Synthesis and molecular docking studies of imines as α-glucosidase and α-amylase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103491

Imine functionality is found in many compounds with important biological activity. Thus, the development of novel synthetic approaches for imines is important. In this work, it is propose an easy, eco-friendly and straightforward synthesis pathway of aryl imines under microwave irradiation catalyzed by Alumina-sulfuric acid. In addition, the in vitro enzymatic inhibition, antioxidant activity and molecular docking studies were performed. The aryl imines were isolated with yields in the range of 37–94%. All aryl imines synthesized were evaluated for in vitro inhibitory potential against α-glucosidase and α-amylase enzymes and the results exhibited that the most of the compounds displayed inhibitory activity against both enzymes. The (E)-1-(4-nitrophenyl)-N-(pyridin-2-yl)methanimine (3d) was 1.15-fold more active than acarbose against α-amylase whilst the (E)-1-phenyl-N-(pyridin-2-yl)methanimine (3c) displayed similar activity that acarbose against α-glucosidase. The molecular docking studies in α-glucosidase and α-amylase reveal that aryl imines mainly establish an H-bond with the R²-subtituent and hydrophobic interactions with the R¹-subtituent. The docking analysis reveals these synthetic aryl imines 3d-i interact in same active site than acarbose drug in both enzymes.

Full text of DOI:10.1016/j.bioorg.2019.103491

Journal Pre-proofs
Synthesis and molecular docking studies of imines as α-glucosidase and α-
amylase inhibitors
Analy Aispuro-Pérez, Juan López-Ávalos, Fernando García-Páez, Julio
Montes-Avila, Lorenzo A. Picos-Corrales, Adrián Ochoa-Terán, Pedro
Bastidas, Sarita Montaño, Loranda Calderón-Zamora, Ulises Osuna-Martínez,
Juan I. Sarmiento-Sánchez
PII:
S0045-2068(19)31327-6
DOI:
https://doi.org/10.1016/j.bioorg.2019.103491
YBIOO 103491
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
12 August 2019
23 November 2019
1 December 2019
Please cite this article as: A. Aispuro-Pérez, J. López-Ávalos, F. García-Páez, J. Montes-Avila, L.A. Picos-
Corrales, A. Ochoa-Terán, P. Bastidas, S. Montaño, L. Calderón-Zamora, U. Osuna-Martínez, J.I. Sarmiento-
Sánchez, Synthesis and molecular docking studies of imines as α-glucosidase and α-amylase inhibitors,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103491
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Synthesis and molecular docking studies of imines as -glucosidase and
-amylase inhibitors
Analy Aispuro-Péreza, Juan López-Ávalosa, Fernando García-Páezb, Julio Montes-Avilaa, Lorenzo A.
Picos-Corralesa, Adrián Ochoa-Teránc, Pedro Bastidasd, Sarita Montañoa; Loranda Calderón-
Zamorae; Ulises Osuna-Martínez*a and Juan I. Sarmiento-Sánchez*b
a Facultad de Ciencias Químico-Biológicas, Universidad Autónoma de Sinaloa, Blvd. de las américas
S/N, 80040, Campus Culiacán, Culiacán, Sinaloa, Mexico.
b
Facultad de Ingeniería Culiacán, Universidad Autónoma de Sinaloa. Blvd. de las américas S/N,
80040, Culiacán, Sinaloa, Mexico. Tel./Fax: +52(667)7134043
c Centro de Graduados e Investigación en Química, Instituto Tecnológico de Tijuana. Blvd. Alberto
Limón Padilla S/N, Mesa de Otay, 22500, Tijuana, B.C., Mexico.
d
Laboratorio de Análisis de Residuos de Plaguicidas, Centro de Investigación en Alimentación y
Desarrollo A.C., Carretera a El Dorado Km 5.5, Campo El Diez, Culiacán Sinaloa, 80129, Mexico.
e Facultad de Biología, Universidad Autónoma de Sinaloa, Blvd. de las américas S/N, 80040, Campus
Culiacán, Culiacán, Sinaloa, México.
____________
* Juan I. Sarmiento-Sánchez and Ulises Osuna-Martínez have contributed equally to this study. For
questions, address to: chemical approach with Prof. Juan Sarmiento (jsarmiento@uas.edu.mx) and
biological activities with Prof. Ulises Osuna (ulises.osuna@uas.edu.mx).

Abstract
Imine functionality is found in many compounds with important biological activity. Thus,
the development of novel synthetic approaches for imines is important. In this work, it is
propose an easy, eco-friendly and straightforward synthesis pathway of aryl imines under
microwave irradiation catalyzed by Alumina-sulfuric acid. In addition, the in vitro enzymatic
inhibition, antioxidant activity and molecular docking studies were performed. The aryl
imines were isolated with yields in the range of 37-94%. All aryl imines synthesized were
evaluated for in vitro inhibitory potential against -glucosidase and -amylase enzymes and
the results exhibited that the most of the compounds displayed inhibitory activity against both
enzymes. The (E)-1-(4-nitrophenyl)-N-(pyridin-2-yl)methanimine (3d) was 1.15-fold more
active than acarbose against -amylase whilst the (E)-1-phenyl-N-(pyridin-2-
yl)methanimine (3c) displayed similar activity that acarbose against -glucosidase. The
molecular docking studies in glucosidase and amylase reveal that aryl imines mainly
establish an H-bond with the R2-subtituent and hydrophobic interactions with the R1-
subtituent. The docking analysis reveals these synthetic aryl imines 3d-i interact in same
active site than acarbose drug in both enzymes.
Keywords: aryl imines; -glucosidase; -amylase; antioxidant activity; docking

