Exploring the dual inhibitory activity of novel anthranilic acid derivatives towards α-glucosidase and glycogen phosphorylase antidiabetic targets: Design, in vitro enzyme assay, and docking studies

Article abstract of DOI:10.3390/molecules23061304

A few new anthranilate diamide derivatives, 3a–e, 5a–c and 7a–d, were designed, synthesized, and evaluated for their inhibitory activity against two interesting antidiabetic targets, α-glucosidase and glycogen phosphorylase enzymes. Different instrumental analytical tools were applied in identification and conformation of their structures like;¹³C NMR,¹H NMR and elemental analysis. The screening of the novel compounds showed potent inhibitory activity with nanomolar concentration values. The most active compounds (5c) and (7b) showed the highest inhibitory activity against α-glucosidase and glycogen phosphorylase enzymes IC₅₀ = 0.01247 ± 0.01 μM and IC₅₀ = 0.01372 ± 0.03 μM, respectively. In addition, in vivo testing of the highly potent α-glucosidase inhibitor (7b) on rats with DTZ-induced diabetes was done and showed significant reduction of blood glucose levels compared to the reference drug. Furthermore, a molecular docking study was performed to help understand the binding interactions of the most active analogs with these two enzymes. The data obtained from the molecular modeling were correlated with those obtained from the biological screening. These data showed considerable antidiabetic activity for these newly synthesized compounds.

Full text of DOI:10.3390/molecules23061304

molecules
Article
Exploring the Dual Inhibitory Activity of Novel
Anthranilic Acid Derivatives towards α-Glucosidase
and Glycogen Phosphorylase Antidiabetic Targets:
Design, In Vitro Enzyme Assay, and Docking Studies
Saleh Ihmaid
Pharmacognosy and Pharmaceutical Chemistry Department, Pharmacy College, Taibah University,
Al-Madinah Al-Munawarah 41477, Saudi Arabia; sihmaid@taibahu.edu.sa
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 26 April 2018; Accepted: 16 May 2018; Published: 29 May 2018
Abstract: A few new anthranilate diamide derivatives, 3a–e, 5a–c and 7a–d, were designed,
synthesized, and evaluated for their inhibitory activity against two interesting antidiabetic targets,
α
-glucosidase and glycogen phosphorylase enzymes. Different instrumental analytical tools were
applied in identification and conformation of their structures like; 13C NMR, 1H NMR and elemental
analysis. The screening of the novel compounds showed potent inhibitory activity with nanomolar
concentration values. The most active compounds (5c) and (7b) showed the highest inhibitory
activity against
IC50 = 0.01372
α
-glucosidase and glycogen phosphorylase enzymes IC50 = 0.01247 0.01
±
µM and
±
0.03 M, respectively. In addition, in vivo testing of the highly potent α-glucosidase
µ
inhibitor (7b) on rats with DTZ-induced diabetes was done and showed significant reduction of
blood glucose levels compared to the reference drug. Furthermore, a molecular docking study was
performed to help understand the binding interactions of the most active analogs with these two
enzymes. The data obtained from the molecular modeling were correlated with those obtained
from the biological screening. These data showed considerable antidiabetic activity for these newly
synthesized compounds.
Keywords: antidiabetic; anthranilate diamide; α-Glucosidase; glycogen phosphorylase; structure–activity
relationships; molecular docking
1. Introduction
Diabetes mellitus disease is a chronic condition in which the human body is unable to produce
adequate insulin hormone type 1 or is a hyperglycemia that is uncontrolled with endogenous
available insulin type 2 and clinically characterized by aberrant elevated levels of glucose in the
blood [
1–
3]. The degree of this problem is exemplified in the fact that type 2 diabetes is now
becoming prevalent in children and adolescents [4,5]. It has become a major danger to global
health, and represents an enormous social and economic problem. According to the World Health
Organization (WHO), diabetes is the third-highest risk factor for premature death, after hypertension
and cigarette smoking (WHO) [
is associated with increasing urbanization, ageing populations, increased sugar intake, obesity,
and low fruit and vegetable consumption [ 11]. Adequate management of this debilitating disease
6]. In most countries, an increased prevalence of type 2 diabetes
7–
in many diabetic patients ultimately requires insulin treatment [12]. Although there are several
treatment options for type 2 diabetes, the long-term efficacy of oral antidiabetic agents has been
limited. It has recently been reported that 21% of patients will fail on metformin treatment within five
years [13,14]. Currently, 14 classes of drugs are available to treat type 2 diabetes mellitus, but only
36% of patients with type 2 diabetes achieve glycemic control with the currently available therapies.
Molecules 2018, 23, 1304; doi:10.3390/molecules23061304
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1304
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Therefore, new treatment options are desperately needed [15]. In the carbohydrate metabolism,
excessive hepatic glucose production is a major component of hyperglycemia and consists of both
glycogenolysis and gluconeogenesis. In the postprandial state glycogenolysis was more than doubled
in diabetic subjects compared to non-diabetics. In addition, diabetics have a 55% reduction in liver
glycogen content compared to normal subjects [16,17]. The enzyme glycogen phosphorylase a (GPa)
is responsible for the release of glucose-1-phosphate from glycogen and is the rate-determining
step in glycogenolysis. GPa has therefore become a target for glucose lowering in type 2 diabetes.
Although several groups have reported small molecule inhibitors of GPa, definitive clinical results
have not been communicated [18–20]. Glucosidase is an enzyme secreted from the intestinal
chorionic epithelium and responsible for carbohydrate hydrolysis [21]. Therefore, glucosidase is
a therapeutic target for many types of antidiabetic drugs having inhibitory activity against it, such as
acarbose, miglitol, and voglibose, which are used in clinic for treatment of type 2 diabetes [22].
Recently, the molecules that were based on anthranilic acid scaffold have gained much attention in
drug discovery and development [23
bioactive molecules. Indeed, the anthranilic acid nucleus serves as a privileged substructure in the
synthesis of amino acid tryptophan and its analogs, and also as a constituent of various alkaloids [26 27].
–25]. Anthranilic acid and its derivatives are constituents of many
,
Both experimental and preclinical studies demonstrated the medicinal properties of anthranilic acid
derivatives, including matrix metalloproteinase inhibition, anticancer, anti-inflammatory, and analgesic
activities [28–32]. In a previous study, the potential antiglycating effect of an anthranilic acid derivative
was identified in multiple stages of a non-enzymatic glycation process [33]. Lately, diamide derivatives
have received significant attention for their glycogen phosphorylase inhibitor activities, especially those
bearing a basic diamide scaffold and pharmacophores [34]. Various diamide derivatives have been
reported for their wide range of pharmacological activities including antidiabetic activity [35–38].
The promising bioactive diversity of this class of diamide compounds urges us to synthesize and
biologically evaluate a series of novel structural variants of anthranilic diamide derivatives as
antidiabetic agents. In the present study, as compared to the reported anthranilic acid potent derivative,
substitution of the substituted aniline derivatives with the pyridyl moiety might lead to the stability
of a compound with consistent lipophilicity. In addition, the terminal amino acid fragment will be
replaced with different aromatic substructures capable of forming hydrogen bonding and aromatic
interactions through various linkers (Figure 1).
HN
COOH
O
NH
HN
O
Cl
IC50 (+glu) = 73 nM
IC50 (-glu) = 460 nM
IC50 (cell) =1290 nM
Figure 1. Cont.

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Figure 1. Structural similarities and pharmacophoric features of reported and designed anthranilate
diamides as antidiabetic agents compared to the reported reference drug.
2. Results and Discussion
2.1. Chemistry
Methyl 2-(picolinamido)benzoate (1) was synthesized with good yield (90–95%) from the reaction
of methyl 2-aminobenzoate with picolinoyl chloride, in the presence of triethylamine (Et3N) as catalyst
in refluxing dimethylformamide (DMF), the according to the previously reported procedure with
no further modification [32] (Scheme 1). The structure of the product (1) was confirmed from the
analysis of their IR, 1H, 13C neuclear magnetic resonance (NMR) spectra in addition to microanalysis
(see experimental Section 3.2).
Scheme 1. Synthesis of methyl 2-(picolinamido)benzoate (1).
The reaction of methyl 2-(picolinamido)benzoate (
sulfa derivatives in the presence of potassium tert-butoxide in acetonitrile led to the
synthesis of N-(2-((4-substiutedbenzyl)carbamoyl)phenyl)picolinamide 3a with yields of 80–95%
and N-(2-((4-sulfasubstitutedphenyl)carbamoyl)phenyl)picolinamide 5a with yields of 80–85%
were confirmed from their infrared (IR),
1) with benzylamine derivatives and
–
e
–
c
(Scheme 2). The structures of the new products 3a–e, 5a–c
1H nuclear magnetic resonance (NMR), and 13C nuclear magnetic resonance (NMR) and microanalysis
(see experimental Section 3.3).
The N-(2-(hydrazinecarbonyl)phenyl)pyridine-2-carboxamide (
methyl 2-(picolinamido)benzoate ( ) with hydrazine hydrate in refluxing ethanol for 8 h, afforded the
targeted N-(2-(hydrazinecarbonyl)phenyl)pyridine-2-carboxamide ( ) in 95% yield according to the
) was confirmed
6), as prepared from the reaction of
1
6
previously reported procedures [32] (Scheme 2). The structure of the new product (
6
by the analysis of their infrared (IR), 1H nuclear magnetic resonance (NMR), and 13C nuclear magnetic
resonance (NMR) spectroscopy in addition to the microanalysis (see experimental Section 3.4).
Schiff bases were synthesized by heating under reflux the acid hydrazide (
aldehydes in the presence of ethanol as solvent and hydrochloric acid (HCl) as catalyst (Scheme 2).
The structures of new Schiff bases 7a
were confirmed by the analysis of their infrared (IR), 1H nuclear
6) with appropriate aromatic
–
d

Molecules 2018, 23, 1304
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magnetic resonance (NMR), and 13C nuclear magnetic resonance (NMR) spectroscopy in addition to
the microanalysis (see experimental Section 3.5).
O
R
3a: H
H2N
3b: OCH3
3c: F
3d: Cl
N
R
H
2a-e
i
NH
R
N
3e: CH3
O
3a-e
O
R
5a: H
NHR
NH2
S
N
O
5b:
O
O
O
S
N
RHN
O
H
N
O
5c:
NH
4a-c
ii
N
S
NH
O
N
O
1
5a-c
H
O
Ar
O
N
NH2
N
N
N
H
NH
H
NH
NH2NH2.H2O
ArCHO
N
iii
O
O
6
7a-d
Ar
7a
7b
7c
7d
Cl
OH
N
,
Cl
OH
Scheme 2. The reactions of the methyl 2-(picolinamido)benzoate (1) with benzylamine derivatives,
sulfa derivatives and aromatic aldehyde derivatives, respectively. Reaction conditions: (
i
) potassium
tert-butoxide in acetonitrile and reflux; (ii) potassium tert-butoxide in acetonitrile and reflux; (iii) EtOH,
HCl, and reflux.
2.2. Biological Screening
2.2.1. α-Glucosidase Inhibitory Assay
All compounds were evaluated with regard to
are reported in (Table 1). Generally, they showed inhibition at various nanomolar concentrations.
Some significant inhibitory effects were detected for the title compounds ( ) and ( ), IC50 = 0.735
and IC50 = 0.416 M, respectively. All tested compounds showed more inhibitory activity than
the parent diamides by a range of IC50 = 0.0124 to IC50 = 0.231 M. The order of inhibitory
behavior of compounds was sorted as follows: benzylamine derivatives 3b 3e 3a 3d 3c
sulfonamide derivatives 5c > 5b > 5a, and last benzylidine series 7b 7a 7d 7c. Among these
compounds, the derivatives of (5c), (7b), (5b) and (3b) showed the most potent anti- -glucosidase
enzyme effect with inhibitory concentrations. It was clear that compound (5c) IC50 = 0.01247
was the most active unique sulfonamide analog, bearing a six-membered aromatic heterocyclic
ring pyridine substructure. Of these active analogs, two selective -glucosidase inhibitors (7a
non-substituted aromatic benzylidine derivative and (3e) 4-methyl benzyl derivative with activity
α-glucosidase inhibition and the resulted data
1
7
µ
µ
>
>
>
>
,
>
>
>
α
µM
α
)

Molecules 2018, 23, 1304
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profile towards this enzyme and less potent against the glycogen phosphorylase one, IC50 = 0.0445 and
IC50 = 0.0708 respectively. However, compounds (7d) IC50 = 0.07842 M and (5a) IC50 = 0.0477
µ
µM
displayed slight equal potency against the two target enzymes (Figure 2A).
Table 1. Inhibitory data of the target compounds to both α-glucosidase and glycogen phosphorylase.
IC50 (µM)
Compounds
Scaffold
α-Glucosidase
Glycogen Phosphorylase
1
0.7355 ± 2.4
0.5415 ± 0.06
N-Furoylanthranilate (NFA)
3a
3b
3c
3d
3e
0.0896 ± 1.52
0.0223 ± 0.08
0.0585 ± 7.57
0.0947 ± 0.68
0.0708 ± 14.2
0.1502 ± 0.07
0.01808 ± 0.84
0.08639 ± 0.02
0.1774 ± 0.05
0.262 ± 0.01
Benzyl NFA
5a
5b
5c
0.0477 ± 0.59
0.0212 ± 0.07
0.01247 ± 0.01
0.4166 ± 11.4
0.07047 ± 0,01
0.0166 ± 0.09
0.02291 ± 0.57
0.5818 ± 0.58
Sulfonamide NFA
NFA-Hydrazine
6
7a
7b
7d
7c
0.0445 ± 12.1
0.0176 ± 0.09
0.07842 ± .05
0.2313 ± 0.57
0.01011 ± 1.81
0.2078 ± 2.57
0.01372 ± 0.03
0.054 ± 5.14
Benzylidine NFA
-
0.0367 ± 2.56
0.0556 ± 0.11
1-Deoxynojirimycin
(A)
(B)
Figure 2. Bar chart of the inhibitory activity data of target compounds sorted from lowest activity to
highest against two antidiabetic drug enzymes compared to the reference drug: (A) α-glucosidase;
(B) glycogen phosphorylase.

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2.2.2. Glycogen Phosphorylase Inhibitory Assay
The new compounds were assayed against rabbit muscle glycogen phosphorylase, as described
in the experimental Section 3.7, and the results are reported in (Table 1). The order of activity
of target compounds assayed against the glycogen phosphorylase enzyme was: benzylamine
derivatives 3b > 3c > 3a > 3d > 3e, sulfonamide derivatives 5b
> 5c > 5a, and last benzylidine series
7b > 7c > 7d > 7a. The inhibitory profile of these compounds ranged between IC50 = 0.01372 and
IC50 = 0.5818 µM. The class benzylidine derivatives are slightly more potent than the sulfonamide
ones and the benzylamine analogs. Two compounds (3c) and (7c) exhibited slight target selectivity by
IC50 = 0.367 and IC50 = 0.0864
µM inhibitory data. The dihydroxy phenyl derivative (7b) is considered
the most potent glycogen phosphorylase inhibitor by IC50 = 0.01372
µM. In contrast the benzylamine
derivative (3e) showed less potency against this target by IC50 = 0.262 µM (Figure 2B).
2.2.3. In Vivo Antidiabetic Screening
The compound (7b) was tested on Sprague-Dawley (SD) rats for the effect on the blood glucose
level, as described in the experimental Section 3.8, and the results are reported in (Table 2) and
(Figure 3). Blood glucose levels were measured after fasting for 15 h (fasting reading), followed by
the administration of a 5-mg meal (reading 1). Moreover, a second day reading after treatment with
normal saline, acarbose, or (7b) compound blood glucose level (reading 2) followed by a 5-mg meal,
was measured. It was shown that diabetic rats exhibited an increase in blood glucose level to above
200 mg/dL. In addition, the blood glucose level after 15 h fasting followed by a meal of 5 mg (reading 1)
was compared to the blood glucose levels under the same conditions, and following the administration
of normal saline, acarbose or compound (7b). For the control non-diabetic non-treated group and
the diabetic non- treated group, no significant difference between (readings 1 and 2) was shown. For
the group of rats treated with acarbose or compound (7b) one hour before a meal, blood glucose
levels decreased from 147
Finally, the target compound (7b) succeeded in decreasing the blood glucose level of diabetic rats
±
2 to 136
±
3.28 and 120
±
0.88 mg/dL to 116
±
0.57, respectively.
better than the reference oral antidiabetic drug, acarbose.
Table 2. In vivo blood glucose data.
Blood Glucose Level (mg/dL)
Groups
Fasting
Reading 1
Reading 2
Control
Diabetic
Acarbose
7b
72 ± 1.72
209 ± 0.66
204 ± 4.5
207 ± 0.88
147 ± 0.88
172 ± 2.72
147 ± 2.0
120 ± 0.88
140 ± 3.17
176 ± 2.90
136 ± 3.28
116 ± 0.57
Figure 3. The bar chart of the effect of reference drug and target compound (7b) on the blood glucose
level in treated and non-treated rats.

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2.3. Molecular Docking Studies and SAR Analyses
To rationalize the experimental results obtained, molecular docking studies were accomplished on
representative compounds (5b) and (7b) against the glycogen phosphorylase site structure (Figure 4A).
The results are presented in (Figure 4) and (Table 3). It was clear that all compound fragments share
in the interaction with the binding pockets. This means the activity of such series are related to both
scaffold and terminal fragments. Two compounds of sulfonamide and benzylidine substructures that
constitute most anti-glycogen phosphorylase inhibitors were docked and compared to the bound
ligand, GSK254. From the data reported, compound GSK254 showed two stable hydrogen bonding
interactions between Arg310 with a terminal carboxylic group. Moreover, the anthranilate C=O
scaffold formed a stable hydrogen bond with the amino acid Gln71. In addition, the compound is
stabilized by an extra hydrophobic interaction with Ile68 through the phenyl fragment (Figure 4B).
The behavior of compound (7b) as a potent inhibitor in the binding pocket showed three consistently
stable hydrogen bonds with the terminal hydroxyl groups, carbonyl linker, and pyridine part with
corresponding amino acid residues Asp76, Tyr738 and Gln71 (Figure 4C). All the modeled drugs
displayed hydrophobic interactions that stabilized the compounds in the binding pockets via the
terminal pyridyl and anthranilate ring with Gln71, Ile68, Phe196, or Arg193 residues. With regards to
compound (5b), two stable hydrogen bonds were formed with the pocket residues Gln71 and Arg193
through the pyridine ring and anthranilate carbonyl function linker (Figure 4D). The scaffold, linker,
and terminal fragments are essential for activity and it is very important to take care during any
optimization processes.
Table 3. Docking data of selected target compounds.
Target Residues
(Distance, Å)
Binding
Energy (dG)
Compounds
5b
Fragment
Interaction
C=O (anthranilate)
Phenyl (sulphonamide)
Gln71 (3.14)
Tyr75, Gln71, Gln72
Arg193 (3.08)
Ile68
H-bonding
Hydrophobic
H-bonding
Hydrophobic
Aromatic stacking
−14.5
Pyridine
Phenyl (anthranilate)
Ala313
–OH
–OH
C=O (anthranilate)
Pyridine
Asp76 (3.1)
Tyr738 (4.65)
Gln71 (3.05)
Phe196
H-bonding
H-bonding
H-bonding
Hydrophobic
Hydrophobic
−15.4
−13.5
7b
Phenyl (anthranilate)
Ile68
Carboxylic gp.
Phenyl (anthranilate)
Carbonyl linker
Arg310 (2.89)
Ile68
Gln71 (3.08)
H-bonding
Hydrophobic
H-bonding
GSK254
The data reported in the table are extracted from MOE program, Chemical Computing Group,
(Montreal, QC, Canada) showing the corresponding amino acid residues in the enzyme pocket,
corresponding fragments of ligands, interaction distances, types of interaction, and their binding
energy to some selected drugs.

Molecules 2018, 23, 1304
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(A)
(B)
(C)
(D)
Figure 4. Docking of selected compounds (7b) and (5b) with glycogen phosphorylase protein target,
PDB (3DD1). 3D and 2D interaction graphs are reported for (A) 3D target structure; (B) bound drug;
GSK254, (C) (7b) and (D) (5b).
3. Materials and Methods
3.1. Chemistry
Infrared spectra were obtained using a schimadzu FT-IR 8201 PC, spectrophotometer
1
(Kyoto, Japan). H and 13C neuclear magnetic resonance (NMR) spectra were obtained using a Bruker
AC 400 NMR spectrometer (Billerica, MA, USA) at 400 and 100 MHz, respectively. All 1H and 13
C
nuclear magnetic resonance (NMR) spectral results are recorded as chemical shifts (d) relative to the

Molecules 2018, 23, 1304
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internal transcranial magnetic stimulation (TMS). Microanalysis was performed by Chemical and
Micro-Analytical Services (CMAS), (Highton, VIC, Australia). Melting point determinations were
carried out using a Stuart Scientific (SMP3) melting point apparatus (Staffordshire, UK) and all melting
points are uncorrected. Reaction courses and product mixtures were routinely monitored by thin-layer
chromatography (TLC) on silica gel pre-coated F254 Merck plates (Darmstadt, Germany).
Starting Materials:
The starting reagents, methyl 2-aminobenzoate, picolinoyl chloride, cesium carbonate, triethylamine,
dimethylformamide (DMF), acetonitrile, substituted benzylamine, substituted sulphonamide,
potassium tert-butoxide, hydrazine hydrate and substituted aryl aldehyde were purchased from
Sigma-Aldrich (St. Louis, MO, USA) and were used as received.
3.2. Synthesis and Characterization of Methyl 2-(Picolinamido)benzoate (1)
According to our previously reported method [32], product
1 was prepared in 82% yield from
the reaction of methyl-2-aminobenzoate (1 mmol) and triethylamine (5 mmol) in dry DMF (10 mL),
2-pyridinecarbonyl chloride (1.2 mmol) was then added drop wise. The reaction mixture was heated
under reflux for 4 h. After cooling, the reaction mixture was poured into ice water and the formed
◦
solid was collected by filtration and recrystallized from ethanol to give (
(KBr)/cm−1 3358, 2955 (N-H), 1710 (C=O), 1698 (C=O); 1H NMR (DMSO-d6)
8.6–7.2 (m, 8H, Ph-H and pyridyl-H), 3.1 (s, 3H, O–CH3); 13C NMR (DMSO-d6)
1
), mp 210–212 C.
ν
max
δ
11.5 (s, 1H, NHCO),
168.8, 162.2, 147.7,
δ
140.2, 137.6, 133.7, 130.6, 126.6, 124.0, 122.6, 122.3, 119.5, 116.3 (Ar-C, C=O), 53.1 (CH2); Found: C, 65.65;
H, 4.79; N, 10.91; C14H12N2O3 requires C, 65.62; H, 4.72; N, 10.93.
3.3. General Procedure for the Synthesis of Diamides 3a–e and 5a–c
In slight modification to our previous reported method to obtain better yields of compounds 3a
and 5a , [32] a mixture of the appropriate benzylamine and/or sulfonamide derivatives (1.5 mmol),
potassium tert-butoxide (6.5 mmol) in acetonitrile (10 mL) was stirred under reflux for two hours,
a solution of methyl 2-(picolinamido)benzoate ( ) (1 mmol) in acetonitrile (10 mL) was added
–e
–
c
1
portion-wise. The reaction mixture was refluxed for overnight until the reaction was judged complete
by TLC and then poured into ice water. The resulting solid thus formed was filtered and recrystallized
from the appropriate solvent.
N-(2-(Benzylcarbamoyl)phenyl)picolinamide (3a). This compound was obtained in 92% yield
◦
(from ethanol), mp 241–242 C.
(DMSO-d6) 11.2 (s, 1H, NHCO), 9.3 (s, 1H, NHCH2), 8.9–7.2 (m, 13H, Ph-H, pyridyl-H and benzyl-H),
3.8 (s, 2H, CH2); 13C NMR (DMSO-d6)
167.8, 162.7, 152.1, 138.1, 137.9, 137.2, 132.6, 131.7, 128.5, 128.4,
ν
max (KBr)/cm−1 3278, 2895 (N-H), 1704 (C=O), 1671 (C=O); 1H NMR
δ
δ
127.0, 126.9, 126.8, 126.5, 124.5, 122.6, 115.3, 122.2, 119.4 (Ar-C, C=O), 44.1 (CH2); Found: C, 72.50;
H, 5.20; N, 12.69; C20H17N3O2 requires C, 72.49; H, 5.17; N, 12.68.
N-(2-((4-Methoxybenzyl)carbamoyl)phenyl)picolinamide (3b). This compound was obtained in 93% yield
◦
(from ethanol), mp 225–227 C.
(DMSO-d6) 10.6 (s, 1H, NHCO), 9.2 (s, 1H, NHCH2) 8.8–6.6 (m, 12H, Ph-H, pyridyl-H and benzyl-H),
3.8 (s, 2H, CH2), 3.8 (s, 3H, O-CH3); 13C NMR (DMSO-d6)
167.8, 162.6, 158.6, 151.2, 147.8, 137.9, 137.6,
ν
max (KBr)/cm−1 3368, 2965 (N-H), 1715 (C=O), 1695 (C=O); 1H NMR
δ
δ
132.3, 131.6, 130.7, 13.5, 130.2, 126.4, 125.0, 124.6, 122.3, 119.5, 114.2, 114.1 (Ar-C, C=O), 55.8 (OCH3),
44.1 (CH2); Found: C, 69.76; H, 5.27; N, 11.59; C21H19N3O3 requires C, 69.79; H, 5.30; N, 11.63.
N-(2-((4-Fluorobenzyl)carbamoyl)phenyl)picolinamide (3c). This compound was obtained in 85% yield
◦
(from ethanol), mp 240–242 C.
(DMSO-d6)
ν
max (KBr)/cm−1 3358, 2955 (N-H), 1725 (C=O), 1695 (C=O); 1H NMR
δ
10.5 (bs, 1H, NHCO), 9.3 (bs, 1H, NHCH2), 8.2–7.1 (m, 12H, Ph-H, pyridyl-H and
benzyl-H), 3.9 (s, 2H, CH2); 13C NMR (DMSO-d6) δ 167.8, 162.7, 160.9, 151.3, 147.9, 137.9, 137.5, 133.2,
132.3, 131.6, 128.5, 128.4, 126.3, 125.0, 124.7, 122.3, 119.1, 115.4, 114.7 (Ar-C, C=O), 44.2 (CH2); Found: C,
68.76; H, 4.67; N, 12.75; C20H16FN3O2 requires C, 68.76; H, 4.62; N, 12.03.

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N-(2-((4-Chlorobenzyl)carbamoyl)phenyl)picolinamide (3d). This compound was obtained in 82% yield
◦
(from ethanol), mp 233–235 C.
(DMSO-d6) 10.6 (s, 1H, NHCO), 8.9 (s, 1H, NHCH2), 8.5–6.9 (m, 12H, Ph-H, pyridyl-H and benzyl-H),
3.9 (s, 2H, CH2); 13C NMR (DMSO-d6)
167.8, 162.7, 151.3, 147.2, 137.9, 137.5, 136.0, 134.7, 134.6, 133.2,
ν
max (KBr)/cm−1 3358, 2955 (N-H), 1710 (C=O), 1698 (C=O); 1H NMR
δ
δ
132.3, 131.6, 128.6, 128.4, 126.8, 125.0, 124.4, 122.1, 119.4 (Ar-C, C=O), 44.4 (CH2); Found: C, 65.66;
H, 4.47; N, 11.47; C20H16ClN3O2 requires C, 65.67; H, 4.41; N, 11.49.
N-(2-((4-Methylbenzyl)carbamoyl)phenyl)picolinamide (3e). This compound was obtained in 80% yield
◦
(from ethanol), mp 275–278 C.
(DMSO-d6) 10.5 (bs, 1H, NHCO), 9.2 (bs, 1H, NHCH2), 8.4–6.9 (m, 12H, Ph-H, pyridyl-H and
benzyl-H), 4.0 (s, 2H, CH2), 2.3 (s, 3H, CH3); 13C NMR (DMSO-d6)
167.8, 162.7, 152.3, 147.2, 138.0,
ν
max (KBr)/cm−1 3358, 2955 (N-H), 1713 (C=O), 1688 (C=O); 1H NMR
δ
δ
137.5, 136.3, 134.2, 132.3, 131.6, 129.0, 128.9, 128.1, 128.0, 126.4, 125.5, 124.9, 122.8, 119.1 (Ar-C, C=O),
44.5 (CH2), 21.5 (CH3); Found: C, 73.05; H, 5.57; N, 12.17; C21H19N3O2 C, 73.03; H, 5.54; N, 12.17.
N-(2-((4-Sulfamoylphenyl)carbamoyl)phenyl)picolinamide (5a). This compound was obtained in 85% yield
◦
(from 1,4-dioxane), mp 188–189 C.
1341, 1152 (S=O); 1H NMR (DMSO-d6)
Ph-H, pyridyl-H and aminosulphonyl ph-H), 3.4 (bs, 2H, NH2); 13C NMR (DMSO-d6)
ν
max (KBr)/cm−1 3357, 3203 (N-H), 1678 (C=O), 1632 (C=O),
δ
11.5 (s, 1H, NHCO), 10.2 (s, 1H, NHCO), 8.6–6.7 (m, 12H,
167.2, 162.9,
δ
151.2, 147.5, 141.3, 137.6, 137.2, 136.5, 132.6, 129.6, 129.4, 127.7, 126.5, 124.5, 123.2, 122.5, 119.6, 118.2,
118.0 (Ar-C, C=O); Found: C, 57.57; H, 4.09; N, 14.11; C18H15N3O5S requires C, 57.57; H, 4.07; N, 14.13.
N-(2-((4-(N-(Pyridin-2-yl)sulfamoyl)phenyl)carbamoyl)phenyl)picolinamide (5b). This compound was
◦
obtained in 83% yield (from acetonitraile), mp 209–211 C.
1675 (C=O), 1640 (C=O), 1341, 1152 (S=O); 1H NMR (DMSO-d6)
SO2NH), 10.5 (s, 1H, NHCO), 8.6–6.7 (m, 16H, Ph-H, pyridyl-H and aminosulphonyl ph-H,
pyridinyl-H); 13C NMR (DMSO-d6)
167.5, 162.4, 152.4, 151.5, 148.2, 147.4, 141.2, 139.1, 137.9, 137.5,
ν
max (KBr)/cm−1 3357, 3203 (N-H),
δ
11.3 (s, 1H, NHCO), 10.8 (s, 1H,
δ
135.3, 132.6, 129.4, 129.7, 127.9, 126.3, 124.6, 123.4, 122.5, 119.5, 118.2, 118.0, 117.4, 109.5 (Ar-C, C=O);
Found: C, 60.90; H, 4.05; N, 14.80; C24H19N5O4S requires C, 60.88; H, 4.04; N, 14.79.
N-(2-((4-(N-(Thiazol-2-yl)sulfamoyl)phenyl)carbamoyl)phenyl)picolinamide (5c). This compound was
◦
obtained in 74% yield (from ethanol), mp 217–219 C.
ν
max (KBr)/cm−1 3348, 3205 (N-H), 1681 (C=O),
δ 11.2 (s, 1H, NHCO), 10.8 (s, 1H, SO2NH),
1
1642 (C=O), 1341, 1152 (S=O); H NMR (DMSO-d6)
10.3 (bs, 1H, NHCO), 8.6–6.3 (m, 14H, Ph-H, pyridyl-H and aminosulphonyl ph-H, thiazole-H);
13C NMR (DMSO-d6)
δ
171.6, 167.3, 162.7, 151.4, 147.9, 141.7, 137.9, 137.5, 137.0, 136.8, 134.9, 132.8,
129.4, 128.9, 127.2, 126.7, 124.5, 123.2, 122.7, 119.5, 118.4, 118.0, 112.1 (Ar-C, C=O); Found: C, 55.09;
H, 3.59 N, 14.58; C22H17N5O4S2 requires C, 55.10; H, 3.57; N, 14.60.
3.4. Synthesis and Characterization of N-(2-(Hydrazinecarbonyl)phenyl)picolinamide (6)
A mixture of methyl 2-(picolinamido)benzoate (1) (1 mmol) and hydrazine hydrate (5 mmol) in
ethanol (20 ml) was refluxed for 8 h. After cooling, the mixture was poured into crushed ice and the
formed solid was filtered off. The crude product was recrystallized from hot water to give compound
(6
) in 95% yield, mp 210–212 ◦C.
1H NMR (DMSO-d6)
pyridyl-H), 4.7 (s, 2H, NH2); 13C NMR (DMSO-d6)
ν
max (KBr)/cm−1 3204–3210 (NH2, NH), 1689 (C=O), 1657 (C=O);
δ
12.4 (s, 1H, NHNH2), 10.2 (s, 1H, NHCO), 8.6–6.7 (m, 7H, ArH, Ph-H and
164.0, 162.6, 151.1, 147.7, 138.0, 137.8, 132.5, 127.3,
δ
126.4, 124.5, 123.7, 122.6, 119.5 (Ar-C, C=O); Found: C, 60.90; H, 4.70; N, 21.88; C13H12N4O2 requires C,
60.93; H, 4.72; N, 21.86.
3.5. General Procedure for the Synthesis of Schiff Bases-Type Hydrazones 7a–d
In slight modification to our previous reported method to obtain better yields of compounds
7a
–d [32] a mixture of N-(2-(hydrazinecarbonyl)phenyl)picolinamide (6) (1 mmol) and the appropriate
aromatic aldehyde (1 mmol) in 20 mL of ethanol and few drops of HCl was refluxed for 3 h.

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After cooling, the reaction mixture was poured into ice water, and the formed solid product was
filtered. The crude product was recrystallized from the appropriate solvent.
(E)-N-(2-(2-Benzylidenehydrazine-1-car◦bonyl)phenyl)picolinamide (7a). This compound was obtained in
83% yield (from ethanol), mp 205–207 C. ν
max (KBr)/cm−1 3248, 2895 (N-H), 1697 (C=O), 1676 (C=O),
1621 (N=N); 1H NMR (DMSO-d6)
δ 11.7 (s, 1H, CONH), 11.3 (s, 1H, CONH), 8.5–6.7 (m, 14H, Ph-H,
pyridyl-H, phenyl-H and H-C=N); 13C NMR (DMSO-d6) δ 165.6, 162.4, 151.8, 147.2, 146.8, 137.9, 137.6,
133.4, 132.6, 131.1, 130.0, 129.7, 128.4, 128.0, 127.4, 126.7, 124.7, 123.6, 122.5, 119.4; (Ar-C, C=O, C=N);
Found: C, 69.75; H, 4.66; N, 16.26; C20H16N4O2 requires C, 69.76; H, 4.68; N, 16.27.
(E)-N-(2-(2-(2,4-Dihydroxybenzylidene)hydrazine-1-carbonyl)phenyl)picolinamide (7b). This compound
was obtained in 81% yield (from ethanol), mp 215–217 ◦C. max (KBr)/cm−1 3380–3252 (O-H), 3248,
ν
2895 (N-H), 1697 (C=O), 1676 (C=O), 1597 (N=N); 1H NMR (DMSO-d6)
CONH), 9.3 (bs, 1H, OH), 8.6–6.4 (m, 13H, Ph-H, pyridyl-H, phenyl-H, and H-C=N, OH-H); 13C NMR
(DMSO-d6) 165.1, 162.4, 162.0, 161.7, 151.5, 147.2, 146.5, 137.9, 137.7, 133.5, 132.7, 127.5, 126.4, 124.5,
δ 11.8 (s, 1H, CONH), 10.5 (s, 1H,
δ
123.3, 122.4, 119.6, 112.5, 109.5, 103.7 (Ar-C, C=O, C=N); Found: C, 63.81; H, 4.33; N, 14.87; C20H16N4O4
requires C, 63.83; H, 4.29; N, 14.89.
(E)-N-(2-(2-(Pyridin-2-ylmethylene)hydrazine-1-carbonyl)phenyl)picolinamide (7c). This compound was
◦
obtained in 80% yield (from ethyl acetate), mp 225–227 C.
ν
max (KBr)/cm−1 3238, 2905 (N-H),
1
1705 (C=O), 1667 (C=O), 1620 (N=N); H NMR (DMSO-d6)
CONH), 8.6–6.7 (m, 13H, Ph-H, pyridyl-H, H-C=N and pyridyl-H); 13C NMR (DMSO-d6)
δ
12.3 (s, 1H, CONH), 11.7 (s, 1H,
165.4,
δ
162.2, 153.3, 151.5, 149.7, 147.7, 144.6, 137.9, 137.5, 136.2, 132.3, 127.9, 127.1, 126.2, 124.8, 123.5, 122.4,
120.1, 119.4 (Ar-C, C=O, C=N); Found: C, 66.09; H, 4.35; N, 20.29; C19H15N5O2 requires C, 66.08; H, 4.38;
N, 20.28.
(E)-N-(2-(2-(2,4-Dichlorobenzylidene)hydrazine-1-carbonyl)phenyl)picolinamide (7d). This compound was
obtained in 75% yield (from toluene), mp 212–214 ◦C.
ν
max (KBr)/cm−1 3248, 2895 (N-H),
1697 (C=O), 1676 (C=O), 1597 (N=N); 1H NMR (DMSO-d6)
δ
11.8 (s, 1H, CONH), 11.3 (s, 1H, CONH),
165.4, 162.6,
8.6–6.4 (m, 12H, ArH, Ph-H, pyridyl-H, phenyl-H, and H-C=N); 13C NMR (DMSO-d6)
δ
151.1, 147.2, 138.7, 137.9, 137.3, 132.8, 132.4, 131.0, 129.5, 129.0, 128.3, 127.7, 127.3, 126.8, 124.6, 123.2, 122.1,
119.4 (Ar-C, C=O, C=N); Found: C, 58.11; H, 3.40; N, 13.54; C20H14Cl2N4O2 requires C, 58.13; H, 3.41;
N, 13.56.
3.6. α-Glucosidase Inhibition Assay
α
-Glucosidase inhibition assay was performed spectrophotometrically.
α-Glucosidase from
Saccharomyces cerevisiae (Abcam, Cambridge, UK) was dissolved in phosphate buffer (pH 6.8, 50 mM).
Test compounds were dissolved in DMSO. In 96-well microtiter plates, 20
of enzyme (20 mU/mL) and 135
After incubation, 25 L of p-nitrophenyl-α-D-glucopyranoside (2 mM, Abcam) was added and change
µ
L of test sample, 20 ◦
µL
µ
L of buffer were added and incubated for 15 minutes at 37 C.
µ
in absorbance was monitored for 20 min at 400 nm. Test compound was replaced by DMSO (10% final)
as control. Deoxynojirimycin hydrochloride (Abcam) was used as a standard inhibitor. The assays
were done in triplicate. IC50 values were calculated from non-linear regression curve of percentage
inhibition versus log compound concentration.
3.7. Glycogen Phosphorylase Enzyme Assay
Rabbit muscle glycogen phosphorylase a (from Abcam, 0.475 mg/ mL) activity was measured
in the direction of glycogen synthesis by the release of phosphate from glucose-1-phosphate using
a 384-well plate at 22 ◦C in 45 mL of buffer containing 50 mM Hepes (pH 7.2), 100 mM KCl, 2.5 mM
EGTA, 2.5 Mm MgCl2, 0.25 Mm glucose-1-phosphate, and 1 mg/mL glycogen with a 30-min incubation
time. Phosphate was measured at 620 nm, 5 minutes after the addition of 150 mL of 1 MHCl containing
10 mg/mL ammonium molybdate and 0.38 mg/mL malachite green. Test compounds were added to

