Cholinergic and neuroprotective drugs for the treatment of Alzheimer and neuronal vascular diseases. II. Synthesis, biological assessment, and molecular modelling of new tacrine analogues from highly substituted 2-aminopyridine-3- carbonitriles

Article abstract of DOI:10.1016/j.bmc.2010.11.040

The synthesis, biological assessment, and molecular modelling of new tacrine analogues 11-22 is described. Compounds 11-22 have been obtained by Friedl?nder-type reaction of 2-aminopyridine-3-carbonitriles 1-10 with cyclohexanone or 1-benzyl-4-piperidone.

Full text of DOI:10.1016/j.bmc.2010.11.040

Bioorganic & Medicinal Chemistry 19 (2011) 122–133
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Cholinergic and neuroprotective drugs for the treatment of Alzheimer
and neuronal vascular diseases. II. Synthesis, biological assessment,
and molecular modelling of new tacrine analogues from highly
substituted 2-aminopyridine-3-carbonitriles
Abdelouahid Samadi a, Carolina Valderas a, Cristóbal de los Ríos b,c, Agatha Bastida a, Mourad Chioua a,
Laura González-Lafuente b, Inés Colmena b, Luis Gandía b, Alejandro Romero b, Laura del Barrio b,
María D. Martín-de-Saavedra b, Manuela G. López b, Mercedes Villarroya b, José Marco-Contelles a,
⇑
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 Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Alcala, 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:
The synthesis, biological assessment, and molecular modelling of new tacrine analogues 11–22 is
described. Compounds 11–22 have been obtained by Friedländer-type reaction of 2-aminopyridine-3-
carbonitriles 1–10 with cyclohexanone or 1-benzyl-4-piperidone. The biological evaluation showed that
some of these molecules were good AChE inhibitors, in the nanomolar range, and quite selective regard-
ing the inhibition of BuChE, the most potent being 5-amino-2-(dimethylamino)-6,7,8,9-tetra-
Received 17 September 2010
Revised 15 November 2010
Accepted 18 November 2010
Available online 26 November 2010
hydrobenzo[1,8-b]-naphthyridine-3-carbonitrile (11) [IC50 (EeAChE: 14 nM); IC50 (eqBuChE: 5.2 lM].
Kinetic studies on the easily available and potent anticholinesterasic compound 5-amino-2-(methoxy)-
6,7,8,9-tetrahydrobenzo[1,8-b]-naphthyridine-3-carbonitrile (16) [IC50 (EeAChE: 64 nM); IC50 (eqBuChE:
This manuscript is dedicated to Professor
Antonio G. García on occasion of his 65th
birthday
9.6 lM] showed that this compound is a mixed-type inhibitor (Ki = 69.2 nM) of EeAChE. Molecular mod-
elling on inhibitor 16 confirms that this compound, as expected and similarly to tacrine, binds at the
catalytic active site of EeAChE. The neuroprotective profile of molecules 11–22 has been investigated
in SH-SY5Y neuroblastoma cells stressed with a mixture of oligomycin-A/rotenone. Compound 16 was
also able to rescue by 50% cell death induced by okadaic acid in SH-SY5Y cells. From these results we con-
clude that the neuroprotective profile of these molecules is moderate, the most potent being compounds
12 and 17 which reduced cell death by 29%. Compound 16 does not affect ACh- nor K+-induced calcium
signals in bovine chromaffin cells. Consequently, tacrine analogues 11–22 can be considered attractive
therapeutic molecules on two key pharmacological targets playing key roles in the progression of Alzhei-
mer, that is, cholinergic dysfunction and oxidative stress, as well as in neuronal cerebrovascular diseases.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
2-Aminopyridin-3-carbonitriles
Tacrine analogues
AChE
BuChE
Inhibition mechanism
Neuroprotection
Molecular modelling
Alzheimer’s disease
Neuronal cerebrovascular diseases
1. Introduction
The cholinergic theory1 suggests that the selective loss of cho-
linergic neurons in AD results in a deficit of acetylcholine (ACh)
Alzheimer’s disease (AD) is an age-related neurodegenerative
process characterized by a progressive loss of cognitive abilities,
such as memory, language skills, attention, disorientation, and
depression.1 Although the etiology of AD is still poorly understood,
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 galantamine are known to improve AD symptoms
by inhibiting AChE, that is, the enzyme responsible for the hydro-
lysis of ACh, thereby rising 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 ace-
tylcholinesterase inhibitor (AChEI) agent binding specifically to the
several factors such as amyloid-b (Ab)2 deposits,
s-protein aggre-
gation, oxidative stress, or low levels of acetylcholine3 are thought
to play significant roles in the pathology of the disease.4 In spite of
the enormous research effort, an efficient strategy for designing
new drugs for the treatment of AD is still lacking.
⇑
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.2010.11.040

A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 122–133
123
Table 1
PAS, affects Ab aggregation in vitro, whereas other catalytic active
site (CAS) inhibitors, such as tacrine, have not a similar effect.8
The multifactorial nature of AD supports new therapeutic strat-
egies. The most current innovative therapeutic approach is based
on the ‘one molecule, multiple targets’ paradigm.9–13 Thus, the mul-
tipotent approach14 includes novel tacrine-melatonin hybrids,15
dual inhibitors of AChE and monoamine oxidase16 or serotonin
transporters,17 potent cholinesterase inhibitors with antioxidant
and neuroprotective properties,18 gallamine-tacrine hybrids bind-
ing at cholinesterases and M2 muscarinic receptors,19 or AChE
inhibitors and tau-hyperphosphorylation regulators.20
Synthesis of tacrine analogues 11–22
R
NH2
NC
Y
X
N
N
Entry Precursor of Product of
R
X
Y
Yield % by MW
irradiation
(classical
general
structure I
general
ref
structure III
heating)
a
121b
224
11
12
H
H
Me2N
CH2 72
Based on these precedents, some years ago we have embarked
in a project focused on the synthesis of multipotent molecules able
to improve the cholinergic neurotransmission, showing also L-type
Ca2+ channel antagonist effects. As a result, we have reported the
chemistry and pharmacology of tacripyrines,21a as potent, selective
(AChE vs BuChE), and mixed-type inhibitors, binding preferentially
at the PAS of AChE, interfering with the Ab pro-aggregating effect
of human AChE (hAChE), and the self-aggregation of Ab1-42; in
addition, tacripyrines were potent Ca2+ antagonists, pass through
blood–brain barrier, and display neuroprotective and antioxidant
properties.21a
In this context, we have recently described the synthesis,
molecular modelling and pharmacological analysis of new, simple,
and readily available 2-aminopyridine-3,5-dicarbonitriles (I), and
2-chloropyridine-3,5-dicarbonitriles (II) (Chart 1).21b From this
work, we concluded that these molecules were modest inhibitors
of AChE and butyrylcholinesterase (BuChE), in the micromolar
range. No clear structure–activity relationship (SAR) could be ob-
tained from these data; but apparently, compounds bearing small
groups such as N,N0-dimethylamino or the pyrrolidine, regardless
of the presence of 2-amino or 6-chloro substituents at the pyridine
ring, preferentially inhibited AChE. The neuroprotective profile of
these molecules was also investigated showing neuroprotection
with values around 30% in SH-SY5Y neuroblastoma cells stressed
with a mixture of oligomycin-A/rotenone.
N
b
CH2 98
N
c
324
13
14
H
H
CH2 91
CH2 78
421b
NH
d
e
f
g
h
i
j
k
l
521b
15
16
17
18
19
20
21
22
Ph Me2N
CH2 47
CH2 95 (51)
CH2 92
CH2 67
CH2 30 (9)
CH2 20
625
H
H
MeO
EtO
Ph MeO
Cl
Ph Cl
726
827
921b
1021b
625
H
H
H
MeO
Cl
NBn 81
NBn 54
921b
R
O
Y
R
NH2
NC
X
CN
NC
(Y= CH2, NBn)
Y
AlCl3, ClCH2CH2Cl,
MW irradiation
or
NH2
N
X
N
N
III
I
TfOSiMe3, AcOEt
(R= H, Ph)
(X= OR, Cl, NR1R2)
Based on these results, and in order to improve the observed
cholinergic and neuroprotective profile, we considered that the
corresponding tacrine analogues III, synthesized from the most po-
tent and neuroprotective molecules of the type I (Chart 1), could
therefore be attractive synthetic targets that would deserve to be
prepared and investigated.
Thus, in this manuscript we describe the successful accomplish-
ment of these objectives, reporting the chemistry and pharmacology
of the new tacrine analogues 11–22 derived from the pharmacologi-
cally active 2-aminopyridine-3,5-dicarbonitriles 1–10 (Table 1) of
family I (Chart 1).21b
Scheme 1. Synthesis of tacrine analogues III by Friedländer-type reaction between
2-aminopyridine-3-carbonitriles
piperidone.
I and either cyclohexanone or 1-benzyl-4-
3,5-dicarbonitriles 1–10 with cyclohexanone in Friedländer-type
reaction conditions (Scheme 1).22 Microwave irradiation was pre-
ferred23 because in some instances (see Table 1, entries f and i),
classical heating conditions gave lower chemical yields. Table 1
shows the structure of the 2-aminopyridine-3,5-dicarbonitriles 1,
4, 5, 9 and 10,21b and the structure of known precursors 2,24 3,24
6,25 7,26 and 8,27 synthesized as described, as well as the structure
of the resulting tacrine analogues with their chemical yields. We
have also prepared tacrines 21 and 22 (Table 1) by reacting both
2-aminopyridine-3,5-dicarbonitriles 625 and 921b with 1-benzyl-
4-piperidone, in modified Friedländer-type reaction conditions
using trimethylsilyl triflate as promotor, and ethyl acetate as sol-
vent (Scheme 1).28 All new compounds showed analytical and
2. Results and discussion
2.1. Chemistry
We have synthesized the differently substituted tacrine ana-
logues 11–20 by reacting the corresponding 2-aminopyridine-
R
R
R
N
NH2
N
NH2
NC
X
CN
NC
X
CN
Cl
NC
X
Y
N
NH2
N
N
I21b
III
II21b
TACRINE
(R= H, Ph;
X= NR1R2, OR, SR)
(Y= CH2, NBn)
(R= H, Ph;
X= NR1R2)
(this work)
Chart 1. The structures of tacrine, 2-aminopyridine-3,5-dicarbonitriles (I), 2-chloropyridine-3,5-dicarbonitriles (II), and the new tacrine analogues (III).

124
A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 122–133
Table 2
Inhibition of AChE from Electrophorus electricus (EeAChE) and bovine erythrocytes (beAChE), and horse serum BuChE (eqBuChE) by tacrine analogues 11–22a
Entry Product of general structure III Structure
IC50
(
lM)
Selectivity eqBuChE/EeAChE
beAChE
EeAChE
eqBuChE
NH2
N
NC
a
11
12
0.10 0.02
0.014 0.001 5.2 0.4
371
N
N
Me2
NH2
NC
N
b
0.16 0.02
0.043 0.006 5.6 0.3
130
N
N
NH2
NC
N
c
13
14
1.0 0.1
0.025 0.005 3.6 0.2
144
87
N
N
NH2
NC
d
1.53 0.04
0.030 0.003 2.6 0.3
N
H
N
N
NH
N
2
e
15
4.4 0.3
0.09 0.02
7.5 0.1
83
NC
Me
2 N
NC
Me
N
NH
2
f
16
17
1.6 0.4
1.0 0.2
0.06 0.01
9.6 0.6
160
O
NC
EtO
N
N
NH2
g
0.050 0.001 >30
>600
N
N
NH2
N
h
18
19
20
21
5
1
0.08 0.01
5.0 0.4
>30
5.1 0.9
64
NC
MeO
NC
Cl
N
NH2
i
>100
>100
>30
>6
N
N
NH2
j
>30
1
NC
Cl
N
N
NH2
NC
MeO
N
k
5
1
0.35 0.04
>100
>286
N
N

A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 122–133
125
Table 2 (continued)
Entry Product of general structure III Structure
IC50
(lM)
Selectivity eqBuChE/EeAChE
beAChE
>100
EeAChE
eqBuChE
>100
NH2
N
NC
N
l
22
0.35 0.09
>286
Cl
N
NH2
m
Tacrine
0.109 0.03 0.027 0.002 0.0052 0.0002 0.19
N
a
Data are expressed as mean SEM of quadruplicates of at least three independent experiments.
spectroscopic data in good agreement with their structures (see
Section 4).
22 for the inhibition of EeAChE, bearing both compounds a chlorine
atom at C-2.
Inhibition experiments using beAChE showed IC50 data mainly
in the micromolar range, being compound 11 the best inhibitor,
2.2. Pharmacology
with an IC50 of 0.1 lM. Hence, these compounds were much more
2.2.1. Anticholinesterasic activity
2.2.1.1. AChE/BuChE inhibitory activity.
active inhibiting eeAChE than beAChE. The reason for this enzy-
matic selectivity could be due to some structural differences, such
as eeAChE being a flexible tetramer,30 while mammal erythrocytes
AChE is presented in globular dimers,31 or because they show dif-
ferent rate of glycosylation.32 Although some authors have not
found any differences in the catalytic behavior of both enzymes,
there are several papers describing a differential inhibitory activity
for eeAChE and mammal erythrocytes AChE with physostigmine,
organophosphorus, and carbamate analogues.33 Otherwise, the fact
that these compounds were better inhibitors of eeAChE, an enzyme
structurally related to the brain enzyme,34 than the peripheral bo-
vine erythrocyte AChE could be beneficial for a potential further
clinical development, taking into account that many of the side ef-
fects of AChE inhibitors are due to the inhibition of the peripheral
enzyme.
Tacrine analogues 11–
22 were evaluated as inhibitors of AChE from Electrophorus electricus
(Ee) and from bovine erythrocytes (be), and BuChE from horse serum
(eq), according to Ellman’s protocol.29 The results are shown in Table
2. From the IC50 values, we conclude that molecules 11–18 are good
EeAChE inhibitors, and quite selective regarding to the inhibition of
eqBuChE, the most potent being 5-amino-2-(dimethylamino)-6,7,8,9-
tetrahydrobenzo[1,8-b]-naphthyridine-3-carbonitrile (11) [IC50 (AChE:
14 nM); IC50 (BuChE: 5.2 lM)]. Note that compared with tacrine, inhib-
itor 11 had similar AChE inhibition profile, but it is less potent BuChE
inhibitor. Regarding eqBuChE, the most potent inhibitor was tacrine
analogue 14 (IC50 = 2.6 0.3 lM).
Structure–activity relationship (SAR) studies show us, firstly,
that the presence of either an hydrogen or a phenyl ring at C-4
has no significant effect on the inhibition of eqBuChE, as IC50 values
for inhibitors 11:15, or 16:18, bearing a similar dimethylamino
(NMe2), or a methoxy (OMe) substituent at C-2, respectively. Re-
moval of the phenyl ring significantly improved AChE inhibition
only in the case of compound 11, as compared with compound
15. Compounds 11–18, bearing an electron-donating group at C2,
were nanomolar-ranged AChEI, being C2-amino-substituted com-
pounds 11–15 slightly more potent than C2-alkoxy-substituted
inhibitors 16–18. AChE inhibition was noticeably affected by the
size of the N2-alkyl substitution, as seen in compounds 11–15.
The shortest N-methyl-substituted compound 11 inhibited AChE
better than compounds having larger alkyl chains at N2. Note also
that the size of the alkoxy-substituent has no significant influence
on the AChE inhibition, as the ethoxy-substituted derivative 17
showed a similar IC50 compared to the methoxy-substituted deriv-
atives 16 or 18. Conversely, and for the inhibition of eqBuChE, on
going from methoxytacrine 16 to ethoxytacrine 17 the AChE activ-
ity is lost, but recovered, in the same range, when a phenyl ring is
located at C4 as in inhibitor 18. Overall, the observed eqBuChE
2.2.1.2. Kinetic analysis for the EeAChE inhibition.
Based on
the previous results, and taking into account the availability based
on the high chemical yield obtained in its synthesis, and the ChE
inhibition profile, due to the potency and selectivity, we selected
inhibitor 5-amino-2-(methyloxy)-6,7,8,9-tetrahydrobenzo[1,8-b]-
naphthyridine-3-carbonitrile (16) [IC50 (EeAChE: 64 nM); IC50
(eqBuChE: 9.6 0.6 mM] to carry out the pertinent kinetic analysis
to determine the Ki and the mechanism for the inhibition of EeAChE.
Among the most active AChEI, compound 16 presented the smallest
substituent at C2. In addition, inhibitor 16 has been isolated in high
chemical yield, and although shows inhibitory potency (IC50
)
slightly lower than other tacrine analogues (see for instance, com-
pounds 12, 13 and 17), these values are in the same order of mag-
nitude. The type of inhibition was elucidated from the analysis of
Lineweaver–Burk reciprocal plots (Fig. 1) showing increasing slopes
(lower Vmax) and slightly increasing intercepts (higher Km) with
higher inhibitory concentration. The graphical analysis of steady-
state inhibition data for compound 16 is shown in Figure 1, where
plots of different inhibitor concentrations almost intersect in x-axis.
This would suggest a non-competitive inhibition, but scaling down
the Lineweaver–Burk plot we can see lines converging just above
the x-axis. This suggests a mixed-type inhibition.35 This type of
inhibition was also confirmed by the fact that the resulting Ki value,
estimated from the slopes of double reciprocal plots versus com-
pound 16 concentrations was 69.2 nM, while the Ki value, esti-
mated from the intercepts in y-axis of double reciprocal plots
versus compound 16 concentrations was 143 nM.
inhibition for molecules 11–20 ranged between 2.6 and >30 lM.
The incorporation of an electron withdrawing substituent, such
as a chlorine atom, at C-2, resulted in a dramatic loss of cholines-
terase activity. Finally, it has been observed that the change of
the methylene group (CH2) at C-7 in inhibitors 16 and 19 by an
NBn (N-benzyl) group in inhibitors 21 and 22, respectively, aug-
mented AChE selectivity. Compared with compound 21, inhibitor
16, bearing also a methoxy group at C-2, was 5.8-fold more active;
but interestingly, inhibitor 19 was found 14-fold less potent than

126
A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 122–133
20
18
16
14
12
10
8
ual residues to the binding energy reveals that major interactions
arise from Trp84, Asp85, Ser 200, Phe330, Tyr 334, Trp423, and
His440.
In summary, the calculations suggest that the ligand is success-
fully docked in the CAS site of the AChE which shows a high stabil-
ity, as supported by MD simulations.
0nM
10nM
30nM
60nM
100nM
300nM
2.2.2. Neuroprotection of compounds 11–22 against rotenone/
oligomycin-A-induced cell death
The exposure of SH-SY5Y cells to a mixture of rotenone plus oli-
gomycin (Rot/Olig) constitutes a good model of oxidative stress
having its origin in mitochondria.37 Rotenone blocks complex I of
mitochondrial electron transport chain and oligomycin-A inhibits
complex V, thus disrupting ATP synthesis; they elicit neurotoxicity
and allow the evaluation of potential neuroprotective drugs used
to treat AD patients, which inhibit AChE, such as galanthamine,
donepezil and rivastigmine. The results shown in Table 3 indicate
that compounds 11–22 showed modest neuroprotective effect
against oxidative stress, and in the same range of the well known
reference antioxidant N-acetylcysteine (NAC).38 Compounds 12
and 17 induced a protection of 29%, while tacrine analogues 22,
13, 14 and 16 protected by 20% (Table 3). However, no clear SAR
has been found in this series of compounds to evaluate or predict
more new potent neuroprotective agents.
6
4
2
0
-4
-2
0
2
4
6
8
10
[ATCh]-1 (mM-1)
Figure 1. Steady-state inhibition of AChE hydrolysis of acetylthiocholine (ATCh) by
compound 16. Lineweaver–Burk reciprocal plots of initial velocity and substrate
concentrations (0.1–1 mM) are presented. Lines were derived from a weighted
least-squares analysis of data.
2.2.1.3. Molecular modelling.
Next, a molecular modelling
study was performed in order to determine the binding mode of
the ligand 16 to the AChE from Torpedo californica. Docking analysis
was performed with AutoDock 4.0 program.36 A box encompassing
both the CAS and the PAS site was defined for the exploration of pos-
sible binding modes in the enzyme. Two different protonation states
were proposed for compound 16, the mode I, without protonation at
pyridineringC, andII, withit. Forbothstates, theresultsshowedthat
the ligand was exclusively docked at the CAS site. The putative bind-
ing mode is analogous to that found in the crystallographic complex
of tacrine bounded at the CAS site of AChE (Fig. 2). To validate and
test the stability of the proposed binding mode (I and II) obtained
by docking, molecular dynamics (MD) simulation have been carried
out. Compound 16 (mode I and II) remain docked along the simula-
tions within the proposed binding pocket and retained its starting
conformation (rmsd <0.5 Å for all non-hydrogen atoms) (see Figs. 3
and 4). The inspection of the van der Walls contributions of individ-
2.2.3. Effect of compound 16 on cells treated with okadaic acid
The Ser/Thr phosphatase inhibitor okadaic acid reproduces a
characteristic of the pathology of AD, that is, s-protein phosphory-
lation in different in vivo39 and in vitro40 models, such as in the hu-
man neuroblastoma cell line SH-SY5Y,39a,41 which is the
mechanism giving rise to neurofibrillary tangles (NFTs). Thus, it
is accepted that okadaic acid-induced toxicity is a good model for
´
the neuronal death occurring in Alzheimers disease and that is
linked to tau-hyperphosphorylation.20a
Cells exposed to okadaic acid 30 nM in the absence of the com-
pound showed 61 3% cell death with respect to control cells
(8.0 0.1%), measured as % of LDH release. Compound 16 was able
to significantly reduce cell death induced by okadaic acid from 0.1
to 1
respect to control). There were no statistically significant differ-
ences between 0.1 and 1 M. Higher doses were not neuroprotec-
tive. Galanthamine, used as reference, protected SH-SY5Y cells by
67% at 0.3
M (data not shown).41b
lM; with a maximum reduction of LDH release 0.1 lM (49%
l
l
2.2.4. Analysis of compound 16 as nAChR blocker or as a
voltage-dependent calcium channels antagonist
As previously documented, and discussed,20,21 the ability of our
tacrine analogues to act as Ca2+ channels blockers to prevent apop-
tosis and neuronal death, was of interest in the search for drugs for
AD. To evaluate these pharmacological activities, we have selected
again compound 16.
To explore the potential of compound 16 as putative nicotinic
receptors (nAChR) blocker or as a voltage-dependent calcium
channels antagonist, changes in cytosolic Ca2+ signals ([Ca2+]c) elic-
ited by application of the nAChR agonist acetylcholine (ACh,
100 l
M) or depolarizing solutions (70 mM K+) were evaluated in
bovine chromaffin cell populations. Figure 5A shows a representa-
tive experiment carried out in bovine chromaffin cells in the ab-
sence (control) and in the presence of 10 lM of inhibitor 16. The
application of ACh (left panel) or K+ (right panel) elicited a sharp
increase in [Ca2+]c that reached a plateau and then tended to slowly
decline along the 40 s of the recording. Incubation of the cells with
compound 16 (10 l
M) did not significantly affect the [Ca2+]c signal.
Figure 5B shows average data obtained by using this type of proto-
col in cells from four different cell cultures and six different con-
centrations of the compound (0.3, 1, 3, 10, 30, and 100 lM); No
Figure 2. Binding mode of 16 and THC to AChE after docking in the CAS site. Some
relevant residues are shown as sticks. Ligands are displayed as sticks with carbon
atoms in gray, (O, red and N, blue). Water molecules are represent as red sticks.

A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 122–133
127
Figure 3. Binding mode of compound 16 to AChE after docking and MD simulation. The C (alpha) traces of the enzyme are displayed as a ribbon. Some relevant residues are
shown as sticks. Ligand is displayed as sticks with carbon atoms in green.
Figure 4. Representation of the binding mode of the ligand 16 in the CAS predicted by the docking and MD simulation. Relevant residues are shown as sticks and colour by
atoms (C, blue). The residues forming the hydrophobic pocket belong to the active site (Trp 84, Trp 334, Phe 330, His 440, Ser 200 and Asp 85). Ligand is colour by atoms (C,
green).
clear effects on either the ACh-elicited (left panel) nor the K+-elic-
ited (right panel) [Ca2+]c signal was observed. These results suggest
that this compound does not behave as a nAChR antagonist nor as a
voltage-dependent calcium channel antagonist. Lack of nicotinic
antagonism of 16 is considered beneficial, as nAChRs antagonists
have been described to impair memory and cognitive functions.
By contrast, Ca2+ channels blockers have shown neuroprotection
in different models of neurodegeneration. Otherwise, the loss of

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A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 122–133
Table 3
Neuroprotection induced by the tacrine analogues (11–22) (1
lM) in human neuroblastoma cells stressed with a
mixture of rotenone/oligomycin-Aa
Entry
Product of general structure III
Structure
% Protection
NH2
NC
a
11
6
Me
2 N
N
N
NH2
NC
b
12
29
N
N
N
NH2
NC
N
c
13
14
19
20
N
N
NH2
N
NC
d
N
H
N
NH2
N
e
15
3
NC
Me2N
NC
N
NH2
f
16
17
23
29
MeO
NC
N
N
NH2
g
EtO
N
N
NH2
N
h
18
12
NC
MeO
N
NH2
NC
Cl
i
19
20
21
4
8
N
Ph
N
NH2
NC
Cl
j
N
N
NH2
NC
MeO
NC
k
N
11
N
N
NH2
N
l
22
20
33
Cl
N
N
m
NAC
a
The results are the averaged values, of at least three experiments, of percentages of MTT reduction compared to
control in absence of drugs. NAC: N-acetylcysteine.

A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 122–133
129
activity over Ca2+ channels has also been found in close analogues
4. Experimental part
4.1. General methods
recently described by our group, which maintained a very interest-
ing neuroprotective profile.20a
Melting points were determined on a Koffler apparatus, and
are uncorrected. 1H NMR and 13C NMR spectra were recorded at
rt 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)) as
internal reference. 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 spec-
trometer with an API-ES ionization source. Elemental analyses
were performed at CQO (CSIC, Spain). TLC was performed on silica
F254 and detection by UV light at 254 nm or by charring with
either ninhydrin, anisaldehyde or phosphomolybdic-H2SO4 dyeing
reagents. Anhydrous solvents were used in all experiments.
Column chromatography was performed on Silica Gel 60
(230 mesh). Reactions under MW irradiation (250 W) were
performed in a CEM Discover system™, equipped with electro-
magnetic sample stirrer, an infrared temperature detector and a
pressure sensor. The reaction was performed in 30 mL glass tube
equipped with septa.
3. Conclusions
In this work, we have described the synthesis, biological assess-
ment, and molecular modelling of new tacrine analogues 11–22.
Compounds 11–22 have been obtained by Friedländer-type reac-
tion of 2-aminopyridine-3-carbonitriles 1–10 with cyclohexanone,
as well as with 1-benzyl-4-piperidone for comparative purposes.
The biological evaluation showed that some of these molecules
were good AChE inhibitors, in the nanomolar range, and quite
selective regarding the inhibition of BuChE, the most potent
being 5-amino-2-(dimethylamino)-6,7,8,9-tetrahydrobenzo[1,8-
b]-naphthyridine-3-carbonitrile (11). Kinetic studies on the easily
available and potent anticholinesterasic compound 5-amino-2-
(methoxy)-6,7,8,9-tetrahydrobenzo[1,8-b]-naphthyridine-3-carbo-
nitrile (16) showed that this compound is a mixed-type inhibitor
(Ki = 69.2 nM) of EeAChE. Molecular modelling on inhibitor 16 con-
firms that this compound, as expected, and similarly to tacrine,
binds at the catalytic active site of AChE. The modest neuroprotec-
tive profile shown by molecules 11–22 has been investigated in
SH-SY5Y neuroblastoma cells stressed with a mixture of oligomy-
cin-A/rotenone. From these results we conclude that the neuropro-
tective profile of these molecules is rather moderate, the most
potent being compounds 12 and 17 which reduced cell death by
29%. However, one of these molecules, compound 16, was able to
rescue by 50% cell death induced by okadaic acid in SH-SY5Y cells.
Very interestingly, compound 16 does not affect ACh- nor K+-
induced calcium signals in bovine chromaffin cells.
4.1.1. General procedure for the synthesis of 6,7,8,9-
tetrahydrobenzo[1,8-b]-naphthyridin-5-amine derivatives III
Method A.21a In a 20 mL glass tube equipped with septa, pre-
cursors (1 equiv) were dissolved in distilled 1,2-dichloroethane.
To this solutions AlCl3 (1.5 equiv) and cyclohexanone (1.5 equiv)
were added. The reaction mixture was stirred for 30 s, and then
exposed to MW irradiation at 95 °C during the time indicated for
each compound. When the reaction was over (TLC analysis:
CH2Cl2/EtOAc, 10:1, v/v), the reaction mixture was diluted with
a solution of THF/water (1:1); then, an aqueous solution of so-
dium hydroxide (10%) was added till pH 11–12. After stirring
for 30 min, the mixture was extracted with CH2Cl2, dried over
anhydrous sodium sulphate, filtered and the solvent evaporated.
The resultant solid was purified by column chromatography
using methanol/dichloromethane mixtures (1–15%) as eluent to
give pure final products. Method B.28 To solution of the precursor
(1 equiv) and the appropriate 1-alkyl-4-piperidone (1.3 equiv) in
dry ethyl acetate, TMSOTf was added dropwise. The reaction
mixture was heated during the time indicated for each com-
pound. After cooling, the precipitate was filtered, dissolved in a
mixture of EtOH/H2O (1:2) and basified with an aqueous solu-
tion of sodium hydroxide 1 M. After extraction with EtOAc, the
organic layer was dried with anhydrous sodium sulfate and
concentrated. The resulting crude was purified by column
chromatography.
To sum up, tacrine analogues 11–22 can be considered attrac-
tive therapeutic molecules on two key pharmacological receptors
playing key roles in the progression of Alzheimer, that is, choliner-
gic dysfunction and oxidative stress, as well as in neuronal cere-
brovascular diseases.
A
+16
Control
+16
Control
10% Fmax
70K+
ACh
B
150
125
100
75
150
125
100
75
4.1.2. 5-Amino-2-(dimethylamino)-6,7,8,9-
tetrahydrobenzo[1,8-b]-naphthyridine-3-carbonitrile (11)
Following the general procedure (Method A), reaction of com-
pound 121b (200 mg, 1.1 mmol), cyclohexanone (166
lL, 1.6 mmol),
50
50
and AlCl3 (211 mg, 1.6 mmol) in Cl(CH2)2Cl (11 mL), after 85 min of
irradiation and column chromatography (1–15% of MeOH in dichlo-
romethane), gave product 11 (207 mg, 72%): mp 250–252 °C; IR
25
25
0
0
ACh
0.3
1
3
10 30 100
70K0.3
1
3
10
30
100
(KBr)
m
3474, 3299, 3217, 2939, 2850, 2207, 1627, 1599, 1540,
[16] (μM)
[16] (μM)
1519, 1394 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d 8.34 (s, 1H), 4.91
(s, 2H), 3.33 (s, 6H), 2.96 (t, J = 5.6 Hz, 2H), 2.49 (t, J = 5.6 Hz, 2H),
1.87 (m, 4H); 13C NMR (75 MHz, CDCl3) d 165.6, 158.5, 148.7,
142.0, 119.6, 109.9, 104.7, 92.8, 41.2 (2C), 34.9, 23.8, 23.1 (2C);
MS (IE) m/z (%): 267 (M+, 72), 252 (90), 238 (100). Anal. Calcd for
Figure 5. Effects of compound 16 on calcium signals in bovine chromaffin cell
populations. Panel A shows representative recordings of the increase of [Ca2+
in
]
c
bovine chromaffin cell populations stimulated with ACh or K+. Data are expressed
as% of Fmax. Panel B shows averaged data obtained in 12 dishes (from four different
cell cultures) upon application of ACh or K+ in the absence or in the presence of
increasing concentrations of compound 16. Data have been normalized with respect
to the initial response to ACh or K+.
C15H17N5: C, 67.39; H, 6.41; N, 26.20. Found: C, 67.16; H, 6.70; N,
25.98.

130
A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 122–133
4.1.3. 5-Amino-2-pyrrolidin-1-yl-6,7,8,9-tetrahydrobenzo[1,8-
b]-naphthyridine-3-carbonitrile (12)
4.1.7. 5-Amino-2-(methyloxy)-6,7,8,9-tetrahydrobenzo[1,8-b]-
naphthyridine-3-carbonitrile (16)
Following the general procedure (Method A), reaction of com-
Following the general procedure (Method A), reaction of com-
pound 224 (200 mg, 0.94 mmol) with cyclohexanone (146
lL,
pound 625 (87 mg, 0.5 mmol) with cyclohexanone (57
lL,
1.4 mmol) and AlCl3 (186 mg, 1.4 mmol) in Cl(CH2)2Cl (9.4 mL)
after 4.5 h, and column chromatography (1% of MeOH in dichloro-
methane), gave product 12 (272 mg, 98%): mp 270–272 °C; IR (KBr)
0.6 mmol) and AlCl3 (113.3 mg, 0.8 mmol) in Cl(CH2)2Cl (5 mL),
after 32 min, and column chromatography (5% of MeOH in dichlo-
romethane) gave product 16 (87 mg, 95%); mp 259–260 °C; IR
m
3473, 3300, 3216, 2648, 2870, 2210, 1627, 1600, 1539, 1481,
(KBr)
m
3351, 3181, 3058, 2229, 1661, 1594, 1540, 1397, 1271,
;
1437, 1365, 1236 cmꢀ1 1H NMR (300 MHz, CDCl3) d 8.30 (s, 1H),
;
1223 cmꢀ1
1H NMR (400 MHz, DMSO-d6) d 9.36 (s, 1H, H4), 7.23
4.90 (s, 2H), 3.86 (dt, J = 6.6, 3.3 Hz, 4H), 2.95 (t, J = 5.7 Hz, 2H),
2.47 (t, J = 5.8 Hz, 2H), 2.02–1.93 (m, 4H), 1.86 (d, J = 5.7 Hz, 5H);
13C NMR (75 MHz, CDCl3) d 164.8, 155.4, 155.0, 148.8, 141.9,
119.6, 108.8, 103.9, 91.3, 49.2 (2C), 34.4, 25.7 (2C), 23.3, 22.7
(2C); MS (IE) m/z (%): 293 (M+, 57), 263 (76), 262 (100), 238 (67),
198 (17). Anal. Calcd for C17H19N5: C, 69.60; H, 6.53; N, 23.87.
Found: C, 69.55; H, 6.28; N, 23.76.
(m, 2H, NH2), 2.84 (m, 2H, CH2, H6), 1.80 (m, 2H, CH2, H9), 1.22
(m, 4H, 2CH2, H7 and H8); 13C NMR (100 MHz, DMSO-d6) d
163.1, 160.8, 154.0, 150.6, 142.1, 116.0, 109.9, 105.9, 91.9, 54.1,
33.7, 23.2, 22.1, 22.0; MS (API-ES+): 255 [(M+H)+], Anal. Calcd for
C14H14N4O: C, 66.13; H, 5.55; N, 22.03. Found: C, 66.07; H, 5.72;
N, 21.98. The free base was dissolved in dry ethyl ether, and HCl/
ethyl ether was added dropwise with stirring. A white precipitate
was formed immediately. The precipitate was separated by filtra-
tion, washed with ether and dried in vacuo to afford a white pow-
4.1.4. 5-Amino-2-piperidin-1-yl-6,7,8,9-tetrahydrobenzo[1,8-b]-
naphthyridine-3-carbonitrile (13)
der: mp 265–268 °C; IR (KBr)
m 3060, 2944, 2234, 1661, 1629,
Following the general procedure (Method A), reaction of com-
1602, 1477, 1408, 1305 cmꢀ1; Anal. Calcd for C14H15ClN4O: C,
57.83; H, 5.20; N, 19.27. Found: C, 57.58; H, 5.48; N, 19.42.
pound 324 (250 mg, 1.1 mmol) with cyclohexanone (170
lL,
1.6 mmol) and AlCl3 (217 mg, 1.6 mmol) in Cl(CH2)2Cl (11 mL),
after 3 h and 45 min, and column chromatography (1–5% of MeOH
in dichloromethane), gave product 13 (310 mg, 91%): mp 240–
4.1.8. 5-Amino-2-(ethyloxy)-6,7,8,9-tetrahydrobenzo[1,8-b]-
naphthyridine-3-carbonitrile (17)
242 °C; IR (KBr)
m
3471, 3307, 3221, 2929, 2852, 2831, 2216,
Following the general procedure (Method A), reaction of com-
1630, 1600, 1542, 1433, 1347, 1281, 1253, 1238 cmꢀ1
;
1H NMR
pound 726 (200 mg, 1.1 mmol) with cyclohexanone (165
lL,
(300 MHz, CDCl3) d 8.62 (s, 1H), 5.54 (s, 2H), 3.68 (s, 4H), 2.98 (t,
J = 5.4 Hz, 2H), 2.49 (t, J = 5.4 Hz, 2H), 1.86 (d, J = 5.1 Hz, 4H), 1.64
(s, 6H); 13C NMR (75 MHz, CDCl3) d 163.3, 159.3, 154.2, 149.7,
142.1, 118.5, 109.8, 104.6, 94.7, 49.6 (2C), 33.6, 26.0 (2C), 24.6,
23.3, 22.5, 22.5; MS (IE) m/z (%): 307 (M+, 99), 278 (100), 265
(41), 251 (38), 224 (44), 198 (38). Anal. Calcd for C18H21N5: C,
70.33; H, 6.89; N, 22.78. Found: C, 70.15; H, 6.88; N, 22.59.
1.6 mmol) and AlCl3 (213 mg, 1.6 mmol) in Cl(CH2)2Cl (10 mL),
after 30 min, and column chromatography (5% of MeOH in dichlo-
romethane), gave product 17 (260 mg, 92%): mp 265–267 °C; IR
(KBr)
m
3333, 3232, 2929, 2851, 2222, 1636, 1614, 1455,
;
1296 cmꢀ1
1H NMR (300 MHz, DMSO-d6) d 9.16 (s, 1H, CH), 6.94
(br s, 2H, NH2), 4.48 (q, J = 7.1 Hz, 2H, CH2), 2.79 (m, 2H, CH2),
2.46 (t, J = 6.0 Hz, 2H, CH2), 1.80 (m, 4H, 2 x CH2), 1.38 (t,
J = 7.1 Hz, 3H, CH3). 13C NMR (75 MHz, DMSO-d6) d 163.0, 160.5,
154.0, 150.6, 142.1, 116.0, 109.9, 105.8, 92.0, 62.5, 33.6, 23.2,
22.1, 22.0, 14.2; MS (IE) m/z (%): 268 (M+, 59), 253 (89), 240
(100), 223 (36). Anal. Calcd for C15H16N4O: C, 67.15; H, 6.01; N,
20.88. Found: C, 67.03; H, 6.28; N, 21.04.
4.1.5. 5-Amino-2-(prop-2-yn-1-ylamino)-6,7,8,9-
tetrahydrobenzo[1,8-b]-naphthyridine-3-carbonitrile (14)
Following the general procedure (Method A), reaction of com-
pound 421b (197 mg, 1.0 mmol) with cyclohexanone (0.15 mL,
1.5 mmol) and AlCl3 (198 mg, 1.5 mmol) in Cl(CH2)2Cl (10 mL),
after 3.5 h, and column chromatography (5% of MeOH and 0.5%
of Et3N in dichloromethane), gave product 14 (215 mg, 78%): mp
4.1.9. 5-Amino-2-methoxy-4-phenyl-6,7,8,9-
tetrahydrobenzo[1,8-b]-naphthyridine-3-carbonitrile (18)
Following the general procedure (Method A), reaction of com-
>230 °C; IR (KBr)
m
3470, 3368, 3294, 3222, 2940, 2214, 1628,
;
1613, 1548, 1520 cmꢀ1
1H NMR (300 MHz, CDCl3) d 8.98 (s, 1H,
pound 827 (300 mg, 1.2 mmol) with cyclohexanone (180
lL,
CH), 7.67 (br s, 1H, NH), 6.48 (s, 4H), 2.98 (t, J = 5.4 Hz, 2H), 2.49
(t, J = 5.4 Hz, 2H), 1.86 (d, J = 5.1 Hz, 4H), 1.64 (s, 6H); 13C NMR
(75 MHz, CDCl3) d 161.4, 155.2, 154.3, 141.1, 141.2, 116.7, 108.5,
103.5, 90.8, 82.0, 72.2, 32.8, 30.0, 23.0, 22.0 (2C); MS (IE) m/z (%):
277 (M+,100), 262 [(MꢀCN)+, 15], 251 (21), 223 (38), 205 (25).
Anal. Calcd for C16H15N5ꢁHClꢁ3/2H2O: C, 56.39; H, 5.62; Cl, 10.40;
N, 20.55. Found: C, 56.50; H, 5.37; Cl, 9.84; N, 20.29.
1.8 mmol) and AlCl3 (237 mg, 1.8 mmol) in Cl(CH2)2Cl (12 mL),
after 12 h, and column chromatography (1–5% of MeOH in dichlo-
romethane), gave product 18 (266 mg, 67%); mp 300–302 °C; IR
(KBr)
m
3515, 3419, 3392, 3036, 2937, 2862, 2223, 1591, 1574,
1547, 1504, 1431, 1379, 1314, 1269, 1161 cmꢀ1
;
1H NMR
(300 MHz, CDCl3) d 7.74–7.52 (m, 3H), 7.54–.33 (m, 2H), 4.41 (br
s, 2H, NH2), 4.21 (s, 3H, CH3), 3.04 (t, J = 5.5 Hz, 2H), 2.31 (t,
J = 5.3 Hz, 2H), 1.88 (d, J = 2.7 Hz, 4H). 13C NMR (75 MHz, CDCl3) d
163.9, 161.6, 156.8, 155.1, 150.0, 136.5, 130.6, 129.6 (2C), 128.0
(2C), 115.0, 111.5, 104.6, 97.2, 55.1, 34.4, 23.4, 22.6, 22.5; MS
(API-ES+): 331 [(M+H)+, 100], 353 [(M+Na)+, 18]. Anal. Cald. for
4.1.6. 5-Amino-2-(dimethylamino)-4-phenyl-6,7,8,9-
tetrahydrobenzo[1,8-b]-naphthyridine-3-carbonitrile (15)
Following the general procedure (Method A), reaction of com-
pound 521b (200 mg, 0.8 mmol), cyclohexanone (118
l
L, 1.1 mmol)
C20H18N4O: C, 72.71; H, 5.49; N, 16.96. Found: C, 72.58; H, 5.20;
and AlCl3 (150 mg, 1.1 mmol) in Cl(CH2)2Cl (6 mL) after 2 h, and
column chromatography (10% of EtOAc and 1% of Et3N in dic-
hlormethane), gave compound 15 (124 mg, 47%): mp 222–
N, 16.70.
4.1.10. 5-Amino-2-chloro-6,7,8,9-tetrahydrobenzo[1,8-b]-
naphthyridine-3-carbonitrile (19)
224 °C; IR (KBr) 3484, 2928, 2210, 1625, 1557, 1390 cmꢀ1 1H
;
NMR (300 MHz, CDCl3) d 7.74–7.46 (m, 3H), 7.49–7.29 (m, 2H),
4.28 (s, 2H), 3.32 (s, 6H), 2.98 (t, J = 5.1 Hz, 2H), 2.26 (t, J = 5.8 Hz,
2H), 1.83 (d, J = 6.5 Hz, 4H); 13C NMR (75 MHz, CDCl3) d 163.4,
158.3, 157.2, 154.6, 149.5, 136.8, 129.9, 129.4 (2C), 128.0 (2C),
116.9, 109.3, 102.8, 94.8, 40.7 (2C), 33.7, 23.0, 22.3, 22.1; MS
(API-ES+): 344 [(M+H)+]. Anal. Calcd for C21H21N5: C, 73.44; H,
6.16; N, 20.39. Found: C, 73.17; H, 6.42; N, 20.10.
Following the general procedure (Method A), reaction of com-
pound 921b (200 mg, 1.1 mmol) with cyclohexanone (174
lL,
1.7 mmol) and AlCl3 (221 mg, 1.7 mmol) in Cl(CH2)2Cl (11.2 mL),
after 4.5 h of irradiation, and column chromatography (1–5% of
MeOH in dichloromethane), gave product 19 (88 mg, 30%): mp
>270 °C; IR (KBr)
m
3351, 3181, 3058, 2229, 1661, 1594, 1540,
;
1397, 1271, 1223 cmꢀ1
1H NMR (300 MHz, DMSO-d6) d 9.36 (s,

A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 122–133
131
1H, H4), 7.23 (br s, 2H, NH2), 2.84 (m, 2H, H6), 1.80 (m, 2H, H9),
1.22 (m, 4H, H7 and H8); 13C NMR (75 MHz, DMSO-d6) d 165.4,
154.6, 151.4, 149.5, 143.0, 116.6, 112.6, 109.6, 102.6, 27.9, 22.7,
20.6, 20.1; MS (API-ES+): 259 [(M+H)+, 100] 281 [(M+Na)+], 353.
Anal. Calcd for C13H11ClN4: C, 60.35; H, 4.29; N, 21.66; Cl, 13.70.
Found: C, 60.18; H, 4.49; N, 21.37; Cl, 13.49.
tion (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-nitrobenzoic acid (DTNB, Sigma
Aldrich, Madrid, Spain). Inhibition curves were made by pre-incu-
bating this mixture with at least nine concentrations of each com-
pound for 10 min. A sample with no compound was always present
to determine the 100% of enzyme activity. After this pre-incubation
period, acetylthiocholine iodide (ATChI, 0.35 mM) or butyrylthi-
ocholine iodide (BuTChI, 0.5 mM) (Sigma Aldrich, Madrid, Spain)
were added, allowing 15 min more of incubation, where the DTNB
produces the yellow anion 5-thio-2-nitrobenzoic acid along with
the enzymatic degradation of ATChI or BuTChI. Changes in absor-
bance were detected at 405 nm in a spectrophotometric plate read-
er (FluoStar OPTIMA, BMG Labtech). Compounds inhibiting AChE
or BuChE activity would reduce the color generation, thus IC50 val-
ues were calculated as the concentration of compound that pro-
duces 50% AChE activity inhibition. Data are expressed as
means SEM of at least three different experiments in
quadruplicate.
4.1.11. 5-Amino-2-chloro-4-phenyl-6,7,8,9-
tetrahydrobenzo[1,8-b]-naphthyridine-3-carbonitrile (20)
Following the General procedure (Method A), reaction of com-
pound 1021b (100 mg, 0.4 mmol) with cyclohexanone (174
lL,
1.7 mmol) and AlCl3 (78 mg, 0.6 mmol) in Cl(CH2)2Cl (5 mL), after
60 h, and column chromatography (from 1% to 4% of MeOH in
dichloromethane), afforded product 20 (30 mg, 20%): mp
>270 °C; IR (KBr)
m
3498, 3334, 3230, 2931, 1725, 1629, 1571,
1540, 1445, 1269, 1223 cmꢀ1
;
1H NMR (300 MHz, DMSO-d6) d
7.68 (d, 2H, 2CH, 20,60-Py), 7.59 (d, 2H, 2CH, 30,50-Py), 5.50 (s, 2H,
NH2), 2.85 (m, 2H, CH2, H6), 2.31 (m, 2H, CH2, H9), 1.78 (m, 4H,
2CH2, H7, H8); 13C NMR (75 MHz, DMSO-d6) 158.1, 156.5, 154.5,
153.9, 147.5, 133.5, 131.6, 130.5 (2C), 127.9 (2C), 113.8, 113.6,
109.6, 106.6, 27.9, 22.7, 20.6, 20.1; MS (API-ES+): 335.2 [(M+1)+,
100], 357.2 [(M+Na)+]. Anal. Calcd for C19H15N4Cl: C, 68.16; H,
4.52; N, 16.73. Found: C, 67.98; H, 4.70; N, 16.91.
4.2.2. Kinetic analysis of the AChE inhibition
To obtain estimates of the competitive inhibition constant Ki,
reciprocal plots of 1/V versus 1/[S] were constructed at different
concentrations of the substrate acetylthiocholine (0.1–1 mM) by
using Ellman’s method.29 Experiments were performed in a trans-
parent 48-well plate, with a final volume of 1 mL, containing each
4.1.12. 5-Amino-7-benzyl-2-methoxy-6,7,8,9-
tetrahydropyrido[2,3-b][1,6]naphthyridine-3-carbonitrile (21)
Following the general procedure (Method B),28 reaction of com-
pound 625 (200 mg, 1.2 mmol) with 1-benzyl-4-piperidone
(0.26 mL, 1.5 mmol) and TMSOTf (0.48 mL, 2.6 mmol) in dry EtOAc
(2.4 mL) after 20 h, and recrystallization from EtOH, gave pure
well DTNB 0.35 mM, 1
give desired final concentration. Reaction was initiated by adding
45 L of AChE (final concentration 0.18 U/mL) at 30 °C to give a fi-
lL DMSO (control) or inhibitor solution to
l
nal. Progress curves were monitored at 412 nm for 2 min in a fluo-
rescence plate reader Fluostar Optima (BMG-technologies,
Germany) absorbance ready. Progress curves were characterized
by a linear steady-state turnover of the substrate and values of a
linear regression were fitted according to Lineweaver–Burk replots
using Origin software. The plots were assessed by a weighted least
square analysis. Determination of Michaelis constant for the sub-
strate ATCh was done at five different concentrations (0.1–1 mM)
product 21 (320 mg, 81%): mp 210–212 °C; IR (KBr)
m 3462,
3375, 2225, 1618, 1552, 1351, 1293 cmꢀ1 1H NMR (300 MHz,
;
CDCl3) d 8.45 (s, 1H), 7.42–7.25 (m, 5H), 4.84 (s, 2H), 4.16 (s, 3H),
3.78 (s, 2H), 3.48 (s, 2H), 3.15 (t, J = 5.8 Hz, 2H), 2.87 (t, J = 5.8 Hz,
2H); 13C NMR (75 MHz, CDCl3) d 162.8, 155.4, 147.7, 140.2,
138.3, 129.6 (2C), 129.1 (2C), 128.1, 116.2, 110.1, 106.6, 95.6,
63.4, 55.6, 51.3, 50.3, 34.6; MS (IE) m/z (%): 345 (M+, 55), 344
(100), 328 (30), 254 (49), 91 (44). Anal. Calcd for C20H19N5O: C,
69.55; H, 5.54; N, 20.28. Found: C, 69.51; H, 5.36; N, 20.02.
to give a value of KM = 0.27 0.02 mM, and Vmax = 1.0 0.1 minꢀ1
Slopes of the reciprocal plots were then plotted against the concen-
tration of 16 (range 0–0.3
M) as described,42 to evaluate Ki data.
.
l
Data analysis was performed with Origin Pro 7.5 software (Origin
4.1.13. 5-Amino-7-benzyl-2-chloro-6,7,8,9-
Lab Corp.).
tetrahydropyrido[2,3-b][1,6]naphthyridin-3-carbonitrile (22)
Following the general procedure (Method B),28 reaction of com-
pound 921b (200 mg, 1.1 mmol) with 1-benzyl-4-piperidone
(0.26 mL, 1.4 mmol) and TMSOTf (0.47 mL, 2.6 mmol) in dry EtOAc
(2.3 mL) after 48 h, and purification by column chromatography
(5% MeOH and 0.5% Et3N in dichloromethane), gave the product
4.2.3. Molecular modelling of inhibitor 16
The X-ray crystal structure of Torpedo californica acetylcholines-
terase (PDB code 1ACJ)43 at 2.80 Å resolution was retrieved from
the Protein Data Bank (http://www.rcsb.org/pdb/). Charges were
assigned to ligand atoms (compound 16) with MOPAC (MOPAC)44
ESP with MNDO method.45 The Lamarckian genetic algorithm
implemented in AUTODOCK 4.046 (http://autodock.scripps.edu/) was
used to generate docked conformations of inhibitor 16 within the
binding site by randomly changing the overall orientation of the
molecule as well as the torsion angles of all routable bonds. A vol-
ume of exploration was defined in the shape of a three-dimen-
sional cubic grid (40 ꢂ 40 ꢂ 40 Å3) with a spacing of 0.375 Å. At
each grid point, the enzyme’s atomic affinity potentials for carbon,
aromatic carbon, oxygen, nitrogen and hydrogen atoms were recal-
culated for rapid intra- and intermolecular energy evaluation of the
docking solutions 16.
22 (211 mg, 54%): mp >300 °C; IR (KBr)
m
3345, 3244, 2845,
2756, 2232, 1629, 1592, 1541, 1347, 1273 cmꢀ1
;
1H NMR
(300 MHz, DMSO-d6) d 9.39 (s, 1H), 7.54–7.16 (m, 5H), 3.75 (s,
2H), 3.49 (s, 2H), 2.93 (t, J = 5.7 Hz, 2H), 2.75 (t, J = 5.7 Hz, 2H);
13C NMR (75 MHz, DMSO-d6) d 162.5, 154.3, 149.2, 149.1, 142.4,
137.9, 128.8 (2C), 128.2, 127.0, 115.9, 110.3, 109.3, 102.20, 61.4,
51.0, 48.7, 33.5; MS (IE) m/z (%): 350 (M+, 20), 348 (45), 258 (48),
91 (100). Anal. Calcd for C19H16ClN5: C, 65.24; H, 4.61; N, 20.02;
Cl, 10.13. Found: C, 64.99; H, 4.37; N, 19.93; Cl, 10.18.
4.2. Pharmacology
4.2.1. Inhibition experiments of AChE and BuChE
4.2.4. Molecular dynamics simulations (MD)
To assess the inhibitory activity of the compounds towards
AChE or BuChE, we followed the spectrophotometric method of Ell-
man,29 using purified AChE from Electrophorus electricus (Type V-S),
bovine erythrocytes (Type XII-S) or BuChE from horse serum
(lyophilized powder) (Sigma Aldrich, Madrid, Spain). The reaction
took place in a final volume of 3 mL of a phosphate-buffered solu-
The MD simulations were carried out using the AMBER 10 suite of
programs.47 The bonded and no bonded parameters were assigned
from those already present in the AMBER database in a way consisted
with the second-generation force field (parm 99).48 The molecular
system was neutralized by the addition of three chlorine ions,49
and immersed in a truncated octahedron of ꢃ3760 TIP3P water

132
A. Samadi et al. / Bioorg. Med. Chem. 19 (2011) 122–133
molecules.50 Periodic boundary conditions were treated using the
smooth particle mesh Ewald method51 with a grid spacing of 1 Å.
The cutoff distance for the non-bonded interactions was 9 Å. The
SHAKE algorithm52 was applied to all bonds and an integration step
of 2.0 fs was used throughout. Solvent molecules and counter ions
were relaxed by energy minimization and allowed to redistribute
around the positionally restrained solute (25 kcal molꢀ1 Å2) during
50 ps of MD at constant temperature (300 K) and pressure (1 atm).
These initial harmonic restraints were gradually decreased in a ser-
ies of progressive energy minimizations until they were com-
pletely removed. The resulting system was heated again from
100 to 300 K during 20 ps and allowed to equilibrate in the absence
of any restraints for 2 ns during which system coordinates were
collected every 2 ps for further analysis. The resulting trajectories
and structures were visually inspected using the computer
additional 24-h period. At the end of the experiments, cell death
was assessed by measuring the activity of lactate dehydrogenase
(LDH) released.
4.2.9. Neuroprotection experiments with rotenone/oligomycin-
A
The neuroprotective effect of the compounds against this toxic
stimulus was evaluated with the method of MTT reduction, at
the concentration of 1
l
M on SH-SY5Y neuroblastoma cells, ex-
M oligomycin-A for 24 h.
posed to 30 M rotenone plus 10
l
l
4.2.10. Isolation and culture of bovine chromaffin cells
Bovine chromaffin cells were isolated from adrenal glands of
adult cows, following standard methods57 with some modifica-
tions.58 Cells were suspended in Dulbeccos modified Eagles med-
´
´
53
graphics program PYMOL and analyzed with the PTRAJ module of
AMBER 10.
ium (DMEM) supplemented with 5% foetal calf serum, 10
cytosine arabinoside, 10
M fluorodeoxyuridine, 50 IU mlꢀ1 peni-
cillin and 50 g/ml streptomycin. Cells were plated at a density
lM
l
l
4.2.5. MM-GBSA binding calculations
of 2 ꢂ 105 cells per well into 96-well plates. Cells were kept for
2 days at 37 °C in a water-saturated incubator, with a 5% CO2/
95% air atmosphere. Experiments were performed at room temper-
ature (24 2 °C).
Effective binding free energy was qualitatively estimated using
the MM-GBSA approach.54 MM-GBSA method approaches free en-
ergy of binding as a sum of molecular mechanics (MM) interaction
term, a salvations contribution thorough a generalized Born (GB)
model, and a surface area (SA) contribution to account for the
non polar part of desolvation. These calculations were performed
for each snapshot from the simulation using the appropriate mod-
ule within AMBER 10 and averaged out.
4.2.11. Measurement of [Ca2+]c in bovine chromaffin cells
Cells were loaded with Krebs-HEPES (in mM: 144 NaCl, 5.9 KCl,
1.2 MgCl2, 2 CaCl2, 11
D-glucose, 10 HEPES, pH 7.4) containing
10 M fluo-4 AM, and 0.2% pluronic acid for 45 min at 37 °C in
l
4.2.6. Quantification of viability by the MTT reduction method
in SH-SY5Y cells
the dark. After this incubation period, cells were washed twice
with Krebs-HEPES at room temperature in the dark. Changes in
fluorescence (excitation 485 nm, emission 520 nm) were measured
using a fluorescent plate reader (Fluostar, BMG Labtechnologies).
Basal levels of fluorescence were monitored before adding stimula-
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-diphenyl-tetrazolium bromide, Sig-
ma Aldrich, Madrid, Spain), as described previously.55 SH SY5Y
cells were seeded into 48-well culture 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 spectrophoto-
metrically measurement at 540 nm. The tetrazolium ring of MTT
can be cleaved by active reductases in order to produce a precipi-
tated formazan derivative. The formazan produced was dissolved
tion solutions (100 l
M ACh or 70 mM K+) by using an automatic
dispenser. After stimulation, changes in fluorescence were mea-
sured for 40 seg. To normalize fluo-4 signals, responses from each
well were calibrated by measuring maximum and minimum fluo-
rescence values. At the end of each experiment, addition of 3% Tri-
ton X-100 (Fmax) was followed by addition of 1 M MnCl2 (Fmin).
Data were calculated as a percentage of Fmax ꢀ Fmin
.
by adding 300 lL DMSO, resulting in a colored compound whose
optical density was measured in an ELISA reader at 540 nm. All
Acknowledgments
MTT assays were performed in triplicate.
A. Samadi thanks CSIC for a I3P-post-doc contract. M. Chioua
thanks Instituto de Salud Carlos III (MICINN) for a ‘Sara Borrell’
post-doctoral contract. J.M.C. thanks MICINN (SAF2006-08764-
C02-01, SAF2009-07271), ISCIII [Red RENEVAS (RD06/0026/0009,
RD06/0026/1002)]), CAM (S/SAL-0275-2006), and CSIC-GRICES
(2007PT-13) financial support. The present work has also been
supported by Fundación Teófilo Hernando, MEC Grants BFI2003-
02722, SAF-2006-08540, SAF2006-1249 and CTQ2005-09365, and
Fundación La Caixa (Barcelona, Spain). L.G. thanks MICINN for sup-
port (SAF 2007-65181). M.G.L. thanks MICINN for support (SAF
2009-12150). M.V. thanks Fundación CIEN for support (Instituto
de Salud Carlos III). Ágatha Bastida thanks Eva Priego (IQM, CSIC)
for the molecular dynamic analysis of compound 16.
4.2.7. Measurement of lactate dehydrogenase activity
Samples of incubation media were collected at the end of a 24 h
period of exposure to the toxic to estimate extracellular LDH, an
indication of cell death.56 LDH activity was also measured in the
cells after treatment with 10% Triton X-100 (intracellular LDH).
LDH activity was measured spectrophotometrically at 490–
620 nm, using a microplate reader (Labsystems iEMS reader MF;
Labsystems, Helsinki, Finland). Total LDH (intracellular plus extra-
cellular) was normalized to 100%; then, the amount of LDH re-
leased to the extracellular medium was expressed as percentage
of this total. Data were normalized by subtracting basal LDH (cells
not subjected to any treatment) to the different treatment groups
in each individual experiment, and the result for rot/oligo group
was normalized to 100% (percentage cell death).
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