New pyrano-1,2,3-triazolopyrimidinone derivatives as anticholinesterase and antibacterial agents: Design, microwave-assisted synthesis and molecular docking study

Article abstract of DOI:10.1016/j.molstruc.2020.128685

This work describes an effective microwave-assisted synthesis of new hybrid regioisomeric molecules N-linked-1,2,3-triazole along with pyranopyrimidinone moiety under copper(I) catalyst, and the evaluation of their anticholinesterase and antibacterial act

Full text of DOI:10.1016/j.molstruc.2020.128685

Journal Pre-proof
New prano-1,2,3-triazolopyrimidinone derivatives as anticholinesterase and
antibacterial agents: Design, microwave-assisted synthesis and molecular docking
study
Maher Cherif, Mabrouk Horchani, Youssef O. Al-Ghamdi, Sami G. Almalki, Yaser E.
Alqurashi, Hichem Ben Jannet, Anis Romdhane
PII:
S0022-2860(20)31010-3
DOI:
https://doi.org/10.1016/j.molstruc.2020.128685
MOLSTR 128685
Reference:
To appear in: Journal of Molecular Structure
Received Date: 17 April 2020
Revised Date: 4 June 2020
Accepted Date: 10 June 2020
Please cite this article as: M. Cherif, M. Horchani, Y.O. Al-Ghamdi, S.G. Almalki, Y.E. Alqurashi, H.
Ben Jannet, A. Romdhane, New prano-1,2,3-triazolopyrimidinone derivatives as anticholinesterase and
antibacterial agents: Design, microwave-assisted synthesis and molecular docking study, Journal of
Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.128685.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier B.V.

Maher Cherif did the synthesis work. Mabrouk Horchani tested the anticholinesterase activity and he
did the docking part. Youssef O. Al-Ghamdi tested the antimicrobial activity. Sami G. Almalki helped
in the interpretation of biological results. Yaser E. Alqurashi guided some biological assays. Hichem
Ben Jannet contributed to the discussion 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 compounds and the biological study, and he completed the
redaction of the article.

Graphical abstract
Ar = C6H5
4-CH3-C6H4
4-Cl-C6H4
O
N
Ar
O
1,2,3-triazole fragment
N
Ar1
N
N
N
Ar1 = C6H5
3-CH3-C6H4
4-CH3O-C6H4
4-Cl-C6H4
Pyranopyrimidinone fragment
1-Naphtyl
Design and Microwave-assisted Synthesis
Anticholinesterase an antibacterial activities
Molecular docking study (Anti-BuChE)

New prano-1,2,3-triazolopyrimidinone derivatives as anticholinesterase
and antibacterial agents: Design, microwave-assisted synthesis and
molecular docking study
Maher Cherifa, Mabrouk Horchania, Youssef O. Al-Ghamdib,*, Sami G. Almalkic, Yaser E.
Alqurashic, Hichem Ben Janneta and Anis Romdhanea,*
aLaboratory of Heterocyclic Chemistry, Natural Products and Reactivity, Team: Medicinal Chemistry
and Natural Products, Faculty of Science of Monastir, University of Monastir, Avenue of
Environment, 5019 Monastir, Tunisia
bDepartment of Chemistry, College of Science Al-zulfi, Majmaah University, Al-Majmaah,
11952, Saudi Arabia
cDepartment of Biology, College of Science Al-zulfi, Majmaah University, Al-Majmaah,
11952, Saudi Arabia
*Corresponding to: Anis Romdhane (Tel.: +21673500279, Fax: +21673500278; E-mail:
anis_romdhane@yahoo.fr). Youssef O. Al-Ghamdi (Tel.: +966164044094, Fax:
+966164044094; E-mail: yo.alghamdi@mu.edu.sa).
Abstract
This work describes an effective microwave-assisted synthesis of new hybrid regioisomeric
molecules N-linked-1,2,3-triazole along with pyranopyrimidinone moiety under copper(I)
catalyst, and the evaluation of their anticholinesterase and antibacterial activities. All of the
obtained structures have been assigned by means of spectroscopic data (NMR, HRMS). The
triazole derivatives 4 showed potent anti-cholinesterase activity such as compound 4a (Ar =
Ar1 = Ph; IC50 = 6.7 ± 0.2 µM). Most of 1,2,3-triazoles derivatives 4 were found to be active
against Pseudomonas savatanoi. Besides, in silico docking studies were also performed to
give rational explanation for their biological activities against BuChE.

Keywords: Pyrimidinone-triazole hybrids, Click Chemistry, microwave-assisted synthesis,
Anticholinesterase activity, Antibacterial activity, molecular docking analysis.
Introduction
Fused pyrimidines continue to attract considerable attention of researchers in different
countries because of their great practical usefulness, primarily, due to a very wide spectrum of
their biological activities. Pyranopyrimidines occupy a special position among these
compounds and they represent important building blocks in synthetic bioactive compounds,
which show antiproliferative [1-2], antiviral [3], antitumor [4], anti-HIV [5], antimicrobial
(Figure 1-A [6] and 1-B [7]) and anti-alzheimer (Figure 2-A [8] and 2-B [9]) activities.
Figure 1. Previously reported antimicrobial compounds
On the other hand, 1,2,3-Triazoles derivatives constitute an important class of heterocyclic
compounds with a wide spread of pharmacological and therapeutic uses which is a privileged
scaffold found in many medicinal active molecules, including natural products exhibiting a
wide range of biological and pharmacological activities such as antityrosinase [10], anti-
inflammatory [11], α-glycosidase inhibitor [12], anticancer [13], antibacterial (Figure: 1-C
[14] and 1-D [15]), and anti-alzheimer properties (Figure: 2-C [16] and 2-D [17]).

Figure 2. Previously reported anti-alzheimer compounds
Moreover, the synthesis of 1,2,3-triazole was widely studied [18-20]. The main for the
synthesis of these five-heterocyclic compounds is the reaction between azides and acetylenes,
originally described by Huisgen [21]. Nevertheless, due to the high activation energy, these
reactions require high temperature, long reaction time and formation of mixture of 1,4- and
1,5-regioisomers [22]. In 2002, it was reported that, Cu catalyzed azide-alkyne cycloaddition
reaction (CuAAC) popularly known as Click-reaction, which proceeds under mild reaction
conditions, hazard-free, leads to 100% regioselectivity (only 1,4-disubstituted regioisomer is
formed) and excellent yielding [23-24]. In the other hand, microwave assisted technique had
become a landmark and significant contribution to preserve the environment by reducing the
waste as well, as it offers an efficient internal heat transfer which reduces the reaction time as
well as increasing the rate of reaction and yield. Many reactions have proven to result in
higher yield and/or selectivity under microwave irradiation compared with conventional
heating [25-26]. Amoung these, the cycloaddition of alkynes and azides [27-29].
In consideration of the above literature data describing biological significance of
pyranopyrimidine and triazole derivatives along with the advantages of microwave irradiation
technology and encouraged by our previous successful synthesis of hybrid molecules which
possessing improved activity [30-32], in the present work, we designed and synthesized some

new hybrid compounds through combination of pyranopyrimidinone moiety with 1,2,3-
triazole pharmacophore (Figure 3) via 1,3-dipolar cycloaddition using various arylazides. A
regiospecific, simple and versatile copper(I)-catalyzed, microwave-assisted procedure for
preparation of a series of 1,2,3-triazole derivatives was developed. The antibacterial and
anticholinesterase activities of prepared compounds were evaluated. Furthermore, the
structure-activity relationship (SAR) was supported by the molecular docking analysis.
Figure 3. General structure of the designed compounds
Experimental
Melting temperatures were determined on an electrothermal 9002 apparatus and were reported
uncorrected. NMR spectra were recorded on a Bruker AC-300 spectrometer at 300 MHz (1H)
and 75 MHz (13C). All chemical shifts were reported as δ values (ppm) relative to residual
non deuterated solvent. Mass spectra were obtained with ESI-TOF (LCT, Waters) using the
reflectron mode in the positive ion mode. Spots were visualized by UV light at 254-365
nm.All reactions were monitored by thin layer chromatography over silica gel-coated TLC
plates (Silica gel 60, F254). Column chromatography was performed with silica gel 60 (particle
size 40-63 μm) with the appropriate eluent. The starting materials 1 prepared according to the
literature [8].

General procedure for synthesis of compound 3a-c
The synthesis of precursor 3 started by the condensation reaction of 2-aminonaphthopyrano-3-
carbonitrile 1 with acetic anhydride in the presence of a catalytic amount of polyphosphoric
acid (PPA) under reflux for 1 h. Then, under argon atmosphere, the obtained
benzopyranopyrimidinone 2 (1g, 1.55 mmol) is dissolved in anhydrous DMF (50 mL).
Propargylbromide bromide (1.5 equiv.) was added in the presence of sodium hydride (2
equiv.). The mixture was stirred for 2 h at room temperature, after completion of the reaction
monitored by TLC, the reaction mixture was poured into ice-cold water, the precipitate was
filtered and washed several times with water. The crude product was purified by column
chromatography to afford pure compounds 3a-c. (See supplementary materials for spectral
data of synthesized compounds 3a-c)
General procedure for the preparation of compound 4a-n
To compound 3 (0.1 g, 0.2 mmol) in anhydrous DMF, we added the appropriate arylazides (1
equiv.) which prepared as described in the literature in the presence of triethylamine (1
equiv.), 5 mol% CuCl was added in one pot and the reaction mixture was irradiated for 5-10
min under 300 W or refluxed for 4-8 hours. After completion of reaction controlled by TLC,
the reaction mixture was extracted with ethyl acetate. The organic layer was dried over
anhydrous Na2SO4, after removal of solvent under reduced pressure, the resulting residue was
purified by silica gel column chromatography (EtOAc/petroleum ether) to give compound 4.
(See supplementary materials for spectral data of synthesized compounds 4a-n)
Biological activity methods
Anticholinesterase activity
BChE was used to hydrolyze butyrylthiocholine (colorless) to butyrate and thiocholine. Then,
thiocholine reacts with DTNB to form 5-mercapto-2-nitrobenzoic acid (5-MNBA), a yellow
product. The rate of 5-MNBA formation, measured spectrophotometrically at 405 nm is

