Inhibition of acetylcholinesterase and butyrylcholinesterase with uracil derivatives: kinetic and computational studies

Article abstract of DOI:10.1080/14756366.2018.1543288

Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) inhibitors are interesting compounds for different therapeutic applications, among which Alzheimer’s disease. Here, we investigated the inhibition of these cholinesterases with uracil derivatives. The mechanism of inhibition of these enzymes was observed to be due to obstruction of the active site entrance by the inhibitors scaffold. Molecular docking and molecular dynamics (MD) simulations demonstrated the possible key interactions between the studied ligands and amino acid residues at different regions of the active sites of AChE and BuChE. Being diverse of the classical AChE and BuChE inhibitors, the investigated uracil derivatives may be used as lead molecules for designing new therapeutically effective enzyme inhibitors.

Full text of DOI:10.1080/14756366.2018.1543288

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Inhibition of acetylcholinesterase and
butyrylcholinesterase with uracil derivatives:
kinetic and computational studies
Huseyin Cavdar, Murat Senturk, Murat Guney, Serdar Durdagi, Gulru Kayik,
Claudiu T. Supuran & Deniz Ekinci
To cite this article: Huseyin Cavdar, Murat Senturk, Murat Guney, Serdar Durdagi, Gulru
Kayik, Claudiu T. Supuran & Deniz Ekinci (2019) Inhibition of acetylcholinesterase and
butyrylcholinesterase with uracil derivatives: kinetic and computational studies, Journal of Enzyme
Inhibition and Medicinal Chemistry, 34:1, 429-437, DOI: 10.1080/14756366.2018.1543288
To link to this article: https://doi.org/10.1080/14756366.2018.1543288
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 429–437
https://doi.org/10.1080/14756366.2018.1543288
RESEARCH PAPER
Inhibition of acetylcholinesterase and butyrylcholinesterase with uracil derivatives:
kinetic and computational studies
Huseyin Cavdara, Murat Senturkb, Murat Guneyc, Serdar Durdagid, Gulru Kayikd, Claudiu T. Supurane
Deniz Ekincif
and
b
aDepartment of Mathematics and Science Education, Education Faculty, Dumlupınar University, Kutahya, Turkey; Department of Basic Sciences
c
of Pharmacy, Pharmacy Faculty, Agri Ibrahim Cecen University, Agri, Turkey; Department of Chemistry, Science and Art Faculty, Agri Ibrahim
d
Cecen University, Agri, Turkey; Department of Biophysics, Computational Biology and Molecular Simulations Laboratory, Bahcesehir University,
e
f
School of Medicine, Istanbul, Turkey; Department of Neurofarba, University of Florence, Sesto Fiorentino (Firenze), Italy; Department of
Agricultural Biotechnology, Agriculture Faculty, Ondokuz Mayis University, Samsun, Turkey
ABSTRACT
ARTICLE HISTORY
Received 13 September 2018
Revised 26 October 2018
Accepted 30 October 2018
Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) inhibitors are interesting compounds for
different therapeutic applications, among which Alzheimer’s disease. Here, we investigated the inhibition
of these cholinesterases with uracil derivatives. The mechanism of inhibition of these enzymes was
observed to be due to obstruction of the active site entrance by the inhibitors scaffold. Molecular docking
and molecular dynamics (MD) simulations demonstrated the possible key interactions between the
studied ligands and amino acid residues at different regions of the active sites of AChE and BuChE. Being
diverse of the classical AChE and BuChE inhibitors, the investigated uracil derivatives may be used as lead
molecules for designing new therapeutically effective enzyme inhibitors.
KEYWORDS
Alzheimer’s disease; uracil
derivatives; acetylcholin-
esterase; butyrylcholinester-
ase; inhibitor; docking; MD
simulations
1. Introduction
catalyses the hydrolysis of ACh, which has an important role in cogni-
tion and memory. The observation of ACh depletion in AD patients
due to the loss of cholinergic neurons constitutes a strategy for their
treatment. Drugs such as tacrine, donepezil, galantamine, and riva-
stigmin are AChE enzyme inhibitors, mainly increasing the amount of
ACh by blocking ACh hydrolysis4. While this strategy works in about
half of the patients for several years, curative therapy continues to be
an unachieved goal4,5. These drugs interact with the active site of the
AChE: tacrine, without altering the structure of the enzyme (being a
reversible inhibitor), whereas rivastigmine changing it:6,7 the carba-
moyl group of rivastigmine was found covalently bound to AChE,
with the rest of the drug in the catalytic site and with its phenol func-
Alzheimer’s disease (AD) is defined as a neurodegenerative condition
characterised by abnormal behaviour, intellectual reduction, being a
major public health problem, especially due to the increasing elderly
population in developed countries1,2. In spite of the fact that AD
pathogenesis has not been clarified as yet, one of the most important
theories was the ‘‘cholinergic hypothesis”3. A defect in the levels of
acetylcholine (ACh) and butyrylcholine (BCh) acting as neuromedia-
tors was observed in the brains of patients with AD. The inhibition of
AChE and BuChE enzymes that hydrolyse ACh and BCh neurotrans-
mitters has become thus a treatment option of AD3. For this reason,
many research groups have conducted investigations of the inhibi-
tory activity for these enzymes involved in AD pathogenesis. AChE
tional group exposed to the solvent7–11
.
O
N
O
N
F
Br
Cl
NH
NH
O
O
O
O
F
N
NH
HO
Cl
NH
O
N
H
O
N
H
OH
OH
HO
HO
Sorivudine
5-FU
Uramustine
Fluoroxyuridine
CONTACT Huseyin Cavdar
huseyin.cavdar@dpu.edu.tr
Education Faculty, Dumlupınar University, Kutahya, Turkey; Claudiu T. Supuran
claudiu.supuran@unifi.it
Department of Neurofarba, University of Florence, Via Ugo Schiff 6, Polo Scientifico, Sesto Fiorentino (Firenze), 50019, Italy
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

430
H. CAVDAR ET AL.
Table 1. IC50 values obtained from AChE and BuChE (mM). Docking scores of corresponding calculations are also shown in the table.
AChE
Docking score
BuChE
Docking score (kcal/mole)
Experimental
resultsa
(mM) IC50
Ligand
Efficiency (XP,
kcal/mol)
Experimental
results
(mM) IC50
Ligand
Glide/SP
(kcal/mol)
Glide/XP
(kcal/mol)
Glide/SP
GOLD (kcal/mol)
Glide/XP
(kcal/mol)
Efficiency (XP, Selectivity
kcal/mol)
Inhibitor
GOLD
indexb
2
3
4
5
6
7
8
9
0.136
0.151
0.088
0.111
0.236
0.191
0.388
0.205
0.136
NA
59.92 ꢀ7.81
56.47 ꢀ6.11
80.97 ꢀ8.38
66.45 ꢀ7.48
52.28 ꢀ6.26
57.52 ꢀ6.32
57.81 ꢀ7.40
59.47 ꢀ7.00
82.62 ꢀ8.92
114.72 ꢀ14.30
ꢀ6.56
ꢀ0.59
ꢀ0.66
ꢀ0.41
ꢀ0.53
ꢀ0.56
ꢀ0.57
ꢀ0.61
ꢀ0.51
ꢀ0.70
ꢀ0.63
0.270
0.292
0.137
0.195
0.345
0.368
0.544
0.443
0.084
NA
44.41 ꢀ8.10
41.36 ꢀ6.76
65.92 ꢀ8.06
55.24 ꢀ7.55
36.20 ꢀ6.70
39.91 ꢀ6.76
42.96 ꢀ7.74
42.01 ꢀ7.63
64.82 ꢀ6.14
72.38 ꢀ7.83
ꢀ6.84
ꢀ0.62
1.98
1.93
1.55
1.75
1.46
1.93
1.40
2.16
0.62
ꢀ5.95
ꢀ7.90
ꢀ6.94
ꢀ5.06
ꢀ5.13
ꢀ6.14
ꢀ5.10
ꢀ11.23
ꢀ17.88
ꢀ7.16
ꢀ7.97
ꢀ6.92
ꢀ5.95
ꢀ6.10
ꢀ6.71
ꢀ5.93
ꢀ4.04
ꢀ7.25
ꢀ0.79
ꢀ0.42
ꢀ0.53
ꢀ0.66
ꢀ0.67
ꢀ0.67
ꢀ0.59
ꢀ0.25
ꢀ0.25
Neostigmine
Donepezil
aMean from at least three determinations. Errors in the range of 3% of the reported value (data not shown).
bSelectivity Index: IC50 of BuChE/IC50 of AChE.
