Design, synthesis, biological evaluation, molecular docking, DFT calculations and in silico ADME analysis of (benz)imidazole-hydrazone derivatives as promising antioxidant, antifungal, and anti-acetylcholinesterase agents

Article abstract of DOI:10.1016/j.molstruc.2020.128527

Ten hydrazone derivatives bearing a (benz)imidazole nucleus were designed, synthesized and evaluated for their antioxidant, antifungal, and anti-acetylcholinesterase activities. All the synthesized compounds (1-10) showed good to excellent antioxidant activity. Among them, compound 10 was found to be the best acetylcholinesterase inhibitor with an IC₅₀ value comparable to that of the galantamine. Compound 5 was found to be the best antifungal agent against Fusarium oxysporum fungal strain when compared to the commercial fungicide carbendazim. DFT calculations, for representative molecules 1 and 6, were also performed to investigate the antioxidant mechanisms, and it was found that SETPT (sequential electron transfer-proton transfer) is the most favorable mechanism in ethanol. Molecular docking studies of the most active compounds were carried out, and results showed reasonable binding modes in the active site of Fusarium oxysporum FGB1 enzyme and acetylcholinesterase. Finally, in silico predictions of ADME and pharmacokinetic parameters indicated that these compounds should have good oral bioavailability.

Full text of DOI:10.1016/j.molstruc.2020.128527

Journal Pre-proof
Design, synthesis, biological evaluation, molecular docking, DFT calculations and
in silico ADME analysis of (benz)imidazole-hydrazone derivatives as promising
antioxidant, antifungal, and anti-acetylcholinesterase agents
Imene Amine Khodja, Houssem Boulebd, Chawki Bensouici, Ali Belfaitah
PII:
S0022-2860(20)30852-8
DOI:
https://doi.org/10.1016/j.molstruc.2020.128527
MOLSTR 128527
Reference:
To appear in: Journal of Molecular Structure
Received Date: 18 February 2020
Revised Date: 19 May 2020
Accepted Date: 25 May 2020
Please cite this article as: I.A. Khodja, H. Boulebd, C. Bensouici, A. Belfaitah, Design, synthesis,
biological evaluation, molecular docking, DFT calculations and in silico ADME analysis
of (benz)imidazole-hydrazone derivatives as promising antioxidant, antifungal, and anti-
acetylcholinesterase agents, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/
j.molstruc.2020.128527.
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Imene Amine Khodja: Investigation; Houssem Boulebd: Conceptualization,
Methodology, Formal analysis, Writing – original draft, Visualization, Writing – review
and editing; Chawki Bensouici: Resources; Ali Belfaitah: Resources.

Design, synthesis, biological evaluation, molecular docking, DFT
calculations and in silico ADME analysis of (Benz)imidazole-hydrazone
derivatives as promising antioxidant, antifungal, and anti-
acetylcholinesterase agents
Imene Amine Khodjaa, Houssem Boulebda,*, Chawki Bensouicib, Ali Belfaitahc
a
Laboratory of Synthesis of Molecules with Biological Interest, University of Frères Mentouri
Constantine 1, Constantine, Algeria.
b Centre de Recherche en Biotechnologie Ali Mendjli Nouvelle Ville, Constantine, Algérie
c
Laboratoire des Produits Naturels d’Origine Végétale et de Synthèse Organique, Faculté des
Sciences Exactes, Campus de Chaabat Ersas, Université des frères Mentouri-Constantine,
Constantine 25000, Algeria
*Corresponding author: E-mail: boulebd.houssem@umc.edu.dz
Abstract
Ten hydrazone derivatives bearing a (benz)imidazole nucleus were designed, synthesized and
evaluated for their antioxidant, antifungal, and anti-acetylcholinesterase activities. All the
synthesized compounds (1-10) showed good to excellent antioxidant activity. Among them,
compound 10 was found to be the best acetylcholinesterase inhibitor with an IC50 value
comparable to that of the galantamine. Compound 5 was found to be the best antifungal agent
against Fusarium oxysporum fungal strain when compared to the commercial fungicide
carbendazim. DFT calculations, for representative molecules 1 and 6, were also performed to
investigate the antioxidant mechanisms, and it was found that SETPT (sequential electron
transfer-proton transfer) is the most favorable mechanism in ethanol. Molecular docking
studies of the most active compounds were carried out, and results showed reasonable binding
modes in the active site of Fusarium oxysporum FGB1 enzyme and acetylcholinesterase.
Finally, in silico predictions of ADME and pharmacokinetic parameters indicated that these
compounds should have good oral bioavailability.
Keywords: (Benz)imidazole; Hydrazone; Acetylcholinesterase; Antioxidant activity;
Docking study; DFT calculations.
1

