Synthesis, crystal structure, biological evaluation, docking study, and DFT calculations of 1-amidoalkyl-2-naphthol derivative

Article abstract of DOI:10.1016/j.molstruc.2020.128179

An eco-friendly and efficient synthesis of N-((2,4-dichlorophenyl)(2-hydroxynaphthalen-1-yl)methyl)acrylamide (NDHA), using phenylboronic acid as catalyst, has been reported. NDHA was fully characterized by various physical and spectroscopic techniques, a

Full text of DOI:10.1016/j.molstruc.2020.128179

Journal Pre-proof
Synthesis, crystal structure, biological evaluation, docking study, and DFT
calculations of 1-amidoalkyl-2-naphthol derivative
Khawla Boudebbous, Houssem Boulebd, Raouf Boulcina, Lamia Bendjeddou, Chawki
Bensouici, Hocine Merazig, Abdelmadjid Debache
PII:
S0022-2860(20)30504-4
DOI:
https://doi.org/10.1016/j.molstruc.2020.128179
MOLSTR 128179
Reference:
To appear in: Journal of Molecular Structure
Received Date: 16 February 2020
Revised Date: 25 March 2020
Accepted Date: 31 March 2020
Please cite this article as: K. Boudebbous, H. Boulebd, R. Boulcina, L. Bendjeddou, C. Bensouici,
H. Merazig, A. Debache, Synthesis, crystal structure, biological evaluation, docking study, and DFT
calculations of 1-amidoalkyl-2-naphthol derivative, Journal of Molecular Structure (2020), doi: https://
doi.org/10.1016/j.molstruc.2020.128179.
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Khawla Boudebbous: Investigation; Houssem Boulebd: Formal analysis, Writing –
original draft, Visualization, Writing – review and editing; Raouf Boulcina:
Investigation; Lamia Bendjeddou: Investigation; Hocine Merazig: Resources;
Chawki Bensouici: Resources; Abdelmadjid Debache: Conceptualization, Validation,
Supervision.

Graphical abstract

Synthesis, crystal structure, biological evaluation, docking study, and DFT
calculations of 1-amidoalkyl-2-naphthol derivative
Khawla Boudebbousa, Houssem Boulebda,*, Raouf Boulcinaa, Lamia Bendjeddoub, Chawki
Bensouicic, Hocine Merazigb, and Abdelmadjid Debachea,*
aLaboratory of Synthesis of Molecules with Biological Interest, University of Frères Mentouri
Constantine 1, Constantine, Algeria.
bUnité de Recherche Chimie de l'Environnement et Moléculaire, Structurale, 'CHEMS',
Faculté des Sciences Exactes, Campus Tidjani Hadem, Université, Frères Mentouri
Constantine 1, 25000 Constantine, Algérie.
cCentre de Recherche en Biotechnologie, Ali Mandjli Nouvelle Ville UV 03 BP E73,
Constantine, Algérie.
*e-mail: boulebd.houssem@umc.edu.dz; a_debache@yahoo.fr
Abstract
An eco-friendly and efficient synthesis of N-((2,4-dichlorophenyl)(2-hydroxynaphthalen-1-
yl)methyl)acrylamide (NDHA), using phenylboronic acid as catalyst, has been reported.
NDHA was fully characterized by various physical and spectroscopic techniques, and the
proposed structure was confirmed by single-crystal X-ray analysis. In vitro antioxidant
activity has been carried out using DPPH, ABTS, Ferric-phenanthroline and CUPRAC assays,
and it was found that NDHA is a potent antioxidant agent. This result has been confirmed by
DFT calculations. The inhibitory potential of the synthesized compound on cholinesterase and
α-glucosidase enzymes was also investigated. The results showed that NDHA is a promising
AChE and α-glucosidase inhibitor. Binding modes between the (R) and (S) enantiomers of
NDHA and the target enzymes were determined using docking studies.
Keywords: 1-Amidoalkyl-2-naphtols; Antioxidant; Anticholinesterase, α-Glycosidase
inhibitors; Crystal structure.

1. Introduction:
Antioxidants are substances that scavenge reactive oxygen metabolites, block their generation
or enhance endogenous antioxidants capabilities[1, 2]. The use of antioxidants may minimize
the neuronal degeneration that can prevent Alzheimer’s disease progression[3]. Alzheimer’s
disease (AD) is a progressive neurodegenerative brain disorder that gradually destroys a
person’s memory and ability to maintain activities of daily living[4]. The most commonly
used treatment for Alzheimer’s disease is medicines known as acetylcholinesterase
inhibitors[5]. On the other hand, type 2 diabetes mellitus is becoming a major health problem
associated with excess morbidity and mortality[6]. Prevention of the disease has become a
major and permanent concern, thus induced the search for new therapeutic agents.
1-Amidoalkyl-2-naphthol has become an important pharmacophore due to the broad
array of biological activities presented by its derivatives, including cardiovascular[7],
hypotensive and bradycardiac[8], as well as antibacterial and antiviral[9] activities. It can be
converted to 1,3-oxazine derivatives, which have attracted strong interest to their potential
biological
activities
such
as
antibiotic[10],
antitumor[11],
antimalarial[12],
antihypertensive[13], antirheumatic[14], antipsychotic[15], antitubercular[16] and
antioxidant[17]. 1-Amidoalkyl-2-naphthol is also found in drugs commonly used in medicine,
especially in those which possess nucleoside antibiotics[18], HIV protease (Ritonavir and
Lopinavir) inhibitors[19], and non-nucleoside reverse transcriptase (Efavirenz) inhibitor[20].
In view of the wide interest in the activity profile of 1-amidoalkyl-2-naphthols in the
development of new synthesis protocols[21, 22] and the search for new bioactive
molecules[23], we describe in this paper a simple and efficient one-pot synthesis of 1-
amidoalkyl-2-naphthol derivative (NDHA), using eco-friendly and green catalyst
(phenylboronic acid), by a three-component condensation reaction of β-naphthol, aryl
aldehyde and acrylamide under thermal solvent-free conditions. This compound was fully
characterized by various physical and spectroscopic techniques, and the proposed structure
was confirmed by single-crystal X-ray analysis. The antioxidant, anticholinesterase, and α-
glucosidase inhibitory activities have been explored. In addition, some in silico studies such
as DFT calculations and molecular modeling have been investigated.

2. Experimental
2.1. Materials and methods
All chemicals were purchased from Sigma-Aldrich and were used without further purification.
The melting point was determined using a digital Electrothermal IA9100 apparatus. FT-IR
spectrum was carried out with a Shimadzu FT/IR-8201 PC spectrophotometer, Kyoto, Japan.
1H and 13C NMR spectra were obtained in DMSO-d6 solvent on a Bruker DPX250
1
spectrometer, Bruker GmbH, Karlsruhe, Germany (250 MHz for H NMR and 62.5 MHz for
13C NMR). Chemical shifts (ꢀ) were reported in parts per million (ppm). The chemical shifts
1
for H and 13C NMR are referenced to DMSO d6 solvent at 2.50 ppm and 39.51 ppm
respectively. Spin multiplicities are abbreviated as follows: broad (br), doublet (d), doublet of
doublets (dd) and triplet (t). Crystal suitable for single crystal X-ray diffraction was selected
using a polarizing 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 (SADABS; 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, Perkin Elmer Multimode Plate Reader EnSpire, USA.
2.2. Synthesis
N-((2,4-dichlorophenyl)(2-hydroxynaphthalen-1-yl)methyl)acrylamide
(NDHA)
was
prepared by the mixture of 2-naphthol (2 mmol), 2,4-dichlorobenzaldehyde (2.4 mmol) and
acrylamide (2.4 mmol) in the presence of a catalytic amount of phenylboronic acid (0.15
mmol). The resulting mixture was magnetically heated at 120 ºC without solvent for 2 h.
After completion, the reaction mixture was allowed to cool 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 chromatography on silica gel using ethyl acetate - hexane (1:2) as
eluent to afford the final pure product and crystallized from 1-buthanol.
N-((2,4-dichlorophenyl)(2-hydroxynaphthalen-1-yl)methyl)acrylamide (NDHA). Yield 92%;
orange crystals; M.P. 227-230 °C; IR (νmax/cm-1): 3390, 2921, 956, 1643, 1610, 1577, 1505,
1321, 1274, 1067, 932, 844, 811, 748, 627; 1H NMR (DMSO-d6, 250 MHz): δ (ppm) 9.96 (br,
1H, OH), 8.96 (d, 1H, J= 7.8 Hz, NH), 8.02 (d, 1H, J= 8.6 Hz, Harom), 7.84-7.77 (m, 2H,
Harom), 7.60-7.14 (m, 7H, Harom), 6.56 (dd, 1H, J= 17.1 Hz, J= 10 Hz, Hethylenic), 6.16 (dd, 1H,
J= 17 Hz, J= 1.6 Hz, Hethylenic), 5.63 (dd, 1H, J= 10.1 Hz, J= 1.8 Hz, Hethylenic); 13C NMR
(DMSO-d6, 62.9 MHz): δ (ppm) 164.1, 153.9, 138.9, 133.1, 132.8, 131.9, 131.4, 131.3, 129.9,
128.7, 128.6, 128.3, 126.7, 126.6, 126.0, 122.5, 119.3, 118.7, 116.1, 47.5.

2.3. Crystal structure analysis
X-ray diffraction data were acquired at 293 K on a Bruker APEX II CCD diffractometer
employing graphite crystal monochromatized MoKα radiation source (0.71073A). Data
collection, indexing with reduction and absorption corrections were carried out using APEX2,
SAINT and SADABS programs, respectively. The structure was solved using direct methods
(SIR92)[24] and refined by full-matrix least-squares techniques on F2 with SHELXL-2014
[25] operating within WinGX[26]. All non-H atoms were refined anisotropically. All H atoms
were positioned geometrically and refined as riding atoms, with N-H = 0.86, O-H = 0.82, C-H
= 0.93 and 0.98 aromatic, methylene, acetylenic and methane respectively, and constrained to
ride on their parent atoms, with U iso (H) = xU eq (C, N, O), where x = 1.5 for hydroxyl H,
and x = 1.2 for all other H atoms. General crystallographic details for the NDHA are provided
in Table 1. The final atomic coordinates, the selected bond distances and bond angles are
presented in Tables S1-S4 (supporting information), respectively. The figures were made
using the Mercury program[27]. Complete crystallographic data for the structural analysis
have been deposited with the Cambridge Crystallographic Data Centre; CCDC reference
number 1966270. These data can be obtained 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).
Table 1. Single-crystal X-ray data and structure refinement details for NDHA.
Compound
Formula
M r
NDHA
C20H15Cl2NO2
372.23
Temperature/K
Wavelegth/Å
Crystal system
Space group, N
a/Å
293
0.71073
Monoclinic
P21/n, 14
8.9004(4)
16.8171(6)
11.8063(5)
90
b/Å
c/Å
α/ °
94.216(2)
90
β/ °
γ / °

V/Å3
1762.37(13)
Z
4
Dc (g cm Å-3 )
1.403
No. of measured, independent and observed
[I > 2σ(I)] reflections
Rint
18852/5371/3684
0.044
Data/restraints/parameters
R1 /wR2 [I > 2σ(I) ]
Goodness-of-fit on F2
Δρmax, Δρmin (e Å−3)
5371/0/226
0.049/0.144
1.04
0.41/-036
2.4. Biological evaluation
2.4.1. Antioxidant activity
•
DPPH free radical scavenging assay. A 0.1 mM solution of DPPH in ethanol was prepared
and 4 mL of this solution was added to 1 mL of sample solutions in ethanol at different
concentrations (3.12, 6.25, 12.5, 25, 50, 100, and 200 µM). Thirty minutes later, the
absorbance was measured at 517 nm. Lower absorbance of the reaction mixture indicated
higher free radical scavenging activity. BHT and BHA, under the same conditions as the
samples and for each concentration, were used as antioxidant standards. The DPPH radical
scavenging activity was calculated using the following equation:
Aꢅꢆꢇꢈꢉꢆꢊ ꢋ Aꢌꢍꢎꢏꢊꢐ
ꢁ ꢂ
DPPH scavenging effect % ꢃ ꢄ
ꢑ ꢒ 100
Aꢅꢆꢇꢈꢉꢆꢊ
Where Acontrol and Asample are the absorbances of the reference and sample obtained from the
UV-visible spectrophotometer, respectively. The result was given as IC50 value (µM)
corresponding to the concentration of 50% inhibition.
Antioxidant activity by ABTS assay. The ABTS•+ solution was prepared by the mixture of 7
mM ABTS with 2.45 mM potassium persulfate using the water as solvent. The mixture was
left in the dark at room temperature for 12 h before use. The ABTS•+ solution was diluted in
ethanol or water to get an absorbance of 0.708±0.025 at 734 nm. 40 µL of sample solution in
ethanol at different concentrations (3.12, 6.25, 12.5, 25, 50, 100, and 200 µM) was added to
160 µL of ABTS•+ solution. The mixture was left at ambient temperature for 10 min, then the
absorbance was measured at 734 nm using a 96-well microplate reader. The experiments were
performed in triplicate. BHA, BHT were used as antioxidant standards for comparison of the
activity. Percentage inhibitions of all samples were calculated using the following equation:

Aꢅꢆꢇꢈꢉꢆꢊ ꢋ Aꢌꢍꢎꢏꢊꢐ
ꢁ ꢂ
ABTS radical scavenging activity % ꢃ ꢄ
ꢑ ꢒ 100
ꢓꢔꢕꢖꢗꢘꢕꢙ
Where Acontrol and Asample are the absorbance of the reference and sample obtained from the
UV-visible spectrophotometer, respectively. BHT and BHA were used as antioxidant
standards. The result was given as IC50 value (µM) corresponding to the concentration of 50%
inhibition.
Cupric reducing antioxidant capacity. 40 µL of the compound solution at different
concentrations (3.12, 6.25, 12.5, 25, 50, 100, and 200 µM) and 50 µL of CuCl2 solution (10
mM) were added into a 96 well round-bottomed plate. Following this, 50 µL of neocuproine
solution (7.5 mM) and 60 µL of NH4Ac buffer (1 M, pH 7.0) solution were given to each
well. After 60 min the absorbance was measured at 450 nm. The reducing capacity of the
compound was compared with those of BHA and BHT. Result was given as A0.5 (µM)
corresponding to the concentration indicating 50% absorbance intensity.
Ferric-phenanthroline assay. 10 µL of the compound solution at different concentrations
(3.12, 6.25, 12.5, 25, 50, 100, and 200 µM), 50 µL of 0.2% FeCl3 solution in ethanol and 30
µL of 0.5% 1,10-phenanthroline solution in ethanol were placed into a 96 well round-
bottomed plate and made up to volume of 110 µL with ethanol. The obtained solution was
mixed and left at room temperature in a dark. After 20 min, the absorbance of an orange-red
solution was measured at 510 nm. The result was given as IC50 value (µM) corresponding to
the concentration of 50% inhibition compared with the absorbance of BHA and BHT.
2.4.2. Anticholinesterase activity
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity was
measured, by the spectrophotometric method developed by Ellman et al[28]. Briefly, 150 µL
of 100 mM sodium phosphate buffer (pH 8.0), 10 µL of sample solution dissolved in
methanol at different concentrations (3.12, 6.25, 12.5, 25, 50, 100, 200, 400, 600, and 800
µM) and 20 µL AChE (5.32 x10-3 U) or BChE (6.85 x10-3U) solution were mixed and
incubated for 15 min at 25°C, and 10 µL of 0.5 mM DTNB [5,5’-dithio-bis(2-nitrobenzoic)
acid] were added. The reaction was then initiated by the addition of 10 µL of
acetylthiocholine iodide (0.71 mM) or butyrylthiocholine chloride (0.2 mM). The hydrolysis
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
enzymatic hydrolysis of acetylthiocholine iodide or butyrylthiocholine chloride, respectively,
at a wavelength of 412 nm, every 5 min for 15 min, utilising a 96-well microplate reader

(Perkin Elmer Multimode Plate Reader EnSpire, USA) in triplicate experiments.
Galanthamine was used as reference compound. The results were given as 50% inhibition
concentration (IC50) and the percentage of inhibition 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:
ꢚꢛꢜ
ꢁ ꢂ
Inhibition of AchE or BChE % ꢃ
ꢒ 100
ꢚ
Where E is the activity of enzyme without test sample, and S is the activity of enzyme with
test sample.
2.4.3. α-glucosidase inhibitory activity
The α-glucosidase capacity of compound was determined using the method described by
Lordan et al[29] with minor modification. Briefly, 50 µL of sample solution at varying
concentration (3.12, 6.25, 12.5, 25, 50, 100, 200, 400, 600, and 800 µM) and 100 µl of α-
glucosidase from Saccharomyces cerevisiae (0.1 U/ml) in phosphate buffer were mixed in a
96-well microplate. After that 50 µL of p-nitrophenyl-α-D-glucopyranoside (PNPG) (5 mM)
was added to each well. The mixture was incubated for 30 min at 37°C and absorbance was
read at 405 nm using a microplate reader. The results were compared with those of Acarbose
and Quercetin. The activity was expressed as percentage inhibition using the following
equation:
A
ꢅꢆꢇꢈꢉꢆꢊ ꢋ Aꢌꢍꢎꢏꢊꢐ
% Inhibition ꢃ ꢄ
ꢑ ꢒ 100
ꢓꢔꢕꢖꢗꢘꢕꢙ
Where Acontrol and Asample are the absorbances of the reference and sample obtained from the
UV-visible spectrophotometer.
2.5. Computational details
Density functional theory (DFT) calculations have been carried out using Gaussian09
software[30]. The B3LYP functional[31, 32] and the 6-311G(d,p) basis set have been used for
all calculations. This approach has been used successfully by several research groups and
good agreement between theory and experiment was found[33-36]. Solvent effect of ethanol
was approximated by the integral equation formalism of polarizable continuum model (IEF-
PCM). All the ground states were confirmed by vibrational frequency analysis (no imaginary
frequency). The numerical descriptors of the antioxidant mechanisms (BDE, IP, PDE, PA,
ETE) have been calculated as described in our previous studies[36-39].

2.6. Molecular modeling methods
In order to investigate the possible binding modes of the (R) and (S) enantiomers of NDHA to
the proteins AChE, BChE and α-glucosidase, calculations were carried out with “Achilles”
Blind Docking Server (http://bio-hpc.eu/). Using a “blind docking” approach, the docking of
the small molecule to the targets is done without a priori knowledge of the location of the
binding site by the system[40]. Figures were drawn using the BIOVIA Discovery Studio
(https://3dsbiovia.com/). The ligand structures have been built and energy minimized using
the program MarvinSketch [ChemAxon ltd, Budapest, Hungry]. The coordinates of hAChE
(PDB ID: 1B41)[41], hBChE (PDB ID: 6EP4)[42] and α-glucosidase (PDB: 5ZCB)[43], were
obtained from the Protein Data Bank (PDB) (https://www.rcsb.org).
3. Results and discussion
3.1. Synthesis
N-((2,4-dichlorophenyl)(2-hydroxynaphthalen-1-yl)methyl)acrylamide
(NDHA)
was
synthesized as outlined in Scheme 1. A mixture of 1 eq. of 2-naphthol, 1.2 eq. of 4-
dichlorobenzaldehyde and 1.2 eq. of acrylamide was heated at 120 ºC under solvent-free
conditions for 2 h in the presence of a catalytic amount of phenylboronic acid (0.075 eq.). The
desired product was obtained after purification with 92% of yield. A plausible mechanism of
the reaction is illustrated in Scheme 2. The proposed molecular structure of NDHA was
1
13
confirmed by FT-IR, H NMR, C NMR spectroscopy techniques and single crystal X-ray
diffraction analysis.
The IR spectrum exhibits broad absorption band at 3395 cm-1 for O-H stretching and a
sharp band at 3057 cm-1 for N-H stretching of amide. The presence of amide group was
apparent from strong absorptions at 1615 cm-1 (C=O stretching) and 1508 cm-1 (C-N
stretching)[44]. The bands around 3000 cm-1 can be attributed to the N-H stretching mode of
the amine group[45]. The strong peak at 1610 cm-1 can be ascribed to the characteristic band
of the ν(C=O) stretching vibration of the lateral chain[45, 46]. The peaks around 1400 and
1600 cm-1 can be ascribed to the stretching modes of the benzene-ring skeleton[45]. The
absorption band around 600 cm-1 belong to the characteristic C-H deformation vibration.
Finally, the peaks at 2921cm-1 can be ascribed to the characteristic band of the C-H bond ν
(C-H).
The NMR spectrum (see Fig. S2 and Fig. S3 in SI) of NDHA was recorded in DMSO-
1
1
d6. The H resonances were assigned on the basis of chemical shifts. The H NMR spectrum

exhibits a broad signal at 9.96 ppm, attributed to the presence of OH group. Two doublet
signals at 8.96 ppm (J=7.8 Hz) and 8.02 ppm (J=8.6 Hz) assigned to NH group and an
aromatic proton, respectively. Two additional signals that appear as multiplets in the range
7.84-7.77 ppm (2H) and 7.60-7.14 ppm (7H) corresponds to the other aromatic hydrogens.
Additionally, ethylenic protons shows three doublets of doublets at 6.56, 6.16 and 5.63 ppm
with characteristic coupling constants of 17.0, 10.1 and 1.8 Hz attributable to trans, cis and
13
germinal coupled proton pairs respectively as in vinyl group. In the C NMR spectrum, the
signal resonated in the downfield region at 164.1 ppm is assigned to the amide carbon. The
appearance of a collection of signals in the region 138.9-116.1 ppm is ambiguously assigned
to aryl carbons. Another signal resonated in the downfield region at 153.9 ppm belongs to C-
OH aryl carbon. In the aliphatic region, the signal at 47.5 ppm is assigned to the sole sp3
hybridized carbon.
Scheme 1. Synthesis of 1-amidoalkyl-2-naphthol derivative (NDHA) catalyzed by PhB(OH)2.

Scheme 2. Proposed mechanism for the synthesis of NDHA.
3.2. Crystal structure
Single-crystal structure analysis shows that NDHA crystallizes in the monoclinic space group
P21/n, with one symmetry-independent molecule in the asymmetric unit (Fig. 1). All bond
lengths and angles in this compound have regular values[47]. The bond distance of C18=O2
is 1.238 (2) Å, which is typical for double bonds[48]. The naphthalene rings systems are
essentially planar with maximum deviations from the mean plane of 0.0359. The dihedral
angle between the fused rings is 3.67 (8)°. The mean plane of the naphthalene ring system
subtends a dihedral angle of 87.26 (5) Å with the dichlorophenyl ring, while the dihedral
angles between the naphthalene ring system and the acrylamide group is 67.69 (12) Å. These
results are in good agreement with those in previously reported structures[49, 50]. Atom O1
deviating by 6.57 (2) Å from the least squares plane of the naphthalene ring. In the solid state,
the molecular units of NDHA are interconnected by intermolecular O1—H1··· O2i and C20-
H20A···O1ii hydrogen bonds with the O2 and O1 atoms from acrynamide and hydroxyl group
respectively to form a one-dimensional chain C(8) and Rꢝꢝ (7) motif [51] running parallel to
the [101] direction (Fig. 2). The N-H group of NDHA is involved in an intramolecular N1-
H1···O1 hydrogen bond with the 2-hydroxy group in the same molecule (Table S4 in SI).
These one-dimensional chains are linked into a two-dimensional layer by C11—H11···O2i
hydrogen bonds. An Rꢟꢞ (24) motif is thus formed by the combination of O1—H1···O2i and

C11—H11···O2i interactions. These sheets unfold along the crystallographic b axis (Fig. 3).
A two-dimensional supramolecular array is additionally stabilized by four C-H···π type
(Table S4 in SI) [H2···Cg2IV = 2.88 Å, and C2-H2···Cg2IV = 131°; Cg2 is the centroid of the
C8–C13 phenantroline ring; H2···Cg4IV = 2.99Å and C2-H2···Cg4IV = 119°; Cg4 is the
centroid of the naphthyl ring; H3 ···Cg3IV = 2.77Å and C3-H3 ···Cg3IV = 142°; Cg3 is the
centroid of the C12-C17 phenantroline ring; symmetry code: (iv) x-1/2, -y-1/2, z-1/2;
H20A···Cg1v = 2.92Å and C20-H20A···Cg1v = 130°; Cg1 is the centroid of the C1-C6
phenyl ring; symmetry code: (v): x-1/2, -y-1/2, z-3/2 ] (Fig. 3). The two-dimensional sheets
are further linked by C5-Cl2...Cg1vii interactions and C20-H20B···Cg3vi hydrogen bonds to
generate a three-dimensional network (Fig. 4), with cg3 is the centroid of the C12–C17
phenantroline ring (H20B ···Cg3vi= 2.88Å and C20-H20B ···Cg3vi = 156°) symmetry code:
(vi) -x+3/2, y-1/2, -z+1/2; vii: 2-x,1-y,1-z.
Fig. 1. The asymmetric unit of title compound, showing the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.

Fig. 2. The one-dimensional chain C(6) formed through O-H···O intermolecular hydrogen
bonds in (1) [Symmetry codes: (i) x-1/2, -y+1/2, z+1/2].
Fig. 3. A view of the two-dimensional network of the title compound, showing the N- H···O
and O-H···O, C-H···O and C-H···.π (blue and red dashed lines) hydrogen bonds. [Symmetry
codes: (i) x-1/2, -y+1/2, z+1/2; (ii) x-1, y, z; (iii) x+1/2, -y+1/2, z-1/2; (iv) x-1/2, -y-1/2, z-1/2;
(v) x-1/2, -y-1/2, z-3/2; (vi) -x+3/2, y-1/2, -z+1/2].

Fig. 4. A view of the three-dimensional network of the title compound, showing the C-Cl···π
interactions (pink dashed lines) and C-H···π hydrogen bonds (purple dashed lines).
[Symmetry codes: (vi) -x+3/2, y-1/2, -z+1/2; vii: 2-x,1-y,1-z].
3.3. In vitro biological evaluation
3.3.1. Antioxidant activity
An approach with multiple assays for evaluating the antioxidant potential of the synthesized
compound would be more informative and even necessary. Therefore, the antioxidant activity
was screened using several complementary assays including, DPPH radical scavenging,
ABTS cation radical scavenging, Ferric-phenanthroline and CUPRAC assays. The obtained
results are tabulated in Table 2. Although NDHA showed moderate antioxidant activity in
DPPH assay (IC50> 200µM), it showed good antioxidant activity in the other assays. In ABTS
assay the IC50 value of NDHA (15.25±0.10 µM) is comparable to that of BHT (IC50:
16.00±0.27 µM) and slightly higher than that of BHA (IC50: 10.18±0.13 µM). In CUPRAC
assay NDHA is slightly less active (A0.50: 47.37±2.20 µM) than the standards BHT and BHA
(A0.50: 36.62±0.27 µM and 24.28±3.22 µM, respectively). Finally, in the Ferric-
phenanthroline assay, NDHA exhibited comparable antioxidant activity (IC50: 8.05±0.40 µM)
to that of the BHA (IC50: 10.00±0.07 µM) and higher than that of the BHT (IC50:
23.40±0.57µM). As can be concluded from the obtained results, the synthesized NDHA could
be considered as a potent antioxidant agent when compared to the standards BHT and BHA.

Table 2. IC50 and A0.5 values in µM of antioxidant activity of NDHA by the DPPH, ABTS
Ferric-phenanthroline and CUPRAC assays.
Ferric-
Compound DPPH assay
ABTS assay
CUPRAC assay
phenanthroline assay
8.05±0.40
>200
15.25±0.10
10.18±0.13
16.00±0.27
47.37±2.20
36.62±0.27
24.28±3.22
NDHA
BHA
22.35±0.25
10.00±0.07
BHT
157.02±0.87
23.40±0.57
* IC50 and A0.50 values (µM) expressed are means ± SD of three parallel measurements (p <
0.05).
3.3.2 Anticholinesterase activity
In search for potent cholinesterase inhibitors, the synthesized compound NDHA was
evaluated in vitro for its AChE and BChE inhibitory activities using a modifier Ellman’s
assay[52]. Galantamine, used for mild Alzheimer’s disease, was used as positive control. The
obtained results are tabulated in Table 3. As can be seen, the obtained IC50 values against
AChE enzyme reveals that NDHA exhibits comparable inhibitory activity (IC50: 25.52±0.13
µM) to that of galantamine (IC50: 21.82±4.00 µM). However, in term of anti-BChE activity, it
has weak inhibitory activity (IC50: 462.67±5.24 µM). These results indicate that NDHA is a
good selective (selectivity 18.12) AChE inhibitor when compared to the standards
Galantamine.
Table 3. Acetylcholinesterase and butyrylcholinesterase inhibitory activities of NDHA.
Compound AChE (IC50 µMa) BChE (IC50 µMa)
Selectivityb
25.52±0.13
21.82±4.00
462.67±5.24
40.72±2.85
18.12
NDHA
Galantamine
1.86
aIC50 Values represent the means ± SD of three parallel measurements (p < 0.05).
bSelectivity for AChE defined as IC50(BChE)/IC50(AChE).
3.3.3. α-glucosidase inhibitory activity
The α-Glucosidases are enzymes specialized in the digestion of sugar. α-glucosidase
inhibitors are considered to be anti-diabetic agents used for type 2 diabetes mellitus, which
work by preventing the digestion of carbohydrates. The α-glucosidase inhibitory activity of
the synthesized compound NDHA was evaluated at lower concentrations (46.875-750 µM),
using the α-glucosidase inhibitors standards Acarbose and Quercetin. The obtained results are

shown in Table 4. According to Table 4, NDHA exhibited high α-glucosidase inhibitory
activity when compared to the standards Acarbose and Quercetin. The IC50 value of NDHA
(49.21±0.61 µM) is about eight times lower than that of Acarbose (426.62±2.46 µM), and
only about four times higher than that of Quercetin (12.35 ± 0.38 µM). These results indicate
clearly that NDHA is a potent α-glucosidase inhibitor and could be considered as promising
anti-diabetic agent.
Table 4. α-glucosidase inhibitory activity of NDHA.
Compound
NDHA
IC50 ± SD (µM)*
49.21±0.61
Acarbose
426.62±2.46
Quercetin
12.35 ± 0.38
*Values expressed are means±S.D. of three parallel measurements.
3.4. In silico investigations
3.4.1. DFT study of the antioxidant activity
It is widely recognized that the antioxidant action can accrue according to three main
mechanisms namely, hydrogen atom transfer (HAT), sequential electron transfer proton
transfer (SETPT), and sequential proton loss electron transfer (SPLET)[37, 53-56]. As
described in our previous studies[36-38], these three mechanisms are characterized by several
thermodynamic indicators such as BDE (bond dissociation enthalpy), IP (ionization
potential), PDE (proton dissociation enthalpy), PA (proton affinity) and ETE (electron
transfer enthalpy).
To have a better understanding of the antioxidant activity of the synthesized NDHA
and in which mechanism its follow to scavenge free radicals, the three mentioned antioxidant
mechanisms have been investigated by computing the thermodynamic descriptors BDE, IP,
PDE, PA and ETE using DFT method at B3LYP/6-311G(d,p) level of theory. As NDHA
have two possible sites (NH and OH bonds) for the antioxidant activity, both these bonds
have been considered in our investigation. For comparison, the thermodynamic descriptors
have also been computed for the standards BHT. As the experiments were conducted in
EtOH, the implicitly of this solvent has been also considered by employing the integral
equation formalism of polarizable continuum model (IEF-PCM). The obtained results are
depicted in Fig. 5 and tabulated in Table S5 in SI.

Fig. 5. Thermodynamic descriptors, BDE (bond dissociation enthalpy), IP (ionization
potential), PDE (proton dissociation enthalpy), PA (proton affinity), and ETE (electron
transfer enthalpy), of the antioxidant mechanisms for NDHA (OH and NH bonds) and BHT
in the gas-phase and EtOH.
As shown in Fig. 5 and Table S5, the BDE values of NDHA OH bond (76.19 and
78.52 kcal/mol) are significantly lower than those of the NH bond (103.06 and 106.86
kcal/mol), suggestion that the OH bond is most reactive via HAT mechanism. By comparison,
DBEs of BHT are about 5 kcal/mol lower than those of the OH bond of NDHA in the two
studied mediums. This indicates that BHT is slightly more reactive than NDHA through HAT
mechanism.
The IP values of NDHA are about 3 kcal/mol and 1 kcal/mol lower than those of BHT
in gas-phase and ethanol, respectively. While the PDE values of BHT are lower than those of
NDHA by about 8 kcal/mol and 4 kcal/mol in gas-phase and ethanol, respectively. This
indicates that both NDHA and BHT have comparable antioxidant activity via SETPT
mechanism in the studied mediums. For the NH bond both IP and PDE are significantly
higher than those of the OH bond. Due to the high solvation enthalpies of electron and proton
in solution, all the IP and PDE values have been decreased in ethanol by about 60 kcal/mol
and 210 kcal/mol, respectively. These observations are consistent with those of previous
studies on phenolic compounds [57, 58].
In gas-phase and ethanol, PA of NDHA-OH (324.53 and 40.98 kcal/mol) are lower
than those of BHT (343.76 and 52.01 kcal/mol), while ETE of BHT (43.89 and 59.13
kcal/mol) are lower than those of NDHA-OH (68.89 and 77.71 kcal/mol), which leads to
comparable antioxidant activity via SPLET mechanism. The NH bond is also not favorable

for this mechanism. In ethanol, and due to the large solvation enthalpies of the proton, all the
PA values are decreased by about 290 kcal/mol. In contrast, ETE values are slightly increased
in ethanol by 20 kcal/mol. These results are also in line with previous studies[54, 59, 60].
Thermodynamically, the antioxidant preferred mechanism can be determined by
comparing the BDE, IP, and PA values[37, 57, 61]. As shown in Fig. 5 and Table S5, for both
NDHA and BHT, the IP and PA values are significantly higher than the BDE values in the
gas-phase, indicating that the HAT is the dominates mechanism in this medium. In ethanol,
the large decrease observed with PA values makes SPLET the more favorable mechanism.
Beside the antioxidant mechanisms (HAT, SPLET, and SPTET), the antioxidant
activity could be characterized by other parameters such as highest occupied molecular orbital
(HOMO), molecular electrostatic potential (MEP) mapping, and spin density distribution [36,
37, 61, 62]. The energy of the HOMO is correlated with the electron-donating ability, while
its distribution determines the sites for the free radical attack. Molecular electrostatic potential
determines the possible sites for free radical attack. Spin density distribution is an indication
of stability of radical after abstraction of H atom. Using the same level of theory as described
above, we have computed all the mentioned antioxidant indicators for NDHA and BHT for
comparison, and the obtained results are shown in Fig. 6.
Fig. 6. HOMO distribution and energy, molecular electrostatic potential (MEP) mapping, and
spin density distribution of NDHA obtained, in the gas-phase, at B3LYP/6-311G(d,p) level of
theory.
As can be seen from Fig. 6, The HOMO of NDHA is mainly distributed on the
naphthyl ring with small contributions on the α,β-unsaturated carbonyl moiety. This suggests

that the OH group is the most privileged site for free radical attack. This result is confirmed
by the MEP, which indicates that the most electron-deficient site for NDHA is located around
the phenolic hydroxyl group. By comparison, HOMO energy of NDHA is comparable to that
of BHT with values of -5.98 eV and -5.74 eV, respectively. This indicates that the electron
donating ability of NDHA is as higher as BHT, which is in agreement with the experimental
results. For example, IC50 values of NDHA in ABTS and Ferric-phenanthroline assays
(15.25±0.10 and 8.05±0.40 µM, respectively) are comparable or lower than those of BHT
(16.00±0.27 and 23.40±0.57 µM, respectively). Finally, spin population for NDHA appears to
be more delocalized for radical issued from the OH bond (0.307) than for that located on the
NH bond (0.708), and comparable to that of the BHT (0.351).
3.4.2. Molecular docking studies
To gain more insights on the suitability of the NDHA to act as AChE, BChE and α-
glucosidase inhibitor, we have performed docking simulations for both the (R) and (S)
enantiomers of NDHA, as well as for the standards galantamine and quercetin for
comparison. The obtained results for the most energetically favorable binding modes of these
compounds are tabulated in Table 6. As can be seen from Table 6, both the enantiomers of
NDHA could favorably interact with AChE, BChE and α-glucosidase enzymes as can be
concluded from their low binding energies ranging from -7.40 kcal/mol to -9.40 kcal/mol. By
comparison, the energies of enantiomers of NDHA with AChE are lower than those with
BChE, which indicates that the interaction with AChE is more favorable than BChE. This
result is in good agreement with the obtained, in vitro, IC50 values (25.52±0.13 µM for AChE
and 462.67±5.24 for BChE).
Table 6. Binding energies in kcal/mol for the (R) and (S) enantiomers of NDHA with AChE
(PDB ID: 1B41), BChE (PDB ID: 6EP4) and α-glucosidase (PDB ID: 5ZCB).
Binding energies (kcal/mol)
Compound
AChE
-9.40
-9.10
-8.30
nd
BChE
-7.90
-8.50
-7.70
nd
α-glucosidase
-7.80
(S)-NDHA
(R)-NDHA
-7.40
Galantamine
Quercitin
nd
-8.20
nd: not determined

The binding poses of the (R) and (S) enantiomers of NDHA at the active site of AChE,
BChE, and α-glucosidase are presented in Fig. 7 and Fig. 8. For the AChE enzyme, the (R)
enantiomer is expected to forms two hydrogen bonds between the OH group and residues
SER203 and GLY121. It forms also, by the naphthyl moiety and the Cl atoms, several π−π
stacking and hydrophobic interactions with HIS447 and TRP86. The (S) enantiomer interacts
favorably with residues SER203, HIS447, GLU202, TYR133, TYR449, TYR337, TRP439,
TRP86, GLY120, and TYR124. SER203 and HIS447 forms two hydrogen bonds with the
carbonyl moiety. TYR133 forms one hydrogen bond with the OH group. GLU202 interacts
with the Cl atom by halogen bond. TYR449, TYR337, TRP439, TRP86, GLY120, and
TYR124 make several π-π stacking and hydrophobic interactions with the Cl, phenyl and
naphthyl moieties. Some of these residues are reportedly involved in ligand-receptor
complexes of Tacrine, Galantamaine, Huperzine A, and Donepezil[63].
For the BChE enzyme, the (R) enantiomer makes three hydrogen bonds between the
OH and carbonyl groups and residues VAL288 and SER287. π−π stacking and hydrophobic
interactions are also expected to form between the Cl and naphthyl moieties and residues
TYR396, LEU286, GLU238, and ARG242. The (S) enantiomer does not show any hydrogen
bond, is stabilized only by hydrophobic interactions with VAL233, PRO230, TRP522, and
PRO401.
Finally, for α-glucosidase enzyme, the (R) enantiomer interacts with GLU141 by
mean of a hydrogen bond between the NH group of NDHA and the carbonyl of GLY141. It
forms also four hydrophobic and two electrostatic interactions with PHE225, PRO223,
GLU141, and LYS290. The (S) enantiomer is stabilized via the OH and NH groups by three
hydrogen bonds with residues GLY271, GLY274, and LYS242, and by hydrophobic
interactions with ASN277 and TRP6 through the phenyl and naphthyl moieties.
As can be concluded from the above discussion, (R) and (S) enantiomers of NDHA
displayed stronger binding affinity towards the AChE, BChE and α-glucosidase enzymes
through multiple hydrogen bonds, π-π stacking and hydrophobic interactions.

Fig. 7. Docking poses of compounds (R)-NDHA and (S)-NDHA in the active site of AChE
and BChE enzymes.
Fig. 8. Docking poses of compounds (R)-NDHA and (S)-NDHA in the active site of α-
glucosidase enzyme.

4. Conclusion
In this work, we have described a simple and efficient green protocol for one-pot preparation
of new 1-amidoalkyl-2-naphthol derivative (NDHA) using ecofriendly and green catalyst.
This compound has been characterized by various physical and spectroscopic techniques
1
including FT-IR, H NMR, 13C NMR and single crystal X-ray diffraction. The in vitro
biological evaluation was revealed that NDHA is a promising antioxidant and potent AChE
and α-glucosidase inhibitor with IC50 values of 8.05±0.40 µM (Ferric-phenanthroline assay),
25.52±0.13 µM, and 49.21±0.61 µM, respectively. The DFT study on the antioxidant activity
suggests that the OH bond is the most favorable site for free radical attack, and thus defines
the antioxidant properties of this compound. Finally, molecular docking studies revealed a
strong interaction between the two enantiomers of NDHA and the active sites of AChE,
BChE and α-glucosidase enzymes, which confirms the in vitro results.
Acknowledgements
We would like to thank MESRS (Ministère de l’Enseignement Supérieur et de la Recherche
Scientifique, Algeria) and DGRST (Direction Générale de la Recherche Scientifique et
Technologique, Algeria) for financial support. H.B. thanks the HPC resources of UCI-UFMC
(Unité de Calcul Intesif) of the university Fréres Mentouri Constantine 1 for the
computational resources used.
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Highlights
•
•
•
•
Eco-friendly and efficient synthesis of 1-amidoalkyl-2-naphthol derivative (NDHA)
has been reported.
Molecular structure of NDHA was full characterized by physical and spectroscopic
techniques, and confirmed by X-ray analysis.
Antioxidant, anti-cholinesterase and α-glucosidase inhibitory activities have been
studies.
In silico studies including DFT calculations and molecular modeling have been
investigated.

Declaration of interests
☒ 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: