Azole antifungal compounds could have dual cholinesterase inhibitory potential according to virtual screening, enzyme kinetics, and toxicity studies of an inhouse library

Article abstract of DOI:10.1016/j.molstruc.2021.130268

Recent advances in cholinesterase inhibitors opened new venues for the treatment of cognitive disorders like Alzheimer's disease. Certain azole antifungals like miconazole were reported to have cholinesterase inhibitory effects and hence ameliorate cognitive deficits. In this study, we tested a set of azole antifungal derivatives selected through virtual screening of an inhouse library for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory effects. Compound 61 showed potent and selective AChE inhibition (IC₅₀ = 8.77 μM). The study also yielded dual AChE/BChE inhibitors in addition to a number of potent AChE inhibitors. Enzyme kinetics assays revealed that AChE inhibitors were competitive inhibitors. All the active compounds were imidazole derivatives and the modeling study showed that imidazole at protonated state contributed greatly to the binding interactions with some key residues of AChE and BChE active site. The active derivatives had negligible cytotoxic effects on murine fibroblast viability. According to our results, compounds featuring the classical scaffold of azole antifungal drugs could hold high potential for anticholinesterase drug design.

Full text of DOI:10.1016/j.molstruc.2021.130268

Journal of Molecular Structure 1235 (2021) 130268
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Azole antifungal compounds could have dual cholinesterase inhibitory
potential according to virtual screening, enzyme kinetics, and toxicity
studies of an inhouse library
Burak Baruta, Suat Sarib,∗, Suna Sabuncuog˘luc, Arzu Özela,d
a Karadeniz Technical University, Faculty of Pharmacy, Department of Biochemistry, Trabzon, Turkey
b Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Sıhhiye, Ankara, Turkey
c Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, Sıhhiye, Ankara, Turkey
d Karadeniz Technical University, Drug and Pharmaceutical Technology Application and Research Center, Trabzon, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Recent advances in cholinesterase inhibitors opened new venues for the treatment of cognitive disorders
like Alzheimer’s disease. Certain azole antifungals like miconazole were reported to have cholinesterase
inhibitory effects and hence ameliorate cognitive deficits. In this study, we tested a set of azole antifungal
derivatives selected through virtual screening of an inhouse library for their acetylcholinesterase (AChE)
and butyrylcholinesterase (BChE) inhibitory effects. Compound 61 showed potent and selective AChE in-
hibition (IC50 = 8.77 μM). The study also yielded dual AChE/BChE inhibitors in addition to a number of
potent AChE inhibitors. Enzyme kinetics assays revealed that AChE inhibitors were competitive inhibitors.
All the active compounds were imidazole derivatives and the modeling study showed that imidazole at
protonated state contributed greatly to the binding interactions with some key residues of AChE and BChE
active site. The active derivatives had negligible cytotoxic effects on murine fibroblast viability. Accord-
ing to our results, compounds featuring the classical scaffold of azole antifungal drugs could hold high
potential for anticholinesterase drug design.
Received 18 January 2021
Revised 21 February 2021
Accepted 5 March 2021
Available online 11 March 2021
Keywords:
Azole antifungal
Virtual screening
Acetylcholinesterase
Butyrylcholinesterase
Enzyme kinetics
Molecular docking
Cytotoxicity
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
Alzheimer disease (AD), as well as that cholinergic activation trig-
gers protein metabolism in amyloid plaques, which in turn led to
Cholinesterase enzymes catalyze hydrolysis of cholinergic neu-
rotransmitters, which leads to termination of cholinergic signal-
ing. Two types of cholinesterases are known to breakdown acetyl-
choline: acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE). AChE is the major type found mainly in neuromuscular
junctions and cholinergic synapses, while BChE is found in liver
and plasma and believed to act as a surrogate for AChE in the
case of reduced AChE activity [1]. Inhibition of cholinesterase en-
zymes has gained popularity with the understanding that cholin-
ergic neuron loss and dysfunction is common among patients with
discovery of anticholinergic drugs, such as donepezil, galantamine
and rivastigmine, for use in AD [2,3]. Expression of BChE is in-
creased in AD, thus dual cholinesterase inhibition is considered im-
portant for neurological disorders with cognitive impairment [4,5].
Although considerable improvement in efficacy in AD has been ob-
tained with cholinesterase inhibitors, currently available AD drugs
are far from ideal.
Azoles are known to serve as effective pharmacophores, im-
prove drug-like properties, and offer diverse options for structural
modifications in drug design efforts, thus they are commonly in-
cluded in druglike chemical space. Although mainly known for
their use as antifungal drugs, azoles’ reported benefits include but
not limited to antiviral, antibacterial, antitubercular, anticonvul-
sant, anti-inflammatory, and neuroprotective effects. In addition,
anticholinesterase actions of azole antifungals (conazoles) have in-
creasingly been reported in recent years (Fig. 1). Jin et al. ob-
served that enilconazole inhibited AChE activity at 0.3 μg/ml by
43% [6]. Likewise, Kolesárová et al. showed tebuconazole-based an-
tifungals possessed dose-dependent AChE and BChE inhibitory ef-
Abbreviations: AChE, (acetylcholinesterase); BChE, (butyrylcholinesterase); MW,
(molecular weight); RB, (number of rotatable bonds); HD, (hydrogen bond donor
count); HA, (hydrogen bond acceptor count); PSA, (polar surface area); PAINS,
(pans-assay interference compounds); DCC, (N,N’-dicyclohexylcarbodiimide); DMAP„
84-dimethylaminopyridine); SI, selectivity index; AChI, (acetylthiocholine iodide);
MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).
∗
Corresponding author.
E-mail address: suat.sari@hacettepe.edu.tr (S. Sari).
https://doi.org/10.1016/j.molstruc.2021.130268
0022-2860/© 2021 Elsevier B.V. All rights reserved.

B. Barut, S. Sari, S. Sabuncuog˘lu et al.
Journal of Molecular Structure 1235 (2021) 130268
Fig. 1. Azole antifungals with cholinesterase inhibitory effects.
fects [7]. More importantly, miconazole was found to be a dual
cholinesterase inhibitor [8] and its ameliorating effects on memory
deficits in mouse model were well established [9], which prompted
efforts to successfully find potent cholinesterase inhibitors in anti-
fungal azole scaffold [10].
tion study was performed by redocking of the co-crystallized lig-
˚
˚
and in each crystal structure. RMSD values of 0.42 A and 0.97 A
were obtained for the co-crystallized ligand of AChE and BChE, re-
spectively, regarding their original conformers, which indicates re-
liability for the docking method.
Nosocomial and drug resistant systemic mycoses are the dead-
liest form of fungal infections, which greatly affect immune com-
promised individuals [11]. Given that elderly population tend to be
immune compromised due to certain chronic disorders and im-
plant usage, and have cognitive disorders at the same time, an-
tifungals with anticholinesterase potential could be beneficial to
these populations.
2.2. Biological activity studies
2.2.1. Cholinesterase inhibition assay
AChE and BChE inhibitory effects of the compounds were eval-
uated according to the method by Ingkaninan et al. with minor
modifications [14]. Galantamine was used as positive control and
DMSO (1%) as negative control. The solutions were freshly pre-
pared in Tris-Cl buffer (50 mM) at pH 8.0. Then, Tris-Cl buffer
pH 8 (50 μL) and 5,5-dithio-bis(2-nitrobenzoic)acid (125 μl, 3 mM)
(Sigma-Aldrich, D8130) were added to the wells. The plates were
incubated for 15 min at room temperature. Afterwards, acetylthio-
choline iodide (AChI) (Sigma-Aldrich, A5751) or butyrylthiocholine
iodide (Sigma-Aldrich, 20820) (25 μl, 15 mM) was added as sub-
strate to begin the enzymatic reaction. The absorbance was mea-
sured at 412 nm using microplate reader (Thermo ScientificTM
MultiskanTM GO Microplate Spectrophotometer). The inhibitory ef-
fects of the compounds were expressed as the concentration which
inhibited 50% of the enzyme activity (IC50). The IC50 values (μM) of
the compounds were calculated according to the following equa-
tion:
As evidence for azole antifungals’ potential for anti-
cholinesterase activity became clearer, we selected
a set of
compounds from our inhouse azole library via virtual screening
and tested them for their AChE and BChE inhibitory effects. The
library included azole antifungal analogues, some of which were
reported for their highly potent anti-Candida activity. Some of the
tested compounds showed potent cholinesterase inhibition, thus
an enzyme kinetics study was performed to determine their Ki
values and inhibition types. Structure activity relationships (SARs)
analysis was made by the help of enzyme kinetics and molecular
modeling output. The active compounds were checked for their
cytotoxic effects on healthy cells, as well.
