Synthesis, in vitro acetylcholinesterase inhibitory activity and molecular docking of new acridine-coumarin hybrids

Article abstract of DOI:10.1016/j.ijbiomac.2017.06.006

A novel series of acridine-coumarin hybrids was synthesized and biologically evaluated for their potential inhibitory effect on both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The newly synthesized derivatives 9a–d have shown higher ac

Full text of DOI:10.1016/j.ijbiomac.2017.06.006

Accepted Manuscript
Title: Synthesis, in vitro acetylcholinesterase inhibitory
activity and molecular docking of new acridine-coumarin
hybrids
Authors: Slavka Hamulakova, Ladislav Janovec, Ondrej
Soukup, Daniel Jun, Kamil Kuca
PII:
S0141-8130(16)32819-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.06.006
BIOMAC 7686
To appear in:
International Journal of Biological Macromolecules
Received date:
Revised date:
Accepted date:
9-12-2016
1-6-2017
2-6-2017
Please cite this article as: Slavka Hamulakova, Ladislav Janovec, Ondrej Soukup,
Daniel Jun, Kamil Kuca, Synthesis, in vitro acetylcholinesterase inhibitory activity
and molecular docking of new acridine-coumarin hybrids, International Journal of
Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.06.006
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1
<AT>Synthesis, in vitro acetylcholinesterase inhibitory activity and molecular docking of
new acridine-coumarin hybrids
a*
<
AU>Slavka Hamulakova ##Email##slavka.hamulakova@upjs.sk##/Email##, Ladislav
a
b,d
b,d
b,c
Janovec , Ondrej Soukup , Daniel Jun , Kamil Kuca
a
<
AFF> Department of Organic Chemistry, Institute of Chemical Sciences, Faculty of
Science, P. J. Safarik University, Moyzesova 11, SK-041 67 Kosice, Slovak Republic;
b
<
AFF> Biomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581,
5
<
00 05 Hradec Kralove, CzechRepublic
AFF> Department of Chemistry, Faculty of Science, University of Hradec Kralove,
c
Rokitanského 62, 500 03 Hradec Kralove, Czech Repulic
d
<
AFF> Department of Toxicology and Military Pharmacy, Faculty Military Helath Sciences,
University of Defense, Trebesska 1575, 500 05 Hradec Kralove, Czech Repulic
Corresponding autor. Tel: +421 55 2342334/421 55 2341197
*
<
(
PA>Fax: +421 55 6222124.
S. Hamulakova)
<ABS-HEAD>Highlights► ▪ Novel acridine-coumarin hybrids have been synthesized. ► ▪
The inhibition activity against human acetylcholinesterase (hAChE) and human
butyrylcholinesterase (hBuChE) of new compounds have been screened. ► ▪ Most of the
tested hybrids exhibited significant inhibition activity with IC50 values in the mikromolar
range. ► ▪ Acridine-coumarin hybrid 9b with seven methylene groups in linker showed the
highest inhibitory activity against hAChE (IC50 = 5.85 µM). ► ▪ Docking studies performed
using some of the compounds are presented.
<
ABS-HEAD>Abstract
<ABS-P>A novel series of acridine-coumarin hybrids was synthesized and biologically
evaluated for their potential inhibitory effect on both acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE). The newly synthesized derivatives 9a-d have shown higher
activity against human AChE (hAChE) compared with 7-MEOTA as the standard drug.
Among them derivative 9b exhibited the most potent acetylcholinesterase inhibitory activity,
with an IC50 value of 5.85 µM compared with 7-MEOTA (IC50 = 15 µM). Molecular
modeling studies were performed to predict the binding modes of compounds 9b, 9c and 9f
with hAChE/hBuChE.
<
KWD>Keywords: Alzheimer´s disease; cholinesterase inhibitors; coumarin; acridine;
tacrine; cholinesterase
H1>1. Introduction
<
Alzheimer´s disease (AD) is a neurodegenerative disorder characterised by progressive
memory loss and numerous cognitive impairments which cause serious health, social and
1
economic problems. The classic characteristic neuropathological hallmarks found in the
brains is the presence of amyloid ß-peptide (Aß) plaque deposits and neurofibrillary tangles
2
(
NFTs), loss of synapses, and degeneration of cholinergic neurons. The cholinergic
hypothesis of AD associates the disease with a deficiency of the neurotransmitter
acetylcholine in the brain, which is related to the activity of the enzyme acetylcholinesterase
3
(
AChE). The most prevailing amyloid hypothesis of AD points to amyloid plaque formation
4
and aggregation of Aß from amyloid ß precursor protein (AßPP). Dual-site inhibitors that
interact simultaneously with catalytic (CAS) and peripheral (PAS) sites at the same time
inhibit the production and aggregation of Aß, and represent new therapeutic agents for
5
effective treatment of AD. The enzyme AChE, which colocalizes with Aß in the senile

2
plaques, can bind to Aß thereby accelerating its aggregation into amyloid fibrils as an early
event during the aggregation process of Aß. Moreover, to the extend that these compounds
are able to block the PAS of AChE, they should be able to inhibit the AChE-induced Aß
4
aggregation.
Figure 1.
Since the first notable AChE inhibitor tacrine (9-amino-1,2,3,4-tetrahydroacridine, 1, Figure
1
) was later found to be hepatotoxic, only three other ChEIs approved by FDA, donepezil,
rivastigmine, and galantamine, and one N-methyl-D-aspartate (NMDA) receptor antagonist
memantine, are used for the treatment of AD today. 6 Against this backdrop were designed
and synthesized new series of potentially more potent and selective of tacrine-based homo-
and hetero-dimers as dual binding site AChEIs, such as tacrine-huperzine A hybrids, tacrine-
donepezil hybrids, tacrine-melatonin hybrids, tacrine-4-aminoquinoline hybrids, tacrine-
ferulic acid hybrids, tacrine-phenylphenanthridinium hybrids, and tacrine-indole hybrids.8
Coumarins are a large class of natural heterocyclic compounds which consist of fused
,4,7
-13
1
4
benzene and α-pyrone rings. Coumarin and its derivatives exhibit anticoagulant, anti-
inflammatory, antibacterial, antifungal, antiviral, anticancer, antihypertensive, antitubercular,
1
5-22
anticonvulsant, antioxidant, and neuroprotective activity.
demonstrated the ability of coumarins to inhibit AChE.
Several studies have
The benzopyrone ring included
3
,5,23-28
in hybrid molecules is significant for the design and synthesis cholinesterase inhibitors which
can simultaneously interact with both the catalytic site (CAS) and peripheral anionic site
(
PAS).29
Based on the dual-site theory various heterodimers involving tacrine-coumarin
hybrids2
4,25,27,30,31
and tacrine-4-oxo-4H-chromene hybrids have been synthesized as anti-
26
Alzheimer drug candidates. The best AChE/BuChE inhibitor among the investigated tacrine-
coumarin hybrids was hybrid 2 (Figure 1) with a 6-methylene linker on position 3 of the
coumarin skeleton, with K of 16.7 nM and 16.1 nM against hAChE and hBuChE
i
2
5
respectively. Tacrine-4-oxo-4H-chromene hybrid 3 produced from 6-chlorotacrine and 5-
hydroxy-7-methoxy-4-oxo-4H-chromene was found to be the most potent hAChE inhibitor of
this series (IC50 = 35 pM), displaying a level of inhibition 10 000-times higher than the parent
2
6
tacrine fragment (Figure 1).
Najafi designed and synthesized a novel series of acridine-chromenone and quinoline-
3
1
chromenone hybrids as novel anti-Alzheimer’s agents. Among them hybrid 4 (Figure 1)
possessing methyl and chlorine groups at the 4-position of the chromenone and 6-position of
the cyclopenta[b]quinoline moieties respectively, showed the most potent inhibitory activity
with IC50 = 16.17 µM. We recently designed and synthesized a series of tacrine-acridine
hybrids with alkylene-piperazine linkers (Figure 1, compounds 5, 6), with inhibition
potencies approximately 170 and 90 times greater respectively (for AChE IC50 = 3 and 6 nM)
than that of the tacrine standard, and derivative 5 was also shown to possess excellent sub-
nanomolar potency for BuChE (IC50 = 20 nM), better than that of tacrine itself. 32
.
As a continuation of our previous research33-36 this study describes the synthesis, docking
studies, and biological evaluation of a series of novel acridine-coumarin hybrids. Despite the
fact that coumarin derivatives and the acridine moiety have separately shown significant
potency in the treatment of Alzheimer´s disease, coumarin-acridine hybrids have not yet been
designed and synthesized as novel cholinesterase inhibitors. Accordingly, we have prepared a
series of compounds combining a coumarin scaffold with 4-carboxamidoacridone moieties,