proportional to the enzymatic activity of BchE in the sample. Human plasma (pool plasma
from samples designated for biochemical analysis) was used as a source of BchE. 100 μL of
compound were added to 100 μL of plasma and the mixture was incubated at 37°C for a
determined time. After incubation, the enzyme activity was measured by Konelab 30 UV
apparatus. The control (plasma and distilled water) was treated in the same conditions.
Galanthamine was used as control positive and all assays were carried out in duplicate. The
anticholinesterase activity was calculated by the following formula: % Inhibition = [(Activity
of control -Activity of sample) / Activity of control] x100. The sample concentration
providing 50% inhibition (IC50) was calculated by plotting inhibition percentages against
concentrations of the sample. To determine the effect of temperature and incubation times,
assays were carried by the same procedure for the same product, at 25°C (fixed incubation
times) and for different times at 37°C [33].
Antibacterial activity
The synthesized products were screened for their antibacterial activity by using the agar disc
diffusion method. NA medium cooled at 45°C was supplemented with a bacterial suspension
(106 CFU/mL) and poured into Petri plates. After solidification, The sterilized paper disc
prepared from the Whatman paper (diameter 6 mm) were placed at the surface of the culture
medium and 20 μL (1000 μg/mL) of the product dissolved in DMSO was dropped onto each
disc. The negative control plates had no product added to the filter paper whereas in the
positive control plates, discs were impregnated with the same volume of ampicillin solution (5
mg/mL). The treated Petri dishes were incubated at 25°C for 48 h. The antibacterial activity
was evaluated by measuring the diameter of the inhibitory zones formed around the discs. The
MIC values for the antibacterial screening were determined with dilution method
(microdilution using 96-well microplates) following the procedure described by Kalpesh et al.
Briefly, all samples were prepared at a concentration of 1 mg/mL by dissolution of the

products in DMSO, for preparing the paper disc for the determination of the minimum
inhibitory concentration (MIC), the sterilized paper discs were dived in each of the different
dilutions prepared from products, for better diffusion of our products the plates were
incubated in refrigerator for 30 min. After that the plates were incubated in the incubator at
37°C for 24 h. The result was interpreted after 24 hrs. The MIC of each sample was defined as
the lowest concentration of that inhibited the bacterial growth. The test was performed
triplicate [34-35].
Molecular docking procedure
The optimization of all the geometries of scaffolds was performed with ACD (3D viewer)
software (http://www.filefacts.com/acd3d-viewer-freeware-info). The crystal structures of
PDB (PDB: 1P0I) was obtained from the RSCB protein data bank [36]. Docking studies were
performed using Autodock 4.2 software [37]. The visualization and analysis of interactions
were performed using Pymol, v. 0.99 (The PyMOLMolecular Graphics System).
Results and Discussion
Chemistry
Our approach to the target hybrid molecules 4a-n first started by the synthesis of 2-
aminonaphthopyrano-3-carbonitriles 1a-c, which were served as key starting material, via the
one-pot, three-component reaction of equimolar amounts of arylaldehyde, malononitrile, and
β-naphthol in aqueous ethanol using CuI as catalyst according to the literature [32]. The
structures of these precursors were established on the basis of their spectroscopic data
according to our previous work (Scheme 1). [8]
Scheme 1. Synthetic pathway to 2-aminonaphtopyrano-3-carbonitriles 1a-c

Mechanistically, the preparation of precursors 1a-c is performed in two steps. The reaction
starts by a Knoevenagel condensation reaction between the intermediate (I), prepared in situ,
and the protonated arylaldehyde in the presence of CuI as catalyst to give the intermediate
(II), then, a Michael addition of β-naphthol at the conjugated C=C bond of (II) after losing a
molecule of water and a molecule of CuI, followed by a concerted intramolecular cyclization
to lead to the precursors 1a-c (Scheme 2).
O
Ar
H
Cu
I
I
N
C
H O
Ar
H
H
The beginning of
new catalytic cycle
NC
Knoevenagel
condensation
NC C C N Cu
H
NC
Ar
CN
I
CuI
CuI
I
CN
NH2
Cu OH
H
CN
O
Ar
C
CN
II
N
Cu
I
1a-c
Ar
I
C
H
NH
Cu
O
OH
Ar
H
- H2O
- CuI
III
Michael
addition
C
CN
N
Cu
I
Scheme 2. Proposed mechanism for the formation of compounds 1a-c
These precursors (1a-c) were further converted into pyranopyrimidinone derivatives 2a-c via
their reaction with acetic anhydride in the presence of a catalytic amount of polyphosphoric
acid (PPA) under reflux for one hour. From a mechanistic point of view, the reaction process
is assumed to follow a two-step pathway. The precursors 1a-c should react with acetic
anhydride to afford, the non-isolable intermediates 1'a-c which cyclize in 1''a-c after a
nucleophilic attack at the nitrile group. Intramolecular rearrangement, type Dimroth [38-39],
of the so formed intermediate led to pyranopyrimidinones 2a-c, which were then N-