1. Introduction
Imines can be prepared by various methods including the classical way which involve the
heating of an aldehyde with an amine in a polar solvent with dehydrating agents or removing
the water azeotropically. Other approaches reported in the literature include condensation
reactions promoted by ultrasound irradiation using SiO2 [1], Mg(ClO4)2 [2], electrochemical
oxidation of benzylic amines [3], SiO2-NaHSO4 [4], aniline with aryl aldehyde in water [5],
basic alumina or poly-4-vinylpyridine [6], SiO2-H3PO4 [7], aerobic oxidation of benzylic
amines using graphite-supported gold nanoparticles [8], primary alcohols with amines using
ruthenium(II) bis(benzimidazole)pyridine complex [9] or copper-aluminum mixed oxide
[10] as catalyst, TiO2-nanotubes under sunlight [11], SnO2-doped silica nanospheres [12], K-
10 clay [13], CaO-activated [14], SiO2-P2O5 [15], MCM-41-SO3H [16], grinding using
H2SO4 as catalyst [17] and p-TsOH acid [18]. Such pathways often have complicated
procedures, moisture sensitive catalysts or reagents, large volume of toxic solvents,
expensive catalysts, high reaction temperatures and long reaction times. Imines are the
intermediates to obtain some compounds as isoxazoline and pyrazolines [19], quinolones
[20], α-alkylidene β-oxoamides [21] and 1,5-amino/ketoalcohols [22]. Imines are an
important class of compounds because they exhibit a variety of applications in organic,
inorganic and medicinal chemistry. The imine moiety is shown in bioactive compounds and
exhibit a broad spectrum of biological activities as antioxidant [17], antidiabetic [23], α-
glucosidase inhibitors [24,25], antimicrobial [26], anticancer [27,28], antifungal [29], anti-
Leishmania tropica [30], among others.
On the other hand, Diabetes mellitus (DM) is a chronic disease characterized by
hyperglycemia. The main strategy for the treatment of diabetes is the drugs consumption to

control the glucose level in the blood. The glucose in the blood originates from the hydrolysis
of carbohydrates and it is catalyzed by enzymes such as -glucosidase and -amylase. The
-glucosidase inhibitors reduce the absorption of glucose from the small intestine, and it is
secreted by the pancreas and salivary glands to hydrolyze the oligosaccharides into simple
sugar. The inhibition of -amylase and -glucosidase delays the glucose absorption,
therefore, the postprandial blood glucose level is lowered, which is considered to be a
therapeutic approach for the treatment of diabetes. Hence, due to the biological activity of
imines and their use in organic field, the design of simple, efficient and eco-friendly ways
for the imine synthesis has a great interest.
Currently, our research group is focused on the development of simple synthetic approaches
for small compounds and the study of their potential biological activity. Recently, we have
reported the synthesis of small compounds and their biological activity [31–35]. Here, we
report an easy and efficient one-step microwave-assisted synthesis of aryl imines catalyzed
by alumina-sulfuric acid (ASA) and their study as -glucosidase and -amylase inhibitors.
2. Result and discussion
2.1 Chemistry
OHC
acid catalyst, W
R2
R1 NH2
R2
+
N
R1
10 min, 120 °C
1
2
3
Scheme 1. Synthetic route of aryl imines.
A series of aryl imines were obtained by reacting aryl amines with aryl aldehydes in the
presence of an acid catalyst (scheme 1). In this regard, the initial efforts were devoted toward

the optimization of reaction conditions. The synthesis of N-benzylidene aniline (3a, BnA)
under microwave irradiation was study as model reaction for this aim. The first studies were
carried out setting the reaction conditions in 120 ºC and 10 minutes using some solvents
(Table 1). These experiments display the higher yield with protic solvents as MeOH, EtOH
and iPrOH (entry 3-5, Table 1) as it was expected. Lehn et. al.[36] report that traces of BnA
were detected in water, nevertheless, the BnA under this approach was obtained in 69% yield
(entry 2, Table 1) and lower time than in previous reports. [5,37]
Table 1. Yields for 3a using different solvents
Entry
solvent
-
H2O
MeOH
EtOH
iPrOH
1,4-dioxane
Benzene
Toluene
EtOAc
DMF
DMSO
THF
CH3CN
Yield (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
68
69
88
87
83
78
78
70
73
76
70
45
78
In a second research step, a screening of commercial supports (entry 1-6, table 2) were used
in order to find an efficient catalyst for the synthesis of imines (using 20 mg of catalyst) and
these experiments showed better yield with acid alumina. Therefore, some tests varying
temperature (80 to 140 ºC), time (2 to 15 minutes) and catalyst amount (10 to 150 mg) were
carried out to establish the optimal conditions with acid alumina under solvent-free condition
(supplementary material). The optimal conditions from these experiments were 120 ºC, 10