1. Introduction
Nitrogen-containing heterocycles such as imidazole and benzimidazole exhibit diverse range
of biological activities[1-5]. These heterocycles cover nearly all ranges of activities, such as
anticancer[6], anti-HIV[7], antitubercular[8, 9], antihepatitis C[10], anti-inflammatory[11],
antibacterial[12], antihypertensive[13], cholinesterase inhibitors[14], antioxidants[15], and
antiprotozoal[16]. In the medical field, a great number of imidazole and benzimidazole-based
compounds as clinical drugs have been extensively used to treat various types of diseases with
high therapeutic potency. For examples (Fig. 1), Emedastine is used as H1 receptor antagonist
in eye drops to alleviate the symptoms of allergic conjunctivitis[17]. Clotrimazole is an
antifungal drug used to treat some dermatophytes, yeasts, and dimorphic as well as
filamentous fungi infections[18]. Carbendazim is a widely used, broad-spectrum
benzimidazole fungicide[19]. Dacarbazine is chemotherapy agent used in the treatment of
melanoma and Hodgkin's lymphoma[20]. Albendazol is an anthelmintic drug used for the
treatment of a variety of parasitic worm infestations[21]. Losartan is an angiotensin II
receptor antagonist used to treat hypertension[22].
On the other hand, the hydrazone functional group presents innumerous applications
in medicinal chemistry[23], organic synthesis[24], supramolecular chemistry[25], dynamic
combinatorial chemistry[26], and others[27]. In medicinal chemistry, compounds bearing this
pharmacophore also exhibit a broad spectrum of biological activities including
antimicrobial[28],
antioxidant[29],
antiproliferative[30],
MAO
Inhibitory[31],
leishmanicidal[32], and antiproliferative[33]. Hydrazone functional group is present in
numbers of commercialized drugs, Fig. 1 shows some examples. Isocarboxazide is a non-
selective irreversible monoamine oxidase inhibitor (MAOI)[34]. Ferimzone is an antifungal
drug used for the control of fungal diseases in rice[35]. Dihydralazine is a smooth muscle
relaxant used to treat high blood pressure by acting as a vasodilator[36]. Mitoguazone is an
anticancer agent used in chemotherapy[37]. Nifuroxazide is an antibiotic used to treat colitis
and diarrhea[38]. Levosimendan is a calcium sensitizer used in the treatment of acute
congestive heart failure.[39]
Our goal in this paper was to incorporate these two independently biologically active
moieties into one molecule to generate compounds with new and/or enhanced biological
activities. In this context, and as a continuation of our previous developments on this
topic[40-44], we have designed and synthesized (Fig. 1) a series of hybrid compounds bearing
imidazole or benzimidazole nucleus and hydazone moiety and evaluated them for their
antioxidant, antifungal, and anti-acetylcholinesterase activities. To gain more insights into the
2

structure-activity relationship of the investigated compounds, some in silico studies such as
DFT calculations, molecular modeling, and ADME predictions have been also performed and
discussed.
Fig. 1. Design of (benz)imidazole-phenylhydrazone hybrids.
2. Experimental section
2.1. Materials and instrumentation
The FTIR spectra were recorded with a JASCO FT/IR-6300typeA spectrometer and only
1
significant absorption band frequencies are cited. H NMR and 13C NMR spectra were
recorded on a Bruker Avance DPX250. The measurements of the diffracted intensities were
recorded on APEX II diffractometer equipped with a two-dimensional detector Kappa CCD
(λKα = 0.71073 Å). Melting points were determined on a Kofler melting point apparatus.
Commercial grade reagents were used as supplied (Alfa Aesar). The starting materials 1-
methylbenzimidazole-2-carbaldehyde and 1-methylimidazole-2-carbaldehyde were prepared
as described in our previous studies[41, 43].
2.2. General procedure for the synthesis of (benz)imidazole-hydrazone derivatives 1-10
The hydrazones 1-10 were prepared by a condensation reaction between phenylhydrazine
derivatives and imidazole or benzimidazole carbaldehydes[45-47]. In a 25-mL Erlenmeyer
flask,
1
mmol of 1-methylbenzimidazole-2-carbaldehyde or 1-methylimidazole-2-
mmol of phenylhydrazine derivative (phenylhydrazine, 4-
carbaldehyde and
1
methoxyphenylhydrazine, 3,4-dimethylphenylhydrazine, 4-chlorophenylhydrazine and (4-
benzylphenyl)hydrazine) were dissolved in 3 ml of ethanol. The reaction mixture was stirred
3