3
and subsequently studied their ability to inhibit AChE and BuChE. Interaction with both CAS
and PAS of AChE was confirmed by docking simulation. These compounds consist of
coumarin and acridine moieties substituted at the 4-position, and linked by alkylenediamide
tethers of different lengths, as shown in Figure 2.
<
H1>2. Experimental methods
<H2>2.1 Materials
All solvents, chemicals and reagents were obtained commercially and used without
purification. Human erythrocyte AChE, human plasmatic BuChE, and 5,5'-dithiobis-(2-
nitrobenzoic acid) [DTNB] were supplied by Sigma-Aldrich, Prague, Czech Republic. Thin-
layer chromatography was performed on Macherey-Nagel Alugram ®Sil G/UV254 plates
and spots were visualized with UV light. Column chromatography was performed using silica
gel from Merck (0.063 - 0.040 mm).
<
H2>2.2 Chemistry
1
13
H NMR (400 MHz) and C NMR (100 MHz) spectra were recorded on a Varian Mercury
Plus NMR spectrometer using CDCl or DMSO-D as solvents with tetramethylsilane as
3
6
internal standard. Chemical shifts, δ, are given in parts per million (ppm), and spin
multiplicities are given as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet) or
m (multiplet). Coupling constants J are expressed in hertz (Hz). Melting points were recorded
on a Boetius hot-plate apparatus and are uncorrected. Yields refer to isolated pure products
and were not maximized. CHN analysis was performed on a Perkin-Elmer 2400 CHN
analyzer.
<H2>2.3 Preparation of acridine-coumarin hybrids 9a-g
The reaction sequences for the syntheses of acridine-coumarin hybrids 9a-g are depicted in
Scheme 1, which is shown in Supplementary Material. Reaction conditions and compound
characterisations are given in Supplementary Material.
<H2>2.4 Measurement of AChE and BuChE activity
The AChE and BuChE inhibitory activity of the tested drugs was determined using Ellman´s
3
7
method and is expressed as IC50, defined as the concentration at which cholinesterase
activity is reduced by 50%. Human recombinant AChE (hAChE; EC 3.1.1.7), human
plasmatic BuChE (hBuChE; EC 3.1.1.8), 5,5´-dithiobis-(2-nitrobenzoic acid) (Ellman´s
reagent, DTNB), phosphate buffer (PB, pH 7.4), acetylthiocholine (ATC), butylthiocholine
(
BTC), and tacrine hydrochloride were purchased from Sigma-Aldrich, Prague, Czech
Republic. Polystyrene cuvettes (Brand GmbH + Co. KG, Denmark) were used for measuring
purposes. All experiments were carried out in 0.1 M KH PO /K HPO buffer, pH 7.4.
2
4
2
4
Enzyme solutions were prepared at 2.0 units/mL in 2 mL aliquots. The assay medium (1 mL)
consisted of 650 µL of 0.1 M phosphate buffer (pH 7.4), 200 µL of 0.01 M DTNB, 25 µL of
enzyme, and 100 µL of 0.01 M substrate (ATC chloride solution). Assay solutions with
-3
-10
inhibitor (10 – 10 M) were preincubated for 5 min. The reaction was initiated by an
immediate addition of 100 µL of substrate. The activity was determined by measuring the
increase in absorbance at 412 nm at 1 min intervals using a Helios-Zeta spectrophotometer
(Thermospectronic, Cambridge UK). Each experiment was carried out in triplicate. The study
using in vitro BuChE was carried out in a method similar to that described above. Origin 6.1
software (Northamption USA) was used for statistical data evaluation.
<H2>2.5 Molecular modelling
Molecular models of derivatives 9b, 9c and 9f were computer-built by means of the building
3
8
options in ACD/ChemSketch. The models were built as 3D structures and saved as mopac
3
9
40
input files using the ACD/3D Viewer. Marvin 5.1.4 2008, ChemAxon was used to
determine the overall protonation of compounds. MOPAC2016 was used to optimise ligand
4
1
geometry (PM7, gnorm 0.1) and to assign partial atomic charges. In order to prepare the