minutes and 100 mg of catalyst. Then, some acid catalysts as SBA-15-sulfuric acid, silica-
sulfuric acid (SSA), alumina-sulfuric acid (ASA), metal hydrogen sulfates and alumina or
acid silica were tested under the optimal conditions (entry 7-23, table 2). The higher yields
were obtained with the alumina catalysts. Noteworthy, the ASA material was the most
efficient catalyst giving a 94% yield during screening for efficiency (entry 10, Table 2).
Table 2. Synthesis of N-benzylidene aniline 3a using various catalysts
Entry
1
catalyst
neutral alumina
basic alumina
acid alumina
Silica gel
Yield (%)a
83
2
3
4
82
88
42
5
6
7
diatomaceous earth
sodium bisulfite
SBA-15
50
78
53
8
9
SBA-15-SO3H
SSA
76
63
10
11
12
13
14
15
16
17
18
19
20
21
22
23
ASA
SiO2-HCl
94
traces
traces
traces
5
53
80
5
82
20
traces
traces
traces
16
SiO2-H2SO4
SiO2-HNO3
SiO2- H3PO4
Al2O3-HCl
Al2O3-H2SO4
Al2O3-HNO3
Al2O3-H3PO4
Zn(HSO4)2
Mg(HSO4)2
Ca(HSO4)2
Ba(HSO4)2
Sn(HSO4)2
ayield obtained using 100 mg of catalyst; SSA -
silica sulfuric acid; ASA - alumina sulfuric acid.
Due to our interest in green chemistry and the above results that showed ASA as the best
catalyst for the solvent-free synthesis of imines, this approach was used to investigate its

generality and feasibility and the synthesis of the desired compounds 3a-m (Table 3) were
produced in 37-94% yields. The structure of the imines were confirmed by NMR and GC-
MS analyses. A structure-yield analysis reveals that lower yields are obtained when R1 is a
cycloalkyl substituent (3b, 3i and 3l, Table 3). If the 4-NO2 analogous are compared (3d-3j),
the highest yields are obtained with the orto-substituted aryl amines having a weak electron-
donating group (EDG) (3e and 3h).
Table 3. Yields of aryl imines 3a-3m under µW irradiation
OHC
R2
R1 NH2
R2
+
N
R1
1
2
3
R1
R2
Yield (%)
94
3a
3b
3c
3d
3e
3f
C6H5
H
C6H11
H
37
2-C5H4N
2- C5H4N
H
44
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
2-OH
4-Cl
54
2-CH3C6H4
3-CH3C6H4
4-CH3C6H4
2,6-(CH3)2C6H3
C6H11
82
63
3g
3h
3i
50
82
40
3j
4-OHC6H4
4-OHC6H4
C6H11
63
3k
3l
82
39
3m
4-CH3C6H4
3-OH
40
On the other hand, the recycling of heterogeneous catalyst is well reported. For this reason,
we address the study to recyclability and reusability of the catalyst toward green chemistry
approach. Upon the end of the reaction, the ASA was separated by filtration, washed with

isopropyl alcohol, dried and activate using µW irradiation under open vessel system (120 ºC,
10 minutes). The reactivated ASA was used in three additional cycles for the synthesis of
compound 3a and did not show a significant loss of yield (87, 85 and 80% yield,
respectively).
2.2 Biological activities
The inhibitory potency of aryl imines 3a-m against -glucosidase and -amylase was
evaluated and compared with acarbose as a reference standard. All the synthesized aryl
imines displayed inhibitory activity against -glucosidase (G), the compounds 3a and 3c
were likewise active than acarbose. It is observed a better activity if R1 is an aromatic ring as
phenyl or pyridyl (3a and 3c) compared with a non-aromatic ring as c-hexyl (3b).
Furthermore, a weak or null inhibition is show with an electron-withdrawing group (EWG)
on R2 position (3d-j), even if pyridine ring is present as R1 substituent (3d). When R1 is a
tolyl, the enzyme inhibition follows the reactivity order meta > para > ortho being 1.54- and
1.79-fold more active, respectively (3e-g). Nonetheless, the p-hydroxyphenyl (R1-
substituent) does not induce inhibition with an EWG as nitro in R2 (3j), whilst an EDG as
hydroxyl (3k) show 1.55-fold less inhibition than acarbose. From these results, we concluded
that aryl imines with an EDG could be more active against GI than those with a EWG as
substituent.
Table 4. Biological activities of synthetic imines
IC50
IC50
%AA
IC50
(10 µg mL-1) a (antioxidant)
(-glucosidase) (-amylase)
b
3a
3b
25.37
-
b
>100
53.87
-
2.990  2.940

b
3c
>100
>100
>100
>100
>100
>100
>100
24.02
35.05
62.15
34.68
53.51
36.32
44.56
-
3.089  2.325
5.224  0.865
2.181  0.644
13.130  2.990
3.635  0.708
3.998  4.080
3.498  0.982
3d
28.53
44.77
37.56
52.25
126.4
35.27
3e
3f
3g
3h
3i
b
b
3j
-
-
3k
62.16
24.38
112.2
61.28
Acarbose
Vitamin C
Gallic acid
8.79
4.08
56.84  3.21
89.24  13.78
All IC50 are given in µg mL-1; a value represents mean  S.D.; b not inhibition
Based on the inhibitory activity against porcine pancreatic α-amylase (A), the compounds
3d-g and 3i displayed the best activity. The compounds 3d, 3e, 3f and 3i were found to be
1.15-, 0.37-, 0.63- and 0.74-fold more active than acarbose, respectively. The highest
enzymatic inhibition was observed if R1 is pyridine and R2 is an EWG (3d), whilst the lack
of substitution in R2 leads to inactive compounds (3a-c). When R1 is a tolyl, the enzymatic
inhibition follows the reactivity order meta > ortho > para being 1.19- and 1.39-fold more
active, respectively (3e-g). Nevertheless, when R1 is a 4-hydroxypheyl there is not inhibition
with a EWG as 4-NO2 in R2 (3j), whereas an EDG as 2-OH in R2 (3k) show 0.83-fold less
inhibition that acarbose. These results suggest that aryl imines substituted in R2 and R1 by
EWG and weak EDG could be more active against A.
On the other hand, all the synthetic imines behave as reducing agents over the DPPH radical,
the 3d and 3f compounds behave as the best antioxidants (Table 4). Based on the structure-

antioxidant activity relationship, the antioxidant activity can be related with the substitution
on the aromatic ring; i.e., if R1 is a non-aromatic ring (3b, 3i and 3l) or 2-pyridyl, the
antioxidant activity increased with a EWG as substituent in the R2 position. The compound
3f displayed the best DPPH activity (13.130  2.990%) at 100 µg mL-1 being 1.51-, 2.75-
and 2.91-fold better than 3d, 3i and 3l, respectively. However, all the synthesized aryl imines
show poor antioxidant activity (IC50 >100 µg mL-1) compared with vitamin C (IC50= 8.79 µg
mL-1) or gallic acid (IC50= 4.08 µg mL-1).
2.3 Molecular docking studies
All the synthetic aryl imines (3a-l) were analyzed by molecular docking studies in order to
find the binding interactions in the active pocket site of the enzyme. The X-ray structures of
human pancreatic α-amylase (αA, pdb code: 5E0F) and human lysosomal acid-α-glucosidase
(αG, pdb code: 5NN8) was selected as template for this purpose. The aryl imines (3a-l)
docked within αA displayed the binding energies (∆Gb) in the range of -5.82 at -7.37 kcal
mol-1 being better than acarbose (-5.45 kcal mol-1). The molecular docking analysis exhibited
several interactions between the inhibitors and the A upon binding in the active site
residues. The binding energies calculated through the docking studies do not always show
the biological response observed in the in vitro tests, the ligand-enzyme interactions could
be used as a guide for the design of new drugs. In this sense, the compound 3d which exhibit
the highest in vitro activity against A (IC50= 28.53 µg mL-1) is bound into the receptor site
via a hydrophilic interaction establishing a H-bond between NO…H-N of Lys200 (2.58 Å,
111.9º) (Figure 1a). Other interactions result from other groups in the molecule, as the
electrostatic interaction with the catalytic amino acid Glu233 (4.37 Å). Similarly, the

enzyme-ligand (E-L) complex for the active compounds 3e-g are stabilized by H-bonds
between the oxygen (NO2) of the R2-substituent with Lys200. In addition, they show other
interactions that stabilize the E-L complex, as the electrostatic interaction with the catalytic
amino acids Glu233. If the in vitro activities of compounds 3e-g are observed, the docking
studies reveals the lack of the H-bond with Ile235 as a probable cause of the decrease in the
activity. Also, the docking parameters display the order 3e > 3g > 3f, suggesting a good
approximation between the docking studies with the in vitro assay. The best docking
parameters are exhibit when R2 is a EWG, as is shown by the 3b and 3i activity. The same
effect is observe for the aryl amines 3c and 3d. The docking studies against A show the aryl
amines 3a-c as inactive compounds because of the absence of conventional H-bonds.
Nonetheless, when R1 is phenyl or pyridyl group another type of interactions were observed
(T-shaped π-π, π-alkyl, π-sigma and stacked π-π). The molecular docking study revealed that
all active compounds docked into the A mainly display a H-bond with the oxygen of the
R2-subtituent, while the R1-subtituent mainly exhibit hydrophobic interactions (T-shaped π-
π, π-alkyl, π-sigma, alkyl and stacked π-π) and the substitution in R1 with weak EDG leads
to the best docking parameters.
On the other hand, the aryl imines (3a-k) docked within α-glucosidase displayed the binding
energies (∆Gb) in the range of -5.26 at -6.22 kcal mol-1 being better than acarbose (-4.42 kcal
mol-1). The aryl amines 3a and 3c displayed the highest in vitro activity against G (IC50=
25.37 and 24.02 µg mL-1, respectively). Besides, the molecular docking against G reveals
that only compound 3c (∆Gb = -5.26 kcal mol-1) form the interaction C=N…H-O of Ser676
(1.91 Å, 163.4º) and a hydrophobic interaction between ring B with Ser676 (3.36 Å).
Otherwise, the aryl amine 3d (∆Gb = -6.19 kcal mol-1) shows a similar activity than acarbose

against G and A enzymes, and it interacts in same binding site. Hence, the docking studies
for 3d in G exhibited the interaction NO…H-N of Arg600 (2.01 Å, 141.2º) and a C-H bond
with Arg600 (3.18 Å), an electrostatic (ring B…O of Asp616 at 3.55 Å) and hydrophobic
interactions (T-shaped π- π with Trp376 and Phe649) (Figure 1b).
The enzyme-ligand (E-L) complex for the active compounds 3e-g and 3i are stabilized by H-
bonds between NO…H-N of Arg600 similar to compound 3d. In addition, they show other
interaction that stabilize the E-L complex as electrostatic (ring B with Asp616), hydrophobic
(T-shaped π-π with Trp376 and Phe649) and a C-H-O (NO…H-C of Arg600) interactions.
The molecular docking studies in G revealed, as in A, that aryl imines mainly displayed
H-bond in the R2-subtituent and hydrophobic interactions on R1-subtituent. In general, the
docking studies show the interaction of these synthetic aryl imines 3d-i in the same active
pocket site that acarbose drug in -glucosidase and -amylase enzymes.
Figure 1. Putative binding interactions of compound 3d against α-amylase (left) and α-
glucosidase (right). Hydrogen, carbon Hydrogen, electrostatic and hydrophobic bonds are
shown as orange, pink, yellow and blue pale dashed line. The atoms of nitrogen, polar
hydrogen and oxygen are represented in blue, white and red, respectively.