2. Experimental
ꢀꢁ
ꢁ
ꢂꢂ
ꢃ
Inhibition
%
( )
=
A
− A
/ A
(
× 100
(
)
)
control
control
compound
2.1. Virtual screening and molecular modeling
Lineweaver-Burk and Dixon plots were performed to investigate
inhibition type and constant (Ki) values of the compounds for AChE
[17,18]. The kinetic analysis was conducted by various AChI con-
centrations in the absence and presence of the compounds
Statistical analysis was performed using Microsoft Excel Win-
dows 10 and GraphPad Prism 5.0. The results were expressed
The inhouse library was prepared and modeled using LigPrep
(2020–2, Schrödinger LLC, New York, NY) and MacroModel (2020–
2, Schrödinger LLC, New York, NY) with conjugate gradients al-
gorithm and OPLS3e forcefield. Enantiomers and ionization states
of the compounds were considered during this process. Molecu-
lar descriptors were calculated using QikProp (2020–2, Schrödinger
LLC, New York, NY) and PAINS check was performed using Swis-
sADME server (www.swissadme.ch) [12]. Calculator Plugins were
used to calculate pKa values (Marvin 17.26.0, 2017, ChemAxon
(http://www.chemaxon.com)). Human AChE (PDB id: 4M0E [13],
as mean
standard deviation (n = 3). The differences between
the results of the compounds and those of galantamine were in-
vestigated by one-way analysis of variance (ANOVA) followed by
Tukey’s tests.
˚
˚
resolution: 2.00 A) and BChE (PDB id: 6I0B [14], resolution: 2.38 A)
were retrieved from the RCSB Protein Data Bank (www.rcsb.org)
[15] and prepared for docking using the Protein Preparation Wiz-
ard of Maestro (2020–2, Schrödinger LLC, New York, NY) [16]. Par-
tial charges, hydrogen atoms, and bond orders, H bonds were as-
signed, ionization and tautomeric states of the residue side chains
were set and water molecules were sampled in this process. Re-
ceptor girds were prepared using Maestro with central coordinates
2.2.2. Cytotoxicity assay
The compounds’ cytotoxic effects on cell viability of L929
(murine fibroblast) cell line were determined using MTT (3-(4,5-
dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide) assay de-
scribed by Ohguro et al. with minor modifications [19]. Following
a 2-week growth, the cells were seeded at 10,000 cells/well con-
centration in 96-well plates and allowed to attach and grow for
24 h. Then the cells were treated with each compound at different
concentrations between 2.5 and 100 μM for 48 h. After the incuba-
tion period, the medium in each well was replaced with 1 mg/ml
MTT solution in 100 μl culture medium. The cells were further in-
cubated at 37 °C for 3 h. Then, 100 μl DMSO was added onto the
cells to dissolve the formazan crystals and the optic density was
measured at 570 nm using a microplate reader (SpectraMax M2,
−17.27, −42.32, 25.90 for AChE and 135.05, 112.49, 40.61 for BChE
3
˚
and with a volume of 8000 A . Molecular docking was performed
by Glide at extra precision mode with 50 runs per ligand. The re-
sults were visually evaluated using Maestro Pose Viewer and 20
compounds with the best docking scores were identified for fur-
ther studies. Before starting the virtual screening process a valida-
2

B. Barut, S. Sari, S. Sabuncuog˘lu et al.
Journal of Molecular Structure 1235 (2021) 130268
Molecular Devices Limited, Berkshire, UK). Cytotoxicity of the com-
pounds were determined by calculating the percentage of cell via-
bility of the treated cells with respect to untreated (control) cells
(% cell viability). Each experiment was performed in duplicate.
triazole) and aryl group (4-chlorophenyl, 2,4-dichlorophenyl, or 2-
naphthyl), an ethylene linker, and a tail group consisting of vari-
ous structures (except for the first 13 derivatives) attached to the
ethylene linker via alcohol ester or oxime ester functional groups
(Fig. 2). The library compounds were previously reported for cer-
tain biological effects, especially for their antifungal properties
[20–27].
