Research Article

Chemical & Pharmaceutical Research

Synthesis, α-Glucosidase Enzymatic Inhibition and Docking Studies of

some Fused Heterocycles
Marco Brito-Arias1*, Paola L. Ramírez-Hernández1, Esmeralda C. García-Barrera1, Mario Perez-
Venegas2, Christian Montiel-Valenciana1 and Leonor P. Rodríguez-Pascual1
1
Departamento de Ciencias Básicas, Unidad Profesional
Interdisciplinaria de Biotecnología del Instituto Politécnico
*
Correspondence:
Marco Brito-Arias, Departamento de Ciencias Básicas, Unidad
Nacional (UPIBI-IPN) Avenida Acueducto s/n La Laguna Ticomán
Profesional Interdisciplinaria de Biotecnología del Instituto
Ciudad de México, México. Politécnico Nacional (UPIBI-IPN), México, E-mail: mbrito@
ipn.mx.
2
Departamento de Química Centro de Investigación y Estudios
Avanzados (Cinvestav-IPN) Avenida Instituto Politécnico Nacional Received: 27 December 2018; Accepted: 02 February 2019
2508 San Pedro Zacatenco Ciudad de México México.

Citation: Marco Brito-Arias, Paola L. Ramírez-Hernández, Esmeralda C. García-Barrera, et al. Synthesis, α-Glucosidase Enzymatic
Inhibition and Docking Studies of some Fused Heterocycles. Chem Pharm Res. 2019; 1(1): 1-6.

ABSTRACT
In the search of heterocyclic ring systems potentially useful as α-glucosidase inhibitors we have synthesized a set of
heterocycles bearing hydroacridone 4-5, hydroxanthone 6, quinazolone 8, benzoyl phtalimide 9 and isoquinolone
10-11. These compounds under study were subjected to enzyme inhibition and compared against reference inhibitor
acarbose observing potent inhibition for benzoyl phtalimide 9, and isoquinolone dione 11. Based on their inhibition
activity, both candidates were selected for docking analysis to determine their best posing, and the interactions
involved between the ligand and the residues. A comparative analysis was established to determine the potential
correlation between the interactions found, the inhibition observed for the candidates and the interactions observed
for the reference inhibitor acarbose.

Keywords providing important information about structural diversity which

Fused heterocycles, α-Glucosidase, Enzymatic inhibition, can be applied in drug discovery and lead modification [1-3].
Molecular docking.
On the other hand, progress in the search of synthetic α-glucosidase
Introduction inhibitors specially having heterocyclic rings [4] such as pyrazole
The glycaemic control is considered a crucial step in the prevention [5], coumarins [6], fused pyrimidines [7], benzo[d]oxazol
and management of diabetes mellitus, and the search of inhibitors [8], bisbenzimidazoles [9], oxadiazole [10], xanthones [11],
targeting the enzymes α-amylase and α-glucosidase responsible 4-quinazolones [12-14], and quinoxaline [15] derivatives has been
for the hydrolysis of starch and oligosaccharides present in the diet also described.
an important strategy in the modulation of the glycaemic profile. It
is well known that α-glucosidase inhibitors slow down the process With the aim of looking for heterocyclic rings within the wide range
of digestion and absorption of carbohydrates by competitively of structural diversity in either natural or synthetic compounds
blocking its activity, leading to the reduction of the glucose displaying α-glucosidase inhibition we proceed to synthesize
concentrations in blood [1]. different fused heterocycles bearing quinolone, isoquinoline,
xanthone, phtalimide and quinazolinone ring systems and those
In the search of active compounds useful as α-glucosidase presenting α-glucosidase inhibition used as lead molecules for a
inhibitors an intensive screening has been undertaken particularly structure-activity relationship program.
from natural resources including microorganisms and medicinal
plants. For instance a recent review reported the evaluation of 61 Results and Discussion
terpenes, 37 alkaloids, 49 quinones, 7 xanthones, 103 flavonoids Chemistry
and 37 phenols either with or without glycosidic moieties, The heterocyclic rings used for the screening analysis were
Chem Pharm Res, 2018 Volume 1 | Issue 1 | 1 of 6
hydroacridinone 4, hydroquinolinone 5, xanthone dione 6,
hydroquinazolone 8, benzoyl phtalimide 9, benzoyl isoquinolone
10 and isoquinoline trione 11 (Figure 1).