4
input files Autodock MGL TOOLS 1.4.5 (revision 30) was used. Docking simulations were
carried out using AUTODOCK VINA ver. 1.0.4
2,43
Molecules of water and other nonenzymatic molecules were removed and hydrogen atoms
added. Gasteiger partial atomic charges for proteins were applied. For the docking
simulation, the energy grid was set at coordinates x 116.4, y 104.3, z -142.6 within the
hAChE (PDB ID: 1B41). For the hBChE (PDB ID: 1P01) dimensions were 30 points × 30
points × 30 points and x 137.9, y 117.5, z 38.7. The dimensions of the active site were 20
points × 20 points × 24 points with spacing of 1.0 Å.
Flexible ligand docking was performed for the compounds. All default parameters were
assigned for docking simulations. The scoring function of AutoDock Vina was used, and for
each run the pose with the lowest binding energy was chosen for visualisation of
4
4
intermolecular ligand-enzyme interactions. Pictures were prepared using Chimera software.
<H1>3. Results and discussion
<H2>3.1 Synthesis of acridine-coumarin hybrids
Target compounds 9a-g were synthesized as shown in Scheme 1 (Supplementary material).
3
6
As starting compounds acetamides 8a-g and 9-oxo-9,10-dihydroacridin-4-carboxylic acid
4
5
(
7) were used to synthesise the desired derivatives 9a-g.
(7-Hydroxy-2-oxo-2H-chromen-4-yl)acetic acid was synthesized using resorcinol and citric
acid in the presence of concentrated sulfuric acid according to the previously reported
4
7
procedure, and was subsequently esterified to its methyl ester. Reaction of (7-hydroxy-2-
oxo-2H-chromen-4-yl)acetic acid methyl ester with the relevant diamine (1,n-diaminoalkane,
n = 6-9; N1-(2-aminoalkyl)-1,n-alkanediamine, n = 2,3; and N1,N3-di(2-aminoethyl)-1,3-
propanediamine) in acetonitrile produced intermediate acetamides 8a-g. 9-Oxo-9,10-
dihydroacridine-4-carboxylic acid (7) was obtained by cyclization of N-(2-
4
5
carboxyphenyl)anthranilic acid (6) in acid solution. Copper-catalysed amination of o-
chlorobenzoic acid with anthranilic acid in 2-methoxyethanol yielded N-(2-
4
8
carboxyphenyl)anthranilic acid (6). The key intermediates 8a-g were then reacted with 9-
oxo-9,10-dihydroacridine-4-carboxylic acid (7) in the presence of 1,1´-carbonyldiimidazole
(CDI) in dry THF or DMF to produce the target acridine-coumarin hybrids 9a-g (Scheme 1).
<H2>3.2 Inhibition of human AChE and BuChE
The IC50 values of all tested compounds for AChE and BuChE are summarized in Table 1.
The IC50 values of compounds 9a-g were determined against human erythrocyte AChE
(
3
hAChE, E.C. 3.1.1.7) and against human plasmatic butyrylcholinesterase (hBuChE, E.C.
.1.1.8) using the method of Ellman et al. The IC50 values suggested that most of the
3
7
designed acridine-coumarin hybrids 9a-g exhibited potent inhibitory activities in the
micromolar range towards both hAChE and hBuChE. The inhibitory effect of hybrids 9a-d
was far stronger on AChE than on BuChE. Among the target compounds, hybrid 9b with
seven methylene groups showed the highest inhibitory activity against hAChE (IC50 = 5.85
µM), with a potency 3-times stronger than that of the reference compound 7-MEOTA (IC50
=
1
5 µM). Compound 9c exhibited the strongest inhibition against hBuChE with an IC50 value
of 32.53 µM.
Table 1
<H2>3.3 Studies of molecular modelling
Docking simulations were performed with compound 9b, the most active against hAChE, and
with derivatives 9c and 9f that had been found to be the most active against hBuChE (for
additional details see Experimental section).
In order to allow sufficient space sampling for these derivatives, all possible ligand structural
combinations were built into the simulations. We used a combination of conformations of the

5
amide -NHCO- bonds. The result of these molecular combinations was a set of four different
static conformations for each derivative under study (see Supplementary data, Fig. S01).
Each of these static conformations of the amide bonds was used as the input geometry for
docking simulations in order to reveal all of the possible interactions between the ligand and
the active site of the enzyme. To determine the level of protonation for the ligands at
4
0
physiological pH, we applied the Marvin software package. Results were summarized in
terms of the ligand’s binding ability.
The pose with the lowest binding energy for derivative 9b with hAChE is depicted in Figure
3
. Plausible π-π binding interactions might be found in PAS of the enzyme between the
coumarin core and the aromatic residue of Trp286. The model proposes an interaction of
amino acids Ser302 and His447 with the amide group of the acridone core. Another
interaction might be expected between amino acids of the enzyme active site and the ligand,
as depicted in Figure 3. The ternary inhibition complex of compound 9c with hBuChE shows
a direct interaction with the catalytic triad via His438 and Ser198 as depicted in Figure 4.
Additional hydrogen bonds as well as potential π-π interactions formed between amino acids
of the catalytic cavity and derivative 9c are revealed in Figure 4. Interactions of derivative 9f
with hBuChE are depicted in Figure 5. The putative ternary complex assumes direct
interaction with His438 and Ser198 as well as additional intermolecular interaction between
the catalytic cavity of the enzyme and derivative 9f.
Binding of compounds 9b and 9c within the catalytic site of the hBuChE is proposed in a
folded state of the ligand facilitated by the long spacer between the acridone and coumarin
moieties.
According to the docking results, multimodal binding of ligands to the enzymes should be
expected. Thus the models discussed above of the ternary inhibitory enzyme-ligand complex
should be considered only as candidates from several possible outcomes, albeit with the
lowest binding energy. Hence conclusions should be drawn with care.
Figure 3.
Figure 4.
Figure 5.
<
H1>4. Conclusion
This study describes the design, synthesis and biological evaluation of a series of new
acridine-coumarin heterodimers as novel anti-Alzheimer’s agents. Some of these studied
compounds showed moderate potency for inhibition of AChE and BuChE in comparison to
the control compounds tacrine and 7-MEOTA. Compound 9b exhibited the highest anti-
AChE activity (IC50 = 5.85 µM). As the docking of compound 9b into hAChE shows, the
acridine ring is localized inside the CAS and the 8-hydroxychromenyl moiety binds to the
PAS as a dual binding inhibitor. Thus, acridine seems to be a possible substitute for tacrine in
the family of dual binding site inhibitors, and this observation opens a new field for further
acridine-based AChEIs.
Acknowledgments
The work was supported by Slovak Grant Agency VEGA (Grant 1/0001/13) and project
NV15-30954A and Long-term developmentproject UHK.

6
The authors are grateful to Ian McColl MD, PhD for assistance with the manuscript.
<
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</BIBL>
Figure;1;1; Caption
International Journal of Biological Macromolecules
Slavka Hamulakova
<Figure>Figure 1. Structures of tacrine 1, tacrine-coumarin 2 - 4 and tacrine-acridine 5, 6
hybrids as anti- Alzheimer’s agents.
<
Figure>Figure 2. Structures of synthesized of acridine-coumarin hybrids 9a-g.
Figure>Figure 3. Top-score docking pose of derivative 9b depicting its putative hydrogen
<
bonds formed with amino acid residues in the active-site gorge of hAChE.
Figure>Figure 4. Top-score docking pose of derivative 9c depicting its putative hydrogen
bonds formed with amino acid residues in the active-site gorge of hBuChE.
Figure>Figure 5. Top-score docking pose of derivative 9f depicting its putative hydrogen
bonds formed with amino acid residues in the active-site gorge of hBuChE.
Table>Table 1 In vitro inhibition of hChEs (hAChE and hBuChE) by acridine-coumarin
hybrids 9a-g and control compounds tacrine and 7-MEOTA.
<
<
<
Compound
X
IC50 (µM) SEM IC50 (µM) SEM
AChEa
7.52
BuChE
42.37
34.33
b
9
a
-(CH
-(CH
-(CH
2
2
2
)
6
)
7
)
8
-
-
-
0.48
0.59
0.94
5.67
3.92
7.37
9
b
5.85
7.39
9
c
32.53

10
9
9
9
9
d
e
f
g
-(CH
2
)
9
-
6.62
238.50
85.30
116.20
0.5
0.55
63.61
1.99
20.14
0.1
58.04
155.40
43.40
67.45
0.023
3.41
23.35
2.52
8.31
0.004
-(CH
-(CH
2
2
)
)
2
-NH-(CH
-NH-(CH
2
)
)
2
-
-
3
2
3
-(CH
2
)
3
-NH-(CH
2
)
2
-NH--(CH
2
)
3
-
Tacrine
7
-MEOTA
15
2.9
21
3.4
a
IC50: Inhibitory concentration of AChE.
IC50: Inhibitory concentration of BuChE.
b
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