Novel pyrano-triazolo-pyrimidine derivatives as anti- α-amylase agents: Synthesis, molecular docking investigations and computational analysis

Article abstract of DOI:10.1016/j.molstruc.2021.130346

A novel series of pyranotriazolopyrimidine derivatives 3a-j was synthesized and characterized by ¹H NMR, ¹³C NMR, and HRMS experimental data. The synthesized compounds were assessed for their inhibitory potential on the α-amylase enz

Full text of DOI:10.1016/j.molstruc.2021.130346

Journal of Molecular Structure 1237 (2021) 130346
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Novel pyrano-triazolo-pyrimidine derivatives as anti- α-amylase
agents: Synthesis, molecular docking investigations and computational
analysis
Amel Hajlaouia, Maha Laajimib, Mansour Znatia, Hichem Ben Janneta, Anis Romdhanea,∗
a University of Monastir, Faculty of Sciences of Monastir, Laboratory of Heterocyclic Chemistry, Natural Products and Reactivity (LR11ES39), Team: Medicinal
Chemistry and Natural Products, Avenue of Environment, 5019 Monastir, Tunisia
b Quantum and Statistical Physics Laboratory, Faculty of Sciences, University of Monastir, Monastir, Tunisia
a r t i c l e i n f o
a b s t r a c t
A novel series of pyranotriazolopyrimidine derivatives 3a-j was synthesized and characterized by 1H NMR,
13 C NMR, and HRMS experimental data. The synthesized compounds were assessed for their inhibitory
potential on the α-amylase enzyme. Results showed that seven of the synthesized compounds displayed
Article history:
Received 18 January 2021
Revised 21 February 2021
Accepted 19 March 2021
Available online 26 March 2021
potent α-amylase inhibitory activity. Compound 3b (IC50 = 2.78
group at triazole ring, exhibited the highest activity followed by 2a, 2b and 3c with IC50 values rang-
ing from 3.15 0.25 to 4.15
0.10 μg/mL, in comparison to the standard acarbose IC50 = 6.84
0.14 μg/mL) bearing a cyanomethyl
Keywords:
Pyranotriazolopyrimidine
α-amylase inhibition
In silico docking study
Pharmacokinetics
1.22 μg/mL). A molecular docking study was performed to investigate the possible inhibitory mecha-
nism at the binding site of the target enzyme which reinforced the observed activity of compounds 2a,
2b, 3b, and 3c. The analysis revealed the strength of intermolecular hydrogen bonding and hydropho-
bic interactions in the ligand-receptor complexes as significant descriptors to rationalize the inhibition
results obtained. Several physicochemical properties related to the pharmacokinetics of the synthesized
derivatives were predicted. These properties were found to lie within the desired limit and we have no-
ticed that all compounds are likely to be orally active as they obeyed Lipinski’s rule of five. HOMO-LUMO
energy gap and some reactivity descriptor’s parameters were evaluated using Density Functional Theory
(DFT) employing B3LYP level with 6-311++G (d,p) basis set.
HOMO-LUMO energy
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
teorism [5,6]. Thus, it is necessary to develop new and safe thera-
peutic agents with low side effects to treat type 2 diabetes.
Type 2 Diabetes mellitus (T2DM) is a metabolic disorder char-
acterized by high blood sugar levels over a prolonged period. Un-
controlled hyperglycemia can cause serious damage to many vital
organs such as heart disease, kidney damage and nerve damage
[1]. A therapeutic approach for treating diabetes in its early stages
is to reduce postprandial hyperglycemia. This is done by retard-
ing glucose absorption through the inhibition of the carbohydrate-
hydrolyzing enzymes, α-amylase and α-glucosidase, in the diges-
tive tract [2,3]. Particularly, α-amylase, is one of the enzymes of
great concern to the medical practitioners and researchers in the
control of T2DM. Inhibitors of this enzyme lead to a reduction in
the glucose absorption rate and, therefore, attenuate the postpran-
dial glucose levels [4]. Acarbose and miglitol are such oral hypo-
glycemic drugs. These inhibitors present certain limitations due to
their side effects, such as abdominal discomfort, diarrhea, and me-
Researchers are now focused on finding new heterocyclic moi-
eties that, if studied systematically, may appear to be potential
candidates for glycosidase inhibitors.
Due to their exceptional chemical and versatile biological pro-
files, heterocyclic systems are considered as an attractive area for
most medicinal chemists and have therefore been the privileged
core of the majority of medicinal compounds [7]. Among these
heterocycles, pyrimidine and triazole rings, which have played a
crucial role in several biological processes and have had a con-
siderable pharmacological and chemical importance as anticancer,
anti-alzheimer, antifungal, antihypertensive, analgesic and poten-
tial herbicidal activities [8,9]. These compounds have frequently
proven their efficiency in different fields of medicinal drug, bio-
chemistry, materials science, and agrochemistry [10–12].
Another one of relevance building blocks in the design of bioac-
tive structures is 4H-Pyran ring [13]. Indeed, pyrans and their
derivatives constitute a promising class of oxygen-containing hete-
rocyclic compounds, which have shown a broad series of biological
∗
Corresponding author.
https://doi.org/10.1016/j.molstruc.2021.130346
0022-2860/© 2021 Elsevier B.V. All rights reserved.

A. Hajlaoui, M. Laajimi, M. Znati et al.
Journal of Molecular Structure 1237 (2021) 130346
Fig. 1. Previously reported antidiabetic compounds.
eluents. Melting points are uncorrected and were determined us-
ing an electrothermal 9002 apparatus. 1H (300 MHz) and 13C (75
MHz) NMR spectra were recorded on a Bruker AC-300 spectrom-
eter, in deuterated CDCl3 and DMSO–d6 using non-deuterated sol-
vents as an internal standard. Chemical shifts (δ) are expressed in
ppm and coupling constants (J) in Hz. High-Resolution Mass Spec-
tra (HRES- MS) were obtained with Micromass LCT (ESI technique,
positive mode) spectrometers.
Fig. 2. The starting reagent and target compounds.
effects, including antitumor [14], anticoagulant [15] and anti-
alzheimer [16] activities. These derivatives have been found in di-
verse biodegradable agrochemicals, pigments, fluorescent reagents,
and photo-active materials [17,18]. It is also important to note that
pyran, triazole, and pyrimidine have been reported to exhibit po-
tential antidiabetic activity [10,19]. In this regard, Channar et al
[20] prepared a series of 1,2,4-triazole derivatives that have shown
promising results against α-amylase, among them compound A
(Fig. 1) has demonstrated a strong α-amylase inhibitory activity
(IC50 = 1.1 mmol/g) [19]. Likewise, compound B has been iden-
tified as a potent anti-diabetic analog with an IC50 value of 54.2
2.02 μM [21]. Fused pyrimidines have also been studied as in-
hibitors for α-amylase such as compound C, showing a percentage
inhibition value of 92% [22].
2.1.1. General procedure for the synthesis of 2-amino-3-cyanopyranes
(1)
A mixture of arylaldehyde (10 mmol), malononitrile (10 mmol),
ethyl acetoacetate (10 mmol) and a catalytic amount of piperidine
was stirred in 30 mL of absolute ethanol at room temperature for
1 h. The solid product obtained was filtered, dried, and purified by
recrystallization from ethanol to afford compounds 1a-c.
2.1.1.1. 2-amino-3-cyano-5-ethoxycarbonyl-6-methyl-4-phenyl-4H-
pyran (1a). White solid, yield: 70%, mp: 194-196°C, 1H NMR
(DMSO-d6, 300 MHz): δ (ppm) = 1.03 (t, 3H, H9, J= 5.2 Hz); 2.32
(s, 3H, H10); 3.97 (q, 2H, H8, J= 5.2 Hz); 4.30 (s, 1H, H4); 6.90
(s, 2H, NH2); 7.14 - 7.33 (m, 5H, Harom). 13C NMR (DMSO-d6,
75 MHz): δ (ppm) = 14.2 (C9); 18.6 (C10); 39.3 (C4); 57.8 (C3);
60.6 (C8); 107.6 (C5); 120.2 (CN); 127.3 (C4’); 127.7 (C2’, 6’); 128.9
(C3’, 5’); 145.2 (C1’); 157.0 (C6); 158.9 (C2); 165.9 (C7). HR-ES-MS
[M+H]+ calcd for (C16 H17 N2O3)+: 285.1161, found: 285.1194.
In the light of these findings, it was considered worthwhile
to synthesize a new condensed pyrimidine scaffold using an effi-
cient and convenient synthetic method: the multicomponent reac-
tion. This latter has been presented as a powerful and remarkable
synthetic approach in the preparation of heterocyclic components
owing to its simple experimentations and its high atom economy
[23]. Thus, we report in this paper the use of α-aminocarbonitriles
1 as starting material for the synthesis of a new series of pyra-
notriazolopyrimidines 3 (Fig. 2). Computational approaches were
carried out to explain some pharmacokinetic effects of compounds
(1, 2 and 3) as well as certain toxicological properties. These com-
pounds were tested for their in vitro α-amylase inhibitory activity.
In silico studies were performed to gain insight toward the inter-
actions of title compounds with the target protein α-amylase. The
HOMO-LUMO energy gap and some reactivity descriptor’s param-
eters were evaluated to simulate the electronic properties of our
compounds through DFT calculations using B3LYP 6-311++G(d,p)
method.
2.1.1.2. 2-amino-3-cyano-5-ethoxycarbonyl-6-methyl-4-(4-
methoxyphenyl)-4H-pyran (1b). White solid, yield: 81%, mp:
130-132°C. 1H NMR (DMSO-d6, 300 MHz): δ (ppm) = 1.13 (t, 3H,
H9, J= 5.3 Hz); 2.37 (s, 3H, H10); 3.78 (s, 3H, H11); 4.05 (q, 2H, H8,
ꢀ
J= 5.3 Hz); 4.42 (s, 1H, H4); 4.54 (s, 2H, NH2); 6.84 (d, 2H, H3 ,
ꢀ
ꢀ
ꢀ
5 , J= 6.4 Hz); 7.13 (d, 2H, H2 , 6 , J= 6.4 Hz). 13C NMR (DMSO-d6,
75 MHz): δ (ppm) = 14.0 (C9); 18.4 (C10); 38.0 (C4); 55.3 (C11);
60.7 (C3); 62.5 (C8); 108.2 (C5); 119.1 (CN); 114.0 (C3’, 5’); 128.6
ꢀ
(C2’, 6’); 136.1 (C1’); 156.3 (C6); 157.4 (C4 ); 158.7 (C2); 166.0 (C7).
ES-MS [M+H]+ calcd for (C17 H19 N2O4)+: 315.1267, found: 315.13.
2.1.1.3. 2-amino-4-(4-chlorophenyl)-3-cyano-5-ethoxycarbonyl-6-
methyl-4H-pyran (1c). White solid, yield: 79%, mp: 177-179°C. 1H
NMR (CDCl3, 300 MHz): δ (ppm) = 1.13 (t, 3H, H9, J= 5.3 Hz);
2.37 (s, 3H, H10); 4.05 (q, 2H, H8, J= 5.3 Hz); 4.44 (s, 1H, H4);
2. Experimental section
ꢀ
ꢀ
ꢀ
2.1. Chemistry
4.62 (s, 2H, NH2); 7.15 (d, 2H, H2 , 6 , J= 6.3 Hz); 7.28 (d, 2H, H3 ,
ꢀ
5 , J= 6.3 Hz). 13C NMR (CDCl3, 75 MHz): δ (ppm) = 13.8 (C9);
Progress of the reactions was monitored by Thin Layer Chro-
matography (TLC) using aluminum sheets of Merck silica gel 60
F254, 0.2 mm. Column chromatography was carried out on silica
gel (70-230 mesh) using chloroform and ethyl acetate mixture as
18.5 (C10); 38.2 (C4); 60.7 (C3); 61.5 (C8); 107.6 (C5); 118.7 (CN);
128.8 (C2’, 6’); 129.0 (C3’, 5’); 133.0 (C4’); 142.4 (C1’); 157.0 (C6);
157.6 (C2); 165.7 (C7). ES-MS [M+H]+ calcd for (C16 H16 ClN2O3)+:
319.0771, found: 319.0782.
2

A. Hajlaoui, M. Laajimi, M. Znati et al.
Journal of Molecular Structure 1237 (2021) 130346
2.1.2. General procedure for the synthesis of iminoethers 2
The mixture of 1 mmol of pyranic derivative 1 and 1 mmol of
triethylorthoformate was added to 25 mL of acetic anhydride and
refluxed for 6 hours. After cooling, the resulting product was fil-
tered and throughly washed with petroleum ether to provide com-
pounds 2a-c.
2.1.3.2. Ethyl
2-(cyanomethyl)-8-methyl-10-phenyl-10H-pyrano[3,2-
e][1,2,4]triazolo[1,5-c]pyrimidine-9-carboxylate (3b). Orange solid,
yield: 58%, mp: 164-166°C. 1H NMR (CDCl3, 300 MHz): δ
(ppm) = 1.21 (t, 3H, H13, J= 5.3 Hz); 2.63 (s, 3H, H14); 4.01
(s, 2H, H15); 4.13 (q, 2H, H12, J= 5.3 Hz); 5.50 (s, 1H, H10); 7.18
- 7.44 (m, 5H, Harom); 9.05 (s, 1H, H5). 13C NMR (CDCl3, 75
MHz): δ (ppm) = 14.0 (C13); 18.7 (C14); 19.0 (C15); 37.8 (C10);
60.8 (C12); 104.3 (C9); 107.1 (C10a); 114.6 (CN); 127.5 (C4’); 128.4
(C3’, 5’); 128.5 (C2’, 6’); 138.2 (C5); 142.3 (C1’); 153.2 (C2); 153.3
(C10b); 159.7 (C8); 160.6 (C6a); 165.6 (C11). ES-MS [M+H]+ calcd
for (C20H18 N5O3)+: 376.1345, found: 376.1419.
2.1.2.1. Ethyl
5-cyano-6-((ethoxymethylene)amino)-2-methyl-4-
phenyl-4H-pyran-3-carboxylate (2a). White solid, yield: 79%,
mp: 94-96°C. 1H NMR (DMSO-d6, 300 MHz): δ (ppm) = 1.03 (t,
3H, H9, J= 5.3 Hz); 1.30 (t, 3H, H13, J= 5.3 Hz); 2.36 (s, 3H, H10);
3.98 (q, 2H, H8, J= 5.3 Hz); 4.30 (q, 2H, H12, J= 5.3 Hz); 4.52
(s, 1H, H4); 7.23 - 7.38 (m, 5H, Harom); 8.54 (s, 1H, H11). 13C
NMR (DMSO-d6, 75 MHz): δ (ppm) = 14.0 (CH3); 14.1 (CH3); 18.6
(C10); 39.2 (C4); 60.6 (C8); 64.5 (C12); 81.7 (C3); 106.6 (C5); 117.7
(CN); 127.9 (C4’); 128.2 (C2’, 6’); 129.1 (C3’, 5’); 143.5 (C1’); 156.3
(C6); 157.7 (C11); 162.3 (C2); 165.7 (C7). ES-MS [M+H]+ calcd for
(C19 H21N2O4)+: 341.144, found: 341.151.
2.1.3.3. Ethyl 2,8-dimethyl-10-phenyl-10H-pyrano[3,2-e][1,2,4]triazolo
[1,5-c]pyrimidine-9-carboxylate (3c). White solid, yield: 61%, mp:
172-174°C. 1H NMR (CDCl3, 300 MHz): δ (ppm) = 1.21 (t, 3H, H13,
J= 6.9 Hz); 2.07 (s, 3H, H15); 2.16 (s, 3H, H14); 4.13 (q, 2H, H12,
J= 6.9 Hz); 5.57 (s, 1H, H10); 7.17 - 7.50 (m, 5H, Harom); 8.97 (s,
1H, H5). 13C NMR (CDCl3, 75 MHz): δ (ppm) = 14.5 (CH3); 19.0
(CH3); 37.2 (C14); 37.6 (C10); 60.3 (C12); 103.5 (C9); 107.3 (C10a);
127.7 (C4’); 128.1 (C3’, 5’); 128.4 (C2’, 6’); 137.6 (C5); 142.7 (C1’);
152.5 (C10b); 153.0 (C2); 159.8 (C8); 165.7 (C6a); 167.1 (C11). ES-
MS [M+H]+ calcd for (C19H19 N4O3)+: 351.138, found: 351.148.
2.1.2.2. Ethyl
5-cyano-6-((ethoxymethylene)amino)-4-(4-
(2b). White
methoxyphenyl)-2-methyl-4H-pyran-3-carboxylate
solid, yield: 69%, mp: 152-154°C. 1H NMR (CDCl3, 300 MHz): δ
(ppm) = 1.14 (t, 3H, H9, J= 7.2 Hz); 1.37 (t, 3H, H13, J= 7.2 Hz);
2.41 (s, 3H, H10); 3.79 (s, 3H, H14); 4.07 (q, 2H, H8, J= 7.2 Hz);
2.1.3.4. Ethyl
8-methyl-10-phenyl-10H-pyrano[3,2-e][1,2,4]triazolo
[1,5-c]pyrimidine-9-carboxylate (3d). White solid, yield: 54%, mp:
120-122°C. 1H NMR (CDCl3, 300 MHz): δ (ppm) = 1.22 (t, 3H, H13,
J= 6.9 Hz); 2.63 (s, 3H, H14); 4.14 (q, 2H, H12, J= 6.9 Hz); 5.55 (s,
1H, H10); 7.17 - 7.47 (m, 5H, Harom); 8.30 (s, 1H, H2); 9.10 (s, 1H,
H5). 13C NMR (CDCl3, 75 MHz): δ (ppm) = 13.5 (C13); 18.5 (C14);
37.5 (C10); 60.3 (C12); 103.1 (C9); 106.7 (C10a); 126.8 (C4’); 127.9
(C3’, 5’); 128.4 (C2’, 6’); 138.1 (C5); 142.1 (C1’); 151.7 (C10b); 152.4
(C2); 156.3 (C8); 159.1 (C6a); 165.3 (C11). ES-MS [M+H]+ calcd for
(C18 H17 N4O3)+: 337.1235, found: 337.1307.
ꢀ
ꢀ
4.38 (q, 2H, H12, J= 7.2 Hz); 4.53 (s, 1H, H4); 6, 85 (d, 2H, H3 ,5 ,
ꢀ
ꢀ
J= 8.7 Hz); 7, 17 (d, 2H, H2 ,6 , J= 8.7 Hz); 8, 23 (s, 1H, H11).
13C NMR (CDCl3, 75 MHz): δ (ppm) = 13.4 (CH3); 13.5 (CH3);
17.9 (C10); 39.3-39.4 (C4, C14); 60.2 (C8); 63.7 (C12); 82.6 (C3);
106.5 (C5); 113.5 (C3’, 5’); 116.9 (CN); 128.4 (C2’, 6’); 134.5 (C1’);
154.9 (C6); 156.8 (C11); 158.4 (C2); 158.5 (C4’); 165.4 (C7). ES-MS
[M+H]+ calcd for (C20H23N2O3)+: 371.1529, found: 371.1622.
2.1.2.3. Ethyl
4-(4-chlorophenyl)-5-cyano-6-((ethoxymethylene)
2.1.3.5. Ethyl
10-(4-methoxyphenyl)-8-methyl-2-phenyl-10H-pyrano
(3e). White
amino)-2-methyl-4H-pyran-3-carboxylate (2c). White solid, yield:
71%, mp: 95-97°C. 1H NMR (DMSO-d6, 300 MHz): δ (ppm) = 1.04
(t, 3H, H9, J= 5.3 Hz); 1.29 (t, 3H, H13, J= 5.3 Hz); 2.37 (s, 3H,
H10); 3.98 (q, 2H, H8, J= 5.3 Hz); 4.30 (q, 2H, H12, J= 5.3 Hz);
[3,2-e][1,2,4]triazol[1,5-c]pyrimidine-9-carboxylate
solid, yield 71%, mp: 171-173°C. 1H NMR (CDCl3, 300 MHz): δ
(ppm) = 1.23 (t, 3H, H13, J= 7.0 Hz); 2.64 (s, 3H, H14); 3.72 (s, 3H,
H15); 4.16 (q, 2H, H12, J= 7.0 Hz); 5.57 (s, 1H, H10); 6.81- 8.29
(m, 9H, Harom); 9.05 (s, 1H, H5). 13C NMR (CDCl3, 75 MHz): δ
(ppm) = 14.1 (C13); 19.1 (C14); 37.0 (C10); 55.2 (C15); 60.7 (C12);
104.2 (C9); 107.2 (C10a); 113.7 (C3’, 5’); 127.8 (C2’’, 6’’); 128.7 (C3’’,
5’’); 129.5 (C2’, 6’); 129.7 (C4”); 130.9 (C1’’); 135.1 (C1’); 138.1
ꢀ
ꢀ
4.56 (s, 1H, 1H, H4); 7.27 (d, 2H, H2 , 6 , J= 6.3 Hz); 7, 42 (d,
ꢀ
ꢀ
2H, H3 , 5 , J= 6.3 Hz); 8.54 (s, 1H, H11). 13C NMR (DMSO-d6, 75
MHz): δ (ppm) =14.1 (CH3); 14.2 (CH3); 18.6 (C10); 39.3 (C4); 60.8
(C8); 64.5 (C12); 81.2 (C3); 106.1 (C5); 117.5 (CN); 129.1 (C2’, 6’);
130.1 (C3’, 5’); 132.5 (C4’); 142.5 (C1’); 156.5 (C6); 158.2 (C11);
162.5 (C2); 165.5 (C7). ES-MS [M+H]+ calcd for (C19 H20ClN2O4)+:
375.1033, found: 375.1043.
ꢀ
(C5); 152.7 (C10b); 153.0 (C8); 158.7 (C4 ); 159.6 (C2); 166.0 (C6a);
167.1 (C11). ES-MS [M+H]+ calcd for (C25H23N4O4)+: 443.1659,
found: 443.1738.
2.1.3.6. Ethyl 10-(4-methoxyphenyl)-2,8-dimethyl-10H-pyrano[3,2-e]
2.1.3. General procedure for the synthesis of pyrimidine derivatives 3
0.7 mmol of iminoether 2 and 1.2 equivalents of hydrazide were
refluxed in dry dioxane (20 mL) in the presence of a few drops of
acetic acid. The solid formed by precipitation was filtered, washed
with petroleum ether, dried and purified by column chromatogra-
phy (CHCl3 /AcOEt: 9/1) to yield the compounds 3.
[1,2,4]triazolo[1,5-c]pyrimidine-9-carboxylate
(3f). White
solid,
yield: 52%, mp: 168-170°C. 1H NMR (CDCl3, 300 MHz): δ
(ppm) = 1.21 (t, 3H, H13, J= 5.2 Hz); 2.56 (s, 3H, H16); 2.60
(s, 3H, H14); 3.75 (s, 3H, H15); 4.13 (q, 2H, H12, J= 5.2 Hz); 5.47
ꢀ
ꢀ
ꢀ
ꢀ
(s, 1H, H10); 6.80 (d, H3 , 5 , J= 6.0 Hz); 7.36 (d, H2 , 6 , J= 6.0
Hz); 8.95 (s, 1H, H5). 13C NMR (CDCl3, 75 MHz): δ (ppm) = 14.0
(C13); 14.6 (CH3); 19.0 (CH3); 36.9 (C10); 55.2 (C15); 60.6 (C12);
104.0 (C9); 107.4 (C10a); 113.7 (C3’, 5’); 129.4 (C2’, 6’); 135.1
2.1.3.1. Ethyl 8-methyl-2,10-diphenyl-10H-pyrano[3,2-e][1,2,4]triazolo
[1,5-c]pyrimidine-9-carboxylate (3a). White solid, yield: 67%, mp:
220-222°C. 1H NMR (CDCl3, 300 MHz): δ (ppm) = 1.21 (t, 3H, H13,
J= 7.1 Hz); 2.65 (s, 3H, H14); 4.16 (q, 2H, H12, J= 7.1 Hz); 5.61 (s,
1H, H10); 7.18 - 8.29 (m, 10H, Harom); 9.04 (s, 1H, H5). 13C NMR
(CDCl3, 75 MHz): δ (ppm) = 14.0 (C13); 19.0 (C14); 37.9 (C10);
60.7 (C12); 104.0 (C9); 106.9 (C10a); 127.2 (C4’); 127.8 (C2’’, 6’’);
128.3 (C3’, 5’); 128.5 (C2’, 6’); 128.7 (C3’’, 5’’); 129.8 (C4’’); 130.8
(C1’’); 138.2 (C5); 142.7 (C1’); 152.7 (C10b); 153.1 (C8); 159.9 (C2);
165.9 (C6a); 167.2 (C11). ES-MS [M+H]+ calcd for (C24H21N4O3)+:
413.1554, found: 413.1620.
ꢀ
(C1’); 137.7 (C5); 152.6 (C10b); 152.8 (C2); 158.7 (C4 ); 159.4 (C8);
165.9 (C6a); 167.3 (C11). ES-MS [M+H]+ calcd for (C20H21N4O4)+:
381.1485, found: 381.1518.
2.1.3.7. Ethyl
10-(4-methoxyphenyl)-8-methyl-10H-pyrano[3,2-e]
[1,2,4]triazolo[1,5-c] pyrimidine-9-carboxylate (3g). White solid,
yield: 62%, mp: 166-168°C. 1H NMR (CDCl3, 300 MHz): δ
(ppm) = 1.23 (t, 3H, H13, J= 5.3 Hz); 2.61 (s, 3H, H14); 3.75
(s, 3H, H15); 4.15 (q, 2H, H12, J= 5.3 Hz); 5.49 (s, 1H, H10); 6.81
ꢀ
ꢀ
ꢀ
ꢀ
(d, H3 , 5 , J= 6.5 Hz); 7.35 (d, H2 , 6 , J= 6.5 Hz); 8.31 (s, 1H, H2);
3

A. Hajlaoui, M. Laajimi, M. Znati et al.
Journal of Molecular Structure 1237 (2021) 130346
9.10 (s, 1H, H5). 13C NMR (CDCl3, 75 MHz): δ (ppm) = 14.0 (C13);
19.0 (C14); 37.1 (C10); 55.1 (C15); 60.7 (C12); 104.6 (C9); 107.3
(C10a); 113.8 (C3’, 5’); 129.4 (C2’, 6’); 135.0 (C1’); 138.4 (C5); 152.1
Where Abscontrol corresponds to the absorbance of the solution
containing only α-amylase and the buffer instead of the com-
pound, and Abssample corresponds to the absorbance of the solution
in the presence of both tested compound and α-amylase. Com-
pound concentration providing 50% inhibition (IC50) was obtained
plotting the inhibition percentage against the concentrations of the
tested compound. The tests were carried out in triplicate.
ꢀ
(C10b); 152.8 (C2); 156.7 (C4 ); 158.7 (C8); 159.2 (C6a); 165.8
(C11). ES-MS [M+H]+ calcd for (C19H19 N4O4)+: 367.1328, found:
367.1362.
2.1.3.8. Ethyl 10-(4-chlorophenyl)-8-methyl-2-phenyl-10H-pyrano[3,2-
e][1,2,4]triazolo[1,5-c]pyrimidine-9-carboxylate (3h). White solid,
yield: 77%, mp: 218-220°C. 1H NMR (CDCl3, 300 MHz): δ
(ppm) = 1.23 (t, 3H, H13, J= 5.3 Hz); 2.65 (s, 3H, H14); 4.15 (q, 2H,
H12, J= 5.3 Hz); 5.58 (s, 1H, H10); 7.26 - 8.27 (m, 9H, Harom); 9.06
(s, 1H, H5). 13C NMR (CDCl3, 75 MHz): δ (ppm) = 14.0 (C13); 19.1
(C14); 37.4 (C10); 60.8 (C12); 103.4 (C9); 106.5 (C10a); 127.8 (C2’,
6’); 128.5 (C2’’, 6’’); 128.7 (C3’’, 5’’); 129.6 (C3’, 5’); 129.8 (C4’’);
131.0 (C4’); 133.1 (C1’’); 138.3 (C5); 141.2 (C1’); 152.6 (C10b); 152.9
(C8); 160.2 (C2); 165.7 (C6a); 167.2 (C11). ES-MS [M+H]+ calcd for
(C24H20ClN4O3)+: 447.1146, found: 447.1176.
2.3. Molecular docking procedure
The 3D crystal structure of human pancreatic α-amylase (HPA)
2QV4.pdb was retrieved from the RCSB Protein Data Bank (http:
//www.rcsb.org/pdb/home/home.do). Water molecules, hetatoms,
and other organic molecules were removed. The polar hydrogens
and Gasteiger charges were then added to the HPA structure.
The optimization of all chemical compounds structures was per-
formed with ACD (3D viewer) software (http://www.filefacts.com/
acd3d-viewer-freeware-info). The molecular docking analysis at
the HPA-binding site was carried out using autodock Vina software
˚
˚
[26,27]. Grid map of 40 × 40 × 40 A dimensions with 1A spacing
was prepared using AutoGrid. The docking results were further an-
alyzed using the Biovia Discovery Studio Visualizer v17.2.0.16349.
2.1.3.9. Ethyl
10-(4-chlorophenyl)-2,8-dimethyl-10H-pyrano[3,2-e]
[1,2,4]triazolo[1,5-c] pyrimidine-9-carboxylate (3i). White solid,
yield: 56%, mp: 134-136°C. 1H NMR (CDCl3, 300 MHz): δ
(ppm) = 1.22 (t, 3H, H13, J= 5.3 Hz); 2.57 (s, 3H, H15); 2.62
(s, 3H, H14); 4.15 (q, 2H, H12, J= 5.3 Hz); 5.50 (s, 1H, H10); 7.25
2.4. Computational details (DFT studies)
ꢀ
ꢀ
ꢀ
ꢀ
(d, H2 , 6 , J= 6.3 Hz); 7.40 (d, H3 , 5 , J= 6.3 Hz); 8.98 (s, 1H,
H5). 13C NMR (CDCl3, 75 MHz): δ (ppm) = 14.0 (C13); 14.6 (CH3);
19.1 (CH3); 37.2 (C10); 60.8 (C12); 103.0 (C9); 106.8 (C10a); 128.6
(C2’, 6’); 129.7 (C3’, 5’); 133.1 (C4’); 138.0 (C5); 141.2 (C1’); 152.5
(C10b); 152.8 (C2); 160.0 (C8); 165.6 (C6a); 167.3 (C11). ES-MS
[M+H]+ calcd for (C19H18 ClN4O3)+: 385.0989, found: 385.0996.
The entire set of calculations have been performed using DFT-
B3LYP method with 6-311++G(d,p) basis set using Gaussian 09
program package [28] and GaussView visualization program [29].
HOMO–LUMO gaps of each compound were calculated by subtract-
ing the LUMO energy value from the corresponding HOMO energy
value. Different parameters have been determined such as chemi-
cal potential (μ), global hardness (η), global softness (S) and elec-
trophilicity (ɷ) according to the following equations [30,31]:
2
2.1.3.10. Ethyl
10-(4-chlorophenyl)-8-methyl-10H-pyrano[3,2-e]
[1,2,4]triazolo[1,5-c] pyrimidine-9-carboxylate (3j). White solid,
yield: 51%, mp: 188-190°C. 1H NMR (CDCl3, 300 MHz): δ
(ppm) = 1.23 (t, 3H, H13, J= 5.2 Hz); 2.63 (s, 3H, H14); 4.15
∼
=
∼
=
μ
(EHOMO + ELUMO)/2; η ELUMO − EHOMO; S = 1/η; ω = μ /2η
3. Results and discussion
ꢀ
ꢀ
(q, 2H, H12, J= 5.2 Hz); 5.53 (s, 1H, H10); 7.26 (d, H2 ,6 , J= 6.1
ꢀ
ꢀ
Hz); 7.39 (d, H3 ,5 , J= 6.1 Hz); 8.31 (s, 1H, H2); 9.12 (s, 1H, H5).
13C NMR (CDCl3, 75 MHz): δ (ppm) = 14.0 (C13); 19.0 (C14); 37.4
(C10); 60.9 (C12); 104.4 (C9); 106.8 (C10a); 128.6 (C2’, 6’); 129.7
(C3’, 5’); 133.2 (C4’); 138.6 (C5); 141.1 (C1’); 152.0 (C10b); 152.8
(C2); 156.7 (C8); 165.5 (C6a); 167.4 (C11). ES-MS [M+H]+ calcd for
(C18 H16 ClN4O3)+: 371.0833, found: 371.0846.
3.1. Chemistry
α-aminocarbonitriles constitute key precursors often used by
chemists for the synthesis of a wide range of nitrogen molecules
due to their high reactivity. Thereby, 2-amino- 3-cyanopyrane 1
was chosen as our starting material. It was prepared via one-pot
three-component reaction of arylaldehyde, malononitrile, and ethyl
acetoacetate according to some previous work [32]. Subsequently,
our strategy for the target systems 3 was initially based on the
construction of functionalized iminoether 2, through the condensa-
tion reaction of precursor 1 with triethylorthoformate under reflux
of acetic anhydride (Scheme 1).
2.2. In vitro α-amylase inhibition assay
α-Amylase inhibitory activity of synthesized compounds was
determined using the assay method described by Kwon, Aposto-
lidis and Shetty [24,25]. Briefly, 500 μL of each test sample at dif-
ferent concentrations was incubated with 500 μL of α-amylase so-
lution (0.5 mg/mL in 0.02 M sodium phosphate buffer; pH 6.9 with
0.006 M NaCl). After pre-incubation at 25°C for 10 min, 500 μL of
starch solution (1%) in 0.02 M sodium phosphate buffer (pH 6.9
with 0.006 M NaCl) was added to each tube. The resulting mix-
tures were then incubated at room temperature (10 min), and the
reaction was stopped using 1 mL of DNS (Dinitro salicylic acid)
reagent. At this time, the test tubes were placed in a water bath
(100°C) for 5 min and cooled until room temperature was attained.
The mixture was then diluted with 10 mL of deionized water, and
the absorbance was determined at 540 nm. The readings were
compared with the control, containing buffer instead of test com-
pounds. Acarbose was used as a positive control. The inhibition of
α-amylase was calculated using the following equation:
Structures of compounds 1 and 2 were confirmed based on
their spectral data. The 1H NMR spectra of compounds 1 indicated
essentially the presence of two singlets related to H4 (δH 4.30-
4.44) and NH2 protons (δH 4.54-6.90). While examining the spectra
of derivatives 2, we observed the disappearance of amino group
protons, the appearance of a singlet relative to the imidic pro-
ton and the presence of signals corresponding to a second ethoxy
group. The 13C NMR spectra of these precursors were in agreement
with the proposed structures. Hence, the analysis of 13C NMR spec-
tra of iminoethers 2 showed mainly the appearance of new signals
relative to carbons C13, C12, and the imidic carbon C11
.
Therefore, a series of new pyrano-triazolo-pyrimidine deriva-
tives 3a-j was prepared by cyclocondensation reaction of the in-
termediate 2 with appropriate hydrazides in the presence of a cat-
alytic amount of acetic acid at reflux of dioxane (Scheme 2). Plausi-
ble pathway implies two successive nucleophilic additions of -NH2
group on the imidic carbon and on the nitrile function supervened
ꢀ
ꢁ
%inhibition of α − Amylase = Abscontrol−Abssample
/
Abs
×100
)
(
control
4

A. Hajlaoui, M. Laajimi, M. Znati et al.
Journal of Molecular Structure 1237 (2021) 130346
Scheme 1. Synthetic pathway to precursors 1 and 2.
Scheme 2. Synthetic pathway to derivatives 3a-j.
ion peaks [M+H] +, which were compatible with the target struc-
Table 1
Scope of hydrazides and the Ar functional group on 2 to synthesize compounds 3.
tures.
Ar
R1
Yield (%)
3.2. In silico pharmacokinetic prediction
3a
3b
3c
3d
3e
3f
Ph
Ph
67
58
61
54
71
52
62
77
56
51
Ph
CH2CN
CH3
H
Ph
Pharmacokinetic (PK) properties are considered important fac-
tors in drug development since they define the characteristics of
an effective oral drug. PKs are mainly related to the chemical de-
scriptors of the molecule. Computer predictions are currently used
in drug discovery programs to investigate an ADME profiling of
new drug candidates to select only drug-like compounds with op-
timal PK [33]. Several physicochemical properties related to the
pharmacokinetics of synthesized derivatives, were predicted for
their compliance with Lipinski’s rule of five using Molinspiration
online software and SwissADME web tool [34–36]. As shown in
Table 2, the prepared compounds were found to have Log P values
less than five, implying good permeability across the cell mem-
brane. It is one of the principal parameters for the estimation of
lipophilicity of chemical compounds [20,37]. All the titled deriva-
tives have weighted less than 500 and thereby predicting their
easy transportation, absorption, and diffusion. It was observed also
that the number of hydrogen bond acceptors and the number of
hydrogen bond donors were less than 10 and 5, respectively. In-
terestingly, it can be anticipated that all products have good oral
bioavailability as they obeyed Lipinski’s rule of five [20]. The topo-
logical polar surface area (TPSA) is another significant factor in
Ph
4-OCH3Ph
4-OCH3Ph
4-OCH3Ph
4-ClPh
4-ClPh
4-ClPh
Ph
CH3
H
3g
3h
3i
Ph
CH3
H
3j
by dehydrocyclization to obtain new heterocycles incorporating the
pyran, triazole and pyrimidine nuclei 3 (Table 1).
Structures of the above compounds were established by spec-
troscopic methods. The 1H NMR spectra of these compounds
showed the disappearance of signals relative to the ethoxy group
of the α-functionalized iminoether 2 and the presence of a sin-
glet (8.95 - 9.12 ppm) corresponding to the imidic proton. The 13C
NMR spectra were also in agreement with the proposed structures
and proved the presence of signals relative to C5, C10b carbons and
those provided by the hydrazide used, in particular, carbon C2. Fur-
ther, the high-resolution mass spectra (ESI-HRMS) of all synthe-
sized compounds have revealed the correct protonated molecular
5

A. Hajlaoui, M. Laajimi, M. Znati et al.
Journal of Molecular Structure 1237 (2021) 130346
Table 2
Prediction of physicochemical properties of the target compounds.
Lipinski’s
TPSAa
n-rotbb
MWc
MVd
LogPe
n-OHNHf
n-ONg
violation
Rule
1a
1b
1c
2a
2b
2c
3a
3b
3c
3d
3e
3f
<500
<5
<5
2
2
2
0
0
0
0
0
0
0
0
0
0
0
0
0
<10
5
<1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
85.35
94.59
85.35
80.93
90.16
78.63
78.63
102.42
78.63
78.63
87.86
87.86
87.86
78.63
78.63
78.63
4
5
4
7
8
7
5
5
4
4
6
5
5
5
4
4
284.31
314.34
318.76
340.38
370.40
374.82
412.45
375.39
350.38
336.35
442.48
380.40
366.38
446.89
384.82
370.80
259.89
285.43
273.42
314.21
339.75
327.74
364.03
326.28
309.18
292.62
389.58
334.73
318.17
377.57
322.72
306.16
2.96
3.02
3.64
3.77
3.82
4.45
4.88
2.84
2.85
2.77
4.94
2.91
2.82
4.89
3.53
3.45
6
5
6
7
6
7
8
7
7
8
8
3g
3h
3i
8
7
7
3j
7
a
Topological polar surface area: TPSA.
Num. of rotatable bonds: n-rotb.
Molecular weight: MW.
b
c
d
e
f
Molecular volume: MV.
Logarithm of partition coefficient between n-octanol and water: LogP.
Num. of hydrogen bond donors: n-OHNH.
g
Num. of hydrogen bond acceptors: n-ON.
Table 3
Predicted ADME properties of the target compounds.
Compounds
BBB
PPB (%)
HIA (%)
Caco-2 (nm/sec)
hERG inhibition
1a
1b
1c
2a
2b
2c
3a
3b
3c
3d
3e
BBB +
BBB +
BBB +
BBB +
BBB +
BBB +
BBB +
BBB +
BBB +
BBB +
BBB +
BBB +
BBB +
BBB +
BBB +
BBB +
76.82
77.20
88.49
83.52
81.73
87.09
99.13
91.62
86.81
89.30
97.22
85.80
89.83
93.25
88.74
89.95
95.85
94.85
97.46
98.51
98.04
99.14
97.92
98.57
99.07
98.78
98.48
99.26
99.14
97.58
98.53
98.69
19.89
22.02
21.37
37.73
43.20
37.48
41.77
37.79
34.60
33.80
45.82
42.12
36.63
34.24
29.05
27.57
WI
WI
WI
WI
WI
WI
WI
WI
WI
WI
WI
WI
WI
WI
WI
WI
3f
3g
3h
3i
3j
∗∗Blood-Brain Barrier (BBB): don’t cross BBB (-); cross BBB (+),
Plasma protein binding (PPB): > 90 (strongly bound)
Human intestinal absorption (HIA, %): 70-100% (well absorbed)
Caco-2 cell permeability: < 4 (low permeability), 4-70 (medium permeability)
hERG inhibition: Human ether-a-go-go related gene channel inhibition; WI: Weak inhibitor
determining the molecular absorption rate. The number of rotat-
able bonds is also considered a good descriptor for suitable drugs.
parameters as mutagenicity, tumorigenicity, irritation, and repro-
ductive or developmental toxicity of the synthesized components
were estimated by OSIRIS Data Warrior [40]. Toxicity risk predic-
tion softwares locate fragments within a molecule that represents
a potential toxicity risk. The results indicated that none of the pre-
sented compounds contained any toxic subcomponents, which en-
sure the safety of all compounds (Table 4). From these various pa-
rameters, it can be concluded that the synthesized derivatives have
shown strong drug-like performance and could be considered as
promising candidates for further development.
Their values for all compounds were in the normal range (TPSA
2
˚
values <140 A and n-rotb <10, as given in Table 2, according to
Veber’s rule [38]). ADME profile of synthesized derivatives was de-
termined using PreADMET database. As indicated in Table 3, the
absorption efficacy of all derivatives was found to be very high
(94.85-99.26%) via the human intestinal tract. They have shown
also medium permeability in the Caco-2 cell model and a posi-
tive result for blood-brain barrier crossing ability. Besides, the be-
havior of the drug depends on absorption and distribution which
can be assessed by Plasma Protein Binding. Here, the molecules
have displayed good binding affinity, especially compounds 3a, 3b,
3e, and 3h. Moreover, inhibition of potassium channels by the hu-
man ether-a-go-go related gene (hERG) leads mainly to QT syn-
drome with fatal ventricular arrhythmia [39]. In the present study,
all prepared compounds showed a weak inhibitory property to the
human hERG and are considered to be relatively safer. Toxicity
3.3. In vitro α-amylase inhibitory activity
All the synthesized compounds (1a-c), (2a-c), and (3a-j) were
subjected to in vitro α-amylase activity. The results, expressed in
IC50 (μg/mL), are illustrated in the Table 5. Seven compounds
2a, 2b, 3a, 3b, 3c, 3e, and 3h exhibited interesting α-amylase
inhibitory activity (IC50 = 9.58
0.17 - 2.78
0.14 μg/mL)
6

A. Hajlaoui, M. Laajimi, M. Znati et al.
Journal of Molecular Structure 1237 (2021) 130346
Table 4
Computational Toxicity Risk parameters.
1a
1b
1c
2a
2b
2c
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
Mut
Tum
Irri
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Rep
Low
Low
Low
Low
Low
Mut: Mutagenicity, Tumo: Tumorigenicity, Irri: Irritation, Rep: Reproductive or developmental toxicity.
Table 5
that the seven synthesized ligands displayed a various type of in-
termolecular interactions with the significant residues of the ac-
tive site (Trp 58, Trp 59, Tyr 62, Gln 63, Tyr 151, Leu 162, Leu 165,
Asp 197, Ala 198, Lys 200, His 201, Glu 233, Ile 235, Asp 300 and
His 305) of the target enzyme [41]. In the first instance, we re-
docked the co-crystallized ligand (acarbose) in the binding site of
α-amylase with the same parameters as used for the docking of
synthesized ligands. The co-crystallized ligand showed seven hy-
drogen bond interactions with the amino-acid residues Trp 59, Ala
106, Gly 164, Asp 197, Asp 300, and His 305 of the target enzyme
(Fig. 3). The value of the binding energy of acarbose was found
to be −7.6 kcal/mol. Analysis of the best-docked poses of imi-
noethers 2a and 2b revealed that they mainly exhibited hydrogen
and hydrophobic interactions. As shown in figure S1, the ethoxy
group’s oxygen linked to the imine function and the pyran ring of
compound 2a have established conventional and Pi-Donor hydro-
gen bonds with His 305 and Tyr 151, respectively. Additionally, the
pyran, phenyl ring, and methyl group of conjugate 2a have inter-
acted through hydrophobic interactions (Pi-Sigma, Pi-Pi T-shaped,
and Pi-Alkyl) with Ile 235, Tyr 151, His 201, Leu 162, Lys 200,
and Ala 198. It was noteworthy that the pyran nucleus, in com-
pound 2b, was involved in an electrostatic interaction with Asp
300, which can play an essential role in stabilizing the inhibitor
in the active site.
α-Amylase inhibitory activity of the synthesized compounds.a
Compound
IC50 (μg/mL)
1a
92.17
42.29
98.15
3.15
1.52i
1.10f
2.42h
0.25ab
0.17ab
3.74j
0.15d
0.14a
0.10b
2.50i
0.17d
1.11e
2.88h
0.09c
3.08h
2.12g
1.22c
1b
1c
2a
2b
2c
3.28
96.00
9.27
3a
3b
3c
2.78
4.15
3d
3e
92.27
9.58
3f
13.00
85.07
6.31
3g
3h
3i
99.07
56.27
6.84
3j
Acarbose
a
Values given are the mean
standard error. Different let-
ters denote significant differences in IC50 values. Statistical sig-
nificance was accepted at P < 0.05. IC50 was calculated using
graph pad prism software.
compared to acarbose (IC50 = 6.84
1.22 μg/mL). Compound 3b
(Ar= Ph and R1= CH2CN) was found to be the most potent ana-
Further, compound 3b demonstrated the highest activity in the
series, displayed numerous strong interactions through residues
that are present on the side chain of the active site of α-amylase,
exhibiting binding energy -8.0 kcal/mol. Interestingly, the amino
acid residues Gln 63 has formed Hydrogen donor interaction with
log in this series (IC50 = 2.78
0.14 μg/mL). The rest of com-
pounds displayed moderate to weak activity. It has been found
that the iminoethers 2a and 2b exhibited good and comparable
inhibitory potency with a IC50 values of 3.15
0.17 μg/mL, respectively, while the starting compounds 1a and 1b
displayed relatively less activity (IC50 = 92.17 1.52 and 42.29
0.25 and 3.28
˚
the cyano-group nitrogen (3.00 A), which stabilizes its binding
affinity with the target receptor, and may therefore be responsi-
ble for its significant in vitro activity. This product was also found
to exhibit crucial hydrophobic contacts: three Pi-Alkyl, a Pi-Pi T-
shaped, and three alkyl interactions with His 201, Leu 162, Leu 165,
Tyr 62, Ala 198, Leu 162, Ile 235, respectively (Fig. 4).
1.10 μg/mL, respectively). This implies the importance of modify-
ing the primary amine function to an imine. Regarding the series
3a-j, the first suggestion is that the incorporation of the pyrimidine
and triazole rings has significantly improved the activity compared
to the starting reagents (1a-c) which showed limited inhibition of
the enzyme. The nature of the aryl moiety and the substituents on
the triazole ring (R1) may influence the inhibitory potential. The
results showed that in the series 3a-d (Ar= Ph), the highest activ-
ity was noted when R1= CH2CN (case of 3b). For the series 3e-
3g (Ar= 4-OCH3Ph), the activity is better with R1 = Ph (case of
Binding manner of 3c, the second most active derivative in
the series 3, revealed that this compound was involved in numer-
ous interactions including Hydrogen Bond, Pi-anion, Pi-sigma, Pi-
Pi T-Shaped, and Pi-alkyl interactions with the side chain residues
Tyr 151, Glu233, Ile235, His201, Lys200, and Leu162. These in-
teractions could be the main reason for stabilizing the inhibitor-
receptor complex and making the derivative second most active
analog among the series. Similarly, hydrophobic interactions and
hydrogen bindings (especially with residue Lys 200), were found as
well in the case of compounds 3a, 3e, and 3h. Electrostatic bonds
were also observed, in the conjugates 3a and 3h, with residues
Asp 197 and Glu 233 (Fig. S2, 3h as an example) that may explain
the lower binding energy values of these compounds compared to
acarbose: -8.9 and -9.2 Kcal/mol, respectively (Table 6).
3e, IC50 = 9.58
ClPh), the most active derivative was found to be 3h with R1= Ph
0.09 μg/mL).
0.17 μg/mL) and for the series 3h-3j (Ar= 4-
(IC50 = 6.31
The presence of a CH2CN group linked to the triazole ring in 3b
could better facilitate its interaction with the enzyme compared
to the phenyl, methyl and hydrogen in the rest derivatives. Such
results highlight the importance of the nature of the substituent
carried by the triazole.
3.4. Molecular docking analysis
3.5. Density functional theory (DFT)-based computations: frontier
molecular orbital analysis
To predict the putative binding mode of the active derivatives
with the target protein α-amylase, a docking study was carried
out into the active site of co-crystal structure PDB: 2QV4, using
Autodock Vina. The docking results of tested inhibitors revealed
DFT-based computation could potentially be an effective tool
in computer-aided drug design and thus, becoming an attractive
method for medicinal studies [42]. The energies of frontier molec-
7

A. Hajlaoui, M. Laajimi, M. Znati et al.
Journal of Molecular Structure 1237 (2021) 130346
Fig. 3. 3D-representation of the docked pose of the co-crystallized ligand (Acarbose) in the active site of α-amylase.
Fig. 4. Predicted binding modes of conjugate 3b in the active site of α-amylase (PDB: 2QV4).
Table 6
molecule. Molecules with a larger EHOMO-LUMO band gap should
be more stable than those having a smaller gap [44]. As indicated
in Table 7, 3a, 3e, and 3h show the three lowest energy gap val-
ues (2.901, 2.918, and 2.912 eV, respectively), suggesting that they
are more reactive and less stable compared to the rest of the com-
pounds.
Binding Energies of tested compounds with α-amylase
enzyme (2QV4).
Compound
Binding energy (kcal/mol)
2a
-7.7
-7.4
-8.9
-8.0
-7.4
-9.4
-9.2
-7.6
2b
3a
The HOMO and LUMO distributions of the active derivatives (2a,
2b, 3a, 3b, 3c, 3e, and 3h) are illustrated in Figs. 5, S3, and S4.
The localization pattern of frontier molecular orbitals in different
positions demonstrates their structural and functional diversity.
As can be seen from figures (5, S3 and S4), strong delocaliza-
tion of the HOMO for compounds 2a and 2b occurs on the pyranic
nucleus, nitrile, and imine groups (donors moieties) while strong
delocalization of the LUMO occurs on the benzene ring (acceptor
moiety). Further, strong delocalization of the HOMO for the other
compounds occurs on the pyranotriazolopyrimidine donor moiety
while strong delocalization of the LUMO occurs on the benzene
ring bonded to triazole (3a, 3e, and 3h) and on the benzene ring
attached to pyran (3b and 3c). Thus, the lowering of the HOMO-
LUMO E-gap is mainly a consequence of the high stabilization of
the LUMO owing to the high electron-accepting capacity of the
electron acceptor group.
3b
3c
3e
3h
Acarbose
ular orbitals are significant parameters in various chemical and
pharmacological processes.
The highest occupied molecular orbital energy EHOMO mea-
sures the electron-donating character of a compound, while the
lowest unoccupied molecular orbital energy ELUMO assesses its
electron-accepting character. HOMO and LUMO orbitals can be
used to explain different types of reactions and predict the most
reactive position in conjugated systems [43]. The energy gap be-
tween HOMO and LUMO is responsible for the stability of the
8

A. Hajlaoui, M. Laajimi, M. Znati et al.
Journal of Molecular Structure 1237 (2021) 130346
Table 7
DFT/B3LYP/6–311++G(d,p) calculations of HOMO-LUMO energy gap, chemical potential, global hardness, global softness,
electrophilicity index and dipole moment of compounds 1, 2 and 3.
E
HOMO(eV)
E
LUMO(eV)
࢞E(eV)
μ (eV)
H(eV)
S (eV)−1
ɷ(eV)
D. M (Debye)
1a
1b
1c
2a
2b
2c
3a
3b
3c
3d
3e
3f
-8.135
-8.124
-8.017
-8.241
-8.240
-8.040
-7.975
-7.991
-7.992
-8.011
-7.978
-7.993
-8.012
-7.976
-7.991
-8.010
-4.725
-4.622
-4.630
-4.838
-4.711
-4.728
-5.073
-4.763
-4.760
-4.762
-5.060
-4.656
-4.646
-5.064
-4.654
-4.660
3.410
3.501
3.384
3.403
3.526
3.311
2.901
3.230
3.232
3.246
2.918
3.339
3.365
2.912
3.337
3.350
-6.430
-6.373
-6.324
-6.541
-6.476
-6.384
-6.524
-6.376
-6.375
-6.386
-6.519
-6.325
-6.329
-6.521
-6.322
-6.335
3.410
3.501
3.384
3.403
3.526
3.311
2.901
3.230
3.232
3.246
2.918
3.339
3.365
2.912
3.337
3.350
0.292
0.286
0.294
0.294
0.282
0.302
0.345
0.310
0.308
0.308
0.343
0.299
0.297
0.342
0.299
0.297
6.063
5.801
5.906
6.286
5.945
6.152
7.331
6.295
6.290
6.280
7.281
5.990
5.944
7.296
5.989
5.988
6.882
7.620
7.502
4.349
3.392
4.948
2.285
5.566
2.229
3.026
3.088
2.223
3.907
2.946
3.019
3.739
3g
3h
3i
3j
Fig. 5. Visualization of frontier molecular orbitals of 3a, 3e and 3h with DFT/B3LYP method using 6-311++G(d,p) basis set.
Moreover, HOMO-LUMO energy values are the basis for calcu-
the global electrophilicity index provides information about stabil-
ity (hardness) and also the electron transfer (chemical potential)
and is an effective descriptor of global chemical reactivity. It is use-
ful for explaining the binding capacity with biomolecules [48,49].