at room temperature for 4h, and then filtered and air-dried. The resulting residue was then
purified by recrystallization in a mixture of ethanol/DMSO to give the pure product.
(E)-1-méthyl-2-((2-phenylhydrazineylidene)methyl)-1H-imidazole (1). Yield: 97%. (yellow
powder). Mp=164°C. IR: νmax 1637 (C=N) cm-1. 1H NMR (250 MHz, DMSO-d6): δ 10.12 (s,
1H, NHHyd), 7.82 (s, 1H, CHHyd), 7.24 (t, J = 7.7 Hz, 2H, Harom), 7.12 (s, 1H, Himid), 7.08 (d, J
= 7.7 Hz, 2H, Harom), 7.00 (s, 1H, Himid), 6.79 (t, J = 7.7 Hz, 1H, Harom), 4.09 (s, 3H, NCH3).
13C NMR (62.5 MHz, DMSO-d6): δ 146.29, 144.67, 130.19, 128.55, 128.18, 124.95, 120.78,
113.39, 36.16.
(E)-2-((2-(4-methoxyphenyl)hydrazineylidene)methyl)-1-methyl-1H-imidazole (2). Yield:
76%. (yellow powder). Mp>260°C. IR: νmax 1648 (C=N) cm-1. 1H NMR (250 MHz, DMSO-
d6): δ 10.53 (s, 1H, NHHyd), 7.88 (s, 1H, CHHyd), 6.98-6.77 (m, 6H, Himid, Harom), 6.86 (t, J =
13
7.2 Hz, 1H), 3.91 (s, 3H, OCH3), 3.69 (s, 3H, NCH3). C NMR (62.5 MHz, DMSO-d6): δ
152.73, 142.79, 139.14, 128.53, 127.81, 123.92, 119.54, 114.74, 113.54, 112.98, 55.32, 35.24
(E)-2-((2-(3,4-dimethylphenyl)hydrazineylidene)methyl)-1-methyl-1H-imidazole (3). Yield:
1
88%. (orange powder). Mp=166°C. IR: νmax 1651 (C=N) cm-1. H NMR (250 MHz, DMSO-
d6): δ 10.12 (s, 1H, NHHyd), 7.08 (s, 1H, CHHyd), 6.54-6.10 (m, 6H, Himid, Harom), 3.74 (s, 3H,
13
NCH3), 1.49 (s, 3H, CH3), 1.44 (s, 3H, CH3). C NMR (62.5 MHz, DMSO-d6): δ 143.32,
138.32, 131.07, 129.09, 125.03, 124.69, 124.40, 117.50, 114.85, 111.03, 35.80, 20.08, 18.95.
(E)-2-((2-(4-chlorophenyl)hydrazineylidene)methyl)-1-methyl-1H-imidazole (4). Yield: 79%.
1
(yellow powder). Mp=248°C. IR: νmax 1638 (C=N) cm-1. H NMR (250 MHz, DMSO-d6): δ
10.12 (s, 1H, NHHyd), 7.88 (s, 1H, CHHyd), 7.39-7.16 (m, 6H, Himid, Harom), 4.03 (s, 3H,
NCH3). 13C NMR (62.5 MHz, DMSO-d6): δ 144.07, 143.55, 130.23, 126.77, 125.17, 123.06,
122.85, 121.24, 115.39, 104.76, 36.02.
(E)-2-((2-(4-(benzyloxy)phenyl)hydrazineylidene)methyl)-1-methyl-1H-imidazole (5). Yield:
1
95%. (yellow powder). Mp=148°C. IR: νmax 1661 (C=N) cm-1. H NMR (250 MHz, DMSO-
d6): δ 10.46 (s, 1H, NHHyd), 7.85 (s, 1H, CHHyd), 7.45-7.21 (m, 6H, Harom), 6.95-6.84 (m, 5H,
13
Harom), 5.03 (s, 2H, CH2), 3.91 (s, 3H, NCH3). C NMR (62.5 MHz, DMSO-d6): δ 151.76,
142.71, 139.28, 137.56, 128.75, 128.44, 127.88, 127.71, 123.95, 115.88, 112.90, 69.65, 35.24
(E)-1-methyl-2-((2-phenylhydrazineylidene)methyl)-1H-benzo[d]imidazole (6). Yield: 92%.
1
(yellow brown). Mp=252°C. IR: νmax 1633 (C=N) cm-1. H NMR (250 MHz, DMSO-d6): δ
10.80 (s, 1H, NHHyd), 8.06 (s, 1H, CHHyd), 7.60 (t, J = 7.6 Hz, 2H, Harom), 7.34-7.10 (m, 6H,
Harom), 6.86 (t, J = 7.2 Hz, 1H, Harom), 4.13 (s, 3H, NCH3). 13C NMR (62.5 MHz, DMSO-d6):
4

δ 148.34, 144.37, 142.68, 136.81, 129.40, 128.89, 122.72, 121.93, 119.91, 119.57, 118.84,
113.05, 112.39, 109.98, 32.12.
(E)-2-((2-(4-methoxyphenyl)hydrazineylidene)methyl)-1-methyl-1H-benzo[d]imidazole
(7).
1
Yield: 80%. (yellow powder). Mp=162°C. IR: νmax 1656 (C=N) cm-1. H NMR (250 MHz,
DMSO-d6): δ 10.79 (s, 1H, NHHyd), 7.99 (s, 1H, CHHyd), 7.58 (t, J = 8.1 Hz, 2H, Harom), 7.29-
7.17 (m, 2H, Harom), 7.05 (d, J = 7.4 Hz, 2H, Harom), 6.93 (d, J = 7.4 Hz, 2H, Harom), 4.12 (s,
3H, OCH3), 3.72 (s, 3H, NCH3). 13C NMR (62.5 MHz, DMSO-d6): δ 153.3, 148.61, 142.73,
138.29, 136.81, 127.53, 122.53, 121.86, 118.71, 114.85, 113.45, 109.87, 55.29, 32.09.
(E)-2-((2-(3,4-dimethylphenyl)hydrazineylidene)methyl)-1-methyl-1H-benzo[d]imidazole (8).
1
Yield: 87%. (brown powder). Mp=232°C. IR: νmax 1653 (C=N) cm-1. H NMR (250 MHz,
DMSO-d6): δ 10.78 (s, 1H, NHHyd), 8.00 (s, 1H, CHHyd), 7.58 (t, J = 8.8 Hz, 2H, Harom), 7.30-
7.18 (m, 2H, Harom), 7.03 (t, J = 8.0 Hz, 1H, Harom), 6.89-6.83 (m, 2H, Harom), 4.12 (s, 3H,
13
NCH3), 2.21 (s, 3H, CH3), 2.15 (s, 3H, CH3). C NMR (62.5 MHz, DMSO-d6): δ 148.50,
142.69, 142.34, 136.97, 136.78, 130.28, 127.88, 127.46, 122.55, 121.85, 118.72, 113.71,
109.88, 109.83, 32.04, 19.86, 18.65.
(E)-2-((2-(4-chlorophenyl)hydrazineylidene)methyl)-1-methyl-1H-benzo[d]imidazole
(9).
1
Yield: 86%. (yellow powder). Mp=240°C. IR: νmax 1642 (C=N) cm-1. H NMR (250 MHz,
DMSO-d6): δ 11.04 (s, 1H, NHHyd), 8.05 (s, 1H, CHHyd), 7.64-7.61 (m, 2H, Harom), 7.35-7.09
(m, 6H, Harom), 4.12 (s, 3H, NCH3). 13C NMR (62.5 MHz, DMSO-d6): δ 148.05, 143.30,
142.63, 136.78, 129.73, 129.20, 123.17, 122.83, 121.98, 118.91, 113.85, 110.04, 32.11.
(E)-2-((2-(4-(benzyloxy)phényl)hydrazineylidène)méthyl)-1-méthyl-1H-benzo[d]imidazole
1
(10). Yield: 94%. (yellow powder). Mp=160°C. IR: νmax 1658 (C=N) cm-1. H NMR (250
MHz, DMSO-d6): δ 11.67 (s, 1H, NHHyd), 7.05 (s, 1H, CHHyd), 6.85-6.82 (m, 1H, Harom),
6.71-6.67 (m, 1H, Harom), 6.71-6.27 (m, 9H, Harom), 5.98 (d, J = 8.6 Hz, 2H, Harom), 4.01 (s,
13
2H, CH2), 2.99 (s, 3H, NCH3). C NMR (62.5 MHz, DMSO-d6): δ 154.21, 137.20, 136.80,
133.09, 130.62, 128.47, 127.87, 127.78, 125.94, 125.03, 115.75, 115.64, 115.24, 113.32,
112.17, 69.55, 31.81.
2.3. In vitro antioxidant evaluation
2.3.1. DPPH free radical scavenging assay
The free radical-scavenging activity was determined spectrophotometrically by the DPPH
assay[48]. In its radical form, DPPH• absorbs at 517nm, but upon reduction by an antioxidant
or a radical species its absorbance decreases. Briefly, a 0.1 mM solution of DPPH• in ethanol
5