propargylated by reaction with propargyl bromide and sodium hydride in anhydrous DMF at
room temperature to give the corresponding N-propargylated dipolarophiles 3a-c (Scheme 3)
[40].
Scheme 3. Synthetic pathway to pyranopyrimidinones 2a-c and 3a-c
The structure of compounds 3a-c was established on the basis of their spectral data. In fact,
1
their H NMR spectra showed the disappearance of the singlet related to the mobile proton (-
NH-) at δH 12.43-12.60 and the observation of two doublet of doublets at δH 4.62-4.69 (J =
2.4 Hz, J = 17.4 Hz) and δH 5.08-5.18 (J = 2.4 Hz, J = 17.4 Hz) attributable to the methylene
protons group (N-CH2-) and a triplet at δH 2.29-2.33 (J = 2.4 Hz) corresponding to the
acetylenic proton (-C≡C-H).
The 13C NMR spectra of compounds 3a-c displayed in particular three new signals at δC
144.1-146.8, δC 144.1-146.8 and δC 144.1-146.8 attributable, respectively to carbons C1, C2
and C3 introduced by the propargyl bromide.

In the above mentioned precursors 3, the triple bond moiety made them valuable key
precursors for the formation of several five-heterocyclic compounds [17, 41-42] and aroused
our interest to explore them in order to obtain the desired hybrid compounds 4a-n.
Thus, the 1,3-dipolair cycloaddition reaction of pyrimidinic precursors 3a-c with various
arylazides in anhydrous DMF under microwave irradiation [43], in the presence of copper (I)
catalyst (CuAAC), generates the corresponding new hybrid molecules 4a-n (Scheme 4). All
reactions were carried out under microwave irradiation (300 W) and completed within 5-10
min. The desired new compounds 4a-n were obtained in yields ranging from 90 to 97%
(Table 1).
Currently, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) is the most commonly
method used for the synthesis of 1,2,3-triazoles. This reaction allowed to yield exclusively the
1,4-disubstituted regioisomer, according to some previous work [23-24, 44-45]
Scheme 4. Synthetic pathway to derivatives 2, 3 and 4.
Mechanistically, formation of copper (I) acetylide I (step A) occurs through a π-alkyne copper
complex intermediate. π-Coordination of alkyne to copper significantly acidifies terminal
hydrogen of the alkyne, bringing it into the proper range to be deprotonated in presence of
TEA and resulting in the formation of a σ-acetylide with a second copper (I) atom.
The added azide is then activated by coordination to copper (step B), thus forming the
intermediate II. In the next step (C), the formation of the first C–N bond takes place, and a
strained copper metallacycle III forms.

This intermediate undergoes a rearrangement to lead to copper (I) triazole intermediate IV
(step D), which in the presence of another molecule of alkyne gives the desired triazole while
regenerating intermediate I (step E) (Schema 5).
Scheme 5. Proposed mechanism for the formation of compounds 4a-n
The structures of compounds 4a-n as 1,4-substituted triazole regioisomers were evidenced on
the basis of their spectroscopic measurements. Their ES-HRMS gave in each case a pseudo-
molecular ion peak [M+H]+, which is consistent with the corresponding molecular formula.
The 1H NMR spectra of these compounds showed in addition of the proton signals introduced
by the dipolarophiles 3a-c except those of the acetylenic proton, a singlet at δH 7.89-8.09
attributable to the proton H-5 of the triazole moiety and signals at the aromatic region (δH
7.00-7.70) relative to the aromatic protons introduced by the arylazides used. These structures
were further supported by 13C and DEPT NMR spectra, which showed all the expected carbon
signals corresponding to the cycloadducts 4a-n, essentially the new signals at δC 114.2-160.1
relative to the aromatic carbons introduced by the azides used and those of the triazoles
formed.

Table 1. Synthesis of compounds 3a-ca and 4a-nb.
Product
3a
Ar
4-MePh
Ph
Ar1
Time (min) Yield (%)c
-
120
120
120
5
92
88
80
97
93
92
92
90
96
94
92
92
91
96
92
94
94
-
-
3b
3c
4-ClPh
Ph
Ph
4a
4-MePh
4-ClPh
Ph
3-MePh
4-MeOPh
4-ClPh
10
10
10
5
4b
4c
4d
4e
4-MePh 1-Naphthyl
4-ClPh
Ph
Ph
10
10
5
4f
3-MePh
4g
4-MePh 4-MeOPh
4h
4i
4-ClPh
Ph
4-ClPh
1-Naphthyl
Ph
10
10
10
10
10
10
4j
4-MePh
4-ClPh
Ph
4k
4l
3-MePh
4-MeOPh
4-ClPh
4m
4n
4-MePh
a in DMF at 120°C b under MW irradiation (300 W) c Isolated yield after purification.
Biological activity
In order to survey the possible biological activities of this class of compounds, all the
prepared compounds were screened for their anticholinesterase and antibacterial activities.
Anticholinesterase activity
All the compounds were further screened for their anticholinesterase activity (Table 2). These
results were expressed as IC50 values as shown in Table 4. Compound 1a, with a phenyl
substituent at C-6 is revealed to be the potent inhibitor of BuChE with an IC50 value of 31.1±
0.2 mg/mL compared to its analogues 1b (4-MePh) and 1c (4-ClPh). In the series 2a-c, the
compound 2a (Ph) still was found to be the most active (IC50= 31.9± 0.3 µM). Indeed, this

