM
25
24
IC50 = 175.1 ± 1.9 µM
IC50 = 130.8 ± 1.1 µM
Figure-5: Structure-activity relationship of compounds 3, 22-25.
Amongst the chloro substituted derivatives, o-chloro substituted compound 26 (IC50 = 160.5 ± 1.7
µM) exhibited four-folds enhanced activity as compared to the standard acarbose (IC50 = 750 ± 10
µM). Switching of chloro group from ortho to para position as in case of derivative 27 (IC50 =
324.7 ± 3.8 µM) results in a two-fold decreased activity which indicates the ortho position is
favorable for the chloro group to interact well with the active site of an enzyme. Incorporation of
methoxy next to chloro in compound 28 (IC50 = 326.5 ± 3.8µM) leads to a two-fold decline in the
inhibitory power might be creating the steric hindrance for the chloro group to interact with the
active site residues (Figure-6).
10

Cl
OMe
Cl
Cl
NC
CN
S
NC
CN
NC
CN
H2N
N
H2N
N
S
H2N
N
S
27
IC50 = 324.7 ± 3.8 µM
26
IC50 = 160.5 ± 1.7 µM
28
IC50 = 326.5 ± 3.8 µM
Figure-6: Structure-activity relationship of compounds 26-28
Limited SAR suggested that the halogens Br, F, Cl, and I and p-hydroxy substitutions played an
important role in the inhibition potential. Furthermore, an extended conjugation also takes part in
enhancing the strength of α-glucosidase inhibition.
Enzyme kinetic studies of α-glucosidase inhibition
Mode of inhibition of the most potent analog 7 was inspected contrary to α-glucosidase activity
with different concentrations of the substrate which is p-nitrophenyl α-D-glucopyranoside (2-10
mM), in the absence and presence of sample (compound 7) at different concentrations (0, 15, 35,
and 55 µM). A Lineweaver-Burk plot was generated to ascertain the type of inhibition. The
Michaelis-Menten constant (Km) value was determined from the plot between reciprocal of the
substrate concentration (1/[S]) and reciprocal of enzyme rate (1/V) over various inhibitor
concentrations. Experimental inhibitor constant (Ki) value was determined by secondary plots of
the inhibitor concentration [I] versus Km.
According to (Figure-7a), the Lineweaver-Burk plot displayed a gradual increase in Km value and
no change in Vmax with the increase of inhibitor concentration which indicates a competitive type
inhibition. The results show that residue 7 compete with the substrate to bind with the active site
of an enzyme. Furthermore, the plot of the Km versus different concentration of inhibitor gave an
approximation of the inhibition constant, Ki of 53 µM (Figure-7b).
11

b)
a)
7
6
5
4
3
2
1
0
30
20
10
0
Concentration of Inhibitor
0 µM
15 µM
35 µM
55 µM
-1.5
-1.0
-0.5
0.0
0.5
1.0
1/[S](PNPG, mM-1)
-60
-40
-20
0
20
40
60
80
-10
Compound (ALI-I-51)
Figure-7: Kinetics of α-glucosidase inhibition by compound (7). (a) The Lineweaver-Burk plot in
the absence and presence of different concentrations of compound (7); (b) The secondary plot
between Km and various concentrations of compound (7). Note: ALI-I-51 = Compound 7
Docking study
Docking study was performed using Autodock Tools (version 1.5.6) in order to elucidate the
interaction modes of the synthetic analogs in the α-glucosidase active site. Since the
crystallographic structure of α-glucosidase from S. cerevisiae (target enzyme in α-glucosidase
inhibition assay) do not report yet, we constructed modeled α-glucosidase by using the crystal
structure of S. cerevisiae isomaltase that has 71% identity and 84% similarity with S. cerevisiae α-
glucosidase [25]. Superimposition structure of standard inhibitor acarbose and most potent analog
7 in the active site of modeled α-glucosidase is shown in Figure-8.
Figure-8: Acarbose (cyan) and the most potent compound 7 (pink) superimposed in the active
site pocket of modeled α-glucosidase.
12

Figure-9: Docked conformer of acarbose in the active site of α-glucosidase.
Figure-9 shows that acarbose interacted with the active site residues Asn241, Ser308, Thr301,
Glu304, Thr307, Arg312, and Gln322 via hydrogen bonds and residue His279 via hydrophobic
interaction. Furthermore, it also created weak interactions with active site residues such as His239
and Val305.
The most active compound 7 interacted with residues Arg312, Glu304, His239, Phe157, His279,
Phe300, and Gln350 (Figure-10a). Sulfur unit of 6-(phenylthio) moiety interacted with His239 and
phenyl ring of this moiety interacted with Glu304 and Arg312 via a π-anion and a hydrophobic
interaction, respectively. Arg312 also formed a hydrophobic interaction with the pyridine ring. 2-
Amino and 5-cyano substituents of the pyridine ring created two hydrogen bonds with Phe157 and
His279. Furthermore, 4-hydroxyphenyl moiety of compound 7 formed a π-π hydrophobic
interaction with Phe300 via phenyl ring and a hydrogen bond with Gln350 via hydroxyl
substituent.
13