3. Conclusion
An easy, efficient and ecofriendly approach for the synthesis for aryl imines using ASA as
heterogeneous catalyst under microwave solvent-free conditions was developed. This
approach affords aryl imines in short time using a simple workup procedure, avoiding
purification methods for the final products. The molecular docking results encourage us to
the design of novel aryl imines with potential antidiabetic activities and with better efficiency
and selectivity. According with the results in the in vitro tests and the molecular docking
studies, the aryl imines 3d, 3f, and 3i have potential for in vivo antidiabetic evaluations.
4. Experimental
4.1 Chemistry
All reagents were purchased in the highest quality available and were used without further
purification. Nuclear Magnetic Resonance of 1H (400 MHz) and 13C (100 MHz) spectra were
recorded on a Bruker 400 MHz Spectrometer in DMSO-d6 with TMS as internal standard.
Chemical Ionization Mass spectra were obtained at a GC-MS Agilent 7890B with ion trap,
and the intensities were reported as a percentage relative to the base peak after the
corresponding m/z value. Melting points were obtained on a Stuart apparatus model SMP30
(calibrated before use with phenolphthalein) and were uncorrected. The values are report as
an average of three separated experiments. All reactions described herein were conducted in
Pyrex tubes sealed with a silicon septum in a single-mode microwave reactor (Discover-SP
model 909150 equipped with an Explorer 12 hybrid model 909505, with 725 W of maximum
power, CEM Corp.) with 100 W of initial power.

Preparation of alumina-sulfuric acid
Alumina-sulfuric acid was prepared following the same methodology reported by Pramanik
et al. [38] The concentration of H+ on alumina-sulfuric acid for this procedure is 3x10-4 mol
g-1.
General procedure for the Imines synthesis
To a microwave reactor vessel (10 mL) was added an aldehyde (1.0 mmol), a primary amine
(1.1 mmol) and alumina-sulfuric acid (100 mg). Mixture was heated at 120 ºC for 10 min,
and then cooled to room temperature. The resulting mixture was filtered using methanol (5
mL), ethyl acetate or DCM and concentrated under vacuum. The solid formed was washed
with diethyl ether (2 x 2 mL) and imines were obtained. Characterization data are similar to
those reported in the literature.
1
(E)-N,1-diphenylmethanimine (3a). lit. [39,40]; yield 94%; Pale yellow solid; H-NMR
(400 MHz, DMSO-d6): δ 8.63 (s, 1H, imidic H), 7.94 (m, 2H, ArH), 7.53 (m, 3H, ArH), 7.43
(dd, 2H, J = 7.3, 6.8 Hz, ArH), 7.29 (m, 3H, ArH); 13C-NMR (100 MHz, DMSO-d6): δ 160.5
(CH, imidic), 151.3 (C-N, Ar), 139.5 (C, Ar), 131.4 (CH, Ar), 129.1(2xCH, Ar), 129.0
(2xCH, Ar), 128.8 (2xCH, Ar), 125.9(CH, Ar), 120.9 (2xCH, Ar). GC-MS (CI) m/z: 182
[M+H]+. (E)-N-cyclohexyl-1-phenylmethanimine (3b). lit. [9]; yield 37%; white solid; 1H-
NMR (400 MHz, DMSO-d6): δ 8.36 (s, 1H, iminic H), 7.87 (d, J = 6.1 Hz, 1H, ArH), 7.71
(m, 1H, ArH), 7.40 (m, 3H, ArH), 3.21 (m, 1H, CH-N), 1.52 (m, 10H, -CH2); 13C-NMR (100
MHz, DMSO-d6): δ 158.2 (CH, imidic), 136.6 (C, Ar), 130.3 (CH, Ar), 128.5 (2xCH, Ar),
127.7 (2xCH, Ar), 68.5 (CH-N), 34.0 (2xCH2), 25.2 (CH2), 24.1 (2xCH2). GC-MS (CI) m/z:
188 [M+H]+.

(E)-1-phenyl-N-(pyridin-2-yl)methanimine (3c). lit. [41]; yield 40%; white solid; 1H-NMR
(400 MHz, DMSO-d6): δ 9.17 (s, 1H, iminic H), 8.51 (ddd, J = 4.8, 1.9, 0.9 Hz, 1H, Py-H),
7.92 (dd, J = 6.8, 1.2 Hz, 2H, ArH), 7.72 (ddd, J = 8.0, 7.3, 1.5 Hz, 1H, Py-H), 7.54 (m, 3H,
ArH), 7.38 (dd, J = 8.4, 1.8 Hz, 1H, Py-H), 7.25 (dd, J = 7.3, 1.22 Hz, 1H, Py-H); 13C-NMR
(100 MHz, DMSO-d6): δ 162.5 (CH, imidic), 160.4 (C-N, Py), 148.7 (CH-N, Py), 138.4 (CH,
Py), 135.5 (C, Ar), 132.0 (CH, Ar), 129.2 (2xCH, Ar), 128.8 (2xCH, Ar), 122.1 (CH, Py),
119.3 (CH, Py). GC-MS (CI) m/z: 183 [M+H]+.
(E)-1-(4-nitrophenyl)-N-(pyridin-2-yl)methanimine (3d). lit. [41]; yield 54%; pale yellow
1
solid; H-NMR (400 MHz, DMSO-d6): δ 9.33 (s, 1H, iminic H), 8.54 (dd, J = 4.9, 1.2 Hz,
1H, Py-H), 8.24 (dd, J = 8.8, 4.1 Hz, 2H, ArH), 8.17 (dd, J = 8.8, 4.1 Hz, 2H, ArH), 7.74 (d,
J = 9.1 Hz, 1H, Py-H), 7.71 (ddd, J = 8.5, 6.9, 1.9 Hz, 2H, Py-H); ); 13C-NMR (100 MHz,
DMSO-d6): δ 160.8 (CH, imidic), 159.7 (C-N, Py), 150.4 (C-NO2, Ar), 148.9 (CH-N, Py),
146.6 (C, Ar), 140.1 (CH, Py), 130.6 (2xCH, Ar), 124.2 (2xCH, Ar), 123.3 (CH, Py), 120.0
(CH, Py). GC-MS (CI) m/z: 228 [M+H]+.
(E)-1-(4-nitrophenyl)-N-(o-tolyl)methanimine (3e). lit. [42]; yield 82%; yellow crystals;
1H-NMR (400 MHz, CDCl3-d): δ 8.47 (s, 1H, iminic H), 8.32 (d, J = 8.6 Hz, 2H, ArH), 8.08
(d, J = 8.6 Hz, 2H, ArH), 7.21 (m, 3H, ArH), 6.96 (dd, J = 7.6, 1.5 Hz, 1H, ArH), 2.39 (s,
13
3H, -CH3); C-NMR (400 MHz, CDCl3-d): δ 156.4 (CH=N), 149.9 (C-N, Ar), 149.2 (C-
NO2, Ar), 141.8 (C-CH3, Ar), 132.6 (C-CH, Ar), 130.5 (2xCH, Ar), 129.3 (2xCH, Ar), 126.8
(CH, Ar), 124.0 (2xCH, Ar), 117.1 (CH, Ar), 17.8 (-CH3). GC-MS (CI) m/z: 241 [M+H]+.
(E)-1-(4-nitrophenyl)-N-(m-tolyl)methanimine (3f). lit. [43]; yield 63%; yellow crystals;
1H-NMR (400 MHz, CDCl3-d): δ 8.47 (s, 1H, iminic H), 8.32 (d, J = 8.6 Hz, 2H, ArH), 8.08
(d, J = 8.6 Hz, 2H, ArH), 7.22 (m, 3H, ArH), 6.97 (dd, J = 7.6, 1.5 Hz, 1H, ArH), 2.39 (s,

3H, -CH3); 13C-NMR (100 MHz, CDCl3-d): δ 156.4 (CH, imidic), 149.9 (C-NO2, Ar), 149.3
(C-N, Ar), 141.8 (2xC, Ar), 132.6 (CH, Ar), 130.6 (CH, Ar), 129.3 (2xCH, Ar), 126.8 (CH,
Ar), 124.0 (2xCH, Ar), 117.2 (CH, Ar), 17.8 (-CH3). GC-MS (CI) m/z: 241 [M+H]+.
(E)-N-(4-nitrophenyl)-1-(p-tolyl)methanimine (3g). lit. [44]; yield 50%; yellow crystals;
1H-NMR (400 MHz, DMSO-d6): δ 8.80 (s, 1H, iminic H), 8.34 (dt, J = 8.8, 2.2 Hz, 2H, ArH),
8.17 (dt, J = 8.8, 2.2 Hz, 1H, ArH), 7.27 (dd, J = 8.5, 3.1 Hz, 4H, ArH), 2.34 (s, 3H, -CH3);
13C-NMR (100 MHz, CDCl3-d): δ 157.5 (CH, imidic), 148.6 (C-N, Ar), 147.8 (C-NO2, Ar),
141.6 (C, Ar), 136.5 (C-CH3, Ar), 129.7 (2xCH, Ar), 129.4 (2xCH, Ar), 123.9 (2xCH, Ar),
121.2 (2xCH, Ar), 20.5 (-CH3). GC-MS (CI) m/z: 241 [M+H]+.
(E)-N-(2,6-dimethylphenyl)-1-(4-nitrophenyl)methanimine (3h). lit. [45]; yield 82%;
yellow crystals; 1H-NMR (400 MHz, DMSO-d6): δ 8.53 (s, 1H, iminic H), 8.39 (d, J = 8.8
Hz, 2H, ArH), 8.21 (d, J = 9.0 Hz, 2H, ArH), 7.09 (dd, J = 7.6, 0.5 Hz, 2H, ArH), 6.98 (t, J
13
= 7.1 Hz, 1H, ArH), 2.09 (s, 6H, 2xCH3); C-NMR (100 MHz, DMSO-d6): δ 161.7 (CH,
imidic), 150.2 (C-N, Ar), 149.0 (C-NO2, Ar), 141.1 (C, Ar), 129.3 (2xCH, Ar), 127.9 (2xCH,
Ar), 126.2 (2xC-CH3, Ar), 123.9 (2xCH, Ar), 123.9 (CH, Ar), 17.8 (-CH3). GC-MS (CI) m/z:
255 [M+H]+.
(E)-N-cyclohexyl-1-(4-nitrophenyl)methanimine (3i). lit. [8]; yield 40%; white solid; 1H-
NMR (400 MHz, DMSO-d6): δ 8.52 (s, 1H, iminic H), 8.28 (d, J = 9.0 Hz, 2H, ArH), 7.98
(d, J = 9.0 Hz, 2H, ArH), 3.32 (m, 1H, CH-N), 1.75 (m, 6H, CH2), 1.25 (m, 4H, CH2); 13C-
NMR (100 MHz, DMSO-d6): δ 157.9 (CH, imidic), 148.3 (C-NO2, Ar), 141.9 (C, Ar), 128.7
(2xCH, Ar), 123.8 (2xCH, Ar), 68.5 (CH-N), 33.8 (2xCH2), 25.1 (CH2), 23.9 (2xCH2). GC-
MS (CI) m/z: 232 [M+H]+.

1
(E)-4-((4-nitrobenzylidene)amino)phenol (3j). lit. [46]; yield 63%; H-NMR (400 MHz,
DMSO-d6): δ 8.76 (s, 1H, iminico H), 8.30 (d, J = 8.8 Hz, 2H, ArH), 8.11 (d, J = 8.8 Hz, 2H,
ArH), 7.30 (d, J = 8.6 Hz, 2H, ArH), 6.84 (d, J = 8.8 Hz, 2H, ArH); 13C-NMR (100 MHz,
DMSO-d6): δ 157.2 (CH=N, imidico), 154.6 (C-OH, Ar), 148.3 (C-NO2, Ar), 142.1 (C-N,
Ar), 141.6 (C, Ar), 129.0 (2xCH, Ar), 123.8 (2xCH, Ar), 123.0 (2xCH, Ar), 115.8 (2xCH,
Ar). GC-MS (CI) m/z: 243 [M+H]+.
(E)-2-(((4-hydroxyphenyl)imino)methyl)phenol (3k). lit. [47]; yield 82%; white solid; 1H-
NMR (400 MHz, DMSO-d6): δ 8.85 (s, 1H, iminic H), 7.59 (dd, J = 7.7, 1.6 Hz, 1H, ArH),
7.47 (ddd, J = 8.6, 7.2, 1.8 Hz, 1H, ArH), 7.15 (d, J = 8.8 Hz, 1H, ArH), 7.02 (d, J = 8.1 Hz,
1H, ArH), 6.97 (t, J = 7.6 Hz, 1H, ArH), 6.85 (d, J = 8.8 Hz, 2H, ArH). GC-MS (CI) m/z:
214 [M+H]+.
(E)-1-(4-chlorophenyl)-N-cyclohexylmethanimine (3l). lit. [3]; yield 40%; white solid; 1H-
NMR (400 MHz, DMSO-d6): δ 8.36 (s, 1H, iminic H), 7.73 (d, J = 8.3 Hz, 2H, ArH), 7.49
(d, J = 8.3 Hz, 2H, ArH), 3.23 (s, 1H, CH-N), 1.71 (m, 5H, CH2), 1.37 (m, 5H, CH2); ); 13C-
NMR (100 MHz, DMSO-d6): δ 157.1 (CH, imidic), 135.1 (C, Ar), 134.8 (C-Cl, Ar), 129.3
(2xCH, Ar), 128.5 (2xCH, Ar), 68.3 (CH-N), 33.9 (2xCH2), 25.1 (CH2), 24.0 (2xCH2). GC-
MS (CI) m/z: 221 [M+H]+.
1
(E)-3-((p-tolylimino)methyl)phenol (3m). lit. [48]; yield 40%; yellow crystals; H-NMR
(400 MHz, CDCl3): δ 13.35 (s, 1H, OH-phenolic), 8.61 (s, 1H, imidic H), 7.36 (t, 2H, ArH),
7.21 (m, 4H, ArH), 7.01 (d, 1H, J = 7.5 Hz, ArH), 6.92 (td, 1H, J = 7.4, 1.2 Hz, ArH), 2.37
(s, 3H, -CH3); 13C-NMR (100 MHz, CDCl3): δ 161.6 (CH, imidic), 161.1 (C-OH, Ar), 145.9
(C-N, Ar), 136.8 (CH, Ar), 132.8 (C-CH3, Ar), 132.1 (C, Ar), 129.9 (2xCH, Ar), 120.9

(2xCH, Ar), 119.3 (CH, Ar), 118.9 (CH, Ar), 117.2 (CH, Ar), 21.0 (-CH3). GC-MS (CI) m/z:
212 [M+H]+.
4.2 α-Amylase inhibitory activity
The α-amylase activity of the synthetic quinazoline-2,4(1H,3H)-diones was assessed as
described by Adisakwattana et al. with slight modification.[49] Porcine pancreatic α-
amylase (EC 3.2.1.1) was dissolved in 0.1 M phosphate buffer at pH 6.9 (concentration of
0.5 mg mL-1). In a 96-well flat-bottom plate, 75 µL of the enzyme solution and 75 µL of
quinazoline-2,4(1H,3H)-diones solution (concentrations of 1, 10, 100, 500, 1000 µg mL-1 in
methanol) were added. After 10 minutes of incubation at 25 °C, 75 µL of the starch solution
(0.1% p/v in 0.1 M phosphate buffer at pH 6.9) was added to start the reaction and the mixture
were incubated for 10 minutes at 25 °C. The reaction was stopped by adding 62.5 µL
dinitrosalicylic (DNS) reagent (1% 3,5-dinitrosalicylic acid, 0.2% phenol, 0.05% Na2SO3
and 1% NaOH in aqueous solution) to the reaction mixture. The mixtures were heated at
100°C for 5 minutes in order to stop the reaction. After cooling to room temperature, the
absorbance was recorded at 580 nm using a microplate reader. The α-amylase inhibitory
activity was expressed as percentage inhibition and was calculated as follows:
퐴푏푠푐표푛푡푟표푙 ― 퐴푏푠푠푎푚푝푙푒푠
% 퐼푛ℎ푖푏푖푡푖표푛 =
푥100
퐴푏푠푐표푛푡푟표푙
Where Abscontrol was the absorbance without sample, Abssamples was the absorbance of the
sample at different concentrations.
4.3 α-Glucosidase inhibitory activity
The α-glucosidase (EC 3.2.1.20) activity of the synthetic quinazoline-2,4(1H,3H)-diones was
assessed as described by Kwon et al. with slight modification. [50] In a 96-well flat-bottom