Figure 3: General scheme for the synthesis of compound 8.

The fused heterocycles 9, 10 and 11 where prepared according to

Figure 4 starting from benzoylphtalimide 9 which was prepared
by reaction of bromoacetophenone with potassium phtalimide in
DMF, and treated with sodium methoxide conditions to afford
benzoyisoquinolone 10, and then converted to the isoquinoline
trione 11 by treatment with acetic anhydride in nitric acid. This
method is an alternative approach to a previous synthesis of
isoquinoline-1,3,4-triones based on strong oxidative conditions of
Figure 1: Fused rings evaluated as α-glucosidase inhibitors. isoquinoline [19].

The synthesis of hydroacridinone 4 was carried out according to

scheme I consisting in the reaction between 2-nitrobenzaldehyde
1 with 2 equivalents of dimedone 2 under basic condition to yield
heterocycle 4 (Figure 2).

Figure 4: General scheme for the preparation of compound 11.

In order to provide a plausible explanation about the formation

of the isoquinolone dione 11 from 10 we propose a step sequence
involving the initial enol protonation through a tautomerism
equilibrium to form an enamine which under acidic medium is
converted to an iminium salt. Addition of a water molecule is
followed by a nucleophilic attack leading to isoquinolin trione
formation as shown in Figure 5.

Figure 2: General scheme for the synthesis of compound 4.

The synthesis of heterocycle 5 was carried out from intermediate

3 [16] which under reducing condition afforded the acridinone 5,
and its structure confirmed by X ray diffraction analysis [17]. The
hydro xanthone 6 was prepared also from intermediate 3 which
was submitted to base treatment with sodium methoxide under
refluxing conditions as described in scheme II. The structure of
this heterocycle was also confirmed by X-ray analysis [18].

The synthesis of quinazolinone 8 was achieved by preparation

of dimeric intermediates 7a-c and final ring formation of the
carbamate intermediate by hydride nucleophilic addition to yield 8 Figure 5: Proposed reaction mechanism for the formation of compound
as shown in Figure 3. 11.

Chem Pharm Res, 2018 Volume 1 | Issue 1 | 2 of 6

α-Glucosidase inhibition Assay Figure 7: Lineweaver-Burk plot for the inhibition Saccharomyces
The α-Glucosidase assay used for the inhibitory determination was cerevisiae α-glucosidase enzyme by compound isoquinolone dione 11.
the colorimetric protocol based on the glycosidic bond cleavage
of the p-nitrophenyl-α-glucopyranoside (p-NPG) and the resulting Next, we proceed to determine the IC50 by plotting the activity
absorbance measured at 405 nm, in the presence of inhibitor. against the inhibitor concentration and the Ki values which were
calculated from the Lineweaver-Burk inhibition equation. The Ki
Assay tubes containing 10μl of 3 mM p-nitrophenyl-α-d- calculated values of compound 9 were 2.80 mM and for compound
glucopyranoside (p-NPG), 4μl of Saccharomyces cerevisiae 11 were of 5.56 mM. The IC50 values for compound 9 and 11 the
α-glucosidase enzyme (9.67U/mL) in 50 mM phosphate buffer highest inhibition potency in the range of inhibition of the reference
(pH 6.8) in a total volume of 500μl where mixed gently and then acarbose (Table 1).
10μl of compounds under evaluation at final concentration 24,
48, 96 and 120 μg/ml were added. Acarbose (Glucobay, Bayer Compund Inhibition mode Ki (mM) IC50 (mM)
Pharmaceuticals) and Dimethyl sulfoxide (DMSO) were used as Competitive
9 2.80 5.6
positive and negative controls respectively. Tubes were incubated inhibition
at 37oC during 3 min, followed by the addition of 500μl 0.1M 11 Mixed inhibition 5.56 3.5
sodium bicarbonate as stopping agent. Absorbance was measured Table 1: Inhibition mode and constant (Ki value) and IC50 value of
at 405 nm in a Genesys 10 S Spectrophotometer (Thermo Fisher compound 9 and compound 11 against Saccharomyces cerevisiae
Scientific). α-glucosidase.

One unit of α-Glucosidase is the amount of enzyme that catalyzes The compounds tested probed to be effective inhibitors against
the hydrolysis of 1.0 µmol of substrate (p-nitrophenol) per minute Saccharomyces cerevisiae α-glucosidase, and compound 9 were
at 37°C. The resulting enzymatic activity was used to build the the most potent with a competitive inhibition mode, explained by
Lineweaver-Burk double reciprocal plot of substrate concentration its structure and dockins studies. The mixed competitive inhibitor
vs velocity, and from the equation the Vmax and Km was compound 11, was less effective in inhibition of α-glucosidase.
determined. Based on the Vmax and Km values observed from the
graphics we conclude that benzoylphtalimide 9 and isoquinolone Docking studies
dione 11 were competitive and mixed inhibitors respectively The glycoside hydrolase (GH), GluI operates via an inverting
(Figure 6 and 7). mechanism and the catalytic acid and base are not definitively
known [20]. Although substrate-binding motif in the rat and
mammalian homologs has been proposed no eukaryotic structures
have been determined, and therefore we can only rely on two
structures which have been solved of prokaryotic GH63: the
Escherichia coli homolog YgjK (PDB code 3D3I), and the T.
Thermophilus homolog TTHA0978 (PDB ID 2Z07). However
neither of these structures is sufficiently similar to mammalian
GluI to act as a realistic model at the atomic level.

Much of what we know about the characteristics of GluI has been

learned from studying the Saccharomyces cerevisiae enzyme,
Cwh41p. Cwh41p and human GluI share 24% overall identity
and from 34 to 59% identity in the catalytically active C-terminal
Figure 6: Lineweaver-Burk plot for the inhibition Saccharomyces
domain, and so similar structures are expected. Yeast and human
cerevisiae α-glucosidase enzyme by compound benzoylphtalimide 9.
GluI share similar substrate specificity, pH optimum, and inhibitor
sensitivity. Thus, the yeast enzyme besides its affordability serves
as a good experimental model to learn more about the structure,
substrate specificity, and enzymatic mechanism of human GluI
[20].

Therefore the protein chosen for the docking studies was

Saccharomyces cerevisiae Cwh41p (Protein Data Bank PDB
ID 4J5T) and the program we use for the docking analysis was
Autodock vina [21] and the visualizer program Discovery [22] to
identify the binding modes interacions ligand-residues (Figure 8
and 9).

Chem Pharm Res, 2018 Volume 1 | Issue 1 | 3 of 6

 Figure 10: Binding mode for the reference inhibitor acarbose in the active
site of Saccharomyces cerevisiae α-glucosidase enzyme (PDB 4J5T).

Acarbose was docked with α-glucosidase PDB ID 4J5T from

Saccharomyces cerevisiae to establish the interactions for the best
pose with affinity -8.0 kcal/mol and as a result it was found for
the best conformation the residues establishing polar contacts were
Arg 428, Asp 568, Glu 361 and Gly 566 as shown in Figure 10.