activity could be interpreted by the nature of the substituent fixed in para position of the
phenyl group in both series 1 and 2.
Moreover, compound 3a (Ph) (IC50= 27.2 ± 0.3 µM) displayed the best activity compared to
its analogues 3b (4-MePh) and 3c (4-ClPh). These results show clearly the effect of the
propargyl moiety introduced to relatively improve the activity of compounds 3a-c compared
with 2a-c.
Nearly all the tested hybrid compounds 4a-n showed strong capacities of cholinesterase
inhibition (IC50 = 6.7 ± 0.2 - 25.5 ± 0.2 µM) than their starting materials 3a-c. These results
show the importance of the introduction of the triazole moiety via the methylene linker to
improve this activity. On the other hand, in all this series, it has been found that compounds
4a (Ar= Ph, Ar1= Ph), 4c (Ar= 4-ClPh, Ar1= 4-MeOPh), 4h (Ar= 4-MePh, Ar1= 4-MeOPh)
and 4m (Ar= Ph, Ar1= 4-MeOPh) were found to be the most active with IC50 values of 6.7 ±
0.2, 8.4 ± 0.4, 7.8 ± 0.2 and 9.1 ± 0.1 µM, respectively. This finding shows clearly the
importance of the nature of the substituent attached to the aryl group.
Table 2. Anticholinesterase activity of compounds 1a-c, 2a-c, 3a-c and 4a-n.
Entry Product
Enty
Product
IC50 (µM)
IC50 (µM)
1
2
3
4
5
6
7
8
31.1 ± 0.2 13
33.6 ± 0.1 14
38.5 ± 0.3 15
31.9 ± 0.3 16
35.3 ± 0.2 17
41.9 ± 0.2 18
27.2 ± 0.3 19
27.9 ± 0.2 20
39.7 ± 0.2 21
6.7 ± 0.2 22
14.6 ± 0.2 23
20.6 ± 0.2
22.9 ± 0.1
12.3 ± 0.2
16.8 ± 0.2
7.8 ± 0.2
16.2 ± 0.2
23.3 ± 0.1
22.1 ± 0.2
11.9 ± 0.2
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4d
4e
4f
4g
4h
4i
4j
4k
9
10
11
4l
4m
4n
9.1 ± 0.1
24.1 ± 0.2
0.38*10-3 ± 0.002*10-3
Galanthamine
Molecular docking studies

To help to understand the observed experimental results of the anticholinesterase activity of
the cycloadducts 4a-n, molecular docking studies were carried out using AutoDock Vina
software [34] to obtain the plausible binding mode of the most active compounds 4a, 4c, 4h
and 4m within the active site of BuChE. As can be seen in Figure 4, the most potent
derivative against BuChE, compound 4a (Ar= Ph, Ar1= Ph) having the lowest binding affinity
(-12.5 Kcal. mol-1) (Table 3) is involved in conventional hydrogen binding with THR-A-120
(green color) by its pyran moiety. Besides, the pyrimidinone pharmacophore is involved in the
Pi-sigma interaction with ASP-A-70 (dark purple color). Moreover, the naphthalene ring is
involved in the Pi-Pi interaction with TRP-A-82 (dark pink color). The presence of a phenyl
ring linked to the pyran scaffold led to the formation of Pi-Alkyl interaction with ALA-A-328
(light pink color) and Pi-Pi stacking with TYR-A-332 (dark pink color). This last amino acid
displayed also a Pi-Pi interaction with the triazole moiety. In addition, the phenyl group
linked to triazole fragment is involved in Pi-Sigma interaction with PRO-A-285 (dark purple
color).
Table 3. Binding energies of promising anticholinesterase agents.
Compounds
Binding Energy (kcal/mol)
-12.5
-11.4
-11.6
-12.4
4a
4c
4h
4m

Figure 4. Binding site of conjugate 4a (most active anticholinesterase agent) within the active
site of BuChE (PDB: 1P0I)
In the case of compounds 40c, 4h and 4m as shown in Figure 5, the orientation of the ligand
in the active site of BuChE shows the same interactions as 4a with THR-A-120, ASP-A-70
and TRP-A-82. Additionally, the insertion of the methoxy group in para-position of the
phenyl moiety attached to the triazole ring displayed carbon-hydrogen bond with GLU-A-283
(grey color) in the case of compound 4c and 4h and a carbon-hydrogen bond with ALA-A-
277 and ASN-A-289 in the case of compound 4m. Moreover, compound 4c facilitated the
formation of the same interactions as 4a with PRO-A-285 and TYR-A-332 but the presence
of an electron withdrawing group such as chlorine atom on the para-position of the phenyl
ring attached to the pyran scaffold of compound 4c led to Halogen interaction with ALA-A-
328 (sky blue color) and Pi-alkyl with PHE-A-329 (light pink color). Similarly for compound
4h while the presence of the methyl group on the para-position of the phenyl ring linked to
the pyran scaffold created a Pi-sigma interaction with PHE-A-329 (dark purple color) in
addition to Alkyl interaction with ALA-A-328 (light pink color) by the tolyl group.