plate, 100 µL of the enzyme solution (1 U mL-1 in 0.1 M of phosphate buffer at pH 7.0) and
50 µL of quinazoline-2,4(1H,3H)-diones solution (concentrations of 1, 10, 100, 500, 1000
µg mL-1 in methanol) were added. After 10 minutes of incubation at 25 °C, 50 µL of 5 mM
p-nitro-phenyl-α-D-glucopyranoside solution in 0.1 M phosphate buffer (pH 7.0) was added
and the mixture were incubated at 25 °C for 5 minutes. After, the absorbance was recorded
at 405 nm using a microplate reader. The α-glucosidase inhibitory activity was expressed as
percentage inhibition and was calculated as follows:
퐴푏푠푐표푛푡푟표푙 ― 퐴푏푠푠푎푚푝푙푒푠
% 퐼푛ℎ푖푏푖푡푖표푛 =
푥100
퐴푏푠푐표푛푡푟표푙
Where Abscontrol was the absorbance without sample, Abssamples was the absorbance of the
sample at different concentrations.
4.4 DPPH-scavenging Activity
The DPPH scavenging activities of the imines were assessed as described by Clarke et. al.
[51] with slight modifications. This method is based on the reduction of DPPH in the
presence of antioxidants, the antioxidant activity is detected as a change from purple to
yellow color in the solution. In a 96-well flat-bottom plate, 20 µL of the imines (0.1 to 100
μg mL-1 in methanol) and 180 µL of DPPH solution (150 µM in methanol) were added. The
mixture was shaken, incubated for 20 min at room temperature in darkness conditions, and
the absorbance was measured at 532 nm. The DPPH-scavenging activity of the imines was
calculated as follows:
퐴0 ― 퐴1
퐷푃푃퐻 ― 푠푐푎푣푒푛푔푖푛푔 푒푓푓푒푐푡 (%) =
푥100
퐴0

Where: A0 was the absorbance of control; and A1 was the absorbance in the presence of the
imines. Vitamin C and gallic acid were used as standard (0.1 to 10 µg mL-1). Calculated
values corresponded to the mean ± standard deviation of two experiment by triplicate and
were determined by GraphPad Prism V6 software.
4.5 Molecular docking [52–54]
The automated molecular docking studies were carried out using AutoDock Tool v1.5.6 and
were guided to active site of the enzyme [55]. First, the aryl imines (ligands) were design
using ChemDraw V16 and Spartan V14 was used for 3D optimization using MMFF94s for
the optimization to lowest energy geometry including water phase for all ligand. Later, the
structures were saved as SYBYL mol2 file format and Gasteiger charges were assigned to
the ligands. Second, the crystal structure for human pancreatic α-amylase (pdb code: 5E0F)
and human lysosomal acid-α-glucosidase (pdb code: 5NN8) was downloaded from RCSB
protein data bank. Before of docking calculations, all water molecules, ligands and cations
were removed from protein. Later, polar hydrogens and the Kollman charges were assigned
for the protein. Then, the grid box site was set for human pancreatic α-amylase in -7.946,
10.438 and -21.863 Å (x, y and z) with a grid of 80, 72 and 66 point (x, y and z). For human
lysosomal acid-α-glucosidase the grid box site was set at -12.175, -35.415 and 88.753 Å (x,
y and z) with a grid of 74, 70 and 90 point (x, y and z) with 0.375 Å spacing. Docking
calculations were performed using Lamarckian genetic algorithm for ligands conformational
searching. The docking for the ligands consisted of a total of 200 runs that were carried out
with a population size of 150 individuals, a maximum of 25 million energies evaluations, a
maximum of 270,000 generations, a gene mutation rate of 0.02 and a crossover rate of 0.8.

Cluster analysis was performed on the docked results using a root mean square (RMS)
tolerance of 0.5 Å.
The conformation with the lowest binding energy was examined by Accelrys, Discovery
Studio Visualizer v17.2.0.16349 [Accelrys Inc., San Diego, CA (2007)] to determine their
binding orientations, molecular modeling, evaluation of the hydrogen bonds and other
interactions.
5. Acknowledgements
We gratefully acknowledge support for this project by Programa de Fomento y Apoyo a
Proyectos de Investigación of Universidad Autónoma de Sinaloa (UAS-PROFAPI, grant No.
PROFAPI2015/273).

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Graphical abstract

Highlights
• Alumina-Sulfuric acid is an efficient catalyst for aryl imines synthesis.
• Aryl imines synthesized displayed significant a-amylase inhibition with IC50 values of
28.53 - 126.40 µg mL-1.
• Aryl imines synthesized displayed significant a-glucosidase inhibition with IC50 values of
24.02 - 62.16 µg mL-1.
• Molecular docking studies were performed to find the binding interactions at the active
pocket site of the enzyme.

Conflict of Interest
November 22th, 2019
Editorial Office
Bioorganic Chemistry Journal
Scientific Electronic Library Online
Dear Editor
We confirm that the manuscript entitled " Synthesis and molecular docking studies of
imines as α-glucosidase and α-amylase inhibitors” has been read and approved by all
authors and that there are no other persons who satisfied the criteria for authorship. We
further confirm that the order of authors listed in the manuscript has been approved by all
of us.
Therefore, we confirm that this manuscript has not been published elsewhere, not been
submitted to, nor is under review at, another journal or other publishing venue. Also, all
authors approved the manuscript and authorize its submission in the Bioorganic Chemistry
Journal.
Moreover, the principal author signing on behalf of all co-authors of the manuscript.
Dr. Juan I. Sarmiento Sánchez
Professor of Engineering School
Autonomous University of Sinaloa
Américas Boulevard, University City
Culiacán, Sinaloa 80040, México
E-mail: jsarmiento@uas.edu.mx