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:
Aꢀꢁꢂꢃꢄꢁꢅ − Aꢆꢇꢈꢉꢅꢊ
DPPH scavenging effect (%) =
× 100
Aꢀꢁꢂꢃꢄꢁꢅ
where Acontrol and Asample are the absorbances of the reference and sample obtained from the
UV–visible spectrophotometer, respectively. The results were given as IC50 (µM)
corresponding to the concentration of 50% of inhibition.
2.3.2. ABTS radical scavenging assay
The ABTS•+ scavenging activity was determined according to the method of Re et al[49], 10
μL aliquot of each tested sample at different concentrations (3.12, 6.25, 12.5, 25, 50, 100, and
200 µM) were added to 1.0 mL of diluted ABTS•+ solution. The ABTS•+ was generated by the
reaction between 7mM ABTS in water and 2.45mM potassium persulfate, 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:
Aꢀꢁꢂꢃꢄꢁꢅ − Aꢆꢇꢈꢉꢅꢊ
ABTS scavenging effect (%) =
× 100
Aꢀꢁꢂꢃꢄꢁꢅ
where Acontrol and Asample are the absorbances of the reference and sample obtained from the
UV–visible spectrophotometer, respectively. The results were given as IC50 (µM)
corresponding to the concentration of 50% of inhibition.
2.3.3. Cupric reducing antioxidant capacity (CUPRAC) assay
The cupric reducing capacity of the compounds was determined by the CUPRAC method[50].
One millilitres of copper (II) chloride solution (0.01 M prepared from CuCl2.2H2O), 1 mL of
ammonium acetate buffer at pH 7.0 and 1 mL of neocaproin solution (0.0075 M) were mixed
to 0.5 mL of samples or standard of different concentrations solution (3.12, 6.25, 12.5, 25, 50,
100 and, 200 µM). The final volume of the mixture was adjusted to 4.1 mL by adding 0.6 mL
6

of distilled water. The resulting mixture was incubated for 1 h at room temperature, and then
the absorbance 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)
corresponding the concentration indicating 0.50 absorbance intensity.
2.3.4. Galvinoxyl free radicals (GOR) scavenging assay
The GOR scavenging activity was determined according to the method of Shi et al.[51]. 160
μL of 0.1 mM ethanolic solution of Galvinoxyl was added to 40 μL of different
concentrations (3.12, 6.25, 12.5, 25, 50, 100, and 200 µM) of compounds 1-10 in ethanol. The
absorbance was read at 428 nm after 120 min incubation in dark at room temperature.
Galvinoxyl solution in ethanol was used as a control. BHT and BHA were used as antioxidant
standards. The results were given as IC50 (µM).
2.4 In vitro antifungal evaluation
The antifungal activity of the synthesized hydrazones 1-10, on the mycelium growth of the
phytopathogenic agent (fusarium oxysporum), is determined by measuring the radial growth
of the fungi on PDA medium (potato dextrose agar) containing the molecule to be tested. A
volume of 1 mL of DMSO containing a different mass for each product for a concentration of
200µM was added to 75 mL of PDA medium at 60°C. Previously sterilized and then
distributed in 3 petri dishes. Similarly, 1 mL of DMSO was added to 75 mL of PDA medium,
and was considered as a positive control. The negative control contains the PDA medium
without any other products.
Experimentally, a disk of 5 mm in diameter is taken from a young fungal culture and
is deposited aseptically in the center of the petri dish containing the PDA medium and the
molecule to be tested. The experiment is replicated 3 times for each treatment. After 6 days of
incubation at 28°C, the mycelial growth of the phytopathogenic agents is measured at
millimetric scale. Results were expressed as the percentage of growth inhibition of each
compound with respect to the mean colony diameters of each fungus grown in control
medium. The inhibition activity was expressed as a percentage and was calculated to the
formula:
C − T
I =
× 100
C
7