A graphical model was made to establish an interaction pattern

between the amino acid residues present in the pocket and the 8
conformations obtained for ligand 9 and 11 having the highest
inhibition, along with the reference inhibitor acarbose and the
Figure 8: Binding mode for compounds benzoylphtalimide 9 in the active natural substrate maltose used as a control.
site of Saccharomyces cerevisiae α-glucosidase enzyme (PDB 4J5T).
As a result we observed that the polar contacts with highest scores
were Arg 428 with 8 for reference inhibitor acarbose and control
maltose, and 6 for compound 11 and 4 for compound 9. Other
residues establishing polar contacts are Asp 568, Trp 391 and Trp
710 having compound 9 the highest proportion of polar contacts
with Trp 710 residue (Figure 11).

Figure 9: Binding mode for compound isoquinolone dione 11 in the active

site of Saccharomyces cerevisiae α-glucosidase enzyme (PDB 4J5T).

Figure 11: Graphical model describing the scores presented by the 8

conformations determined for compound 9, 11, acarbose and maltose.

Experimental
Solvents were purchase as analytical degree and all intermediates
were purified by column chromatography using silica gel 60 and
hexane-ethyl acetate at different gradients as elution system. Thin
layer chromatography were carried out on pre-coated aluminum
sheets silica gel with fluorescent indicator UV254 (Macherey-Nagel,
Germany). 1Н and 13С NMR were recorded on Varian Mercury
300 MHz using CDCl3 or DMSO-d6 as internal standard. High
resolution mass spectra were performed by positive electrospray
ionization micrOTOF-Q II 10392 analyzer.

Bis-(1E,1’E)-(2-nitrobenzyl)hydrazone (7a)
In a 50 ml round flask was dissolved 2-nitrobenzaldehyde 1 (0.5
g, 3.30 mmoles) in pyridine (7 mL) and hydrazine hydrochloride
(0.7 g, 6.78 mmol) was added and the reaction heated under reflux
during 4 hrs. The pyridine was evaporated under diminished
pressure and the solid dissolved in CH2Cl2 30 mL, washed with
water, dried over sodium sulfate and evaporated to yield 0.82 g
Chem Pharm Res, 2018 Volume 1 | Issue 1 | 4 of 6
(83 %) of a yellow solid. 1H NMR spectrum (CDCl3): δ 7.65 (t, 10 and dissolved with a mixture of acetic acid-acetic anhydride
2H, J = 6.9,), 7.75 (t, 2H, J = 6.9,), 8.09 (d, 2H, J = 7.5), 8.29 (d, 3mL (1:1 v/v) with stirring. The reaction was cooled in ice-water
2H, J = 7.5), 9.13 (s, 2H). 13C NMR (CDCl3) δ 124.80, 128.67, bath and nitric acid previously cooled was added dropwise during
129.60, 131.27, 133.60, 157.82, 158.51. HRMS (ESI-pos, m/z) 30 min. The reaction was poured into a beaker containing ice to
C14H10N4O4 [M + H]+ Found 299.0778 Calculated 298.0702. induce precipitation. The yellow solid was filtered and washed
with cold water to yield 0.123 g (yield 74.5%) of compound 11.
Bis-(1E,1’E)-(2-aminobenzyl)hydrazone (7b) 1
HNMR of 11 in DMSO-d6: δ 7.87 (m, 2H), 8.03 (dd, 1H), 8.11
In a 50 mL round-bottom flask the nitro dimer 7a (0.4 g, 1.34 (dd, 1H), 12.00 (s, NH). 13CNMR DMSO-d6 δ 127.1, 128.5, 130.2,
mmoles) was dissolved in ethanol (7 mL) and stirred until 132.7, 134.4, 135.3, 157.9, 163.6, 175.9.
dissolution. Then palladium on activated carbon 10 % (30 mg) was
added and the reaction stirred under hydrogen atmosphere during 6 Acknowledgment
h and filtered in a short column packed with celite using methanol Authors are grateful to COFAA-IPN and SIP-IPN for the financial
as elution system. The product was concentrated on rotavapor support provided for the development of this research.
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© 2018 Marco Brito-Arias, et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License

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