Synthesis, biological evaluation, theoretical investigations, docking study and ADME parameters of some 1,4-bisphenylhydrazone derivatives as potent antioxidant agents and acetylcholinesterase inhibitors

Article abstract of DOI:10.1007/s11030-020-10064-8

Abstract: Five 1,4-bisphenylhydrazone derivatives (1–5) were successfully synthesized and evaluated for their antioxidant and acetylcholinesterase inhibitory activities. The antioxidant activity has been carried out using DPPH, ABTS, CUPRAC and superoxide radical scavenging methods. All the compounds showed a very good antioxidant activity compared to that of the standards used. Compound 1 was found to be the best antioxidant agent with IC₅₀ values lower or comparable to that of the standards. The acetylcholinesterase inhibitory activity has been evaluated using a modified Ellman’s assay. The obtained results indicate that compound 2 is the best acetylcholinesterase inhibitor with a low IC₅₀ value comparable to that of the galantamine. In addition, DFT calculations have been performed to determine in which mechanism the synthesized hydrazones follow to scavenge free radicals. Molecular docking study was performed for compound 2, and its interaction modes with the enzyme acetylcholinesterase were determined. As a result, a strong interaction between this compound and the active site of AChE enzyme was revealed. Finally, ADME properties of the synthesized compounds were also studied and showed good drug-like properties. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11030-020-10064-8

Molecular Diversity
https://doi.org/10.1007/s11030-020-10064-8
ORIGINAL ARTICLE
Synthesis, biological evaluation, theoretical investigations, docking study
and ADME parameters of some 1,4‑bisphenylhydrazone derivatives
as potent antioxidant agents and acetylcholinesterase inhibitors
Imene Amine Khodja1 · Houssem Boulebd1
Received: 22 December 2019 / Accepted: 26 February 2020
© Springer Nature Switzerland AG 2020
Abstract
Five 1,4-bisphenylhydrazone derivatives (1–5) were successfully synthesized and evaluated for their antioxidant and acetylcho-
linesterase inhibitory activities. The antioxidant activity has been carried out using DPPH, ABTS, CUPRAC and superoxide
radical scavenging methods. All the compounds showed a very good antioxidant activity compared to that of the standards
used. Compound 1 was found to be the best antioxidant agent with IC50 values lower or comparable to that of the standards.
The acetylcholinesterase inhibitory activity has been evaluated using a modifed Ellman’s assay. The obtained results indicate
that compound 2 is the best acetylcholinesterase inhibitor with a low IC50 value comparable to that of the galantamine. In addi-
tion, DFT calculations have been performed to determine in which mechanism the synthesized hydrazones follow to scavenge
free radicals. Molecular docking study was performed for compound 2, and its interaction modes with the enzyme acetylcho-
linesterase were determined. As a result, a strong interaction between this compound and the active site of AChE enzyme was
revealed. Finally, ADME properties of the synthesized compounds were also studied and showed good drug-like properties.
Graphic abstract
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-020-10064-8) contains
supplementary material, which is available to authorized users.
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

Molecular Diversity
Keywords Hydrazone · Acetylcholinesterase · Antioxidant activity · Docking study · DFT calculations · ADME parameters
atherosclerosis, myocardial infarction, sickle cell disease,
vitiligo, autism and others [18, 19].
Introduction
Although the antioxidant properties of compounds
derived from hydrazone functional group have been widely
studied, few works describe a fundamental theoretical
study of the structure–activity relationship of these com-
pounds [20, 21]. The main aim of this research work is the
synthesis and the study of the antioxidant properties of a
series of aryl compounds bearing two hydrazone functions
in one molecule. Firstly, the antioxidant activity has been
evaluated in vitro by using series of assays including DPPH
(2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis(3-
ethylbenzothiazoline-6-sulfonic acid), CUPRAC (cupric ion
reducing antioxidant capacity) and DMSO alkaline. Then, a
theoretical study based on DFT (density functional theory)
calculations for the most active compound has been carried
out in order to better understand the antioxidant activity of
this family of compounds. Antioxidant mechanisms, fron-
tier molecular orbitals and molecular electrostatic potential
(MEP) mapping have been investigated. To further explore
the multifunctional properties of the synthesized hydra-
zones, their acetylcholinesterase inhibitory activity has
been also investigated. This enzyme is a primary target for
symptomatic improvement in Alzheimer’s disease [22]. The
obtained experimental results were validated by molecular
docking study. Finally, in silico ADME (absorption, dis-
tribution, metabolism and excretion) parameters of all the
compounds have been determined to evaluate their pharma-
cokinetic properties.