Figure 5. Binding site of conjugate 4c, 4h, and 4m (most effective anticholinesterase agents)
within the active site of BuChE (PDB: 1P0I).
Antibacterial activity
The compounds 1a-c, 2a-c, 3a-c and 4a-n were further screened for their antibacterial activity
against three microorganisms namely Pseudomonas syringae pv syringae, P. savatanoi and
Agrobacterium tumefasciens assessed by the inhibition zone diameter (IZ) and by Minimum
inhibitory concentration (MIC) values. The results did not show any significant activity of
compounds 1a-c, 2a-c and 3a-c against the above strains used.
The results given in Table 4 showed that most 1,2,3-triazole hybrids 4 showed antibacterial
activity against the used bacteria with IZ and MIC values of 7-12 mm and 500-250 µg/mL,

respectively. The antibacterial effect of most of these hybrid molecules 4 showed the
importance of the added triazole ring to the pyrimidinone 2 via the methylene linker to
generate activity. The highest activity against P. syringae pv syringae in the series 4 was
obtained with compound 4m (Ar = Ph, Ar1 = 4-MeOPh) followed by 4f (Ar = 4-ClPh, Ar1 =
Ph).
Most 1,2,3-triazole hybrids 4 showed comparable activity against P. savatanoi with IZ and
MIC values of 7-10 mm and 500-250 µg/mL, respectively. The different combinations Ar/Ar1
used did not lead to interesting activities against this starin. The highest activity against A.
tumefaciens was displayed by compound 4l (Ar = 4-ClPh, Ar1 = 3-MePh) followed by 4g (Ar
= Ph, Ar1 = 3-MePh). The existence of the common 3-MePh group attached to the triazole
moiety in both derivatives could be at the origin of this comparable activity.
Table 4. Antibacterial activity of the synthesized compounds 4a-n.
Pseudomonas syringae
Pseudomonas
savatanoi
IZ
Agrobacterium
tumefasciens
pv. syringae
product
4a
IZa
MICb
MIC
500±6
500±6
250±5
500±6
250±3
250±3
250±3
250±4
250±4
250±3
250±7
250±5
250±5
IZ
MIC
c
8.0±0.1
9.0±0.2
8.1±0.1
8.0±0.1
8.0±0.1
11.1±0.1
8.0±0.1
7.5±0.1
9.5±0.2
8.5±0.1
10.0±0.1
9.0±0.1
12.0±0.1
250±2
250±2
250±2
250±3
250±3
250±2
250±2
500±3
250±5
250±5
250±5
250±2
250±3
7.0±0.1
7.0±0.1
8.0±0.1
7.5±0.2
9.0±0.1
8.5±0.1
8.0±0.1
10.0±0.1
8.5±0.2
7.0±0.1
7.5±0.1
8.0±0.1
8.0±0.2
-
-
-
-
4b
4c
-
-
-
-
4d
4e
9.5±0.1
8.5±0.1
11.0±0.1
10.0±0.2
-
250±1
250±6
250±5
250±4
-
4f
4g
4h
4i
-
-
4j
-
-
4k
4l
12.0±0.1
9.0±0.2
250±5
250±5
4m

10.0±0.2
26.0±0.2
250±4
9.0±0.1
250±5
7.5±0.1
500±5
4n
Ampicillind
DMSO
22.5±0.1
26.0±0.1
-
-
-
a,
b(IZ): Inhibition zone in mm and (MIC) Minimal Inhibition Concentration in µg/mL were the
means of three replicates (mean ± SD, n = 3);
c- no inhibition zone observed;
dpositive control (5 mg/mL).
Conclusion
In this paper, we have successfully designed and synthesized a new series of
pyranopyrimidinones linked 1,2,3-triazoles via regiospecific 1,3-dipolar cycloaddition
reaction catalyzed by Cu(I) (CuAAC) and microwave-assisted of pyranopyrimidinone
derivatives 3a-c, which obtained in two steps from 2-aminonaphthopyrano-3-carbonitriles 1a-
c, with some arylazides. All the newly synthesized derivatives 4a-n have been tested for their
anticholinesterase and antibacterial activities. Mostly, we noticed that all the tested hybrid
compounds 4a-o showed strong capacities of cholinesterase inhibition than their starting
materials 3a-c, which shows the importance of the introduction of the triazole moiety via the
methylene linker to improve this activity. Molecular Docking analysis of the most potent anti-
BuChE compounds 4a, 4c, 4h and 4m confirmed that they have been well fitted in the active
site of this enzyme via desirable interactions. In the other hand, target compounds 4a-n were
screened for their antibacterial activity against three starins of pathogenic bacteria. Biological
results revealed that some of these compounds demonstrated excellent to moderate
antimicrobial activities and showed the importance of the added triazole ring to the
pyranopyrimidinone 2a-c via the methylene linker to generate activity. Moreover, compounds
4m and 4f showed the highest inhibitory activity against P. syringae pv syringae, whereas
compounds 4l and 4g displayed the most significant activity against A. tumefaciens.

