Synthesis, pharmacological assessment, and molecular modeling of 6-chloro-pyridonepezils: New dual AChE inhibitors as potential drugs for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2013.06.021

6-Chloro-pyridonepezils are chloropyridineedonepezil hybrids designed by combining the N-benzylpiperidine moiety present in donepezil with the 2-chloropyridine-3,5-dicarbonitrile heterocyclic ring system, both connected by an appropriate polymethylene lin

Full text of DOI:10.1016/j.ejmech.2013.06.021

European Journal of Medicinal Chemistry 67 (2013) 64e74
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, pharmacological assessment, and molecular modeling
of 6-chloro-pyridonepezils: New dual AChE inhibitors as potential
drugs for the treatment of Alzheimer’s disease
,
b
b
c
Abdelouahid Samadi a , Mario de la Fuente Revenga , Concepción Pérez , Isabel Iriepa ,
*
Ignacio Moraleda c, María Isabel Rodríguez-Franco b, 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 e 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:
6-Chloro-pyridonepezils are chloropyridineedonepezil hybrids designed by combining the N-benzylpi-
peridine moiety present in donepezil with the 2-chloropyridine-3,5-dicarbonitrile heterocyclic ring
system, both connected by an appropriate polymethylene linker. 6-Chloro-pyridonepezils 1e8 were
prepared by reaction of 2,6-dichloro-4-phenylpyridine-3,5-dicarbonitrile (13) [or 2,6-dichloropyridine-
3,5-dicarbonitrile (14)] with suitable 2-(1-benzylpiperidin-4-yl)alkylamines (9e12). The biological
evaluation showed that these new compounds are cholinesterase inhibitors, in the submicromolar range,
Received 17 April 2013
Received in revised form
31 May 2013
Accepted 4 June 2013
Available online 25 June 2013
one of them (6) being a potent hBuChE inhibitor (IC50 ¼ 0.47 ꢀ 0.08
m
M). 6-Chloro-pyridonepezils 4, 7 and
Keywords:
hAChE
8 are potent hAChE inhibitors showing IC50 in the 0.013e0.054
m
M range. Particularly, 6-chloro-pyr-
idonepezil 8 is 625-fold more selective for hAChE than for hBuChE and compared to donepezil is equi-
potent for the inhibition of hAChE. Molecular modeling investigation on 6-chloro-pyridonepezils 4, 6e8
supports its dual AChE inhibitory profile, by binding simultaneously at the catalytic active and at pe-
ripheral anionic sites of the enzyme. The in vitro Blood Brain Barrier (BBB) and theoretical ADME analysis
of 6-chloro-pyridonepezils 1e8 have been carried out. Overall, compound 8, is a permeable potent and
selective dual AChEI that can be considered as a good candidate with potential impact for further
pharmacological development in Alzheimer’s therapy.
hBuChE
Dual AChE inhibitors
6-Chloro-pyridonepezils
In vitro blood brain barrier
Molecular modeling
ADME
Alzheimer’s disease
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
acetyltransferase and acetylcholinesterase (AChE) enzyme activity
[2,3].
Alzheimer’s disease (AD) is the most prevalent neurodegener-
ative disorder [1]. AD is characterized by the gradual development
of forgetfulness, progressing to disturbances in language, disori-
entation, 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
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 the
cognitive decline associated with advanced age and AD [4]. Thus,
drugs capable of inhibiting AChE might potentiate central cholin-
ergic 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. For a while the treatment with AChEI
was reported to produce only symptomatic improvement, having
no effect in the course of the disease [6]. However, other studies
senile plaques, consisting principally of amyloid-b (Ab), and intra-
cellular neurofibrillary tangles, which are composed of phosphor-
ylated tau protein. Moreover, the basal nucleus of Meynert
undergoes profound neuron loss, the neocortex exhibits a loss of
cholinergic fibers and receptors, and a decrease of both choline
* Corresponding authors. Fax: þ34 91 5644853.
E-mail addresses: samadi@iqog.csic.es, asamadi1972@gmail.com, samadi72@
yahoo.com (A. Samadi), iqoc21@iqog.csic.es (J. Marco-Contelles).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.021

A. Samadi et al. / European Journal of Medicinal Chemistry 67 (2013) 64e74
65
indicated that AChE interacts with A
ment close to the PAS, thus promoting A
Moreover, AChE-A complexes increase A
b
by a hydrophobic environ-
Recently, we have described the multipotent donepezileindole
ASS234 [30] and the donepezilepyridine ASS280 [31] hybrids (Chart
1), which have in common the propargylamine group. These two
compounds are monoamine oxidase (MAO) and cholinesterase (ChE)
inhibitors. The donepezilepyridine hybrid I was also reported as a
dual ChE inhibitor [32], whose biological and molecular modeling
analysis showed that this molecule acts as dual AChEI, binding
simultaneously to the CAS and PAS of the enzyme. Finally, related
2-aminopyridine-3,5-dicarbonitrile II and 2-chloropyridine-3,5-
dicarbonitrile III (Chart 1) have been investigated in this laboratory
as potential AChEIs [33]. It is interesting to highlight that related 2-
amino-4-arylpyridine-3,5-dicarbonitrile derivatives (i.e. structures
such as the LUF derivatives and BAY60-6583, similar to those of the
compounds of the present study), have received much attention as
adenosine receptor ligands [34].
b
fibril formation [7,8].
b
b
-dependent neurotox-
icity [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 com-
pounds 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 path-
ogenesis [12].
Considering the non-cholinergic aspects of AChE related to the
PAS [7e9] in associating with A
of new antidementia drugs emerged. Peripheral or dual site in-
hibitors of AChE may simultaneously alleviate the cognitive deficit
in AD patients and prevent the assembly of Ab, which will delay the
b
, an attractive target for the design
Herein, we describe the synthesis, pharmacological evaluation
[AChE and butyrylcholinesterase (BuChE) inhibition], and the
in vitro Blood Brain Barrier (BBB) permeability, using the PAMPA
protocol, of 6-chloro-pyridonepezils 1e8 (Table 1), their theoretical
ADME analysis as well as the molecular modeling of 6-chloro-pyr-
idonepezils 6, and 8. These new hybrids (IV) were designed by
combining the N-benzylpiperidine moiety of donepezil with the
2-chloropyridine moiety present in compound III [33] (Chart 2).
neurodegenerative process [13]. This strategy was pursued for
several medicinal chemists who have been developing new com-
pounds with dual AChE inhibitory activity [14e17], based on well
known AChEIs such as tacrine [18], (E)-2-(4-(((7-(diethylamino)-
2,4-dioxochroman-3-ylidene)methyl)amino)phenyl)-N0-(1,2,3,4-
tetrahydroacridin-9-yl)acetohydrazide, a high-affinity, fluorescent
tacrine-derived cholinesterase inhibitor able to bind to amyloid
structures, described by Gütschow and co-workers [19], riva-
stigmine [20], donepezil [21] and galanthamine [22]. A recent
clinical study with moderate-to-severe Alzheimer’s disease pa-
tients determined that continued treatment with donepezil could
improve cognition and function for even severe patients [23].
Several classes of donepezil hybrids such as donepeziletacrine
[24,25], donepezileaminoacids [26], indanone hybrids [27], done-
pezileaminothienoquinoline [28], or indanone and aurone de-
rivatives [29] 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.
2. Results and discussion
2.1. Chemistry
The synthesis of 2-{[(1-benzylpiperidin-4-yl)methyl]amino}-6-
chloro-4-phenylpyridine-3,5-dicarbonitrile 1e4 and 2-{[(1-
benzylpiperidin-4-yl)methyl]amino}-6-chloropyridine-3,5-
dicarbonitrile 5e8 was easily achieved in one-step reaction from
commercially available amine 9, or known amines 10 [35], 11 [31a]
and 12 [32], with 2,6-dichloro-4-phenylpyridine-3,5-dicarbonitrile
13 [36] and 2,6-dichloropyridine-3,5-dicarbonitrile 14 [37],
respectively (Scheme 1). In these reactions, in some instances, di-
mers 2b and 5b, obtained in 25% and 21% of yield, were probably
formed due to a concentration of amine in reaction solvent (THFe
ethanol). However, no effort was dedicated to improve the yield of
Chart 1. multipotent e General structure of the multipotent MAO/ChE inhibitors ASS234 and ASS280 and the donepezil-pyridine hybrids of type I-III.

66
A. Samadi et al. / European Journal of Medicinal Chemistry 67 (2013) 64e74
Table 1
b
IC50
PAMPA-BBB assay for 6-chloro-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 10ꢁ6 cm sꢁ1
Compd.
R
n
hAChE
hBuChE
S.Ic
Pe
Prediction
1
2a
3
4
5a
6
7
8
Ph
Ph
Ph
Ph
H
H
H
H
Ph
H
0
2
3
4
0
2
3
4
2
0
9.43 ꢀ 0.43
4.40 ꢀ 0.34
3.86 ꢀ 0.62
0.031 ꢀ 0.008
5.35 ꢀ 0.38
0.31 ꢀ 0.05
0.054 ꢀ 0.004
0.013 ꢀ 0.002
9.77 ꢀ 0.78
7.30 ꢀ 0.17
0.01 ꢀ 0.002
0.8 ꢀ 0.1
>10
>10
e
nd
nd
nd
nd
e
9.41 ꢀ 0.11
5.62 ꢀ 0.95
>10
0.47 ꢀ 0.08
8.39 ꢀ 0.20
8.13 ꢀ 0.41
>10
2.43
181
e
10.12 ꢀ 0.59
10.31 ꢀ 0.86
7.39 ꢀ 0.54
8.52 ꢀ 0.71
5.98 ꢀ 0.09
6.99 ꢀ 0.07
nd
cnsþ
cnsþ
cnsþ
cnsþ
cnsþ
cnsþ
nd
1.52
155
625
e
2b
5b
>10
e
5.00 ꢀ 0.11
cnsþ
Donepezil
2.5 ꢀ 0.07
250
e
e
e
IIId
>10
e
e
nd: not determined for solubility problems.
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.
b
c
PBS:EtOH (70:30). Data are the mean ꢀ SD of three independent experiments.
Selectivity index ¼ hBuChE IC50/hAChE IC50
.
d
Inhibition was done using EeAChE and eqBuChE enzymes.
the corresponding derivatives 1, 2a and 5a, as the secondary
products could be of interest from the pharmacological point of
view. All the new compounds were fully characterized by spec-
troscopic techniques (mass spectrometry, FT-IR, and 1D and 2D
NMR) and elemental analysis (see Experimental part).
Comparing to the previously published donepezil derivatives,
6-chloro-pyridonepezil 8 is the most active among all the published
indanone hybrids [27] in the same range as donepezil, in the same
range as donepeziletacrine hybrids [24,25], and more active than
donepezilepyridine hybrid I (Chart 1) [32], differing only in the
chlorine atom. Compounds 1, 2a, 3, 5a, 2b and 5b showed a similar
inhibitory power compared to donepezileamino acid derivatives
[26]. These data suggest that substitution of the amino group by the
chlorine atom increased the inhibitory activity around 5-fold.
Concerning the structureeactivity relationships (SAR), we
conclude that 6-chloro-pyridonepezils bearing a phenyl group at C4
position on the pyridine ring were slightly less active than com-
pounds bearing a proton at C4. The hBuChE inhibitory potency
remains similar (compare 6-chloro-pyridonepezils 3 and 4 with 7
and 8). Very interestingly, the change of the length in the linker
produces an interesting increase of the inhibitory potency on going
from n ¼ 0 to n ¼ 4. For example, compound 4 (n ¼ 4) was found
304-fold more active than 1 (n ¼ 0), and compound 8 (n ¼ 4) was
found 411-fold more potent than 5a (n ¼ 0).
2.2. In vitro evaluation of human cholinesterase inhibition
The in vitro activity of the 6-chloro-pyridonepezils 1e8 against
hAChE and hBuChE was determined using the Ellman’s method
[38]. For comparative purposes, donepezil and chloropyridine III
[33] were used as reference compounds. From the IC50 data shown
in Table 1, we conclude that all compounds are potent cholines-
terase inhibitors. Some of them are selective for hAChE. The
most potent hAChE inhibitors were compounds 4, 6, 7 and 8 in
the nanomolar range, showing IC50 from 0.013
ꢀ
0.017 to
0.31 ꢀ 0.05 M. Comparing to compound III, also bearing the
m
2-chloropyridine ring, 6-chloro-pyridonepezils 4, 6, 7 and 8 are more
active in both hAChE and hBuChE enzymes. These data suggest that
the incorporation of the N-benzylpiperidine fragment to the
chloropyridine system increases the inhibitory potency for both
ChE enzymes. Compared to donepezil, compound 8 proved to be
almost equipotent for hAChE, and 4-fold less active for hBuChE,
while compound 6 was 3-fold less active for hAChE and 5-fold more
active for hBuChE.
2.3. In vitro bloodebrain barrier permeation assay
To evaluate the brain penetration we used the PAMPA-BBB
method described by Di et al. [39] and subsequently optimized by
Rodríguez-Franco et al. for molecules with limited water-solubility
Chart 2. General structure of denepezil, 2-choropyridine III and the novel dual ChE inhibitors IV.

A. Samadi et al. / European Journal of Medicinal Chemistry 67 (2013) 64e74
67
Scheme 1. Synthesis of the 6-chloro-pyridonepesils 1e8.
[40e45]. The in vitro permeability (Pe) values of a selection of new
compounds through a lipid extract of porcine brain were deter-
mined by using PBS: ethanol (70:30) (Table 1). In the same assay, 11
commercial drugs of known CNS penetration were also tested and
their experimental values were compared to reported values, giv-
ing a good lineal correlation, Pe (exp.) ¼ 0.6551 Pe (bibl.) þ 2.2142
(R2 ¼ 0.9136). From this equation and taking into account the limits
established by Di et al. for bloodebrain barrier permeation [39]
we found that compounds with permeability values above
4.8 ꢂ 10ꢁ6 cm sꢁ1 could penetrate into the CNS, whereas molecules
with permeability values below 3.5 ꢂ 10ꢁ6 cm sꢁ1 were predicted
to be a low BBB permeation. All tested compounds showed good
permeability values (Table 1) and thus, they could cross the BBB and
reach its biological targets located in the CNS.
The binding mode of inhibitor 6 is shown in Fig. 1. As in done-
pezilepyridine hybrids of type I (chart 1), it has been observed that
the cationep interaction of this cationic inhibitor with the
p-electron-rich active site is not the driving force for the binding.
This compound is oriented along the active-site gorge, extending
from the catalytic site at the bottom, to the peripheral anionic site
at the top, via aromatic stacking interactions with various aromatic
acid residues. The hydrophobic interaction between 6 (the piper-
azine and the alkyl linker) and the rich aromatic contents (Tyr341,
Phe338, Phe297, Phe295 and Trp286) along the gorge could
potentially direct the pyridine ring to penetrate deep into the cat-
alytic anionic site and oxyanion hole regions in the choline-binding
site. The interaction is further intensified by two hydrogen-bonding
interactions between the ligand and the residues in the active site.
This includes a hydrogen bond between the amino group and the
amino acid residue Tyr337. The cyano group can establish H-bond
interaction with the hydroxyl group of Tyr133. The pyridine ring
2.4. Molecular modeling of compounds 4, 6e8
To identify the molecular determinants responsible for the
binding mode of the studied compounds with AChE protein, com-
pounds 4, 6e8 were submitted to molecular modeling studies. We
have chosen compound 6 with better activity inhibition than
donepezil for BuChE and 8 with equivalent activity to donepezil for
AChE. To evaluate the influence on ChE inhibitory potency and
selectivity of the linkers of different length, compound 7 has been
studied. Finally, we have included compound 4 in order to explain
the lower but good activity of this compound with a phenyl moiety
with respect to compound 8.
Docking simulations were carried out via the Autodock Vina [46]
program. Docking experiments employed full ligand flexibility and
partial protein flexibility focused at the ligand binding site. We used
the determined X-ray crystallographic structure of hAChE com-
plexed with fasciculin-II (PDB: 1B41) and blind docking protocols
were performed for the detection of possible binding sites and
modes of peptide ligands by scanning the entire surface of protein
targets.
interacts with Trp86 by means of a
pep stacking. The benzyl group
interacts by means of T-shaped interaction with the indole
pep
ring of Trp286. The hydrogen atom at the piperidine C4 is oriented
toward Phe338 and Phe295.
Modification of the mid-gorge spacer modulated enzyme
selectivity and improved potency. Compound 7 with n ¼ 3 showed
the same binding profile as compound 6 (n ¼ 2) with an increase
in AChE activity and an activity decrease at BuChE. We then
investigated computationally the binding of compound 7 with
AChE to provide structural insights and potential to functional
modifications.
As depicted in Fig. 2, the phenyl fragment of the ligand was well
accommodated in the PAS through a pep stacking of this ring and
indole moiety of Trp286. This interaction was reinforced by a
bifurcated hydrogen bond between the secondary amino group of 7
and Asp74 and Tyr124. In addition, the pyridine part of the ligand
was oriented to the anionic site (AS). The interactions responsible
for stabilization of the pyridine in the active site included
pep
The quality of docking results can be affected by the simplifi-
cation of treating protein structures as rigid entities. Selective
residue flexibility is an option available on several molecular
docking tools, including Autodock Vina used in this work, where
only selected residues are allowed to be flexible. This approach is
promising as it attempts to provide a more realistic protein envi-
ronment while preventing an escalation of the computer power
need. In this study eight residues were selected from the active-site
gorge of the hAChE structure: Trp286, Tyr124, Tyr337 and Tyr72,
Asp74, Thr75, Trp86, and Tyr341. These eight residues delineate the
shape of the gorge entry and lining, as their motion may signifi-
cantly enlarge the gorge mouth to facilitate ligand access to the
catalytic site.
stacking and a hydrogen bonding. The interaction was formed
pep
between the pyridine ring and the indole moiety of Trp86 and the
hydrogen bonding interaction was between the cyano group and
the hydroxyl group of Tyr133.
The visual inspection of the top scored pose of compound 8
(n ¼ 4) reveals that the ligand is arranged at the active-site gorge in
a position similar to that found for compound 6 (n ¼ 2) and 7 (n ¼ 3)
(Figs.1 and 2). It has been observed that pep interactions played an
important role in stabilizing the complex. The benzyl group in-
teracts by means of T-shaped interaction with Tyr72 and a
face-to-face interaction with Trp286 (PAS). The hydrophobic
pep
pep
interaction between 8 (the piperidine and the alkyl linker) and the
rich aromatic residues (Tyr124, Tyr337, Phe338 and Tyr341) along

68
A. Samadi et al. / European Journal of Medicinal Chemistry 67 (2013) 64e74
Fig. 1. Binding mode of inhibitor 6 at the active site of hAChE. (A) Compound 6 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 (light blue). Different subsites of the active site were colored: catalytic triad (CT) in green, oxyanion hole (OH) in pink, anionic sub-site (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. (B) Intermolecular hydrophobic interactions of 6 with hAChE in 2D flattened space generated by LIGPLOT [47]. Purple stick models present the inhibitor and hy-
drophobic contacts with the ligand are presented by red semi-circles with radiating spokes. Hydrogen bonds are green dashed lines. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
the gorge could direct the pyridine ring to penetrate into the cat-
alytic anionic site and oxyanion hole regions in the choline-binding
site (Fig. 3). These interactions could possibly help to dock the
and Asp74. The cyano group forms a hydrogen bond with the hy-
droxyl group of Tyr133. The hydrogen atom at the piperidine C4 is
oriented toward Tyr341. According to the above description, ligands
6, 7 and 8 bind to hAChE in an extended conformation from the
mouth to the central region of the active site gorge, and hydro-
phobic interactions of the chloro group with two aminoacids
(Ser203 and His447) of the catalytic triad residues have been found.
These results suggest that the ligands could favorably interact with
the PAS and with the catalytic site of AChE.
pyridine moiety of the ligand to the catalytic anionic site by
pep
stacking interaction between Trp86 (AS) and the pyridine moiety.
Additionally, 8 is able to form hydrogen bonds between the sec-
ondary amine function and the hydroxyl group of Tyr337 residue
Our docking studies have revealed that, compound 8, possessed
the optimal spacer length that allows it strongly interacting with
hAChE. This inhibitor is able to better bind to the central pocket and
peripheral site than compounds 6 and 7.
The main structural difference of compounds 5ae8 with respect
to the compounds 1e4 is the presence of the phenyl group at po-
sition 4 of the pyridine ring, the tether length for these compounds
was in the range of zero to four, with the optimum equaling four.
We also obtained a docking model for the complex between AChE
and compound 4, in order to explain why the substitution of a
hydrogen atom by a phenyl group decrease the activity. Compound
4 was a simple modification of compound 8. It contained a phenyl
group in the pyridine moiety. Due to this substituent, compound 4
was arranged in different way along the active-site gorge than
compounds 6, 7 and 8.
As can be seen in Fig. 4, compound 4 also showed a binding
mode with an extended conformation and it can simultaneously
bind at both the PAS and the CAS of AChE. At the top of the gorge,
two edge-to-face
pep interactions between the phenyl ring and
Fig. 2. Binding mode of inhibitor 7 at the active site of hAChE. (A) Compound 7 is
rendered as sticks and illustrated in violet. The side chains conformations of the mobile
residues are illustrated in the same color as the ligand (light violet). Different subsites
of the active site were colored: catalytic triad (CT) in green, oxyanion hole (OH) in pink,
anionic sub-site (AS) in orange, except Trp86, acyl binding pocket (ABP) in yellow, and
peripheral anionic subsite (PAS) in violet. Black dashed lines are drawn among atoms
involved in hydrogen bond interactions. Hydrogen bonds are black dashed lines. (For
interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
Tyr72 side chain and Trp286 indole ring were identified. In the
middle of the gorge, the nitrogen atom from nitrile group forms a
hydrogen bond with the hydroxyl group of the Tyr124, which forms
an additional hydrogen bond with the hydrogen atom of the pro-
tonated piperidine ring. Near the bottom of the gorge, the phenyl
ring of the benzyl group stacks against the Trp86 indole ring and
hydrophobic interactions of this phenyl group with two aminoacids

A. Samadi et al. / European Journal of Medicinal Chemistry 67 (2013) 64e74
69
Fig. 3. Binding mode of inhibitor 8 at the active site of hAChE. (A) Compound 8 is rendered as sticks and illustrated in red. The side chains conformations of the mobile residues are
illustrated in light pink. 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 light pink. Black dashed lines are drawn among atoms involved in hydrogen bond interactions. (B)
Intermolecular hydrophobic interactions of 8 with hAChE in 2D flattened space generated by LIGPLOT [47]. Purple stick models present the inhibitor and hydrophobic contacts with
the ligand are presented by red semi-circles with radiating spokes. Hydrogen bonds are green dashed lines. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
(Ser203 and His447) of the catalytic triad residues have been found.
It can be postulated that this compound, due do its large size at the
pyridine moiety, is unable to enter by this side into the narrow
active site gorge of the AChE receptor and hence the benzyl group
was oriented toward the CAS.
secondary amino group is a donor bifurcated hydrogen bond
making hydrogen bonds with both the oxygen atom of the Tyr332,
and the carboxylate group of the Asp70, located in the PAS. The
nitrogen atom of the cyano group is bridged to Tyr332 via hydrogen
bonding. The pyridine moiety formed
pep stacking with Trp82.
The interaction of compounds 4, 6, 7 and 8 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 above. Docking results reveal that hBuChE
could effectively accommodate compounds 4, 6e8 inside the active
site gorge (Fig. 5).
Compounds 6e8 were arranged in a similar way, but their
conformations were bent, not linear, as for AChE. Close examination
of the first shell of residues surrounding 6 and 8 reveals that the
Besides, the phenyl moiety interacts with Trp231 by means of hy-
drophobic interactions. In this orientation, hydrophobic in-
teractions with the catalytic triad residues, Ser198 and His438 were
found (Fig. 6). The hydrogen at the piperidine C4 is oriented toward
Thr120. As can be seen in Fig. 5, compound 7 established all the
interactions mentioned above, but the secondary amino group did
not establish hydrogen bond interactions with Tyr332.
The best docked poses of compounds 4 and 8 were superposed
and showed to be similarly docked in the gorge of BuChE. The
detailed picture of the superimposed docking poses of 4 and 8
is shown in Fig. 7. The aromatic parts of the inhibitor form
Fig. 4. Binding mode of inhibitor 4 at the active site of hAChE. (A) Compound 4 is
rendered as sticks and illustrated in green. The side chains conformations of the mobile
residues are illustrated in the same color as the ligand (light green). Different subsites
of the active site were colored: catalytic triad (CT) in green, oxyanion hole (OH) in pink,
anionic sub-site (AS) in orange, except Trp86, acyl binding pocket (ABP) in yellow, and
peripheral anionic subsite (PAS) in light green. Black dashed lines are drawn among
atoms involved in hydrogen bond interactions. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Binding modes of inhibitors 6, 7 and 8 at the active site of hBuChE. The com-
pounds are rendered as sticks, compound 6 is illustrated in blue, compound 7 in yellow
and compound 8 in red. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)

70
A. Samadi et al. / European Journal of Medicinal Chemistry 67 (2013) 64e74
Fig. 6. Schematic diagram of proteineligand interactions generated by LIGPLOT [47]. (A) Interactions of compound 6 with BuChE. (B) Interactions of compound 8 with BuChE.
Brown sticks models present the residues of the enzymes, purple stick models present the inhibitor, hydrogen bonds are green dashed lines and hydrophobic contacts with the
ligand are presented by red semi-circles with radiating spokes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)
characteristic aromatic interactions in the enzyme. One aromatic
moiety (benzyl group) is engaged in hydrophobic interactions with
Trp231, Ser198 and His438 as it had been observed for compounds
6e8. The second aromatic fragment (phenylpyridine moiety)
occupied a similar position to that occupied by the pyridine moiety
in compounds 6e8. In fact, compound 4 still preserves the already
observed hydrogen bond between the nitrogen atom of the cyano
group and the hydroxyl group of Tyr332. The phenyl ring attached
to the pyridine moiety was perpendicularly stacked between Trp82
and Phe329 and positioned in an aromatic pocket comprised of
Tyr430, Tyr440 and Tyr332.
extended conformation whereas they bind to BuChE with a folded
conformation (Fig. 8). For compound 6, the energy gap in hBuChE is
approximately 20 kcal/mol higher than the calculated for the
hAChE. In the case of compound 8 the value is increased to 48 kcal/
mol. This energetic penalty can be, at least in part, the reason for
why both ligands show low hBuChE inhibitory activity, with com-
pound 8 being more selective.
2.5. Theoretical ADME analysis of 6-chloro-pyridonepezils 1e8
Finally, the ADME (Absorption, Distribution, Metabolism and
Excretion) properties were calculated using the QikProp [48],
which predicts required principle and physiochemical descriptors
of possible drug compounds (see Supplementary material).
The bioactive conformations for inhibitors 4, 6e8 in AChE were
different from those in BuChE. The ligands bind to AChE with an
About 45 physically significant descriptors and pharmacologi-
cally relevant properties of these compounds were predicted and
some of the important properties were analyzed. All the com-
pounds showed significant values for the properties analyzed and
showed drug-like characteristics based on Lipinski’s rule of five (see
Table 1). The drugs used for neurological disorder treatment are
generally CNS acting drugs. CNS drugs show values of molecular
Fig. 8. (A) Superposition of the bioactive conformations for compound 8 in hAChE (red
sticks) and in hBuChE (pink sticks). (B) Superposition of the bioactive conformations
for compound 6 in hAChE (blue sticks) and in hBuChE (pale blue sticks). (For inter-
pretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)
Fig. 7. Top-scored docking poses representation within hBuChE active site for 4 (violet)
and 8 (red). (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)

A. Samadi et al. / European Journal of Medicinal Chemistry 67 (2013) 64e74
71
weight, HB donor, acceptors and rotatable bonds, etc., in general, in
4.1.2. 2-((1-Benzylpiperidin-4-yl)amino)-6-chloro-4-
a smaller range than general therapeutics. Factors that are relevant
to the success of CNS drugs were analyzed. CNS drugs show values
of MW <450, HB donor <3, HB acceptors <7, QPlogPo/w <5, PSA
<90, number of rotatable bonds <8 and hydrogen bonds <8 [49].
The compounds 1, 5a, and 6e8 satisfied all the characteristic of CNS
acting drugs. The QPLogPo/w values and the molecular weight of
compounds 2a, 3, 4 and 2b exceeded those of CNS drugs. The sol-
ubility of organic molecules in water has a significant impact on
many ADME-related properties. Only compounds 1, 5a, and 6e8
present solubility values within the limits, while the rest of the
compounds show values between 7.3 and 8.4.
phenylpyridine-3,5-dicarbonitrile (1) [31a]
Following the General procedure, reaction of 2,6-dichloro-4-
phenylpyridine-3,5-dicarbonitrile 13 (300 g, 1.1 mmol) in a mixture
of THF/EtOH (10/5 mL) was added 4-amine-1-benzylpiperidine 9
(0.22 mL, 1.1 mmol) and triethylamine (0.335 mL, 2.2 mmol). The
reaction was heated at reflux for 2 h and 30 min. The reaction was
concentrated and the residue was purified by column chromatog-
raphy (hexane:EtOAc, from 2:1 to 1:1) to gave compounds 1 [31a] as
a white solid (339 mg, 81%). Mp 185e187 ꢃC; IR (KBr)
n
3436, 3315,
2224, 1588, 1557 cmꢁ1 1H NMR (400 MHz, CDCl3)
;
d 7.61e7.50 (m,
5H, Ph), 7.39e7.26 (m, 5H, Bn), 5.76 (d, J ¼ 7.6 Hz, 1H, NH), 4.21e4.06
(m, H, CH), 3.56 (s, 2H, CH2Ph), 2.89 (d, J ¼ 11.9 Hz, 2H, CH2), 2.24 (t,
J ¼ 10.6 Hz, 2H, CH2), 2.05 (d, J ¼ 10.9 Hz, 2H, CH2), 1.72e1.56 (m, 2H,
3. Conclusions
CH2); 13C NMR (100 MHz, CDCl3)
d 159.9 (C4), 157.7 (C6), 157.5 (C2),
138.2 (C100), 133.0 (C10), 131.3 (C40), 129.2 (C30’’), 129.2 (C30), 128.5
(C20), 128.2 (C200), 127.3 (C400), 114.7, 114.5 (2ꢂ CN), 98.0 (C5), 94.4
(C3), 63.0 (CH2Ph), 51.9 (2ꢂ CH2), 49.5 (CH), 31.9 (2ꢂ CH2); MS (ESþ)
m/z (%): 428 [M þ H]þ; Anal. Calcd. for C25H22ClN5: C, 70.17; H, 5.18;
N, 16.37; Cl, 8.28. Found: C, 69.96; H, 5.31; Cl, 8.30; N, 16.09.
To sum up, 6-chloro-pyridonepezils 1e8 were synthesized and
evaluated against hAChE and hBuChE, using donepezil as a refer-
ence compound. Most of the assayed compounds showed high ChE
inhibitory activity, the most potent, 6-chloro-pyridonepezil 8, was
equipotent to donepezil. Compounds 1, 2a, 5a, 2b and 5b were
extremely selective for hAChE, but 6-chloro-pyridonepezils 2e4 and
6e8 showed high activity for both hAChE and hBuChE. Concerning
SAR, compound 8 (n ¼ 4), bearing a hydrogen at C4, was more
active than the analog 4 (n ¼ 4) bearing a phenyl group at C4. The
BBB-PAMPA study indicates that all tested compounds would
probably cross the BBB by passive diffusion. Finally, the docking
analysis confirmed that these new chloro-pyridonepezils behave as
dual hAChE inhibitors, binding simultaneously to the CAS and PAS
of the enzyme, showing thus potential for the treatment of AD.
Experiments toward this objective are being now investigated and
will be reported in due course.
4.1.3. 2-((2-(1-Benzylpiperidin-4-yl)ethyl)amino)-6-chloro-4-
phenylpyridine-3,5-dicarbonitrile (2a) and 2,6-bis((2-(1-
benzylpiperidin-4-yl)ethyl)amino)-4-phenylpyridine-3,5-
dicarbonitrile (2b)
Following the General procedure, reaction of 2,6-dichloro-4-
phenylpyridine-3,5-dicarbonitrile 13 (383 mg, 1.398 mmol) in a
mixture of THF/EtOH (10/5 mL) with 2-(1-benzylpiperidin-4-yl)
ethanamine 10 (300 mg, 1.398 mmol) and triethylamine (0.43 mL,
2.795 mmol). After 15 h, and column chromatography (2% of MeOH
in DCM) afforded compounds 2a and 2b. 2a: white solid (334 mg,
65%); Rf ¼ 0.45 (10% of MeOH in DCM); mp 180e3 ꢃC; IR (KBr)
n 3336,
2933, 2918, 2223, 1586, 1452 cmꢁ1 1H NMR (400 MHz, CDCl3)
;
d
7.56e7.49 (m, 5H, Ph), 7.40e7.19 (m, 5H, Bn), 5.96 (t, J ¼ 5.3 Hz, 1H,
4. Experimental part
NH), 3.60 (m, 2H, CH2NH), 3.51 (s, CH2Ph), 2.90 (d, J ¼ 11.2 Hz, 2H),
1.97 (t, J ¼ 10.6 Hz, 2H), 1.71 (m, 2H), 1.60 (m, 2H), 1.35 (m, 3H); 13
C
4.1. General methods
NMR (100 MHz, CDCl3) d 159.6 (C4ePy), 158.3 (C2ePy), 157.4 (C6e
Py), 138.2 (C1eBn),132.8 (C10ePh), 131.1 (C40ePh),129.1 (2C, 2ꢂ CH),
129.0 (2C, 2ꢂ CH), 128.3 (2C, 2ꢂ CH), 128.1 (2C, 2ꢂ CH), 126.9 (C4,
Bn), 114.5 and 114.4 (2ꢂ CN), 97.7 (C5ePy), 90.5 (C3ePy), 63.3
(CH2Ph), 53.5 (2C, 2ꢂ CH2), 39.8 (CH2NH), 35.7 (CH2(CH2NH)), 33.2
(CH), 32.0 (2C, 2ꢂ CH2); MS (ES) m/z (%): 456 [M þ H]þ, 478
[M þ Na]þ. Anal. Calcd. for C27H26ClN5: C, 71.12; H, 5.75; Cl, 7.78; N,
15.36. Found: C, 70.89; H, 6.01; Cl, 7.34; N, 15.33. 2b: white solid
(220 mg, 25%); Rf ¼ 0.24 (10% of MeOH in DCM); mp 209e11 ꢃC; IR
Melting points were determined on a Koffler apparatus, and are
uncorrected. 1H NMR and 13C NMR spectra were recorded in CDCl3
or DMSO-d6 at 300, 400 or 500 MHz and at 75, 100 or 125 MHz,
respectively, using solvent peaks [CDCl3: 7.27 (D), 77.2 (C) ppm;
D2O: 4.60 ppm and DMSO-d6: 2.49 (D), 40 (C) ppm] as internal
reference. The assignment of chemical shifts was based on standard
NMR experiments (1H, 13C-DEPT, 1H, 1HeCOSY, gHSQC, gHMBC).
Mass spectra were recorded on a GC/MS spectrometer with an API-
ES ionization source. Elemental analyses were performed at CNQO
(CSIC, Spain). TLC were performed on silica F254 and detection
by UV light at 254 nm or by charring with either ninhydrin,
anisaldehyde or phosphomolybdiceH2SO4 drying reagents. Anhy-
drous solvents were used in all experiments. Column chromatog-
raphy was performed on silica gel 60 (230 mesh). Chromatotron
separations were performed on a Harrison Research Model 7924
Chromatotron equipped with a UV Lamp. The circular disks were
coated with Kieselgel 60 PF254 (E. Merck). All known compounds
showed NMR data identical to those described in the literature.
(KBr)
(400 MHz, CDCl3)
n
3338, 2924, 2203, 1586, 1567, 1536, 736, 698 cmꢁ1; 1H NMR
d
7.49 (s, 5H, Ph), 7.34e7.22 (m, 10H, Bn), 5.66 (t,
J ¼ 5.6 Hz, 2H, 2ꢂ NH), 3.56e5.51 (m, 4H, 2ꢂ CH2NH), 3.49 (s, 4H, 2ꢂ
CH2Ph), 2.88 (d, J ¼ 11.5 Hz, 4H, 2ꢂ CH2), 1.95 (t, J ¼ 10.6 Hz, 4H, 2ꢂ
CH2), 1.68 (d, J ¼ 9.3 Hz, 4H, 2ꢂ CH2), 1.60e1.55 (m, 4H, 2ꢂ
CH2CH2NH), 1.37e1.26 (m, 6H, 2ꢂ CH2 and 2ꢂ CH); 13C NMR
(100 MHz, CDCl3)
d
159.75 (C2 and C6),158.9 (C4),138.6 (2ꢂ C1eBn),
134.7 (C10ePh), 130.6 (C, CePh), 129.4 (4C, Bn), 129.0 (2C, CePh),
128.5 (2C, Ph), 128.4 (4C, Bn), 127.2 (2C, Bn), 117.2 (2ꢂ CN), 80.8 (C3
and C5), 63.7 (2ꢂ CH2Ph), 53.8 (4C, 4ꢂ CH2), 39.5 (2ꢂ CH2NH), 36.3
(2ꢂ CH2(CH2NH)), 33.6 (2ꢂ CH), 32.4 (4C, 4ꢂ CH2); MS (ESIþ) m/z:
638 [M þ H]þ, 660 [M þ Na]þ. Anal. Calcd. for C41H47N7: C, 77.20; H,
7.43; N, 15.37. Found: C, 76.97; H, 7.40; N, 15.45.
4.1.1. General procedure for the synthesis of compounds (1e8)
2,6-Dichloro-4-phenylpyridine-3,5-dicarbonitrile 13 or 2,6-
dichloropyridine-3,5-dicarbonitrile 14 was suspended in
a
4.1.4. 2-((3-(1-Benzylpiperidin-4-yl)propyl)amino)-6-chloro-4-
phenylpyridine-3,5-dicarbonitrile (3)
Following the General procedure, reaction of 2,6-dichloro-4-
phenylpyridine-3,5-dicarbonitrile 13 (61 mg, 0.25 mmol) with 3-
(1-benzylpiperidin-4-yl)propan-1-amine 11 (58 mg, 0.25 mmol) and
mixture of THF/EtOH (2:1, v:v). The corresponding amines 9e12,
followed by triethylamine, were then added. The mixture was then
heated under reflux. After cooling, the solvent was removed in
vacuum and the resulting crude submitted to flash column chro-
matography, to give compounds 1e8.
triethylamine (76 mL) in EtOH/THF (2/4, 5 mL), after 1 h, and column

72
A. Samadi et al. / European Journal of Medicinal Chemistry 67 (2013) 64e74
chromatography (from 0% to 5% of MeOH in CH2Cl2) gave product 3
(55 mg, 53%) as a white solid: Rf ¼ 0.42 (CH2C2/MeOH, 20/1, v/v); mp
heated at reflux for 1 h and then for overnight at room temperature.
The reaction was concentrated and the residue was purified by col-
umn chromatography (from 2.5 to 5% of MeOH in DCM). to give
compound 6 [31a] (301 mg, 70%) as a white solid; Anal. Calcd. for
172e4 ꢃC; IR (KBr)
n ;
3436, 2236, 1582, 1561, 1540, 1476, 1385 cmꢁ1
1H NMR (500 MHz, CDCl3)
d 7.52e7.42 (m, 5H, Ph), 7.27e7.17 (m, 5H,
Ph), 5.83 (t, J ¼ 5.3 Hz, 1H, NH), 3.52e3.48 (m, 2H, CH2eNH), 3.47 (s,
C21H22ClN5: C, 66.39; H, 5.84; Cl, 9.33; N, 18.44. Found: C, 66.14; H,
5.79; Cl, 9.41; N, 18.31.
2H, CH2Ph), 2.86 (d, J ¼ 9.9 Hz, 2H), 1.94e1.89 (m, 2H), 1.64e1.57 (m,
4H),1.29e1.19 (m, 5H); 13C NMR (126 MHz, CDCl3)
d 159.6 (C4), 158.3
(C2), 157. 4 (C6), 138.0 (C100), 132.8 (C10), 131.2 (C40), 129.3 (C300),
129.1 (C30), 128.3 (C20), 128.2 (C200), 127.0 (C400), 114.6, 114.5 (2ꢂ CN),
97.8 (C5), 90.6 (C3), 63.3 (CH2Ph), 53.6 (2ꢂ CH2), 42.5 (CH2NH), 35.2
(CH), 33.4 (CH2e(CH2)2NH), 32.0 (2ꢂ CH2), 26.3 (CH2eCH2NH); MS
(IE) m/z (%): 91 (100) [PhCH2]þ, 188 (8) [BnepiperidineeCH2]þ, 202
(34) [BnepiperidineeCH2CH2]þ, 216 (8) [Bnepiperidinee(CH2)3]þ,
378 (5) [M ꢁ Bn]þ, 392 (2) [M ꢁ Ph]þ, 468 (6) [M ꢁ H]þ; HRMS:
Calcd. for C28H29ClN5 (M þ H)þ: 470.2111. Found: 470.2119. Anal.
Calcd. for C28H28ClN5: C, 71.55; H, 6.00; Cl, 7.54; N, 14.90. Found: C,
71.3; H, 5.87; Cl, 7.83; N, 14.76.
4.1.8. 2-((3-(1-Benzylpiperidin-4-yl)propyl)amino)-6-
chloropyridine-3,5-dicarbonitrile (7)
Following the General procedure, reaction of 2,6-dichlorop-
yridine-3,5-dicarbonitrile 14 (51 mg, 0.258 mmol) with 3-(1-
benzylpiperidin-4-yl)propan-1-amine 11 (60 mg, 0.258 mmol) and
triethylamine (78 mL) in EtOH/THF (1/4, 5 mL), after 1 h, and column
chromatography (AcOEt/CH2Cl2 from 1/5 to 1/3, v/v) gave product 7
(59 mg, 58%) as a white solid: Rf ¼ 0.29 (CH2C2/MeOH, 20/1, v/v); mp
143e5 ꢃC; IR (KBr)
n ;
3344, 2927, 2224, 1602, 1590, 11,395 cmꢁ1 1H
NMR (500 MHz, CDCl3)
d 7.83 (m, 1H, CH4), 7.39e7.24 (m, 5H, Ph),
5.94 (t, J ¼ 5.5 Hz,1H, NH), 3.70 (s, 2H, CH2Ph), 3.53 (td, J ¼ 7.3, 5.7 Hz,
4.1.5. 2-((4-(1-Benzylpiperidin-4-yl)butyl)amino)-6-chloro-4-
phenylpyridine-3,5-dicarbonitrile (4)
2H, CH2eNH) 3.09 (d, J ¼ 12.2 Hz, 2H), 2.16 (t, J ¼ 11.2 Hz, 2H), 1.79e
1.57 (m, 4H), 1.53e1.21 (m, 5H); 13C NMR (126 MHz, CDCl3)
d 158.1
Following the General procedure, reaction of 2,6-dichloro-4-
phenylpyridine-3,5-dicarbonitrile 13 (71.7 mg, 0.261 mmol) with
4-(1-benzylpiperidin-4-yl)butan-1-amine 12 (60 mg, 0.243 mmol)
and triethylamine (74 mL) in EtOH/THF (2/4, 6 mL), after 7 h, and
column chromatography (AcOEt/CH2Cl2 from 1/5 to 1/3, v/v) gave
(C2), 156.4 (C6), 145.9 (C4), 137.7 (C100), 129.4 (C300), 128.2 (C200), 127.1
(C400),114.5 and 114.1 (2ꢂ CN), 97.1 (C5), 90.7 (C3), 62.5 (CH2Ph), 53.3
(2ꢂ CH2), 42.3 (CH2NH), 34.7 (CH), 33.1 (CH2e(CH2)2NH), 31.7 (2ꢂ
CH2), 26.3 (CH2eCH2NH); MS (IE) m/z (%): 91 (100) [PhCH2]þ, 188
(12) [BnepiperidineeCH2]þ, 202 (37) [BnepiperidineeCH2CH2]þ,
216 (9) [Bnepiperidinee(CH2)3]þ, 302 (8) [M ꢁ Bn]þ, 316 (5)
[M ꢁ Ph]þ, 392 (5) [M ꢁ H]þ, 393 (6), [M]þ. HRMS: Calcd. for
product 4 (155 mg, 97%) as a white solid: Rf ¼ 0.47 (CH2C2/MeOH, 10/
1, v/v); mp 90e2 ꢃC; IR (KBr)
n 3421, 3306, 2930, 2223, 1591, 1552,
1449 cmꢁ1; 1H NMR (500 MHz, CDCl3)
d
7.51e7.43 (m, 5H, Ph), 7.26e
C
C
22H25ClN5 (M þ H)þ: 394.1798. Found: 394.1802; Anal. Calcd. for
7.16 (m, 5H, Ph), 5.80 (t, J ¼ 5.4 Hz, 1H, NH), 3.54e3.50 (m, 2H, CH2e
NH), 3.43 (s, 2H, CH2Ph), 2.82 (d, J ¼ 10.98 Hz, 2H),1.87 (t, J ¼ 10.5 Hz,
2H), 1.61e1.55 (m, 4H), 1.36e1.30 (m, 2H), 1.25e1.13 (m, 5H); 13C
22H24ClN5: C, 67.08; H, 6.14; Cl, 9.00; N, 17.78. Found: C, 66.79; H,
6.22; Cl, 9.23; N, 18.02.
NMR (126 MHz, CDCl3)
d
159.6 (C4), 158.4 (C2), 157.5 (C6), 138.3
4.1.9. 2-((4-(1-Benzylpiperidin-4-yl)butyl)amino)-6-chloropyridine-
3,5-dicarbonitrile (8)
Following the General procedure, reaction of 2,6-dichloro-
pyridine-3,5-dicarbonitrile 14 (44 mg, 0.223 mmol) with 4-(1-
benzylpiperidin-4-yl)butan-1-amine 12 (55 mg, 0.223 mmol) and
(C100), 132.8 (C10), 131.2 (C40), 129.2 (C300), 129.1 (C30), 128.3 (C20),
128.1 (C200), 126.9 (C400), 114.6 and 114.5 (2ꢂ CN), 97.8 (C5), 90.6 (C3),
63.4 (CH2Ph), 53.8 (2ꢂ CH2), 42.2 (CH2NH), 36.0 (CH2e(CH2)3NH),
35.5 (CH), 32.2 (CH2eCH2NH), 29.2 (2ꢂ CH2), 23.9 (CH2e(CH2)2NH);
MS (IE) m/z (%): 91 (100) [PhCH2]þ, 188 (12) [BnepiperidineeCH2]þ,
202 (22) [BnepiperidineeCH2CH2]þ, 216 (75) [Bnepiperidinee
(CH2)3]þ, 392 (37) [M ꢁ Bn]þ, 406 (19) [M ꢁ Ph]þ, 482 (44) [M ꢁ H]þ,
483 (36), [M]þ. HRMS: Calcd. for C29H31ClN5 (M þ H)þ: 484.2268.
Found: 484.2285. Anal. Calcd. for C29H30ClN5: C, 71.96; H, 6.25; Cl,
7.32; N, 14.47. Found: C, 71.94; H, 5.94; Cl, 7.10; N, 14.62.
triethylamine (68 mL) in EtOH/THF (2/4, 6 mL), after 1 h, and column
chromatography (from 1% to 5% of MeOH in CH2Cl2) gave product 8
(30 mg, 33%) as a white solid: Rf ¼ 0.2 (CH2C2/MeOH, 20/1, v/v); mp
140e2 ꢃC; IR (KBr) 3413, 3329, 2920, 2227, 1599, 158, 1396 cmꢁ1
n ;
1H NMR (500 MHz, CDCl3)
d 7.83 (m, 1H, CH4), 7.33e7.24 (m, 5H,
Ph), 5.81 (t, J ¼ 5.2 Hz, 1H, NH), 3.57e3.53 (m, 4H, CH2e
NH þ CH2Ph), 2.93 (d, J ¼ 10.3 Hz, 2H, CH2epiperidine), 1.98 (t,
J ¼ 9.7 Hz, 2H, CH2epiperidine), 1.66e1.60 (m, 4H, CH2CH2e
NH þ CH2epiperidine), 1.41e1.35 (m, 2H), 1.31e1.27 (m, 5H); 13C
4.1.6. 2-((1-Benzylpiperidin-4-yl)amino)-6-chloropyridine-3,5-
dicarbonitrile (5a) [31a] and 2,6-bis((1-benzylpiperidin-4-yl)
amino)pyridine-3,5-dicarbonitrile (5b) [31a]
NMR (126 MHz, CDCl3)
d 158.1 (C2), 156.4 (C6), 145.9 (C4), 137.7
Following the General procedure, reaction of 2,6-dichloro-4-
phenylpyridine-3,5-dicarbonitrile 14 (300 mg, 1.51 mmol) in a
mixture of THF/EtOH (10/5 mL) was added 4-amine-1-
(C100), 129.4 (C300), 128.2 (C200), 127.1 (C400), 114.5 and 114.1 (2ꢂ CN),
97.1 (C5), 90.5 (C3), 63.2 (CH2Ph), 53.7 (2ꢂ CH2), 42.1 (CH2NH), 35.9
(CH2e(CH2)3NH), 35.4 (CH), 32.0 (CH2eCH2NH), 29.1 (2ꢂ CH2), 23.8
(CH2e(CH2)2NH); MS (IE) m/z (%): 91 (100) [PhCH2]þ, 202 (12) [Bne
piperidineeCH2CH2]þ, 216 (25) [Bnepiperidinee(CH2)3]þ, 316 (12)
[M ꢁ Bn]þ, 330 (5) [M ꢁ Ph]þ, 406 (9) [M ꢁ H]þ, 407 (5), [M]þ.
HRMS: Calcd. for C23H27ClN5 (M þ H)þ: 408.1955. Found: 408.1957.
Anal. Calcd. for C23H26ClN5: C, 67.72; H, 6.42; Cl, 8.69; N, 17.17.
Found: C, 67.54; H, 6.70; Cl, 9.01; N, 17.08.
benzylpiperidine
9 (0.31 mL, 1.51 mmol) and triethylamine
(0.46 mL, 3 mmol). The reaction was stirred overnight at room
temperature. After completion, the reaction was concentrated and
the residue was purified by column chromatography (hex-
ane:EtOAc, 1:1) to afford 5a and 5b. 5a [31a]: white solid (399 mg,
75%). Anal. Calcd. for C19H18ClN5: C, 64.86; H, 5.16; Cl, 10.08; N,
19.91. Found: C, 64.59; H, 5.45; Cl, 9.40; N, 19.68. 5b [31a]: white
solid (161 mg, 21%); Anal. Calcd. for C31H35N7: C, 73.63; H, 6.98; N,
19.39. Found: C, 73.39; H, 7.11; N, 19.21.
4.2. Pharmacology
4.2.1. Inhibition experiments of AChE and BuChE
4.1.7. 2-((2-(1-Benzylpiperidin-4-yl)ethyl)amino)-6-
chloropyridine-3,5-dicarbonitrile (6) [31a]
A solution of 2,6-dichloropyridine-3,5-dicarbonitrile 14 (300 mg,
1.515 mmol) in a mixture of THF/EtOH (10/5 mL) was added 2-(1-
benzylpiperidin-4-yl)ethanamine 10 (330 mg, 1.515 mmol) and
triethylamine (0.7 mL, 4.545 mmol). The reaction mixture was
To assess the inhibitory activity of the compounds toward AChE
or BuChE, we followed the spectrophotometric method of Ellman
[38], using purified AChE from human erythrocytes (min. 500 units/
mg protein in buffered aqueous solution) and BuChE from human
serum (3 units/mg protein, lyophilized powder) (Sigma Aldrich,
Madrid, Spain). Compounds were evaluated in 100 mM phosphate

A. Samadi et al. / European Journal of Medicinal Chemistry 67 (2013) 64e74
73
buffer, pH 8.0, at 30 ꢃC using acetylthiocholine and butyrylth-
iocholine (0.4 mM) as substrates, respectively. 5,5-Dithio-bis(2-
nitrobenzoic) acid (DTNB, Ellman’s reagent, 0.2 mM) was used,
and the values of IC50 were calculated by UV spectroscopy from the
absorbance changes at 412 nm as the concentration of compound
that produces 50% AChE activity inhibition. Data are expressed
as means ꢀ s.e.m. of at least three different experiments in
quadruplicate.
(x ¼ 116.546; y ¼ 110.33; z ¼ ꢁ134.181). Default parameters were
used except num_modes, which was set to 40. The AutoDock Vina
docking procedure used was previously validated [52].
For hBuChE (1P0I) docking calculations were performed
following the same protocol described before for hAChE. All dock-
ꢀ
ings were performed as blind dockings where a cube of, 75 A with
ꢀ
grid points separated 1 A, was positioned at the middle of the
protein (x ¼ 137.985; y ¼ 122.725; z ¼ 38.78). Default parameters
were used except num_modes, which was set to 40. In order to find
the best poses for compounds 6 and 8, the proper energies of the
docked poses along with the analysis of molecular feature of pro-
teineligand interactions, were selected as filter criteria to reject
other poses. Finally, the docking results generated were directly
loaded into Discovery Studio, version 2.1.
4.2.2. In vitro bloodebrain barrier permeation assay
Prediction of the brain penetration was evaluated using a par-
allel artificial membrane permeation assay (PAMPA), in a similar
manner as described previously [39,44,45]. Commercial drugs,
phosphate-buffered saline solution at pH 7.4 (PBS), and dodecane
were purchased from Sigma, Aldrich, Acros, and Fluka. Millex filter
units (PVDF membrane, diameter 25 mm, pore size 0.45 mm) were
Acknowledgments
acquired from Millipore. The porcine brain lipid (PBL) was obtained
from Avanti Polar Lipids. The donor microplate was a 96-well filter
A. Samadi thanks CSIC for JAE-Doc post-doctoral contract.
JMC thanks MICINN (SAF2009-07271; SAF2012-33304), and COST
EU (CM1103 Action) for support. M.I.R-F. acknowledges the finan-
cial support from the Spanish Ministry of Economy and Competi-
tiveness (SAF2012-31035), CSIC (PIE-201280E074), and Fundación
de Investigación Médica Mutua Madrileña Automovilística
(AP103952012). M.F.R. thanks CSIC for a PhD fellowship (JAE
Program).
plate (PVDF membrane, pore size 0.45
microplate was an indented 96-well plate, both from Millipore. The
acceptor 96-well microplate was filled with 180 L of PBS/ethanol
(7:3), and the filter surface of the donor microplate was impreg-
mm), and the acceptor
m
nated with
4 mL of porcine brain lipid (PBL) in dodecane
(20 mg mLꢁ1). Compounds 1e8 were dissolved in PBS/ethanol (7:3)
at 1 mg mL-1, filtered through a Millex filter, and then added to the
donor wells (180 mL). The donor filter plate was carefully put on the
acceptor plate to form a sandwich, which was left undisturbed for
4 h at 25 ꢃC. After incubation, the donor plate is carefully removed,
and the concentration of compounds in the acceptor wells was
determined by UV spectroscopy. Every sample is analyzed at five
wavelengths, in four wells, and at least in three independent runs,
and the results are given as the mean (standard deviation). In each
experiment, 15 quality control standards of known BBB perme-
ability were included to validate the analysis set.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.06.021.
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