where I = inhibition rate in %; C = radial growth of phytopathogenic agent in mm on PDA
medium with DMSO (control); T = the radial growth, in mm, of the phytopathogenic agent on
PDA medium containing the molecule to be tested.
To identify the lowest inhibitory concentration, the test was repeated with
concentrations of 800µM, 400µM, 200µM, 100µM, and 50µM. The same result was obtained
which means that the efficiency threshold concentration can be lower.
2.5. Inhibition of acetylcholinesterase
AChE inhibitory activity was measured using quantitative colorimetric assay using a 96-well
microplate reader according to the method described by Rhee et al[52] based on Ellman’s
method[53]. The enzyme hydrolyses the substrate acetylthiocholine resulting in the product
thiocholine which reacts with Ellman’s reagent: 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB)
to produce 2-nitrobenzoic-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which can be
detected at 412 nm. In this method, 150 μL of 100 mM sodium phosphate buffer (pH 8.0), 10
μL of test solution at different concentrations (3.12, 6.25, 12.5, 25, 50, 100, and 200 µM) and
20 μL of AChE from Electrophorus 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) were added. The reaction was
then initiated by the addition of 10μL of acetylthiocholine iodide (0.71 mM). The hydrolysis
of this substrates was monitored spectrophotometrically 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 buffers, pH 8) using the formula:
Aꢀꢁꢂꢃꢄꢁꢅ − Aꢆꢇꢈꢉꢅꢊ
Percentage of inhibition (%) =
× 100
Aꢀꢁꢂꢃꢄꢁꢅ
where Acontrol and Asample are the absorbances of the reference and sample obtained from the
UV–visible spectrophotometer, respectively.
2.6. Docking study
In order to investigate the possible binding modes of compound 5 and 10 (the most active
compounds) to the enzymes FGB1 (Guanine nucleotide-binding protein beta) and hAChE
(human acetylcholinesterase), respectively, molecular docking studies 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[54]. Figures were drawn using the BIOVIA Discovery
8

Studio (https://3dsbiovia.com/). The ligand structures have been built and energy minimized
using the program Gaussian09 [M06-2X/6-311++G(d,p)]. Due to the absence of X-ray crystal
structure of FGB1 in Protein Data Bank, homology model was carried out to determine the
3D coordination of FGB1. Using SWISS-MODEL tools (https://swissmodel.expasy.org),
Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 (PDB ID: 3SN6)[55] was
selected as the best template, which shown an identity of 67.46%. The modeled protein
structure was checked and validated using the Ramachandran plot[56]. The coordinates of
human AChE (PDB ID: 4EY6)[57], were obtained from the Protein Data Bank (PDB).
2.7. Computational details
Density functional theory (DFT) calculations have been carried out using Gaussian 09
software [58]. The M06-2X functional[59] and the 6-311++G(d,p) basis set have been used
for all calculations. The reliability of DFT/M06-2X method for calculations of reaction
energies involving free radicals has been confirmed by previous studies [60, 61]. Solvent
effects of ethanol was approximated by the Truhlar’s SMD solvation model[62]. All the
ground states were confirmed by vibrational frequency analysis (no imaginary frequency).
Thermodynamic descriptors of the antioxidant mechanism (BDE, IP, PDE, PA, and ETE)
have been calculated as described in our previous studies[44, 63-68].
3. Results and discussion
3.1. Synthesis
The (benz)imidazole-hydrazone derivatives 1-10 were synthesized as outlined in Scheme 1.
The starting materials 1-methylimidazole-2-carbaldehyde and 1-methybenzimidazole-2-
carbaldehyde were prepared as described in our previous studies[41, 43]. The reaction of an
equimolar quantity of 1-methyl(benz)imidazole-2-carbaldehyde and appropriate
phenylhydrazine derivative in ethanol at room temperature (4h), gave the corresponding
imidazole or benzimidazole phenylhydrazone derivatives (1-10) in good to excellent yields
1
(76-97%). Structures of all prepared compounds were confirmed by IR, H NMR and 13C
NMR analysis and their data are reported in the experimental section. Among the synthesized
compounds, the molecular structure of compound 6 was confirmed by single crystal X-ray
diffraction analyses (Fig. 2). X-ray data has been deposited at the Cambridge crystallographic
data center with the CCDC number 1984681. The obtained structure confirms the (E)-
configuration of the hydrazone function.
9

Scheme 1: Synthesis of (benz)imidazole-hydrazone derivatives 1-10. Reagents and
conditions: (a) EtOH, room temp., 4h.
Fig. 2. ORTEP plot of the X-ray crystal structure of compound 6. Displacement ellipsoids are
drawn at the 40 % probability level.
10

3.2. Evaluation of biological and antioxidant properties
3.2.1. Evaluation of antioxidant activity
In vitro evaluation. The antioxidant activity of the synthesized compounds (1-10) was
determined using DPPH, CUPRAC, ABTS and GOR assays. The IC50 and A0.50 values were
determined for all compounds and presented in Table 1.
Table 1. Determination of antioxidant activity of compounds 1-10 by DPPH, ABTS,
CUPRAC, and GOR assays.
DPPH
assay
CUPRAC
assay
ABTS assay
IC50 µM*
GOR assay
IC50 µM*
Compound
IC50 µM*
41.7±2.2
51.8±2.6
22.4±1.2
59.9±1.2
47.8±2.1
82.4±3.4
55.2±0.9
60.4±1.9
55.8±4.0
40.4±0.9
70.8±6.6
26.0±1.9
A0.50 µM*
31.5±2.3
32.8±1.6
31.8±1.7
17.8±0.4
49.5±0.4
20.1±0.8
24.1±0.9
20.7±0.6
27.7±1.9
41.9±1.1
53.4±4.8
16.5±0.9
14.8±0.3
14.8±1.0
14.4±0.6
7.3±0.8
22.4±0.2
36.0±1.6
3.2±0.3
<3.12
30.9±0.9
30.6±0.4
54.0±1.0
31.4±0.5
29.9±0.6
33.8±0.7
27.0±0.2
30.7±0.4
25.2±0.5
33.0±0.5
29.3±0.2
48.5 ±0.1
1
2
3
4
5
6
7
8
<3.12
9
3.6±0.1
7.2±1.7
8.2±0.4
10
BHT
BHA
* Values expressed are means±S.D. of three parallel measurements. (p<0.05).
In DPPH assay, all tested compounds have good antioxidant activity, compound 3
shows the lowest IC50 value among the synthesized hydrazones (22.4±3.2 μM) with higher
antioxidant activity than that of the standard BHT (70.8±6.6 μM) and comparable to that of
the standard BHA (26.0±1.7 μM). Except compound 6 (IC50= 82.4±3.4 μM), all the prepared
hydrazones exhibit better antioxidant activity (IC50 between 22.4±3.2 and 60.4±1.9 μM) than
that of the standard BHT (70.8±6.6 μM). The effect of the heterocycle ring (imidazole or
benzimidazole) on the antioxidant activity can be analyzed by comparing the activity of
compounds 1-5 and compounds 6-10. In general, the imidazole and benzimidazole derivatives
11

