Pyridonepezils, new dual AChE inhibitors as potential drugs for the treatment of Alzheimer's disease: Synthesis, biological assessment, and molecular modeling

Article abstract of DOI:10.1016/j.ejmech.2012.09.030

The synthesis, biological assessment and molecular modeling of new pyridonepezils 1-8, able to inhibit human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBuChE), are described. The new compounds have been designed as hybrids resulting from a conjunctive approach that combines the N-benzylpiperidine moiety, present in donepezil, and the 2-amino-6- chloropyridine heterocyclic ring system, connected by an appropriate polymethylene linker. Compounds 1-8 were prepared by reaction of 2-amino-6-chloro-4-phenylpyridine-3,5-dicarbonitrile (13) [or 2-amino-6-chloropyridine-3,5-dicarbonitrile (14)] with 2-(1-benzylpiperidin-4- yl)alkylamines (9-12). The biological evaluation of molecules 1-8 showed that compounds 1-6 are potent AChE inhibitors, in the submicromolar, while compounds 7 and 8 are on the nanomolar range, the most potent, 2-amino-6-((3-(1- benzylpiperidin-4-yl)propyl)amino)pyridine-3,5-dicarbonitrile (7), showing a IC₅₀ (hAChE) = 9.4 ± 0.4 nM. Inhibitors 2-8 are permeable as determined in the PAMPA assay. Compared to donepezil, compound 7 is in the same range of inhibitory activity for hAChE, and 703-fold more selective for hAChE than for hBuChE. Molecular modeling investigation on pyridonepezil 7 supports its dual AChE inhibitory profile, binding simultaneously at the catalytic active and at peripheral anionic sites of the enzyme. The theoretical ADME analysis of pyridonepezils 1-8 has been carried out. Overall, compound 7, a potent and selective dual AChEI, can be considered as a candidate with potential impact for further pharmacological development in Alzheimer's therapy.

Full text of DOI:10.1016/j.ejmech.2012.09.030

European Journal of Medicinal Chemistry 57 (2012) 296e301
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Pyridonepezils, new dual AChE inhibitors as potential drugs for the treatment of
Alzheimer’s disease: Synthesis, biological assessment, and molecular modeling
,
b
b
b
Abdelouahid Samadi a , Martín Estrada , Concepción Pérez , María Isabel Rodríguez-Franco ,
*
Isabel Iriepa c, Ignacio Moraleda c, Mourad Chioua a, José Marco-Contelles a
,
*
a Laboratorio de Química Médica, Instituto de Química Orgánica General (CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain
b Instituto de Química Médica (IQM-CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain
c Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Alcalá, Ctra. Barcelona, Km. 33.5, 28817, Alcalá de Henares, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2012
Received in revised form
20 September 2012
Accepted 24 September 2012
Available online 29 September 2012
The synthesis, biological assessment and molecular modeling of new pyridonepezils 1e8, able to inhibit
human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBuChE), are described. The new
compounds have been designed as hybrids resulting from a conjunctive approach that combines the N-
benzylpiperidine moiety, present in donepezil, and the 2-amino-6-chloropyridine heterocyclic ring
system, connected by an appropriate polymethylene linker. Compounds 1e8 were prepared by reaction
of 2-amino-6-chloro-4-phenylpyridine-3,5-dicarbonitrile (13) [or 2-amino-6-chloropyridine-3,5-
dicarbonitrile (14)] with 2-(1-benzylpiperidin-4-yl)alkylamines (9e12). The biological evaluation of
molecules 1e8 showed that compounds 1e6 are potent AChE inhibitors, in the submicromolar, while
compounds 7 and 8 are on the nanomolar range, the most potent, 2-amino-6-((3-(1-benzylpiperidin-4-
yl)propyl)amino)pyridine-3,5-dicarbonitrile (7), showing a IC50 (hAChE) ¼ 9.4 ꢀ 0.4 nM. Inhibitors 2e8
are permeable as determined in the PAMPA assay. Compared to donepezil, compound 7 is in the same
range of inhibitory activity for hAChE, and 703-fold more selective for hAChE than for hBuChE. Molecular
modeling investigation on pyridonepezil 7 supports its dual AChE inhibitory profile, binding simulta-
neously at the catalytic active and at peripheral anionic sites of the enzyme. The theoretical ADME
analysis of pyridonepezils 1e8 has been carried out. Overall, compound 7, a potent and selective dual
AChEI, can be considered as a candidate with potential impact for further pharmacological development
in Alzheimer’s therapy.
Keywords:
Pyridonepezils
hAChE
hBuChE
Dual AChE inhibitors
In vitro blood brain barrier
Molecular modeling
ADME
Alzheimer’s disease
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
a decrease of both choline acetyltransferase and acetylcholines-
terase (AChE) enzyme activity [2,3].
Alzheimer’s disease (AD) is the most prevalent neurodegen-
erative disorder [1]. AD is characterized by the gradual develop-
ment of forgetfulness, progressing to disturbances in language,
disorientation, and mutism. The symptomatic course of the
disease is generally five or more years of stepwise decline in
memory and attention span [2]. Typical pathological hallmarks are
extracellular senile plaques, consisting principally of amyloid-
Since the symptoms of AD were associated with an altered
cholinergic function, research has been focused on the basal fore-
brain cholinergic system [3]. As a result, the cholinergic hypothesis
was developed, which postulated that a loss of cholinergic function
in the central nervous system contributed significantly to cognitive
decline associated with advanced age and AD [4]. Thus, drugs
capable of inhibiting AChE might potentiate central cholinergic
function, therefore improving cognition and perhaps even some of
the behavioral problems experienced by AD patients [5].
AChE inhibitors (AChEI) may inhibit AChE via a competitive
mechanism, by interacting with the catalytic active site (CAS) of the
enzyme, via a non-competitive mechanism, by binding with the
peripheral anionic site (PAS), or via both mechanisms, by exerting
a dual binding AChE inhibition [5]. For a while the treatment with
AChE inhibitors (AChEI) was reported to produce only symptomatic
improvement, having no effect in the course of the disease [6].
b
(Ab), and intracellular neurofibrillary tangles, which are
composed of phosphorylated tau protein. Moreover, the basal
nucleus of Meynert undergoes profound neuron loss, the
neocortex exhibits a loss of cholinergic fibers and receptors, and
* Corresponding authors. Fax: þ34 91 5644853.
E-mail addresses: samadi@iqog.csic.es (A. Samadi), iqoc21@iqog.csic.es
(J. Marco-Contelles).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.030

A. Samadi et al. / European Journal of Medicinal Chemistry 57 (2012) 296e301
297
However, other studies indicated that AChE interacts with A
hydrophobic environment close to the PAS, thus promoting A
formation [7,8]. Moreover, AChEeA complexes increase
b
by an
fibril
Ab-
Herein, we describe the synthesis, pharmacological evaluation
[AChE and butyrylcholinesterase (BuChE) inhibition, Blood Brain
Barrier (BBB) permeability using the PAMPA protocol, theoretical
ADME analysis], and molecular modeling of pyridonepezils 1e8
(Table 1), a novel class of highly potent donepezileaminopyridine
hybrids of type I (Chart 1). These new hybrids were designed by
combining the N-benzylpiperidine moiety of donepezil with 2-
aminopyridine moiety of compound II [31] (Chart 1).
b
b
dependent neurotoxicity [9]. These reports arose a new interest in
AChEI, demonstrating their ability to enhance the release of non-
amyloidogenic soluble derivatives of amyloid precursor protein
(APP) in vitro and in vivo, and possibly to slow down the formation
of amyloidogenic compounds in the brain [10]. The increase of
soluble APP (APPs) was also consistent with AChE inhibition [11].
Numerous clinical trials have shown the safety and efficacy of
AChEIs in the treatment of AD. Besides, there is growing evidence
from preclinical studies indicating that these agents can attenuate
neuronal damage and death from cytotoxic insults, and therefore
might affect AD pathogenesis [12].
Considering the non-cholinergic aspects of AChE related to the
PAS [7e9] in associating with Ab, an attractive target for the design
of new antidementia drugs emerged. Peripheral or dual site
inhibitors of AChE may simultaneously alleviate the cognitive
deficit in AD patients and prevent the assembly of Ab, which will
delay the neurodegenerative process [13]. This strategy was
pursued for several medicinal chemists who have been developing
new compounds with dual AChE inhibitory activity [14e17], based
on well known AChEIs, such as tacrine [18], rivastigmine [19],
donepezil [20] and galanthamine [21]. A recent clinical study of
patients with moderate-to-severe Alzheimer’s disease found that
continued treatment with donepezil could improve cognition and
function for even severe patients [22].
2. Results and discussion
2.1. Chemistry
The synthesis of pyridonepezils 2-amino-6-((2-(1-benzylpiper-
idin-4-yl)alkyl)amino)-4-phenylpyridine-3,5-dicarbonitriles 1e4,
and 2-amino-6-((2-(1-benzylpiperidin-4-yl)alkyl)amino)pyridine-
3,5-dicarbonitriles 5e8 was easily achieved by reaction of readily
available precursors, such as commercial 9, known 10 [32] and 11
[30b], or new 12, amines, with 2-amino-6-chloro-4-phenylpy
ridine-3,5-dicarbonitrile 13 [33] and 2-amino-6-chloropyridine-
3,5-dicarbonitrile 14 [34], respectively (Scheme 1) (see
Supplementary material).
Compound 12 was prepared starting from alcohol 15 [29].
Reaction of 15 with DPPA, DBU and sodium azide in DMF gave azide
16. Hydrolysis of azide 16 in the presence of triphenylphosphine/
water/THF gave amine 12 (Scheme 2).
Several classes of donepezil hybrids, such as donepeziletacrine
[23,24], donepezileaminoacids [25], indanone hybrids [26],
donepezileaminothienoquinoline [27], or indanone and aurone
derivatives [28], have been developed as dual binding site AChE
inhibitors. The success of the dual binding site strategy is evidenced
by the increased AChE inhibitory potency of these hybrids
compared to the parent compounds from which they have been
designed.
Based on this rational design, we have recently described the
highly multipotent donepezileindole ASS234 [29] and the
donepezilepyridine hybrid ASS280 [30] (Chart 1), showing that
these molecules act as dual AChEI, binding simultaneously the CAS
and PAS of the enzyme. The simple aminopyridine II has been also
reported [31] (Chart 1).
2.2. In vitro evaluation of human cholinesterase inhibition
The in vitro activity of the pyridonepezils 1e8 against hAChE and
hBuChE was determined using Ellman’s method [35]. For compar-
ative purposes, donepezil was used as reference compound. The
obtained IC50 values are summarized in Table 1. The IC50 values
show that most of these molecules are potent, in the nanomolar
range. Some of them are selective hAChE inhibitors. The most
potent inhibitors were pyridonepezils 7 and 8 [IC50 (hAChE) ¼ 9.4e
70 nM]. Compared to donepezil, compound 7 was in the same
range of inhibitory activity for hAChE, and 2.6-fold less active than
donepezil for hBuChE. However, compounds 2, 3, 4, 6 and 8 were
3.7-, 1.6-, 4.3-, 23-, and 1.5-fold more active against hBuChE,
respectively. Inhibitors 1 and 5 (both with n ¼ 0) were extremely
Chart 1. General structure of the multipotent MAO/ChE inhibitors ASS234, ASS280, donepezil and the novel dual ChE inhibitors I and 2-aminopyridine derivative II.

298
A. Samadi et al. / European Journal of Medicinal Chemistry 57 (2012) 296e301
Table 1
b
IC50
PAMPA-BBB assay for pyridonepezils 1e8 with their predictive penetration in the CNS.
(m ) in the
M)a for the inhibition of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBuChE) and experimental permeability (Pe 10ꢂ6 cm sꢂ1
Compd.
R
n
hAChE
hBuChE
S.Ic
Pe
Prediction
1
2
3
4
5
6
7
8
Ph
Ph
Ph
Ph
H
H
H
H
0
2
3
4
0
2
3
4
8.8 ꢀ 0.4
>10
e
Insoluble
34.0 ꢀ 0.3
31.6 ꢀ 1.6
25.7 ꢀ 0.3
17.0 ꢀ 0.3
14.4 ꢀ 0.8
20.8 ꢀ 1.1
21.2 ꢀ 0.7
e
Insoluble
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
CNSþ
e
0.39 ꢀ 0.04
0.24 ꢀ 0.05
0.12 ꢀ 0.02
3.1 ꢀ 0.8
0.67 ꢀ 0.04
1.5 ꢀ 0.08
0.58 ꢀ 0.04
>10
0.26 ꢀ 0.07
6.6 ꢀ 0.7
1.7 ꢀ 0.5
2.5 ꢀ 0.07
>10
1.7
6.3
4.8
e
0.22 ꢀ 0.08
0.0094 ꢀ 0.0004
0.070 ꢀ 0.005
0.010 ꢀ 0.002
3.5 ꢀ 0.2
1.2
703
24
250
e
Donepezil
IId
e
e
a
The in vitro test compound concentration required to produce 50% inhibition of hAChE and hBuChE. The result (IC50) is the mean ꢀ SEM of three independent experiments.
PBS:EtOH (70:30). Data are the mean ꢀ SD of three independent experiments.
b
c
Selectivity Index ¼ hBuChE IC50/hAChE IC50
.
d
Inhibition was done using EeAChE and eqBuChE enzymes.
selective for hAChE, but derivatives 2e4 and 6 showed equipotent,
high activity for both hAChE and hBuChE. Compounds 7 and 8 were
found to be 703- and 24-fold more selective for hAChE than for
hBuChE, respectively.
brain penetration of pyridonepezils 1e8, we used the PAMPA-BBB
method as reported [36e38]. The in vitro permeabilities values
(Pe) of these pyridonepezils and 15 commercial drugs through
a lipid extract of porcine brain were determined using a mixture of
PBS:EtOH (70:30). Assay validation was made by comparing the
experimental permeability with the reported values for these
commercial drugs, which gave a good lineal correlation, Pe
(exptl) ¼ 1.24 Pe (bibl) þ 1.98 (R2 ¼ 0.93). Except compound 1,
which presents low solubility in the experimental conditions, all
tested pyridonepezils showed good permeability values (Pe ranges
from 17.0 ꢁ 10ꢂ6 to 34.0 ꢁ 10ꢂ6 cm sꢂ1), as the known CNS drugs
used in the assay validation, pointing out that these molecules
would cross the BBB by passive diffusion (Table 1).
Compared to compound II as reference, bearing also the 2-
aminopyridine ring, most of compounds 1e8 are more active in
both enzymes hAChE and hBuChE. These data suggest that the
incorporation of N-benzylpiperidine fragment to the aminopyridine
system increase the inhibitory potency for both ChE enzymes.
Compared to the already published donepezil derivatives,
compound 7 proved to be the most active within all the published
series of indanone hybrids [26], and in the same order of magnitude
of donepeziletacrine hybrids [23,24], while compounds 1e6 and
donepezileaminoacid derivatives [25] showed a similar inhibitory
power.
2.4. Molecular modeling of compound 7
Concerning the structure-activity relationships (SAR), the
presence of a phenyl group at C4 decreases the AChE inhibitory
activity. Thus, inhibitor 7 (n ¼ 3) was found to be 25-fold more
active than compound 3 (n ¼ 3) bearing a phenyl group at C4.
Regarding the effect of the linker, for compounds 1e4 bearing
a phenyl group at C4, the inhibition of hAChE increases 74-fold on
going from n ¼ 0 to n ¼ 4. In the absence of phenyl group at C4, as in
compounds 5e8, the inhibition of hAChE increases 334-fold on
going from n ¼ 0 to n ¼ 3, and 45-fold on going from n ¼ 0 to n ¼ 4.
To shed light in the effective AChE binding mode of the novel
AChE inhibitors here reported, compound 7 was submitted to
molecular modeling studies. Docking simulations were carried out
with the software Autodock Vina [39] using hAChE (PDB:1B41).
The docking procedure was applied to the whole protein target
(“blind docking”). To account for side chain flexibility during
docking, flexible torsions in the ligands were assigned, and the
acyclic dihedral angles were allowed to rotate freely. It is well
known that protein conformational flexibility is an important
aspect of ligand binding; thus, the incorporation of protein struc-
tural flexibility into the ligand binding procedure has been
commonly used in our previous studies [30b]. Accordingly, Trp286,
Tyr124, Tyr337 and Tyr72 residues were prescribed flexible in these
2.3. In vitro bloodebrain barrier permeation assay
The ability of a drug to penetrate the Blood Brain Barrier (BBB) is
of fundamental importance in drug design. In order to evaluate the
Scheme 1. Synthesis of pyridonepezils 1e8.

A. Samadi et al. / European Journal of Medicinal Chemistry 57 (2012) 296e301
299
Scheme 2.
works. However, when the ligand is large, this motion by itself is
not sufficient to enlarge the gorge to enhance ligand access to the
active center. Therefore, Asp74, Thr75, Trp86, and Tyr341 receptor
residues were also selected to be flexible during docking simula-
tion. These eight residues delineate the shape of the gorge entry
and lining, as their motion may significantly enlarge the gorge
mouth to facilitate ligand access to the catalytic site.
The binding mode of inhibitor 7 is shown in Fig. 1. It can be seen
that the ligand can favorably interact with both the catalytic pocket
and the PAS of the enzyme. In particular, the following major
interactions, responsible for the inhibiting profile of the selected
molecule, could be identified: (i) the benzyl group interacts by
catalytic triad residue Ser198 and the PAS, while mode II accom-
modates the ligand interacting with the PAS. A close up of mode I in
the vicinity of the catalytic triad, His438, Ser198, Glu325, uncovers
a likely critical interaction where the cyano group (in ortho position
with respect to the amino group) is hydrogen bonded to the Ser198
side chain, whereas the amino group can establish three hydrogen
bonds with the side chain of Ser287 and the backbone of Leu286.
Besides, the other cyano group is doubly hydrogen bonded to the
Thr120 and to the protonated piperidine ring by an intramolecular
hydrogen bond. Finally, the ligand interacts with Trp82 located in
the PAS, forming a face-to-face
pep interaction with the benzyl
moiety. Close examination of the first shell of residues surrounding
7 in mode II reveals a hydrogen bond interaction between the
primary amino group and the hydroxyl group of Tyr332, located in
the PAS, besides, the pyridine moiety interacts with Trp82 by
means of pep stacking. In this orientation, no interactions with the
catalytic triad residues were found.
By comparing the bioactive conformations for inhibitor 7, the
energy gap in hBuChE is approximately 29 kcal/mol higher than the
calculated for the hAChE. Thisenergetic penaltycanbe, atleast inpart,
thereasonforwhycompound7showslowhBuChEinhibitoryactivity.
means of
p
e
p
stacking with the indole ring of Trp286; (ii) the
stacking; (iii)
pyridine ring interacts with Trp86 by means of a
pep
the cyano group can establish H-bond interaction with the hydroxyl
group of Tyr133; (iv) the secondary amino group donates a proton
to the hydroxyl group of Tyr124 and forms a hydrogen bond; (v) the
interaction of the primary amino group with Ser203 seems to be
very crucial, because donates a proton to His447, an important step
during the hydrolysis of ACh. Formation of this hydrogen bond
probably masks the ability of Ser203 to participate in catalysis.
The interaction of this compound with hBuChE has also been
carried out. hBuChE (PDB: 1P0I) was taken for the study. The
preparation of the protein and the docking protocol applied was the
same as that mentioned earlier. Major differences between AChE
and BuChE are restricted to those residues that line the active site
cleft. Docking results reveal that compound 7 has two major pre-
dicted binding modes at the active site of the enzyme (Fig. 2). Mode
I, places the ligand in the binding pocket interacting with both the
2.5. Theoretical ADME analysis of pyridonepezils 1e8
Finally, a series of theoretical calculations allowed us to describe
the ADME (Absorption, Distribution, Metabolism, and Excretion)
properties of compounds 1e8 within the organism. All these four
criteria influence the drug levels, its kinetics and exposure to the
tissues, and hence the performance and pharmacological activity of
Fig. 1. Binding mode of inhibitor 7 at the active site of hAChE. Compound 7 is rendered as sticks and illustrated in blue. The side chains conformations of the mobile residues are
illustrated in the same color as the ligand. Different subsites of the active site were colored: catalytic triad (CT) in green, oxyanion hole (OH) in pink, anionic subsite (AS) in orange,
except Trp86, acyl binding pocket (ABP) in yellow and peripheral anionic subsite (PAS) in blue. Black dashed lines are drawn among atoms involved in hydrogen bond interactions.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

300
A. Samadi et al. / European Journal of Medicinal Chemistry 57 (2012) 296e301
Fig. 2. Two major binding modes of inhibitor 7 at the active site of hBuChE. Mode I (a): The compound is rendered as sticks and illustrated in red; mode II (b): The compound is
rendered as sticks and illustrated in blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
a drug. The ADME properties were calculated by QikProp (version
3.5, Schrödinger, LLC, New York, NY, 2012). About 45 physically
significant descriptors and pharmacologically relevant properties
of these compounds were predicted and some of the important
properties were analyzed. Lipinski’s rule of five (RO5) [40] is a rule
of thumb to evaluate the drug likeness of a molecule based on some
molecular descriptors representing ADME properties (see Table S1,
Supplementary material). All the compounds (1e8) showed
significant values for the properties analyzed and showed drug-like
characteristics based on Lipinski’s rule of five (see Table S1).
Compounds used for neurological disorders treatment are gener-
ally CNS acting drugs. CNS drugs show values of molecular weight,
HB donor, acceptors and rotatable bonds, etc., in general, in
a smaller range than general therapeutics. Factors that are relevant
to the success of CNS drugs were also analyzed. Except compound
4, all the compounds fulfill molecular weight, show a log Po/w < 5
and the number of hydrogen bonds donors and acceptors are in the
limits. The solubility (QPlogS) of organic molecules in water has
a significant impact on many ADME-related properties like uptake,
distribution, transport, and eventually bioavailability. Seven
compounds (1e2, 4e8) present solubility values within the limits
(ꢂ6.5e0.5), while compounds 3 and 4 show values of ꢂ6.9
and ꢂ7.3, respectively, being in the limits of aqueous solubility.
Acknowledgments
A. Samadi thanks CSIC for I3P post-doctoral contract. M. Estrada
thanks COLCIENCIAS (Colombia) for a PhD fellowship. M. Chioua
thanks Instituto de Salud CarlosIII (MICINN) fora “Sara Borrell” post-
doctoral contract. JMC thanks MICINN (SAF2006-08764-C02-01;
SAF2009-07271; SAF2012-33304). M.I. Rodríguez-Franco thanks
MICINN for grants SAF2006-01249 and SAF2009-13015-C02-01.
Appendix A. Supplementary material
Supplementary material related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2012.09.030.
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To sum up, we have reported the synthesis and cholinesterase
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shows that all tested compounds would cross the BBB by passive
diffusion. Work is now in progress in our laboratory to prepare
and evaluate new pyridonepezils of type I, and the results will
report in due course.
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