3. Results and discussion
Physicochemical and pharmacokinetic parameters play an im-
portant role in high attrition rates in clinical drug studies; thus, the
library was first evaluated for druglikeness using a set of descrip-
tors, namely molecular weight (MW), number of rotatable bonds
3.1. Virtual screening of the inhouse library
The inhouse library includes 114 azole derivatives sharing the
classical azole antifungal scaffold: an azole ring (imidazole or 1,2,4-
Fig. 2. The inhouse azole library used in the current study.
3

B. Barut, S. Sari, S. Sabuncuog˘lu et al.
Journal of Molecular Structure 1235 (2021) 130268
Table 1
Top-scoring 23 compounds according to the consensus scores (kcal/mol).
ꢀꢀ
aCalculated by the formula “(AChE score + BChE score)/2 for each compound.
4

B. Barut, S. Sari, S. Sabuncuog˘lu et al.
Journal of Molecular Structure 1235 (2021) 130268
Table 2
a
Calculated molecular descriptors and PAINS check for the selected compounds.
b
Comp.
RB (0–15)
MW (30.0 – 725.0)
HD (0.0 – 6.0)
HA (2.0 – 20.0)
LogP (–2.0 – 6.5)
PSA (7.0 – 200.0)
PAINS
61
22
16
26
63
55
25
77
51
81
17
27
76
47
52
59
18
98
49
28
65
71
50
6
6
7
6
7
6
9
6
7
6
8
6
6
6
8
5
7
7
7
6
6
6
6
389.3
339.8
305.8
374.2
513.4
417.3
395.8
407.3
387.3
406.2
319.8
408.7
403.3
374.2
417.3
411.3
319.8
432.5
450.3
353.8
424.3
386.2
437.3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
4.0
5.5
5.5
5.5
4.0
4.0
7.5
4.5
4.0
5.0
5.5
5.5
4.0
5.5
6.0
4.0
5.5
4.0
5.5
5.5
5.5
5.5
4.0
5.5
3.7
3.0
4.2
8.2
6.2
3.8
5.5
5.6
4.1
3.7
4.6
5.8
4.1
4.1
5.7
3.4
6.9
5.7
4.0
5.1
4.0
6.5
47.4
61.4
61.8
61.4
47.5
47.4
87.9
47.5
48.3
93.4
63.2
60.2
46.7
62.7
72.0
46.8
60.4
45.4
62.7
61.3
62.0
73.3
47.4
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
a
Ideal values ranges are provided in parentheses for each descriptor.
Outliers are highlighted.
b
(RB), hydrogen bond donor and acceptor count (HD and HA), logP,
and polar surface area (PSA), which are cited as the most relevant
descriptors for orally drugs [28,29]. Another factor that undermines
drug design efforts is pan-assay interreference compounds (PAINS)
phenomenon, in which compounds possessing non-specific reac-
tive moieties may mislead drug research by in vitro false positive
results. Compounds with poor pharmacokinetics and non-specific
reactive moieties can be predicted and discarded in early drug de-
sign stages. The inhouse library mainly complied with the molec-
ular descriptors criteria and included no PAINS (see Supporting In-
formation for details).
proper carboxylic acid in the presence of a coupling agent and a
transacylation catalyst, N,N’-dicyclohexylcarbodiimide (DCC) and 4-
dimethylaminopyridine (DMAP), respectively (see Supporting Infor-
mation for further details) [30].
3.3. Anticholinesterase activity and enzyme kinetics studies
In the enzyme activity assay, the IC50 values of the selected 23
compounds and galantamine were determined against AChE and
BChE (Table 3). A selectivity index (SI) was calculated to under-
Then, a structure based virtual screening was performed for the
library using molecular docking with the active site of human AChE
and BChE structures and docking scores were assigned for each
compound with both structures. In order to prioritize virtual dual
inhibitors, the compounds were ranked according to their con-
sensus scores. Consensus score was defined as the average of the
docking scores from enzymes, so that those having better scores
with both enzyme structures were ranked higher. Top 20% of the
library (23 compounds) according to the consensus scores were se-
lected for further studies (Table 1). The scores indicate predicted
binding affinity to the enzyme; thus, higher absolute values mean
stronger predicted binding to the enzyme and better activity was
expected for such compounds.
Table 3
IC50 values (μM) and SI indexes of the tested compounds.
a
Compound
AChE
BChE
SI index
b
61
8.77
0.20
1.38
151.29
142.03
239.65
>250
159.36
222.16
>250
144.04
88.18
>250
>250
>250
50.71
97.48
86.41
51.59
132.55
87.29
>250
>250
61.24
118.14
84.90
45.05
4.38
2.40
5.03
17.25
2.99
3.53
–
22
47.37
67.86
63.02
27.00
57.82
52.66
51.93
50.03
42.70
88.18
49.75
49.44
164.46
53.72
49.27
166.62
162.28
174.86
56.43
16.48
57.11
54.15
24.88
16
2.77
2.73
1.00
3.30
2.55
2.01
3. 22
2.80
5.60
2.69
2.44
26
c
63
2.99
4.85
5.9
55
3.84
–
25
77
2.86
2.77
1.76
–
51
3.08
81
17
–
The selected compounds were in compliance with the values
ranges of the calculated descriptors defined by QikProp except 63
and 98, which had too high LogP values (Table 2) (See Supporting
Information for details).
27
–
76
2.00
3.73
2.09
1.30
1.03
0.59
1.61
1.04
0.79
0.53
–
47
7.00
2.22
2.90
6.75
52
59
18
4.80
3.2. Synthesis of the selected compounds
98
5.90
6.60
3.20
49
28
1.50
–
The compounds in Table 1 were synthesized according to the
b
65
0.20
2.85
3.20
0.35
3.02
3.71
2.06
1.57
1.81
˙
scheme in Fig. 3 as previously reported [20,21,23]. Imidazole was
71
4.44
N-alkylated with proper 1-aryl-2-halogenoethanone derivative to
obtain 1, 3, and 4, through which 6–8 was synthesized by re-
duction with sodium borohydride (NaBH4), and 9 and 11 was
synthesized by condensation with hydroxylamine hydrochloride
(NH2OH.HCl) at basic medium. The title compounds were afforded
by Steglich esterification of the alcohol and oxime derivatives with
50
2.05
1.00
Galantamine
a
Calculated by the formula IC50 BChE/IC50 AChE for each compound to show se-
lectivity toward either enzyme. Not calculated for those with IC50 BChE >250 μM.
b
p <0.0001.
c
not significant.
5

B. Barut, S. Sari, S. Sabuncuog˘lu et al.
Journal of Molecular Structure 1235 (2021) 130268
Fig. 3. Synthesis of the selected compounds. Reagents and conditions: (i) imidazole, DMF, 0–5 °C → rt; (ii) NaBH4, MeOH, 0–5 °C; (iii) NH2OH.HCl, EtOH, pH 14, ref.; (iv)
RCOOH, DCC, DMAP, DCM, 0–5 °C → rt.
Table 4
stand whether or not a given compound was selective towards ei-
Ki values (μM) and inhibition types of the selected
ther enzyme. Lower IC50 values indicate better activity and SI val-
compounds against AChE.
ues >1 indicate selectivity towards AChE, while SI values close to
Compound
Inhibition type
Ki
are interpreted as dual inhibition. Thus, the results clearly showed
that most of the tested compounds were AChE inhibitors. Com-
pound 61 was the most potent in the series and, along with 65,
showed better AChE inhibitory effect than galantamine, an AChE
inhibitor used in AD. 63 was also notable for its AChE IC50 value
close to galantamine. All these three compounds were selective to
AChE, 61 was highly AChE-selective with a single-digit IC50 value
against AChE and almost no effect on BChE. The activity profile of
these compounds was similar as AChE selective drugs galantamine
and donepezil [31].
61
63
65
81
competitive
competitive
competitive
competitive
10.20
3.40
0.25
0.15
10.60
23.00
0.20
0.50
Burk plots of the compounds indicated that Km increased and Vmax
kept the same upon increasing concentrations of the compounds
against AChE, indicating competitive inhibition (Fig. 4).
Two compounds (59 and 76) showed notable inhibitory effects
against BChE with IC50 values around 50 μM. The same com-
pounds inhibited AChE at very similar IC50 values, which showed
that, although not very potent, 59 and 76 were dual cholinesterase
inhibitors, a similar profile as rivastigmine [4]. 65 also showed but
moderate BChE inhibition.
3.4. SARs and interactions of the compounds with the cholinesterases
The virtual screening study favored imidazole derivatives over
triazole derivatives, which was consistent with the fact that azole
antifungals with known cholinesterase inhibitory effects are all im-
idazole antifungals. The imidazole derivatives in the library were
modelled as neutral and imidazolium species and the latter were
scored higher because the imidazolium was better at making polar
contacts, which was also observed in our previous studies [32].
Lineweaver-Burk and Dixon plots were created to determine the
kinetic data for 17, 21, 19 and 42, which were the most potent
derivatives against AChE. According to the Dixon plots, 63 had the
best Ki value (Table 4), which also had the best docking score with
AChE (See Supporting information for the Dixon plots). Lineweaver-
6

B. Barut, S. Sari, S. Sabuncuog˘lu et al.
Journal of Molecular Structure 1235 (2021) 130268
Fig. 4. Lineweaver-Burk plots of the compounds.
Fig. 5. Binding modes and interactions of 61 (A), 63 (B), and 65 (C) with AChE. Ligands are represented as color stick and balls, amino acid residues as gray sticks, protein
back bone as white cartoons, and binding interactions as color dashes (yellow for H bond, blue for π-π, green for π-cation, magenta for salt bridge, and violet for halogen
bond).
Cholinesterase active site features four main domains: a periph-
eric anionic site (PAS) lining the rim of the active site entrance,
an acyl- and choline-binding pocket in the middle of the active
site gorge, which accommodate the acyl and choline portions of
acetylcholine, respectively, and a catalytic triad close to the bottom
of the gorge made up of a Ser-Glu-His-motif, which is responsible
for the catalytic function of these enzymes. Among the best AChE-
inhibitor derivatives, 61 showed a unique binding mode in AChE
active site interacting with the key residues of all the four do-
mains (Fig. 5A). In this binding mode of 61, the 2,4-dichlorophenyl
moiety interacted with the PAS residues Tyr72, Asp74, and Tyr124,
while the tail group perfectly fit in the acyl-binding pocket mak-
ing key interactions with Phe338 and Tyr341, and the imidazolium
moiety made π-π interaction with Trp86, salt bridge with Glu202,
and π-cation interaction with Tyr337, the choline-binding domain
residues, as wells a H bond with His447, a catalytic triad residue
(see Supporting Information for details of the binding interac-
tions). According to the crystallographic studies, π-cation interac-
tion with Tyr337 and π-stacking with Trp86 are typical properties
of donepezil-AChE complex and galantamine is known for making
H bond with Glu202 [33]. But these drugs are not known to di-
rectly contact the catalytic triad. 63 and 65 occupied the active site
gorge with the tail stretching deeper while the azole and aryl moi-
eties mainly engaged with the PAS (Fig. 5B and C). Although not
7

B. Barut, S. Sari, S. Sabuncuog˘lu et al.
Journal of Molecular Structure 1235 (2021) 130268
Fig. 6. Binding modes and interactions of 59 (A, C) and 76 (B, D) with AChE (A, B) and BChE (C, D). Ligands are represented as color stick and balls, amino acid residues as
gray sticks, protein back bone as white cartoons, and binding interactions as color dashes (yellow for H bond, blue for π-π, green for π-cation, and violet for halogen bond).
effective as 61, both compounds contacted the acyl- and choline-
binding domains. 63, having quite a long tail, was able to interact
with the catalytic triad (Fig. 5B).
with the catalytic triad was possible. The pKa of the of the conju-
gate acid of the imidazole protonated at the 3rd position was cal-
culated 6.77 for 59, 61, 63, and 76, and 6.74 for 65 making these
derivatives partly ionized at physiological medium (36.91% for 59,
61, 63, and 76, and 35.37% for 65). The tail was the best effective
when it occupied the acyl-binding pocket. The active compounds
in the series usually had linear-shaped tail with aromatic groups.
Those with bulky rings or only aliphatic groups for tail, such as 18
and 98, receptively, were mostly inactive. Most of the active deriva-
tives incorporated 2,4-dichlorophenyl as aryl group, which was jus-
tified by the halogen bonds observed in the binding mode of some
compounds.
BChE active site, although features the same four domains, is
larger than AChE. This is due to the presence of a number of
residues with smaller side chains in BChE active instead of their
corresponding aromatic residues in AChE. For example, Phe295 and
Phe297 in AChE acyl-binding pocket corresponds to Leu286 and
Val288, respectively [34]. The dual inhibitors of our study, 59 and
76, were found to engage mainly with the PAS and mid-gorge
residues of AChE, just like 63 and 65 (Fig. 6A and B). The BChE
active site, on the other hand, was able to accommodate 59 and
76 deeper with the imidazolium reaching close to the catalytic
triad, the tail and the aryl groups occupying the acyl- and choline-
binding pockets, similar as 61 in the AChE active sate (Fig. 6C and
D). Trp82 emerged as a key residue for both compounds, which
was established to be a crucial residue for BChE inhibition [35–
37]. Another important residue was Glu197, with which 76 made
H bond via the imidazolium [34].
3.5. Cytotoxic effects of the compounds
Four selected compounds were tested for their cytotoxic effects
on healthy murine fibroblast cells. Three AChE-selective (61, 63,
and 65) and one dual inhibitor (76) was included in the assay in
a wide concentration range including their active concentrations.
The viability values of the fibroblast cells were above 80% in the
presence of the compounds at 40 μM, indicting host safety (Fig. 7).
The docking results clearly showed the importance of imidazole
at protonated state, which was more useful when close contacts
8

B. Barut, S. Sari, S. Sabuncuog˘lu et al.
Journal of Molecular Structure 1235 (2021) 130268
Fig. 7. Cell viability values (%) of murine fibroblasts treated with 61, 63, 65, and 76 at concentrations ranging between 2.5–100 μM.
However, 76 caused a decline in cell viability at ≥50 μM (See Sup-
Acknowledgments
porting Information for details).
This study was funded by grants from Hacettepe University
(grant number: 014 D09 301 001-703, TPT-2015-6794, TDK-2017–
14965, TPT-2018–16666).
4. Conclusion
Cholinesterase inhibitory effects of azole fungicides are known
but there is very limited study on this field. In view of these find-
ings, we identified a set of azole derivatives sharing the azole an-
tifungal scaffold with anticholinesterase effects from an inhouse
compound library. Although the virtual screening campaign aimed
detecting potentially dual AChE/BChE inhibitors, the selected com-
pounds were mostly AChE selective. One of them (61) were a
single-digit μM inhibitor 17 times more potent against AChE than
BChE. Enzyme kinetics studies showed that the most active deriva-
tives competitive inhibitors of AChE. On the other hand, two com-
pounds proved dual cholinesterase inhibitors with moderate ac-
tivity. All the selected compounds were imidazole derivatives like
those reported in the literature and the modeling studies elab-
orated the importance of imidazolium for the interactions with
the catalytic triad, as well as the tail group with the acyl-binding
pocket. Cytotoxicity studies confirmed the safety of the selected
derivatives. Our study suggests a strong proof that compounds fea-
turing the classical scaffold of azole antifungal drugs could hold
high potential for anticholinesterase drug design regardless of their
antifungal profiles, since the active compound set includes deriva-
tives both with and without antifungal effects.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130268.
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10