Acknowledgements
The authors are grateful to Miss Amna Benzarti, NMR service at the Faculty of Monastir,
University of Monastir for the NMR analysis and to the Ministry of Higher Education and
Scientific Research of Tunisia for financial support (LR11ES39).
References
[1] A. Bouattour, M. Fakhfakh, S. Abid, L. Paquin, R. Le Guével, A. Corlu, S. Ruchaud, S. Bach, H.
Ammar, J. P. Bazureau, Arkivoc (2017) 291-302.
[2] F. F. Alblewi, R. M. Okasha, Z. M. Hritani, H. M. Mohamed, M. A. A. El-Nassag, A. H. Halawa,
A. Mora, A. M. Foudad, M. A. Assirid, A. M. Al-Diesd, T. H. Afifi, A. M. El-Agrody, Bioorg.
Chem. 87 (2019) 560-571.
[3] A. H. C. Shamroukh, M. E. A. C. Zaki, E. M. H. C. Morsy, F. M. C. Abdel-Motti, Arch. Pharm.
340 (2007) 236-243.
[4] F. F. Alblewi, R. M. Okasha, A. A. Eskandrani, T. H. Afifi, H. M. Mohamed, A. H. Halawa, A. M.
Fouda, A. M. Al-Dies, A. Mora, A. M. El-Agrody, Molecules 24 (2019) 1060-1677.
[5] N. A. Al-Masoudi, H. H. Mohammed, A. M. Hamdy, O. A. Akrawi, N. Eleya, A. Spannenberg,
Pannecouque, P. Langer, Z. Naturforsch. 68b (2013) 229 – 238.
[6] H. M. Aly, M. M. Kamal, Eur. J. Med. Chem. 47 (2012) 18-23.
[7] M. A. Abozeid, M. R. El-Kholany, L. A. Abouzeid, A. H. Abdel-Rahman, E. I. El-Desoky, J.
Heterocyclic. Chem. 56 (2019) 2922-2933.
[8] A. Romdhane, M. T. Martin, A. Ben Said, A. Jabrane, H. Ben Jannet, J. Tun. Chem. Soc. 14 (2012)
127-135.
[9] Y. Bouazizi, A. Romdhane, H. Ben Jannet, Eur. J. Chem. 5 (2014) 457-462.
[10] M. B. Tehrani, P. Emani, Z. Rezaei, M. Khoshneviszadeh, M. Ebrahimi, N. Edraki, M. Mahdavi,
B. Larijani, S. Ranjbar, A. Foroumadi, M. Khoshneviszadeh, J. Mol. Struct. 1176 (2019) 86-93.
[11] A. Haque, M. F. Hsieh, S. I. Hassan, M. S. H. Faizi, A. Saha, N. Dege, J. A. Rather, M. S. Khan,
J. Mol. Struct. 1146 (2017) 536-545.

[12] Z. Bakherad, M. M. Khanaposhtani, H. S. Aliabadi, S. Rezaei, A. Fassihi, M. Bakherad, H.
Rastegar, M. Biglar, L. Saghaie, B. Larijani, M. Mahdavi, J. Mol. Struct. 1192 (2019) 192-200.
[13] K. Bozorov, J. Zhao, H. A. Aisa, Bioorg. Med. Chem. 27 (2019) 3511-3531.
[14] S. Sharma, M. Saquib, S. Verma, N. N. Mishra, P. K. Shukla, R. Srivastava, Y. S. Prabhakar, A.
K. Shaw, Eur. J. Med. Chem. 83 (2014) 474-489.
[15] R. Suresh, S. Muthusubramanian N. Paul, N. Kalidhasan V. Shanmugaiah, Med. Chem. Res. 23
(2014) 4367-4375.
[16] A. Kaur, S. Mann, A. Kaur, N. Priyadarshi, B. Goyal, N. K. Singhal, D. Goyal, Bioorg. Chem. 87
(2019) 572–584.
[17] M. Zayane, A. Rahmouni, M. D. Remadi, M. Ben Mansour, A. Romdhane, H. Ben Jannet, J.
Enzyme Inhib. Med. Chem. 31 (2016) 1566-1575.
[18] Z. X. Wang, H. L. Qin, Chem. Commun. 19 (2003) 2450-2451.
[19] W. J. Evans, E. Montalvo, T. M. Champagne, J. W. Ziller, A. G. DiPasquale, A. L. Rheingold, J.
Am. Chem. Soc. 130 (2008) 16-17.
[20] D. Amantini, F. Fringuelli, O. Piermatti, F. Pizzo, E. Zunino, L. Vaccaro, J. Org. Chem. 70
(2005) 6526-6529.
[21] H. Rolf, S. Günter, M. Leander, Chem. Ber. 100 (1967) 2494-2507.
[22] A. A. Ali, M. Konwar, M. Chetia, D. Sarma, Tetrahedron. Lett. 57 (2016) 5661-5665.
[23] V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Chem. Int. Ed. 41 (2002) 2596-
2599.
[24] C. W. Tornøe, C. Christensen, M. Meldal, J. Org. Chem. 67 (2002) 3057-3064.
[25] P. Lidström, J. Tierney, J. Wathey, J. Westman, Tetrahedron. 57 (2001) 9225-9283.
[26] G. W. Kabalka, A. R. Mereddy, Tetrahedron. Lett. 47 (2006) 5171-5172.
[27] D. Ashok, S. Gundu, V. K. Aamate, M. G. Devulapally, Mol. Divers. 22 (2018) 769-778.
[28] M. Sarasija, D. Ashok, Shivaraj, Indian. J. Heterocycl. Chem. 22 (2012) 5-8.
[29] M. Aarjane, S. Slassi, B. Tazi, M. Maouloua, A. Amine, J. Chem. Sci. 131(2019) 1-11.
[30] I. Filali, J. Bouajila, M. Znati, F. B. El Garah, H. Ben Jannet, J. Enz. Inhibe Med. Chem. 30
(2015) 371-376.

[31] A. Romdhane, A. Ben Said, M. Cherif, H. Ben Jannet, Med. Chem. Res. 25 (2016) 1358-1368.
[32] M. Zayane, A. Romdhane, M. D. Remadi, H. Ben Jannet, J. Chem. Sci. 127 (2015) 1619-1626.
[33] A. Ben Nejma, A. Nguir, H. Ben Jannet, M. A. Hamza, A. Daïch, M. Othman, A. M. Lawson,
Bioorg. Med. Chem. Lett. 25 (2015) 1665-1670.
[34] I. Aissa, V. D. Nimbarte, A. Zardi-Bergaoui, M. Znati, G. Flamini, R. Ascrizzi, H. Ben Jannet,
Chem. Biodivers.16(4) 2019, e1800648. doi: 10.1002/cbdv.201800648.
[35] M. Horchani, A. Hajlaoui, A.H. Harrath, L. Mansour, H. Ben Jannet, A. Romdhane, J. Mol.
Struct. 1199 (2020) https://doi.org/10.1016/j.molstruc.2019.127007
[36] Y. Nicolet, O. Lockridge, P. Masson, J. C. Fontecilla-Camps, F. Nachon, J. Biol. Chem. 278
(2003) 41141-41147.
[37] O. Trott, A. J. Olson, J. Comput. Chem. 3(2010) 455-461.
[38] M. Cherif, M. Debbabi, S. Chortani, A. Romdhane, H. Ben Jannet, Turk. J. Chem 42 (2018)
1623-1639.
[39] F. K. Mohammed, A. Y. Soliman, A. S. Sawy, M. G. Badre, J. Chem. Pharm. Res. 1(2009), 213-
224.
[40] D. Hesek, M. Lee, B. C. Noll, J. F. Fisher, S. Mobashery, J. Org. Chem. 74 (2009) 2567-2570.
[41] A. Rahmouni, S. Souiei, M.A. Belkacem, A. Romdhane, J. Bouajila, H. Ben Jannet, Bioorg.
Chem. 66 (2016) 160-168.
[42] I. Filali, A. Romdhane, M. Znati, H. Ben Jannet, J. Bouajila, Med. Chem. 12 (2), 2016, 184-190.
[43] D. Ashok, D.M. Gandhi, G. Srinivas, A.V. Kumar, Med. Chem. Res. 23 (2014) 3005-3018.
[44] K. Chouaïb, F. Hichri, A. Nguir, M. D. Remadi, N. Elie, D. Touboul, H. Ben Jannet, M.A.
Hamza, Food. Chem. 183 (2015) 8-17.
[45] (a) B. T. Worrell, J. A. Malik and V. V. Fokin, Science, 2013, 340, 457; (b) R. Berg and
B. F. Straub, Beilstein J. Org. Chem., 2013, 9, 2715.

Highlights
A series of new 1,2,3-triazole linked pyranopyrimidinones 4a-n was regiospecifically
synthesized using microwave irradiation
The structures of the new hybrid compounds were performed by spectroscopic means.
All compounds were screened in vitro for their antibacterial and anticholinesterase
potentialities.
Most of 1,2,3-triazoles derivatives 4 were found to be active against Pseudomonas
savatanoi.
Docking studies of some pyranopyrimidinone-1,2,3-triazoles gave rational explanation
for their activity against BuChE.

Declaration of interests
X
☐The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: