In silico to in vitro screening of hydroxypyridinones as acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.01.080

We have previously shown the improved acetylcholinesterase inhibitory activity of a model hydroxypyridinone compound transforming the hydroxyl group on the main ring into an N,N-dimethylcarbamate group; in the course of that study we developed a computational model to screen compounds for enzymatic activity. Herein we report development of second generation libraries. Candidates that adhere to drug-like criteria from a virtual library of compounds were tested using computational docking studies. Synthesis and characterization of chosen test compounds and their acetylcholinesterase inhibitory activity are presented.

Full text of DOI:10.1016/j.bmcl.2016.01.080

Accepted Manuscript
In silico to in vitro screening of hydroxypyridinones as acetylcholinesterase in-
hibitors
Maria A. Telpoukhovskaia, Brian O. Patrick, Cristina Rodríguez-Rodríguez,
Chris Orvig
PII:
S0960-894X(16)30094-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.01.080
BMCL 23545
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
8 December 2015
26 January 2016
29 January 2016
Please cite this article as: Telpoukhovskaia, M.A., Patrick, B.O., Rodríguez-Rodríguez, C., Orvig, C., In silico to
in vitro screening of hydroxypyridinones as acetylcholinesterase inhibitors, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.01.080
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Graphical Abstract
In silico to in vitro screening of
hydroxypyridinones as acetylcholinesterase
inhibitors
Leave this area blank for abstract info.
Maria A. Telpoukhovskaia, Brian O. Patrick, Cristina Rodríguez-Rodríguez* and Chris Orvig*

Bioorganic & Medicinal Chemistry Letters
In silico to in vitro screening of hydroxypyridinones as acetylcholinesterase
inhibitors
†
‡*
*
Maria A. Telpoukhovskaia, Brian O. Patrick, Cristina Rodríguez-Rodríguez and Chris Orvig
Medicinal Inorganic Chemistry Group, Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1, Canada
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
We have previously shown the improved acetylcholinesterase inhibitory activity of a model
hydroxypyridinone compound transforming the hydroxyl group on the main ring into an N,N-
dimethylcarbamate group; in the course of that study we developed a computational model to
screen compounds for enzymatic activity. Herein we report development of second generation
libraries. Candidates that adhere to drug-like criteria from a virtual library of compounds were
tested using computational docking studies. Synthesis and characterization of chosen test
compounds and their acetylcholinesterase inhibitory activity are presented.
Keywords:
hydroxypyridinone
dimethylcarbamate
acetylcholinesterase
inhibitor
2
009 Elsevier Ltd. All rights reserved.
virtual library screening
8
8
Curing Alzheimer’s disease (AD) remains an elusive
In(III), and Ga(III) in the solid states. Hhpp is a close relative
of Hppp, with a hydroxyl group off the phenyl N-substituent of
the HPO ring (Fig. 1), and we have studied Ga(III) chelation to
therapeutic goal, while the number of patients who get the bleak
1
prognosis increases with the rise in life expectancy. The
9
formulation of the amyloid cascade hypothesis, which states that
the deposits of the protein amyloid-beta are the key in AD
development, has contributed to establishing many research
avenues, the outcomes of which have been tested in clinical
the glucose conjugate of Hhpp on the phenyl substituent. In
fact, this molecular scaffold has attracted many investigators to
develop radiopharmaceuticals for diagnostic imaging and
radiotherapy; for example, HPO ligands able to coordinate Ga
and Ga have been investigated in vivo.
6
7
2
68
10,11
trials; unfortunately, it has yet to yield a successful treatment.
On the other hand,
Other related areas of research include studies with antioxidant
compounds and metal chelators such as the drug PBT2 (5,7-
the benefits of incorporating a glucose in the molecular structure
(for brain uptake and cytotoxicity considerations) have also been
investigated by conjugation to hydroxyl of HPO ring; for
3
dichloro-2-((dimethylamino)-methyl)-8-hydroxyquinoline),
4
125
which is under investigation in clinical trials. With the success
instance, rat brain uptake of I radiolabeled glycosylated Hppp
12
of this ligand in preclinical studies, and taking into account the
multifactorial nature of AD, chelators that possess antioxidant
and/or amyloid-beta binding functionalities are being explored
analogue was investigated.
With the observation that the neurotransmitter acetylcholine is
depleted in patients suffering from AD due to loss of cholinergic
neurons came a strategy for their treatment. Drugs such as
rivastigmine (Fig. 1), tacrine, donepezil, and galantamine act as
acetylcholine esterase inhibitors, essentially increasing the
5
within the medicinal inorganic chemistry community.
Our group takes advantage of the versatility of the
hydroxypyridinone (HPO) family in three ways – its promiscuity
toward metals (by acting as a binder in different stoichiometries),
its ability to accommodate various N-substituents without
influencing metal binding activity, as well as the ability to be
transformed into a prodrug through installation of a masking
group onto the hydroxyl group. For instance, the chelation of
8
13
amount of acetylcholine by blocking its breakdown. While this
strategy only works in less than half of the patients for a couple
1
4
of years, ameliorative treatment remains the only option in the
1
3
clinic. The mode of action of these drugs remains in their ability
to interact within the active site of the AChE enzyme: tacrine acts
without altering the structure of the enzyme (reversible
6
7
7
Hppp (Fig. 1) has been studied with Cu, Zn, Fe, Al(III),
—
——
*
Corresponding author. Tel.: +1-604-827-1833; fax: +1-604-822-7894; e-mail: cristina.rodriguez@ubc.ca
Corresponding author. Tel.: +1-604-822-4449; fax: +1-604-822-2847; e-mail: orvig@chem.ubc.ca
*
†
‡
Present address: Gladstone Institute of Neurological Disease, 1650 Owens Street, San Francisco, CA, 94158, USA.
Present address: Department of Physics & Astronomy, University of British Columbia, 6224 Agricultural Road, Vancouver,
BC, V6T 1Z1, Canada.

scaffold. In the first approach, the metal binding site is left intact
and the carbamate is moved to be positioned on the N-substituent
of the HPO (Fig. 1). In the second approach, the carbamate is
attached to the HPO ring by the benzyl ether linker spacer (Fig.
1
) that had been shown by Cohen and coworkers to be eliminated
19
upon enzymatic removal of an acetyl group. Based on these
designs, we created a virtual library consisting of 80 compounds.
After filtering out unsuitable candidates, we performed docking
studies and chose three model compounds Chpp, Cchpp, and
Cbppp (derivatives of Hppp and Hhpp) to synthesize and
perform in vitro acetylcholinesterase inhibitory activity studies.
Overall, we aim to continue the development of the
multifunctional HPO scaffold by exploring its novel inhibitory
functionality with the aid of computational studies.
In the first step to create a virtual library of molecules,
SciFinder was consulted to compile a pool of HPOs that had been
synthesized and characterized. In the next step, the N,N-dimethyl
carbamate was attached to either the main ring of the HPO with a
benzyl ether linker, or to the N-substituent (Fig. 1). This resulted
in a virtual database of 80 molecules total, with 40 in each half of
the library (Figs. S1 and S2).
Fig. 1 Top: HPO pro-ligands Hppp and Hhpp, neurotransmitter
acetylcholine. Bottom: the first generation compound (Cppp), as well as
two second generation designs.
15
16
inhibitor), while rivastigmine permanently modifies it.
The initial database was prefiltered to create a smaller
database that captures the diversity of the entire set, while
reducing the computational demands of the virtual screening
process. In the first stage, the database was filtered to discard
those molecules deemed unlikely to be suitable drug candidates
Notably, the aftermath of the inhibition has been crystallized, and
solid state structure analysis reveals that the carbamoyl group of
the drug is covalently attached to the enzyme, with the rest of
rivastigmine in the catalytic site with the phenol functional group
1
6
exposed.
20
according to Lipinski’s parameters. Lipinski’s rules are some of
the guiding parameters that researchers use to filter out
potentially problematic compounds: molecules were selected for
low molecular weight (under 500), appropriate lipophilicity (with
logP under 5), low number of hydrogen acceptor (under 10) and
donor (under 5) atoms. Subsequently, the parameter log BB was
calculated with Clark’s equation (eqn 1) incorporating parameters
calculated using the online chemoinformatics software
Following a previously reported strategy that imbues a metal
1
7
chelate with acetylcholinesterase inhibitory activity,
we
functionalized Hppp with dimethyl carbamate to yield Cppp
18
(
Fig. 1). We found that the acetylcholinesterase inhibitory
activity of Cppp (Fig. 1) is improved compared to the parent
compound Hppp and through kinetic studies we determined that
18
Cppp is a reversible inhibitor. The computational studies
illustrated how the compound interacts with the enzyme to
molinspiration
(http://www.molinspiration.com),
which
18
achieve the observed activity. Because it acts as a reversible
inhibitor, the metal chelating functionality is not accessible for
Cppp.
approximates LogP with miLogP value.
logBB = - 0.0148 TPSA + 0.152 clogP + 0.139
(1)
Log BB is a way to predict passive permeability of
compounds through the BBB, which takes into account
topological polar surface area (TPSA) and octanol-water partition
The new approach detailed in this work consists of two
modifications to conserve the metal chelating core of the HPO
Fig. 2 Selected compounds for computational docking analysis.

Fig. 3 Top-scored docking poses for the lead compounds a) Cbppp, b) Chpp, and c) Cchpp, in TcAChE.
coefficient (logP), one measure of lipophilicity. Compounds with
oxygen of the pyridinone scaffold (Fig. S4) stabilizing the
carbamate moiety far from the catalytic site and therefore
blocking any possible hydrolysis reaction. Overall, after this
analysis we conclude that all these inhibitors should act in a
reversible manner.
21
logBB < -1 were discarded. As well, compounds containing
reactive functional groups (like aliphatic ketones and aliphatic
esters) were removed from the library due to their instability in
22
serum and reactivity toward proteins and other biomolecules.
As a result of this filtering process, we obtained 59 molecules,
from which 15 structurally diverse compounds were selected for
an in-depth molecular docking analysis in order to understand the
mode of interaction with the AChE enzyme (Fig. 2). Structure-
based molecular docking analysis has been used for rationalizing
ligand-AChE interactions and understanding the observed
Further analysis was performed on Cbppp, Cchpp, and
Chpp. As stated before, the main differences between them are
the number of carbamate motifs and their position. Molecular
docking simulations predicted that Cbppp, Cchpp, and Chpp
present similar interactions and conformations (Fig. 3). In terms
of ligand-receptor interactions, the HPO scaffold does not
participate as an active group to guide the molecule inside the
active site of the AChE. On the contrary, the presence of Cp-
cresol motif is responsible for the interaction in the acyl pocket
forming hydrogen bonds with the amide groups (NH) of the
backbone of the enzyme (Phe288 and Arg289) (Fig. 3). This
interaction may prevent the compounds from going into the
catalytic triad.
23–25
inhibitory activity.
Molecular modeling using the 3D
crystallographic structure of AChE from Torpedo californica
(
TcAChE) co-crystallized with tacrine (PDB 1ACJ) as a receptor
18
was performed, as detailed in our previously published work.
Note that the in vitro assays were performed using the Electric
eel AChE; a comparative sequence analysis of both enzymes
shows a high degree of amino acids conservation as shown in
Fig. S3. Thus, we can consider the active site very similar in both
enzymes. The binding site of TcAChE revealed a narrow and
deep cavity lined by aromatic side chains. The dynamic motion
of Phe330 and Trp279 residues is apparent after analysing the
several available crystal structures of AChE bound to different
ligands. Therefore, a molecular docking protocol was performed
allowing random rotation of Phe330 and Trp279 as validated
previously with tacrine and a first generation of carbamoyl-
Among all docking conformations, Cbppp had the best
docking score of -5.59 kcal/mol, followed by compounds Cchpp
and Chpp with predicted binding affinities of -5.31 and -4.96
kcal/mol, respectively. Cbppp was found to form two favourable
π-π stacking interactions: (i) between the phenyl ring of Cbppp
and the indole ring of Trp84; and (ii) the HPO scaffold and the
Phe330 amino acid both at the anionic site of AChE (Fig. 3).
Similarly, docking simulations revealed that the best-scored
docking conformation for Cchpp showed a binding pattern very
similar to that of Cbppp (Fig. 3); however, the π- π stacking
interactions were not observed (Fig. 3). Chpp showed an
additional hydrogen bonding interaction with Asp72 (Fig. 3). In
order to study which part of the molecule is responsible for the
observed inhibition, we performed docking and experimental
analysis of Cp-cresol (Fig. 2). In all the poses obtained from
docking, Cp-cresol shows interactions with the aforementioned
amino acids in the acyl pocket (Phe288 and Arg289), thus we
assume that it is unlikely that this motif is responsible for the
difference in observed binding affinity. The trend of ligand
affinity has good correlation with the experimental in vitro
results, as detailed below.
1
8
derivatized HPO. An open conformation of Phe330 on binding
of the TcAChE with rivastigmine was reported in its X-ray
structure (PDB code 1GQR). In this work, according to docking
studies, Phe330 also adopts an open conformation when the
studied compounds bind to TcAChE.
Molecular docking analysis was performed on the 15 HPO
molecules (Fig. 2) that were obtained as a result of the virtual
screening process. For each molecule, the top-scored pose
(lowest binding energy) in the most stable cluster was considered
as the most favourable docking conformation (Figs. 3 and S4).
According to docking results, each molecule interacts with
TcAChE via hydrogen bonding and π-stacking, with locations in
both the peripheral and the active sites. Due to their molecular
similarity, the binding of molecules to AChE present common
patterns (Figs. 3 and S4). In most cases, the interaction is
between with the carbamate moiety of the molecules and the
backbone formed by the amino acids Phe288 and Arg289 at a
bond length of ~ 2.0 Å in the acyl pocket of the enzyme. In other
cases, π-π stacking interactions are observed with aromatic
residues e.g. Phe330 and Trp84 (Cbppp and 1) or Tyr334 (9)
As a conclusion, based on the most stable conformations, we
predict that the interactions observed with molecular docking
calculations lead to reversible AChE inhibitors. The best
interaction is shown for those molecules from the library of
compounds in which the carbamate moiety is positioned on the
HPO ring using a spacer. Interestingly, the presence of two
carbamate motifs in the molecule does not result in improved
interaction. When all docking results were analyzed, a few
conformations close to the active site of TcAChE (His440,
(Figs. 3 and S4) located in the anionic, and the peripheral sites.
Some of these molecules, particularly 4, 5, and 6 show two
hydrogen bonds between Ser200 and His440 and the carbonyl

Scheme 1 Left: Synthetic routes for Chpp. Right: Synthetic route for Cbppp.
Ser200 and Glu327) for compounds Cbppp, Chpp, and Cchpp
were observed. We set out to test these molecules in vitro and to
assess their inhibition mode with acetylcholinesterase.
activity: 219.9 μM (Hppp), 347.6 μM (Chpp), 252.3 μM
(Cchpp), and 216.5 μM (Cbppp). It was also noted that the
carbamated fragment Cp-cresol has inhibitory activity that is
about one order of magnitude less: 1282 μM.
Synthesis of compound Chpp followed the synthetic routes in
Scheme 1. The product could be obtained using two routes. In the
first, Hhpp is reacted with N,N-dimethylcarbamoyl chloride,
To investigate the mode of action of these inhibitors, substrate
turnover (of acetylthiocholine iodide, ATCI) was monitored over
60 minutes. We determined that compared to control (no
inhibitor present), the compounds Chpp and Cbppp presented
similar profiles – that is, the turnover rate remained nearly
constant over time (Fig. 4). This profile is similar to what we
observed with previous generation compound, Cppp, as well as
reference compound tacrine that is a well-known reversible
18
resulting in both singly and doubly carbamated compounds,
Chpp and Cchpp, that were separated by column
chromatography. The second route avoids the doubly carbamated
by-product by starting with a benzyl protected starting material,
Bnhpp. After carbamation, BnChpp must be debenzylated, and
while both these routes result in Chpp, the second requires two
extra steps. Identity and purity of Chpp and Cchpp were
18
inhibitor.
1
13
established with H, C NMR spectroscopies (Figs. S5-8),
HRMS, and EA. Starting materials Hhpp and Bnhpp, as well as
Chpp were analyzed by X-ray crystallography and their solid
state structures appear in Fig. S9. Crystal refinement data and
selected bonds and angles can be found in Tables S1 and S2. As
can be seen from Table S2, HPO ring retains its bond distances
across the three compounds.
Another piece of evidence suggesting that the inhibition
observed from Chpp and Cbppp is reversible is based on
analysing drug activation extracts by mass spectrometry and
TLC. Solely peaks corresponding to masses of and bands of
Chpp and Cbppp were observed after 1 or 24 hours of
incubation with AChE. In another set of experiments, dilute
4 3
Fe(ClO ) was added to reaction vials with AChE and Chpp or
Compound Cbppp was synthesized using the route in Scheme
Cbppp. Consistent with previous results, no metal chelation to
Cbppp was observed. On the other hand, the advantage of Chpp
– in that it retains metal chelation became apparent as a
Chpp:iron complex was observed by mass spectrometry.
18
19
1
. First, p-cresol is carbamated and brominated using NBS,
19
and then it is reacted
with Hppp. The product was
1
13
characterized by H and C NMR spectroscopies (Figs. S10 and
S11) and HRMS, and HPLC was used to provide an assessment
of its purity (Fig. S12).
In conclusion, a virtual library for a second generation of
carbamated HPOs was prepared following two different
strategies: (i) inserting a linker between the carbamate and the
HPO and (ii) positioning the carbamate moiety on the N-
substituent of the HPO. After pre-filtering the initial database, we
selected 15 compounds to study by molecular docking.
Molecular docking studies provided valuable insight into the
The synthetic intermediate Cp-cresol was of particular
interest as it is a fragment of the final products Cbppp and
Chpp. Since we aimed to include this compound in in vitro and
1
13
computational studies, we characterized it fully by H, C NMR
spectroscopies (Figs. S13 and S14), HRMS, and EA. As well, a
single crystal of Cp-cresol was studied by X-ray crystallography
(Table S3, Fig. S9) to reveal the expected delocalization of the
electrons from the C8-O2 double bond to result in shortening of
C8-N1 bond (1.35 Å compared to 1.45 Å for the N1-C10 and N1-
C9 bonds), giving it partial double bond character. One of the N-
methyl groups is nearly planar, with the torsion angle of -1.5° for
O2-C8-N1-C10, while the other methyl group is out of the plane
by 10°. The partial double bond character translates into the
inability of the C8-N1 bond to rotate at room temperature,
resulting in inequivalent N-methyl groups that are observed with
NMR spectroscopy (Figs. S13 and S14).
Fig. 4 Left: Inhibition kinetics of Chpp (blue) and Cbppp (green) with
eelAChE: enzymatic activity vs. time, as compared to a control with no
inhibitor (red). Right: Table of IC50 values for in vitro eelAChE activity
for Hppp, Chph, Cchpp, Cbppp, Cp-cresol, tacrine, rivastigmine, and
Cppp.
In vitro acetylcholinesterase activity was established for the
synthesized compounds Chpp, Cchpp, Cbppp, as well as for the
fragment Cp-cresol (Figs S15 and S16). As can be seen in Figure
4
, all compounds have similar acetylcholinesterase inhibitory

activity of the second generation of carbamated HPO derivatives.
It was revealed that the compounds interact in such a way that
prohibits them from entering the active site, which is why the
inhibitory action is reversible. Therefore, in cases where metal
binding is advantageous, an HPO such as Chpp with an
unmasked metal chelating site can be used. Cbppp, Chpp, and
Cchpp were synthesised, and their identities and purities were
12. Schugar, H.; Green, D. E.; Bowen, M. L.; Scott, L. E.; Storr, T.;
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established with H and C NMR spectroscopy, HRMS, and EA,
as well as X-ray crystallography studies for Chpp. It was then
confirmed experimentally that these compounds act as reversible
inhibitors of AChE. Acetylcholinesterase inhibition is currently
one of the only ways to help Alzheimer’s disease patients with
memory problems. HPOs such as Chpp can act as both metal
binders and AChE inhibitors, thus combining two potentially
important aspects of AD therapy in one compound.
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Acknowledgments
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5. Kochi, A.; Eckroat, T. J.; Green, K. D.; Mayhoub, A. S.; Lim, M. H.;
Garneau-Tsodikova, S. Chem. Sci. 2013, 4, 4137.
We thank the Alzheimer Society of Canada for a doctoral
award (M.A.T.), the Natural Sciences and Engineering Research
Council of Canada (NSERC) for a Discovery Grant (C.O.) and a
PGSD-2 award (M.A.T.), and the Canadian Institutes of Health
Research (CIHR) Proof of Principle program (C.O.). C.O.
acknowledges the Canada Council for the Arts for a Killam
Research Fellowship (2011-2013) and the Alexander von
Humboldt Foundation for a Forschungspreis. As well, we thank
Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR)
from Generalitat de Catalunya (Beatriu de Pinós (BP-DGR-
26. Luo, W.; Li, Y.-P.; Tan, J.-H.; Gu, L.-Q.; Huang, Z.-S. J. Enzyme Inhib.
Med. Chem. 2011, 26, 706.
2009)) for postdoctoral fellowship funding (C.R.R.), and the staff
of UBC Chemistry analytical services for help with
characterization of compounds. Finally, we thank Caterina
Ramogida for analysis of compounds by HPLC.
Supplementary Data
Supplementary data (Figs. S1-S16, Tables S1-S3, and X-ray
crystal data in CIF format) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.0000.00.000. Crystallographic data for Hhpp, Bnhpp,
Chpp, and Cp-cresol can be found free of charge at
http://www.ccdc.cam.ac.uk from the Cambridge Crystallographic
Data Centre (CCDC 988955-988958, respectively).
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