have comparable antioxidant activity, with the exception of compounds 1 and 6 (IC50:
41.7±2.2 vs 82.4±3.4 μM) and compounds 3 and 8 (IC50: 22.4±3.2 vs 60.4±1.9 μM).
In ABTS assay, all tested compounds exhibit also a high-antioxidant activity, the
benzimidazole derivatives 7-10 are the best antioxidant agents (IC50 ≤ 3.6±0.1 μM) followed
by compound 4 (IC50= 7.3±0.8 μM). These compounds have higher or comparable
antioxidant activity than that of the standards BHT and BHA (IC50: 7.2±1.7 μM and 8.2±0.4
μM, respectively).
In CUPRAC assay, all the prepared hydrazones exhibit a better or comparable
antioxidant activity (A0.5 between 17.8±0.4 and 49.5±0.4 μM) than that of the standard BHT
(53.4±4.8 μM). The best result was obtained with compound 4, which shows an A0.5 (17.8±0.4
μM) comparable to that of the standard BHA (16.5±0.9 μM), and approximately three times
less than that of the standard BHT (53.4±4.8 μM).
Finally, In GOR assay, all compounds show a high antioxidant activity with IC50
values in the range of 25.2±0.5−54.0±1.0 μM. The best result was obtained with compound 9,
which shows an IC50 value (25.2±0.5 μM) two times less than that of the standard BHA (48.4
±0.1 μM) and comparable to that of the standard BHT (29.3±0.2 μM). Except compounds 3,
all the synthesized hydrazones exhibit higher antioxidant activity (IC50 between 25.2±0.5 μM
and 33.0±0.5 μM) than that of the standards BHA (48.4 ±0.1 μM) and comparable to that of
the standard BHT (29.3±0.2 μM).
In summary, from the results of the antioxidant evaluation by DPPH, ABTS,
CUPRAC and GOR assays the following observations can be derived:
-
-
-
-
The studied assays for measuring antioxidant activity are not correlated with each
other. This result can be explained by the different mechanisms of action of the assays.
With the exception of the ABTS assay, the other assays have a small dynamic range in
the data. This may also be an explanation for the non-correlation of the results.
All the prepared hydrazone derivatives exhibit high antioxidant activity towards
DPPH, ABTS, GOR and CUPRAC assays.
In DPPH assay, compound 3 is the best antioxidant with higher antioxidant activity
than that of the standards BHA and BHT (IC50 equal to that of the BHA and four times
less than that of the BHT).
-
In ABTS assay, compounds 7-10 are the best antioxidants with higher antioxidant
activity than the standards BHA and BHT.
12

-
-
In CUPRAC assay, the best results were obtained with compound 4 which shows
higher antioxidant activity than that of the standards BHA and BHT (A0.50 equal to that
of the BHA and three times less than that of the BHT)
In GOR assay, compound 7 is the best antioxidant with comparable or higher
antioxidant activity than that of the standards BHA and BHT (IC50 equal to that of the
BHT and two times less than that of the BHA).
DFT calculations. As described in our previous studies[44, 63-68], antioxidants scavenge
free radicals through three main mechanisms, namely hydrogen atom transfer (HAT),
sequential electron transfer proton transfer (SETPT), and sequential proton loss electron
transfer (SPLET)[69-72]. These mechanisms are characterized by several thermodynamic
descriptors such as BDE (bond dissociation enthalpy), IP (ionization potential), PDE (proton
dissociation enthalpy), PA (proton affinity) and ETE (electron transfer enthalpy). HAT is
characterized by BDE value, SPLET is characterized by IP and PDE values, and finally
SETPT is characterized by PA and ETE values. The lower the values of the thermodynamic
descriptors, the higher the antioxidant activity.
In order to have a better understanding of the antioxidant properties of the synthesized
hydrazones, and in which mechanism they follow to scavenge free radicals, all the mentioned
thermodynamic descriptors (BDE, IP, PDE, PA, and ETE) have been computed for
compounds 1 and 6, as representative compounds, using DFT method at M06-2X/6-
311++G(d,p) level of theory. Since the experimental study has been performed in solution,
the implicitly of ethanol has been also considered. The obtained results are presented in Fig. 3
and tabulated in Table S1 (supporting information). Analyzing of the obtained results show
that both compounds 1 and 6 have comparable values of the thermodynamic descriptors. For
example, the difference in BDE values is only about 1 kcal/mol. This indicates that
compounds 1 and 6 have comparable radical scavenging activity. By comparing BDE, IP and
PA values, it is clearly observed that PA is significantly lower than the other thermodynamic
descriptors. For example, PA of compound 1 is about 30 kcal/mol lower than BDE and 45
kcal/mol lower than IP. Hence, it can be concluded that the SETPT mechanism is more
favorable than the other mechanisms in ethanol. These results agree well with previous
studies on phenolic compounds[70, 73, 74]
13