The higher electrophilicity index of studied compounds (especially
3a (7.331 eV), 3e (7.281 eV), and 3h (7.296 eV)), will have higher
binding interactions.
lating some global chemical reactivity parameters such as chemical
potential (μ), global hardness (η), global softness (S), electrophilic-
ity index (ω) and dipole moment (D.M). These quantum chemical
descriptors, computed at B3LYP/6-311++G(d,p) level, are presented
in Table 7.
Therefore, the energy gap of HOMO–LUMO is used to determine
the hardness and the softness of the molecules. Large gaps denote
a hard molecule and small gaps signify a soft molecule. Softness
is defined as the reciprocal of hardness and hence, the reactivity
of a molecule rises with the increase in the softness [45]. 3a, 3e,
and 3h show the lowest values of chemical hardness and energy
gap, and so, the three highest softness values (0.345, 0.343, and
0.342 eV, respectively), proving that they are softer and most reac-
tive species.
4. Conclusion
In this paper, we have synthesized a series of new pyranotri-
azolopyrimidine 3a-j by reacting the α-functionalized-iminoether
2, previously prepared from α-aminocarbonitrile 1, with a series
of hydrazides. Pharmacokinetics and toxicological predictions have
revealed their drug-like properties, presenting good ADMET prop-
erties and showing good oral bioavailability as they obeyed Lipin-
ski’s rule of five. The newly prepared compounds 1-3 have been
tested for in vitro α-amylase activity. Among them, 2a, 2b, 3a, 3b,
3c, 3e, and 3h exhibited interesting α-amylase inhibitory activity,
The dipole moment is another significant electronic parameter
that is due to the non-uniform distribution of charges on the dif-
ferent atoms in a given molecule [46]. It is an indicator of drug-
receptor interaction, which facilitates hydrogen-bond interactions
[33,47]. All synthesized compounds show comparatively higher
dipole moments varying from 2.223 to 7.620 Debey. The values of
the dipole moment promote intermolecular interactions. Besides,
and 3b was found to be the most active one (IC50= 2.78
0.14
mg/mL). In silico studies were performed to gain insight toward the
interactions of title compounds with the target protein. The bind-
9

A. Hajlaoui, M. Laajimi, M. Znati et al.
Journal of Molecular Structure 1237 (2021) 130346
ing modes of active compounds showed that hydrophobic and hy-
drogen bonds interactions with important residues such as Gln 63,
Tyr 151, Leu 162, Leu 165, Asp 197, Ala 198, Lys 200, His 201, Glu
233, Ile 235, Asp 300, and His 305, appeared significant for high
potency. A theoretical DFT/B3LYP study has been carried out to ex-
plain the reactivity of the different compounds. The use of the re-
activity parameters of HOMO-LUMO energies has been discussed.
As we saw, 3a, 3e, and 3h showed the lowest values of chemi-
cal hardness, energy gap, and the three highest softness values, in
comparison with the other molecules, making them the most reac-
tive species in the series. This study will be useful for further lead
optimization and developing new α-amylase inhibitors.
[12] P. Yadav, K. Lal, L. Kumar, A. Kumar, A. Kumar, A.K. Paul, R. Kumar, Designed
chalcone-1,2,3-triazole conjugates as potential antimicrobial agents synthe-
sis, crystal structure and antimicrobial potential of some fluorinated chal-
cone-1,2,3-triazole conjugates, Eur. J. Med. Chem. 155 (2018) 263–274.
[13] A. Maleki, Z. Varzi, F. Hassanzadeh-Afruzi, Preparation and characterization of
an eco-friendly ZnFe2O4@alginic acid nanocomposite catalyst and its applica-
tion in the synthesis of 2-amino-3-cyano-4H-pyran derivatives, Polyhedron 171
(2019) 193–202.
[14] D. Kumar, P. Sharma, H. Singh, K. Nepali, G.K. Gupta, S.K. Jaina, F. Ntie-Kang,
The value of pyrans as anticancer scaffolds in medicinal chemistry, RSC Adv. 7
(2017) 36977–36999.
[15] M. Zayane, A. Rahmouni, M.D. Remadi, M. Ben Mansour, A. Romdhane,
H.Ben Jannet, Design and synthesis of antimicrobial, anticoagulant, and anti-
cholinesterase hybrid molecules from 4-methylumbelliferone, J. Enzyme Inhib.
Med. Chem. 31 (2016) 1566–1575.
[16] Y. Bouazizi, A. Romdhane, H.Ben Jannet, Synthesis of new 3,4-dihydropy-
rano[c]chromene derivatives and their evaluation as acetyl cholinesterase in-
hibitors, Eur. J. Chem. 5 (2014) 457–462.
Author Statement
[17] B. Maleki, M. Baghayeri, S.A.J. Abadi, R. Tayebee, A. Khojastehnezhad, Ultra-
sound Promoted Facile One Pot Synthesis of Highly Substituted Pyran Deriva-
tives Catalyzed by Silica-Coated Magnetic NiFe2O4 Nanoparticles-Supported
Amel Hajlaoui did the synthesis work and the Docking party.
Maha Laajimi performed the DFT study. Mansour Znati tested the
α-amylase activity. Hichem Ben Jannet contributed to the discus-
sion of the results and completed the redaction of the manuscript.
Anis Romdhane was the supervisor of the present work, he also
checked the structure determination of the synthesized com-
pounds and the biological study, and he completed the redaction
of the article.
H
14 [NaP5W30O110 ] under Mild Conditions, RSC Adv 6 (2016) 96644–96661.
[18] S. Wang, Q. Qi, C. Li, G. Ding, S.H. Kim, Photoswitching of bisthienylethene
using 2D-π-A type pyran-based fluorescent dye for rewritable optical storage,
Dyes Pigm 89 (2011) 188–192.
[19] S. Hameed, F.Seraj Kanwal, R. Rafique, S. Chigurupati, Abdul Wadood, A. Ur
Rehman, V. Venugopal, U. Salar, M. Taha, K.M. Khan, Synthesis of benzotria-
zoles derivatives and their dual potential as α-amylase and α-glucosidase in-
hibitors in vitro: Structure-activity relationship, molecular docking, and kinetic
studies, Eur. J. Med. Chem. 183 (2019) 111677.
[20] P.A. Channar, A. Saeed, F.A. Larik, S. Rashid, Q. Iqbal, M. Rozi,
S. Younis, J. Mahar, Design and synthesis of 2,6-di(substituted
phenyl)thiazolo[3,2-b]-1,2,4-triazoles as α-glucosidase and α-amylase in-
hibitors, co-relative Pharmacokinetics and 3D QSAR and risk analysis, Biomed.
Pharmacother. 94 (2017) 499–513.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
[21] H. Kashtoh, M.T. Muhammad, J.J.A. Khan, S. Rasheed, A. Khan, S. Perveen,
K. Javaid, K.M.Khan Atia-tul-Wahab, M.I. Choudhary, Dihydropyrano [2,3-c]
pyrazole: Novel in vitro inhibitors of yeast α-glucosidase, Bioorg. Chem. 65
(2016) 61–72.
[22] A. Yousefi, R. Yousefi, F. Panahi, S. Sarikhani, A. Zolghadr, A. Bahaoddini,
A. Khalafi-Nezhad, Novel curcumin-based pyrano[2,3-d]pyrimidine anti-ox-
idant inhibitors for α-amylase and α-glucosidase: Implications for their
pleiotropic effects against diabetes complications, Int. J. Biol. Macromol. 78
(2015) 46–55.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130346.
[23] M. Amirnejad, M.R. Naimi-Jamal, H. Tourani, H. Ghafuri, A facile solvent-free
one-pot three-component method for the synthesis of 2-amino-4H-pyrans and
tetrahydro-4H-chromenes at ambient temperature, Monatsh. Chem. 144 (2013)
1219–1225.
References
[24] Y.-I. Kwon, E. Apostolidis, K. Shetty, In vitro studies of eggplant (Solanum me-
longena) phenolics as inhibitors of key enzymes relevant for type 2 diabetes
and hypertension, Bioresour. Technol. 99 (2008) 2981–2988.
[25] Z. Toobaei, R. Yousefi, F. Panahi, S. Shahidpour, M. Nourisefat, M.M. Dorood-
mand, A. Khalafi-Nezhad, Synthesis of novel poly-hydroxyl functionalized acri-
dine derivatives as inhibitors of α-Glucosidase and α-Amylase, Carbohydr. Res.
411 (2015) 22–32.
[1] G. Wang, Z. Peng, J. Wang, X. Li, J. Li, Synthesis, in vitro evaluation and
molecular docking studies of novel triazine-triazole derivatives as potential
α-glucosidase inhibitors, Eur. J. Med. Chem. 125 (2017) 423–429.
[2] H. Kashtoh, M.T. Muhammad, J.J.A. Khan, S. Rasheed, A. Khan, S. Perveen,
K. Javaid, K.M.Khan Atia-tul-Wahab, M.I. Choudhary, Dihydropyrano [2,3-c]
pyrazole: Novel in vitro inhibitors of yeast α-glucosidase, Bioorg. Chem. 65
(2016) 61–72.
[3] R. Tundis, M.R. Loizzo, F. Menichini, Natural Products as α-Amylase and
α-Glucosidase Inhibitors and their Hypoglycaemic Potential in the Treatment
of Diabetes: An Update, Mini-Rev. Med. Chem. 10 (2010) 315–331.
[4] R.R. Ruddarraju, G. Kiran, A.C. Murugulla, R. Maroju, D.K. Prasad, B.H. Ku-
mar, V. Bakshi, N.S. Reddy, Design, synthesis and biological evaluation of
theophylline containing variant acetylene derivatives as α-amylase inhibitors,
Bioorg. Chem. 92 (2019) 103–120.
[26] O. Trott, A.J. Olson, Software News and Update AutoDock Vina: Improving the
Speed and Accuracy of Docking with a New Scoring Function, Efficient Opti-
mization, and Multithreading, J. Comput. Chem. 31 (2010) 455–461.
[27] http://autodock.scripps.edu/
[28] M.J. Frisch, G.W. Trucks, H.B. Schlegel, et al., Gaussian 09, Revision B.01, Gaus-
sian, Inc., Wallingford, CT, 2010.
[29] R.I. Dennington, T. Keith, J. Millam, GaussView, Version 5.0.8, Semichem. Inc.
Shawnee Mission, KS, 2008.
[30] S. Chortani, H. Edziri, M. Manachou, Y.O. Al-Ghamdi, S.G. Almalki,
Y.E. Alqurashi, H.Ben Jannet, A. Romdhane, Novel 1,3,4-oxadiazole linked
benzopyrimidinones conjugates: Synthesis, DFT study and antimicrobial
evaluation, J. Mol. Struct. 1217 (2020) 128357.
[5] M.S. Ganesan, K.K. Raja, K. Narasimhan, S. Murugesan, B.K. Kumar, Design,
synthesis, α-amylase inhibition and in silico docking study of novel quinoline
bearing proline derivatives, J. Mol. Struct. 1208 (2020) 127873.
[6] R. Rafique, K.M. Khan, Kanwal Arshia, S. Chigurupati, Abdul Wadood, A. Ur
Rehman, A. Karunanidhi, S. Hameed, M. Taha, M. al-Rashida, Synthesis of new
indazole based dual inhibitors of α-glucosidase and α-amylase enzymes, their
in vitro, in silico and kinetics studies, Bioorg. Chem. 94 (2020) 103–195.
[7] M. Dhamej, P. Gupta, Synthetic heterocyclic candidates as promising
α-glucosidase inhibitors: An overview, Eur. J. Med. Chem. 176 (2019) 343–377.
[8] T. Umar, S. Gusain, M.K. Raza, S. Shalini, J. Kumar, M. Tiwari, N. Hoda, Naph-
thalene-triazolopyrimidine hybrid compounds as potential multifunctional an-
ti-Alzheimer’s agents, Bioorg. Med. Chem. 27 (2019) 3156–3166.
[9] M. Cherif, M. Horchani, Y.O. Al-Ghamdi, S.G. Almalki, Y.E. Alqurashi, H.Ben Jan-
net, A. Romdhane, New prano-1,2,3-triazolopyrimidinone derivatives as anti-
cholinesterase and antibacterial agents: Design, microwave-assisted synthesis
and molecular docking study, J. Mol. Struct. 1220 (2020) 128685.
[10] F. Panahi, R. Yousefi, M.H. Mehraban, A.K. Nezhad, Synthesis of new pyrim-
idine-fused derivatives as potent and selective antidiabetic α-glucosidase in-
hibitors, Carbohydr. Res. 380 (2013) 81–91.
[11] A. Romdhane, A. Ben Said, M. Cherif, H. Ben Jannet, Design, syn-
thesis and anti-acetylcholinesterase evaluation of some new pyra-
zolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives, Med. Chem. Res. 25
(2016) 1358–1368.
[31] L.R. Domingo, M. Ríos-Gutiérrez, P. Pérez, Applications of the Conceptual Den-
sity Functional Theory Indices to Organic Chemistry Reactivity, Molecules 21
(2016) 748.
[32] A. Romdhane, H.Ben Jannet, Synthesis of new pyran and pyranoquinoline
derivatives, Arab. J. Chem. 10 (2017) S3128–S3134.
[33] M. Shahinozzaman, N. Taira, T. Ishii, M.A. Halim, M.A. Hossain, S. Tawata,
Anti-Inflammatory, Anti-Diabetic, and Anti-Alzheimer’s Effects of Prenylated
Flavonoids from Okinawa Propolis: An Investigation by Experimental and Com-
putational Studies, Molecules 23 (2018) 2479.
[34] Z. Lv, W. He, X. Tian, J. Kang, Y. Liu, Y. Peng, L. Zheng, Q. Wang, W. Yu,
J. Chang, Design, synthesis, and biological evaluation of new N4-Substituted
2’-deoxy-2’-fluoro-4’-azido cytidine derivatives as potent anti-HBV Agents, Eur.
J. Med. Chem. 101 (2015) 103–110.
[35] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and compu-
tational approaches to estimate solubility and permeability in drug discovery
and development settings, Adv. Drug Deliv. Rev. 23 (1997) 3–25.
[36] A. Daina, O. Michielin, V. Zoete, SwissADME:
a free web tool to evaluate
pharmacokinetics, druglikeness and medicinal chemistry friendliness of small
molecules, Sci. Rep. 7 (2017) 42717.
10

A. Hajlaoui, M. Laajimi, M. Znati et al.
Journal of Molecular Structure 1237 (2021) 130346
[37] J. Ashraf, E.U. Mughal, A. Sadiq, N. Naeem, S.A. Muhammad, T. Qousain,
M.N. Zafar, B.A. Khan, M. Anees, Design and synthesis of new flavonols as dual
ɑ-amylase and ɑ-glucosidase inhibitors: Structure-activity relationship, drug–
likeness, in vitro and in silico studies, J. Mol. Struct. 1218 (2020) 128458.
[38] D.F. Veber, S.R. Johnson, Hung-Yuan Cheng, B.R. Smith, K.W. Ward, K.D. Kop-
ple, Molecular Properties That Influence the Oral Bioavailability of Drug Can-
didates, J. Med. Chem. 45 (2002) 2615–2623.
[39] M.M. Matin, M.S. Hasan, M. Uzzaman, M.M.H. Bhuiyan, S.M. Kibria, M.E. Hos-
sain, M.H.O. Roshid, Synthesis, spectroscopic characterization, molecular dock-
ing, and ADMET studies of mannopyranoside esters as antimicrobial agents, J.
Mol. Struct. 1222 (2020) 128821, doi:10.1016/j.molstruc.2020.128821.
[40] T. Sander, J. Freyss, M. von Korff, C. Rufener, DataWarrior: An Open-Source
Program For Chemistry Aware Data Visualization And Analysis, J. Chem. Inf.
Model. 55 (2015) 460–473.
[41] K. Rehman, T.A. Chohan, I. Waheed, Z. Gilani, M.S.H. Akash, Taxifolin prevents
postprandial hyperglycemia by regulating the activity of α-amylase: Evidence
from an in vivo and in silico studies, J. Cell. Biochem. 120 (2019) 425–438.
[42] H. Tandon, T. Chakraborty, V. Suhag, A Brief Review on Importance of DFT In
Drug Design, Res Med Eng Sci 7 (2019) 791–795.
[43] N. Choudhary, S. Bee, A. Gupta, P. Tandon, Comparative vibrational
spectroscopic studies, HOMO–LUMO and NBO analysis of N-(phenyl)-2,2-
dichloroacetamide, N-(2-chloro phenyl)-2,2-dichloroacetamide and N-(4-chloro
phenyl)-2,2-dichloroacetamide based on density functional theory, Comput.
Theor. Chem. 1016 (2013) 8–21.
[44] B. Amul, S. Muthu, M. Raja, S. Sevvanthi, Spectral, DFT and molecular docking
investigations on Etodolac, J. Mol. Struct. 1195 (2019) 747–761.
[45] F. Azam, N.H. Alabdullah, H.M. Ehmedat, A.R. Abulifa, I. Taban, S. Upadhyayula,
NSAIDs as potential treatment option for preventing amyloid β toxicity in
Alzheimer’s disease: an investigation by docking, molecular dynamics, and DFT
studies, J Biomol Struct Dyn 36 (2017) 2099–2117.
[46] A.M. Mansour, N.T. Abdel Ghani, Hydrogen-bond effect, spectroscopic and
molecular structure investigation of sulfamethazine Schiff-base: Experimen-
tal and quantum chemical calculations, J. Mol. Struct. 1040 (2013) 226–
237.
[47] E.J. Lienx, Z-Ru Guo, R-Li Li, C-Tang Su, Use of Dipole Moment as a Parameter
in Drug-Receptor Interaction and Quantitative Structure-Activity Relationship
Studies, J. Pharm. Sci. 71 (1982) 641–655.
[48] S. Alyar, Tü. S¸ en, C. S¸ en, H. Alyar, S¸ . Adem, Üü.Ö. Özdemir, Synthesis, spec-
troscopic characterizations, enzyme inhibition, molecular docking study and
DFT calculations of new Schiff bases of sulfa drugs, J. Mol. Struct. 1185 (2019)
416–424.
[49] R. Parthasarathi, V. Subramanian, D.R. Roy, P.K. Chattaraj, Electrophilicity index
as a possible descriptor of biological activity, Bioorg. Med. Chem. 12 (2004)
5533–5543.
11