Novel C-2 symmetric molecules as α-glucosidase and α-amylase inhibitors: Design, synthesis, kinetic evaluation, molecular docking and pharmacokinetics

Article abstract of DOI:10.3390/molecules24081511

A series of symmetrical salicylaldehyde-bishydrazine azo molecules, 5a–5h, have been synthesized, characterized by ¹H-NMR and ¹³C-NMR, and evaluated for their in vitro α-glucosidase and α-amylase inhibitory activities. All the synthesized compounds efficiently inhibited both enzymes. Compound 5g was the most potent derivative in the series, and powerfully inhibited both α-glucosidase and α-amylase. The IC₅₀ of 5g against α-glucosidase was 0.35917 ± 0.0189 μM (standard acarbose IC₅₀ = 6.109 ± 0.329 μM), and the IC₅₀ value of 5g against α-amylase was 0.4379 ± 0.0423 μM (standard acarbose IC₅₀ = 33.178 ± 2.392 μM). The Lineweaver-Burk plot indicated that compound 5g is a competitive inhibitor of α-glucosidase. The binding interactions of the most active analogues were confirmed through molecular docking studies. Docking studies showed that 5g interacts with the residues Trp690, Asp548, Arg425, and Glu426, which form hydrogen bonds to 5g with distances of 2.05, 2.20, 2.10 and 2.18 ?, respectively. All compounds showed high mutagenic and tumorigenic behaviors, and only 5e showed irritant properties. In addition, all the derivatives showed good antioxidant activities. The pharmacokinetic evaluation also revealed promising results.

Full text of DOI:10.3390/molecules24081511

molecules
Article
Novel C-2 Symmetric Molecules as α-Glucosidase
and α-Amylase Inhibitors: Design, Synthesis, Kinetic
Evaluation, Molecular Docking and Pharmacokinetics
1
1,
1
1
2
Danish Shahzad , Aamer Saeed * , Fayaz Ali Larik
, Pervaiz Ali Channar , Qamar Abbas ,
3
1
4
5
Mohamed F. Alajmi , M. Ifzan Arshad , Mauricio F. Erben
Hussain Raza 5 , Sung-Yum Seo 5 and Hesham R. El-Seedi 6
, Mubashir Hassan ,
1
Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan;
danish.scientist.shahzad@gmail.com (D.S.); fayazali@chem.qau.edu.pk (F.A.L.);
mrpervaiz@gmail.com (P.A.C.); ifzan_ifzan@yahoo.com (M.I.A.)
Department of Physiology, University of Sindh, Jamshoro 76080, Pakistan; qamar.abbas.qau@gmail.com
Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia;
malajmii@ksu.edu.sa
2
3
4
5
CEQUINOR (UNLP, CONICET-CCT La Plata), Departamento de Química, Facultad de Ciencias Exactas,
Universidad Nacional de La Plata, Boulevard 120 e/60 y 64 N 1465, La Plata 1900, Argentina;
erbenmf@gmail.com
◦
Department of Biological Sciences, College of Natural Sciences, Kongju National University,
5
6 Gongjudehak-Ro, Gongju, Chungnam 32588, Korea; mubashirhassan_gcul@yahoo.com (M.H.);
hussain_solangi@yahoo.com (H.R.); dnalove@kongju.ac.kr (S.-Y.S.)
Pharmacognosy Group, Department of Medicinal Chemistry, Biomedical Center (BMC), Uppsala University,
SE-751 23 Uppsala, Sweden; Hesham.El-Seedi@fkog.uu.se
6
*
Correspondence: aamersaeed@yahoo.com; Tel.: +92-51-9064-2128; Fax: +92-51-9064-2241
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 8 March 2019; Accepted: 2 April 2019; Published: 17 April 2019
Abstract: A series of symmetrical salicylaldehyde-bishydrazine azo molecules, 5a–5h, have been
1
13
synthesized, characterized by H-NMR and C-NMR, and evaluated for their in vitro α-glucosidase
and -amylase inhibitory activities. All the synthesized compounds efficiently inhibited both
enzymes. Compound 5g was the most potent derivative in the series, and powerfully inhibited
both -glucosidase and -amylase. The IC50 of 5g against -glucosidase was 0.35917 0.0189
standard acarbose IC50 = 6.109 0.329 M), and the IC50 value of 5g against -amylase was 0.4379
M (standard acarbose IC50 = 33.178 2.392 M). The Lineweaver-Burk plot indicated that
-glucosidase. The binding interactions of the most active
α
α
α
α
±
µM
±
(
0
±
µ
α
.0423
µ
±
µ
compound 5g is a competitive inhibitor of
α
analogues were confirmed through molecular docking studies. Docking studies showed that 5g
interacts with the residues Trp690, Asp548, Arg425, and Glu426, which form hydrogen bonds to 5g
with distances of 2.05, 2.20, 2.10 and 2.18 Å, respectively. All compounds showed high mutagenic
and tumorigenic behaviors, and only 5e showed irritant properties. In addition, all the derivatives
showed good antioxidant activities. The pharmacokinetic evaluation also revealed promising results
Keywords: bis-azo Schiff bases; dual inhibitor; α-glucosidase inhibitor; α-amylase; antioxidant; SAR;
chemo-informatics; kinetic mechanism; molecular docking
1
. Introduction
Diabetes mellitus (DM) is a metabolic disorder that poses a serious threat to human health across
the globe [ ]. In particular, type 2 diabetes mellitus (T2DM) is widely encountered in aged people [ ].
An imbalance in the transport of glucose is the main factor leading to diabetes [ ]. An abrupt increase
1
2
3
Molecules 2019, 24, 1511; doi:10.3390/molecules24081511
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1511
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in the glucose level is the major cause of T2DM. Changes in social norms and a lack of proper education
regarding a balanced diet are among the main causes of DM [ ]. According to the World Health
Organization, at least 2.8% of the world’s population suffered from T2DM in 2000, and it is estimated
that by 2030, this percentage will almost double [ ]. The drugs approved by the U.S. FDA for the
treatment of diabetes can be categorized into five classes: Biguanides, thiazolidinediones, sulfonylureas,
meglitinides and -glucosidase inhibitors (AGIs) [ ]. The efficacy and sustainability of some drugs are
not sufficient. Therefore, the identification of more effective and safer anti-diabetic drugs is of great
4
5
α
6
®
®
®
importance. Several AIGs, such as acarbose (Glucobay ), voglibose (Volix , Basen ) and miglitol
®
(Glyset ) (Figure 1), can reversibly inhibit
α
-glucosidase, consequently delaying the absorption of
sugars from the gut, and they have been used clinically for the treatment of DM [7].
Figure 1. Structures of some of the available α-glucosidase inhibitors.
There commercially available AGIs are associated with certain drawbacks, such as
requiring tedious, multistep syntheses; for example voglibose is prepared via a 13-step
procedure from (
−
) shikimic acid, and miglitol is prepared in 13 steps from (R)-methyl
2-benzamido-3-((tert-butyldimethylsilyl)oxy)propanoate. Moreover, these AGIs also show some
serious adverse effects, including causing flatulence and diarrhoea [8].
To control postprandial glucose levels in T2DM patients, AGIs must be administered. AGIs assist
in controlling the progress of hyperglycaemia and hyperinsulinaemia by diverting the workload from
pancreatic cells. Postprandial hyperglycaemia (PPHG), if not cured properly and in a timely manner,
can lead to life-threatening complications, such as cardiovascular disease and hypertension-related
disorders [9]. Acarbose was the first AGI and was made commercially available in 1990. Japanese
regulatory agencies approved voglibose as a new AGI in 1994, and two years later, American regulatory
agencies approved miglitol as a potent anti-diabetic agent [10]. Among these three AGIs (acarbose,
voglibose and miglitol), acarbose has promising effects on several intestinal enzymes; for example, it
mainly inhibits
10%) [11]. -Glucosidase is a membrane-bound enzyme found in intestinal cells that catalyses the
production of glucose from a carbohydrate source [12] and -Glucosidase cleaves -glycosidic bonds.
α-amylases, including glucoamylase (90%), sucrose (65%), maltase (60%) and isomaltase
(
α
α
α
Glucosidase inhibitors can be used as first-line drugs for the treatment of T2DM, because they can
lower the rate of carbohydrate absorption and suppress postprandial hyperglycaemia [13–17].
Schiff bases, also known as imines or azomethines, are considered a privileged class of organic
compounds and have extensive applications in the fields of biology [18,19], catalysis [20], materials
chemistry [21] and inorganic [22] and analytical chemistry [23]. These compounds possess a wide
range of biological activities, such as antibacterial, antifungal, antimalarial, anticancer, antitubercular,
anti-inflammatory, and antiviral activities [24]. Schiff bases are key precursors in the synthesis of a
wide variety of metal complexes, and Schiff base-metal complexes are stable in nature and are useful in
non-linear optics (NLOs). Salicylaldehyde-derived Schiff bases have attracted substantial attention,
because they exhibit promising effects against microbes [25].

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Azo dyes are key chromophores in the chemical industry as dyes and pigments [26], food
additives [27], indicators [28], radical initiators [29] and therapeutic agents [30]. Azo azomethine
compounds are commonly prepared by the condensation of a primary amine or hydrazine with an
aromatic aldehyde or ketone-linked –N=N– chromophore [31]. Azo ligands are considered robust
and chemically stable compounds, and they have been thoroughly investigated in the dye and
photoelectrochemical fields [32]. Besides a number of negative features attached to azo compounds,
such as toxicity and non-biodegradability, there are several favorable aspects that make them medically
attractive compounds. These include the specific azoreduction in vivo, site specific drug delivery of
polymeric azo compound in the colon diseases, such as colitis and irritable bowel syndrome Reductive
degradation and subsequent splitting of the azo bond Occur in the colon, and therefore they are
highly site-specific. Similarly, the azo pro-drugs are reduced to the corresponding amines that are
exact therapeutics, for example, 5-aminosalicylic acid derivatives that exhibit anti-inflammatory and
cytoprotective potency [33]. FP-21399 is another azo molecule which exhibits anti-HIV potency by
inhibiting HIV envelope glycoprotein-mediated membrane fusion that precedes virus infiltration
into the cell [34]. There are a limited number of studies on the anti-diabetic and pharmacodynamic
properties of salicylaldehyde-derived bis-azo Schiff base ligands.
Herein, we develop a synthetic route to construct new bis-azo ligands using salicylaldehyde as
a key precursor. The final products contain a bis-azo group, four aromatic rings, two rings bearing
hydroxyl groups and two rings bearing electron-withdrawing substituents.
These ligands were selected for two reasons: (i) To provide new acyclic salicylaldehyde-based
bis-azo-containing Schiff base ligands, and (ii) to investigate their inhibitory activities against
α-amylase
and -glucosidase to uncover their antioxidant potential, the kinetics of their enzyme inhibition, their
α
pharmacodynamics (chemo-informatics and Lipinski’s rule validation), their initial structure-activity
relationships and their binding affinities by exploiting molecular docking.
2
. Results and Discussion
The synthesis of the bis-azo dyes containing azomethine is depicted in Scheme 1, and the structures
and yields of the compounds are shown in Scheme 2. Appropriately substituted anilines (1a
used as the starting materials, and the amine group was converted into the diazonium salt using
NaNO and drops of HCl. The diazonium salts (2a ) were then coupled with salicylaldehyde
. The aldehyde groups of 4a were converted to the
–h) were
–
h
2
under mildly basic conditions to afford 4a
–
h
–h
corresponding imine with hydrazine hydrate using ethanol to afford the desired products (5a–h) in
encouraging yields.
Scheme 1. The synthetic route towards novel symmetrical compounds (5a–h).

Molecules 2019, 24, 1511
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Scheme 2. Structures and yields of synthesized symmetrical molecules (5a–h).
The synthesized compounds were characterized through H-NMR and 13C-NMR spectroscopy.
The signal at approximately 9.56 ppm was attributed to the azomethine proton. The phenolic proton
appeared at 5–6 ppm. The signals appearing at 7–8 ppm were attributed to protons on the phenyl
ring. The signals in the 13C-NMR spectra from 160–170 ppm were assigned to the carbon of the imine
1
2
moiety. The carbons bearing electron-withdrawing groups appeared downfield, and the sp carbons of
the phenyl rings appeared from approximately 140–120 ppm.
Azomethine-linked salicylaldehyde is widely recognized as a bioactive moiety. These newly
synthesized, bis-azo salicylaldehyde-based Schiff bases inhibit α-glucosidase and α-amylase to various
degrees. To elucidate the electronic influence on enzyme inhibition, substrates representing almost
all possible substitution patterns of the azo group on the phenyl ring were explored. The search for
new, potent and safe anti-diabetic agents will remain an important and challenging task for medicinal
chemists. Synthesized derivatives 5a–h are structurally similar to each other, and they only differ
in the position of the electron-withdrawing group on the phenyl ring. However, these ligands all
share a common feature: The presence of two hydroxyl groups, which are pivotal to the inhibition
of
α-glucosidase and α-amylase. The azomethine moiety is considered one of the most intriguing
structural entities in medicinal chemistry, because it can interact with the target protein of the enzyme
via non-covalent interactions by serving as a hydrogen bond acceptor. Interestingly, compound 5g
,
which is a non-halogenated derivative in this series, exhibited the highest inhibitory potential against
both enzymes (α-glucoside and α-amylase), and it was a dual inhibitor of these two diabetes-promoting
enzymes [35]. The IC50 values of 5g were found to be several folds better than those of acarbose, which
was used as a standard (Table 1).

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Table 1. α-Glucosidase and α-amylase inhibitory activities of compounds 5a–h.
Inhibitor
α-Glucosidase (µM)
α-Amylase (µM)
5
a
0.547 ± 0.0289
5.345 ± 0.2826
8.061 ± 0.4263
19.521 ± 1.0321
0.367 ± 0.01941
1.841 ± 0.09738
0.359 ± 0.0189
0.400 ± 0.0211
6.109 ± 0.329
8.541 ± 0.653
28.373 ± 2.171
9.5482 ± 0.730
9.7183 ± 0.863
28.4828 ± 2.081
4.2861 ± 0.328
0.4379 ± 0.042
0.5902 ± 0.012
33.178 ± 2.392
5
b
5
c
5
d
5
e
5
f
5
g
5
h
Acarbose
Values are expressed as the mean ± SEM; SEM = Standard Error of the Mean.
Nitro-substitution is found in a number of marketed drugs such as Azomycin, Nifurtimox,
Benznidazole, Tinidazole, Fexinidazole, Ventetoclax, Delamanid, Entacapone etc. Similarly, there are
several therapeutic agents containing a nitro group with bioreductive potentials, such as Paclitaxel
prodrug, Tarloxotinib, BTZ043, Evofosfamide, CB-1954 and KS119 [36]. Nevertheless, the nitro group
is considered as both a pharmacophore and a toxicophore (hepatotoxic, mutagenic). Compound 5g
possessed a nitro group at the para position of the phenyl ring and showed the most potent inhibition.
Compound 5d, which possessed a chloride at the para position of the phenyl ring, showed the weakest
inhibition in the case of α-glucosidase, indicating that the nature of the electronic group substantially
influenced the inhibitory effect. A nitro group can induce great charge density by pulling electrons
from the aromatic ring via a resonance effect, while the substituent on compound 5d can also shift the
electron cloud via an inductive effect, but the charge separation will be greater in the case of 5g. In the
case of α-amylase inhibition, compound 5e displayed the weakest inhibition, which can be attributed
to the presence of a sulfonamide group at the para position of the phenyl ring. All the compounds
showed significant inhibition and can serve as lead molecules in the design of DM inhibitors.
2
.1. Kinetic Analysis
To understand the inhibition mechanism of the synthesized compounds on α-glucosidase, an
inhibition kinetics study was performed. The inhibition type and an inhibition constant of the most
potent compound, 5g, based on our IC50 results, were determined. In our evaluation of the kinetics
of the enzyme, the Lineweaver-Burk plot of 1/V versus 1/[S] in the presence of different inhibitor
concentrations gave a series of straight lines. The Lineweaver-Burk plot of compound 5g showed
that Vmax remains the same without significantly affecting the slopes. K
concentration, while Vmax remains essentially constant. This behavior indicates that 5g inhibits the
-glucosidase in a competitive manner (Figure 2A). Second, as shown in Figure 2B, the plot of the
slope against the concentration of 5g provided the EI dissociation constant. Ki was calculated from the
concentration of 5g versus the slope, and K was found to be 3.865 µM.
m
increases with increasing
α
i
2
.2. Free Radical Scavenging
The DPPH free radical scavenging ability of each of the synthesized compounds was evaluated.
Compounds 5d
,
5f and 5g did not show significant scavenging potencies (%); however, the other
g/mL (Figure 3).
compounds showed significant radical scavenging potentials at a concentration of 100
µ

Molecules 2019, 24, 1511
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Figure 2. Lineweaver-Burk plots for the inhibition of
concentrations of 5g were 0.00, 0.15, and 0.3 M; the concentrations of the substrate (p-nitrophenyl-
glucopyranoside) were 10, 5, 2.5, 1.25, 0.625, and 0.3125 mM. ( ) The plots of the slope versus inhibitor
concentration are shown as insets and were used to determine the inhibition constants.
α
-glucosidase by compound 5g (
A
) The tested
µ
α
-D
B
Figure 3. Free radical scavenging activity (%) of the synthesized compounds; values are presented as
the mean ± SEM. All compound concentrations were 100 µg/mL.
2
.3. Structural Assessment of α-Amylase and α-Glucosidase
α
-Amylase (EC#: 3.2.1.1) and
are actively involved as receptor molecules and can be used as targets for therapeutics for the treatment
of diabetes. Porcine pancreatic -amylase contains 496 residues with 22% helices, 30% sheets and
7% coil. -Glucosidase comprises 811 amino acids with 35% helices, 25% sheets and 38% coil. X-ray
diffraction studies were used to confirm the structures of -amylase and -glucosidase to resolutions
of 1.85 Å and 2.04 Å, respectively (Figure 4). The Ramachandran plots of -amylase and -glucosidase
indicated that 97.1% and 97.6% of residues, respectively, were present in the well-resolved regions.
The Ramachandran graphs showed the high accuracy of the phi ( ) and psi ( ) angles among the
-amylase and -glucosidase are provided
α-glucosidase (EC#: 3.2.1.106) are a class of hydrolyzed proteins that
α
β
4
α
β
α
α
α
α
ϕ
ψ
coordinates of target proteins. The Ramachandran graphs of
in Supplementary Figure S1A,B, respectively.
α
α

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Figure 4. The crystal structures of α-amylase and α-glucosidase.
2
.4. Chemo-Informatics and Lipinski’s Rule of Five (RO5) Assessments of the Ligands
The predicted properties, such as the molecular polar surface area (PSA), molar volume, density,
polarizability, surface tension, molar refractivity and Lipinski rule violations, were evaluated to
determine drug likeness Prior research has shown that PSA is a significant parameter for predicting
drug absorption in drug discovery [37]. The molar refractivity and molecular lipophilicity of drug
molecules are important for receptor binding, bioavailability and cellular uptake. Standard values
3
2
for molar refractivity (40 to 130 cm ), molecular weight (160 to 480 g/mol) and PSA (<89 Å ) have
been reported [38]. The predicted results showed that the molar refractivity and PSA values were
comparable to the standard values as mentioned in Table 2. RO5 analysis was used to justify the
therapeutic potential of the synthesized compounds. Hydrogen-bonding affinity is an important
parameter for evaluating drug permeability [39], and the values of HBA > 10 and HBD > 5 for the
ligands result in poor permeation in the body. Our chemo-informatics analyses showed that these
ligands possess
their logP values (
examples of RO5 violations among existing drugs [40
≤
10 HBA and < 5 HBD, which suggest good penetration within the body. Moreover,
2.10) were also comparable to the standard values (< 5). However, there are many
−
,41]. The predicted properties are presented in
Table 2.
Table 2. Cheminformatics Properties.
Properties
5a
5b
5c
5d
5e
5f
5g
5h
Mol. Weight (g/mol)
No. HBA
632.14
16
584.01
8
516.09
8
516.09
8
638.10
16
652.04
14
540.15
12
516.09
8
No. HBD
6
2
2
2
6
2
4
2
Mol. LogP
6.77
0
11.01
0
9.82
0
10.06
0
4.42
0
6.26
0
7.82
0
10.06
0
No of SC
Mol. Vol (A )
3
536.68
154.84
1.65
61.38
−1.49
477.50
150.61
1.47
59.70
−0.51
446.16
141.40
1.36
56.05
−0.67
448.00
141.40
1.36
56.05
−0.54
510.16
158.32
1.57
62.76
−1.22
515.19
N/A
N/A
N/A
−1.33
474.01
143.52
1.45
56.89
−1.16
448.15
141.40
1.36
56.05
−0.73
Molar Refractivity
Density
Polarizability
Drug Likeness Score
Abbreviation: LogP = Lipophilicity of partition coefficient, SC = stereo centers.

Molecules 2019, 24, 1511
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2
.5. Lead Optimization and Lipophilicity Values
To further evaluate the lead optimization potential of the synthesized compounds. The ligand
efficiency (LE), lipophilic ligand efficiency (LLE) and lipophilicity-corrected ligand efficiency (LELP)
values of all the synthesized compounds were predicted using the Data Warrior tool. Lipophilicity
is a fundamental property for improving the efficacy of lead compounds and identifying drug
candidates [42
values for LE, LLE and LELP on the basis of cLogP values [44]. Suggested acceptable standard
values have been reported for LE (>~0.30 kcal/mole/HA), LLE (>~0.5 kcal/mol), and LELP ( 10< to
10) [44 45]. The predicted LE values of the synthesized compounds were comparable to the standard
–44]. Hopkins studied the lipophilicities of different compounds and predicted standard
−
<
,
values. Furthermore, all the compounds showed mutagenic and irritant effects. All the compounds
showed potent mutagenic and tumorigenic behavior, whereas only 5e showed irritant properties. The
predicted values for all compounds along with their mutagenic and irritant effects are presented in
Table 3.
Table 3. Predicted ligand efficacy values.
Ligands
cLogP
LE
LLE
LELP
Mutagenic
Tumorigenic
Irritant
5
a
4.8122
10.923
9.7106
9.7106
7.027
1.5448
6.6554
9.7106
0.24806
0.28743
0.30535
0.30535
0.25421
0.28781
0.28044
0.30535
3.5055
−2.960
−1.697
−1.697
1.1262
7.2633
1.5214
−1.697
19.399
38.001
31.802
31.802
27.642
5.3675
23.732
31.802
high
high
high
high
high
high
high
high
high
high
high
high
high
high
high
high
none
none
none
none
high
none
none
None
5
b
5
c
5
d
5
e
5
f
5
g
5
h
2
.6. Molecular Docking Analysis
The docked ligand-protein complexes were analysed based on their minimum energy values. The
compounds showed good binding energy values against -amylase and -glucosidase (Figure 5). The
α
α
energy values gave the binding potentials of synthesized compounds against the target proteins. The
results showed that 5e was the most active compound with the best binding energy values for both
α
-amylase and
α
-glucosidase (
−
10.70 and
−
11.50 kcal/mol, respectively) among the tested derivatives.
Complexes with 5g docked showed comparable docking energy values of
−
9.30 and 9.70 kcal/mol,
−
respectively. The docking energy values of all the docked complexes were calculated using Equation (1),
as mentioned in the Supplementary Materials. Prior research has shown that the standard error for
Autodock is 2.5 kcal/mol. However, the predicted energy value for each of the docked complexes was
less than the standard energy value. Although the synthesized compounds all had the same basic core,
most ligands possess good energy values, and no large energy differences were observed. Compound
5
g
was the most active in the in vitro analysis; therefore, a structure-activity relationship analysis was
performed to evaluate its actual binding within the target proteins.
2
.7. Structure-Activity Relationship (SAR) Analysis
The active binding region in -amylase has been reported previously [46]. The
results showed that 5g binds within the active binding region of the target protein and forms three
hydrogen bonds. The residues in the active site of -amylase that actively participate in hydrogen
α
α-amylase docking
α
bonding are Gln63, His201, and Asn301. The benzyl oxygen in 5g forms a hydrogen bond with Gln63
with a bonding distance of 2.73 Å. Moreover, the hydroxyl and oxygen moieties on the benzene of 5g
form hydrogen bonds with His201 and Asn301 with bond distances of 2.26 and 1.90 Å, respectively.
Our docking results were well correlated with a previously published article, confirming the accuracy
of our docking results [47]. In
α-glucosidase docking, Trp690, Asp548, Arg425, and Glu426 were the
most active residues, as they formed hydrogen bonds to 5g with distances of 2.05, 2.20, 2.10 and 2.18 Å,