5-Fluorouracil (5-FU) is an uracil analogue used as an antineo- assay), 50 mL DTNB (0.5 mM), 50 mL acetylthiocholine iodide/S-
plastic drug (antimetabolite). 5-FU interferes with DNA synthesis
by blocking DNA polymerase and thymidylate synthetase
enzymes. 5-FU and its metabolites have several different mecha-
nisms of action. In vivo, 5-FU is converted to the active metabolite
5-fluoroxyuridine monophosphate (5-FUMP); replacing U, 5-FUMP
incorporates into RNA and inhibits RNA processing, thereby inhib-
iting cell growth. Fluoroxyuridine is used to treat malignant neo-
plasms of the liver and gastrointestinal tract and hepatic
metastases. Sorivudine is a uridine derivative with potent antiviral
activity against herpes simplex and varicella zoster viruses.
Sorivudine acts by inhibiting DNA polymerase by converting it
into triphosphate form in cells. Uramustine, a uracil derivative, is
an alkylating antineoplastic agent used in lymphatic malignancies
butyrylthiocholine chloride (10 mM), and 10 mL enzyme (0.28 units
mL for the AChE assay and 0.32 units/mL for the BuChE assay).
The reaction was initiated upon addition of the enzyme. The reac-
tion system was prepared at room temperature in a quartz
cuvette. The blank reading was composed of all chemicals except
the inhibitor16,17
.
The absorbance of the reaction mixture was measured at
412 nm within 5 min from the start of the reaction on a Thermo
Scientific Evolution 200 Series (UV-VIS) spectrophotometer
(Thermo Fischer Scientific, Waltham, MA, USA). The absorbance for
each reaction mixture was measured three times within 5 min of
adding the enzyme and the results are reported as mean stan-
dard deviation. The inhibition properties are reported as IC50 val-
ues which were determined graphically from inhibition curves of
log inhibitor concentration vs. percent of inhibition. IC50 values
represent the concentration of inhibitor required for 50% inhib-
that causes mainly gastrointestinal and bone marrow damage12,13
.
In this study, the in vitro inhibition properties and in silico calcula-
tions of these uracil derivatives 2–9 in their interaction with AChE
and BuChE were investigated.
ition of the enzyme16–20
.
2. Materials and methods
2.3. In silico studies
2.1. Chemistry
2.3.1. Ligand and protein preparation
1-Acetyl-1H-pyrimidine-2,4-dione (2), 5-bromo-1H-pyrimidine-2,4-
dione (3), 5-Bromo-1-(toluene-4-sulfonyl)-1H-pyrimidine-2,4-dione
(4), 5-Bromo-1-methanesulfonyl-1H-pyrimidine-2,4-dione (5). Uracil
Maestro Molecular Modeling Package21 was used for protein and
ligand preparations. First, AChE (PDB ID: 4EY7)22 and BuChE (PDB
ID: 5DYW)23 crystal structures were retrieved from the protein
data bank, then AChE and BuChE amino acid sequences were
downloaded in UniProt24 to crosscheck and fix the unresolved res-
idues in the crystal structures. Crosslink Proteins tool in Maestro
was utilised to fill the missing amino acid residues in implicit solv-
ent environment. “A” chain of each crystal structures was used for
further steps. The missing elements in the proteins (e.g. hydrogen
atoms and missing atoms) were added by Protein Preparation
Wizard module25. Water molecules near 5 Å of the ligands were
kept and other water molecules were removed. The pKa predic-
tion and protonation state of ligands21,26 was predicted at pH 7.
PROPKA was used to assign the protonation states of the protein
residues at pH 7. Subsequently, restrained minimisation (with
0.30 Å RMSD heavy atom convergence) was realised for the sys-
tems with OPLS3 force field. Ligands were drawn with 3D Builder
tool and subsequently Ioniser module in conjunction with LigPrep
tool of Maestro molecular modelling suite was used for compound
derivatives 2–5 were synthesised according to ref 1114 5-
.
Flourouracil (6), 6-methyluracil (7), 1,3-Dimethyluracil (8), 5-
Hydroxymethyluracil (9), and other chemicals were obtained com-
mercially from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).
2.2. Biological activities
The AChE and BuChE enzymes inhibitory activities with the target
uracil derivative 2–9 were determined by using the Ellman
method15 Neostigmine was used as the reference drug in this
.
study. The IC50 values obtained for compounds 2–9 are summar-
ised in Table 1.
1 mg of each inhibitor was dissolved in 1 ml DMSO and then
diluted to various concentrations with deionised water. To deter-
mine the cholinesterase inhibition activity, six serial dilutions of
the inhibitors were measured. The reaction system was composed
of 5–60 mL inhibitor sample, 200 mL buffer (1 M, pH 8.0: Tris-HCl
buffer for the AChE assay and phosphate buffer for the BuChE preparation and energy optimisation with OPLS3 force field.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
431
O
O
O
Br
Br
1.BuLi
2.MsCl
Br2
NH
NH
NH
DMF
N
H
O
N
H
O
N
O
SO2CH3
1
3
5
.
piridin/Ac2O
u
B
Li
1
2.TsCl
O
O
N
Br
NH
O
NH
O
N
Ac
SO2PhCH3
2
4
O
O
O
O
Br
Br
Br
NH
NH
NH
NH
N
O
N
O
N
O
N
H
O
Ac
SO2CH3
PhCH
SO2
3
2
3
5
4
O
O
O
O
F
N
NH
HO
NH
NH
N
O
N
H
O
N
H
N
H
O
O
6
7
8
9
Figure 1. 2D structures of compounds 2–9.
2.3.2. Ligand docking
was specified as orthorhombic. Explicit water molecules (SPC)
were used in the preparation of the system, and also 0.15 M NaCl
ion concentrations were added to it for the neutralisation of the
(i) GoldScore scoring function implemented in GOLD (Genetic
Optimization for Ligand Docking, v.5.3) docking programme27 was
used in order to obtain the predicted binding poses for protein-
ligand complexes and binding energies of the studied ligands
towards AChE and BuChE proteins. Protein binding sites of AChE
and BuChE targets were defined according to their co-crystallised
ligands allowing to cover the whole ligand binding cavity regions
during the docking simulations. 50 poses were generated for each
ligand where protein residues were treated as rigid bodies and
ligands were treated flexible. Water molecules were set in toggle
and spin states at the surrounding ligand sites. Search efficiency
was set to 100% while 10,000 and 125,000 minimum and max-
imum operation values were selected, respectively. Early termin-
ation was turned off and diverse solution generation selection
was invoked.
ꢀ
system. In MD simulations, NPT ensemble at 310 K with Nose-
Hoover temperature coupling and at constant pressure of 1.01 bar
via Martyna-Tobias-Klein pressure coupling was provided26. All the
systems were prepared and put through the MD simulations by
using Desmond programme employing the OPLS2005 force field
and RESPA integrator28 There were no constraints on the gener-
.
ated systems and the initial velocity values are used as default.
The prepared system was subjected to 100 ns of MD simula-
tions run.
3. Results and discussion
In an earlier report from our group, the inhibitory ability of com-
pounds 2–9 on human carbonic anhydrase (hCA) was investi-
gated14. Some of these uracil derivatives demonstrated good to
(ii) In addition, Glide/SP and Glide/XP docking algorithms in
Maestro were also used for flexible ligand docking simula-
moderate inhibition profiles against hCA
I
and hCA II14,32
tions28–31 Protein grid generation calculation steps (prior to dock-
.
.
Inhibitors of carbonic anhydrase (CA) have been carried out in
many therapeutic applications, especially antiglaucoma activity. It
was thus decided to screen them against AChE and BuChE. AChE
and BChE inhibitors are used in the treatment of many neurode-
ing) and both standard (SP) and extra precision (XP) docking
settings were used with default values. Docking simulation boxes
were defined from the centroids of their crystal ligand binding
sites and maximum 50 poses were requested for each ligand.
generative diseases, especially Alzheimer’s disease8–11 Compounds
.
2–9 (Figure 1), possessing different functional groups on the pyr-
2.3.3. MD simulations
imidine scaffold, were evaluated for their inhibitory activity of
AChE and BuChE by means of the Ellman’s colorimetric assay15
The top-docking scored poses of molecule 4 complexed with
AChE and BuChE were used in the MD simulations. The buffer size
.
Neostigmine, commercially available cholinesterase inhibitor was
of the system box was set to 10 ꢁ 10 ꢁ 10 Å3, and the box shape used as the reference compound.

432
H. CAVDAR ET AL.
Figure 2. (Top) 3D representation of compound 4 at the binding pocket of AChE (Glide/XP) (left) and GoldScore (right); 2D ligand interaction diagram of the top dock-
ing pose of donepezil (Glide/XP); (bottom) 2D ligand interactions diagrams of top docking poses of compound 4 at the binding pocket of AChE using Glide/SP, Glide/
XP and GoldScore from left to right, respectively. Green and purple lines represent p-p stacking and hydrogen bonding interactions, respectively.
The concentration of the uracil derivatives (inhibitors 2–9)
required to inhibit 50% of AChE and BuChE activity was calculated
from various inhibitor concentrations and reported in Table 1. A
comparison of the IC50 values of 2–9 indicated that their inhib-
ition was mixed in nature, IC50 values of the inhibitors ranged
from 0.088 to 0.388 mM for AChE and from 0.137 to 0.544 mM for
BuChE. The results demonstrated that the compounds showed
IC50 values weaker compared to the reference compounds neo-
stigmine (IC50 AChE ¼ 0.136 mM and IC50 BuChE ¼ 0.084 mM)
against both AChE and BuChE. The strongest inhibition was
observed with 4 (IC50 ¼ 0.088 mM) against AChE but was 1.54-fold
active compared to neostigmine. Compound 4 (IC50 ¼ 0.137 mM)
exhibited the strongest inhibition of BuChE; however, 1.63-fold
less active compared to neostigmine. Thus, a computational study
was performed in order to rationalizse the observed inhibitory
activities. Compunds 2–9 docking scores ranged from ꢀ5.06 to
ꢀ7.90 kcal/mol for AChE and ꢀ5.93 to ꢀ7.97 kcal/mol for BuChE
(Glide/XP results).
Both GOLD and Glide docking results fit to experimental find-
ings (Table 1). Scores of top docking poses of compound 4 show
higher scores compared to other molecules. In GOLD, higher
GoldScore Fitness scoring values represent tighter binding interac-
tions. Results also show ligand efficiency scores (LIE) of studied
molecules. In order to escape the affinity-biased selection and
optimisation towards larger ligands, Hopkins et al.33recommended
to assess binding affinity in relation to number of heavy atoms in
a molecule and introduced the term ligand efficiency (the average
affinity contribution per atom is considered) instead of consider-
ing the affinity of the whole compound. This provides a way to
compare the affinity of molecules corrected for their size. In our
case, we used Glide/XP scores for the calculation of ligand effi-
ciency scores (ligand efficiency: GlideScore/number of heavy
atoms). Results show that compound 3 has top-scored LIE values
both in AChE and BuChE.
Figure 2 represents the 2 D and 3 D ligand interaction diagrams
of top-docking poses of compound 4 as well as a well-known
AChE inhibitor donepezil at the binding pocket of the target. Both
molecules interact common active site residues at the AChE; such
as Phe295, Trp86, and Trp286. However, as compared to top-poses
of Glide SP and Glide XP, top docking pose of GoldScore has an
alternative orientations at the binding pocket. The Br-uracil frag-
ment locates between the Ser125 and Glu202 residues where this
orientation allows compound 4 to make hydrogen bonding inter-
action within Glu202 and p-p stacking interaction with Trp86
(Figure 2). For BuChE, both three docking algorithms predict iden-
tical binding orientation of compound 4 (Figure 3) where Trp82
and Tyr 332 form p-p stacking interaction with the aromatic rings
of the ligand while Glu197 and His438 residues make hydrogen
bonding interactions with the Br-uracil fragment of the molecule.
In order to investigate the structural and dynamical profiles of
molecule 4 at the binding pockets of AChE and BuChE, MD simu-
lations were performed for the top-docked poses attained from
Glide/XP and GoldScore for AChE and Glide/XP for BuChE, using
Desmond. Figures 4 and 5 show a timeline representation of the
interactions and contacts (H-bonds, hydrophobic, ionic, water
bridges) of compound 4 at the binding pockets of AChE and
BuChE. The top panel shows the total number of specific contacts
the AChE makes with the molecule 4 over the course of the tra-
jectory. The bottom panel represents which residues interact with
the ligand 4 in each trajectory frame. Some residues make more
than one specific contact with the ligand, which is represented by
a darker shade of orange, according to the scale to the right of
the plot. Mostly observed contacts at AChE are from Trp286,
Phe295, Arg296, Phe338, and Tyr341. Corresponding interactions
were Trp82, Glu197, Tyr332, His438, and Tyr441 at the BuChE.
Interactions that occur more than 30.0% throughout the simula-
tion are also shown Figures 4 and 5. The ligand torsions plot sum-
marises the conformational evolution of every rotatable bond in
the ligand 4 throughout the simulation (Figure 6). The top panel

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
433
Figure 3. Superposition of top-docking poses of compound 4 at BuChE binding site, generated by Glide/SP (wheat), Glide/XP (blue), and GoldScore (pink).
Figure 4. Timeline representation of the interactions and contacts throughout the MD simulations of 4 at the binding pocket of AChE. Protein-ligand interactions are
monitored throughout the MD simulations. These interactions are categorsed into four types: Hydrogen Bonds, Hydrophobic, Ionic, and Water Bridge. Interactions that
occur more than 30.0% of the simulation time in the selected trajectory are shown in 2D interaction diagram.
shows the 2D schematic of a ligand with colour-coded rotatable
AChE and BuChE are enzymes which play an important role
bonds. Each rotatable bond torsion is accompanied by a dial plot in memory and cognition. They catalyse the hydrolysis of acetyl-
and bar plots of the same colour. The radial plots describe the choline causing a loss of communication between nerve cells.
conformational change of the torsion throughout the MD simula- This leads to a loss of brain function and causes AD. Treatment
tions. The beginning of the simulation is in the centre of the of AD relies on the restoration of the level of acetylcholine8–11
.
radial plot and the time evolution is plotted radially outwards. Pharmaceutical research has thus been focusing on cholinester-
The bar plots summarise the data on the dial plots, by showing ase inhibitors as treatments for cognitive disorders. Commercially
the probability density of the torsion. Results show that rotatable available medicines for AD suffer from drawbacks such as
bonds are quite stable throughout the simulations. The histogram gastrointestinal upset and bioavailability problems and therefore
plot and torsional analysis of ligand give detailed information into new cholinesterase inhibitors are continuously being investi-
the conformational change of the ligand 4 at the binding sites of gated. We thus screened uracil derivatives 2–9 for their inhibi-
of AChE and BuChE.
tory activity.

434
H. CAVDAR ET AL.
Figure 5. Timeline representation of the interactions and contacts throughout the MD simulations of 4 at the binding pocket of BuChE. Protein-ligand interactions are
monitored throughout the MD simulations. These interactions are categorised into four types: Hydrogen Bonds, Hydrophobic, Ionic, and Water Bridge. Interactions that
occur more than 30.0% of the simulation time in the selected trajectory are shown in 2D interaction diagram.
Figure 6. A schematic of detailed ligand atom (molecule 4) interactions with the AChE (top) and BuChE (bottom) residues. Interactions that occur more than 30.0% of
the simulation time throughout the MD simulations.
Uracil derivative 8 (IC50 ¼ 0.388 mM) showed the least potent position is more important for inhibition activity compared to
inhibitory activity against AChE. Decreasing the number of methyl toluene-4-sulfonyl groups present in the molecule. A more in-
groups on the aromatic ring showed an improvement of the IC50 depth study will be done to investigate this theory. To determine
values obtained, 7 (0.191 mM) with methyl group demonstrated a the importance of the toluene-4-sulfonyl group on inhibitory
2.03-fold decrease of inhibition activity while 9 (0.205 mM) with activity, other seven uracils were compared to compound 8.
hydroxymethyl group showed a 1.89-fold decrease of inhibition Adding a mesylate group (4) to toluene-4-sulfonyl group (5) the
activity compared to compound 8. However, compound
4
inhibitory activity decreased 1.26-fold, suggesting that the tolu-
(0.088 mM) possessing 1-(toluene-4-sulfonyl) group showed better ene-4-sulfonyl moiety is an important functional group for enzyme
inhibitory activity compared to other seven molecules. The differ- activity. Compound 4 exhibited a 1.71-fold stronger inhibitory pro-
ence between the other tested uraciles and 4 is that this molecule file compared to uracil 3.
is a more voluminous derivative. The stronger inhibition capability
In the case of BuChE, uracil derivative 4 showed the most
of uracil derivative 4 may suggest that the compound’s geometry promising activity with an IC50 value of 0.137 mM. This is in agree-
is more suitable for enzyme interaction when (toluene-4-sulfonyl) ment with the results observed for N1 position toluene-4-sulfonyl
group is N1. These results may indicate that the substituent group substituted uracil. 3 (IC50 ¼ 0.292 mM) is a slightly weaker

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
435
inhibitor (2.13-fold) compared to uracil derivative 4 but possessed
better inhibitory potential compared to the other compounds
tested (see entry 6–9). 4 was a slightly better inhibitor (1.42-fold)
compared to 5 (0.195 mM), this once again supports the N1 tolu-
ene-4-sulfonyl substituted 5 Br-uracil theory as discussed before.
The weakest inhibitor amongst the set of compounds was 8 with
an IC50 value of 0.544 mM. Tested uracil derivatives (2–9) showed
similar results for both AChE and BuChE.
derived semicarbazones as dual inhibitors of monoamine
oxidase and cholinesterase: effect of the size of aryl binding
site. J Enzyme Inhib Med Chem 2018;33:37–57. (d) Gulc¸in I,
Abbasova M, Taslimi P, et al. Synthesis and biological evalu-
ation of aminomethyl and alkoxymethyl derivatives as car-
_
bonic
anhydrase,
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and
butyrylcholinesterase inhibitors. J Enzyme Inhib Med Chem
2017;32:1174–82. (e) Mocan A, Zengin G, Simirgiotis M, et al.
Functional constituents of wild and cultivated Goji (L. bar-
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profile, and computational studies. J Enzyme Inhib Med
Chem 2017;32:153–68.
4. Conclusions
As discussed, the screening led to interesting results and can help
with the development of more effective drugs to slow down or
stop AD. We will expand the study to explore the structure activ-
ity relationship of uracil derivatives 2–9, in addition, a comparison
of these uracil derivatives with other aromatic compounds will be
investigated. These compounds could also be used as precursors
or building blocks in the preparation of much more effective
drug molecules.
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4. (a) Telpoukhovskaia MA, Patrick BO, Rodrıguez-Rodrıguez C,
Orvig C. In silico to in vitro screening of hydroxypyridinones
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Acknowledgements
This study was financed by Agri Ibrahim Cecen University
Scientific Research Council, (project no: FEF.15.007) for (MS
and MG).
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
5. (a) Schneider LS. Training of Alzheimer’s disease with cholin-
esterase inhibitors. Clin Geriatr Med 2001;17:337–58. (b)
Claudiu T. Supuran
http://orcid.org/0000-0003-4262-0323
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