Fig. 3. Thermodynamic descriptors of the antioxidant mechanisms for compounds 1 and 6
calculated at M06-2X/6-311++G(d,p) level of theory in EtOH.
3.2.2. Evaluation of antifungal activity
In vitro evaluation. The antifungal activity of the synthesized hydrazones 1-10 was evaluated
in vitro against Fusarium oxysporum fungal strain at a concentration of 200 µM using the
benzimidazole derivative “carbendazim (car.)” as positive control. The genus Fusarium is a
taxonomic group of filamentous, cosmopolitan fungi that can be both beneficial and harmful
to humans, animals, and plants[75]. Fusarium oxysporum is the most important
phytopathogen, which has been shown to infect a variety of food and ornamental plants[76,
77]. The obtained results of the evaluation of the antifungal activity are shown in Fig. 4.
14

Fig. 4. Antifungal activity of compounds 1-10 against Fusarium oxysporum fungal strain.
Carbendazim (Car.) was used as a positive control.
Examination of the obtained results reveals that the target compounds have different
levels of antifungal activity against the fungi tested at 200 μM. The imidazole derivatives 1-5
(17.0-57.7%) are clearly more reactive than their benzimidazole analogues 6-10 (5.1-50.7%).
The best results were obtained with imidazole derivatives 4 (R=Cl) and 5 (R=OCH2Ph), and
benzimidazole 6 (R=OMe), which displayed inhibitory rates of 47.5%, 57.7% and 50.7%,
respectively. Compared to that of the commercial fungicide carbendazim, these compounds
have good antifungal activity. Compounds 2, 3, and 7 were shown a moderate antifungal
activity with inhibitory rates ranging from 26.0% to 38.6%. While, the other compounds (1
and 8-10) were displayed inhibitory rates ranging from 5.1% to 17.0%, reflecting a relatively
low antifungal activity. Based on these results, we calculated the IC50 values for the most
active compounds 4-6 and standard carbendazim using concentrations of 100 to 800 µM (Fig.
4). As can be seen from Fig. 4, the three studied compounds (4-6) were shown a good
antifungal activity when compared to that of the commercial fungicide carbendazim.
Compound 5 (198.4±1.7 µM) and, relatively, compound 6 (203.6±0.9 µM) have an IC50 value
comparable to that of carbendazim (194.8±1.6 µM). These results indicate that compounds 4-
6 are promising antifungal agents against Fusarium oxysporum fungal strain.
Molecular docking study. In order to rationalize the promising in vitro results obtained for
the most active compound (5), molecular docking study was carried out with Guanine
15

nucleotide-binding protein beta (FGB1) as the target receptor[78]. FGB1 is one of the most
important membrane proteins of F. oxysporum fungal and it is implicated in various
biological processes, including gene expression, cellular function and metabolism such as
cAMP level, heat resistance, colony morphology and conidia formation[79-81]. These
biological processes make FGB1 a potential target to develop potent anti-F. oxysporum
agents.
The molecular docking study of the interaction of the most active compound (5) as
ligand and FGB1 as receptor was performed using Blind Docking Server. Due to the absence
of X-ray crystal structure of FGB1 in Protein Data Bank, homology model was carried out to
determine the 3D coordination of FGB1 (see Experimental section for details). To validate the
approach used, we have also performed a molecular docking study for the standard
carbendazim, and the obtained results for the most energetically favorable binding mode of
this compound are tabulated in Table S2 in SI. Fig. 5 shows the most energetically favorable
binding mode of compound 5 at FGB1 enzyme, and Table S3 in SI summarizes all the
molecular docking binding interactions. The obtained results reveal that both compound 5 and
carbendazim could favorably interacts with the enzyme FGB1 as can be concluded from their
low binding energies of −8.30 kcal/mol and −6.80 kcal/mol, respectively. In this pose,
compound 5 forms two hydrogen bonds between the NH of the hydrazone and the carbonyl
groups of residues Gln214 and Ile169. It also forms three hydrophobic interactions with
Leu209, Arg167, and Pro123 by means of the phenyl moieties. These results indicate that
compound 5 could interact favorably with the FGB1 enzyme and forms stable complex,
which could be considered as an explication of its antifungal activity.
Fig. 5. Binding mode of compound 5 at the active site of FGB1 (template PDB ID: 3SN6).
16

3.2.3. Evaluation of acetylcholinesterase inhibitory activity
In vitro evaluation. Acetylcholinesterase (AChE) is an enzyme that catalyzes the hydrolysis
reaction of acetylcholine to choline and acetic acid. This reaction is necessary to allow the
cholinergic receptors to return to their resting state after activation. AChE is recognized as a
primary target for symptomatic improvement of Alzheimer's disease[82]. The capacity of the
synthesized hydrazones 1-10 to inhibit the enzyme AChE was evaluated using Ellman’s
assay[53]. Galantamine, used for mild Alzheimer’ disease, was used as positive control. The
results are expressed as IC50 values and are tabulated in Table 2. As can be seen, all the
investigated imidazole-hydrazones 1-5 are weak AChE inhibitors with IC50 values > 200 µM.
On the other hand, their benzimidazole analogues 6-10 are moderate to good inhibitors with
IC50 values ranging from 11.8±0.1 µM to 61.8±1.7 µM. Compounds 9 and 10, bearing
respectively 4-Cl and 4-OCH2Ph groups, are the best AChE inhibitors with IC50 values
(11.8±0.1 µM for 9, and 13.1±1.2 µM for 10) comparable to that of the Galantamine (8.9±1.2
µM). Compound 7, bearing 4-OMe group, is the least active AChE inhibitor with an IC50
value of 61.8±1.7 μM. Accordingly, hydrozones 6-10 could be considered as promising
acetylcholinesterase inhibitors.
Table 2. IC50 values for the inhibition of AChE for compounds 1-10. Values expressed are
means±S.D. of three parallel measurements. (p<0.05).
AChE
Compound
IC50 µM
>200
>200
1
2
>200
3
>200
4
>200
5
48.3 ± 1.5
61.8 ± 1.7
22.5 ± 0.9
13.1 ± 1.2
11.8 ± 0.1
8.9 ± 0.1
6
7
8
9
10
Galantamine
17

Molecular docking study. A docking study was performed in order to investigate the
interaction modes of the most active compound (10) with human acetylcholinesterase
(hAChE, PDB code: 4EY6). The most energetically favorable binding mode of compound 10
at the active site of hAChE is shown in Fig. 6, and all the molecular docking binding
interactions are summarized in Table S4 in SI. As results, the most favorable binding mode of
compound 10 is characterized by a binding energy of –9.00 kcal/mol. In this pose compound
10 is located in the PAS and no interactions with the catalytic active site (CAS) were found.
As can be observed in Fig. 6, the hydrazone group of compound 10 forms two hydrogen
bonds with residue Ser293. The benzimidazole nucleus and the phenyl moieties forms several
π-π stacking with residues Gln291, Trp286, Tyr341 and Gnt604. Two hydrophobic
interactions are also found with residues Leu289 and Trp286. Some of these residues are
reportedly involved in ligand-receptor complexes of Tacrine, Galantamaine, Huperzine A,
and Donepezil[57].
Fig. 6. Binding mode of compound 10 at the active site of hAChE (PDB ID: 4EY6).
3.4. ADME analysis
In drug development, 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. In
order to evaluate the ADME properties of the synthesized hydrazones 1-10, their
pharmacokinetic parameters were calculated by using Molinspiration online property
calculation toolkit (available at: http://www.molinspiration.com.). All the obtained parameters
are presented in Table 3. According to Lipinski's rule[83], in general, an orally active drug
has no more than one violation of the following criteria: (i) No more than 5 hydrogen bond
18

donors (n-OHNH). (ii) No more than 10 hydrogen bond acceptors (n-ON). (iii) A molecular
weight (MW) less than 500 D and (iv) An octanol-water partition coefficient (milogP) not
greater than 5. As observed in Table 3, except compounds 8-10 which show one violation, all
the hydrazones did not shown any violation of Lipinski's rule. In addition, the percentage of
absorption of the compounds has been calculated and interesting values have been obtained
for all the compounds (90.79-94.05%)[84]. These results suggested that the synthesized
hydrazones have a good ADME parameters and can be considered as drug candidates.
The BBB (blood brain barrier) permeability is another important parameter, which
affects the biological activity results. Drugs that specifically target the central nervous system,
such as cholinesterase inhibitors, must cross the blood brain barrier. The BBB permeability of
the synthesized Hydrazones was evaluated by using the CBLigand-BBB prediction server
(available at: http://www.cbligand.org) and the obtained results are presented in Table 3.
Accordingly, all the compounds were found to be BBB-positive, which is required for the
acetylcholinesterase activity.
Table 3. In silico some physicochemical and pharmacokinetic parameters of the synthesized
(benz)imidazole-hydrazone derivatives 1-10
Comp.
miLogP
<5
TPSA (oA)
MW
nON
nOHNH
n-rotb
MV
%ABS
vio.
<1
0
BBB
<500
<10
4
<5
1
2.92
2.98
3.75
3.60
4.58
4.43
4.49
5.25
5.11
6.08
42.22
51.45
42.22
42.22
51.45
42.22
51.45
42.22
42.22
51.45
200.25
230.27
228.30
234.69
306.37
250.31
280.33
278.36
284.75
356.43
3
4
3
3
6
3
4
3
3
6
188.89
214.43
222.01
202.42
286.08
232.88
258.42
266.00
246.41
330.07
94.05
90.79
94.05
94.05
90.79
94.05
90.79
94.05
94.05
90.79
+
+
+
+
+
+
+
+
+
+
1
2
5
1
0
4
1
0
3
4
1
0
4
5
1
0
5
4
1
0
6
5
1
0
7
4
1
1
8
4
1
1
9
4
1
1
10
Percentage of absorption (%ABS); Topological polar surface area (TPSA); Number of rotatable bonds (n-rotb); Molecular
weight (MW); Molecular volume (MV); Logarithm of partition coefficient between n-octanol and water (miLogP); Number
of hydrogen bond donors (n-OHNH); Number of hydrogen bond acceptors (n-ON); Lipinski’s violation (vio);
4. Conclusion
In this paper, ten hydrazone derivatives bearing an imidazole or benzimidazole nucleus have
been designed, synthesized and evaluated for their antioxidant, antifungal, and anti-
19

acetylcholinesterase activities. It was found that all the synthesized hydrazones are potent
antioxidants. The study of the antioxidant mechanisms of representative molecules suggests
that SPLET is the most favorable mechanism in ethanol. Among the investigated hydrazones,
compounds 5 and 10 were shown good to excellent antifungal and anti-acetylcholinesterase
activities, respectively. In addition, the docking results reveled that these compounds inhibited
AChE and FGB1 enzymes through interactions including H-bonds, π-π stacking, and
hydrophobic interaction. Finally, in silico ADME studies have demonstrated that these
compounds have a good pharmacokinetic profile.
On the basis of our investigations, hydrazones 1-10 with antioxidant activity and some
of them antifungal and anti-acetylcholinesterase activities, providing promising starting points
for the design and development of new potent biological active compounds.
Acknowledgements
We would like to thank MESRS (Ministère de l’Enseignement Supérieur et de la Recherche
Scientifique, Algeria) and DGRSDT (Direction Générale de la Recherche Scientifique et du
développement Technologique, Algeria) for financial support, as well as 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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25

Highlights
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-
-
-
Ten hydrazone derivatives bearing (benz)imidazole nucleus were synthesized.
Antioxidant, antifungal, and anti-AChE activities have been investigated.
DFT calculations of the antioxidant mechanisms have been performed.
In silico molecular docking and ADME studies have be carried out.

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: