Multipotent drugs with cholinergic and neuroprotective properties for the treatment of Alzheimer and neuronal vascular diseases. I. Synthesis, biological assessment, and molecular modeling of simple and readily available 2-aminopyridine-, and 2-chloropyridine-3,5-dicarbonitriles

Article abstract of DOI:10.1016/j.bmc.2010.06.095

The synthesis, molecular modeling, and pharmacological analysis of new multipotent simple, and readily available 2-aminopyridine-3,5-dicarbonitriles (3-20), and 2-chloropyridine-3,5-dicarbonitriles (21-28), prepared from 2-amino-6-chloropyridine-3,5-dicarbonitrile (1) and 2-amino-6-chloro-4- phenylpyridine-3,5-dicarbonitrile (2) is described. The biological evaluation showed that some of these molecules were modest inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), in the micromolar range. The 2-amino (3, 4), and 2-chloro derivatives 21-23, 25, 26 were AChE selective inhibitors, whereas 2-amino derivatives 5, 14 proved to be selective for BuChE. Only inhibitor 24 was equipotent for both cholinesterases. Kinetic studies on compound 23 showed that this compound is a mixed-type inhibitor of AChE showing a K_i of 6.33 μM. No clear SAR can be obtained form these data, but apparently, compounds bearing small groups such as the N,N′-dimethylamino or the pyrrolidino, regardless of the presence of a 2-amino, or 6-chloro substituent in the pyridine ring, preferentially inhibit AChE. Molecular modeling on inhibitors 4, 5, 22, and 23 has been carried out to give a better insight into the binding mode on the catalytic active site (CAS), and peripheral anionic site (PAS) of AChE. The most important differences in the observed binding relay on the modifications of the group at C2, as the amino group forms two hydrogen bonds that direct the binding mode, while in the case of compounds with a chlorine atom, this is not possible. The neuroprotective profile of these molecules has been investigated. In the LDH test, only compounds 26, 3, 22, and 24 showed neuroprotection with values in the range 37.8-31.6% in SH-SY5Y neuroblastoma cells stressed with a mixture of oligomycin-A/rotenone, but in the MTT test only compound 17 (32.9%) showed a similar profile. Consequently, these compounds can be considered as attractive multipotent therapeutic molecules on two key pharmacological receptors playing key roles in the progress of Alzheimer, that is, cholinergic dysfunction and oxidative stress, and neuronal vascular diseases.

Full text of DOI:10.1016/j.bmc.2010.06.095

Bioorganic & Medicinal Chemistry 18 (2010) 5861–5872
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Multipotent drugs with cholinergic and neuroprotective properties for the
treatment of Alzheimer and neuronal vascular diseases. I. Synthesis,
biological assessment, and molecular modeling of simple and readily
available 2-aminopyridine-, and 2-chloropyridine-3,5-dicarbonitriles
a
a
a
Abdelouahid Samadi a, José Marco-Contelles a, , Elena Soriano , Mónica Álvarez-Pérez , Mourad Chioua ,
Alejandro Romero b, Laura González-Lafuente b, Luis Gandía b, José M. Roda c, Manuela G. López b,
Mercedes Villarroya b, Antonio G. García b,d, Cristóbal de los Ríos b
*
a Laboratorio de Radicales Libres y Química Computacional (IQOG, CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain
b Instituto Teófilo Hernando, and Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo 4, 28029 Madrid, Spain
c Unidad de Investigación Cerebrovascular, Cirugía Experimental, Hospital Universitario La Paz, Madrid, Spain
d Servicio de Farmacología Clinica, Hospital Universitario de la Princesa, c/Diego de León 62, 28006 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis, molecular modeling, and pharmacological analysis of new multipotent simple, and readily
available 2-aminopyridine-3,5-dicarbonitriles (3–20), and 2-chloropyridine-3,5-dicarbonitriles (21–28),
prepared from 2-amino-6-chloropyridine-3,5-dicarbonitrile (1) and 2-amino-6-chloro-4-phenylpyri-
dine-3,5-dicarbonitrile (2) is described. The biological evaluation showed that some of these molecules
were modest inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), in the micro-
molar range. The 2-amino (3, 4), and 2-chloro derivatives 21–23, 25, 26 were AChE selective inhibitors,
whereas 2-amino derivatives 5, 14 proved to be selective for BuChE. Only inhibitor 24 was equipotent for
both cholinesterases. Kinetic studies on compound 23 showed that this compound is a mixed-type inhib-
Received 24 March 2010
Revised 22 June 2010
Accepted 29 June 2010
Available online 3 July 2010
Keywords:
2-Aminopyridine
2-Chloropyridines
AChE
itor of AChE showing a Ki of 6.33 lM. No clear SAR can be obtained form these data, but apparently, com-
pounds bearing small groups such as the N,N0-dimethylamino or the pyrrolidino, regardless of the
presence of a 2-amino, or 6-chloro substituent in the pyridine ring, preferentially inhibit AChE. Molecular
modeling on inhibitors 4, 5, 22, and 23 has been carried out to give a better insight into the binding mode
on the catalytic active site (CAS), and peripheral anionic site (PAS) of AChE. The most important differ-
ences in the observed binding relay on the modifications of the group at C2, as the amino group forms
two hydrogen bonds that direct the binding mode, while in the case of compounds with a chlorine atom,
this is not possible. The neuroprotective profile of these molecules has been investigated. In the LDH test,
only compounds 26, 3, 22, and 24 showed neuroprotection with values in the range 37.8–31.6% in
SH-SY5Y neuroblastoma cells stressed with a mixture of oligomycin-A/rotenone, but in the MTT test only
compound 17 (32.9%) showed a similar profile. Consequently, these compounds can be considered as
attractive multipotent therapeutic molecules on two key pharmacological receptors playing key roles
in the progress of Alzheimer, that is, cholinergic dysfunction and oxidative stress, and neuronal vascular
diseases.
BuChE
Kinetic analysis
Inhibition mechanism
Neuroprotection
Molecular modeling
Alzheimer’s disease
Neuronal vascular diseases
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
gation, oxidative stress or low levels of acetylcholine3 are thought
to play significant roles in the pathology of the disease.4 In spite of
Alzheimer’s disease (AD) is an age-related neurodegenerative
process characterized by a progressive loss of cognitive abilities,
such as memory, language skills, disorientation, attention, and
depression.1 Although the etiology of AD is still poorly understood,
the enormous research effort, an efficient strategy for designing
new drugs for the treatment of AD is still lacking.
The cholinergic theory1 suggests that the selective loss of cho-
linergic neurons in AD results in a deficit of acetylcholine (ACh)
in specific regions of the brain that mediate learning and memory
functions.5 Consequently, three acetylcholinesterase (AChE) inhib-
itors have been approved for commercial use. Thus, donepezil, riv-
astigmine, and galanthamine are known to improve AD symptoms
several factors such as amyloid-b (Ab)2 deposits,
s-protein aggre-
* Corresponding author. Tel.: +34 91 5622900; fax: +34 91 5644853.
E-mail address: iqoc21@iqog.csic.es (J. Marco-Contelles).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.095

5862
A. Samadi et al. / Bioorg. Med. Chem. 18 (2010) 5861–5872
by inhibiting AChE, that is, the enzyme responsible for the hydro-
lysis of ACh, thereby raising the levels of ACh in the synaptic cleft.6
Recently, a renewed interest for AChE inhibitors has been stimu-
lated by the potential role of AChE in accelerating the formation
of amyloid fibrils in the brain and forming stable complexes with
Ab.7 This role involves the peripheral anionic binding site (PAS)
of AChE, as noted by the fact that propidium iodide, a potent AChEI
agent binding specifically to the PAS, affects Ab aggregation in
vitro, whereas other catalytic active site (CAS) inhibitors such as
tacrine have not a similar effect.8
the in vitro pharmacology of the enantiomers as well as the in vivo
animal models are now being investigated.29b
In this context, and in order to validate the proposed binding
mode,29a and to simplify the structure of the lead compound
(RL2/101), we have now carried out the synthesis, the biological
evaluation, and molecular modeling of a number of 2-aminopyri-
dine-3,5-dicarbonitriles (I), and 2-chloropyridine-3,5-dicarbonitr-
iles (II) (Chart 2), as two new families of very simple, extremely
easy available molecules, from which we have identified com-
pounds showing moderate, but selective AChE versus BuChE inhib-
itory activity, and a significant neuroprotective profile. In this
report we describe these new results.
The multifactorial nature of AD supports new therapeutic strate-
gies. The most current innovative therapeutic approach is based on
the ‘one molecule, multiple targets’ paradigm.9–13 Thus, the multi-
potent approach14 includes novel tacrine–melatonin hybrids,15 dual
inhibitors of AChE and monoamine oxidase16 or serotonin transport-
ers,17 potent cholinesterase inhibitors with antioxidant and neuro-
protective properties,18 gallamine–tacrine hybrids binding at
cholinesterases and M2 muscarinic receptors,19 NO-donor–tacrine
hybrids as hepatoprotective drugs focusing on AD20a or fluorescent
tacrine–coumarin hybrids.20b
2. Results and discussion
2.1. Chemistry
The synthesis of 2-aminopyridine-3,5-dicarbonitrile (I), and 2-
chloropyridine-3,5-dicarbonitrile (II) type of compounds has been
carried out starting from the readily available 2-amino-6-chloro-
pyridine-3,5-dicarbonitrile (1)30 and 2-amino-6-chloro-4-phenyl-
pyridine-3,5-dicarbonitrile (2).31 Compounds 1 and 2 are very
well functionalized for the synthesis of molecules of type I and
II, based on presumably simple aromatic nucleophilic substitution
for incorporating new substituents at C6, followed by transforma-
tion of the amino into the chloro group (Chart 2).
The reaction of compounds 1 and 2 with several selected com-
mercially available amines (dimethylamine, pyrrolidine, piperidine,
7-azabicyclo[2.2.1]heptane,32 4-hydroxypiperidine, ethanolamine,
propargylamine, 4-benzylpiperidine, and benzylamine), gave the
expected C6 substituted 2-aminopyridine-3,5-dicarbonitriles (3,
4,33 5,33 6–11, 12,34 13–15) (Table 1).
Similarly, the reaction of 2-amino-6-chloropyridine-3,5-dicar-
bonitrile (1)30 with sodium methoxide in methanol gave the
known compound 1635 (Table 2). For the synthesis of compounds
17 and 18, 2-amino-6-methoxy-pyridine-3,5-dicarbonitrile (16)
was reacted with Na/EtOH, and 3-phenylpropanol with Na in
DMF, respectively (see Section 4).
2-Amino-4-phenyl-6-(phenylthio)pyridine-3,5-dicarbonitrile
(19) and 2-amino-6-mercapto-4-phenylpyridine-3,5-dicarbonitrile
(20) (Chart 3) were prepared as described in the literature.37
The C6 substituted 2-aminopyridine-3,5-dicarbonitriles have
been transformed into the corresponding new 2-chloropyridine-
3,5-dicarbonitriles (21–28) (Table 3) by Sandmeyer reaction38
using the conditions reported by Doyle.39
On the other hand, it is known that Ca2+ overload is the main
factor that triggers the processes leading to cell death. Thus, it
has been shown that calcium dysfunction, involved in the patho-
genesis of AD, augments Ab formation and
s hyperphosphoryla-
tion.21,22 Moreover, calcium entry through L-type Ca2+ channels
(Cav 1.1–1.4) causes both calcium overload and mitochondrial dis-
ruption, which leads to the activation of the apoptotic cascade and
cell death.23 In fact, nimodipine, a neuronal L-type Ca2+ channel
blocker, protects neurons from death evoked by focal cerebral
ischemia.24 Since 1,4-dihydropyridines (DHPs) are compounds that
selectively block L-type voltage-dependent Ca2+ channels (VDCCs),
hybrid molecules that combine an AChEI and a DHP, such as ta-
crine and nimodipine (Chart 1), might represent a promising ap-
proach to the treatment of AD. Support to this therapeutic
strategy comes from the fact that bis(7)-tacrine attenuates Ab-elic-
ited neuronal apoptosis by regulating L-type calcium channels.25
Besides inhibition of AChE and blockade of VDCCs, compounds able
to prevent the oxidative stress might have increased therapeutical
value, since oxidative damage precedes the appearance of other
pathological hallmarks of AD.26–28
Based on these precedents, some years ago we have embarked
in a project targeted to the synthesis of multipotent molecules able
to interact with the cholinergic system, showing also L-type Ca2+
channel antagonism effect. As a result, we have reported the chem-
istry and pharmacology of tacripyrines (Chart 1),29 and identified a
new lead compound such as ‘p-methoxytacripyrine’ (RL2/101)
(Chart 1) as a potent and selective AChE versus BuChE and
mixed-type inhibitor, binding preferentially at the PAS of AChE.
Accordingly, RL2/101 interferes with the pro-aggregation Ab effect
of human AChE (hAChE), and is a mild inhibitor of self-aggregation
of Ab42. In addition, RL2/101 is a potent Ca2+antagonist that per-
meates the blood–brain barrier (BBB) and displays neuroprotective
and antioxidant properties.29a The racemate has been resolved, and
The new compounds have been conveniently characterized by
their analytical and NMR spectroscopic data (see Section 4).
2.2. Pharmacology and computational chemistry
2.2.1. AChE/BuChE inhibitory activity
2-Amino-3,5-dicyanopyridines (3–20) and 2-chloro-3,5-dicy-
anopyridines (21–28) were evaluated as inhibitors of AChE from
OMe
O2N
OMe
O
O
NH2
NH2
EtO2C
O
O
N
H
N
N
H
N
Tacrine
Nimodipine
" -metoxytacripyrine"
p
(RL2/101)
Chart 1. Tacripyridines as hybrid molecules based on the juxtaposition of tacrine and nimodipine.

A. Samadi et al. / Bioorg. Med. Chem. 18 (2010) 5861–5872
5863
R
N
R
R
NC
X
CN
NC
Cl
CN
NC
X
CN
Cl
"nucleophile"
Sandmeyer-type
reaction
N
NH2
NH2
N
1 R = H
2 R = Ph
I
II
(R= H, Ph;
X= NR1R2, OR, SR)
(R= H, Ph;
X= NR1R2)
Chart 2. The target 2-aminopyridine-(I), and 2-chloropyridine-3,5-dicarbonitriles (II).
Table 1
Synthesis of 6-N-substituted 2-aminopyridine-3,5-carbonitriles (3–18)
Ph
NC
RS
CN
R
R
NC
Cl
CN
NC
X
CN
N
NH2
XH, Et3N
THF/EtOH
19
R= Ph; 20 R= H
N
NH2
N
NH2
1 R = H
3 15
-
Chart 3.
2 R = Ph
Entry
a
Product(ref)
R
X
Yield (%)
Table 3
Synthesis of 6-N-substituted 2-chloropyridine-3,5-dicarbonitriles (21–28)
3
H
Me2N
98
95
N
433
H
H
R
R
b
c
ONO
NC
X
CN
Cl
NC
X
CN
N
533
98
90
CuCl2
N
N
NH2
N
MeCN, 65 ºC
d
e
6
7
H
H
21 28
-
1 R = H
2 R = Ph
N
99
Entry
a
Product
R
X
Yield (%)
HO
HO
21
H
Me2N
56
84
f
8
9
H
H
99
99
NH
NH
N
b
c
22
23
H
H
g
N
72
66
NH
h
i
10
H
97
75
62
N
11
Ph
Ph
Me2N
d
24
H
N
j
1234
e
f
25
26
Ph
Ph
Me2N
67
64
N
1334
Ph
97
N
k
N
N
g
27
28
Ph
Ph
60
50
l
14
15
Ph
Ph
16
91
N
m
BnNH2
h
Electrophorus electricus (Ee), and BuChE from equine serum, accord-
ing to Ellman’s protocol.40a
From the IC50 data shown in Tables 4 and 5, we conclude that
most compounds in these series were inactive, the 2-amino-3,5-
dicyanopyridines were in general less potent than the 2-chloro-
3,5-dicyanopyridines, and between those compounds being active
for AChE or BuChE inhibition, that is, 3–5, 14; 21–23, 25, 26, a
Table 2
Synthesis of 6-O-substituted 2-aminopyridine-3,5-dicarbonitriles (16–18)
NC
Cl
CN
NC
CN
NC
X
CN
NaOMe
MeOH
XOH, Na
N
NH2
EtOH or DMF
MeO
N
NH2
N
NH2
1
16
17 18
,
Entry
Product
X
Yield (%)
a
b
1635
1736
MeO
EtO
96
91
O
c
18
76

5864
A. Samadi et al. / Bioorg. Med. Chem. 18 (2010) 5861–5872
Table 4
Inhibition of AChE from Electrophorus electricus (EeAChE) and equine serum butyrylcholinesterase (eqBuCEh) by 2-aminopyridine-3,5-dicarbonitriles (3–20)a
R
NC
CN
X
N
NH2
Entry
a
Product
R
X
IC50
(
l
M) EeAChE
IC50 (lM) eqBuChE
3
H
Me2N
22.7 0.9
3.5 0.2
>100
>100
N
b
c
4
5
H
H
N
>100
>100
26
1
N
d
e
6
7
H
H
>100
N
>100
>100
HO
HO
f
8
9
H
H
>100
>100
>100
>100
NH
NH
g
N
h
i
10
11
12
H
>100
>100
>100
>100
>100
>100
Ph
Ph
Me2N
N
j
N
k
l
13
14
Ph
Ph
>100
>100
>100
N
3.8 0.5
m
n
o
15
16
17
Ph
H
H
BnNH2
MeO
EtO
>100
>100
>100
>100
>100
>100
O
p
18
H
>100
>100
q
r
19
20
Ph
Ph
PhS
HS
>100
>100
>100
>100
a
Data are expressed as mean SEM of at least three different experiments in quadruplicate.
Table 5
complete selectivity was observed regarding one of the cholinester-
ases. The 2-amino-3, 4, and 2-chloro-derivatives 21–23, 25, 26 were
AChE selective inhibitors, whereas 2-amino derivatives 5, 14 proved
to be selective for BuChE. Compound 24 (Table 5) was the only
inhibitor that was equipotent for AChE/BuChE. When active, they
were modest cholinesterase inhibitors, in the micromolar range.
From these results, several structure–activity relationships
(SAR) can be established as follows. In the 2-amino-3,5-dicyano-
pyridine family (Table 4), and for those bearing an hydrogen as
the substituent at C4, only compounds 3, 4, and 5 were active, with
4 being the most potent inhibitor, showing also a complete selec-
Inhibition of AChE from Electrophorus electricus (EeAChE) and equine serum butyr-
ylcholinesterase (eqBuCEh) by 2-chloropyridine-3,5-dicarbonitriles (21–28)a
R
NC
X
CN
Cl
N
Entry Product
R
X
IC50 (lM) EeAChE IC50 (lM) eqBuChE
a
21
22
H
Me2N
5.0 0.8
0.8 0.1
>100
N
b
H
H
>100
>100
N
tivity for AChE inhibition (IC50 for AChE 3.5 lM, and >100 lM for
c
23
24
9.5 0.4
BuChE). The incorporation of a second ‘fused-five-membered ring
system’ into the active N-substituted pyrrolidine derivative 4, or
the addition of a hydroxyl group (or a phenylmethyl chain) at C40
in 5, resulted in inactive molecules such as 6, 7, and 10, respec-
tively (Table 4). We have also observed that the N-substitution
produced also inactive molecules (see compounds 8 and 9 in Table
4). Curiously, the incorporation of a phenyl group at C4 in inhibi-
tors 3, 4, and 5 dramatically reduced the inhibition, as none of
the resulting molecules 11–13 was active; conversely, the inactive
molecule 6 becomes now the moderate and specific BuChE inhibi-
tor 14 (Table 4). Finally, the N-benzylated derivative 15, the oxy-
genated 16–18, or the sulfur-containing molecules 19, 20 lacked
any cholinergic activity (Table 4).