Hydrazone is an azomethine group characterized by the
triatomic structure C=N–N. This functional group can be
obtained by simple condensation between hydrazine or its
derivatives and aldehydes or ketones. The presence of spe-
cifc functionalities at the hydrazine or the carbonyl com-
pound promotes the formation of very attractive systems
such as chemosensors [1, 2], adsorbents [3], dyes [4, 5]
and catalysts [6]. Molecules derived from hydrazone pre-
sent a wide range of biological activities such as anticancer
[7], antimicrobial [8, 9], anti-infammatory [10], antifun-
gal [11], antitubercular [12] and antiviral [13]. Hydrazone
functional group is present also in a number of anticancer,
antifungal, antibiotic and antihypertensive commercial
drugs such as mitoguazone, ferimzone, nifuroxazide and
dihydralazine (Fig. 1).
Hydrazone-based compounds are widely studied for
their antioxidant properties, and several studies have shown
that this family of compounds has high antioxidant activ-
ity [14–17]. In the medicinal point of view, compounds
with potent antioxidant properties are very attractive can-
didates for drug development, because the oxidative stress
is well known to be involved in several human diseases
such as cancer, Parkinson’s disease, Alzheimer’s disease,
Experimental section
Materials and instrumentation
The FTIR spectra were recorded with a JASCO FT/
IR-6300 type A spectrometer, and only signifcant absorp-
1
tion band frequencies are cited. H NMR and 13C NMR
spectra were recorded on a Bruker Avance DPX250.
Melting points were determined on a Kofler melting
point apparatus. Commercial-grade reagents were used
as supplied: terephthalaldehyde 98% (Alfa Aesar), phe-
nylhydrazine 97% (Alfa Aesar), 4-chlorophenylhydrazine
97% (Alfa Aesar), 4-methoxyphenylhydrazine 99% (Alfa
Aesar), 3,4-dimethylphenylhydrazine 98% (Alfa Aesar),
[4-(benzyloxy)phenyl]hydrazine 98% (Alfa Chemistry).
Fig. 1 Some commercial drugs derived from hydrazone scafold
1 3

Molecular Diversity
7.06–7.02 (m, 4H, Haromatic), 6.96–6.92 (m, 4H, Haromatic),
4.95 (s, 4H, Hbenzylic). 13C NMR (62.5 MHz, DMSO-d6):
δ 151.71, 139.51, 137.55, 135.29, 135.17, 128.42, 127.72,
125.70, 119.41, 115.81, 112.99, 69.66.
General procedure for the synthesis
of 1,4‑bisphenylhydrazone derivatives 1–5
In a 25-mL Erlenmeyer fask, 1 mmol of terephthalal-
dehyde and 2 mmol of phenylhydrazine derivative were
dissolved in 3 ml of ethanol. The reaction mixture was
stirred for 4 h at room temperature; then, it was fltered
and air-dried. The resulting residue was then purifed by
recrystallization in a mixture of ethanol/DMSO to give
the pure product.
In vitro antioxidant evaluation
DPPH free radical scavenging assay
The free radical scavenging activity was determined spectro-
photometrically by the DPPH assay [23]. In its radical form,
DPPH· absorbs at 517 nm, but upon reduction by an antioxi-
dant or a radical species its absorbance decreases. Briefy, 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 diferent 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 mix-
ture 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:
1,4‑bis((E)‑(2‑phenylhydrazineylidene)methyl)benzene
(1) Yield: 87%. (Yellow crystals) Mp > 260 °C. IR: νmax
1628 (C=N) cm−1. 1H NMR (250 MHz, DMSO-d6): δ 10.53
(brs, 2H, NH), 7.90–7.87 (m, 2H, Haromatic), 7.69–7.66 (m,
4H, 2Haromatic, 2CHimine), 7.24 (t, 4H, J=7.4 Hz, Haromatic),
7.12–7.04 (m, 4H, Haromatic), 6.80–6.74 (m, 2H, Haromatic).
13C NMR (62.5 MHz, DMSO-d6): δ 145.20, 136.18, 135.35,
129.20, 125.92, 118.85, 112.04.
1,4‑bis((E)‑(2‑(4‑methoxyphenyl)hydrazineylidene)methyl)
benzene (2) Yield: 85%. (Yellow crystals) Mp = 259 °C.
1
IR: νmax 1642 (C=N) cm−1. H NMR (250 MHz, DMSO-
d6): δ 10.21 (brs, 2H, NH), 7.90 (s, 2H, CHimine), 7.38–7.31
(m, 2H, Haromatic), 7.24–7.18 (m, 2H, Haromatic), 7.17–7.08
(m, 4H, Haromatic), 7.04–6.96 (m, 4H, Haromatic), 3.76 (s, 6H,
OMe). 13C NMR (62.5 MHz, DMSO-d6): δ 142.04, 141.08,
129.17, 129.07, 124.36, 123.76, 122.91, 122.47, 121.60,
114.34, 113.98, 35.63.
AControl − ASample
DPPH scavenging effect (%) =
× 100
AControl
where Acontrol and Asample are the absorbances of the reference
and sample obtained from the UV–visible spectrophotom-
eter, respectively. The results were given as IC50 (µM) cor-
responding to the concentration of 50% of inhibition.
1,4‑bis((E)‑(2‑(3,4‑dimethylphenyl)hydrazineylidene)methyl)
benzene (3) Yield: 91%. (Yellow crystals) Mp>260 °C. IR:
νmax 1633 (C=N) cm−1. 1H NMR (250 MHz, DMSO-d6): δ
10.36 (brs, 2H, NH), 7.82–7.79 (m, 2H, Haromatic), 7.63–7.62
(m, 4H, 2Haromatic, 2CHimine), 7.00–6.81 (m, 6H, Haromatic),
2.20 (s, 3H, Me), 2.14 (s, 3H, Me). 13C NMR (62.5 MHz,
DMSO-d6): δ 143.28, 136.74, 135.38, 135.33, 130.14,
126.26, 125.77, 113.41, 109.58, 19.88, 18.67.
ABTS radical scavenging assay
The ABTS·+ scavenging activity was determined according
to the method of Re et al. [24]; 10 μL aliquot of each tested
sample at diferent concentrations (3.12, 6.25, 12.5, 25, 50,
100 and 200 µM) was added to 1.0 mL of diluted ABTS·+
solution. The ABTS·+ was generated by the reaction between
7 mM ABTS in water and 2.45 mM potassium persulfate and
stored in the dark at room temperature for 12 h. The ABTS·+
solution was diluted to get an absorbance of 0.703 0.025
at 734 nm with ethanol which was used as a control. After
10 min, the absorbance was measured at 734 nm. BHT and
BHA, under the same conditions as the samples and for
each concentration, were used as antioxidant standards. The
ABTS radical scavenging activity was calculated using the
following equation:
1,4‑bis((E)‑(2‑(4‑chlorophenyl)hydrazineylidene)methyl)ben-
zene (4) Yield: 89%. (Yellow crystals) Mp > 260 °C. IR:
1
νmax 1625 (C=N), 680 (C–Cl) cm−1. H NMR (250 MHz,
DMSO-d6): δ 10.58 (brs, 2H, NH), 7.93–7.87 (m, 2H,
Haromatic), 7.74–7.66 (m, 4H, 2Haromatic, 2CHimine), 7.28–7.25
(m, 4H, Haromatic), 7.11–7.03 (m, 4H, Haromatic). 13C NMR
(62.5 MHz, DMSO-d6): δ 144.14, 137.04, 135.33, 129.04,
126.11, 122.11, 113.5.
AControl − ASample
1,4‑bis((E)‑(2‑(4‑(benzyloxy)phenyl)hydrazineylidene)methyl)
benzene (5) Yield: 94%. (Yellow crystals) Mp=258 °C. IR:
νmax 1632 (C=N) cm−1. 1H NMR (250 MHz, DMSO-d6): δ
10.30 (brs, 2H, NH), 7.81–7.80 (m, 2H, Haromatic), 7.62–7.60
(m, 4H, 2Haromatic, 2CHimine), 7.47–7.32 (m, 8H, Haromatic),
ABTS scavenging effect (%) =
× 100
AControl
1 3

Molecular Diversity
where Acontrol and Asample are the absorbances of the reference
and sample obtained from the UV–visible spectrophotom-
eter, respectively. The results were given as IC50 (µM) cor-
responding to the concentration of 50% of inhibition.
2-nitrobenzoic-5-mercaptothiocholine and 5-thio-2-ni-
trobenzoate which can be detected at 412 nm. In this method,
150 μL of 100 mM sodium phosphate bufer (pH 8.0), 10 μL
of test solution at diferent concentrations (3.12, 6.25, 12.5,
25, 50, 100 and 200 µM) and 20 μL of AChE from Electro-
phorus electricus (5.32×10−3 units) solutions were mixed
and incubated for 15 min at 25 °C, and 10 μL of 0.5 mM
(DTNB) was added. The reaction was then initiated by the
addition of 10 μL of acetylthiocholine iodide (0.71 mM).
The hydrolysis of this substrate was monitored spectropho-
tometrically at a wavelength of 412 nm, every 5 min for
15 min in triplicate experiments. The results were given as
IC50(µM), and the percentage of inhibition was determined
by the comparison of reaction rates of samples relative to the
blank sample (methanol in phosphate bufers, pH 8) using
the formula:
Cupric reducing antioxidant capacity (CUPRAC) assay
The cupric reducing capacity of the compounds was
determined by the CUPRAC method [25]. One milliliter
of copper (II) chloride solution (0.01 M prepared from
CuCl2.2H2O), 1 mL of ammonium acetate bufer at pH 7.0
and 1 mL of neocuproine solution (0.0075 M) were mixed
to 0.5 mL of samples or standard of diferent concentration
solutions (3.12, 6.25, 12.5, 25, 50, 100 and 200 µM). The
fnal volume of the mixture was adjusted to 4.1 mL by add-
ing 0.6 mL of distilled water. The resulting mixture was
incubated for 1 h at room temperature, and then, the absorb-
ance of the solution was measured at 450 nm by the use of
a spectrophotometer against blank and BHT and BHA as
standards. The results were given as A0.5 (μM) correspond-
ing the concentration indicating 0.50 absorbance intensity.