The second most active compound 13 established three hydrogen bonds with Asp408, Arg312,
and Asn241 through 2-amino and 3-cyano substituents of pyridine ring and 3-methoxy group of
4-bromo-3,5-dimethoxyphenyl moiety, respectively (Figure-10b). Pyridine ring and phenyl ring
of 4-bromo-3,5-dimethoxyphenyl moiety also formed hydrophobic interactions with Arg312. The
latter moiety also established hydrophobic interactions with His239 via 4-bromo group and phenyl
ring as well as weak interactions with Glu304 and Ser308 via 5-mertoxy group. 6-(Phenylthio)
moiety of compound 9 interacted with Phe158 (π-sulfur) and Asp349 (π-anion).
(a)
(b)
Figure-10: Docked conformer of the most potent compounds 7 (a) and 13 (b) in the active site of
α-glucosidase enzyme.
14

Conclusion
This piece of research work describes the synthesis and in vitro screening of diversely
functionalized pyridine derivatives for α-glucosidase inhibitory activity. Kinetics and molecular
docking studies were conducted on most potent compounds to support the findings. It was found
that many compounds displayed multifold enhanced inhibitory strength than the standard acarbose,
especially, halogens Br, F, Cl, and I and p-hydroxy substituted compounds as well as compounds
with extended conjugation. Kinetic studies of the most potent analogue revealed a competitive
type of inhibition. Docking studies identified many structural features that played important role
during binding with the active site of enzyme.
Experimental
Materials and Methods
Reagents were purchased from Sigma-Aldrich, USA and were of analytical grades. Thin-layer
chromatography was performed on pre-coated silica gel, GF-254. Spots were visualized under
ultraviolet light at 254 and 366 nm. Mass spectra were recorded on electron impact (EI) on MAT
1
13
312 and MAT 113D mass spectrometers. The H- and C-NMR were recorded on Bruker AM
machines, operating at 300 and 400 MHz. The chemical shift values are presented in ppm (δ)
relative to tetramethylsilane (TMS) as an internal standard and the coupling constant (J) are in Hz.
Symbols in NMR description such as s, d, t, dd, m, and ovp, corresponds to singlet, doublet, triplet,
doublet of doublet, multiplet, and overlapping multiplet, respectively.
General procedure for the syntheses of functionalized 2-amino-4-(substituted)phenyl-6-
phenylsulfanylpyridine-3,5-dicarbonitriles 1-28
Malononitrile (1 mmol) was first dissolved in EtOH (7 mL) into a 100 mL round-bottomed flask,
than aryl aldehyde (1 mmol) was added into it followed by 3 drops of triethylamine. The reaction
mixture was refluxed for 1 h and progress was monitored via TLC analysis (Hexane and Ethyl
acetate (8:2)). After intermediate formation, malononitrile (1 mmol), thiophenol (1 mmol), and
triethylamine (3 drops) were added in the reaction mixture and refluxed for approximately 4 h and
course of the reaction was monitored by TLC analysis. On completion, reaction mixture was
allowed to cool to room temperature which resulted to precipitates formation. The crude product
was collected by filtration and crystallized from EtOH to get the pure product. The purity of the
15