Molecules 2018, 23, 1304
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the assay in 5 mL of 14% DMSO. Compounds were tested against a deoxynojirimycin hydrochloride
(Abcam) standard in 11 point concentration response curve in duplicate on two separate occasions.
Data were analyzed using GraphPad Prism v.4.03, GraphPad Software, (La Jolla, CA, USA).
A nonlinear regression (curve fit) analysis with a sigmoidal dose response equation (variable slope)
was applied to generate IC50 and hill slope values. The reported IC50 had a hill slope between 0.7 and
1.2 and a Z0 value of ~0.8. Compounds were screened with maximal concentrations of 222 mM.
The assay was carefully monitored for signs of compound insolubility. The results are presented as
mean values from three determinations.
3.8. In Vivo Antidiabetic Screening
◦
Twenty male Sprague–Dawley (SD) rats were maintained in an air-conditioned room (25 C)
at a constant humidity (55–60%). All rats were provided with a standard rat diet containing
60% carbohydrate (w/w), 2% fat (w/w), 17.5% protein (w/w), 8% fiber (w/w), and water ad libitum.
The experiments were performed in accordance with the internationally accepted standard ethical
guidelines for laboratory animal use and care, as described in the European Community guidelines.
The Institutional Animal Ethics Committee of Al-Azhar University approved the study protocols
(approval No: 409/432, 3/2018). Diabetes was induced in 12 Sprague–Dawley (SD) male rats by
a single intraperitoneal injection of 40 mg/kg streptozocin (STZ) dissolved in 0.1 M/L citrate buffer
at a pH of 4.5. Three rats were injected with normal saline and vehicle only to serve as negative
controls. The remaining diabetic rats (nine) were identified by an increase in blood glucose more
than 200 ± 10 mg/dL after one week. Blood glucose levels were measured using One Touch Ultra
2 glucometer (Lifescan inc., Wayne, USA) every week for the following two weeks to confirm the
diabetic status. Rats were 6–8 weeks of age and the average weight when starting the experiment was
200
±
20 g. Twelve rats were randomly assigned to four groups. Group 1: non-diabetic non-treated
(control). Group 2: Diabetic non-treated. Group 3: Diabetic and administrated acarbose before meals.
Group 4: Diabetic and administrated our new compound before a meal. Briefly; the first group was
injected with an IP vehicle only. The other three groups were injected with streptozocin (STZ) and
developed diabetes. The second group was diabetic and untreated. The third group was diabetic rats
treated with acarbose at 40 mg per 100 g body weight 30 min. before meal using oral tube. The fourth
group administrated 40 mg/100 g body weight of the test compound (7b). Rats were fasted over
15 h and blood sugar levels were measured (pretreatment). For each rat, 5 mg standard rat diet was
supplied to each rat and blood glucose levels were measured to detect increases after 1 h. Rats were
fed again with the same amount after 8 h. Food was removed to allow animals to fast again for 15 h.
On the following day, groups 1 and two were not given any treatment, while group 3 rats were given
acarbose (positive control) and group 4 were given the test compound (7b). Blood glucose levels were
measured in tail vein blood samples taken from fasting rats (time point 0), then designated groups
are fed either acarbose or the test compound, suspended in PBS and delivered using an oral tube.
After half an hour, the rats were given five mg. Blood samples were taken after 1 h. Blood glucose
was expected to rise after meal and the effect of the drugs was evaluated according to their abilities to
control post-meal excursions.
3.9. Molecular Docking
Molecular docking simulation was done for the selected potent target compounds into the
three-dimensional complex of the biological target including the crystal structure of glycogen
phosphorylase (PDB code: 3DD1) at 2.6 Å resolution focusing on the AMP site [39] was carried out
using the AutoDock software package (version 4.0) (La Julla, California, USA) as implemented through
the graphical user interface AutoDock Tools (ADT) [40]. Prior to the calculations, crystallographic
water and ligand molecules were removed from the X-ray structure. Hydrogen atoms were added to
the structure with the molecular operating environment (MOE, 2012) (2012) [41] and atomic partial
charges were calculated using AutoDock Tools. Selected active anthranilate compounds were docked

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into the active site of the target to predict compound binding mode. For flexible docking, AutoDock
standard parameter settings were applied. High-scoring binding poses were selected on the basis of
visual inspection.
4. Conclusions
A series of N-pyridyl anthranilate derivatives were designed, synthesized, and tested for
screening of their inhibitory activity against two promising antidiabetic α-glucosidase and glycogen
phosphorylase targets. The design of such compounds involves hybrids of benzylamine, sulfonamides
and benzylidine fragments linked to the acidic part of the anathranilic acid scaffold. Most of the tested
diamide compounds exhibited potent α-glucosidase and glycogen phosphorylase inhibitory effect with
nanomolar concentrations. The most effective derivatives are (5b) and (7b) with terminal sulfapyridine
and dihydroxy substituted phenyl fragments. Furthermore, the most active compound (7b), was tested
for decreasing blood glucose level, and the data proved that these novel compounds could decrease
the elevated abnormal glucose level better than the reference drug. Extensive molecular docking
studies were applied for an investigation of the structure–activity relationship of these compounds.
It is anticipated that new inhibitors developed using these techniques will soon be seen in the clinic as
effective antidiabetic drugs.
Acknowledgments: The author gratefully acknowledges support from the Deanship of Scientific Research at
Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia. The author also gives thanks to the chemical
computing group for the Molecular Operating Software (MOE), 2012.
Conflicts of Interest: The author declares no conflict of interest.
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