AControl − ASample
Percentage of inhibition (%) =
× 100
AControl
where Acontrol and Asample are the absorbances of the reference
and sample obtained from the UV–visible spectrophotom-
eter, respectively.
Superoxide radical scavenging activity (DMSO alkaline
assay)
Computational details
Density functional theory (DFT) calculations have been
carried out using Gaussian09 software [29]. The B3LYP
functional [30, 31] and the 6-311G(d,p) basis set have been
used for all calculations. This approach has been used suc-
cessfully by several research groups, and good agreement
between theory and experiment was found [32–36]. Solvent
efect of ethanol was approximated by the integral equation
formalism of polarizable continuum model (IEF-PCM). All
the ground states were confrmed by vibrational frequency
analysis (no imaginary frequency). The numerical descrip-
tors of the antioxidant mechanisms (BDE, IE, PDE, PA,
ETE) have been calculated as follows [37–39]:
Superoxide was generated according to the alkaline DMSO
method described by Hyland et al. [26]. Superoxide radical
(O·2−) is generated by the addition of sodium hydroxide to air-
saturated DMSO. The generated superoxide remains stable
in solution and reduces nitroblue tetrazolium (NBT) into
formazan dye at room temperature which can be measured at
560 nm. The fnal volume used (1.4 mL) for the absorbance
measurement was 0.1 mL of NBT (1 mg/mL) was added to
the reaction mixture containing 1 mL of alkaline DMSO
(1 mL DMSO containing 5 mM NaOH in 0.1 mL water) and
0.3 mL of the tested sample at various concentrations (3.12,
6.25, 12.5, 25, 50, 100 and 200 µM). Tocopherol (TOC) and
tannic acid (TAC) were used as positive control. The results
were given as IC50 (μM) corresponding to the concentration
of 50% of inhibition.
⋅
⋅
BDE = H(R ) + H(H ) − H(R − H)
(
)
IE = H RH+⋅ + H(e−) − H(R − H)
Inhibition of acetylcholinesterase
(
)
(
)
⋅
PDE = H(R ) + H H+ − H RH+⋅
AChE inhibitory activity was measured using quantita-
tive colorimetric assay using a 96-well microplate reader
according to the method described by Rhee et al. [27]
based on Ellman’s method [28]. The enzyme hydro-
lyzes the substrate acetylthiocholine resulting in the
product thiocholine which reacts with Ellman’s reagent:
5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) to produce
(
)
PA = H(R−) + H H+ − H(R − H)
ETE = H(R ) + H(e−) − H(R−)
⋅
1 3

Molecular Diversity
⋅
where H(R ) is the enthalpy obtained by geometry optimi-
Results and discussion
zation of the radicals formed after abstracting of H atom
⋅
from the NH bond, H(H ) is the enthalpy of a single H atom
Chemistry
at the B3LYP/6-311G(d,p) level, H(R − H) is the enthalpy
(
)
of the neutral molecule, H RH+⋅ is the enthalpy of radical
The 1,4-bisphenylhydrazone derivatives were easily pre-
pared from terephthalaldehyde and phenylhydrazine
derivatives as presented in Scheme 1. The reaction of one
equivalent of terephthalaldehyde with two equivalents of
phenylhydrazine derivative (phenylhydrazine, 4-methoxy-
phenylhydrazine, 3,4-dimethylphenylhydrazine, 4-chloro-
phenylhydrazine and (4-benzylphenyl)hydrazine) at room
temperature for 4 h in ethanol gives the corresponding
1,4-bisphenylhydrazone derivatives 1–5 in excellent yields
(85–94%). The molecular structures of the synthesized
compounds were confrmed by IR, 1H NMR and 13C NMR
spectroscopies.
(
)
cation. H(e−) is the enthalpy of single electron, H H+ is
the enthalpy of proton, and H(R−) is the enthalpy of charged
molecule after abstracting of a proton from the NH bond.
The calculated enthalpies of the hydrogen (H·), electron (e−)
and proton (H+) in gas phase and solvent environment were
taken from the literature [40–44].
Molecular docking study
In order to investigate the possible binding modes of com-
pound 2 (the most active compound) to the protein AChE,
calculations were carried out with “Achilles” Blind Docking
server, available at: http://bio-hpc.eu/. Using a “blind dock-
ing” approach, the docking of the small molecule to the tar-
gets is carried out without a priori knowledge of the location
of the binding site by the system [45]. Figures were drawn
using the Chimera program [46]. The ligand structures have
been built and energy-minimized using the program Marvin-
Sketch [ChemAxon ltd, Budapest, Hungry]. The coordinates
of AChE (PDB ID: 1C2B) were obtained from the Protein
Data Bank (PDB).
Antioxidant activity
In vitro investigation of the antioxidant activity
The antioxidant activity of the synthesized hydrazones was
evaluated using a series of assays. Firstly, assays based on
two oxidants, the radical 2,2-diphenyl-1-picrylhydrazyl
(DPPH) and the radical cation 2,20-azino-bis-(3-ethylben-
zothiazoline-6-sulfonic acid (ABTS·+), have been used to
evaluate the radical scavenging properties of the synthesized
compounds. In these assays, the total antioxidant activity of
a molecule is deduced from its ability to inhibit the DPPH·
or ABTS·+ radicals [47]. DMSO alkaline assay has been
used to evaluate the potential of the investigated molecules
to scavenge superoxide radical. In this method, the anti-
oxidant reacts with O·2− to generate the stable O2 molecule
[26]. Finally, the cupric ion reducing capability has been
ADME parameters and BBB permeability
ADME properties were predicted using Molinspiration
online property calculation toolkit available at: http://www.
molinspiration.com. The BBB permeability was evaluated
by using the CBLigand-BBB prediction server available at:
http://www.cbligand.org.
Scheme 1 Synthesis and struc-
tures of 1,4-bisphenylhydrazone
derivatives (1–5)
1 3

Molecular Diversity
elucidated by CUPRAC assay. This method measures the
ability of an antioxidant to reduce Cu2+ to Cu+ [48]. Butyl-
ated hydroxytoluene (BHT) and butylated hydroxyanisole
(BHA) were used as positive controls for DPPH, ABTS and
CUPRAC assays and α-tocopherol (TOC) and tannic acid
(TAC) for DMSO alkaline assay. The IC50 and A0.50 val-
ues were determined for all compounds and are reported in
Fig. 2 and Table S1 in SI.
order of the antioxidant activity by DMSO alkaline assay
is 1>4>5>2>3. Finally, in CUPRAC assay compound 1
has shown the best result with A0.50 value (30.12 0.81 µM)
slightly lower than that of the BHT (43.66 0.87 µM) and
slightly higher than that of the BHA (20.19 0.19 µM). The
order of the antioxidant activity is 1>3>4>5>2.
Overall, among the studied compounds, the hydrazone 1
(non-substituted compound) was found to be the best anti-
oxidant agent. This compound shows a higher or comparable
antioxidant activity than BHT and BHA in DPPH, ABTS
and CUPRAC assays, as well as a higher antioxidant activity
than TOC and TAA in DMSO alkaline assay. On the other
hand, compound 2 (bearing methoxy group) was found to
be the least reactive antioxidant agent among the synthe-
sized compounds. The other compounds (3–5) have rela-
tively comparable antioxidant activity, especially in DPPH,
ABTS and CUPRAC assays. As can be concluded from the
obtained results, the synthesized hydrazones 1–5 are anti-
oxidants as potent as the standards BHT, BHA, TAA and
TOC. These results are in line with those of other reported
hydrazone-based compounds [49–51].
An examination of DPPH, ABTS, CUPRAC and DMSO
alkaline assays results, as shown in Fig. 2 and Table S1,
reveals the following observations: All the tested compounds
1–5 exhibited a high antioxidant activity when compared to
the standards with IC50 and A0.50 values<72.27 0.46 µM.
In DPPH assay, all the studied compounds have shown a
higher antiradical activity than the standard BHT (IC50:
19.90 0.51- 68.55 1.74 vs 81.29 1.19 µM). Compound
1 followed by compound 3 is the best antioxidant agents
with IC50 values (26.75 0.05 µM and 30.12 1.20 µM,
respectively) about three times less than that of the BHT
(81.29 1.19 µM) and comparable to that of the BHA
(31.79 0.41 µM). Compounds 4 (IC50 =40.08 0.54 µM)
and 5 (IC50 =39.90 0.51 µM) have comparable IC50 val-
ues slightly higher than that of the BHA (31.79 0.41 µM).
Among the studied compound, the least reactive antioxi-
dant was found to be compound 2 with an IC50 value of
68.55 1.74 µM. The antioxidant activity of compounds 1–5
in DPPH assay can be ranked in the order: 1>3>5>4>2.
In ABTS assay, the same order was observed, and the best
result was obtained with compound 1, which shows an IC50
value (9.76 0.07 µM) lower than that of the BHT and
BHA (15.85 0.30 and 10.04 0.10 µM, respectively). In
DMSO alkaline assay, the best results were observed with
compounds 1 and 4, and these compounds have shown
a higher antioxidant activity than the standards TAA
(9.71 0.16 µM) and TOC (24.36 0.86 µM) with IC50 val-
ues of 5.38 0.21 µM and 7.67 0.68 µM, respectively. The
Theoretical evaluation of radical scavenging mechanisms
It is widely established that antioxidants scavenge free radi-
cals following several mechanisms [52]. The most studied
in literature are: hydrogen atom transfer (HAT), sequential
electron transfer proton transfer (SETPT) and sequential
proton loss electron transfer (SPLET) [36, 53–56]. In the
HAT mechanism, hydrogen atom transfers from the antioxi-
dant to the free radical in one step. The SETPT mechanism
involves two steps: the initial formation of a radical cation
by the transfer of an electron from the antioxidant to the free
radical and a proton transfer from the radical cation to the
anion. The SPLET mechanism involves the dissociation of a
proton from the antioxidant followed by an electron transfer
Fig. 2 Antioxidant activity of compounds 1–5 by DPPH, ABTS, CUPRAC and DMSO alkaline assays. Values expressed are mean S.D. of
three parallel measurements. (p<0.05)
1 3

Molecular Diversity
to the free radical. The HAT mechanism is characterized
by the BDE (bond dissociation enthalpy) value that corre-
sponds to the ability of an antioxidant to donate its hydrogen
atom and consequently form a radical. The lower the BDE
value, the better the antioxidant properties. The SETPT
mechanism is characterized by the IP (ionization potential)
value, which represents the enthalpy required to remove an
electron from the neutral molecule and the PDE (proton dis-
sociation enthalpy) value that corresponds to the enthalpy of
deprotonation of the antioxidant radical cation. The lower
the IP and PDE values, the higher the antioxidant activity.
Finally, the SPLET mechanism is characterized by the PA
(proton afnity) value, which corresponds to the enthalpy of
the dissociation of a proton from the neutral molecule and
the ETE (electron transfer enthalpy) value that corresponds
to the enthalpy of the transfer of an electron from the anti-
oxidant anion to the free radical. The lower PA and ETE
values are characteristic of higher antioxidant activity.
In order to have a better understanding of the antioxidant
properties of the synthesized hydrazones 1–5 and in which
mechanism they follow to scavenge free radicals, the three
main antioxidant mechanisms (HAT, SETPT and SPLET)
have been investigated. Using DFT method at B3LYP/6-
311G(d,p) level of theory, we have calculated the numerical
descriptors of the antioxidant mechanisms (BDE, IP, PDE,
PA and ETE) for compound 1, as representative compound,
and for BHT for comparison. The implicitly of ethanol has
been also considered by employing the integral equation
formalism of polarizable continuum model (IEF-PCM).
The obtained results are depicted in Fig. 3 and tabulated in
Table S2 in SI.
Fig. 3 Thermodynamic descriptors, BDE (bond dissociation
enthalpy), IP (ionization potential), PDE (proton dissociation
enthalpy), PA (proton afnity) and ETE (electron transfer enthalpy),
of the antioxidant mechanisms for compound 1 and BHT in the gas
phase and EtOH
the PA values are dramatically decreased by about 290 kcal/
mol in ethanol due to the large solvation enthalpies of the
proton. In contrast to IP, PDE and PA values, ETE val-
ues are slightly increased in ethanol by 10 kcal/mol. These
results are also in line with previous studies [54, 59, 60].
Thermodynamically, the antioxidant preferred mecha-
nism can be determined by comparing the BDE, IP and PA
values, in which IP and PA values are related to the frst
steps of SETPT and SPLET mechanisms, respectively [57,
61]. As shown in Fig. 3 and Table S2, for both compound 1
and BHT, the IP and PA values are signifcantly higher than
the BDE values in the gas phase, indicating that the HAT
is the dominate mechanism in this medium. In ethanol, the
large decrease observed with PA values makes SPLET the
more favorable mechanism.
As shown in Fig. 3 and Table S2, both compound 1
and BHT have a small BDE values ranging from 70.42 to
76.45 kcal/mol, indicating that both O–H and N–H functions
are easy to break and form stable radicals. BDE values of
BHT are about 5 kcal/mol lower than that of compound 1 in
the two studied mediums. The IP values of compound 1 are
about 25 kcal/mol and 15 kcal/mol lower than that of BHT
in the gas phase and ethanol, respectively, while the PDE
values of BHT are lower than that of compound 1 by about
20 kcal/mol and 30 kcal/mol in the gas phase and ethanol,
respectively. This indicates that both compound 1 and BHT
have a comparable antioxidant activity via SETPT mecha-
nism. It should be noted that all the IP and PDE values are
decreased in ethanol by about 60 kcal/mol and 210 kcal/
mol, respectively. These decreases are due to the high sol-
vation enthalpies of electron and proton in solution, which
is in line with the results from previous studies on phenolic
compounds [57, 58].
It is well known that the energy and distribution of high-
est occupied molecular orbitals (HOMO) of an antioxidant
are correlated with its antioxidant activity [61]. Molecules
with lower HOMO energy are less likely to donate elec-
trons [62]. Moreover, the high electronic density of distri-
bution of HOMO determines the sites for the free radical
attack. Using the same level of theory as described above,
we have calculated the energies and distribution of HOMO
orbitals of compound 1 and BHT in the gas phase (Fig. 4).
As shown in Fig. 4, the HOMO of compound 1 is delocal-
ized over the whole molecule including the two hydrazone
functions, while the HOMO of BHT is only located on the
phenol group. In addition, HOMO energy of compound 1 is
slightly higher than that of BHT with values of − 5.07 eV
and −5.74 eV, respectively. This indicates that the electron
donating ability of compound 1 is slightly better than that
Compound 1 and BHT have a comparable PA and ETE
values in the studied mediums, indicating that both com-
pounds have also a comparable antioxidant activity via
SPLET mechanism. As observed with IP and PDE values,
1 3

Molecular Diversity
Fig. 4 HOMO orbital distribution and energy as well as the molecu-
lar electrostatic potential (MEP) mapping of compound 1 (left) and
BHT (right) in the gas phase
Fig. 5 IC50 values for the inhibition of acetylcholinesterase for com-
pounds 1–5 and galantamine
of BHT. These results are in good agreement with the cal-
culated IP values.
5, bearing, respectively, 4-Cl and 4-OCH2Ph, are the least
reactive AChE inhibitors among the studied hydrazones,
with IC50 values higher than 200 μM. A comparison of the
AChE inhibitory activity of hydrazones 1–5 with some data
from the literature reveals that compound 2 is among the
best AChE inhibitor of this family of compounds [65–67].
Molecular electrostatic potential (MEP) mapping is
another parameter that characterizes the antioxidant prop-
erties of the investigated compounds [33, 63, 64]. The more
positive sites are the privileged sites for free radical attack.
MEP mapping results calculated at B3LYP/6-311G(d,p)
level of theory for compound 1 and BHT are illustrated in
Fig. 4. The nucleophilic and electrophilic sites are expressed
in term of diferent color codes; a deep red color indicates an
electron-rich site, whereas deep blue indicates an electron-
defcient site. As presented in Fig. 4, the most electron-rich
sites for compound 1 and BHT are located around the nitro-
gen and the oxygen atoms, respectively, whereas the most
electron-defcient sites are one the hydrogen atoms of the
hydrazone function and the phenolic hydroxyl group, respec-
tively. These results confrm that the antioxidant properties
of compound 1 and BHT are due to the hydrazone functions
and the phenolic hydroxyl group, respectively.
Molecular docking study
Molecular docking studies are widely employed to explore
the binding energy and to validate molecular mechanisms for
ligands at the active site of a protein. In order to investigate
the interaction modes of the synthesized hydrazones with
AChE (PDB code: 1C2B), the most active compound (2)
was subjected to molecular docking study using Blind Dock-
ing server. Figure 6 shows the most energetically favorable
binding mode of compound 2 at the active site of AChE,
and Table S4 in SI summarizes all the molecular docking
binding interactions. To validate the approach used, we have
also performed a molecular docking study for galantamine,
and the obtained results for the most energetically favorable
binding mode of this compound are tabulated in Table S5 in
SI. The obtained results revealed that the most energetically
favorable binding mode of compound 2 has a binding energy
of − 9.2 kcal/mol. This energy is slightly lower than that
obtained with galantamine (−8.30 kcal/mol). In this pose,
compound 2 forms six hydrogen bonds, four bonds between
the hydrazone functions and the residues Ser293, Tyr337
and Tyr124, and two bonds between the methoxy group
and the residues His446 and Ser203. Also, the substituted
aromatic moieties of compound 2 interact with Phe279,
Trp86, Tyr337, Phe338, Phe295, Trp286 and Leu289 by
weak π–π stacking and hydrophobic interaction. Finally,
the central aromatic nucleus forms hydrophobic interac-
tions with Phe338 and Tyr341. Some of these residues are
Acetylcholinesterase inhibitory activity
In vitro acetylcholinesterase inhibitory activity
The capacity of the synthesized hydrazones 1–5 to inhibit
Electrophorus electricus acetylcholinesterase (AChE) was
evaluated using a modifer Ellman’s assay [28]. Galantamine
(Gal.), used for mild Alzheimer’s disease, was used as posi-
tive control. The obtained results are presented in Fig. 5
and Table S3 in SI. As can be seen, among the investigated
hydrazones, compounds 1, 2 and 3 have shown a moder-
ate to excellent activity when compared to the galantamine.
Compound 2, bearing a methoxy group, is the best AChE
inhibitor with an IC50 value (9.18 0.40 µM) comparable to
that of the galantamine (8.90 1.16 µM). Compounds 4 and
1 3

Molecular Diversity
Fig. 6 Binding mode of com-
pound 2 at the active site of
AChE (PDB code: 1C2B)
reportedly involved in ligand–receptor complexes of tacrine,
galantamaine, huperzine A and donepezil [68]. From these
results, it is clear that compound 2 possess a high bind-
ing afnity against acetylcholinesterase and can inhibit its
activity, which is in good agreement with the experimental
observations.
addition, the percentage of absorption of the compounds
has been calculated [71] and interesting values have been
obtained for all the compounds (85.19–91.73%). Accord-
ingly, it can be suggested that the synthesized hydrazones
may be have a good pharmacokinetic profle and can be con-
sidered as drug candidates.
The BBB (blood–brain barrier) permeability is another
important parameter that affects the biological activity
results. Drugs that specifcally target the central nervous
system, such as cholinesterase inhibitors, must cross the
blood–brain barrier. The BBB permeability of the synthe-
sized hydrazones was evaluated by using the CBLigand-
BBB prediction server, and the obtained results are pre-
sented in Table 1. The obtained results revealed that all the
compounds are BBB-positive, which is required for the ace-
tylcholinesterase activity.
Prediction of ADME parameters and BBB
permeability
Good pharmacological activities are not enough for a com-
pound to become a drug candidate. It is well known that
absorption, distribution, metabolism and excretion (ADME)
properties of a molecule are one of the main reasons of its
failure in clinical trials [69]. After the evaluation of the
antioxidant and acetylcholinesterase inhibitor activities of
the synthesized hydrazones, we have studied their ADME
properties by using Molinspiration online property calcula-
tion toolkit. All the obtained parameters are presented in
Table 1. According to Lipinski’s rule [70], in general, an
orally active drug has no more than one violation of the fol-
lowing criteria: (1) No more than 5 hydrogen bond donors
(n-OHNH). (2) No more than 10 hydrogen bond acceptors
(n-ON). (3) A molecular weight (MW) less than 500 D. (4)
An octanol–water partition coefcient (milogP) not greater
than 5. As shown in Table 1, all the tested hydrazones ft-
ted Lipinski’s rules by possessing no more than one viola-
tion, except compound 5 which showed two violations. In
Conclusion
Five 1,4-bisphenylhydrazone derivatives have been synthe-
sized and evaluated for their antioxidant and acetylcholinest-
erase inhibitory activities. All the compounds showed a very
good antioxidant activity compared to the standards used.
Among them, compound 1 proved to be the best antioxidant
and compound 2 was the best inhibitor of acetylcholinester-
ase. From DFT calculation, the HAT mechanism was found
to be the dominated mechanism in the gas phase, whereas
Table 1 In silico some
Comp. miLogP TPSA (°A) MW
n-ON n-OHNH n-rotb MV
%ABS vio. BBB
<1
physicochemical and
<5
<500
<10
<5
pharmacokinetic parameters
of the synthesized
1
2
3
4
5
7.59
7.70
8.81
8.66
9.36
48.78
67.25
48.78
48.78
67.25
314.39
374.44
370.50
383.28
526.64
4
6
4
4
6
2
2
2
2
2
6
8
6
6
12
298.19 91.73
349.28 85.19
364.43 91.73
325.26 91.73
492.58 85.19
1
1
1
1
2
+
+
+
+
+
1,4-bisphenylhydrazone
derivatives 1–5
%ABS percentage of absorption, TPSA topological polar surface area, n-rotb number of rotatable bonds,
MW molecular weight, MV molecular volume, miLogP logarithm of partition coefcient between n-octanol
and water, n-OHNH number of hydrogen bond donors, n-ON number of hydrogen bond acceptors, vio
Lipinski’s violation, BBB blood–brain barrier permeability
1 3

Molecular Diversity
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the SPLET is the thermodynamically favorable pathway in
ethanol. Finally, molecular docking study revealed a strong
interaction between compound 2 and the active sites of
AChE. In silico ADME studies have demonstrated that these
compounds have a good pharmacokinetic profle.
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hydrazones derived from cyclic β-diketone for cytotoxic and anti-
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Acknowledgements We would like to thank MESRS (Ministère de
l’Enseignement Supérieur et de la Recherche Scientifque, Algeria)
and DGRST (Direction Générale de la Recherche Scientifque et Tech-
nologique, Algeria) for fnancial support, as well as the HPC resources
of UCI-UFMC (Unité de Calcul Intesif) of the university Fréres Men-
touri Constantine 1 for the computational resources used. We also
thank Dr. Bensouici Chawki (Centre de recherche en Biotechnologie
CRBt, Constantine, Algeria) for his help in the biological evaluation.
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Afliations
Imene Amine Khodja1 · Houssem Boulebd1
1
* Houssem Boulebd
Laboratory of Synthesis of Molecules with Biological
boulebd.houssem@umc.edu.dz
Interest, University of Frères Mentouri Constantine 1,
Constantine, Algeria
1 3