final products was checked by TLC analysis in different solvent systems to ensure absence of any
residual impurity and the products were found to be pure.
2-Amino-4-phenyl-6-(phenylthio)pyridine-3,5-dicarbonitrile (1) [26]
Yellow Solid; Yield: 72%; M.P.: 255-257 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.78 (s, 2H, NH),
7.61 (ovp, 8H, H-2, H-3, H-4, H-5, H-6, H-3ʹ, H-4ʹ, H-5ʹ), 7.50 (ovp, 2H, H-2ʹ, H-6ʹ); EI MS m/z
(% rel. abund.): 328 (M+, 96), 327 (100), 295 (4), 284 (6), 251 (2), 165 (9), 138 (2), 109 (4), 77
(6), 44 (16).
2-Amino-4-(3-nitrophenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (2) [27]
Brown Solid; Yield: 55%; M.P.: 215-216 ºC; 1H-NMR (300 MHz, DMSO-d6) δ 8.50 (s, 1H, H-2),
8.44 (dd, J4,2 = 1.6 Hz, J4,5 = 10.8 Hz, 1H, H-4), 8.06 (d, J6,5 = 10.4 Hz, 1H, H-6), 7.92 (ovp, 3H,
H-5, NH), 7.61 (ovp, 2H, H-3ʹ, H-5ʹ), 7.51 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ); EI MS m/z (% rel. abund.):
373 (M+, 100), 372 (72), 343 (11), 326 (40), 310 (5), 266 (3), 190 (3), 163 (5), 109 (6).
4-([1,1'-Biphenyl]-4-yl)-2-Amino-6-(phenylthio)pyridine-3,5-dicarbonitrile (3)
Off White Solid; Yield: 60%; M.P.: 175-177 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.89 (d, J9,8 =
J11,12 = 8.0 Hz, 2H, H-9, H-11), 7.81 (s, 1H, NH), 7.79 (d, J8,9 = J12,11 = 7.6 Hz, 2H, H-8, H-12),
7.66 (d, J2,3 = J6,5 = 8.4 Hz, 2H, H-2, H-6), 7.62 (ovp, 2H, H-3ʹ, H-5ʹ), 7.53 (ovp, 5H, H-3, H-5,
H-2ʹ, H-4ʹ, H-6ʹ), 7.37 (t, J10(9,11) = 7.6 Hz, 1H, H-10); 13C-NMR (DMSO-d6, 100 MHz): δC 166.1,
159.6, 158.2, 141.9, 138.8, 134.7, 134.7, 132.8, 129.6, 129.4, 129.4, 129.1, 129.1, 129.0, 129.0,
128.1, 127.1, 126.8, 126.8, 126.8, 126.8, 115.3, 115.0, 93.2, 86.9; EI MS m/z (% rel. abund.): 404
(M+, 100), 403 (93), 327 (16), 241 (3), 202 (7), 152 (3), 108 (2), 76 (5); HRESI-MS Calcd for
C25H16N4S: m/z = 404.1086, Found 404.1096.
2-Amino-6-(phenylthio)-4-(o-tolyl)pyridine-3,5-dicarbonitrile (4)
White Solid; Yield: 55%; M.P.: 185-187 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.83 (s, 2H, NH),
7.62 (ovp, 2H, H-3ʹ, H-5ʹ), 7.50 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 7.45 (ovp, 2H, H-11, H-12), 7.44 (t,
13
J10(9,11) = 7.6 Hz, 1H, H-10), 7.29 (d, J9,10 = 7.6 Hz, 1H, H-9), 2.17 (s, 3H, CH3); C-NMR
(DMSO-d6, 125 MHz): δC 165.8, 159.4, 159.0, 134.7, 134.7, 134.6, 133.9, 130.4, 129.9, 129.6,
129.4, 129.4, 128.1, 127.0, 127.0, 126.2, 114.8, 114.5, 93.8, 87.6; EI MS m/z (% rel. abund.): 342
(M+, 100), 341 (76), 327 (26), 265 (87), 238 (5), 206 (5), 179 (8), 152 (7), 140 (6), 77 (7) ); HRESI-
MS Calcd for C20H14N4S: m/z = 342.0943, Found 342.0939.
16

2'-Amino-6'-(phenylthio)-[3,4'-bipyridine]-3',5'-dicarbonitrile (5) [28]
Light Yellow Crystalline Solid; Yield: 60%; M.P.: 305-306 ºC; 1H-NMR (400 MHz, DMSO-d6) δ
8.77 (ovp, 2H, H-2, H-4), 8.05 (dd, J6,2 = 2.0 Hz, J6,5 = 8.0 Hz, 1H, H-6), 7.89 (s, 2H, NH), 7.64
(ovp, 3H, H-5, H-3ʹ, H-5ʹ), 7.50 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ); EI MS m/z (% rel. abund.): 329 (M+,
94), 328 (100), 312 (2), 301 (4), 285 (4) 263 (2), 166 (3), 139 (2), 109 (4), 77 (4).
2-Amino-4-(2-fluorophenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (6) CAS # 391666-95-
8
Yellow Solid; Yield: 55%; M.P.: 195-197 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.90 (s, 2H, NH),
7.67 (ovp, 5H, H-3, H-4, H-6, H-3ʹ, H-5ʹ), 7.50 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 7.43 (t, J5(4,6) = 7.6 Hz,
1H, H-5); EI MS m/z (% rel. abund.): 346 (M+, 98), 345 (100), 328 (12), 327 (41), 320 (6), 302
(7), 291 (2), 251 (2), 183 (9), 173 (6), 163 (3), 146 (5), 136 (4), 109 (6), 77 (6).
2-Amino-4-(4-hydroxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (7) [29]
Brown Solid; Yield: 55%; M.P.: 312-314 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H, OH),
7.69 (s, 2H, NH), 7.60 (ovp, 2H, H-3ʹ, H-5ʹ), 7.49 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 7.39 (d, J2,3 = J6,5 =
8.8 Hz, 2H, H-3, H-5), 6.92 (d, J3,2 = J5,6 = 8.4 Hz, 2H, H-3, H-5); EI MS m/z (% rel. abund.): 344
(M+, 91), 343 (100), 327 (11), 300 (4), 181 (4), 109 (3).
2-Amino-4-(3-hydroxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (8) [30]
White Crystalline Solid; Yield: 58%; M.P.: 335-337 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 9.86 (s,
1H, OH), 7.73 (s, 2H, NH), 7.60 (ovp, 2H, H-3ʹ, H-5ʹ), 7.49 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 7.36 (t,
J5(4,6) = 8.0 Hz, 1H, H-5), 6.95 (d, J2,6 = 0.8 Hz, 1H, H-2), 6.93 (dd, J6,2 = 0.8 Hz, J6,5 = 6.4 Hz, 1H,
H-6), 6.89 (dd, J4,6 = 1.6 Hz, J4,5 = 10.4 Hz, 1H, H-4); EI MS m/z (% rel. abund.): 344 (M+, 100),
343 (85), 327 (16), 286 (4), 161 (45), 160 (83), 133 (11), 104 (10), 77 (9).
2-Amino-4-(4-hydroxy-3-methoxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (9) [31]
White Solid; Yield: 62%; M.P.: 227-229ºC; 1H-NMR (300 MHz, DMSO-d6) δ 9.67 (s, 1H, OH),
7.69 (s, 2H, NH), 7.60 (ovp, 2H, H-3ʹ, H-5ʹ), 7.49 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 7.16 (dd, J2,6 = 2.1
Hz, 1H, H-2), 7.00 (dd, J6,2 = 2.1 Hz, J6,5 = 8.1 Hz, 1H, H-6), 6.93 (d, J5,6 = 8.1 Hz, 1H, H-5), 3.80
(s, 3H, OCH3); EI MS m/z (% rel. abund.): 374 (M+, 100), 373 (79), 358 (13), 357 (9), 343 (13),
330 (8).
17

2-Amino-4-(3-hydroxy-4-methoxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile
(10)
[31]
Yellow Solid; Yield: 65%; M.P.: 215-217 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 9.42 (s, 1H, OH),
7.69 (s, 2H, NH), 7.60 (ovp, 2H, H-3ʹ, H-5ʹ), 7.49 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 7.09 (d, J6,5 = 8.0
Hz, 1H, H-6), 6.95 (ovp, 2H, H-2, H-5), 3.84 (s, 3H, OCH3); EI MS m/z (% rel. abund.): 374 (M+,
100), 373 (79), 357 (16), 343 (13), 327 (21), 303 (10), 282 (9).
2-Amino-4-(4-fluoro-3-methoxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (11)
CAS # 1353657-31-4
Intense Yellow Solid; Yield: 53%; M.P.: 193-195 ºC; 1H-NMR (300 MHz, DMSO-d6) δ 7.81 (s,
2H, NH), 7.60 (ovp, 2H, H-3ʹ, H-5ʹ), 7.50 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 7.45 (ovp, 2H, H-2, H-5),
7.16 (m, 1H, H-6); EI MS m/z (% rel. abund.): 376 (M+, 100), 375 (88), 361 (29), 360 (34), 345
(61), 333 (23), 332 (45), 313 (11), 252 (8), 188 (15), 170 (10), 109 (14), 77 (15).
2-Amino-4-(4-hydroxy-3-iodo-5-methoxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile
(12)
Yellow Solid; Yield: 60%; M.P.: 245-247 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 10.17 (s, 1H, OH),
7.73 (s, 2H, NH), 7.58 (ovp, 2H, H-3ʹ, H-5ʹ), 7.49 (ovp, 4H, H-6, H-2ʹ, H-4ʹ, H-6ʹ), 7.22 (s, 1H, H-
13
2), 3.84 (s, 3H, OCH3); C-NMR (DMSO-d6, 100 MHz): δC 165.9, 159.6, 156.9, 148.1, 146.5,
134.7, 134.7, 130.2, 129.6, 129.4, 129.4, 127.2, 126.2, 115.4, 115.1, 112.6, 93.4, 87.0, 84.3, 53.3;
EI MS m/z (% rel. abund.): 500 (M+, 1.2), 199 (10), 198 (100), 171 (5), 158 (3), 145 (2), 144 (8),
133 (2), 116(1), 92 (8), 67 (5), 66 (3) ); HRESI-MS Calcd for C20H13IN4O2S: m/z = 499.9825,
Found 499.9804.
2-Amino-4-(4-bromo-3,5-dimethoxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (13)
Light Yellow Solid; Yield: 62%; M.P.: 195-197 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.82 (s, 2H,
13
NH), 7.60 (ovp, 2H, H-3ʹ, H-5ʹ), 7.50 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 6.98 (s, 2H, H-1, H-6); C-
NMR (DMSO-d6, 100 MHz): δC 165.9, 159.5, 157.9, 156.5, 156.5, 134.7, 134.7, 134.4, 134.4,
129.7, 129.4, 129.4, 115.2, 114.9, 105.4, 105.4, 101.8, 93.3, 87.1, 56.7, 56.7; EI MS m/z (% rel.
abund.): 466 (M+, 97), 468 (M+2, 100), 451 (6), 435 (14), 357 (5), 341 (5), 329 (10), 301 (8);
HRESI-MS Calcd for C21H15BrN4O2S m/z = 466.0069, Found 466.0099.
18