Molecules 2019, 24, 1511
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respectively (Figure 6). Based on our docking and kinetic results, it can be hypothesized that 5g actively
binds in the active region of the target protein and has great potential in the treatment of diabetes.
Figure 5. Energy graph of both
α-amylase and α-glucosidase docking (the green bar shows α-amylase,
and the blue bars show α-glucosidase).
Figure 6. Docking of 5g with α-amylase and α-glucosidase.
3
. Experimental
3
.1. Methods and Materials
The R values were determined using aluminium pre-coated silica gel plates (Kiesel 60 F254) from
f
Merck (Darmstadt, Germany). Melting points were measured in open capillaries using a Stuart melting
1
point apparatus (SMP3) and are uncorrected. H-NMR spectra were determined from DMSO or CDCl
3
solutions at 300 MHz using a Bruker AM-300 spectrophotometer, and the 13C-NMR spectra were

Molecules 2019, 24, 1511
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determined at 75 MHz using a Bruker 75 MHz NMR from DMSO-d or CDCl solutions. Elemental
6
3
analyses were performed on a LECO CHNS-932 Elemental Analyzer (LECO Corporation, Saint Joseph,
MI, USA). All the chemicals used for the syntheses of the compounds were commercially obtained and
were used without additional purification.
3
.2. General Procedure for the Synthesis of Phenolic Azo Dyes (4a
–4j) and Their Condensation with Hydrazine
Dihydrate (5a–5j)
Suitably substituted anilines (1a
concentrated HCl with stirring at a temperature of 0–5 C. A solution of NaNO (0.01 mL) in 10 mL of
–
1h) (0.01 mol) were dissolved in 20 mL of water and 3.5 mL of
◦
2
water was promptly added to the aniline solution with continuous and vigorous stirring. The reaction
mixture was stirred for 1 h while maintaining the temperature in the same range. After 1 h, the reaction
mixture was checked for the completeness with a paper chromatogram using water as the mobile
phase. The dried chromatogram was sprayed with a solution of p-N,N-dimethyl aminobenzaldehyde
in ethanol. Upon completion of the reaction, the diazonium salts (2a–2h) were stored in a freezer.
Salicylaldehyde
3 (0.01 mL) was dissolved in water (15 mL) with K CO (1.5 g) and kept
2 3
◦
in an ice bath at 0–5 C with stirring. The diazo solution was added dropwise to the stirred
solution of salicylaldehyde over 45 min, and the pH was maintained above 8. The progress of the
reaction was monitored by paper chromatography using H-acid solution in alkaline media. Upon
◦
completion, the solids were isolated by filtration and dried in an oven at 70 C for 3 h to afford the
4
-(benzeneazo)salicylaldehyde derivatives (3a–3h) in 85–92% yields. When mixtures of regioisomers
were obtained, they were separated by column chromatography. Then, 0.01 mol (0.5 g) of hydrazine
dihydrate (N H4 2H O) was slowly added to a solution of 0.02 mol of the appropriate 4-(benzeneazo)
·
2
2
salicylaldehyde derivative (5a–5h). After refluxing the reaction mixture for 3 h, the mixture was cooled,
and the precipitate was collected by filtration. The precipitate was washed several times with ethanol,
◦
crystallized from ethanol, and dried at 50 C overnight (yields > 70%).
(
(
E)-2,2’-((1E,1’E)-Hydrazine-1,2-diylidenebis(methanylylidene))bis(4-((E)-(2,4-dinitrophenyl)diazenyl)phenol)
5a)
◦
Light yellow crystalline solid, mp = 275 C, Yield 95%, R = 0.45 (n-Hexane: Ethyl acetate 6:4),
f
1
H-NMR (DMSO-d , 300 MHZ); δ (ppm) 11.81 (s, 2H, Ar-OH), 9.17 (s, 2H, =N-H), 8.69 (d, 2H, Ar-H,
6
J = 1.9 Hz), 8.48 (d, 2H, Ar-H, J = 1.99 Hz), 8.01 (dd, 2H, Ar-H, J = 8.7 Hz, J = 2.1 Hz), 7.94 (dd, 2H,
Ar-H, J = 8.6 Hz, J = 1.7 Hz), 7.87 (d, 2H, Ar-H, 8.9 Hz), 7.21 (d, 2H, Ar-H, J = 9 Hz), 13C-NMR (75 MHz
DMSO-d6)
δ
(ppm), 161.94 (C=O), 161.83, 152.17, 150.62, 145.58, 144.87, 127.68, 127.21, 125.48, 122.35,
1
2
19.57, 118.27, 118.11,Anal. Calcd. for C H N O : C, 49.69; H, 2.57; N, 22.29; found: C, 49.60; H
.60; N, 22.25.
26
16 10 10
(E)-2,2’-((1E,1’E)-Hydrazine-1,2-diylidenebis(methanylylidene))bis(4-((E)-(2,3-dichlorophenyl)diazenyl)phenol)
(5b)
◦
Dark yellow crystalline solid, mp= 222 C, Yield 89%, R = 0.62 (n-Hexane: Ethyl acetate 6:4),
f
1
H-NMR (CDCl , 300 MH );
δ
(ppm) 11.82 (s, 2H, Ar-OH), 9.15 (s, 2H, =N-H), 8.40 (d, 2H, Ar-H,
J = 2.0 Hz), 7.96 (dd, 2H, Ar-H, J = 8.7 Hz, J = 2.1 Hz), 7.89–7.59 (m, 6H, Ar-H), 7.21 (d, 2H, Ar-H,
J = 8.92 Hz), 13C-NMR (75 MHz CDCl3)
(ppm), 161.96 (C=O), 161.84, 154.19, 150.61, 130.11, 133.21,
28.64, 127.21, 125.48, 122.36, 119.53, 118.11, 116.21 Anal. Calcd. for C H Cl N O : C, 53.27; H, 2.75;
3
Z
δ
1
26
16
4
6
2
N, 14.34; found: C, 53.20; H, 2.83; N, 14.40.
E)-2,2’-((1E,1’E)-Hydrazine-1,2-diylidenebis(methanylylidene))bis(4-((E)-(2-chlorophenyl)diazenyl)phenol)
5c)
(
(
◦
Dark reddish-orange crystalline solid, mp = 195 C, Yield 85%, R = 0.75 (n-Hexane: Ethyl acetate
f
1
6
:4), H-NMR (CDCl , 300 MH );
δ
(ppm) 11.81 (s, 2H, Ar-OH), 9.14 (s, 2H, =N-H), 8.38 (d, 2H, Ar-H,
3
Z
J = 2.0 Hz), 7.92 (dd, 2H, Ar-H, J = 8.60 Hz, J = 2.0 Hz), 7.89–7.49 (m, 8H, Ar-H), 7.20 (d, 2H, Ar-H,
J = 8.98 Hz), 13C-NMR (75 MHz CDCl3)
(ppm) 161.90 (C=O), 161.82, 152.12, 150.51, 127.66, 127.21,
δ

Molecules 2019, 24, 1511
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1
25.47, 132.22, 124.22, 129.26,128.38, 119.58, 118.11Anal. Calcd. for C H C N O : C, 60.36; H, 3.51;
26 18 l2 6 2
N, 16.24; found: C, 60.42; H, 3.59; N, 16.31.
(
(
E)-2,2’-((1E,1’E)-Hydrazine-1,2-diylidenebis(methanylylidene))bis(4-((E)-(4-chlorophenyl)diazenyl)phenol)
5d)
◦
Light yellow crystalline solid, mp = 205 C, Yield 86%, R =0.70 (n-Hexane: Ethyl acetate 6:4);
f
1
H-NMR (CDCl , 300 MH );
δ
(ppm) 11.81 (s, 2H, Ar-OH), 9.14 (s, 2H, =N-H), 8.37 (d, 2H, Ar-H,
J = 2.1 Hz), 7.92 (dd, 2H, Ar-H, J = 8.61 Hz, J = 2.1 Hz), 7.79 (dd, 8H, Ar-H, J = 8.51 Hz), 7.19 (d, 2H,
Ar-H, J = 8.98 Hz), 13C-NMR (75 MHz CDCl3)
(ppm) 162.89 (C=O), 161.83, 152.11, 150.59, 136.21,
24.11, 129.21, 127.19, 125.46, 119.57, 118.10, Anal. Calcd. for C H C N O : C, 60.36; H, 3.51; N,
6.24; found: C, 60.44; H, 3.60; N, 16.29.
,4’-((1E,1’E)-(((1E,1’E)-Hydrazine-1,2-diylidenebis(methanylylidene))bis(4-hydroxy-3,1-phenylene))
bis(diazene-2,1-diyl))dibenzenesulfonamide (5e)
Dark orange crystalline solid, mp = 225 C, Yield 76%, R =0.70 (Chloroform: Ethanol 9:1);
(ppm) 11.83 (s, 2H, Ar-OH), 9.12 (s, 2H, =N-H), 8.38 (d, 2H, Ar-H,
J = 2.0 Hz), 7.99 (dd, 2H, Ar-H, J = 8.6 Hz, J = 2.0 Hz), 7.81 (m, 8H, Ar-H), 7.55 (s, 4H), 7.21 (d, 2H,
Ar-H, J = 8.94 Hz), 13C-NMR (75 MHz DMSO-d6)
(ppm) 161.93 (C=O), 161.82, 152.16, 150.59, 145.61,
27.65, 127.19, 125.46, 122.35, 119.58, 118.09, Anal. Calcd. for C H N O S : C, 51.48; H, 3.66; N, 18.47;
3
Z
δ
1
1
26
18 l2
6
2
4
◦
f
1
H-NMR (DMSO-d , 300 MH );
δ
6
Z
δ
1
26
22
8
6 2
S, 10.57; found: C, 51.42; H, 3.64; N, 18.43; S, 10.52.
Sodium 4,4’-((1E,1’E)-(((1E,1’E)-hydrazine-1,2-diylidenebis(methanylylidene))bis(4-hydroxy-3,1-phenylene))
bis(diazene-2,1-diyl))dibenzenesulfonate (5f)
◦
Bright yellow crystalline solid, m.p = 240 C, Yield 80 %, R = 0.55 (Chloroform: Ethanol 9:1),
f
1
H-NMR (DMSO-d , 300 MH );
δ
(ppm) 11.84 (s, 2H, Ar-OH), 9.16 (s, 2H, =N-H), 8.40 (d, 2H, Ar-H,
6
Z
J = 2.1 Hz), 7.99 (dd, 2H, Ar-H, J = 8.7 Hz, J = 2.1 Hz), 7.82 (m, 8H, Ar-H), 7.23 (d, 2H, Ar-H, J = 9 Hz),
13
C-NMR (75 MHz DMSO-d6)
δ (ppm) 161.96 (C=O), 161.84, 152.17, 150.61, 145.59, 127.67, 127.20,
1
25.48, 122.36, 119.59, 118.11, Anal. Calcd. forC H N Na O S : C, 47.85; H, 2.78; N, 12.88; S, 9.83;
26
18
6
2
8 2
Found: C, 47.81; H, 2.72; N, 12.83; S, 9.78.
(E)-2,2’-((1E,1’E)-Hydrazine-1,2-diylidenebis(methanylylidene))bis(4-((E)-(4-nitrophenyl)diazenyl)phenol) (5g)
◦
Dark brown crystalline solid, m.p =215 C, Yield 85%, R =0.45 (n-Hexane: Ethyl acetate 6:4),
f
1
H-NMR (DMSO-d , 300 MHZ); δ (ppm) 11.82 (s, 2H, Ar-OH), 9.14 (s, 2H, =N-H), 8.40 (d, 2H, Ar-H,
6
J = 1.9 Hz), 7.98 (dd, 2H, Ar-H, J = 8.7 Hz, J = 2.1 Hz), 7.98 (dd, 8H, Ar-H), 7.21 (d, 2H, Ar-H, J = 8.9 Hz),
13
C-NMR (75 MHz DMSO-d6)
δ (ppm) 161.95 (C=O), 161.82, 152.14, 150.59, 145.57, 127.67, 127.19,
1
25.46, 122.35, 119.56, 118.09, Anal. Calcd. for C H N O : C, 57.99; H, 3.37; N, 20.81; found: C, 57.91;
26
18
8
6
H, 3.29; N, 20.89.
(
(
E)-2,2’-((1E,1’E)-Hydrazine-1,2-diylidenebis(methanylylidene))bis(4-((E)-(3-chlorophenyl)diazenyl)phenol)
5h)
◦
Dark yellow crystalline solid, m.p = 210 C, Yield 87%, R = 0.65 (n-Hexane: Ethyl acetate 6:4),
f
1
H-NMR (CDCl , 300 MHZ);
δ
(ppm) 11.80 (s, 2H, Ar-OH), 9.08 (s, 2H, =N-H), 8.39 (d, 2H, Ar-H, J =
.9.0 Hz), 7.98 (dd, 2H, Ar-H, J = 8.59 Hz, J = 2.1 Hz), 8.12-7.76 (dd, 8H, Ar-H, J = 8.61 Hz), 7.21 (d, 2H,
Ar-H, J = 8.90 Hz), 13C-NMR (75 MHz CDCl3)
(ppm) 161.96 (C=O), 161.84, 152.18, 150.61, 133.09,
30.89, 130.24, 127.20, 125.48, 123.44, 121.02, 119.59, 118.11, Anal. Calcd. for C H C N O : C, 60.36;
3
1
δ
1
26
18 l2
6
2
H, 3.51; N, 16.24; found: C, 60.44; H, 3.59; N, 16.32.

Molecules 2019, 24, 1511
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3
.3. Biological Activity Methods
3
.3.1. α-Glucosidase Inhibition Assay
The α-glucosidase inhibitory activities of the synthesized compounds were evaluated following
the method described by Saleem et al., 2014 [33]. Briefly, solutions of the substrate (5 mM,
p-nitrophenyl- -d-glucopyranoside, Sigma, St. Louis, MO, USA) and -glucosidase (Sigma, St. Louis,
α
α
MO, USA) were prepared in 100 mM phosphate buffer (pH 7.0) containing 0.2 g/L NaN and 2 g/L
3
bovine serum albumin. The assay mixtures contained the compound (inhibitors), substrate and enzyme
solutions. First, 10 µL of the test compound (dissolved in 1% DMSO) and 50 µL of the enzyme solution
were added to a 96-well microplate, and the assay plate was incubated for 5 min at room temperature.
◦
Then, 50
µ
L of the substrate solution was added, and the mixtures were incubated for 10 min at 37 C.
Finally, the reactions were terminated by adding 100
µL of sodium carbonate solution (100 mM). The
blank was prepared by adding all the components except the enzyme. The absorbance of was reaction
mixture was recorded at 405 nm using a microplate reader (OPTIMax, Tunable Micro Plate Reader,
Sunnyvale, CA, USA). Acarbose was used as a reference drug. All the reactions were performed in
triplicate and repeated three times. Percentage inhibition was calculated using the following equation,
Inhibition (%) = [(B − S)/B] × 100
(1)
Here, B and S are the absorbances of the blank and sample, respectively. The IC50 values were
calculated by the program Prism 5.0 (GraphPad, San Diego, CA, USA).
3
.3.2. α-Amylase Inhibition Assay
The -amylase inhibitory activity of sinigrin was determined according to a previously reported
method [33] with slight modifications. Briefly, 40 L of a solution of the test inhibitor and 40 L of
enzyme solution ( -amylase from porcine pancreas (Sigma A3176-500KU, 089K1661) in a buffer of 0.02
M sodium phosphate at pH 6.9 with 0.006 M sodium chloride) were added to a 1.5-mL Eppendorf tube
α
µ
µ
α
and incubated for 10 min at room temperature. Then, 40
added to the pre-incubated tubes, and the solutions were incubated for an additional 10 min at 25 C.
µ
L of a starch solution (1% in DMSO) was
◦
Then, 100 µL of DNSA colouring reagent (10 g of sodium potassium tartrate, 1 g of 3,5-dinitrosalicylic
acid and 20 mL of 2 N NaOH to a final volume of 100 mL in distilled water) was added, and the mixture
was incubated in a boiling water bath for 5 min. After cooling to room temperature, the reaction
mixture was diluted to 500 µL using distilled water, and the absorbance was recorded at 540 nm. For
non-enzymatic reactions, the assays were performed with a blank containing all of the components
except the enzyme. The values of the % inhibition and IC50 were calculated from the results of the
assays using the same procedure as that described for the α-glucosidase inhibition assays.
3
.3.3. Free radical Scavenging Assay
Radical scavenging activity was determined by modifying a previously reported 2,2-diphenyl-
1
(
-picrylhydrazyl (DPPH) assay [34
150 M), 20 L of the test compounds solution at increasing concentrations, and enough DMSO to
bring the volume of solution in each well to 200 L. The reaction mixture was then incubated for
0 min at room temperature. Ascorbic acid (vitamin C) was used as a reference inhibitor. The assay
,35]. The assay solution consisted of 100 µL of DPPH solution
µ
µ
µ
3
measurements were carried out using a micro plate reader (OPTIMax, Tunable) at 520 nm. The reaction
rates were compared, and the percentages of inhibition caused by the presence of the test inhibitors
were calculated. Each concentration was analysed in three independent experiments run in triplicate.
3
.3.4. Kinetic Study of α-Glucosidase
Based on the IC50 values, the most potent inhibitor, 5g, was selected for kinetic analysis. A series
of experiments was performed to calculate the inhibition kinetics of 5g. The tested concentrations of

Molecules 2019, 24, 1511
13 of 16
5
g
were 0.00, 0.15 and 0.3. The concentration of substrate, p-nitrophenyl-
α
-d-glucopyranoside, was
between 0.3125 and 10 mM in all the kinetics experiments. The pre-incubation and measurement times
were the same as those discussed in the -glucosidase inhibition assay procedure. The maximal initial
α
velocity was determined from the initial linear portion of absorbance in the first five minutes following
the addition of the enzyme at 30 s intervals. The type of inhibition of the enzyme was assessed by
preparing Lineweaver-Burk plots of the inverse of the velocities (1/V) versus the inverse of the substrate
−1
concentration 1/[S] mM . The EI dissociation constant, Ki, was determined by the secondary plot of
/V versus the inhibitor concentration.
1
3
.4. Computational Methodology
3
.4.1. Selection of Target Proteins from the PDB
The three-dimensional (3D) structures of
α-amylase and α-glucosidase, with PDBIDs of 1DHK and
4
J5T, respectively, were retrieved from the Protein Data Bank (PDB) (www.rcsb.org). The energies of the
target proteins were minimized with an Amber force field by using a conjugate gradient algorithm in
UCSF Chimera 1.10.1 (Pettersen et al., 2006). The overall protein architecture and statistical percentages
of helices,
003). The Discovery Studio 4.1 Client (D. Studio, 2008) was used as a visualizing tool to generate
graphical depictions of the target proteins.
β-sheets, coils and turns were retrieved from the online server VADAR 1.8 (Willard et al.,
2
3
.4.2. In-Silico Design of Ligand Structures
The synthesized ligands were sketched using the ACD/ChemSketch tool and minimized
using UCSF Chimera 1.10.1. The basic biological properties, such as molecular weight (g/mol),
numbers of hydrogen bond acceptors and donors (HBA/D), LogP, number of stereo centres,
3
molecular volume (A ), molar refractivity density, polarizability and drug likeness score were
evaluated using the online computational tools Molsoft (http://www.molsoft.com/) and Molinspiration
(
http://www.molinspiration.com/). Moreover, Lipinski’s rule of five was also considered to evaluate
the efficacy of the ligand structures. Furthermore, the ligand efficiency (LE), lipophilic ligand efficiency
LLE) and lipophilicity-corrected ligand efficiency (LELP) values of all the ligands were calculated
(
using the Data Warrior tool. Moreover, the mutagenic and irritant risks were assessed using the Data
Warrior tool.
3
.4.3. Ligand-Based Docking Simulation
Molecular docking experiments were conducted on all the synthesized ligands against α-amylase
and
parametric values for
values for all coordinates were adjusted as x = 77.93, y = 68.98, and z = 103.65, respectively. Similarly,
for -glucosidase, the grid values were adjusted as center_x = 102.22, center_y = 40.10, center_z
18.02, while the size values were adjusted as size_x = 57.60, size_y = 59.83 and size_z = 72.29,
α
-glucosidase using the PyRx docking tool (Dallakyan and Olson, 2015). The grid box-centred
α
-amylase were adjusted as x = 18.44, y = 20.91, z = 8.22, while the size
−
−
α
=
respectively. A default exhaustiveness value = 8 was used in both docking studies to maximize the
binding conformational analysis. The docking poses (100 numbers of runs) for each docking study
were adjusted to obtain the best docking results. The selected compound was docked separately
and evaluated based on the lowest binding energy (Kcal/mol) and the structure-activity relationship.
Graphical depictions of all the docking complexes were prepared using Discovery Studio (2.1.0).
4
. Conclusions
A series of new bis-azo dyes based on Schiff bases (5a
through elemental and spectroscopic techniques. The abilities of the synthesized compounds to inhibit
-glucosidase and -amylase were evaluated. Compounds 5a 5h all significantly inhibited both of
these enzymes. Derivatives 5a 5h also showed good antioxidant activities. Kinetic studies were
–
5h) were synthesized and characterized
α
α
–
–

Molecules 2019, 24, 1511
14 of 16
performed to elucidate the modes of inhibition of
α
-amylase and
α
-glucosidase, and the most potent
derivative, 5g, was a dual inhibitor of these enzymes via competitive inhibition. The binding modes of
ligands with the target proteins were explored by molecular docking studies. Pharmacokinetics and
chemo-informatics evaluations revealed that the properties of the molecules were similar, and a few of
the derivatives in this series can serve as lead molecules in the design of drugs for the treatment of DM.
Supplementary Materials: Supplementary Materials are available online.
Author Contributions: Conceptualization, A.S.; methodology, P.A.C., D.S.; software, Q.A; validation, M.H.,
H.R., M.I.A. and S.-Y.S.; formal analysis, M.F.E.; investigation, P.A.C.; resources, H.R.E.; data curation, Q.A.;
writing—original draft preparation, D.S., F.A.L.; writing—review and editing, A.S., H.R.E.; visualization, A.S.;
supervision, A.S.; project administration, S.-Y.S.; funding acquisition, H.R.E.-S., M.F.A.
Funding: This research was funded by International Scientific Partnership Program (ISPP), grant number ISPP-126.
The APC was funded by ISPP.
Acknowledgments: The authors wish to express their appreciation to the International Scientific Partnership
Program (ISPP) at King Saud University for funding this research work through an ISPP-126 grant awarded to
H.R.E.-S. and M.F.A.
Conflicts of Interest: The authors declare no conflicts of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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