Synthesis, in vitro and in silico studies of naphto-1,3-oxazin-3(2H)-one derivatives as promising inhibitors of cholinesterase and α-glucosidase

Article abstract of DOI:10.1016/j.molstruc.2020.129103

Various substituted naphto-1,3-oxazin-3(2H)-one derivatives have been prepared by a three-component one-pot condensation reaction of 2-naphthol, aromatic aldehydes, and urea using phenylboronic acid as catalyst. The molecular structures of the synthesized

Full text of DOI:10.1016/j.molstruc.2020.129103

Journal of Molecular Structure 1225 (2021) 129103
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Synthesis, in vitro and in silico studies of naphto-1,3-oxazin-3(2H)-one
derivatives as promising inhibitors of cholinesterase and α-glucosidase
Khawla Boudebbousa, Houssem Boulebda,∗, Chamseddine Derablia,b, Lamia Bendjeddouc,
Chawki Bensouicid, Dominique Harakate, Hocine Merazigc, Abdelmadjid Debachea,∗
a Laboratory of Synthesis of Molecules with Biological Interest, Frères Mentouri Constantine 1 University, 25000 Constantine, Algeria
b National Higher School of Biotechnology “Toufik Khaznadar”, Ali Mendjeli University City, 25100 Constantine, Algeria
c Research Unit of Environmental Chemistry and Structural Molecular Chemistry, CHEMS, Faculty of Exact Sciences, Tidjani Hadem Campus, Frères Mentouri
Constantine 1 University, 25000 Constantine, Algeria
d National Research Centre for Biotechnology, 25000 Constantine, Algeria
e Reims Champagne-Ardenne University and CNRS, Reims Institute of Molecular Chemistry, UMR 7312, Reims, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Various substituted naphto-1,3-oxazin-3(2H)-one derivatives have been prepared by a three-component
one-pot condensation reaction of 2-naphthol, aromatic aldehydes, and urea using phenylboronic acid
as catalyst. The molecular structures of the synthesized compounds were characterized by physical
and spectroscopic techniques and, for compound 4b, by single crystal X-ray diffraction analysis. The
cholinesterase and α-glucosidase inhibitory activities of the titled compounds have been investigated in
vitro. The obtained results revealed that some of the synthesized compounds are highly active towards
both AChE/BChE and α-Glucosidase with IC50 values lower than those of the standards galanthamine and
acarbose. In Silico studies such as Hirshfeld surface, docking study, DFT calculations, and ADME proper-
ties have been also performed in order to get insights into the molecular structure, chemical reactivity,
structure-activity relationship, and bioavailability of the synthesized compounds.
Received 27 July 2020
Revised 16 August 2020
Accepted 18 August 2020
Available online 18 August 2020
Keywords:
Naphthoxazinone
Phenylboronic acid
Cholinesterase
α-Glucosidase
DFT calculations
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
most of the drugs approved by the Food and Drug Administration
(FDA) for the AD therapy are AChE inhibitors including tacrine, ri-
Alzheimer’s disease (AD) is chronic neurodegenerative illness
with a complex pathogenesis [1,2]. Due to complexity of the dis-
ease, only symptomatic treatment is available up to present. AD
is frequently affiliated with damage of cholinergic system in the
brain, characterized by progressive memory loss, with decline in
language and finally even losing control of bodily functions, are
due, among other causes, progressive accumulation of β amyloid
proteins (Aβ) together with tau (β)-protein aggregation leading
to shrinkage and death of neurons [3]. The researchers are fo-
cused on cholinergic hypothesis because it has become the most
leading theory to explain etiology of the disease. Furthermore, al-
most all hypotheses had devoted that increase the level of acetyl-
choline in cholinergic synapses in the brain by inhibiting acetyl-
cholinesterase (AChE) could effectively improve the cognitive func-
tions and memory ability [4]. Nowadays, cholinesterase (ChE) be-
came one of major targets in the current therapy of AD [5], and
vastigmine, donepezil, and galantamine but they have severe ad-
verse effects such as hepatotoxicity and anxiety [6,7].
Diabetes mellitus (DM) is another chronic metabolic disor-
der, characterized by excessive levels of glucose in the blood [7],
resulted from defects in the action or secretion of insulin. α-
Glucosidase, a membrane bound enzyme present in the epithe-
lial wall of the small intestine, is responsible for digestion of
carbohydrate and release of glucose. In human, this metabolic
disorder results in serious health complication associated with
various diseases including cardiovascular, neuropathy problems,
retinopathy, amputations, impaired wound healing and cancer [8].
α-Glucosidase enzyme is the restorative target to treat diabetes
[9] and its inhibitors play an important role for treatment of DM
by decreasing sugar level in the bloodstream [10,11]. Currently, α-
glucosidase inhibitors such as acarbose, voglibose, and miglitol are
approved for the treatment of type-2 diabetes, but they are asso-
ciated with common undesirable effects including gastrointestinal
diseases flatulence, and diarrhea [12,13].
∗
Corresponding authors.
On the other hand, naphthoxazine derivatives and their ana-
logues exhibit a variety of interesting biological properties such
E-mail
addresses:
boulebd.houssem@umc.edu.dz
(H.
Boulebd),
a_debache@yahoo.fr (A. Debache).
https://doi.org/10.1016/j.molstruc.2020.129103
0022-2860/© 2020 Elsevier B.V. All rights reserved.

2
K. Boudebbous, H. Boulebd and C. Derabli et al. / Journal of Molecular Structure 1225 (2021) 129103
as 5-HT ligands [14], platelet fibrinogen receptor antagonists [15],
protein kinase [16], HIV inhibitor [17], anti-viral [18], antimicro-
bial [19], antitumor [20], antimalarial [21], hypertensive [22], and
antiarrhythmic [23]. In addition, molecules with 1,3-amino oxy-
gen functions have been reported to have a variety of biologi-
cal and pharmacological activities, including nucleoside antibiotics
[24] and HIV protease inhibitors [25], and are frequently found in
biologically active natural products [26]. The search for new pro-
cedures for the synthesis of naphthoxazine derivatives remains a
constant preoccupation of organic chemists with the aim of im-
proving yields, reaction times and, as far as possible, respecting
the environment. Currently the best synthesis method for naph-
thoxazinone derivatives is the multicomponent reaction between
2-naphthol, aromatic aldehydes and urea in the presence of a va-
riety of catalysts such as p-TSA [27], [bmim]Br [28], tetramethy-
lammonium hydroxide [29], silica-bonded S-sulfonic acid [30], Thi-
amine hydrochloride [31], TiCl4 [32], Cu [33], and ZnO [34].
1-phenyl-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-one (4a). White
solid (75%). IR (KBr): ν (cm−1) 3217, 2924, 2848, 2355, 1666, 1624,
1512, 1436, 1265; 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 9.74 (d,
J= 2.7 Hz, 1H, NH), 7.99 (d, J= 9.0 Hz, 1H, Ar), 7.95 (dd, J=7.2 Hz,
J= 1.8 Hz, 1H, Ar), 7.82 (d, J= 7.8 Hz, 1H, Ar), 7.51-7.43 (m, 2H,
Ar), 7.39 (d, J= 8.7 Hz, 1H, Ar), 7.33-7.22 (m, 5H, Ar), 6.20 (d, J=
2.7 Hz, 1H, CHsp3); 13C NMR (DMSO-d6, 75 MHz): δ (ppm) 149.3,
–
147.4, 142.9, 130.4, 130.2, 129.0, 128.9, 128.6, 128.0, 127.4, 127.0,
125.1, 123.1, 116.9, 114.1, 53.8.
1-(3-Bromo-4-methoxy-phenyl)-1,2-dihydro-naphtho[1,2-e][1,3]
oxazin-3-one (4b). White crystals (72%). IR (KBr): ν (cm−1) 3221,
3144,2964,1732, 1517, 1439, 1224; 1H NMR (DMSO-d6, 250 MHz):
–
δ (ppm) 8.67 (d, J= 2.6 Hz, 1H, NH), 7.87–7.81 (m, 2H, Ar), 7.58
(d, J= 8.4 Hz, 1H, Ar), 7.47–7.34 (m, 3H, Ar), 7.25 (d, J= 8.9 Hz, 1H,
Ar), 7.1 (dd, J= 8.4 Hz, J= 1.9 Hz, 1H, Ar), 6.8 (d, J= 8.5 Hz, 1H,
Ar), 6.0 (d, J= 2.6 Hz, 1H, CHsp3), 3.76 (s, 3H, OCH3); 13C NMR
(DMSO-d6, 62.9 MHz): δ (ppm) 155.1, 149.4, 147.4, 135.9, 131.5,
130.3, 130.1, 128.8, 128.4, 127.2, 124.8, 122.5, 119.1, 116.6, 112.9,
112.1, 111.1, 55.9, 53.1; HRMS (ESI+): m/z [M+Na]+calculated for
C19 H14 NO3NaBr 406.0055, found 406.0045.
–
–
In continuation of our work focused on the development of
novel heterocyclic compounds that may be useful in the treatment
of chronic disorders such as Alzheimer’s disease or diabetes mel-
litus, we are focusing on naphthoxazinone derivatives [35–38]. In
this study, we aimed to investigate the in vitro AChE, BChE, and
α-glucosidase inhibitory activities of a series of naphthoxazinone
derivatives to determine their potential for the treatments of AD
and DM. Furthermore, In Silico studies such as Hirshfeld surface,
docking study, DFT calculations, and ADME properties were per-
formed in order to support the experimental results.
1-Biphenyl-4-yl-1,2-dihydro-naphtho[1,2-e][1,3]oxazin-3-one (4c).
White-yellow crystals (66%). IR (KBr): ν (cm−1) 3222, 3140, 2924,
1731, 1513, 1437, 1222; 1H NMR (DMSO-d6, 250MHz): δ (ppm) 7.90
(d, J= 8.8 Hz, 1H), 7.88 (d, J= 8.8 Hz, 1H), 7.65–7.58 (m, 2H), 7.55–
7.43 (m, 7H), 7.42–7.29 (m, 4H), 7.10 (s, 1H), 6.20 (s, 1H); 13C NMR
(DMSO-d6, 62.9 MHz): δ (ppm) 150.8, 147.7, 141.6, 140.7, 140.3,
131.1, 130.7, 129.4, 129.0, 128.9, 128.2, 127.6, 127.6, 127.2, 125.4,
123.0, 117.2, 112.6, 55.7; HRMS (ESI+): m/z [M+Na]+calculated for
C24H17 NO2Na 374.1157, found 374.1151.
2. Experimental section
1-(thiophen-2-yl)-1, 2-dihydro-3H-naphtho[1,2-e][1,3]oxazin-3-one
(4d). Green solid (68%). IR (KBr): ν (cm−1) 3258, 3166, 2925, 1743,
1703, 1515, 1389, 1219, 1175, 725; 1H NMR (DMSO-d6, 250 MHz):
δ (ppm) 8.96 (d, J= 2.9 Hz, 1H, NH), 7.92-7.84 (m, 3H, Ar),7.52-7.40
(m, 2H, Ar), 7.28 (dd, J=5.2 Hz, J=1.1 Hz, 1H, Ar), 7.09 (d, J= 3.2 Hz,
1H, Ar), 6.90 (dd, J= 4.9 Hz, J=3.6 Hz, 1H, Ar), 6.46 (d, J= 2.9 Hz,
1H, -CHsp3); 13C NMR (DMSO-d6, 62.9MHz): δ (ppm) 149.5, 147.1,
146.5, 130.2, 130.1, 128.8, 128.4, 127.2, 126.6, 125.7, 125.3, 124.9,
122.6, 116.6, 114.0, 48.9; HRMS (ESI+): m/z [M+H]+calculated for
C16 H12NO2S 282.0589, found 282.0587.
2.1. Materials and equipments
The reagents and solvents are used without any further purifi-
cation. The melting points were determined using a digital Elec-
trothermal IA9100 apparatus. IR spectra were obtained in the form
of potassium bromide pellets (KBr) Shimadzu FT IR-8201 PC spec-
trometer. 1H and 13C NMR spectra were recorded on a Bruker
Advance DPX spectrometer at 250.13 and 62.5 MHz respectively
with the TMS as internal reference. Mass spectrometry experi-
ments were performed in positive mode, on a Synapt-G2 HDMS
Traveling Wave-Ion Mobility (TWIMS) instrument equipped with
an electrospray ionization source (Waters, Manchester, UK). Kiesel-
gel F254 (Merck) plates have been used in TLC. Crystal suitable
for single crystal X-ray diffraction was selected using a polar-
izing microscope. The crystal was coated with Paratone oil and
mounted on a loop for data collection and the lattice parameters
were determined using a Bruker APEX II CCD diffractometer (SAD-
ABS; Sheldrick, 2002). All activities were established with a thermo
Electron Corporation spectrophotometer type Helios Delta. The
measurements were carried out on a 96-well microplate reader,
PerkinElmer Multimode Plate Reader EnSpire, USA.
1-(4-ethylphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-one
(4e).
Beige solid (60%). IR (KBr): ν (cm−1) 3216, 3143, 2962, 2925, 1731,
1513, 1386, 1218, 808;1H NMR (DMSO-d6, 250 MHz): δ (ppm) 8.85
(d, J= 2.9 Hz, 1H, NH), 8.00–7.92 (m, 2H), 7.83 (dd, J= 8.1 Hz, J=
1.3 Hz, 1H, Ar), 7.51–7.36 (m, 3H, Ar), 7.24 (d, J= 8.1 Hz, 2H, Ar),
–
7.15 (d, J= 8.1 Hz, 2H, Ar), 6.17 (d, J= 2.9 Hz, 1H, CHsp3), 2.51
(q, J= 7.5 Hz, 2H, CH2), 1.10 (t, J= 7.5 Hz, 3H, CH3); 13C NMR
(DMSO-d6, 62.9MHz): δ (ppm) 149.4, 147.4, 143.6, 140.3, 130.4,
130.1, 128.9, 128.6, 128.3, 127.3, 126.9, 125.1, 123.1, 116.9, 114.2,
53.6, 27.8, 15.5.
–
–
1-(2-bromophenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-one (4f).
Orange-yellow solid (65%). IR (KBr): ν (cm−1) 3251, 3147, 2921,
1723, 1516, 1381, 1221, 742; 1H NMR (DMSO-d6, 250 MHz): δ
(ppm) 8.95 (d, J= 2.2 Hz, 1H, NH), 8.03 (d, J=8.9 Hz, 1H, Ar),
7.97 (d, J=7.5 Hz, 1H, Ar), 7.70 (dd, J= 7.9 Hz, J= 1.2 Hz, 1H, Ar),
7.58-7.39 (m, 4H, Ar), 7.33-7.19 (m, 2H, Ar), 7.13 (dd, J= 7.4 Hz, J=
2.2. Synthesis
1.5 Hz, 1H, Ar), 6.48 (d, J= 2.5 Hz, 1H, CHsp3);13C NMR (DMSO-
–
2.2.1. Synthesis of 1,2-Dihydro-1-arylnaphtho[1,2-e][1,3]oxazine-3-one
(4a-f)
d6, 62.9MHz): δ (ppm) 148.6, 147.8, 141.2, 133.3, 130.7, 130.5,
130.3, 129.7, 129.1, 128.9, 128.8, 127.7, 125.2, 122.4, 122.2, 116.9,
112.9, 54.1; HRMS (ESI+): m/z [M+H]+calculated for C18 H13NO2Br
354.0130, found 354.0128.
Phenylboronic acid (0.15 mmol) was added to a mixture of 2-
naphthol (2.0 mmol), aldehyde (2.4 mmol), and urea (2.4 mmol).
The resulting mixture was magnetically heated at 120 °C without
solvent for the appropriate time. After completion (TLC), the reac-
tion mixture was allowed to warm to room temperature, then 5
ml of iced water was added while maintaining stirring for 30 min.
The solid product was filtered and purified by column chromatog-
raphy on silica gel (Ethyl acetate: n-Hexane, 1: 2) to afforded the
title compounds.
2.3. Crystal structure analysis
X-ray diffraction data were acquired at 293 K on a Bruker
APEX II CCD diffractometer employing graphite crystal monochro-
matized MoKα radiation source (0.71073A). Data collection,

K. Boudebbous, H. Boulebd and C. Derabli et al. / Journal of Molecular Structure 1225 (2021) 129103
3
Table 1
ration online property calculation toolkit (available at: http://www.
molinspiration.com).
Single-crystal X-ray data and structure refinement details for 4b.
Compound
4b
2.5. Anticholinesterase activity assay
Formula
M r
C
19H14BrNO3
384.21
Temperature/K
293
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)
inhibitory activity was measured, by the spectrophotometric
method developed by Ellman et al [53]. Briefly, 150 μL of 100 mM
sodium phosphate buffer (pH 8.0), 10 μL of sample solution dis-
solved in methanol at different concentrations and 20 μL AChE
(5.32 × 10−3 U) or BChE (6.85 × 10−3U) solution were mixed
and incubated for 15 min at 25°C, and 10 μl of 0.5 mM DTNB
˚
Wavelength/A
0.71073
Monoclinic
P21/c, 14
11.4377(5)
17.1812(7)
9.1941(4)
90
Crystal system
Space group, N
˚
a/A
˚
b/A
˚
c/A
α/ °
β/ °
γ / °
112.806(2)
90
[5,5-dithio-bis(2-nitrobenzoic) acid] were added. The reaction was
´
3
˚
then initiated by the addition of 10 μL of acetylthiocholine iodide
(0.71 mM) or butyrylthiocholine chloride (0.2 mM). The hydroly-
sis of these substrates were monitored spectrophotometrically by
the formation of yellow 5-thio-2-nitrobenzoate anion, as the result
of the reaction of DTNB with thiocholine, released by the enzy-
matic hydrolysis of acetylthiocholine iodide or butyrylthiocholine
chloride, respectively, at a wavelength of 412 nm, every 5 min for
15 min, utilizing a 96-well microplate reader (Perkin Elmer Multi-
mode Plate Reader EnSpire, USA) in triplicate experiments. Galan-
tamine was used as reference compound. The results were given
as 50% inhibition concentration (IC50) and the percentage of inhi-
bition of AChE or BChE was determined by comparison of reaction
rates of samples relative to blank sample (methanol in phosphate
buffer, pH 8) using the equation below:
V/A
1665.52(12)
4
Z
−3
˚
Dc (g cm A
)
1.532
μ
2.48
No. of measured, independent and observed
[I ≥ 2σ(I)] reflections
Absorption correction
Tmin, Tmax
19920/5066/2781
Multi-scan
0.633, 0.746
0.040
Rint
Data/restraints/parameters
R1 /wR2 [I > 2σ(I)]
Goodness-of-fit on F2
5066/0/217
0.046/0.126
1.01
−3
˚
ꢀρmax, ꢀρmin (e A
)
0.79/-070
indexing with reduction and absorption corrections were carried
out using APEX2, SAINT and SADABS programs, respectively. The
structure was solved using direct methods (SIR92) [39] and refined
by full-matrix least-squares techniques on F2 with SHELXL-2014
[40] operating within WinGX [41]. All non-H atoms were refined
anisotropically. All H atoms were positioned geometrically and re-
E − S
x 100
Inhibition of AchE or BChE %
=
( )
E
Where E is the activity of enzyme without test sample, and S is
the activity of enzyme with test sample.
–
–
fined as riding atoms, with N H = 0.86, C H = 0.93, 0.96 and
0.98 aromatic, methylene, acetylenic and methane respectively, and
constrained to ride on their parent atoms, with U iso (H) = 1.2U
eq (C, N). General crystallographic details for compound 4b are
provided in Table 1. The final atomic coordinates, selected bond
distances, and bond angles are presented in Tables S1–S3 (sup-
porting information), respectively. The crystal of compound 4b be-
long to monoclinic systems with a centrosymmetric space group of
P21/c. The figures were made using the Mercury and POV-Ray pro-
grams [42]. Complete crystallographic data for the structural analy-
sis have been deposited with the Cambridge Crystallographic Data
Centre; CCDC reference number 1982885. These data can be ob-
tained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html
(or from the Cambridge Crystallographic Data Centre, 12 Union
Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223 336033; e-mail:
deposit@ccdc.cam.uk).
2.6. Determination of the α-glucosidase activity
The α-glucosidase capacity of compound was determined using
the method described by S. Lordan et al [54] with minor modifica-
tion. Briefly, 50 μL of sample solution at varying concentration and
100 μL of α-glucosidase from Saccharomyces cerevisiae (0.1 U/ml)
prepared in phosphate buffer were mixed in a 96-wellmicroplate.
After 50 μL of p-nitrophenyl-α-D-glucopyranoside (PNPG) (5 mM)
was then added to each well. The mixture was incubated for 30
min at 37°C and absorbance was read at 405 nm using a mi-
croplate reader. The results were compared with those of acarbose
and quercetin. The activity was expressed as percentage inhibition
using the following equation:
ꢀ
ꢁ
Acontrol − Asample
Acontrol
% Inhibition =
× 100
Where Acontrol and Asample are the absorbances of the reference and
2.4. Computational details
sample obtained from the UV visible spectrophotometer.
Molecular structure of compound 4b (representative molecule)
was optimized using DFT calculations starting from the crys-
tal structure coordinates. The Becke’s three parameters Lee-Yang-
Parr exchange correlation functional (B3LYP) [43,44] and the 6-
311++G(d,p) basis set have been used for all calculations [45].
The accuracy of this methodology has been confirmed by previ-
ous studies [46]. Vibrational frequency analysis was employed to
confirm the ground state (no imaginary frequency). Electronega-
tivity, chemical softness, chemical hardness, and electrophilic in-
dex were calculated from the frontier molecular orbitals energies
method as reported in our previous studies [47–50]. All calcula-
tions have been performed using Gaussian09 software [51]. Crys-
tal Explorer 17.5 software has been used to generate the Hirshfeld
surface [52]. ADME properties were calculated by using Molinspi-
2.7. Statistical analyses
All the experimental results are mentioned as
a mean
standard deviation of three trials. Significant differences between
means were determined by Student’s-t test, p<0.05 were regarded
as significant. IC50 values, calculated from the concentration-effect
linear regression.
2.8. Docking study methods
Docking studies of the R and S enantiomers of the most ac-
tive compounds (4b and 4c) to the proteins AChE, BChE, and α-
glucosidase were carried out with “Achilles” Blind Docking Server

4
K. Boudebbous, H. Boulebd and C. Derabli et al. / Journal of Molecular Structure 1225 (2021) 129103
Scheme 1. Synthesis of naphthoxazinone 4a-4f catalyzed by phenylboronic acid.
Table 2
Solvent-free synthesis of β-naphthalene condensed oxazinone derivatives in the
presence of phenylboronic acid.
Time Yield Melting point (˚C)
Compound Ar
(h)
(%)∗
Measured Reported
4a
4b
4c
4d
4e
4f
C6H5
3
2
4
3
4
4
75
72
66
68
60
65
218-220
216-218
232-233
208-209
215-217
243-245
217-218 [59]
3-Br-4-OMe-C6H3
4-phenyl-C6H4
2-thiophenaldehyde
4-Ethyl-C6H4
New
203-206 [59]
208-210 [60]
210-213 [61]
New
2-Br-C6H4
∗
Isolated pure product
(http://bio-hpc.eu/). Using a “blind docking” approach, the dock-
ing of the small molecule to the targets is done without a pri-
ori knowledge of the location of the binding site by the system
[55]. Figures were drawn using the BIOVIA Discovery Studio (https:
//3dsbiovia.com/). The ligand structures have been built and energy
minimized using Gaussian09 program (B3LYP/6-311++G(d,p)). The
coordinates of hAChE (PDB ID: 4EY6) [56], hBChE (PDB ID: 4BDS)
[57], and α-glucosidase (PDB: 5ZCB) [58], were obtained from the
Protein Data Bank (PDB) (https://www.rcsb.org).
Fig. 1. The asymmetric unit of compound 4b, showing the atom-numbering
scheme. Displacement ellipsoids are drawn at the 50% probability level.
Table 3
˚
Hydrogen-bond geometry (A, °) of compound 4b.
D-H…A
D-H
H…A
D…A
D-H…A
N1-H1…O3i
0.8600
0.9300
0.9300
0.9300
2.0400
2. 8500
2.7800
2.9200
2.8823(1)
3.6568(2)
3.6832(2)
3.7501(2)
167.00
146.00
164.00
143.00
3. Results and discussion
C12-H12…Br1ii
C10-H10…Cg2iii
C19-H19…Cg4iv
3.1. Chemistry
Symmetry codes : (i) -x+1, -y+1, -z+2; (ii) -x,- y+1, -z+1; (iii) x, y,
z-1; (iv) -x+1, -y, -z
The naphthoxazinone derivatives 4a-f were synthesized via
the multicomponent cyclocondensation reaction between aromatic
aldehydes (1a-f), 2-naphthol (2), and urea (3) in the presence of
phenylboronic acid under solvent-free conditions (Scheme 1). Wa-
ter was added to the reaction mixture and simply filtering, pro-
vides the crude product, which was purified by column chromatog-
raphy on silica gel (Ethyl acetate: n-Hexane, 1:2) to afford the pure
compounds. As results of our experiments we found that aromatic
aldehydes containing both electron-withdrawing groups (includ-
ing halide groups) or electron-donating groups (such as ethyl or
alkoxyl groups) reacted well to give the corresponding naphthox-
azinone derivatives in good yields. The results are summarized in
Table 2. The molecular structures of the synthesized compounds
were characterized by physical and spectroscopic techniques (see
Experimental section) and, for compound 4b, by single crystal X-
ray diffraction analysis.
parable with those previously reported [63]. The naphthalene
rings systems are essentially planar with maximum deviations
from the mean plane of 0.0106 °. The dihedral angle between
the fused rings is 3.67 (8)°. The mean plane of the naphtha-
lene ring system subtends a dihedral angle of 82.14 (8)° with
the bromo methoxy phenyl ring, while the dihedral angles be-
tween the naphthalene ring system and the six-membered ring
O(2)/C(16)/N(1)/C(19)/C(14)/C(15) is slightly puckered [64] (6.40
˚
(12) A). In the solid state, the molecular units of 4b are in-
i
ii
–
–
terconnected by intermolecular N1 H1···O3 and C12 H12…Br1
hydrogen bonds [symmetry code: (i)-x+1, -y+1, -z+2, (ii) –x, -
y+1, -z+1] (Table 3) which may result in the formation of the
dimeric chains running extended to [101] direction with R22(8)
and R2 (18) motifs [65]. These chains are linked to each other
2
iii
˚
–
3.2. Structural description of compound 4b
by two hydrogen bonds C H···π type [H10···Cg2 = 2.78 A, and
iii
–
–
C10 H10···Cg2 = 164° with Cg2 is the centroid of the C1 C6
iv
˚
Single-crystal structure analysis shows that compound 4b crys-
tallizes in the monoclinic space group P21/c, with one symmetry-
independent molecule in the asymmetric unit (Fig. 1). All bond
lengths and angles in 4b have regular values [62] and com-
phenyl ring; H19…Cg4iv = 2.92 A, and C19-H19···Cg4 = 143° with
Cg4 is the centroid of the C8-C13C14C15C16C17 naphthalene ring,
symmetry code: (iii) x, y, z-1, (iv) –x+1, -y, -z] thus generating a
three-dimensional network (Fig. 2).

K. Boudebbous, H. Boulebd and C. Derabli et al. / Journal of Molecular Structure 1225 (2021) 129103
5
Fig. 2. A view of the three-dimensional network of compound 4b, showing hydrogen bonds.
Table 4
Selected calculated and experimental bond lengths
˚
(A) and angles (°) for compound 4b.
Experimental
Calculated
˚
Bond lengths (A)
Br1-C5
1.890(2)
1.361(3)
1.392(3)
1.218(3)
1.527(3)
1.459(4)
1.358(3)
1.910
1.376
1.382
1.203
1.529
1.473
1.373
O2-C18
O2-C15
O3-C18
C1-C19
C19-N1
C14-C15
Angles (°)
C18-N1-C19
C18-O2-C15
O2-C15-C16
N1-C18-O2
N1-C19-C1
Dihedral angle (°)
N1—C19—C1—C6
127.9(2)
119.6(2)
114.3(2)
117.9(2)
110.0(2)
125.7
121.1
114.8
115.3
111.7
71.09
82.59
3.4. DFT calculations
Theoretical calculations using DFT method at B3LYP/6-
311++G(d,p) level of theory have been performed in order to
get insights into the electronic properties, chemical reactivity, and
molecular structure of the synthesized compounds. Compound 4b,
of which we have its experimental coordinates, was chosen as a
representative molecule. Firstly, a full geometry optimization has
been carried out, and the most stable structure has been compared
with the experimental results obtained by single-crystal structure
analysis. The atom-by-atom superimposition of the optimized
molecular structure of compound 4b on the crystal structure, and
selected calculated and experimental geometry parameters are
shown in Fig. 5 and Table 4, respectively. As can be seen, the
theoretical and experimental structures are perfectly aligned. The
bond lengths and angles of the two structures have nearly the
same values. The only difference was observed for the dihedral
angle of the phenyl ring, which is slightly deviated by about 10°.
After successfully optimized the molecular structure of com-
pound 4b, we then calculated, at the same level of theory, some
electronic and structural properties such as frontier molecular or-
bitals (FMO), molecular electrostatic potential mapping, atomic
charge distributions, and physicochemical descriptors.
Fig. 3. The 3D Hirshfeld surface of compound 4b mapped over dnorm in the range
–0.5534 to 1.6107 a. u.
3.3. Hirshfeld surface analysis
The Hirshfeld surface of compound 4b mapped over dnorm
have been studied in order to quantify the intermolecular con-
tacts of the crystal structure using Crystal Explorer 17.5 software
[52]. The three-dimensional Hirshfeld surface and its related two-
dimensional fingerprints are presented in Figs. 3 and 4, respec-
tively. As shown from the Hirshfeld surface, compound 4b forms
mainly two strong intermolecular hydrogen bonds between the
–
–
NH bond (N1 H1) and the carbonyl group (C18 O3) with dis-
tances shorter than Van der waals (Vdw) radii (represented by
red spots). It forms also several weak intermolecular bonds type
CH…O, CH…Br, CH…C with distances nearly equal to the sum of
Vdw radii (represented by white areas). The contribution of the
different types of contacts are presented as 2D fingerprint plots
in Fig 4. As can be seen, the different types of contacts have
nearly comparable contributions. The highest contribution was ob-
served for the H…C/C…H (28.0%) contacts followed by H…H/H…H
(26.0%), and finally H…O/O…H (23.7%).
The study of the energy and distribution of frontier molecular
orbitals provides valuable insights into chemical stability and reac-
tivity as well as potential biological mechanisms of organic com-
pounds [45,47,48,50,66–68]. In terms of chemical reactivity, the

6
K. Boudebbous, H. Boulebd and C. Derabli et al. / Journal of Molecular Structure 1225 (2021) 129103
Fig. 4. The 2D fingerprints of compound 4b resolved into, H…O/O…H, H…H/H…H, and H…C/C…H contacts (blue areas).
distribution of FMO could be used to determine the sites of elec-
trophilic and nucleophilic attacks. HOMO (highest occupied molec-
ular orbital) is the easiest orbital to donate electrons and deter-
mines the sites for nucleophilic attacks, while LUMO (lowest unoc-
cupied molecular orbital) is the easiest orbital to accept electrons
and determines the sites for electrophilic attacks. The energy gap
of HOMO-LUMO could be used to estimate the chemical stability
and reactivity of molecules. The calculated HOMO-LUMO energies
and distributions of compound 4b are depicted in Fig 6. The ob-
tained results show that both the HOMO and LUMO are mainly
distributed on the naphtyle moiety with small distributions on
the phenyl and oxazinone rings. This indicates that the electronic
charges are mainly located on the naphtyle moiety. The values of
HOMO (-6.39 eV) and LUMO (-1.78 eV) and their energy gap (4.61
eV) indicate a high kinetic stability and low chemical reactivity of
compound 4b [69,70].
It is well established that several physicochemical properties
such as vibrational spectroscopy, stability, chemical reactivity, elec-
trostatic potential, dipole moment, and solubility are mainly de-
termined by the distribution of electrons within molecules [71,72].
The atomic charge distribution of compound 4b has been calcu-
lated using the Mulliken atomic charges method. The obtained re-
sults are presented in Table S4 in SI and visualized in Fig. 7. As
shown, the highest negative charges were found to be localized
around the oxygen atom of the carbony group, and the nitrogen
atom with partial charges of -0.305 and -0.225 respectively. While,
the highest positive charges were found for carbons 14, 18, and
Fig. 5. Atom-by-atom superimposition of the optimized molecular structure of
compound 4b (red) on the crystal structure (green).

K. Boudebbous, H. Boulebd and C. Derabli et al. / Journal of Molecular Structure 1225 (2021) 129103
7
Fig. 6. HOMO and LUMO of compound 4b computed at B3LYP/6-311++G(d,p) level
of theory in the gas-phase.
Fig. 7. Mulliken atomic charges (A) and molecular electrostatic potential (B) of
compound 4b computed at B3LYP/6-311++G(d,p) level of theory in the gas-phase.
17 and the hydrogen atom of the NH bond with partial charges of
0.848, 0.569, 0.427, and 0.335 respectively.
3.5. Biology evaluation
Molecular electrostatic potential (ESP) was also computed to
visualize the electronic charge distribution of compound 4b.
Fig. 7 shows the obtained 3D mapped molecular electrostatic po-
tential. As can be seen, the obtained results are in good agreement
with Mulliken atomic charges distribution. The highest negative
charges are mainly localized around the oxygen atom of the car-
bonyl group, suggesting that this atom should be the most privi-
leged site for nucleophilic attack. Whereas, the highest positive re-
gions are around the hydrogen of the NH bond and the carbon C14,
indicating that these regions should be the most favorable sites for
electrophilic attacks.
3.5.1. Evaluation of AChE and BChE inhibition
The inhibitory potencies of selected compounds 4b-4f toward
AChE and BChE were evaluated using Ellman’s assay [53] as de-
scribed previously in detail. The inhibition studies were carried out
and experimental results were reported in Table 5. Galantamine
was used as a control for its ability to inhibit both ChEs. The
results of AChE inhibition revealed that all selected compounds
showed good to moderate degree of inhibition with IC50 values
ranging from 9.30 to 137.33 μM. The 4-phenyl-C6H4 derivative 4c
with IC50 value of 9.30 0.10 μM was found to be the most potent
compound against AChE. This compound was 2.3 times more po-
tent than the standard drug galantamine. It should be noted that
compounds 4b and 4f showed IC50 values in the same range. The
least active compounds were found to be 4e and 4d, with IC50 val-
ues equal to 125.16 and 137.33 μM, respectively.
Analyzing the structure-activity relationship (SAR), we could
observe that the nature of substituents on the aromatic ring plays
a significant role on the inhibitory potency of AChE. The best result
was obtained with the biphenyl derivative 4c substituent at para
position. Also, as observed with compounds 4b and 4f (bromine-
substituted), the 2-bromo or 3-bromo-4-methoxy substituents on
phenyl ring improved anti-AChE activity. In contrast, the insertion
of thiophenyl or ethyl group at the ortho or para position resulted
in a noticeable decrease in the inhibitory activity.
Physicochemical indicators such as electronegativity (χ), chem-
ical hardness (η), chemical softness (S), and electrophilic index (ω)
are important quantitative properties that characterizes the chem-
ical stability and reactivity of organic compounds [73–75]. χ indi-
cates the capacity of a molecule to attract electrons. η and S are in-
dications of resistance to charge transfer. ω is defined as a measure
of energy lowering associated with a maximum amount of elec-
tron flow between a donor and an acceptor [73–75]. All the men-
tioned physicochemical indicators were calculated for compound
4b using the frontier molecular orbitals method. The obtained re-
sults are shown in Table S5 in SI. It was found that compound
4b presents a chemical hardness value of 2.31 eV and a chemi-
cal softness value of 0.22 eV, suggesting a good kinetic stability
and low chemical reactivity. In terms of electronegativity and elec-
trophilic index, compound 4b is characterized by relatively high
values when compared to some typical antioxidants [47]. This in-
dicates that the electron acceptance capacity of compound 4b is
better than its electron donation capacity.
In terms of inhibitory activity against BChE, all the tested
compounds showed mild to moderate activities with IC50 values
in the range of 2.30–240.45 μM. The best result was obtained
with compound 4b, which shows an IC50 value (2.30 0.04 μM)

8
K. Boudebbous, H. Boulebd and C. Derabli et al. / Journal of Molecular Structure 1225 (2021) 129103
Table 5
Cholinesterase and α-Glucosidase inhibitory activities of selected compounds.
Compound
AChEIC50
21.24
SD (μM)a
BChEIC50
SD (μM)a
Selectivityb
α-GlucosidaseIC50
SD (μM)a
4b
0.01
0.10
0.72
0.65
0.37
4.00
2.30
0.04
0.60
0.24
2.21
4,38
2.85
0.11
1.99
0.55
1.92
10.81
1.87
-
49.63
41.07
95.27
77.63
42.53
Nt
1.81
0.76
0.89
0,52
1,01
4c
9.30
18.56
75.03
240.45
178.97
40.72
Nt
4d
137.33
125.16
16.56
21.82
Nt
4e
4f
Galantaminec
Ascarbosec
Quercetinc
426.62
12.35
2.46
0.38
Nt
Nt
-
a
Values expressed are means
S.D. of three parallel measurements.
b
c
Selectivity ratio: IC50 (BChE)/ IC50 (AChE)
Reference compounds. Nt: Not tested.
Table 6
17.7 times lower than that of the reference drug galantamine
2.85 μM). From the SAR point of view, there
was almost similarity between BChE and AChE. All the tested com-
pounds 4b-4f acted as selective AChE inhibitors, with selectivity
index ranging from 0.11 to 10.81.
Binding energies in kcal/mol of the (R)- and (S)-enantiomers of the
most active compounds (4b and 4c) and the standards galantamine
and quercetin with AChE (PDB ID: 4EY6), BChE (PDB ID: 4BDS), and
α-glucosidase (PDB: 5ZCB).
(IC50 = 40.72
Binding energies (kcal/mol)
Compound
AChE
BChE
α-Glucosidase
3.5.2. Evaluation of α-glucosidase inhibitory activity
(R)-4b
Nd
–9.0
–9.6
Nd
Nd
(S)-4b
Nd
Nd
The in vitro α-glucosidase inhibitory activity of the synthe-
sized compounds 4b-4f were carried out according to the proto-
col of S. Lordan et al with minor modification [54], using quercetin
and acarbose as reference compounds for comparative purposes.
The obtained results are presented in Table 5. It was found that
all the tested compounds are inhibitors of α-glucosidase with a
varying efficiency, having IC50 values ranging from 41.07 0.76 to
95.27 0.89 μM. Compound 4c having biphenyl group was found
to be the most active compound in the series. The second most
(R)-4c
–11.7
–13.1
–10.1
Nd
–8.0
–8.2
Nd
(S)-4c
Nd
Galantamine
Quercetin
–9.2
Nd
–8.2
Nd: not determined
AChE inhibitors such as Galantamaine, Huperzine A, Tacrine, and
Donepezil [56].
The best poses of the interactions of the R and S enantiomers
of compound 4b with BChE enzyme are presented in Fig. 9. As can
be seen, both the enantiomers of compound 4b interact by inter-
molecular hydrogen bonds, π-π stacking, and hydrophobic inter-
acts with residues GLY117, HIS438, and ALA328. The R enantiomer
of compound 4b forms also a hydrogen bond with the residue
TRP82. The residues GLY117, HIS438, and TRP82 are located in the
oxyanion hole, catalytic triad, and anionic site of the active site of
BChE respectively, which could explain the low IC50 value of com-
pound 4b [76,77].
active compound is 4f (42.53
1,01 μM) having a bromo at the
position 2 of phenyl ring. Similarly, inhibitory activity of com-
pounds 4b, 4e, and 4d was significantly higher than that of acar-
bose (IC50 = 426.62
2.46 μM), with IC50 value of 49.63, 77.63
and 95.27 μM, respectively. By comparison, all of the tested com-
pounds were found to be more potent than acarbose and less po-
tent than quercitin.
3.6. Molecular docking studies
As for the α-glucosidase enzyme, the enantiomers of compound
4b do not interact with the same amino acid residues (Fig. 10).
The R enantiomer is expected to form three hydrogen bonds with
GLU408 and LYS334 as well as two hydrophobic interactions with
ASN333 and LUS373. While, the S enantiomer forms two hydro-
gen bonds with HIS108 and TYR68, one carbon hydrogen bond
with ASP67, and several hydrophobic interactions with LYS69 and
GLU180.
Docking studies have been performed for the most active com-
pounds (4b and 4c) in order to rationalize the permission experi-
mental results. The interactions of both the R and S enantiomers of
compound 4b with the active site of AChE (PDB ID: 4EY6), as well
as the two isomers of compound 4c at the active site of BChE (PDB
ID: 4BDS) and α-glucosidase (PDB: 5ZCB) have been investigated.
For comparative purposes, the docking study has been also per-
formed for the standards galantamine and quercetin. The obtained
results of the most energetically favorable binding modes of these
compounds at the active sites of the studied enzymes are shown
in Table 6. As seen from the table, the obtained binding energies
are in good agreement with the in vitro studies. The interactions of
all the studied compounds with the target receptors are character-
ized by low binding energies ranging from −8.0 kcal/mol to −13.1
kcal/mol, comparable or lower than those of the studied standards.
The binding mode of the R and S enantiomers of compound
4b and 4c at the active site of AChE, BChE, and α-glucosidase
are depicted in Figs. 8–10. As shown in Fig. 8, both the enan-
tiomers of compound 4c interact with the active site of AChE
by forming several π-π stacking between TRP86, TYR 124, and
TYR337 residues and the phenyl and naphtyl moieties of com-
pound 4c. The (R) enantiomer form also a strong intermolecu-
lar hydrogen bond with the residue GLY122. These residues are
involved in ligand-receptor interaction of several commercialized
As can be concluded from the above discussion, the expected
interactions, together with the predicted binding affinity for the
potent molecules (4b and 4c) suggest that these compounds
should show significant AChE, BChE, and α-glucosidase inhibitory
activities, which is in good agreement with the in vitro studies.
3.7. In silico ADME analysis
In drug development, good pharmacological activity is not
enough for a compound to become a drug candidate because so
many compounds fail in clinical trials due to poor pharmacokinetic
properties. There are four main components of pharmacokinetics:
absorption, distribution, metabolism and excretion (ADME). These
are used to explain the varied characteristics of different drugs
in the body. In order to evaluate the pharmacokinetic properties
of the synthesized compounds 4a-4f, the ADME parameters of all
the compounds have been calculated using Molinspiration software

K. Boudebbous, H. Boulebd and C. Derabli et al. / Journal of Molecular Structure 1225 (2021) 129103
9
Fig. 8. Docking poses of compounds (R)-4c (A) and (S)-4c (B) in the active site of AChE enzyme.
Fig. 9. Docking poses of compounds (R)-4b (A) and (S)-4b (B) in the active site of BChE enzyme.
Fig. 10. Docking poses of compounds (R)-4c (A) and (S)-4c (B) in the active site of α-glucosidase enzyme.
and the obtained results are tabulated in Table S7 in SI. According
to the Lipinski’s rule [78], each potential drug should have no more
than one violation of the following criteria: (1) molecular mass less
than or equal to 500 daltons (optimum around 300 daltons); (2)
number of hydrogen bond acceptors less than or equal to 10 (op-
timum of approximately 5); (3) number of hydrogen bond donors
less than or equal to 5 (optimum of 2); and (4) lipophilic character
measured by the logarithm of the partition coefficient P, ie log P
greater than or equal to -2 and less than or equal to 5 (optimum
around 3). An examination of the obtained results reported in Ta-
ble S7 indicates that all the studied compounds perfectly respect
the Lipinski’s rule (no more than one violation of the mentioned
criteria). In addition, all the compounds were shown an excellent
percentage of absorption ranging from 92.58 % to 95.77 %, which
indicates that they are easily absorbed by the human body [79].
These results suggest that compounds 4a-4f have excellent phar-
macokinetic properties and could be considered as promising drug
candidates.

10
K. Boudebbous, H. Boulebd and C. Derabli et al. / Journal of Molecular Structure 1225 (2021) 129103
4. Conclusion
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In this work we have reported an environmentally friendly
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phenylboronic acid as catalyst. Molecular structures of the synthe-
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value lower than that of galantamine. While, compound 4b pre-
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CRediT authorship contribution statement
Khawla Boudebbous: Investigation. Houssem Boulebd: Soft-
ware, Formal analysis, Writing - original draft, Visualization, Writ-
ing - review & editing. Chamseddine Derabli: Writing - origi-
nal draft. Lamia Bendjeddou: Investigation. Chawki Bensouici:
Resources. Dominique Harakat: Resources. Hocine Merazig: Re-
sources. Abdelmadjid Debache: Conceptualization, Supervision.
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Supérieur et de la Recherche Scientifique, Algeria) and DGRSDT
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