2-Amino-4-(2-bromo-4,5-dimethoxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (14)
Yellow Solid; Yield: 65%; M.P.: 230-232 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.86 (s, 2H, NH),
7.61 (ovp, 2H, H-3ʹ, H-5ʹ), 7.51 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 7.33 (s, 1H, H-3), 7.17 (s, 1H, H-6),
3.85 (s, 3H, OCH3), 3.76 (s, 3H, OCH3); 13C-NMR (DMSO-d6, 100 MHz): δC 165.6, 159.3, 158.0,
150.4, 148.3, 134.8, 134.8. 129.7, 129.4, 129.4, 126.8, 126.6, 115.4, 114.6, 114.3, 112.8, 111.1,
94.3, 88.2, 56.0, 56.0; EI MS m/z (% rel. abund.): 466 (M+, 100), 468 (M+2, 91), 371 (30), 343
(21), 329 (20), 301 (17), 278 (4), 231 (22), 229 (22), 203 (14), 172 (6), 110 (17), 109 (14), 83 (24)
44 (17); HRESI-MS Calcd for C21H15BrN4O2S m/z = 466.0111, Found 466.0099.
2-Amino-4-(3-bromo-4,5-dimethoxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (15)
White Solid; Yield: 61%; M.P.: 228-230 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.89 (s, 2H, NH),
7.64 (ovp, 2H, H-3ʹ, H-5ʹ), 7.53 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 7.36 (s, 1H, H-2), 7.19 (s, 1H, H-6),
3.88 (s, 3H, OCH3), 3.79 (s, 3H, OCH3); 13C-NMR (DMSO-d6, 100 MHz): δC 165.9, 159.5, 158.3,
150.2, 147.2, 134.8, 134.8, 129.7, 129.4, 129.4, 126.8, 126.3, 115.4, 114.5, 114.0, 113.7, 111.1,
94.7, 87.3, 56.0, 56.0; EI MS m/z (% rel. abund.): 466 (M+, 100), 468 (M+2, 91), 371 (30), 343
(21), 329 (20), 301 (17), 278 (4), 231 (22), 229 (22); HRESI-MS Calcd for C21H15BrN4O2S m/z =
466.0111, Found 466.0099.
2-Amino-6-(phenylthio)-4-(2,3,4-trimethoxyphenyl)pyridine-3,5-dicarbonitrile (16)
Light Yellow Solid; Yield: 70%; M.P.: 213-215 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.40 (s, 2H,
NH), 7.61 (ovp, 2H, H-3ʹ, H-5ʹ), 7.49 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 7.07 (s, 1H, H-6), 6.96 (s, 1H,
13
H-5), 3.87 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.75 (s, 3H, OCH3); C-NMR (DMSO-d6, 75
MHz): δC 159.4, 156.3, 155.2, 150.1, 134.8, 134.8, 129.6, 129.4, 129.4, 127.1, 127.1, 127.1, 124.2,
124.2, 120.2, 115.1, 114.8, 107.8, 94.5, 88.3, 61.2, 60.5, 55.9; EI MS m/z (% rel. abund.): 418 (M+,
100), 417 (35), 403 (11), 387 (11), 371 (19), 354 (38), 343 (25), 329 (19), 317 (18), 294 (9), 144
(10); HRESI-MS Calcd for C22H18N4O3S m/z = 418.1104, Found 418.1100.
2-Amino-6-(phenylthio)-4-(3,4,5-trimethoxyphenyl)pyridine-3,5-dicarbonitrile (17) [32]
º
1
Off White Solid; Yield: 62%; M.P.: 237-239 C; H-NMR (300 MHz, DMSO-d6) δ 7.77 (s, 2H,
NH), 7.60 (ovp, 2H, H-3ʹ, H-5ʹ), 7.50 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 6.91 (s, 2H, H-1, H-6); EI MS
m/z (% rel. abund.): 418 (M+, 100), 403 (70), 343 (35), 331 (10), 317 (19), 300 (11), 289 (18), 172
(9).
19

2-Amino-4-(2,4-dimethoxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (18) [31]
Light Brown Solid; Yield: 60%; M.P.: 215-217 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.67 (s, 2H,
NH), 7.60 (ovp, 2H, H-3ʹ, H-5ʹ), 7.49 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 7.28 (d, J6,5 = 8.4 Hz, 1H, H-6),
6.75 (d, J3,5 = 2.0 Hz, 1H, H-3), 6.69 (dd, J5,3 = 1.2 Hz, J5,6 = 8.4 Hz, 1H, H-4); EI MS m/z (% rel.
abund.): 389 (M+, 55), 388 (100), 387 (75), 373 (10), 357 (25), 311 (65), 279 (7), 194 (11), 151
(9).
2-Amino-4-(3,4-dimethoxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (19) [33]
º
1
Off White Solid; Yield: 65%; M.P.: 262-264 C; H-NMR (400 MHz, DMSO-d6) δ 7.72 (s, 2H,
NH), 7.60 (ovp, 2H, H-3ʹ, H-5ʹ), 7.49 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 7.19 (d, J2,6 = 1.2 Hz, 1H, H-2),
7.12 (ovp, 2H, H-5, H-6); EI MS m/z (% rel. abund.): 388 (M+, 100), 387 (27), 373 (7), 357 (8),
342 (7), 330 (6), 276 (8).
2-Amino-4-(3,5-dimethoxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (20)
Reddish Brown Solid; Yield: 62%; M.P.: 223-225 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.76 (s,
2H, NH), 7.60 (ovp, 2H, H-3ʹ, H-5ʹ), 7.50 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 6.68 (ovp, 3H, H-2, H-4, H-
13
6), 3.79 (s, 6H, OCH3); C-NMR (DMSO-d6, 75 MHz): δC 165.9, 160.4, 159.5, 158.3, 158.3,
135.6, 134.8, 134.8, 129.6, 129.4, 129.4, 127.1, 127.1, 115.1, 114.8, 106.5, 106.5, 101.6, 93.3,
87.0, 55.4, 55.4; EI MS m/z (% rel. abund.): 388 (M+, 100), 357 (19), 342 (3), 329 (5), 276 (2),
194 (4), 165 (3), 151 (1), 109 (1), 77 (2); HRESI-MS Calcd for C22H18N4O3S m/z = 388.0093,
Found 388.0094.
2-Amino-4-(4-ethoxy-3-methoxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (21)
White Solid; Yield: 65%; M.P.: 223-225 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.71 (s, 2H, NH),
7.60 (ovp, 2H, H-3ʹ, H-5ʹ), 7.49 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ), 7.19 (d, J2,6 = 1.6 Hz, 1H, H-2), 7.10
(m, 2H, H-5, H-6), 4.12 (q, JCH2,CH3 = 6.8 Hz, 2H, CH2), 3.79 (s, 3H, OCH3), 1.37 (t, JCH3 CH2 =
,
6.8 Hz, 3H, OCH3); 13C-NMR (DMSO-d6, 125 MHz): δC 166.0, 159.7, 158.3, 149.6, 148.3, 134.7,
134.7, 129.6, 129.4, 129.4, 127.2, 125.6, 121.5, 115.5, 115.2, 112.4, 112.2, 63.7, 55.6, 14.6; EI
MS m/z (% rel. abund.): 402 (M+, 100), 373 (61), 358 (8), 343 (10), 330 (6), 313 (5), 276 (5);
HRESI-MS Calcd for C22H18N4O2S m/z = 402.1145, Found 402.1150.
20

