Journal of Molecular Structure 1209 (2020) 127902

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Journal of Molecular Structure

journal homepage: http://www.elsevier.com/locate/molstruc

Synthesis, biological evaluation and molecular docking studies of

novel 2-alkylthiopyrimidino-tacrines as anticholinesterase agents and
their DFT calculations
Chamseddine Derabli a, b, Houssem Boulebd b, Ahmed B. Abdelwahab c,
Celia Boucheraine a, Sarah Zerrouki a, Chawki Bensouici d, Gilbert Kirsch e,
Raouf Boulcina b, f, *, Abdelmadjid Debache b
a
National Higher School of Biotechnology “Toufik Khaznadar” University City Ali Mendjeli, 25100, Constantine, Algeria
b
Laboratory of Synthesis of Molecules with Biological Interest, Fr

eres Mentouri Constantine 1 University, 25000, Constantine, Algeria
c
Plant Advanced Technologies (PAT), 19 avenue de la For^ et de Haye, 54500, Vandoeuvre-l
es-Nancy, France
d
Biotechnology Research Center, Constantine, Algeria
e
L2CM, Lorraine University, 1 Boulevard Arago, 57070, France
f
Faculty of Technology, Batna 2 University, 05000, Batna, Algeria

a r t i c l e i n f o a b s t r a c t

Article history: To search for effective and selective inhibitors of cholinesterases (AChE and BuChE), a series of poly-
Received 18 January 2020 functionalized Tacrine-derived compounds specifically 2-(alkylthio)-4-aryl-6,7,8,9-tetrahydropyrimido
Accepted 13 February 2020 [4,5-b]quinolin-5-amines were designed an synthesized via Friedlander reaction. The structures of the
Available online 14 February 2020
newly synthesized compounds were confirmed on the basis of their spectral data (1H NMR, 13C NMR) and
elemental analyses (CHNS). Compounds 3a-h were evaluated for their abilities to inhibit AChE and BChE.
Keywords:
The obtained biological results revealed that some synthesized compounds displayed higher anti-
Alzheimer’s disease
cholinesterase activity in comparison to Galantamine. Among them, compound 3d bearing S-ethyl and
2-Alkylthiopyrimidino-Tacrines
Inhibitors of cholinesterases
4-chlorophenyl moieties showed the most potent activity against AChE/BuChE with IC50s values of 4,32
Molecular docking and 15,10 mM, respectively. The anti-AChE activity of 3d was 5-fold more than that of reference drug
DFT calculations Galantamine. Moreover, molecular docking studies were performed for the most active derivatives, 3d
and 3c, in which binding mode between these compounds and the receptors were determined. Density
functional theory (DFT) method at B3LYP/6e311þþG (d,p) level of theory was employed to gain insights
into the molecular structure of the target compounds. Molecular electrostatic potential (MEP) mapping,
reactivity indices such as electronegativity, electrophilic index, softness and hardness as well as frontier
molecular orbitals HOMO-LUMO have been investigated for 3a-3c as representative compounds. In
addition, their antiradical activity has been predicted by computing bond dissociation enthalpies (BDEs).
© 2020 Elsevier B.V. All rights reserved.

1. Introduction interested in this pathology, which affects about 60e70% of elderly

The human brain is so complex that scientists are constantly
researching its anatomy, physiology and dysfunctions at the root of
the various diseases that can destabilize it, the most important of
which are neurodegenerative diseases. In view of the gravity and
extent of the progression of Alzheimer’s disease (AD), scientists are
* Corresponding author. Laboratory of Synthesis of Molecules with Biological

res Mentouri Constantine 1 University, 25000, Constantine, Algeria.
Interest, Fre
E-mail addresses: boulcinaraouf@yahoo.fr, r.boulcina@univ-batna2.dz
(R. Boulcina).
people with dementia [1,2]. AD is a significant problem for old
people worldwide, it is one of the neurodegenerative diseases that
affects the elderly by inducing irreversible memory loss [3]. This
disease characterized by neurofibrillary degeneration with a
continuous, progressive and irreversible decline in cognitive func-
tion until the ability to perform daily tasks is completely lost [4,5].
From a pathophysiological point of view, b-amyloid plaques and
neurofibrillary tangles are the major pathological signs in the brain
of AD patients [6]. Also, there is no cure for AD, only four drugs have
been approved for AD therapy: Memantine, a N-methyl-D-aspar-
tate receptor antagonist [7,8]; three of them are

https://doi.org/10.1016/j.molstruc.2020.127902
0022-2860/© 2020 Elsevier B.V. All rights reserved.
2 C. Derabli et al. / Journal of Molecular Structure 1209 (2020) 127902
O without further purification. All solvents used for spectroscopic and
R2 NH2
NH2 O N
N H
A
R1
N S N N
HO
Tacrine 3a-h
Galantamine
O NH2
O O N Elemental analyses were carried out on a Microanalyzer Flash
N O
O N
Donepezil Rivastigmine Memantine
Fig. 1. Drugs for Alzheimer’s disease and the structure of compounds 3a-h described in
this work.
acetylcholinesterase inhibitors (AChEIs) including Donepezil,
Rivastigmine, and Galantamine [9], able to restore the neuro-
transmitter acetylcholine levels (Fig. 1).
Tacrine, the first marketed AChEI which significantly improved
certain functions of recognition, reasoning, perception, attention,
decision-making and many others.
Indeed, it restarted brain chemistry by restoring neurotrans-
mission at the synapses by protecting acetylcholine and butyr-
ylcholine neurotransmitters. Despite the effectiveness of the
treatment, the patients who took it quickly developed rather
serious side effects: hepatotoxicity, heart problems, neuropsychic
disorders, digestive disorders, etc [10,11], and since then many re-
searchers have been interested in the development of Tacrine an-
alogues by minimizing adverse reactions as much as possible
[12e17].
In this context, we have recently reported the synthesis and
pharmacological studies of a series of readily available Tacrine-
pyranopyrazole [18] and alkylbis(4-amino-5-cyanopyrimidine)
derivatives [19], as promising agents for AD therapy. In our efforts
to contribute to the development of new compounds that may be
useful in the treatment of AD, we are focusing on the synthesis of
new Tacrine analogues which result from exchanging the benzene
ring (A) with a potentially active pharmacophore: 2-
alkylthiopyrimidine. In fact, pyrimidine derivatives and their
analog exhibit a variety of pharmacological activities, including
antitumoral properties [20]. They can be used as anticancer [21],
antimicrobial [22], antitubercular agents [23] and so forth. As our
continuous interest in the synthesis of the biologically important
heterocycles, herein we describe the synthesis of a novel series of
highly functionalized 2-(alkylthio)-4-aryl-6,7,8,9-
tetrahydropyrimido [4,5-b]quinolin-5-amines as cholinesterase
inhibitors. In addition, molecular docking analysis was also per-
formed to disclose the binding interaction template of the most
active inhibitors to the amino acid residues composing active site of
the AChE and BChE enzymes and the findings are presented below.
Furthermore, optimized molecular geometry, frontier molecular
orbitals HOMO-LUMO, molecular electrostatic potential (MEP)
mapping and reactivity indices (electronegativity, electrophilic in-
dex, softness and hardness) have been investigated for 3a-3c as
representative compounds using DFT/B3LYP method. At the same
level of theory, the antiradical properties of 3a-3c have been also
examined by computing bond dissociation enthalpies (BDEs).
2. Experimental section
2.1. Materials and equipments
All chemicals were purchased from the Sigma-Aldrich and used
synthesis studies were reagent grade and further purified by liter-
ature methods. Melting points were determined on an Electro-
thermal capillary fine control apparatus. 1H and 13C NMR spectra
were recorded on a Bruker Avance 400 instrument at 400 and
100 MHz respectively, in CDCl3 or DMSOed6. Chemical shifts (d) are
given in part per million downfield from TMS as an internal stan-
dard for 1H and 13C NMR. Coupling constants (J) values were indi-
cated in Hz. The chemicals were used as obtained commercially.
EA1112 CHNS/O Thermo Electron.
2.2. Synthesis and characterization
2.2.1. Synthesis of 2-(Alkylthio)-4-aryl-6,7,8,9-tetrahydropyrimido
[4,5-b]quinolin-5-amine (3)
In a 100 mL round bottom flask, a mixture of 3-amino-2-
cyanopyrimidine (1, 1 mmol), aluminum chloride (2 mmol) and
cyclohexanone (2, 2 mmol) was added into adequate volume of dry
1,2-dichloroethane (DCE) and stirred at reflux for 24 h (Monitored
by TLC) After completion, water was added and the mixture was
basified with 10% sodium hydroxide solution to pH ¼ 8e9. After
stirring for 30 min, the precipitate was filtered and washed with
water. Purification by recrystallization in ethyl acetate afforded the
title compounds.
2.2.2. Spectral characterization
4-(4-Chlorophenyl)-2-(methylthio)-6,7,8,9-tetrahydropyrimido
[4,5-b]quinolin-5-amine (3a).
Compound 3a was obtained as a yellow solid (41%). M.p:
251e254  C; 1H NMR (400 MHz, CDCl3) d (ppm) 7.44 (m, 4H), 4.49
(br s, 2H, NH2), 2.97 (t, J ¼ 6.0 Hz, 2H), 2.64 (s, 3H, SCH3), 2.32 (t,
J ¼ 6.0 Hz, 2H), 1.86e1.81 (m, 4H).13C NMR (100 MHz, CDCl3)
d (ppm) 170.3, 165.8, 165.6, 158.3, 149.1, 137.4, 136.5, 130.0, 129.8,
129.6, 129.4, 111.9, 103.7, 34.5, 23.5, 22.4, 14.4. Anal. calcd for
C18H17ClN4S: C, 60.58; H, 4.80; N, 15.70; S, 8.99; Found: C, 60.91; H,
4.67; N, 15.35, S, 9.32.
4-(4-Methoxyphenyl)-2-(methylthio)-6,7,8,9-
tetrahydropyrimido [4,5-b]quinolin-5-amine (3b):
Compound 3b was obtained as a yellow solid (58%). M.p:
176e178  C. 1H NMR (400 MHz, CDCl3) d(ppm): 7.43 (dd, J ¼ 6.8,
2.4 Hz, 2H), 6.96 (dd, J ¼ 6.8, 2.0 Hz, 2H), 4.58 (br s, 2H, NH2), 3.81 (s,
3H, OeCH3), 2.97 (t, J ¼ 6.0 Hz, 2H), 2.64 (s, 3H, SeCH3), 2.32 (t,
J ¼ 6.4 Hz, 2H), 1.86e1.81 (m, 4H).13C NMR (100 MHz, CDCl3):
d (ppm): 170.3, 166.6, 165.4, 161.1, 158.4, 149.5, 132.6, 131.2, 130.2,
114.9, 111.6, 103.8, 55.5, 34.4, 26.8, 23.5, 22.5, 22.4, 14.4. Anal. calcd
for C19H20N4OS: C, 64.75; H, 5.72; N, 15.90; S, 9.10; Found: C, 64.35;
H, 5.21; N, 15.45, S, 9.47.
2-(Methylthio)-4-(3-nitrophenyl)-6,7,8,9-tetrahydropyrimido
[4,5-b]quinolin-5-amine (3c):
Compound 3c was obtained as a orange brown solid (55%). M.p:
210e216  C. 1H NMR (400 MHz, CDCl3) d (ppm): 8.39 (s, 1H),
8.32(dd, J ¼ 8.0, 1.6 Hz, 1H), 7.88 (dd, J ¼ 7.6, 1.2 Hz, 1H), 7.67 (t,
J ¼ 8.0 Hz, 1H), 4.38 (br s, 2H, NH2), 3.0 (t, J ¼ 5.6 Hz, 2H), 2.63 (s,
3H), 2.36 (t, J ¼ 6.0 Hz, 2H), 1.87e1.82 (m, 4H). 13C NMR (100 MHz,
CDCl3): d (ppm): 170.5166.5, 163.9, 158.3, 148.6, 148.4, 140.5, 134.8,
130.2, 124.9, 124.1, 112.4, 103.6, 34.5, 23.5, 22.3, 14.5. Anal. calcd for
C18H17N5O2S: C, 58.84; H, 4.66; N, 19.06; S, 8.73; Found: C, 58.59; H,
5.03; N, 18.81, S, 8.95.
4-(4-Chlorophenyl)-2-(ethylthio)-6,7,8,9-tetrahydropyrimido
[4,5-b]quinolin-5-amine (3d).
Compound 3d was obtained as an orange brown (57%). M.p
216e219  C.1H NMR (400 MHz, CDCl3) d (ppm) 7.44 (m, 4H), 4.47
(br s, 2H, NH2), 3.28 (q, J ¼ 7.2 Hz, 2H), 2.97 (t, J ¼ 6.0 Hz, 2H), 2.32 (t,
J ¼ 6.0 Hz, 2H), 1.85e1.81 (m, 4H), 1.36 (t, J ¼ 7.2 Hz, 3H).13C NMR
 C. Derabli et al. / Journal of Molecular Structure 1209 (2020) 127902
(100 MHz, CDCl3) d (ppm) 169.9, 165.8, 165.6, 158.3, 149.0, 137.5,
136.5, 130.0, 129.4, 111.9, 103.7, 34.5, 25.3, 23.5, 22.4, 22.3, 14.4. Anal.
calcd for C19H19ClN4S: C, 61.53; H, 5.16; N, 15.11; S, 8.65; Found: C,
61.87; H, 5.38; N, 14.85, S, 8.45.
2-(Butylthio)-4-(4-chlorophenyl)-6,7,8,9-tetrahydropyrimido
[4,5-b]quinolin-5-amine (3e):
Compound 3e was obtained as a yellow solid (58%). M.p:
230e232  C. 1H NMR (400 MHz, CDCl3) d (ppm): 7.53 (m, 4H), 4.56
(br s, 2H, NH2), 3.39 (t, J ¼ 7.2 Hz, 2H), 3.06 (t, J ¼ 6.4 Hz, 2H), 2.41 (t,
J ¼ 6.0 Hz, 2H), 1.95e1.90 (m, 4H), 1.77 (qt, J ¼ 7.2 Hz, 2H), 1.52 (qt,
J ¼ 7.6 Hz, 2H), 0.96 (t, J ¼ 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3):
d (ppm): 170.2, 165.8, 165.5, 158.3, 149.0, 137.5, 136.5, 130.1, 129.4,
111.8, 103.7, 34.5, 30.9, 23.5, 22.4, 22.3, 22.1, 13.8. Anal. calcd for
C21H23ClN4S : C, 63.22; H, 5.81; N, 14.04; S, 8.04; Found: C, 63.49; H,
6.09; N, 14.39, S, 7.89.
2-(Butylthio)-4-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrimido
[4,5-b]quinolin-5-amine (3f):
Compound 3f was obtained as a yellow solid (39%). M.p:
188e190  C. 1H NMR (400 MHz, CDCl3) d (ppm): 7.53 (dd, J ¼ 6.8,
2.0 Hz, 2H), 7.05 (dd, J ¼ 6.8, 2.0 Hz, 2H), 4.81 (br s, 2H, NH2), 3.90 (s,
3H, OeCH3), 3.37 (t, J ¼ 7.2 Hz, 2H), 3.07 (t, J ¼ 6.0 Hz, 2H), 2.43 (t,
J ¼ 6.0 Hz, 2H), 1.95e1.92 (m, 4H),1.76 (qt, J ¼ 7.2 Hz, 2H), 1.50 (qt,
J ¼ 7.2 Hz, 2H), 0.95 (t, J ¼ 7.2 Hz, 3H).13C NMR (100 MHz, CDCl3):
d (ppm): 170.3, 166.6, 164.9, 161.2, 158.2, 149.7, 131.1, 130.3, 114.6,
111.6, 104.5, 103.8, 55.5, 34.2, 30.9, 30.8, 23.5, 22.4, 22.3, 22.1, 19.7,
13.8. Anal. calcd for C22H26N4OS: C, 66.97; H, 6.64; N, 14.20; S, 8.13;
Found: C, 66.93; H, 6.57; N, 14.20, S, 7.40.
2-(Butylthio)-4-(3-nitrophenyl)-6,7,8,9-tetrahydropyrimido
[4,5-b]quinolin-5-amine (3g).
Compound 3g was obtained as a yellow solid (62%). M.p:
>260  C.1H NMR (400 MHz, CDCl3) d (ppm) 8.48 (d, J ¼ 2.0 Hz, 1H),
8.41 (dd, J ¼ 8.4, 1.2 Hz, 1H), 7.96 (d, J ¼ 7.6 Hz 1H), 7.75 (t, J ¼ 6.0 Hz,
1H), 4.45 (br s, 2H, NH2), 3.39 (t, J ¼ 7.2 Hz, 2H), 3.08 (t, J ¼ 6.0 Hz,
2H), 2.44 (t, J ¼ 6.0 Hz, 2H), 1.95e1.88 (m, 4H), 1.68 (qt, J ¼ 7.2 Hz,
2H), 1.27 (qt, J ¼ 7.2 Hz, 2H), 0.99 (t, J ¼ 7.2 Hz, 3H). 13C NMR
(400 MHz, CDCl3) d (ppm) 170.3, 166.4, 163.9, 158.3, 148.5, 148.3,
140.5, 134.9, 130.1, 124.8, 124.1, 112.3, 103.6, 34.5, 30.8, 23.5, 22.3,
14.2. Anal.calcd for C212H23N5O2S: C, 61.59; H, 5.66; N, 17.10; S, 7.83;
Found: C, 61.87; H, 5.92; N, 17.85, S, 7.54.
4-(4-Chlorophenyl)-2-(octylthio)-6,7,8,9-tetrahydropyrimido
[4,5-b]quinolin-5-amine (3h):
Compound 3h was obtained as a yellow solid (51%). M.p:
164e167  C. 1H NMR (400 MHz, CDCl3) d(ppm7.44 (m, 4H), 4.46 (br
s, 2H, NH2), 3.29 (t, J ¼ 7.2 Hz, 2H), 2.97 (t, J ¼ 6.0 Hz, 2H),2.32 (t,
J ¼ 6.0 Hz, 2H), 1.86e1.81 (m, 4H), 1.68 (qt, J ¼ 7.6 Hz, 2H), 1.40 (qt,
J ¼ 7.6 Hz, 2H),1.24e1.18 (m, 8H), 0.80 (t, J ¼ 6.8 Hz, 3H).13C NMR
(400 MHz, CDCl3): d(ppm): 170.3, 165.8, 165.5, 158.3, 149.0, 137.5,
136.5, 130.1, 129.4, 111.8, 103.8, 34.5, 31.8, 31.1, 29.2, 29.0, 28.8, 23.5,
22.7, 22.4, 22.3, 14.1. Anal. calcd for C25H31ClN4S: C, 65.98; H, 6.87;
N, 12.31; S, 7.05; Found: C, 66.23; H, 6.54; N, 12.70, S, 6.73.
2.3. Method for cholinesterase activity assay
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)
inhibitory activity was measured, by the spectrophotometric
method developed by Ellman, G.L., et al. [24] Briefly, 150 ml of
100 mM sodium phosphate buffer (pH 8.0), 10 ml of sample solution
dissolved in methanol at different concentrations and 20 ml 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 ml of 0.5 mM DTNB[5,50 -
dithio-bis(2-nitrobenzoic) acid] were added. The reaction was then
initiated by the addition of 10 ml 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
3
the reaction of DTNB with thiocholine, released by the enzymatic
hydrolysis of acetylthiocholine iodide or butyrylthiocholine chlo-
ride, respectively, at a wavelength of 412 nm, every 5 min for
15 min, utilizing a 96-well microplate reader (PerkinElmer Multi-
mode Plate Reader EnSpire, USA) in triplicate experiments. Gal-
anthamine 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 re-
action rates of samples relative to blank sample (methanol in
phosphate buffer, pH 8) using the formula:
ES
Inhibition of AChE or BChE ð%Þ ¼  100
E
Where E is the activity of enzyme without test sample, and S is the
activity of enzyme with test sample.
2.4. Molecular modeling
AutodockVina 1.1.1 [25] was the software of choice to generate
the binding modes of the re-docked ligands and the tested com-
pounds.The 2D structure was illustrated by ChemBioDraw Ultra
14.0. ChemBio3D Ultra 14.0from the same package was the tool of
obtaining the 3D configuration. The most stable conformers were
proposed by energy minimization algorithm MOPAC which oper-
ates inside VEGA ZZ 3.1.1.42enviroment [26]. All the crystal struc-
tures were downloaded in PDB format from the Protein Data Bank
web site [27]. The proteins were prepared for docking by deletion of
the unwanted molecules and the water using VEGA ZZ 3.1.1.42.The
energy minimization of proteins were performed by YASARA server
[28]. The residues of the binding sites in the active site of acetyl-
cholinesterase were identified as Trp86, Tyr124, Ser203, Glu202,
Tyr337, Phe338, His447, and Tyr449.The backbone of the binding
cavity of butyrylcholinesterase has the following flexible amino
acids, Asn68, Ile69, Asp70, Trp82, Gln119, Thr120, Ser198, Trp231,
leu286, Ser287, Val288, phe329, Tyr332, phe398, Trp430, Met437,
His438 and Tyr440. The flexible residues were allowed to move
freely during the processed flexible docking. The docking grid was
sized to enclose the co-crystallized molecule and the flexible resi-
dues. For acetylcholinesterase (4ey6) [29] the center of the grid box
was: X ¼ 8.2, Y ¼ 61.5, Z ¼ 23.8 and the size was 17 *17 *17 Å.
Crystal structure AchE (4bdt) [30] has the following spatial center
of the docking box X ¼ 2.22, Y ¼ 34.6, Z ¼ 52.3 and the size was
17*20*18 Å. For butyrylcholinesterase (5dyw) [31] the grid box size
was 28 *22*22 Å and the center of box: X ¼ 137.1 Y ¼ 114.3, Z ¼ 39.9.
Pymol software was the visualization tool for the ligands/protein
complex [32].
2.5. Computational details
Density functional theory (DFT) calculations have been carried
out using Gaussian09software [33]. The B3LYP functional [34,35]
and the 6e311þþG(d,p) basis set have been used for all calcula-
tions. All the ground states were confirmed by vibrational fre-
quency analysis (no imaginary frequency). Electronegativity,
chemical softness, chemical hardness and electrophilic indexwere
calculated based on HOMO and LUMO energiesas reported in the
literature [36e40]. Thebond dissociation enthalpy (BDE) have been
calculated as reported in our previous works [41,42].
3. Results and discussion
3.1. Chemistry
The target compounds 3a-h were synthesized via Friedlander
4 C. Derabli et al. / Journal of Molecular Structure 1209 (2020) 127902

reaction as shown in Scheme 1. First, the straightforward conden- Table 1

sation between 2-alkylthiouronium halides and arylidenemalono- Cholinesterase inhibition efficacy of compounds 3a-h.

nitriles in the presence of potassium carbonate afforded the Compound IC50 (mM) ± SD/% inhibitiona Selectivity index
corresponding alkylthiopyrimidine derivatives 1a-h, as previously AChE BuChE AChEc BuChEd
investigated in our laboratory [43]. Condensation of the starting
3a 8,76 ± 0,22 19,97 ± 0,86 2,28 0,44
pyrimidines 1a-h with cyclohexanone 2 in the presence of AlCl3 in
3b 5,85 ± 0.28 6,78 ± 0,51 1,16 0,86
dry 1,2 dichloromethane gave eight new compounds 3a-h, with 3c 4,68 ± 0,73 21,71 ± 0,41 4,64 0,21
moderate to good yields (39e62%). These compounds were purified 3d 4,32 ± 0,87 15,10 ± 0,19 3,49 0,29
by recrystallization in ethyl acetate and characterized by 1H NMR, 3e 13,16 ± 0,14 115,82 ± 5,95 8,80 0,11
13 3f 4,94 ± 0,42 2,74 ± 0,74 0,55 1,80
C NMR, and elemental analyses (CHNS).
3g 69,19 ± 4,99 121,24 ± 1,40 1,75 0,57
3h 48,94 ± 4,05 151,38 ± 22,2 3,09 0,32
3.2. Biological evaluation Galantamineb 21,81 ± 1,15 40,72 ± 2,85 1,87 0,53
a
IC50 values represent the means ± SD of three parallel measurements (p < 0.05).
The inhibitory proprieties of target compounds 3a-h were per- b
Reference compound.
formed by the spectroscopic method described by Ellman’s [24] c
Selectivity for AChE is defined as IC50 BuChE (mM)/IC50 AChE (mM).
using Galantamine as the standard. Most of the compounds showed
d
Selectivity for BuChE is defined as IC50 AChE (mM)/IC50 BuChE (mM).

potent inhibitory activity against AChE with IC50s values ranging

from 4,32 to 69,19 mM. Compound 3d with IC50 value of 4.32 mM
was found to be the most potent compound against AChE, this
compound was 5-fold more potent than reference drug Galant-
amine with IC50 value of 21.81 mM (Table 1). For comparison pur-
poses, among compounds 3a, 3b and 3c having all the S-methyl
group, with IC50s values of 8,76, 5,85 and 4,68 mM respectively,
molecule 3c with a 4-nitro group on the benzene ring is more active
than the other two compounds 3a and 3b with 4-chloro and 4-
methoxy groups on the benzene ring respectively. On the other
hand, compounds 3e, 3f and 3g with IC50s values of 13,16, 4,94 and
69,19 mM respectively which have the S-ethyl group, it was noticed
that compound 3f with a 4-methoxy group on the benzene ring is
more active than compounds 3e and 3g with 4-chloro and 4-nitro
substituents respectively. In addition, compound 3d was more
potent than 3h with IC50 value of 48,94 mM by 11-fold improve-
ment. The difference in efficacy is due probably to the length of S-
alkyl chain attached to the thiopyrimidine motiey.
The BChE results revealed IC50 values ranging from 2.74 to
151.38 mM. The most active compounds are 3f and 3b with IC50s of
2.74 mM and 6.78 mM respectively which are more potent than
Galanthamine with value of 40.72 mM. Both compounds have a 4-
methoxy group linked to the phenyl, which leads to deduce that
it is responsible for improving BChE’s inhibitory activity. By
comparing compounds having the same S-alkyl chain, 3a, 3b and 3c
with IC50s values of 19,97, 6,78 and 21,71 mM respectively, are
molecule 3b with a 4-methoxy group is more effective than the
other two compounds 3a and 3c with 4-chloro and 4-nitro groups
respectively. The calculation of the selectivity index allows the
evaluation of the degree of affinity that the synthesized compounds
have with AChE and BChE. Experimental results revealed that all
compounds showed relatively more inhibitory activity to AChE as
R2 O R2
NH2
CN 2
N N
R1 AlCl3, DCE R1
S N NH2 S N N
reflux, 24h
1a-h 3a-h
3a (R1 = CH3, R2 = Cl) (41%) 3e (R1 = C4H9, R2 = Cl) (58%)
3b (R1 = CH3, R2 = OCH3) (58%) 3f (R1 = C4H9, R2 = OCH3) (39%)
3c (R1 = CH3, R2 = NO2) (55%) 3g (R1 = C4H9, R2 = NO2) (62%)
3d (R1 = C2H5, R2 = Cl) (57%) 3h (R1 = C8H17, R2 = Cl) (51%)
Scheme 1. The structures of reported Tacrine-derived AChE/BChE inhibitors.
compared to BuChE with a factor ranging from 1.16 to 8.80 except
3c which on the contrary has an affinity for BChE.
3.3. In silico studies
The suggested binding modes of the active candidates were
proposed by molecular docking study. The protein data bank was
accessed for obtaining the 3D structure of the enzymes [27]. Two
proteins of human acetylcholinesterase were downloaded for the
purpose of achieving a comparative study. One of them (pdb code:
4ey6) [29] has Galantamine as a native ligand which was the
reference for the biological study. The other one (pdb code: 4bdt)
[30] was co-crystallized with the Tacrine like compound (huprine
w), so it could offer a model for protein interaction with our com-
pounds which have Tacrine core in their structures. As a conse-
quence we can identify the residues which may be involved in the
stabilization of the tested compounds. The spatial distribution of
the amino acids within the active sites was different between the
two proteins. It was evident that the amino acids move freely to
give a maximum fitting of the ligands. Energy minimization of the
protein to restore the original distribution of the amino acids in the
binding site to their supposed natural orientation was essential.
After the energy minimization of the two proteins, the active sites
showed a good matching of the orientation inside the pocket be-
tween each other.
The noticed difference in the orientation of the amino acids in
the binding pocket which provoked by the interaction with the
bound structure implied the importance of the allowance the free
rotation of the flexible amino acids inside the protein gorge.
Henceforth the flexible docking was the technique of choice to
calibrate the process and for the docking of the tested compounds.
It was postulated that the resulted orientation of the ligand and the
surrounding environment of the flexible amino acids residues may
superimposed with that of the non-minimized protein.
The validation of the docking process was conducted by drawing
the native ligand using in 2D form. The 2D structure was trans-
formed into 3D and the best conformer was picked up. The re-
docking of the native molecules for both examples was per-
formed by AutodockVina [25]. The output of the theoretical inter-
action between Galantamine and the protein showed a pattern
similarity to that of the crystal structure. The ligand was super-
imposed over the co-crystallized one as well as the surrounding
amino acids with a narrow margin of deviation (Fig. 2 a). For
huprine w, although the orientation of the ligand and the flexible
amino acid backbone of the pocket wasn’t superimposed, we could
notice that the spatial interaction between the ligand and the
amino acids was preserved. The hydrophobic interactions between
 C. Derabli et al. / Journal of Molecular Structure 1209 (2020) 127902
the benzene ring of the pyrimidine scaffold and Tyrosine 337 and
the fused alicyclic ring with the tryptophan 86 were maintained
while the hydrogen bond interaction was shifted from Ser203 to
Trp86 (Fig. 2 b).
The same docking parameter was applied for the assayed
compound. Rigid and flexible dockings were performed where the
flexible docking introduced more reliable results than the rigid and
more compatible with biological study. The orientation of Tacrine
core of all compounds (with exception of 3c and 3g) was found to
be well oriented to give a good hydrophobic field toward Trp86 in
addition to hydrogen bonds with Tyr124 (Fig. 3 c).
The presence of the nitro group on 3c and 3g may lead to an
inverse orientation in which the nitro group made hydrogen bonds
with Gly121. Trp86 was parallel in this case to the phenyl ring
which offered a reduced surface area for the hydrophobic interac-
tion which might be compensated by the hydrogen bond interac-
tion from the nitro group and pi-interaction. This effect is suggested
to potentiate the efficacy of 3c while it might have a negative
impact on 3g. The decrease in the inhibitory action could be
attributed to the hindrance between the long side chain of the
compound and the inner wall of the pocket. This Impact could be
seen also in case of 3h which showed the lowest affinity in both
rigid and flexible models. The length of side chain seemed to have a
key role in the interaction which may be repulsive in case of 3g and
3h while might be an additive hydrophobic site in case of 3f (Fig. 3
d).
The pocket of BuChE (Pdb code: 5dyw) [31] is larger than that of
AChE, and the docking was a challenge. The rigid docking modeling
provided results not correlated to the biological studies. The flex-
ible docking still the better choice in this case. The binding affinity
provided by flexible docking was more reasonable in a relative to
the biological study comparing to the rigid one. The interesting
result obtained from the calibration of the docking tool was satis-
factory to consider the docking results as consistent. Fig. 4.
The native ligand oriented in a similar binding mode displayed
by the non-minimized crystal structure of the enzyme. Some
displacement in the position of Trp82 could be noticed which
possibly occurred to maintain the hydrophobic and the pi-inter-
action toward the lateral benzene ring. Similarly Tyr440 was moved
in to adjust the new position of the same benzene ring which could
a b
Fig. 2. The molecular docking study of AChE a)The re-docking result of the native ligand (inside the blue mesh) with crystallized Galantamine (salmon color) is showing the near
positioning between the generated orientation of the flexible amino acids residues (blue sticks) and the crystallized one (salmon sticks) of AchE (Pdb code: 4ey6) b) in silico
proposed binding mode of huprine w and with the free rotated amino acids of the gorge relatively to the crystallized structure (salmon color) of AchE pdb code: 4bdt.
5
result in preserving the hydrophobic interaction. The hydrogen
bonding interaction between the sulfite moiety and Thr120 was
identical in both crystallized and re-docked models. One binding
mode from the docking of the compounds under investigation was
proposed. This pose was selected as it afforded the maximum
number of hydrogen bonds between the most active inhibitor 3f
and the protein. On the other hand it was the most frequent binding
mode between all poses generated by the automated flexible
docking. In the selected binding mode the phenyl ring directed
toward the outside of the binding cavity and pi-interaction with the
phenyl ring of Tyr332 represented a possibility. Another stabiliza-
tion could be provided by hydrogen bond interaction with Asp70.
The hydrophobic interaction of the compounds with the protein
seemed to be more limited in comparison with the native ligand
which may describe the high IC50 of most of them obtained from
the in vitro assay.
3.4. Theoretical investigations
After having successfully synthesized and evaluated in vitro the
biological activity of the target Tacrine analogues, we next envis-
aged to gain insights about the reactive nature and reactivity in-
sights of tacrines 3a-3c, as representative compounds, by using
density functional theory (DFT) calculations. Optimized molecular
geometries obtained by theoretical methods are useful to explain
the three-dimensional structures of the investigated compounds.
The molecular structures of 3a, 3b and 3c have been optimized at
B3LYP/6-311Gþþ(d,p) level of theory. The obtained structures are
shown in Fig. 5. As can be seen, the structures of 3a-3c are non-
planar due to the presence of the NH2 group of the pyridine ring.
The substituted benzenes are inclined by 53.67, 46.45 and 50.85
for 3a, 3b and 3c, respectively. The fused pyridine and pyrimidine
rings adopt quasi-planar geometries for all the studied compounds.
Reactivity indices such as electronegativity, electrophilic index,
softness and hardness are excellent tools that characterize the
reactivity of the target compounds [44,45]. Electronegativity is a
measure of the tendency to attract electrons in a chemical bond.
Electrophilicity index is related to the energy lowering associated
with a maximum amount of electron flow between a donor and an
acceptor [39,46]. The chemical softness and hardness are measures
6 C. Derabli et al. / Journal of Molecular Structure 1209 (2020) 127902

c d

Fig. 3. The molecular docking study of AChE c) 3c binding mode (blue) within the flexible amino acid residues of the binding site (salmon) d) 3d (blue) interaction with the flexible
amino acids backbone of the pocket (salmon color).

Fig. 4. Docking results of BuChE (5dyw) a) Re-docked native ligand (blue) and the flexible amino acid in comparison with the original orientation (salmon color). b) Docking result
of 3f showing the interaction with the movable amino acid residues of the active site (salmon stick).

Table 2
Reactivity indices of 3a-3c obtained at B3LYP/6e311þþG(d,p)level of theory.

Reactivity indices 3a 3b 3c

Electronegativity 4.20 3.98 4.91

Softness 0.27 0.25 0.38
Hardness 1.87 1.97 1.31
Electrophilic index 4.72 4.04 9.24

Fig. 5. Optimized geometries of 3a-3c obtained at B3LYP/6-311Gþþ(d,p) level of
theory.
of resistance to charge transfer. The reactivity indices of compounds
3a-3c were calculated in the gas-phase and are given in Table 2.
An examination of the obtained results reveals that 3a-3c have
comparable values of electronegativity, softness and hardness in
the range of 3.98e4.91, 1.31e1.97 and 0.25e0.38, respectively. 3c
has the highest electrophilicity index value (9.24), whereas 3a and
3b have comparable values (4.04 and 4.72, respectively).These ob-
servations clearly shown that all the studied compounds prefer to
act as electron donor rather than electron acceptor. In terms of
electrophilic index, the least reactive compound as electron donor
is 3c, whereas the other compounds have a comparable reactivity.
Molecular electrostatic potential (MEP) mapping is a very useful
approach to explore the reactivity of the investigated compounds.
MEP mapping results for 3a, 3b and 3c are illustrated in Fig. 6. The
nucleophilic and electrophilic sites are expressed in term of
different color codes; a deep red color indicates an electron-rich
site, whereas deep blue indicates an electron-deficient site. As
shown in Fig. 6, the most electron-rich sites of 3a-3c are located
around the two adjacent nitrogen atoms of the pyridine and py-
rimidine rings, where as the most electron-deficient sites are one
 C. Derabli et al. / Journal of Molecular Structure 1209 (2020) 127902
Fig. 6. Lowest unoccupied molecular orbital (a), highest occupied molecular orbital (b)
and molecular electrostatic potential (c) of 3a-3c calculated at B3LYP/6-311Gþþ(d,p)
level of theory.
the NH2 groups. These results suggest that these sites are the
preferred sites for electrophilic and nucleophilic attacks,
respectively.
Highest occupied molecular orbital (HOMO) and lowest unoc-
cupied molecular orbital (LUMO), can provide some useful infor-
mation for the reactivity of molecules as well as electronic
transitions within molecules [47,48]. In terms of antiradical activity,
the energy and distribution of HOMO are important parameters.
The shape of the HOMO determines the sites for free radical attack,
whereas, its energy is related to the ability of donating electrons.
Molecules with lower HOMO energy are less likely to donate
electrons [44]. The calculated frontier molecular orbitals energies
and distributions in the gas phase for 3a, 3b and 3c are presented in
Fig. 6. The results showed that the LUMOs of 3a and 3b are
distributed nearly on the entire skeleton including the pyridine,
pyrimidine and benzene rigs, while that of 3c is only localized on
the benzene ring. HOMOs of all the investigated compounds are
located on the pyridine, pyrimidineas well as NH2 and SMe groups.
From these results, it is clear that the NH2 groups of 3a-3c are po-
tential sites for free radical attack. By comparing the HOMOs en-
ergies, theelectron donating ability of the studied compound can be
ranged in the following order: 3b > 3a > 3c.
The antiradical potential of the investigated tacrines could also
be estimated by computing the bond dissociation enthalpy (BDE) of
the respective NH bond. BDE corresponds to the ability of an
antioxidant to donate its hydrogen atom and consequently form a
radical [49]. Lower BDE values indicate weaker NeH bonds, from
which the hydrogen is more easily extracted [50]. The calculated
BDE values at B3LYP/6e311þþG(d,p)level of theory in the gas-
phase for 3a, 3b and 3c, as representative compounds, were
found to be, 92.16 kcal/mol 91.65 kcal/moland 93.23 kcal/mol,
respectively. These values are comparable to that of the aniline
(90.83 kcal/mol) calculated at the same level of theory. These re-
sults indicate that the studied Tacrine analogues have a comparable
antiradical potential than that of the aniline. This latter isconsid-
ered as a privileged scaffold for the antiradical activity [51,52].
Based on BDE values the sequence of the antiradical potential can
be ranged in the following order 3b > 3a > 3c. This order is in line
7
with HOMOs results.
4. Conclusion
In the present study, we report the development of a novel se-
ries of substituted 2-alkylthiopyrimidino-Tacrines 3a-h carried out
by applying Friedlander reaction of 3-amino-2-cyanopyrimidines
1a-h with cyclohexanone in the presence of AlCl3 in 1,2 dichloro-
ethane. All compounds were evaluated for their potential to inhibit
commercially available electric eel. acetylcholinesterase (AChE) and
horse serum butyrylcholinesterase (BChE). The experimental re-
sults showed that most compounds were highly active towards
AChE (IC50s ranging from 4.32 to 69.19 mM) and few towards BChE
with IC50s in the range of 2.74e151.38 mM. Also, molecular docking
studies was in parallel to the in vitro results, for the purpose of
assisted in explaining the structure-activity relationships of this
type of compounds. Finally, theoretical calculations by means of
DFT/B3LYP method have been carried out in order to gain insights
about the molecular structure and reactivity insights of tacrines 3a-
3c as representative compounds.
Acknowledgments
We thank MESRS (Ministe
re de l’Enseignement Supe rieur et de
la Recherche Scientifique) and DGRST (Direction Ge  ne
rale de la
Recherche Scientifique et Technologique), Algeria for financial
support. H.B. thanks the HPC resources of UCI (Unite  de Calcul

res Mentouri Constantine 1 University for the used
Intesif) of the Fre
computational resources.
References
[1] C. Patterson, World Alzheimer Report 2018-The State of the Art of Dementia
Research: New Frontiers, Alzheimer’s Disease International, London, UK, 2018.
[2] R.J. Castellani, R.K. Rolston, M.A. Smith, Alzheimer disease, Dis. Mon. 56 (2010)
484e546.
[3] P. Mishra, A. Kumar, G. Panda, Anti-cholinesterase hybrids as multi-target-
directed ligands against Alzheimer’s disease (1998-2018), Bioorg. Med.
Chem. 27 (2019) 895e930.
[4] P. Thomas, A. Pesce, J.P. Cassuto, Maladie d’Alzheimer, 01 ed., MASSON, PARIS,
1989.
[5] H. Hippius, G. Neundo €rfer, The discovery of Alzheimer’s disease, Dialogues
Clin. Neurosci. 5 (2003) 101e108.
[6] D.J. Selkoe, M.B. Podlisny, Deciphering the genetic basis of Alzheimer’s dis-
ease, Annu. Rev. Genom. Hum. Genet. 3 (2002) 67e99.
[7] M. Racchi, M. Mazzucchelli, E. Porrello, C. Lanni, S. Govoni, Acetylcholines-
terase inhibitors: novel activities of old molecules, Pharmacol. Res. 50 (2004)
441e451.
[8] K. Spilovska, F. Zemek, J. Korabecny, E. Nepovimova, O. Soukup, M. Windisch,
K. Kamil, Adamantane - a lead structure for drugs in clinical practice, Curr.
Med. Chem. 23 (2016) 3245e3266.
[9] F. Zemek, L. Drtinova, E. Nepovimova, V. Sepsova, J. Korabecny, J. Klimes,
K. Kuca, Outcomes of Alzheimer’s disease therapy with acetylcholinesterase
inhibitors and memantine, Expet Opin. Drug Saf. 13 (2014) 759e774.
[10] F.E. Shirley, R.M. Dawson, Tacrine: a pharmacological review, Prog. Neurobiol.
36 (1991) 257e277.
[11] P.B. Watkins, H.J. Zimmerman, M.J. Knapp, S.I. Gracon, K.W. Lewis, Hepato-
toxic effects of tacrine administration in patients with Alzheimer’s disease,
J. Am. Med. Assoc. 271 (1994) 992e998.
[12] M. Esquivias-Pe rez, E. Maalej, A. Romero, F. Chabchoub, A. Samadi, J. Marco-
Contelles, M.J. Oset-Gasque, Nontoxic and neuroprotective b-naphthotacrines
for Alzheimer’s disease, Chem. Res. Toxicol. 26 (2013) 986e992.
[13] L. Ismaili, B. Refouvelet, M. Benchekroun, et al., Multitarget compounds
bearing tacrine- and donepezil-like structural and functional motifs for the
potential treatment of Alzheimer’s disease, Prog. Neurobiol. 151 (2017) 4e34.
[14] Y. Dgachi, O. Sokolov, V. Luzet, et al., Tetrahydropyranodiquinolin-8-amines as
new, non hepatotoxic, antioxidant, and acetylcholinesterase inhibitors for
Alzheimer’s disease therapy, Eur. J. Med. Chem. 126 (2017) 576e589.
[15] Y. Dgachi, O.M. Bautista-Aguilera, M. Benchekroun, et al., Synthesis and bio-
logical evaluation of benzochromenopyrimidinones as cholinesterase in-
hibitors and potent antioxidant, Non-Hepatotoxic Agents Alzheimer’s Dis.
Molecules 21 (2016) 634e649.
[16] H. Boulebd, L. Ismaili, H. Martin, A. Bonet, M. Chioua, J. Marco Contelles,
A. Belfaitah, New (benz)imidazolopyridino tacrines as nonhepatotoxic,
cholinesterase inhibitors for Alzheimer disease, Future Med. Chem. 9 (2017)
8 C. Derabli et al. / Journal of Molecular Structure 1209 (2020) 127902
723e729.
[17] H. Boulebd, L. Ismaili, M. Bartolini, A. Bouraiou, V. Andrisano, H. Martin,
A. Bonet, I. Moraleda, I. Iriepa, M. Chioua, Imidazopyranotacrines as non-
hepatotoxic, selective acetylcholinesterase inhibitors, and antioxidant agents
for Alzheimer’s disease therapy, Molecules 21 (2016) 400.
[18] C. Derabli, I. Boualia, R. Boulcina, C. Bensouici, G. Kirsch, A. Debache, A cascade
synthesis, in vitro cholinesterases inhibitory activity and docking studies of
novel Tacrine-pyranopyrazole derivatives, Bioorg. Med. Chem. Lett 28 (2018)
2481e2484.
[19] I. Boualia, C. Derabli, R. Boulcina, C. Bensouici, M. Yildirim, A. Birinci Yildirim,
E. Mokrani, A. Debache, Synthesis, molecular docking studies, and biological
evaluation of novel alkyl bis(4-amino-5-cyanopyrimidine) derivatives, Arch.
Pharm. Chem. Life. Sci. (2019), https://doi.org/10.1002/ardp.201900027.
[20] A. Thiriveedhi, R. Venkata Nadh, N. Srinivasu, et al., Design, synthesis and anti-
tumour activity of new pyrimidine-pyrrole appended triazoles, Toxicol. Vitro
60 (2019) 87e96.
[21] S.A. Elmetwally, K.F. Saied, I.H. Eissa, E.B. Elkaeed, Bioorg. Chem. Design,
synthesis and anticancer evaluation of thieno[2,3-d]pyrimidine derivatives as
dual EGFR/HER2 inhibitors and apoptosis inducers, Bioorg. Chem. (2019),
https://doi.org/10.1016/j.bioorg.2019.102944.
[22] G. Slifirski, M. Krol, J. Kleps, et al., Synthesis of new 5,6,7,8-tetrahydropyrido
[1,2-c]pyrimidine derivatives with rigidized tryptamine moiety as potential
SSRI and 5-HT1A receptor ligands, Eur. J. Med. Chem. 180 (2019) 383e397.
[23] P. Liu, Y. Yang, Y. Tang, T. Yang, et al., Design and synthesis of novel pyrimi-
dine derivatives as potent antitubercular agents, Eur. J. Med. Chem. 163
(2019) 169e182.
[24] G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Feather-Stone, Biochem. Phar-
macol. 7 (1961) 88e95.
[25] O. Trott, A.J. Olson, AutoDockVina: improving the speed and accuracy of
docking with a new scoring function, efficient optimization, and multi-
threading, J. Comput. Chem. 31 (2010) 455e461.
[26] A. Pedretti, L. Villa, G. Vistoli, Vega - an open platform to develop chemo-bio-
informatics applications, using plug-in architecture and script programming,
J. Comput. Aided Mol. Des. 18 (2004) 167e173.
[27] RCSB protein data bank - RCSB pdb. http://www.rcsb.org/pdb/home/home.do.
(Accessed 15 February 2017).
[28] E. Krieger, K. Joo, J. Lee, et al., Improving physical realism, stereochemistry,
and side-chain accuracy in homology modeling: four approaches that per-
formed well in CASP8, Proteins 9 (2009) 114e122.
[29] J. Cheung, M.J. Rudolph, F. Burshteyn, et al., Structures of human acetylcho-
linesterase in complex with pharmacologically important ligands, J. Med.
Chem. 55 (2012) 10282e10286.
[30] F. Nachon, E. Carletti, C. Ronco, et al., Crystal structures of human cholines-
terases in complex with huprine W and tacrine: elements of specificity for
anti-Alzheimer’s drugs targeting acetyl- and butyryl-cholinesterase, Biochem.
J. 453 (2013) 393e399.
[31] U. Kosak, B. Brus, D. Knez, et al., Development of an in-vivo active reversible
butyrylcholinesterase inhibitor, Sci. Rep. 6 (2016) 39495.
[32] Schrodinger, The PyMOL Molecular Graphics System, November 2015, Version
1.8.
[33] M.J. Frisch, G. Trucks, H.B. Schlegel, G. Scuseria, M. Robb, J. Cheeseman,
G. Scalmani, V. Barone, B. Mennucci, G. Petersson, Gaussian 09, Revision A. 1,
vol 27, Gaussian Inc. Wallingford CT, 2009, p. 34.
[34] A.D. Becke, Density-functional exchange-energy approximation with correct
asymptotic behavior, Phys. Rev. 38 (6) (1988) 3098.
[35] P.C. Hariharan, J.A. Pople, The influence of polarization functions on molecular
orbital hydrogenation energies, Theor. Chim. Acta 28 (1973) 213e222.
[36] R.G. Pearson, Absolute electronegativity and hardness correlated with mo-
lecular orbital theory, Proc. Natl. Acad. Sci. Unit. States Am. 83 (1986) 8440.
[37] R.G. Pearson, Maximum chemical and physical hardness, J. Chem. Educ. 76
(1999) 267.
[38] R.G. Pearson, Absolute electronegativity and hardness: application to inor-
ganic chemistry, Inorg. Chem. 27 (1988) 734e740.
[39] P.K. Chattaraj, U. Sarkar, D.R. Roy, Electrophilicity index, Chem. Rev. 106 (6)
(2006) 2065e2091.
[40] J.E. Bartmess, Thermodynamics of the electron and the proton, J. Phys. Chem.
98 (1994) 6420e6424.
[41] H. Boulebd, Comparative study of the radical scavenging behavior of ascorbic
acid, BHT, BHA and Trolox: experimental and theoretical study, J. Mol. Struct.
1201 (2020) 127210.
[42] H. Boulebd, DFT study of the antiradical properties of some aromatic com-
pounds derived from antioxidant essential oils: CeH bond vs. OeH bond, Free
Radic. Res. (2019) 1e10.
[43] C. Derabli, R. Boulcina, G. Kirsch, A. Debache, Rapid access to novel 2-
alkylthiopyrimidine derivatives and attempt of their Tacrine analogs syn-
thesis, Synth. Commun. 49 (2019) 395e403.
[44] K. Sadasivam, R. Kumaresan, Antioxidant behavior of mearnsetin and myr-
icetin flavonoid compounds d a DFT study, Spectrochim. Acta Mol. Biomol.
Spectrosc. 79 (2011) 282e293.
[45] R. Praveena, K. Sadasivam, V. Deepha, R. Sivakumar, Antioxidant potential of
orientin: a combined experimental and DFT approach, J. Mol. Struct. 1061
(2014) 114e123.
[46] R.G. Parr, L.v. Szentpaly, S. Liu, Electrophilicity index, J. Am. Chem. Soc. 121
(1999) 1922e1924.
[47] A. Nataraj, V. Balachandran, T. Karthick, Molecular structure, vibrational
spectra, first hyperpolarizability and HOMOeLUMO analysis of p-ace-
tylbenzonitrile using quantum chemical calculation, J. Mol. Struct. 1038
(2013) 134e144.
[48] H. Boulebd, Y.D. Lahneche, I.A. Khodja, M. Benslimane, A. Belfaitah, New Schiff
bases derived from benzimidazole as efficient mercury-complexing agents in
aqueous medium, J. Mol. Struct. 1196 (2019) 58e65.
[49] C. Giacomelli, F.d.S. Miranda, N.S. Gonçalves, A. Spinelli, Antioxidant activity of
phenolic and related compounds: a density functional theory study on the
OeH bond dissociation enthalpy, Redox Rep. 9 (2004) 263e269.
[50] P. Trouillas, P. Marsal, D. Siri, R. Lazzaroni, J.-L. Duroux, A DFT study of the
reactivity of OH groups in quercetin and taxifolin antioxidants: the specificity
of the 3-OH site, Food Chem. 97 (2006) 679e688.
[51] M. Gizdavic-Nikolaidis, J. Travas-Sejdic, P.A. Kilmartin, G.A. Bowmaker,
R.P. Cooney, Evaluation of antioxidant activity of aniline and polyaniline, Curr.
Appl. Phys. 4 (2004) 343e346.
[52] E. Bendary, R.R. Francis, H.M.G. Ali, M.I. Sarwat, S. El Hady, Antioxidant and
structureeactivity relationships (SARs) of some phenolic and anilines com-
pounds, Ann. Agric. Sci. 58 (2013) 173e181.