N
d
H
75
10
3
2
24
2
e
f
25
26
Ph Me2N
>100
>100
N
Ph
5.6 0.9
N
g
27
28
Ph
>100
>100
>100
N
h
Ph
>100
a
Data are expressed as mean SEM of at least three different experiments in
quadruplicate.

A. Samadi et al. / Bioorg. Med. Chem. 18 (2010) 5861–5872
5865
For the 2-chloro-3,5-dicyanopyridines, most of the compounds
were active, and completely selective for AChE (Table 5). Com-
pound 22 was the most potent inhibitor with a N-pyrrolidine nu-
mine has Ki of 428 nM,40b and a KI infinite, while for propidium
iodide, a cholinergic agent binding specifically at PAS, the values
are Ki = 8.6
lM, and KI = 8.9 l
M.40c
cleus at C6 (IC50: 0.8 lM). Only products 27 and 28, bearing a
phenyl at C4, and piperidine or a 7-azabiclo[2.2.1]heptane substi-
2.2.3. Molecular modeling
tuent at C6, showed no inhibition at all.
In order to carry out the docking analyses, an area of EeAChE
that included both CAS and PAS was selected in order to evaluate
the relative binding at both active sites. Each docking analysis gave
100 solutions. Consequently, from the resulting values we can con-
clude that preferential binding will be observed for the active site
with the most populated cluster. In Table 6 we show the energy
score and the cluster populations for the docking solutions, at
CAS and PAS, for compounds 4 (R = H; R1 = NH2; XH = pyrrolidine),
5 (R = H; R1 = NH2; XH = piperidine), 23 (R = H; R1 = Cl; XH = piper-
idine), and 22 (R = H; R1 = Cl; XH = pyrrolidine).
The comparison between 2-amino-3,5-dicyanopyridines
(Table 4) and 2-chloro-3,5-dicyanopyridines (Table 5) seems appro-
priate. For those bearing an hydrogen at C4, compounds 21 (C6:
NMe2) and 22 (C6: pyrrolidine) are more potent at AChE than their
analogous 3 and 4. Similarly, for those with a phenyl ring at C4, com-
pounds 25(C6: NMe2) and 26 (C6: pyrrolidine)are active, while their
analogues 11 and 12 were not. Now, compounds 27 and 13 are inac-
tive, as well as compound 28, while its analogue 14 was active and
selective for BuChE.
To sum up, no clear SAR can be obtained from these data, but
apparently, compounds bearing small groups such as the N,N0-
dimethylamino or the pyrrolidino, regardless of the presence of a
2-amino, or 6-chloro substituent in the pyridine ring, do inhibit
AChE.
As shown from these data, the population cluster for inhibitors
4, 23 and 22 were the highest in the CAS, with values equal to 99,
94, and 93, respectively.
Thus, we have selected compound 4 as a model, and in Figure 1
we show its putative binding in the CAS and PAS. In the CAS, the aro-
Consequently, to find possible answers to this puzzling situa-
tion, we have carried out the kinetics of the inhibition for the active
compound 23, and docking analyses on the active molecules using
the automated program Autodock (see below).
matic ring establishes p-stacking interactions with Trp86 and
Tyr337, whereas the amino group forms two hydrogen bonds with
Asp74 and Tyr341. This orientation allows the pyrrolidine ring to
be well accommodated in the lipophilic pocket formed by Trp439,
Met443, Pro446, and Tyr449 (Fig. 1).
In the case of inhibitor 5, bearing a piperidine ring the steric
interactions are more important and likely prevent a better binding
at the CAS.
The ligand in most populated clusters for compound 8 shows a
hydrogen bond between the hydroxyl group and the pyridinic
nitrogen in a pseudo-seven membered cycle, that prevents an easy
accommodation in the hydrophobic pocket.
In the PAS, inhibitor 4 forms two hydrogen bonds with Asp74
and Tyr72, the aromatic ring laying between the indole ring of
Trp286, and the phenol of Tyr341. The pyrrolidine ring points to
the inside gorge of the enzyme, while the nitrile groups are ori-
ented towards the outside part of enzyme, and should be solvated
(Fig. 1).
2.2.2. Kinetic study of AChE inhibition by compound 23
The mechanism of inhibition of EeAChE was investigated with
compound 23 (Table 5), a moderate inhibitor of EeAChE (Table
5). The inhibitory constant, Ki (the inhibition constant for
competitive inhibition) was determined from the analysis of
Lineweaver–Burk plots, which show both increasing slopes (lower
Vmax) and intercepts (higher Km) with increasing inhibitor concen-
tration, indicating mixed-type inhibition. The graphical analysis of
steady-state inhibition data for inhibitor 23 is shown in Chart 4. A
Ki of 6.33
concentration of 23. A KI, defined as the inhibition constant for the
noncompetitive inhibition, of 17.2 M was determined from rep-
lM was estimated from the plots of the slope versus the
l
lotting intercepts of Lineweaver–Burk versus concentration of
inhibitor. As this value was very close to that found for Ki, we pro-
pose that compound 23 would show a mixed-type inhibition with
a slight preference for the binding to the PAS of AChE. For compar-
ative purposes, note that a competitive inhibitor such as galantha-
For inhibitor 23, bearing a chlorine atom at C2, and a piperidine
ring at C6, in the CAS, the absence of the amino group results in a
new binding mode based on stereoelectronic interactions. Accord-
ingly, no interactions are now observed with Asp74 and Tyr341,
but p-stacking interactions with Trp86 and Tyr337 are still possi-
50
ble, allowing the piperidine ring to be accommodated out of the
hydrophobic pockets where the steric interactions are less
demanding (Fig. 2).
Control
40
30
20
10
0
In the PAS, the aromatic ring is located between Trp286 y
1uM
Tyr341, establishing stabilizing
p-stacking interactions, whereas
3uM
10uM
30uM
90uM
the piperidine ring points to the bottom of the AChE gorge. How-
ever, and comparing with inhibitor 4, the absence of an amino
group, being substituted by a chlorine atom in 23, allows the Cl
atom to be oriented to the outer side of the enzyme showing sta-
bilizing interactions by solvation (Fig. 3).
To sum up, the most important differences in the binding in the
active sites relay on the modifications of the group at C2: the ami-
no group forms two hydrogen bonds that direct the binding mode,
while in the case of compounds with a chlorine atom, this is not
possible.
2.2.4. Neuroprotective effect of compounds 3–28 against
rotenone/oligomycin-A-induced death of human neuro-
blastoma SH-SY5Y cells
-4000 -2000
0
2000 4000 6000 8000 10000
[ATCh]-1 (M-1)
The mixture of rotenone plus oligomycin-A (rot/olig) blocks
mitochondrial electron transport chain complexes I and V, respec-
tively,41 inducing cell death by oxidative stress. The neuroprotec-
tive effect of compounds 3–28 against this toxic stimulus was
Chart 4. Steady-state inhibition of acetylcholinesterase (AChE) hydrolysis of
acetylthiocholine (ATCh) by compound 23. Lineweaver–Burk reciprocal plots of
initial velocity and substrate concentrations are presented. Lines were derived from
a weighted least-squares analysis of data points.

5866
A. Samadi et al. / Bioorg. Med. Chem. 18 (2010) 5861–5872
Table 6
The energy scores and cluster populations for the docking solutions, at the CAS and PAS sites of acetylcholinesterase (AChE), for compounds 4, 5, 22 and 23
R
NC
X
CN
R1
N
Product
Catalytic site (CAS)
Peripheral anionic site (PAS)
Score (kcal/mol) Population of cluster (100)
Score (kcal/mol)
Population of cluster (100)
4
5
23
22
ꢀ3.84
ꢀ3.60
ꢀ4.82
ꢀ4.55
99
79
94
93
ꢀ3.50
ꢀ3.84
ꢀ4.29
ꢀ4.11
1
21
5
5
evaluated with the methods of LDH release42 and MTT metabolic
reduction43 methods, at the concentration of 1
M, in SH-SY5Y
neuroblastoma cells, exposed to 30 M rotenone plus 10 M oligo-
l
l
l
mycin-A for 24 h. As shown in Tables 7 and 8, the neuroprotective
Table 7
Neuroprotection induced by compounds 3–20 (1 lM) in human neuroblastoma cells
stressed with a mixture of rotenone/oligomycin-Aa
R
NC
X
CN
NH2
Figure 1. Binding of inhibitor 4 in the CAS and PAS of AChE. The ligand appears as
balls and sticks, with the carbon atoms in green.
N
Product
R
X
% Protection (at 1 lM)
LDH
36.2
MTT
12.9
3
4
H
H
Me2N
N
nd
20.1
10.2
N
5
6
H
H
14.8
N
nd
0
3
N
7
H
15.1
HO
HO
8
9
H
H
3
21.4
17.7
NH
NH
nd
Figure 2. Binding of inhibitor 23 in the CAS and PAS of AChE. The ligand appears as
N
balls and sticks, with the carbon atoms in pink.
10
H
nd
19.1
N
11
12
Ph
Ph
nd
0
N
N
13.1
11.1
13
14
Ph
Ph
10.7
nd
2.7
0.7
N
NH
15
Ph
nd
10.6
16
17
H
H
MeO
EtO
nd
10.9
11
32.9
O
18
H
nd
0
S
19
20
Ph
Ph
3.84
14.1
11.2
5.8
SH
*
p <0.05. nd = not determined.
Figure 3. Superposition of the preferred ligand orientation for inhibitor 4 (X = NH2),
and 23 (X = Cl) in the PAS, showing the part of the ligand that points to the outer
site of enzyme.
a
Each experiment has been repeated three times. The results are the averaged
values.

A. Samadi et al. / Bioorg. Med. Chem. 18 (2010) 5861–5872
5867
Table 8
23 have been carried out to give a better insight into the binding
mode of the active molecules in the CAS/PAS active sites of AChE.
The most important differences in binding to active sites relay on
the modifications of the group at C2, as the amino group forms
two hydrogen bonds that direct the binding mode, while in the
case of compounds with a chlorine atom, this is not possible. Cho-
linergic inhibitors 3, 17, 22, 24, and 26 showed also the highest
neuroprotection values in the LDH/MTT tests for the rotenone/oli-
gomycin-A assay, affording neuroprotection around 30%. Conse-
quently, these compounds might be further developed to
attractive multipotent molecules acting on two key pharmacolog-
ical mechanisms having a relevant role in the pathogenesis of Alz-
heimer’s disease, that is, cholinergic dysfunction and oxidative
stress, and neuronal vascular diseases.
Neuroprotection induced by compounds 21–28 (1 lM) in human neuroblastoma cells
stressed with the combination of rotenone/oligomycin-Aa
R
NC
X
CN
Cl
N
Product
R
X
% Protection (at 1 lM)
LDH
nd
MTT
6
21
22
H
H
Me2N
N
32.0
nd
7.5
N
23
24
H
H
14.7
N
31.6
7.5
4. Experimental part
4.1. General methods
25
26
Ph
Ph
Me2N
nd
0
N
37.7
7.5
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 ref-
erence. The assignment of chemical shifts is based on standard
NMR experiments (1H, 13C-DEPT, 1H, 1H-COSY, 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 detec-
tion by UV light at 254 nm or by charring with either ninhydrin,
anisaldehyde or phosphomolybdic-H2SO4 dyeing reagents. Anhy-
drous solvents were used in all experiments. Column chromatogra-
phy was performed on Silica Gel 60 (230 mesh).
N
27
28
Ph
Ph
nd
nd
0
N
12.9
*
p <0.05. nd = not determined.
a
Each experiment has been repeated three times. The results are the averaged
values.
profile of the target molecules ranged from moderate to low for
both LDH and MTT determinations. In the LDH test, compounds
26, 3, 22, and 24 were the ones that showed more neuroprotection
with values in the range 32–38%. In the MTT test only compound
17 (32.9%) showed a similar profile. This difference could be due
to the fact that LDH is measuring necrotic cell death, while MTT
is measuring mitochondrial damage and apoptotic cell death.
Regarding the families of compounds, it seems again, as observed
for the cholinergic inhibition, that the 2-chloropyridine-3,5-dicar-
bonitriles are more potent than the 2-aminopyridine-3,5-dicarbo-
nitriles. In particular, the selective AChE inhibitors 3, 22, 24, and
26 showed also the highest neuroprotection values in the LDH
experiment, whereas the selective BuChE inhibitor 5 showed a
modest neuroprotective profile. Conversely, the inactive choliner-
gic compound 18 showed modest activity in the MTT assay
(32.9%) and low activity in the LDH test (10.9%).
4.1.1. General procedure for the synthesis of 6-N-substituted
2-amino-3,5-dicyanopyridines
The corresponding 6-amino-2-chloropyridine-3,5-dicarboni-
trile (1 or 2) was suspended in a 3:1 mixture of THF/EtOH
(15 mL/mmol of starting pyridine). An excess of the secondary
amine (see Table 1, entries a–m) was then added, followed by tri-
ethylamine (1.0 equiv). The mixture was then heated under reflux.
After cooling, the solvent was removed in vacuum and the result-
ing crude submitted to flash column chromatography, to give com-
pounds 3–15.
4.1.1.1. 2-Amino-6-dimethylamino-pyridine-3,5-dicarbonitrile
(3). Following the general procedure, reaction of 6-amino-2-chlo-
ropyridine-3,5-dicarbonitrile (1) (0.4 g, 1.685 mmol), with dimeth-
ylamine (33% in ethanol) (0.8 mL) in THF/EtOH (10:5, 15 mL), after
1 h, gave product 3 (0.305 g, 98%) as a white solid: Rf = 0.52 (CH2C2/
3. Conclusions
In this work we have synthesized a series of simple, and readily
available 2-aminopyridine-3,5-dicarbonitriles (3–22), and 2-chlo-
ropyridine-3,5-dicarbonitriles (21–28) derived from 2-amino-6-
chloropyridine-3,5-dicarbonitrile (1) and 2-amino-6-chloro-4-
phenylpyridine-3,5-dicarbonitrile (2). The biological evaluation
showed that some of these molecules were modest AChE/BuChE
inhibitors, in the micromolar range; however, some compounds
were highly selective for AChE (i.e., 2-amino derivatives 3, 4; 2-
chloro derivatives 21–23, 25, 26), and for BuChE (2-amino deriva-
tives 5, 14); no inhibitor caused inhibition of both cholinesterases
AcOEt, 10:1, v/v); mp 170–172 °C; IR (KBr)
m
3408, 3350, 3240,
3037, 2212, 1666, 1600, 1549, 1509, 1484, 1423, 1403,
1286 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d 7.73 (s, 1H, CH), 5.28 (s,
2H, NH2), 3.30 (s, 6H, 2 ꢁ CH3); 13C NMR (75 MHz, CDCl3) d 159.1
(C6), 159.0 (C2), 149.9 (C4), 118.9, 116.5 (2 ꢁ CN), 81.3, 80.3 (C3,
C5), 40.3 (2 ꢁ CH3); MS (EI) m/z (%): 172 (61) [MꢀCH3]+, 187
(100) [M]+. Anal. Calcd for C9H9N5: C, 57.74; H, 4.85; N, 37.41.
Found: C, 57.61; H, 4.80; N, 37.24.
with IC50 of less than 50
proved that this is a mixed-type inhibitor showing a Ki of
6.33 M. No clear SAR can be obtained form these data, but it
lM. Kinetic studies on compound 23
4.1.1.2. 2-Amino-6-(pyrrolidin-10-yl)pyridine-3,5-dicarbonitrile
(4). Following the general procedure, reaction of 6-amino-2-chlo-
ropyridine-3,5-dicarbonitrile (1) (357 mg, 2 mmol) with pyrroli-
dine (0.2 mL, 2.4 mmol, 1.2 equiv) and NEt3 (0.28 mL, 2 mmol,
1 equiv) in THF/EtOH (3:1, 30 mL), after 90 min, gave the known
compound 433 (402 mg, 95%).
l
seems that compounds bearing small groups such as the N,N0-
dimethylamino or the pyrrolidino, regardless of the presence of a
2-amino, or 6-chloro substituent in the pyridine ring, preferentially
inhibit AChE. Docking analysis on selected inhibitors 4, 5, 22, and

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A. Samadi et al. / Bioorg. Med. Chem. 18 (2010) 5861–5872
4.1.1.3. 2-Amino-6-(piperidin-10-yl)pyridine-3,5-dicarbonitrile
(5). Following the general procedure, reaction of 6-amino-2-chlo-
ropyridine-3,5-dicarbonitrile (1) (0.3 g, 1.685 mmol) with piperi-
dine (0.33 mL, 3.37 mmol) in THF/ EtOH (10:5, 15 mL), after
90 min, gave compound 533 (0.375 g, 98%).
4.1.1.7. 2-Amino-6-propargylamine-pyridine-3,5-dicarbonitrile
(9). Following the general procedure, reaction of 6-amino-2-chlo-
ropyridine-3,5-dicarbonitrile (1) (0.36 g, 2.022 mmol) with propar-
gylamine (0.166 mL, 2.427 mmol) and triethylamine (0.3 mL) in
EtOH/THF (5:10, 15 mL), after 1 h, gave product 9 (0.394 g, 99%)
as a white solid: Rf = 0.21 (CH2C2/AcOEt, 10:1, v/v); mp 216–
4.1.1.4. 2-Amino-6-(70-azabicyclo[2.2.1]heptan-70-yl)pyridine-
3,5-dicarbonitrile (6). Following the general procedure, reaction
of 6-amino-2-chloropyridine-3,5-dicarbonitrile (1) (57 mg, 0.32
mmol) with 7-azabicyclo[2.2.1]heptane hydrochloride32 (52 mg,
0.39 mmol) and NEt3 (0.05 mL, 0.39 mmol, 1.2 equiv) in THF/EtOH
(3:1, 4.8 mL), after 36 h, and flash column chromatography (40%
hexane in DCM), gave compound 6 (69 mg, 90%) as a solid: mp
218 °C; IR (KBr)
m
3449, 3339, 3225, 2211, 1631, 1603, 1570,
1512, 1483 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d 7.69 (s, 1H, CH),
5.61 (s, 1H, NH), 5.46 (s, 2H, NH2), 4.26 (dd, J = 5.3, 2.5 Hz, 2H,
CH2), 2.29 (t, J = 2.5 Hz, 1H, C„CH); 13C NMR (75 MHz, DMSO-d6)
d 161.3 (C6), 158.9 (C2), 148.8 (C4), 117.4, 117.2 (2 ꢁ CN), 82.1,
80.2 (C3, C5), 79.3 (C„CH), 73.3 (C„CH), 30.4 (CH2); MS (ES) m/
z (%): 159 (75) [MꢀCH2C„CH+H]+, 198 (100) [M+H]+. Anal. Calcd
for C10H7N5: C, 60.91; H, 3.58; N, 35.51. Found: C, 60.68; H, 3.85;
N, 35.42.
189–191 °C; IR (KBr)
m 3469, 3347, 2208, 1627, 1599, 1527, 1460,
1360, 1138 cmꢀ1 1H NMR (CDCl3, 300 MHz) d 7.69 (s, 1H, H4),
;
5.39–5.18 (br s, 2H, NH2), 5.04 (s, 2H, H10, H40), 1.93–1.70 (m,
4H, H20, H30, H50, H60), 1.58–1.44 (m, 4H, H20, H30, H50, H60); 13C
NMR (CDCl3, 75 MHz) d 160.0 (C6), 157.9 (C2), 148.9 (C4), 118.4,
116.5 (2 ꢁ CN), 83.1, 81.0 (C3, C5), 57.6 (C10, C40), 29.4 (C20, C30,
C50, C60); MS (ES) m/z [M+1]+ 240.2/241.2. Anal. Calcd for
4.1.1.8. 2-Amino-6-(4-benzylpiperidin-10-yl)pyridine-3,5-dicar-
bonitrile (10). Following the general procedure, reaction of 6-
amino-2-chloropyridine-3,5-dicarbonitrile (1) (0.3 g, 1.685 mmol)
with 4-benzylpiperidine (0.6 mL, 3.371 mmol) in THF/EtOH (10:5,
15 mL), after 12 h, and flash column chromatography (CH2C2/
AcOEt, 20:1, v/v), afforded compound 10 (0.515 g, 97%): Rf = 0.60
C13H13N5: C, 65.25; H, 5.48; N, 29.27. Found: C, 64.98; H, 5.39; N,
29.04.
(CH2C2/AcOEt, 20:1, v/v); mp 107–109 °C; IR (KBr)
m
3439, 3341,
;
3227, 2930, 2844, 2214, 1599, 1529, 1494, 1456 cmꢀ1
1H NMR
4.1.1.5. 2-Amino-6-(4-hydroxy-piperidin-10-yl)pyridine-3,5-di-
carbonitrile (7). Following the general procedure, reaction of 6-
amino-2-chloropyridine-3,5-dicarbonitrile (1) (0.3 g, 1.685 mmol)
with 4-hydroxypiperidine (0.34 g, 3.371 mmol) in THF/EtOH (10:5,
15 mL), after 12 h, and flash column chromatography (CH2C2/MeOH,
8:1, v/v), gave compound 7 (0.405 g, 99%): Rf = 0.49 (CH2C2/MeOH,
(300 MHz, DMSO-d6) d 8.1 (s, 1H, CH), 7.36 (br s, 2H, NH2), 7.28
(m, 2H, Har), 7.18 (m, 3H, Har), 4.46 (d, J = 13.3 Hz, 2H, CH2),
2.93 (dt, J = 13.2, 2.21 Hz, 2H, CH2), 2.52 (d, J = 7.2 Hz, CH2-Ph),
1.81 (m, 1H, CH), 1.63 (dd, J = 13.2, 2.79, 2H, CH2), 1.2 (m, 2H,
CH2); 13C NMR (100 MHz, DMSO-d6) d 159.3 (C6), 159.0 (C2),
150.5 (C4), 139.9 (C), 129.0 (2 ꢁ CHph), 128.1 (2 ꢁ CHph), 125.8
(CHph), 118.4, 116.4 (2 ꢁ CN), 79.8, 79.7 (C3, C5), 46.9
(2 ꢁ CH2pip), 42.0 (CHpip), 37.34 (CHpip), 31.6 (2 ꢁ CH2pip); MS
(ES+), m/z (%): 211 (61) [MꢀBnꢀNH2]+; 224 (25) [MꢀBn]+; 317
(100) [M]+. Anal. Calcd for C19H19N5: C, 71.90; H, 6.03; N, 22.07.
Found: C, 71.79; H, 6.16; N, 22.20.
8:1, v/v); mp 176–177 °C; IR (KBr)
2939, 2923, 2219, 2196, 1645, 1592, 1529, 1488, 1474,
1457 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d 8.01 (s, CH), 7.38 (br,
m 3488, 3442, 3339, 3228,
;
2H, NH2), 4.79 (d, J = 4.5 Hz, 1H, OH), 4.09 (m, 2H, CH2), 3.75 (m,
1H, CH), 3.39 (m, 2H, CH2), 1.80 (m, 2H, CH2), 1.40 (m, 2H, CH2);
13C NMR (100 MHz, DMSO-d6) d 159.4 (C6), 158.9 (C2), 150.5 (C4),
118.4, 116.4 (2 ꢁ CN), 79.7, 79.6 (C3, C5), 65.2 (CHpip), 44.2
(2 ꢁ CH2 pip), 34.0 (2 ꢁ CH2 pip); MS (IE) (%): 243.1 (100) [M]+,
225 (40) [MꢀH2O]+. Anal. Calcd for C12H13N5O: C, 59.25; H, 5.39;
N, 28.79. Found: C, 59.27; H, 5.36; N, 28.57.
4.1.1.9. 2-Amino-6-dimethylamino-4-phenylpyridine-3,5-dicar-
bonitrile (11). Following the general procedure, reaction of 2-
amino-6-chloro-4-phenylpyridine-3,5-dicarbonitrile (2) (0.31 g,
1.217 mmol) with dimethylamine (33% in ethanol) (0.33 mL) in
THF/EtOH (10:5, 15 mL), after 1 h, give product 11 (0.24 g, 75%)
as a solid: Rf = 0.63 (CH2C2/AcOEt, 10:1, v/v); mp 251–253 °C; IR
4.1.1.6. 2-Amino-6-(2-hydroxyethylamine)pyridine-3,5-dicar-
bonitrile (8). Following the general procedure, reaction of 6-ami-
no-2-chloropyridine-3,5-dicarbonitrile (1) (0.3 g, 1.685 mmol)
with ethanolamine (0.2 mL, 3.371 mmol) in THF/EtOH (10:5,
15 mL), after 90 min, and flash column chromatography (CH2Cl2/
(KBr)
m
3473, 3320, 3221, 2210, 1624, 1586, 1570, 1550, 1515,
1491, 1420, 1400, 1228 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d 7.43–
7.54 (m, 5H, CH-ar), 735 (s, 2H, NH2), 3.21 (s, 6H, 2 ꢁ CH3); 13C
NMR (75 MHz, DMSO-d6) d 162.4 (C6), 160.6 (C2), 159.8 (C4),
135.9 (C), 130.3 (CH), 129.1 (2 ꢁ CH), 129.0 (2 ꢁ CH), 118.5,
116.8 (2 ꢁ CN), 81.0, 80.6 (C3, C5), 40.4 (2 ꢁ CH3); MS (EI) m/z
(%): 262 (110) [MꢀH]+, 263 (50) [M]+. Anal. Calcd for C15H13N5:
C, 68.42; H, 4.98; N, 26.60. Found: C, 68.19; H, 4.90; N, 26.31.
MeOH, 8:1, v/v), gave compound
(CH2C2/MeOH, 8:1, v/v); mp 180–182 °C; IR (KBr)
3229, 2944, 2207, 1638, 1609, 1568, 1512, 1489 cmꢀ1
8
(0.338 g, 99%): Rf = 0.55
3383, 3333,
1H NMR
m
;
(400 MHz, DMSO-d6) d 8.02 (s, 1H, CH), 7.33 (br s, 2H, NH2), 7.23
(t, J = 5.26 Hz, 1H, NH), 4.72 (t, J = 5.26 Hz, 1H, OH), 3.5 (m, 2H,
CH2), 3.42 (m, 2H, CH2); 13C NMR (100 MHz, DMSO-d6) d 160.7
(C6), 158.9 (C2), 147.9 (C4), 116.9, 116.8 (2 ꢁ CN), 79.5, 77.8 (C3,
C5), 59.2 (CH2), 43.1 (CH2); MS (ES) m/z (%): 143.0 (3)
[MꢀOH(CH2)2NH]+, 186.0 (7) [MꢀOH]+, 204.0 (100) [M+H]+, 205
(14) [M+2H]+, 266 (10) [M+Na]+. To a solution of compound 8
(50 mg, 1.685 mmol) in MeOH (10 mL) was added dropwise a re-
cently prepared solution of MeOH saturated with HCl. The mixture
was stirred at rt overnight. The precipitate was filtered, washed
with ether and dried to give compound 8. HCl: mp 183–184 °C;
1H NMR (300 MHz, DMSO-d6) d 8.022 (s, 1H, CH), 7.33 (br s, 2H,
NH2), 7.23 (m, 2H, Har), 4.36 (br s, 2H, OH+NH), 3.50 (t,
J = 5.2 Hz, 2H, CH2), 3.42 (t, J = 5.39 Hz, CH2). Anal. Calcd for
C9H10ClN5O: C, 45.10; H, 4.21; Cl, 14.79; N, 29.22. Found: C,
44.98; H, 4.42, Cl, 14.40; N, 29.16.
4.1.1.10. 2-Amino-6-(pyrrolidin-10-yl)-4-phenylpyridine-3,5-di-
carbonitrile (12). Following the general procedure, reaction of 6-
amino-2-chloro-4-phenylpyridine-3,5-dicarbonitrile (2) (509 mg,
2 mmol) with pyrrolidine (0.2 mL, 2.4 mmol, 1.2 equiv) and NEt3
(0.28 mL, 2 mmol, 1 equiv) in THF/EtOH (3:1, 30 mL), after
90 min, and flash column chromatography (100% DCM), gave
known product 1234 (360 mg, 62%).
4.1.1.11. 2-Amino-6-(piperidin-10-yl)-4-phenylpyridine-3,5-di-
carbonitrile (13). Following the general procedure, reaction of
2-amino-6-chloro-4-phenylpyridine-3,5-dicarbonitrile (2) (0.63 g,
2.48 mmol) with piperidine (0.37 mL, 3.72 mmol) in THF/ EtOH
(20:10, 30 mL), after 2 h, gave product 13 (0.726 g, 97%) as a solid:
mp 199–200 °C; IR (KBr)
m 3474, 3325, 3221, 2202, 1624, 1583,

A. Samadi et al. / Bioorg. Med. Chem. 18 (2010) 5861–5872
5869
1567, 1535, 1489, 1449, 1290, 1022 cmꢀ1
;
1H NMR (CDCl3,
(0.313 g, 2.198 mmol), and sodium (79 mg, 3.448 mmol) in dry
DMF (10 mL) was stirred at reflux for 2 h. Then, cooled water
was added and the precipitate was separated by filtration and
washed with water. The yellow solid was purified by column chro-
matography (CH2Cl2/AcOEt, 40:1, v/v), and recrystallized from eth-
anol to obtain compound 18 (0.227 g, 76%): Rf = 0.5 (CH2C2/AcOEt,
400 MHz) d 7.52–7.50 (m, 5H, Ph), 5.40 (br s, 2H, NH2), 3.79 (m,
4H, 2 ꢁ CH2, piperidine), 1.70 (m, 6H, 3 ꢁ CH2, piperidine); 13C
NMR (CDCl3, 100 MHz) d 162.1 (C6), 151.0 (C2), 159.3 (C4), 134.7
(C, Ph), 130.3 (CH, Ph), 128.6 (2 ꢁ CH, Ph), 128.5 (2 ꢁ CH, Ph),
117.6, 116.4 (2 CN), 83.4, 81.5 (C3, C5), 49.0 (2 ꢁ CH2, piperidine),
25.8 (2 ꢁ CH2, piperidine), 24.3 (CH2, piperidine); MS (ES) m/z (%):
304 (100) [M+H]+, 326 (79) [M+Na]+, 629 (8) [2M+Na]+. Anal. Calcd
for C18H17N5: C, 71.27; H, 5.65; N, 23.09. Found: C, 70.99; H, 5.54;
N, 22.93.
20:1, v/v); mp 140–142 °C; IR (KBr)
m 3433, 3329, 3231, 2219,
1657, 1607, 1544, 1476, 1452 cmꢀ1 1H NMR (400 MHz, CDCl3) d
;
7.82 (s, 1H, CH), 7.32–7.19 (m, 5H, CH-arom), 5.6 (s, 2H, NH2),
4.35 (t, J = 6.5 Hz, 2H, CH2), 2.78 (t, J = 7.7 Hz, 2H, CH2), 2.12 (m,
CH2); 13C NMR (100 MHz, CDCl3) d 165.7 (C6), 160.5 (C2), 147.3
(CH), 140.7 (C, Ph), 128.4 (2 ꢁ CH, Ph), 128.4 (2 ꢁ CH, Ph), 126.0
(CH, Ph), 115.2, 114.4 (2 ꢁ CN), 86.2, 83 (C3, C5), 67.14 (CH2),
31.71 (CH2), 92.82 (CH2); MS (IE), m/z (%): 91 (96), 118 (100),
160 (6) [MꢀPh(CH2)3]+; 278 (30) [M]+. Anal. Calcd for C16H14N4O:
C, 69.05; H, 5.07; N, 20.13. Found: C, 69.20; H, 5.18; N, 20.42.
4.1.1.12. 2-Amino-6-(70-azabicyclo[2.2.1]heptan-70-yl)-4-phen-
ylpyridine-3,5-dicarbonitrile (14). Following the general proce-
dure, reaction of 6-amino-2-chloro-4-phenylpyridine-3,5-dicarbo-
nitrile (2) (82 mg, 0.32 mmol) with 7-azabicyclo[2.2.1]heptane
hydrochloride32 (52 mg, 0.39 mmol) and NEt3 (0.05 mL, 0.39 mmol,
1.2 equiv) in THF/EtOH (3:1, 4.8 mL), after 36 h, and flash column
chromatography (1:1 hexane/DCM), gave compound 14 (16 mg,
4.1.2. General procedure for the synthesis of 2-chloro-3,5-
dicyanopyridines
16%), as a solid: mp 184–186 °C; IR (KBr)
m
3432, 3337, 3233,
2210, 1637, 1557, 1523, 1497, 1451 cmꢀ1
;
1H NMR (CDCl3,
The corresponding pyridine and CuCl2 (1.5 equiv) were dis-
solved in dry MeCN under argon. iso-Amylnitrite (1.5 equiv) was
added, and the mixture heated at 65 °C until TLC analysis showed
complete reaction. The solution was acidified (2 N HCl) to pH 3 and
extracted with DCM. After drying the organic layer with Na2SO4,
the solvent was removed in vacuum and the crude product purified
by flash column chromatography using hexane–DCM mixtures.
300 MHz) d 7.61–7.42 (m, 5H, Ph), 5.45–5.27 (br s, 2H, NH2), 5.08
(s, 2H, H10, H40), 1.97–1.79 (m, 4H, H20, H30, H50, H60), 1.60–1.48
(m, 4H, H20, H30, H50, H60); 13C NMR (CDCl3, 75 MHz) d 161.7
(C6), 160.0 (C2), 158.9 (C4), 134.7 (C, Ph), 130.5, 128.9, 128.6
(CH, Ph), 117.8, 116.6 (2 ꢁ CN), 84.1, 82.2 (C3, C5), 57.9 (C10, C40),
29.3 (C20, C30, C50, C60); MS (ES) m/z [M+1]+ 316.0/317.0, [M+23]+
338.0/339.0. Anal. Calcd for C19H17N5: C, 72.36; H, 5.43; N, 22.21.
Found: C, 72.08; H, 5.59; N, 21.99.
4.1.2.1. 2-Chloro-6-(dimethylamino)pyridine-3,5-dicarbonitrile
(21). Following the general procedure, reaction of 2-amino-6-
(dimethylamino)pyridine-3,5-dicarbonitrile (3) (0.187 g, 1 mmol)
with CuCl2 (0.2 g, 1.5 mmol) and iso-amylnitrite (0.2 mL, 1.5 mmol),
in acetonitrile (8 mL), after 2 h, gave product 21 (0.115 g, 56%) as a
4.1.1.13. 2-Amino-6-benzylamino-4-phenylpyridine-3,5-dicar-
bonitrile (15). Following the general procedure, reaction of 2-
amino-6-chloro-4-phenylpyridine-3,5-dicarbonitrile (2) (0.305 g,
1.2 mmol) with benzylamine (0.17 mL, 1.56 mmol) and triethyl-
amine (0.5 mL, 3.6 mmol) in THF/EtOH (10:5, 15 mL), after 2 h,
gave compound 15 (0.355 g, 91%) as solid: mp 180–182 °C; IR
solid: mp 155–157 °C; IR (KBr)
m
2226, 1599, 1559, 1494, 1424,
1407, 1280 cmꢀ1
;
1H NMR (CDCl3, 300 MHz) d 7.91 (s, 1H, H-4),
3.41 (s, 6H, 2 ꢁ CH3); 13C NMR (CDCl3, 75 MHz) d 157.1 (C6), 154.2
(C2), 149.8 (C4), 116.2, 114.4 (2 ꢁ CN), 96.5 (C5), 88.7 (C3), 40.4
(2 ꢁ CH3); MS (EI) m/z (%) 177 (39), 191 (51) [MꢀCH3]+, 206 (37)
[M]+. Anal. Calcd for C9H7ClN4: C, 52.31; H, 3.41; Cl, 17.16; N,
27.11. Found: C, 52.60; H, 3.54; Cl, 16.80; N, 27.02.
(KBr)
m ;
3463, 3338, 2203, 1625, 1587, 1563 cmꢀ1 1H NMR (CDCl3,
400 MHz) d 7.52 (s, 5H, Ph), 7.39–7.31 (m, 5H, Ph), 5.94 (t,
J = 5.3 Hz, H, NH), 5.50 (br s, 2H, NH2), 4.69 (d, J = 5.69 Hz, 2H,
CH2); 13C NMR (CDCl3, 100 MHz) d 160.9 (C6), 159.3 (C2), 159.3
(C4), 137.4 (C, Bn), 134.1 (C, Ph), 130.5 (CH, Ph), 128.8 (CH, Ph),
128.8 (CH, Bn), 128.2 (CH, Ph), 127.8 (CH, Bn), 127.7 (CH, Bn),
116.4, 116.3 (2 ꢁ CN), 82.6, 80.7 (C3, C5), 45.3 (CH2); MS (EI) m/z
(%): 91 (69), 248 (4) [MꢀPh]+, 324 (100) [MꢀH]+, 325 (73) [M]+.
Anal. Calcd for C20H15N5: C, 73.83; H, 4.65; N, 21.52. Found: C,
73.59; H, 4.47; N, 21.80.
4.1.2.2. 2-Chloro-6-(pyrrolidin-10-yl)pyridine-3,5-dicarbonitrile
(22). Following the general procedure, 2-amino-6-(pyrrolidin-10-
yl)pyridine-3,5-dicarbonitrile (4) (43 mg, 0.2 mmol), with CuCl2
(40.3 mg, 0.3 mmol, 1.5 equiv) and iso-amylnitrite (40.3 lL,
0.3 mmol, 1.5 equiv), in acetonitrile (1.6 mL), after 12 h, provided
compound 22 [(39 mg, 84%; flash column chromatography (40%
4.1.1.14. 2-Amino-6-methoxypyridine-3,5-dicarbonitrile (16). A
mixture of 6-amino-2-chloropyridine-3,5-dicarbonitrile (1) (0.3 g,
1.685 mmol) and NaOMe (0.12 g, 2.191 mmol) in methanol
(20 mL) was heated at reflux and stirred for 90 min. Then, the reac-
tion was cooled and 10 mL of water was added. The precipitate was
filtered, washed and dried to afford known compound 1635
(0.283 g, 96.5%).
DCM in hexane)]: mp 168–170 °C; IR (KBr)
m
2223, 1596, 1550,
1486, 1409, 1335, 1276, 1063 cmꢀ1
;
1H NMR (CDCl3, 300 MHz) d
7.88 (s, 1H, H4), 4.15–3.56 (br s, 4H, H20, H50), 2.06 (s, 4H, H30,
H40); 13C NMR (CDCl3, 75 MHz) d 155.2 (C6), 155.0 (C2), 149.6
(C4), 117.0, 115.0 (2 CN), 96.3, 89.1 (C3, C5), 50.0 (C20, C50), 29.9
(C30, C40); MS (ES) m/z [M+1]+ 233.1/234.9. Anal. Calcd for
C11H9ClN4: C, 56.78; H, 3.90; Cl, 15.24; N, 24.08. Found: C, 56.50;
H, 4.01; Cl, 15.27; N, 24.29.
4.1.1.15. 2-Amino-6-ethoxy-pyridine-3,5-dicarbonitrile (17). A
mixture of 2-amino-6-methoxy-pyridine-3,5-dicarbonitrile (16)
(0.4 g, 2.298 mmol), sodium (0.52 g, 22.98 mmol) in dry EtOH
(25 mL) was stirred at reflux for 2 h. Then, cooled water was added
and the precipitate was separated by filtration and washed with
water. The yellow solid was purified by column chromatography
(CH2Cl2/AcOEt, 20:1, v/v), and recrystallized from ethanol to afford
known compound 1736 (0.393 g, 91%).
4.1.2.3. 2-Chloro-6-(piperidin-1-yl)pyridine-3,5-dicarbonitrile
(23). Following the general procedure, reaction of 2-amino-6-
(piperidin-1-yl)pyridine-3,5-dicarbonitrile (5) (0.3 g, 1.32 mmol),
with CuCl2 (0.236 g, 1.98 mmol) and iso-amylnitrite (0.266 mL,
1.98 mmol), in acetonitrile (10 mL), after 12 h, gave compound 23
(0.234 g, 0.951 mmol, 72%): mp 113–114 °C; IR (KBr)
m
2926, 2227,
2218, 1714, 1599, 1544, 1486, 1406, 1336, 1251 cmꢀ1
;
1H NMR
(CDCl3, 400 MHz) d 7.88 (s, 1H, H4), 3.91 (m, 4H, 2 ꢁ CH2), 1.71 (m,
6H, 3 ꢁ CH2); 13C NMR (CDCl3, 100 MHz) d 156.8 (C6), 154.4 (C2),
149.9 (C4), 116.2, 114.4 (2 ꢁ CN), 96.5, 89.3 (C3, C5), 48.6
(2 ꢁ CH2), 25.8 (2 ꢁ CH2), 23.9 (CH2); MS (IE), m/z(%): 84 (47), 217
4.1.1.16. 2-Amino-6-(3-phenylpropoxy)pyridine-3,5-dicarboni-
trile (18). To
a solution of 2-amino-6-methoxy-pyridine-3,5-
dicarbonitrile (16) (0.2 g, 1.149 mmol), 3-phenyl-propanol

5870
A. Samadi et al. / Bioorg. Med. Chem. 18 (2010) 5861–5872
(100), 246 (79) [M]+. Anal. Calcd for C12H11ClN4: C, 58.42; H, 4.49; Cl,
14.37; N, 22.71. Found: C, 58.25; H, 4.37; Cl, 14.3; N, 22.76.
4.1.2.8. 2-Chloro-6-(70-azabicyclo[2.2.1]heptan-70-yl)-4-phenyl-
pyridine-3,5-dicarbonitrile (28). Following the general procedure,
2-amino-6-(70-azabicyclo[2.2.1]heptan-70-yl)-4-phenylpyridine-3,5-
dicarbonitrile (14) (20.6 mg, 0.065 mmol) with CuCl2 (13.1 mg,
0.098 mmol) and iso-amylnitrite (13.1 lL, 0.098 mmol), in acetoni-
trile (0.8 mL), after 6 h, afforded compound (28) (11 mg, 50%), after
4.1.2.4. 2-Chloro-6-(70-azabicyclo[2.2.1]heptan-70-yl)pyridine-
3,5-dicarbonitrile (24). Following the general procedure, 2-ami-
no-6-(70-azabicyclo[2.2.1]heptan-70-yl)pyridine-3,5-dicarbonitrile
(6) (47.9 mg, 0.2 mmol), with CuCl2 (40.3 mg, 0.3 mmol, 1.5 equiv)
flash column chromatography (30% DCM in hexane), as a solid: mp
and iso-amylnitrite (40.3
lL, 0.3 mmol, 1.5 equiv), in acetonitrile
195–197 °C; IR (KBr) m ;
2227, 1560, 1499, 1480 cmꢀ1 1H NMR
(1.6 mL), after 90 min, gave compound 24 [(34 mg, 0.131 mmol,
66%, flash column chromatography (30% DCM in hexane)], as a so-
(CDCl3, 300 MHz) d 7.66–7.39 (m, 5H, Ph), 5.23 (s, 2H, H10, H40),
2.04–1.81 (m, 4H, H20, H30, H50, H60), 1.74–1.55 (m, 4H, H20, H30,
H50, H60); 13C NMR (CDCl3, 75 MHz) d 162.6 (C6), 156.1, 156.0 (C2,
C4), 133.5 (C, Ph), 131.1, 129.1, 128.6 (CH, Ph), 116.2, 114.8
(2 ꢁ CN), 98.4, 90.6 (C3, C5), 58.4 (C10, C40), 29.4 (C20, C30, C50, C60);
MS (ES) m/z [M+1]+ 335.2/337.2/336.2. Anal. Calcd for C19H15ClN4:
C, 68.16; H, 4.52; Cl, 10.59; N, 16.73. Found: C, 67.90; H, 4.69; Cl,
10.34; N, 17.07.
lid: mp 124–126 °C; IR (KBr)
m
3068, 2948, 2230, 1594, 1520, 1574,
1454, 1403, 1260, 1204, 1163 cmꢀ1
;
1H NMR (CDCl3, 300 MHz) d
7.86 (m, 1H, H4), 5.18 (s, 2H, H10, H40), 2.01–1.77 (m, 4H, H20,
H30, H50, H60), 1.73–1.53 (m, 4H, H20, H30, H50, H60); 13C NMR
(CDCl3, 75 MHz) d 155.6 (C6), 155.3 (C2), 149.3 (C4), 116.4, 114.8
(2 ꢁ CN), 97.4, 90.1 (C3, C5), 58.2 (C10, C40), 29.4 (C20, C30, C50,
C60); MS (ES) m/z [M+1]+ 259.0/261.0/260.0, [M+23]+ 281.0/
283.0/282.0. Anal. Calcd for C13H11ClN4: C, 60.35; H, 4.29; Cl,
13.70; N, 21.66. Found: C, 60.18; H, 4.31; Cl, 13.17; N, 21.54.
4.2. Pharmacology
4.2.1. Measurements of AChE and BuChE inhibition
4.1.2.5. 2-Chloro-6-(dimethylamino)-4-phenylpyridine-3,5-di-
carbonitrile (25). Following the general procedure, reaction of
2-amino-6-(dimethylamino)-4-phenylpyridine-3,5-dicarbonitrile
(11) (0.288 g, 1.095 mmol) with CuCl2 (0.218 g, 1.642 mmol) and
iso-amylnitrite (0.22 mL, 1.642 mmol), in acetonitrile (10 mL), after
12 h, gave product 25 (0.207 g, 67%) as a solid: mp 158–160 °C; IR
To assess the inhibitory activity of the compounds towards
AChE and BuChE, we followed the spectrophotometric method of
Ellman,40a using purified AChE from Electrophorus electricus (type
V-S), human erythrocytes (buffered aqueous solution) or BuChE
from equine serum (lyophilized powder) (Sigma–Aldrich, Madrid,
Spain). The reaction took place in a final volume of 3 mL of a phos-
phate-buffered solution (0.1 M) at pH 8, containing 0.035 U of
AChE or 0.05 U of BuChE, and 0.35 mM of 5,50-dithiobis-2-nitro-
benzoic acid (DTNB, Sigma–Aldrich, Madrid, Spain). Inhibition
curves were made by pre-incubating this mixture with at least
nine concentrations of each compound for 10 min. A sample with
no compound was always present to determine the 100% of en-
zyme activity. After this pre-incubation period, acetylthiocholine
iodide (0.35 mM) or butyrylthiocholine iodide (0.5 mM) (Sigma–
Aldrich, Madrid, Spain) were added, allowing an additional
15 min incubation period in which DTNB produces the yellow an-
ion 5-thio-2-nitrobenzoic acid along with the enzymatic degrada-
tion of acetylthiocholine iodide or butyrylthiocholine iodide.
Changes in absorbance were detected at 405 nm in a spectrophoto-
metric plate reader (FluoStar OPTIMA, BMG Labtech). Compounds
inhibiting AChE or BuChE activity would reduce the color genera-
tion; thus IC50 values were calculated as the concentration of com-
pound that produces 50% AChE inhibition of the enzyme activity.
Data are expressed as mean SEM of at least three different exper-
iments in quadruplicate.
(KBr)
m ;
2223, 1583, 1571, 1486, 1420, 1410, 1271, 1184 cmꢀ1 1H
NMR (CDCl3, 300 MHz) d 7.58–7.46 (m, 5H, Ph), 3.43 (s, 6H,
2 ꢁ CH3); 13C NMR (CDCl3, 75 MHz) d 163.0 (C6), 158.4 (C2),
154.5 (C4), 133.4 (C, Ph), 130.8 (CH, Ph), 128.8 (2 ꢁ CH, Ph),
128.4 (2 ꢁ CH, Ph), 115.8, 114.5 (2 ꢁ CN), 97. (C3), 89.1 (C5), 40.9
(2 ꢁ CH3); MS (EI) m/z (%) 281 (100) [MꢀH]+, 282 (36) [M]+. Anal.
Calcd for C15H11ClN4: C, 63.72; H, 3.92; Cl, 12.54; N, 19.82. Found:
C, 63.55; H, 4.03; Cl, 12.42; N, 19.69.
4.1.2.6. 2-Chloro-6-(pyrrolidin-10-yl)-4-phenylpyridine-3,5-di-
carbonitrile (26). Following the general procedure, 2-amino-6-
(pyrrolidin-10-yl)-4-phenylpyridine-3,5-dicarbonitrile (12) (57.9
mg, 0.200 mmol), with CuCl2 (40.3 mg, 0.3 mmol, 1.5 equiv) and
iso-amylnitrite (40.3 lL, 0.3 mmol, 1.5 equiv), in acetonitrile
(1.6 mL), after 17 h, provided compound 26 [(39.5 mg, 0.128 mmol,
64%; flash column chromatography (1:1 DCM/hexane)] as a solid:
mp 210–212 °C; IR (KBr)
m
2223, 2211, 1580, 1562, 1535, 1482,
;
1454, 1335, 1265, 1248 cmꢀ1
1H NMR (CDCl3, 300 MHz) d 7.63–
7.39 (m, 5H, Ph), 4.08–3.76 (br s, 4H, H20, H50), 2.05 (s, 4H, H30,
H40); 13C NMR (CDCl3, 75 MHz) d 161.9 (C6), 155.0, 154.3 (C2,
C4), 132.8 (C, Ph), 130.0, 128.1, 127.6 (CH, Ph), 115.4, 114.0
(2 ꢁ CN), 96.5, 88.3 (C3, C5), 49.3 (C20, C50), 28.8 (C30, C40); MS
(ES) m/z [M+1]+ 309.0/311.0/310.0, [M+23]+ 331.0/333.0/332.0.
Anal. Calcd for C17H13ClN4: C, 66.13; H, 4.24; Cl, 11.48; N, 18.15.
Found: C, 65.98; H, 4.36; Cl, 10.88; N, 18.01.
4.2.2. Kinetic analysis of AChE inhibition
To obtain estimates of the competitive inhibition constant Ki, re-
ciprocal plots of 1/V versus 1/[S] were constructed at different con-
centrations of the substrate acetylthiocholine (0.1–1 mM) by using
Ellman’s method.40a The plots were assessed by a weighted least
square analysis. Slopes of these reciprocal plots were then plotted
4.1.2.7. 2-Chloro-4-phenyl-6-(piperidin-1-yl)pyridine-3,5-dicar-
bonitrile (27). Following the general procedure, reaction of 2-
amino-6-(piperidin-10-yl)-4-phenylpyridine-3,5-dicarbonitrile
(13) (0.315 mg, 1.038 mmol) with CuCl2 (0.21 g, 1.578 mmol) and
iso-amylnitrite (0.21 mL, 1.578 mmol), in acetonitrile (10 mL), after
12 h, gave product 27 (0.198 mg, 60%) as a solid: mp 113–114 °C;
against the concentration of compound 23 (range 0–90 lM) in a
weighted analysis and Ki was determined as the intercept on the
negative x-axis. For the determination of the noncompetitive inhibi-
tion constant (KI), intercepts of the reciprocal plots were also plotted
against the concentration of compound 23 (range 0–90 lM) in a
weighted analysis and KI was determined as the intercept on the
negative x-axis. Data analysis was performed with Origin Pro 7.5
software (Origin Lab Corp.).
IR (KBr)
m ;
2937, 2862, 2219, 1584, 1566, 1484, 1471, 1260 cmꢀ1
1H NMR (CDCl3, 300 MHz) d 1.75 (m, 6H), 3.92 (m, 6H), 7.47–
7.56 (m, 5H); 13C NMR (CDCl3, 75 MHz) d 163.0 (C6), 158.5 (C4),
154.8 (C2), 133.5 (C), 130.9, 128.9, 128.5 (CH-Ph), 115.7, 114.1
(2 ꢁ CN), 97.83 (C3), 90.1 (C5), 49.33 (C20, C60), 25.89 (C30, C50),
23.9 (C40); MS (ES) m/z 323.3, [M+23]+ 345.2. Anal. Calcd for
4.2.3. Quantification of viability of SH-SY5Y cells using the
mitochondrial probe MTT
Cell death and neuroprotection afforded by synthesized com-
pounds was studied in human neuroblastoma cell line, a kind gift
from Dr. F. Valdivieso from the Centro de Biología Molecular
C18H15ClN4: C, 66.98; H, 4.68; Cl, 10.98; N, 17.36. Found: C,
67.12; H, 4.71; Cl, 11.20; N, 17.22.

A. Samadi et al. / Bioorg. Med. Chem. 18 (2010) 5861–5872
5871
(Madrid, Spain). SH-SY5Y cells were maintained in a 1:1 mixture of
F-12 Nutrient Mixture (Ham12) and Eagle’s minimum essential
medium (EMEM) (Sigma–Aldrich, Madrid, Spain) supplemented
with 15 non-essential amino acids, 1 mM sodium pyruvate, 10%
heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin,
sional grid with a spacing of 0.375 Å. In a second round of docking
experiments, we built smaller grids around both potential binding
sites (0.200 Å). A distance-dependent dielectric function was used
in the computation of electrostatic interactions. After docking, the
solutions were clustered in groups with root mean square devia-
tions less than 1.0 Å. The clusters were ranked by the lowest en-
ergy representative of each cluster.
and 100 lg/mL streptomycin (reagents from Invitrogen, Madrid,
Spain). Cultures were seeded into flasks containing supplemented
medium and maintained at 37 °C in a humidified atmosphere of 5%
CO2 and 95% air. For assays, SH-SY5Y cells were subcultured in 48-
well plates at a seeding density of 1 ꢁ 105 cells per well. Cells were
treated with the drugs before confluence in EMEM with 1% FBS. Cells
were used at a low passage number (<13). Cell viability, virtually the
mitochondrial activity of living cells, was measured by quantitative
colorimetric assay with MTT [3-(4,5 dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] (Sigma–Aldrich, Madrid, Spain), as
described previously.43 SH-SY5Y cells were seeded into 48-well cul-
ture plates and allowed to attach. MTT was added to all wells (5 mg/
mL) and allowed to incubate, in the dark at 37 °C for 2 h followed by
cell lysis and spectrophotometric measurement at 540 nm. The tet-
razolium ring of MTT can be cleaved by active reductases in order to
produce a precipitated formazan derivative. The formazan produced
Acknowledgments
A.S. thanks CSIC for an I3P-post-doc contract. M.C. thanks Institu-
to de Salud Carlos III (MICINN) for a ‘Sara Borrell’ post-doctoral
contract. J.M.C. thanks MICINN (SAF2006-08764-C02-01; SAF2009-
07271), and CSIC-GRICES (2007PT-13) financial support. This work
was partly supported by Grants RD06/0026/1002 (RENEVAS), ISCIII,
MICINN, and S/SAL-0275-2006, Comunidad de Madrid, Spain, to
J.M.C., A.G.G., M.V., and M.G.L. Also by following Grants to A.G.G.:
(1) SAF2006-03589, MICINN; (2) PI016, Fundación C.I.E.N., ISCIII;
and (3) NDE07/09, Agencia Lain Entralgo, Comunidad de Madrid,
Spain. The present work has also been supported by Fundación Teó-
filo Hernando, MEC Grants BFI2003-02722, SAF-2006-08540,
SAF2006-1249 and CTQ2005-09365, and Fundación La Caixa (Barce-
lona, Spain).
was dissolved by adding 200 lL DMSO, resulting in a colored com-
pound whose optical density was measured in an ELISA reader at
540 nm. All MTT assays were performed in triplicate.
Supplementary data
4.2.4. Measurement of cell death and neuroprotection taking as
indicator of the release of lactate dehydrogenase (LDH)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.095. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
LDH activity was spectrophotometrically measured using a
Cytotoxicity Cell Death kit (Roche-Boehringer. Mannheim, Ger-
many) according to the manufacturer’s indications. Total LDH
activity was defined as the sum of intracellular and extracellular
LDH activity; released LDH was defined as the percentage of extra-
cellular compared to total LDH activity. At the end of the 24-h incu-
bation period with oligomycin/rotenone, samples were collected to
estimate extracellular LDH as indication of cell death.42 Intracellu-
lar LDH activity was measured in the cells after incubation with
10% Triton X-100. LDH activity was measured at 490–620 nm,
using a microplate reader (Labsystems iEMS reader MF; Labsys-
tems, Helsinki, Finland). Total LDH (intracellular plus extracellular)
was normalized to 100%; then, the amount of LDH released to the
extracellular medium was expressed as percentage of this total.
Data were normalized by subtracting basal LDH (cells not sub-
jected to any treatment) to the different treatment groups in each
individual experiment, and the result for rotenone/oligomycin-A
group was normalized to 100% (percentage cell death).
References and notes
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3. Cummings, J. L. Rev. Neurol. Dis. 2004, 1, 60.
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*
the B3LYP/6-31G basis set. Partial atomic charges were then ob-
tained using the RESP methodology.45 As target, the X-ray crystal-
lographic structure of the hAChE–fasciculin complex (PDB code
1B41)46 was used. Fasciculin was removed from the structure,
truncated residues were reconstructed, and missing residues were
modeled. Docking was performed with the program AutoDock
4.0.47 A box encompassing both catalytic and peripheral binding
sites was defined for the exploration of possible binding modes.
The Lamarckian genetic algorithm (LGA) implemented in AutoDock
was then used to generate docked conformations of the ligand by
randomly changing the overall orientation of the molecule as well
as the torsion angles of all rotatable bonds. Default settings were
used except for number of runs, population size, and maximum
number of energy evaluations, which were fixed at 100, 100, and
250.000, respectively. Rapid intra- and intermolecular energy eval-
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atomic affinity potential for aliphatic and aromatic carbon, oxygen,
nitrogen, and hydrogen atoms precalculated in a three-dimen-
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