2-Amino-4-(naphthalen-1-yl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (22) [34]
Solid Brown Crystalline; Yield: 65%; M.P.: 295-297 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 8.13
(d, J9,8 = 8.0 Hz, 1H, H-9), 8.07 (d, J2,3 = 7.6 Hz, 1H, H-2), 7.69 (ovp, 3H, H-4, H-3ʹ, H-5ʹ), 7.62
(ovp, 4H, H-3, H-6, H-7, H-8), 7.50 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ); EI MS m/z (% rel. abund.): 378
(M+, 89), 377 (100), 345 (7), 301 (7), 269 (3), 215 (8), 188 (8).
2-Amino-4-(naphthalen-2-yl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (23) [35]
Yellow Solid; Yield: 68%; M.P.: 315-317 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 8.15 (s, 1H, H-
4), 8.12 (s, 1H, H-7), 8.06 (m, 2H, H-9, H-10), 7.84 (s, 2H, NH), 7.68 (ovp, 5H, H-2ʹ, H-3ʹ, H-4ʹ,
H-5ʹ, H-6ʹ), 7.51 (ovp, 3H, H-2, H-5, H-6); EI MS m/z (% rel. abund.): 378 (M+, 100), 377 (98),
334 (5), 215 (5), 189 (8), 160 (9), 147 (7).
2-Amino-4-(3-(benzyloxy)-4-methoxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (24)
Yellow Solid; Yield: 67%; M.P.: 225-227 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.73 (s, 2H, NH),
7.60 (ovp, 2H, H-3ʹ, H-5ʹ), 7.49 (ovp, 4H, H-2ʹ, H-4ʹ, H-6ʹ, H-5), 7.41 (t, J9(8,10) = J10(9,11) = J11(12,10)
= 7.6 Hz, 3H, H-9, H-10, H-11), 7.36 (ovp, 2H, H-8, H-12), 7.18 (ovp, 2H, H-5, H-6), 5.07 (s, 1H,
CH2), 3.84 (s, 3H, OCH3); 13C-NMR (DMSO-d6, 125 MHz): δC 166.0, 159.7, 158.2, 150.7, 147.5,
136.6, 134.8, 134.8, 129.6, 129.4, 129.4, 128.4, 128.4, 128.0, 127.9,127.9, 127.2, 125.8, 122.0,
115.5, 115.2, 113.9, 111.8, 70.4, 55.6; EI MS m/z (% rel. abund.): 464 (M+, 63), 447 (10), 433
(16), 374 (33), 360 (55), 342 (11), 302 (9), 276 (12), 92 (26), 91 (100); HRESI-MS Calcd for
C27H20N4O2S m/z = 464.1287, Found 464.1307.
2-Amino-4-(4-(benzyloxy)phenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (25) CAS #
1019063-42-3
White Solid; Yield: 62%; M.P.: 207-209 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.73 (s, 2H, NH),
7.60 (ovp, 2H, H-2, H-6), 7.52 (ovp, 7H, H-2ʹ, H-3ʹ, H-4ʹ, H-5ʹ, H-6ʹ, H-8, H-12), 7.43 (t, J9(10,8)
=
J11(10,12) = 7.2 Hz, 2H, H-9, H-11), 7.36 (t, J10(11,9) = 7.2 Hz, 1H, H-10), 7.20 (d, J3,2 = J5,6 = 8.8
Hz, 2H, H-3, H-5), 5.18 (s, 2H, CH2); EI MS m/z (% rel. abund.): 434 (M+, 78), 343 (10), 314 (4),
288 (2), 92 (15), 91 (100), 65 (7).
21

2-Amino-4-(2-chlorophenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (26) CAS # 1414218-
12-4
White Solid; Yield: 63%; M.P.: 235-237 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.93 (s, 2H, NH),
7.70 (d, J6,5 = 8.0 Hz, 1H, H-6), 7.62 (ovp, 3H, H-3, H-4, H-5), 7.59 (ovp, 2H, H-3ʹ, H-5ʹ), 7.50
(ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ); EI MS m/z (% rel. abund.): 362 (M+, 100), 361 (91), 328 (23), 327
(87), 326 (11), 325 (6), 310 (6), 299 (6), 218 (5), 163 (4), 136 (2), 109 (7), 77 (7).
2-Amino-4-(4-chlorophenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (27) [36]
Solid Light Yellow Crystalline; Yield: 65%; M.P.: 230-232 ºC; 1H-NMR (300 MHz, DMSO-d6) δ
7.84 (s, 2H, NH), 7.67 (d, J2,3 = J6,5 = 11.2 Hz, 2H, H-2, H-6), 7.60 (ovp, 4H, H-3, H-5, H-3ʹ, H-
5ʹ), 7.50 (ovp, 3H, H-2ʹ, H-4ʹ, H-6ʹ); EI MS m/z (% rel. abund.): 364 (M+, 31), 362 (M+2, 100),
361 (83), 327 (23), 318 (4), 199 (17), 163 (9), 109 (7), 77 (14), 65 (5), 50 (16).
2-Amino-4-(2-chloro-3-methoxyphenyl)-6-(phenylthio)pyridine-3,5-dicarbonitrile (28)
White Solid; Yield: 45%; M.P.: 305-307 ºC; 1H-NMR (400 MHz, DMSO-d6) δ 7.90 (s, 2H, NH),
7.62 (ovp, 2H, H-3ʹ, H-5ʹ), 7.52 (ovp, 4H, H-6, H-2ʹ, H-4ʹ, H-6ʹ), 7.34 (d, J5,4 = 8.0 Hz, 1H, H-5),
13
7.09 (d, J4,5 = 8.0 Hz, 1H, H-5), 3.93 (s, 3H, OCH3); C-NMR (DMSO-d6, 75 MHz): δC 165.9,
159.3, 156.4, 154.8, 134.8, 134.8, 134.2, 129.8, 129.4, 129.4, 128.6, 126.7, 121.2, 119.0, 114.4,
114.1, 114.0, 93.5, 87.6, 56.3; EI MS m/z (% rel. abund.): 392 (M+, 100), 376 (4), 357 (42), 342
(22), 326 (7), 314 (18), 286 (3), 248 (2), 109 (3), 77 (3); HRESI-MS Calcd for C20H13ClN4OS m/z
= 392.0479, Found 392.0499.
Molecular Docking Study
To prepare a homology model of α-glucosidase, we used the method described by Imran et al. [37,
38]. At first, a search was carried out using SWISS-MODEL to identify a protein in the PDB with
a high sequence similarity with S. cerevisiae α-glucosidase. As a result, isomaltase from S.
cerevisiae (PDB code 3A4A), with 72% identical and shares 85% similarity with the S. cerevisiae
α-glucosidase, was selected. Next, this enzyme was subjected through sequence alignment and
homology model using automated homology modeling pipeline SWISS-MODEL (managed by
Swiss Institute of Bioinformatics) and the quality of the obtained homology model was verified
using PROCHECK [39]. The 3D structures of the acarbose and selected synthesized inhibitors
were built by MarvineSketch 5.8.3, 2012, ChemAxon (http://www.chemaxon.com) and converted
22

to pdbqt coordinate using Auto dock Tools. The pdbqt coordinate of modeled α-glucosidase was
produced using the same software. Also, the pdbqt coordinate of protein was procured using the
Auto Dock Tools. The water molecules and the inhibitors were removed from protein. Then, using
Auto Dock Tools, polar hydrogen atoms were added, Koullman charges were assigned, and the
obtained protein structure was used as an input file for the AUTOGRID program. In AUTOGRID
for each atom type in the ligand, maps were calculated with 0.375ꢀÅ spacing between grid points
and the center of the grid box was placed at xꢀ=ꢀ12.5825, yꢀ=ꢀ−7.8955, zꢀ=ꢀ12.519. The dimensions
of the active site box were set at 40ꢀ×ꢀ40ꢀ×ꢀ40ꢀÅ. Flexible ligand dockings were accomplished for
the selected compounds. Each docked system was carried out by 50 runs of the AUTODOCK
search by the Lamarckian genetic algorithm (LGA). The best poses of the selected compounds
were selected for analyzing the interactions between enzyme and the inhibitors. The results were
visualized using BIOVIA Discovery Studio v.3.5.
Acknowledgement: The authors are thankful to the Pakistan Academy of Sciences for providing
financial support to Project No. (5-9/PAS/440).
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Synthesis,
and
Screening of 2-Amino-4-Aryl-6-(phenylthio) pyridine-3,5-
In vitro
In silico
dicarbonitriles as Novel -Glucosidase Inhibitors

Muhammad Ali, Khalid Mohammed Khan, Mohammad Mahdavi, Abdul Jabbar, Shahbaz
Shamim, Uzma Salar, Muhammad Taha, Shahnaz Parveen, Bagher Larijani, Mohammad Ali
Faramarzi
H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University
of Karachi, Karachi-75270, Pakistan
SH
O
R
R
H
N
R
Et3N, EtOH
Reflux, 5 h
NH4OAc
NC
H2N
CN
S
CN
CN
EtOH, Reflux, 2 h
N
N
N
N
Intermediate
1-28
OH
Br
2-Amino-4-aryl-6-(phenylthio) pyridine-
3,5-dicarbonitrile analogs
MeO
OMe
R
NC
H2N
CN
NC
CN
S
NC
CN
S
N
S
H2N
N
7
13
H2N
N
1-28
IC50 = 55.6 ± 0.3 µM
IC50 = 92.7 ± 0.7 µM
IC50 = 55.6 ± 0.3 - 474.2 ± 5.0 µM
Acarbose = IC50 = 750 ± 10 µM
Docked conformer of the most potent
Docked conformer of the most potent
compound 13
compound 7
28

Highlights:





2-Amino-4-aryl-6-(phenylthio)pyridine-3,5-dicarbonitriles 1-28 were synthesized.
Compounds were subjected to in vitro α-glucosidase inhibitory activity.
Compounds 1-3, 7, 9, 11-14, and 16 showed many folds increased inhibitory potential.
Structure-activity relationship (SAR) was established.
Kinetic studies showed the competitive type inhibitory mechanism by the most potent
molecule 7.

In silico study was performed to decipher the binding interaction of compound 7 with the
active pocket of α-glucosidase enzyme.
29