New tacrine-dihydropyridine hybrids that inhibit acetylcholinesterase, calcium entry, and exhibit neuroprotection properties

Article abstract of DOI:10.1016/j.bmc.2008.07.005

In this communication, we describe the synthesis and biological evaluation of tacripyrimedones 1-5, a series of new tacrine-1,4-dihydropyridine hybrids bearing the general structure of 11-amino-12-aryl-3,3-dimethyl-3,4,5,7,8,9,10,12-octahydrodibenzo[b,g][1, 8]naphthyridine-1(2H)-one. These multifunctional compounds are moderately potent and selective AChEIs, with no activity toward BuChE. Kinetic analysis and molecular modeling studies point out that the new compounds preferentially bind the peripheral anionic site of AChE. In addition, compounds 1-5 show an excellent neuroprotective profile, and a moderate blocking effect of L-type voltage-dependent calcium channels due to the mitigation of [Ca²⁺] elevation elicited by K⁺ depolarization. Therefore, they represent a new family of molecules with potential therapeutic application for the treatment of Alzheimer's disease.

Full text of DOI:10.1016/j.bmc.2008.07.005

Bioorganic & Medicinal Chemistry 16 (2008) 7759–7769
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New tacrine-dihydropyridine hybrids that inhibit acetylcholinesterase,
calcium entry, and exhibit neuroprotection properties
d
e
Rafael León a,b, , Cristóbal de los Ríos a,b, José Marco-Contelles a, , Oscar Huertas , Xavier Barril ,
*
*
F. Javier Luque d, , Manuela G. López , Antonio G. García b,c, Mercedes Villarroya b,
b
*
*
a Laboratorio de Radicales Libres (IQOG, CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain
b Instituto Teófilo Hernando, Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo 4, 28029 Madrid, Spain
c Servicio de Farmacología Clínica, Hospital Universitario de la Princesa, C/Diego de León 62, 28006 Madrid, Spain
d Departament de Fisicoquímica and Institut de Biomedicina (IBUB), Facultat de Farmàcia, Universitat de Barcelona, Avda. Diagonal 643, Barcelona, Spain
e Institució Catalana de Recerca i Estudis Avançats (ICREA), Departament de Fisicoquímica and Institut de Biomedicina, Facultat de Farmàcia, Universitat de Barcelona,
Avda. Diagonal 643, Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
In this communication, we describe the synthesis and biological evaluation of tacripyrimedones 1–5, a
series of new tacrine-1,4-dihydropyridine hybrids bearing the general structure of 11-amino-12-aryl-
3,3-dimethyl-3,4,5,7,8,9,10,12-octahydrodibenzo[b,g][1,8]naphthyridine-1(2H)-one. These multifunc-
tional compounds are moderately potent and selective AChEIs, with no activity toward BuChE. Kinetic
analysis and molecular modeling studies point out that the new compounds preferentially bind the
peripheral anionic site of AChE. In addition, compounds 1–5 show an excellent neuroprotective profile,
and a moderate blocking effect of L-type voltage-dependent calcium channels due to the mitigation of
[Ca2+] elevation elicited by K+ depolarization. Therefore, they represent a new family of molecules with
potential therapeutic application for the treatment of Alzheimer’s disease.
Received 6 February 2008
Revised 24 June 2008
Accepted 2 July 2008
Available online 8 July 2008
Keywords:
Tacrine-dihydropyridine hybrids
AChE
BuChE
Kinetic analysis
Ó 2008 Elsevier Ltd. All rights reserved.
Inhibition mechanism
Voltage-dependent calcium channels
Neuroprotection
Molecular modeling
1. Introduction
ments Ab formation,5a and hyperphosphorylation.5b Ca2+ entry
s
through the L-subtype of high-voltage activated Ca2+ channels
(Cav1.1–1.4) causes both Ca2+ overload and mitochondrial disrup-
tion, which activate the apoptotic cascade and cell death.6 Hence,
blocking the entrance of Ca2+ through calcium channels could be
a good strategy to prevent cell death.
Alzheimer’s disease (AD) is an age-related neurodegenerative
disease characterized by progressive memory loss and other cogni-
tive impairments. Although the etiology of AD is not well known,
there are diverse factors such as amyloid-b (Ab) deposits, s-protein
aggregation, oxidative stress, or low levels of acetylcholine (ACh)
that are thought to play significant roles in the disease.1 The cho-
linergic theory of AD suggests that the selective loss of cholinergic
neurons in AD results in a deficit of ACh in specific regions of the
brain that mediate learning and memory functions.2 The primary
approach for treating AD has, therefore, aimed at increasing the
ACh levels in the brain by using acetylcholinesterase inhibitors
(AChEIs) such as donepezil, galantamine, or rivastigmine.3 On the
other hand, Ca2+ overload seems to be the main factor initiating
the processes leading to cell death. Several lines of evidence show
that Ca2+ dysfunction, involved in the pathogenesis of AD,4 aug-
The current multi-target approach in drug design7 for the treat-
ment of AD includes novel tacrine–melatonin hybrids,8a dual
inhibitors of AChE and monoamine oxidase (MAO),8b dual AChEI
and serotonin transporters inhibitors,8c potent AChEIs with antiox-
idant and neuroprotective properties,8d or gallamine–tacrine hy-
brids designed to bind the cholinergic and M2 muscarinic
receptors.8e
Since 1,4-dihydropyridines (DHPs) are compounds that selec-
tively block L-type Ca2+ channels, we considered the synthesis
and the pharmacological study of new multipotent hybrid com-
pounds, based on an AChEI and a DHP, such as tacrine and nimodi-
pine (Chart 1).9 Besides inhibition of AChE and blockade of voltage-
dependent calcium channels (VDCC), we were also interested in
compounds targeted to prevent oxidative stress. Recent research
has demonstrated that oxidative damage is an event that precedes
* Corresponding authors. Tel.: +34 91 5622900; fax: +34 91 5644853.
E-mail addresses: iqoc21@iqog.csic.es (J. Marco-Contelles), fjluque@ub.edu (F.
Javier Luque).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.005

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R. León et al. / Bioorg. Med. Chem. 16 (2008) 7759–7769
X
NH2
O
NH2
N
EtO
N
N
H
Tacrine (AChEI)
Tacripyrines (I) (ref. 13)
NO2
X
O
O
O
NH2
N
OMe
O
O
N
N
H
H
Nimodipine (1,4-DHP)
Tacripyrimedones (II) (this work)
Chart 1.
the appearance of other pathological hallmarks of AD.10 Thus,
drugs that scavenge reactive oxygen species (ROS) may have a par-
ticular therapeutic efficacy.11,12
activity for the novel tacrine-DHP hybrids tacripyrimedones 1–5
(Scheme 1).
Based on this strategy, we have recently reported preliminary
studies on the synthesis and biological activity of ethyl esters of 5-
amino-4-aryl-1,4,6,7,8,9-hexahydro-2-methyl-benzo[b][1,8]naph-
thyridine-3-carboxylic acid (I) (Chart 1) that we have named as
tacripyrines, as the first examples of tacrine-DHP hybrids described
in the literature.13 Prompted by the interesting biological profile of
these compounds (very selective and potent AChEIs, excellent
neuroprotective profile and moderate Ca2+ channel blockade ef-
fects), we have now prepared new analogs of the general structure
II (Chart 1), for which we propose the short name of tacripyrime-
dones, where we have incorporated a functionalized cyclohexane
ringannulated ontotheDHP-ring present in type I compounds, in or-
der to start a general structure–activity relationship (SAR) analysis
targeted to improve the biological activities found in the lead-type
compounds I.13
2. Results and discussion
2.1. Synthesis of 11-amino-12-aryl-3,3-dimethyl-3,4,5,7,8,9,10,12-
octahydrodibenzo[b,g][1,8]naphthyridine-1(2H)-ones (1–5)
The synthesis of tacripyrimedones 1–5 was easily achieved, in
excellent yields, using the Friedländer reaction14 between 2-ami-
no-4-aryl-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carbonitriles (6–10)15 and cyclohexanone, under standard condi-
tions (Scheme 1).16 Precursors 6–10, readily available compounds
in multigram quantities, are prepared by refluxing equimolar
amounts of the corresponding arylidenemalononitrile, dimedone,
and an excess of ammonium acetate in acetic acid as solvent.15
Compounds 1–5 are racemic octahydrobenzo[b,g][1,8]naph-
thyridines substituted at C12 with an aromatic ring, incorporating
different types of substituents at C40 (1–4) or a 40-pyridyl ring (5).
These molecules have been conveniently characterized by their
spectroscopic and analytical data. The 1H and 13C NMR data (see
Section 4) are in good agreement with the values found for related
Accordingly, in this work we report the synthesis and biological
evaluation, including AChE/BuChE (butyrylcholinesterase) inhibi-
tion, kinetic analysis, and molecular modeling of the AChE inhibi-
tion, blockade of cytosolic Ca2+ elevation, and the neuroprotective
X
X
AlCl3,
O
O
NH2
N
O
1,2-dichloroethane
reflux
CN
N
N
NH2
H
H
1
6
7
8
9
(97%)
X= C-H
X= C-F
2 (90%)
3 (96%)
4 (83%)
X= C-Me
X= C-OMe
10 X= N
5
(88%)
Scheme 1.

R. León et al. / Bioorg. Med. Chem. 16 (2008) 7759–7769
7761
compounds,13,15 and the assignments are based on standard NMR
experiments (1H–1H COSY, HMQC, and HMBC).
activity,15b had significantly higher AChE IC50 values than those rel-
ative to 1–5, which were nearly 100-fold more potent (Table 2),
even though the only structural difference was the replacement
of the oxygen atom at C4 in 19–22 by the N–H unit in 1–5 (Chart
2). These results agree with previous works from our laboratory13
where the insertion of the –NH– unit in the heterocyclic ring en-
hanced the AChE inhibitory activity for type I compounds (about
100-fold more active than compounds 15–18). SAR study of these
derivatives and analogs also pointed out the importance of the NH
unit present in the dihydropyridine ring.
Previous studies from our laboratories concluded that com-
pounds 15 and 16 proved to be non-competitive inhibitors of AChE
(Chart 2),19 which suggest interaction at the PAS of AChE. This
assumption was supported by molecular modeling studies, which
revealed a large structural distortion upon forcing binding of 16
at the catalytic site of AChE.19a Moreover, since compounds 1–5
are bulkier than compound 16 due to the tetracyclic system, access
to the AChE catalytic site through the narrow gorge would be more
difficult, thus supporting the feasibility of the interaction with the
PAS of the enzyme. On the other hand, even though AChE and
BuChE share most of the structural and physicochemical proper-
ties,21a BuChE does not possess a peripheral site. This structural
difference would thus explain why compounds 1–5 are strong
inhibitors of AChE, but have no relevant activity on BuChE.
2.2. Pharmacology
2.2.1. Studies of AChE/BuChE inhibition
According to standard methodology17,18 (see Section 4), tacripy-
rimedones 1–5 have been evaluated as possible inhibitors of AChE
from both Electrophorus electricus (electric eel) and human erythro-
cytes, and of BuChE from human serum. The IC50 values corre-
sponding to compounds 1–5, and those observed for related
AChEIs 11–14 as well as for tacrine, which is used as reference
compound,13 are shown in Table 1.
Regardless of the AChE origin (Ee or human erythrocytes), com-
pounds 1–5 were submicromolar AChE inhibitors with similar po-
tency for the inhibition of both AChEs, as noted by IC50 values
ranging from 0.22 to 0.60
2.80 M (for hAChE), which were also similar to the IC50 value
determined for tacrine (0.18 and 0.15 M, respectively). Com-
lM (for EeAChE) and from 0.37 to
l
l
pounds 1–5 were much more selective than tacrine versus AChE;
none of the new tacrine-DHP hybrids inhibited this enzyme (the
hBuChE/hAChE ratio varies from >36 to >270; Table 1).
Considering the type of substituent at C40 in the aromatic ring,
the substitution of the hydrogen in the parent compound (1) by an
electron-withdrawing group (40-F, 40-pyridyl) reduced the EeAChE
inhibition by a factor of 1.6–1.8, while the incorporation of an elec-
tron-donating substituent (Me and OMe) led to a 1.4-fold increase
in the AChE inhibition. Similar trends were found for the depen-
dence of the hAChE inhibition on the substituent at the aromatic
ring. Finally, all the new derivatives 1–5 were less active than com-
pounds 11–1413 (Table 1) on AChE by a factor ranging from 2.5
(3.5) for the pair of compounds 3 and 13 to 10 (8.2) for the pair
2 and 12 in EeAChE (hAChE).
2.2.2. Mechanism of AChE inhibition
One of the hallmarks of AD lies in the formation of senile pla-
ques by accumulation of the Ab peptide. Different studies have
proven that AChE binds to Ab inducing the formation of fibrils21b
and that the PAS of the enzyme is involved in catalyzing the aggre-
gation.22 Ligands like propidium iodide that bind to the PAS are
known to block Ab aggregation.22 Consequently, inhibitors which
are able to bind the PAS can be of utmost interest for their potential
use for the treatment of AD. AChE possesses an active site at which
substrates like ACh and acetylthiocholine (ATCh) are hydrolyzed,
and a PAS, which is spatially distinct from the active center and
We have reported that compounds 17 and 1820 (Chart 2)
showed slightly lower (from two to fourfold less) AChE inhibitory
potencies (Table 2). Compounds 19–22, which did not show BuChE
Table 1
Inhibition of AChE and BuChE (IC50) by compounds 1–5a and 11–1413
X
X
O
O
NH2
N
NH2
N
EtO
N
H
N
H
1-5
11-14
X
IC50
(
l
M) EeAChEb
IC50
(
lM) hAChE
IC50
(l
M) hBuChEc
Selectivityd
Tacrine13
1
2
3
—
0.18 0.01
0.32 0.02
0.54 0.04
0.23 0.01
0.22 0.02
0.6 0.3
0.080 0.001
0.052 0.009
0.091 0.004
0.045 0.005
0.15 0.01
0.71 0.03
1.6 0.2
0.67 0.07
0.37 0.01
2.8 0.1
0.12 0.21
0.19 0.03
0.17 0.01
0.11 0.01
0.036
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.24
>141
>63
>149
>270
>36
>819
>518
>592
>952
C-H
C-F
C-Me
C-OMe
N
H
F
Me
OMe
4
5
1113
1213
1313
1413
a
IC50 values are means SEM of at least three independent measurements.
Ee, Electrophorus electricus.
h, human.
b
c
d
hBuChE/hAChE.

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R. León et al. / Bioorg. Med. Chem. 16 (2008) 7759–7769
OMe
X
O
NH2
N
NH2
N
NH2
EtO2C
EtO2C
N
H
N
O
N
N
1-5
15
16
X
N
NH2
N
NH2
N
O
NH2
N
EtO2C
EtO2C
O
N
O
19-22
17
18
Chart 2.
Table 2
4 and propidium (Fig. 1B and C, respectively), lines crossing the x
axis in the same point reveal unchanged Km and decreased Vmax
at increasing inhibitor concentrations. This is a typical trend for
non-competitive inhibition, and indicates that these compounds
bind to a site different to the catalytic one.26 Since propidium
has been described as a selective ligand for the AChE PAS,23 the
same behavior is therefore expected for compound 4.
The hypothesis of the binding at the AChE PAS for tacripyrime-
dones 1–5 is also consistent with their BuChE/AChE selectivity (Ta-
ble 1), since binding at the PAS is mediated by the residue Trp279
(EeAChE) present at the entrance of the gorge in AChE, but absent
in BuChE.
Inhibition of AChE by compounds 1–5 and 15–22a
X
IC50 EeAChE (lM)
Tacrine13
1
2
3
4
—
0.18 0.01
0.32 0.02
0.54 0.04
0.23 0.01
0.22 0.02
0.60 0.30
0.87 0.20
0.82 0.40
3.00 0.30
1.40 0.40
49.0 1.00
70.0 5.00
52.0 2.00
4.10 3.00
C-H
C-F
C-Me
C-OMe
X = N
—
—
—
—
H
5
1519
1619
1720
1820
1915c
2015c
2115c
2215c
F
2.2.3. Molecular modeling
Me
OMe
The AChE binding mode of tacripyrimedones 1–5 (Scheme 1)
was explored by means of docking studies performed for 4 in
hAChE using the rDock program (see Section 4). To verify the reli-
ability of the docking protocol, docking was also extended to ta-
crine and propidium taking advantage of the known X-ray
crystallographic binding mode of these compounds at the catalytic
and peripheral sites, respectively, of AChE. The docking results con-
firmed the capability of the method to reproduce the experimental
binding mode (see Figs. 2 and 3), thus giving confidence to the
binding mode predicted for 4.
Attempts to dock 4 in the catalytic site of hAChE were unsuc-
cessful, as expected from our previous molecular dynamics studies
performed for 15 and 1619a (Chart 2), which pointed out that the
catalytic site of AChE is not flexible enough to easily accommodate
those compounds. In contrast, it was successfully docked in the
PAS, which is in agreement with the non-competitive inhibition
mechanism exhibited by 4. According to the putative binding mode
of the R-enantiomer of 4 (see Fig. 4), the inhibitor is well stacked
against the indole ring of Trp286 (hAChE, with a distance from
the central aminopyridine unit of 4 to the midpoint of the indole
ring of Trp286 of ꢀ3.6 Å), while forming hydrogen-bonds with sev-
eral residues. Thus, the NH group of the 1,8-naphthyridine unit is
hydrogen-bonded to the hydroxyl group of Tyr72, which in turn
is hydrogen-bonded to the carboxylate group of Asp74 (the latter
group might also form hydrogen-bond interactions with Tyr341
and Thr83). The oxygen atom of the carbonyl group is correctly
positioned to hydrogen-bond to the imidazole ring of His287, thus
mimicking a similar interaction found between one of the NH2
a
IC50 values are means SEM of at least three independent measurements.
where selective inhibitors can bind with high affinity and specific-
ity.23 Hydrolysis of ACh proceeds through the formation of an acyl-
enzyme intermediate, and inhibitors that bind the PAS can also af-
fect steady-state kinetics.24
Based on the preceding considerations and on the hypothesis
that the rather wide shape of molecules such as 1–5 might impair
the entrance into the AChE gorge and favor the binding at the PAS,
we studied the steady-state kinetics of compounds 1–5 as well as
that of tacrine and propidium. The graphical analysis of the steady-
state inhibition data can give information about the binding mode
of the selected compounds, and allow the measurement of their Ki,
as well.
The kinetic characterization of these AChE inhibitors was car-
ried out as follows. Reactions were measured in the presence of
different concentrations of substrate and data were analyzed
according to the Linewever–Burk method (Fig. 1). Five different
concentrations of each inhibitor were used. Values of Ki were cal-
culated from the slope of a secondary plot (see Section 4 and Table
3).
The Ki value found for tacrine agrees with the data reported in
the literature,25 and the reciprocal plots describing the AChE inhi-
bition (Fig. 1A) show both increasing slopes and increasing inter-
cepts with higher inhibition concentration,
indicates mixed inhibition. In the reciprocal plots for compound
a pattern that

R. León et al. / Bioorg. Med. Chem. 16 (2008) 7759–7769
7763
Figure. 2. Two views of the predicted binding mode of tacrine at the catalytic site of
the AChE, taking advantage of the X-ray crystallographic structures of the Torpedo
californica AChE complex (PDB entry 1ACJ). Docked and X-ray structures of the
ligand are colored by atom and in gray, respectively. For the sake of clarity,
hydrogen atoms are omitted.
Figure. 1. Double reciprocal plots of the reaction velocity versus acetyltiocholine
(ATCh) concentration in the presence and absence of inhibitor. The study was
performed in 0.1 M PBS solution, pH 8 and five different concentrations of substrate.
Concentrations of tacrine, compound 4, and propidium were as follows. Tacrine:
Control (j) [I] = 0, (d) [I] = 5 nM, (N) [I] = 15 nM, (.) [I] = 30 nM, (ꢀ) [I] = 60 nM, (?)
[I] = 500 nM. Compound 4: Control (j) [I] = 0, (d) [I] = 5 nM, (N) [I] = 15 nM, (.)
[I] = 30 nM, (ꢀ) [I] = 60 nM, (?) [I] = 120 nM. Propidium: Control (j) [I] = 0, (d)
assisted by a water molecule as the distance between the interact-
ing groups of inhibitor and Ser293 amounts to ꢀ6.4 Å. Finally, the
40-methoxybenzene unit is pointing toward the solvent, adopting a
similar arrangement as the aliphatic chain containing the quater-
nary ammonium group in propidium, thus avoiding steric clashes
with residues at the PAS.
[I] = 0,5 nM, (N) [I] = 1 lM, (.) [I] = 10 lM, (ꢀ) [I] = 30 lM, (?) [I] = 100 lM. (A)
Lineweaver–Burk study of tacrine. Insert: slope (j) of the double reciprocal plots
versus [tacrine]. (B) Lineweaver–Burk study of compound 4. Insert: slope (j) of the
double reciprocal plots versus [4]. (C) Lineweaver–Burk study of propidium. Insert:
slope (j) of the double reciprocal plots versus [propidium]. Data are expressed as
mean SEM of at least four experiments.
It is worth-noting that these features allow us to explain the
100-fold increase in the inhibitory potency observed when com-
paring compounds 1–4 to 19–22 (see above and Table 2); this
difference can be ascribed to the loss of the hydrogen-bond
interaction formed between the inhibitor and Tyr72 upon
replacement of the NH moiety by O. Furthermore, since a similar
change in the inhibitory potency is observed for compounds 11–
14 relative to 15–18, it can be speculated that tacripyrines I bind
to the PAS of AChE mimicking the binding mode of 4. This bind-
ing mode would also explain the relative insensitivity of the
inhibitory potency of compounds 1–4 to the presence of the sub-
stituent in position 40 or to the replacement of the benzene ring
by a pyridine in 5 (see Table 1). Finally, the slightly lower po-
tency of compounds 1–4 relative to 11–14 likely reflects a de-
crease in the flexibility of the carbonyl group to interact with
groups in propidium with His287 (PDB entry 1N5R). Finally, the
amino group is properly oriented toward the hydroxyl and car-
bonyl groups of Ser293, even though those interactions should be
Table 3
IC50 for inhibition of AChE, Ki’s and type of inhibition for tacrine, propidium, and
compounds 1–5 with EeAChE
IC50 AChE (
lM)
Ki SE (nM)
Type of inhibition
Tacrine
1
2
3
4
5
0.18
0.32
0.54
0.23
0.22
0.60
11.0
20.2 0.12
61.3 3.21
106.0 4.67
38.9 1.85
41.9 2.52
157.0 4.76
6800 60
Mixed
Non-competitive
Non-competitive
Non-competitive
Non-competitive
Non-competitive
Non-competitive
Propidium

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R. León et al. / Bioorg. Med. Chem. 16 (2008) 7759–7769
Figure. 3. Two views of the predicted binding mode of propidium at the peripheral
site of the AChE, taking advantage of the X-ray crystallographic structures of the
mouse AChE complex (PDB entry 1N5R). Docked and X-ray structures are colored
by atom and in gray, respectively. For the sake of clarity, hydrogen atoms are
omitted.
Figure. 4. Representation of the putative binding modes of R (top) and S (bottom)
enantiomers of compound 4 (colored by atom) in the PAS of hAChE (shown by
ribbon). Selected residues of the protein are also displayed (Trp286 (hAChE), Tyr72,
Asp74, His287, and Ser293; Thr75 is also shown in the bottom plot). Favorable
hydrogen-bond interactions are shown by blue lines, and unfavorable contacts are
shown by red lines (see text for details). For the sake of clarity, hydrogen atoms are
omitted.
His287. On the other hand, the lack of a tryptophan residue
mimicking Trp286 (hAChE) in the peripheral site of hBuChE (re-
placed by Ala277), as well as the replacement of Tyr72 in hAChE
by Asn68 in hBuChE, could also explain the large AChE/BuChE
selectivity of these compounds.
2.3. Effects of new compounds on the changes of [Ca2+]c evoked
by K+
Since our therapeutic strategy pursues the design of new multi-
potent drugs for the treatment of Alzheimer’s disease, we have
investigated the effect of compounds 1–5 on the elevations of
[Ca2+]c induced by high K+, as well as their neuroprotective actions
against free radicals or Ca2+ overload induced by high extracellular
K+ concentration.
Finally, binding of the S-enantiomer of 4 (see Fig. 4) appears to
be less favorable. Thus, even though the inhibitor is stacked against
Trp286 (hAChE) and the 40-methoxybenzene unit has a similar ori-
entation in the binding modes of R- and S-enantiomers, the inhib-
itor is not as buried in the PAS as the R-enantiomer. Moreover, the
alternative hydrogen-bond interactions formed between the 1,8-
naphthyridine NH and NH2 groups with Thr75 and His287 are
counterbalanced by unfavorable contacts, such as those due to
the carbonyl and pyridine nitrogen groups of S-4, which are point-
ing toward the Trp286 (hAChE) carbonyl (Oꢁ ꢁ ꢁO distance of ꢀ3.9 Å)
and Tyr72 oxygen (Nꢁ ꢁ ꢁO distance of ꢀ2.9 Å) groups, respectively.
Overall, these results suggest that the AChE inhibitory potency
of tacripyrimedones 1–5 should be ascribed to the R-enantiomer,
whose binding mode in the PAS provides a basis to explain the
non-competitive inhibition of these compounds and the main con-
clusions of the SAR study.
To study a possible blockade of voltage-dependant Ca2+ chan-
nels (VDCC) by these compounds, which could prevent excess
Ca2+ entry to the cells, SH-SY5Y human neuroblastoma cells, previ-
ously loaded with the fluorescent dye Fluo-4AM, were incubated in
the presence of the compounds for 10 min and then stimulated
with a concentrated solution of KCl, so that the final concentration
was 70 mM K+. Changes in fluorescence as a consequence of the
[Ca2+]c increase elicited by high K+ concentrations were measured
in a fluorescence plate-reader FluoStar Optima (BMG).
As shown in Table 4, the percentage of inhibition of [Ca2+]c in-
crease by compounds 1–5 was statistically significant. In fact, com-

R. León et al. / Bioorg. Med. Chem. 16 (2008) 7759–7769
7765
Table 4
Table 5
Effect of compounds 1–5 on the [Ca2+
]
c increase elicited by 70 mM K+ in SH-SY5Y cells
Cell viability in the presence of compounds 1–5 (0.3
nimodipine (0.3
protection
l
M), nimodipine (0.3
l
M), and
(% inhibition with respect to a control without any drug)
l
M) expressed as % LDH released in the presence of 70 mM K+ and %
X
X
O
NH2
N
O
NH2
N
N
H
N
H
1-5
1-5
X
% Inhibition in SH-SY5Y cells
X
LDH release (% vehicle)
% Protection
Nimodipine15b
—
C-H
C-F
C-Me
C-OMe
N
45.5 6.1***
24.63 3.55 ns
32.88 3.65**
30.95 3.25**
31.04 4.05**
45.11 2.99***
1
2
3
4
5
Tacrine13
90.4 4.1 ns
75.00 2.24***
68.9 2.6***
66.5 2.5***
64.2 0.0***
65.5 2.0***
70.3 2.3***
13.4 7.2
35.9 2.8
40.7 3.5
41.7 2.9
46.4 1.5
44.1 3.7
39.7 2.7
Nimodipine13
1
2
3
4
5
C-H
C-F
C-Me
C-OMe
N
All new compounds 1–5 and nimodipine were tested at the concentration of
0.3 M.
l
Data are expressed as means SEM of at least three different cultures in quadru-
Data are expressed as means SEM of at least three different cultures in quadru-
plicate. LDH released was calculated for each individual experiment considering
100% the extracellular LDH released in the presence of vehicle with respect to the
total. To calculate % protection, LDH release was normalized as follows: In each
individual quadruplicate experiment, LDH release obtained in non-treated cells
(basal) was subtracted from the LDH released upon 70 K+ treatment and normalized
to 100% and that value was subtracted from 100 ***p < 0.001; ns, not significant.
plicate; *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.
pound 5 bearing the 40-pyridyl ring gave a 45% blockade, which is
higher than that found for the parent compound 1 and similar to
that obtained for nimodipine.
2.4. Neuroprotection
The neuroprotective effect of compounds 1–5 was determined on
SH-SY5Y cells exposed during 24 h to a medium with a depolarizing
concentration of KCl (70 mM), which induces Ca2+ overload and the
Table 6
Cell viability in the presence of compounds 1–5 (0.3 lM), catalase (50 U/mL), tacrine
(0.3
l
M), and nimodipine (0.3
l
M) expressed as % LDH released in the presence of
60
l
M H2O2 and % protection
consequent cell death. Drugs, at the concentration of 0.3 lM, were
administered 24 h before the incubation of cells with high K+
(70 mM; hypertonic) and maintained for an additional 24 h. There-
after, release of lactic dehydrogenase (LDH) into the medium was
measured as a parameter of cell death.27 Basal release of LDH by
the cells was subtracted from the values obtained after incubation
with 70 mM K+ in the presence or absence of the compounds.
Compounds 1–5 proved to be strong neuroprotectants, with
protection (%) values higher than those measured for tacrine or
nimodipine (see Table 5). Interestingly, the neuroprotection was
independent of the type of the substituent at the aromatic ring at
C12, the best compound being molecule 3 (46%) with a 40-Me
group at C12.
Regarding neuroprotection against free radicals, compounds 1–
5 were much less efficient than catalase, a neuroprotectant against
free radical-induced toxicity, which was taken as a positive control.
However, the compounds were more efficient than tacrine, which
had any neuroprotecting effect (Table 6) and were slightly better
than nimodipine. Compound 2, which bears a 40-F group at the aro-
matic ring, afforded the highest protection (around 47%).
To further determine if the protection against H2O2-induced
toxicity could be due to a possible reactive oxygen species (ROS)
scavenging effect of the compounds, we measured the reduction
of fluorescence caused by the fluorescent dye H2DCFDA in the pres-
X
O
NH2
N
N
H
1-5
X
LDH release (% vehicle)
% Protection
Catalase13
—
—
—
C-H
C-F
C-Me
C-OMe
N
18.93 1.77***
100.34 3.37 ns
75.42 2.12***
86.44 3.69**
64.39 1.24***
84.35 3.39***
69.02 2.72***
69.94 2.08***
88.34 2.80
0
Tacrine13
Nimodipine13
36.03 2.82
17.40 4.85
46.84 1.53
20.17 4.48
40.73 3.49
39.66 2.80
1
2
3
4
5
Data are expressed as means SEM of at least three different cultures in quadru-
plicate. LDH released was calculated for each individual experiment considering
100% the extracellular LDH released in the presence of vehicle with respect to the
total. To calculate % protection, LDH release was normalized as follows: In each
individual quadruplicate experiment, LDH release obtained in non-treated cells
(basal) was subtracted from the LDH released upon 60 lM H2O2 treatment and
normalized to 100% and that value was subtracted from 100. **p < 0.01; ***p < 0.001;
ence of 60 lM H2O2 alone or in the presence of compounds 1–5.
None of the compounds tested decreased the fluorescence induced
ns, not significant.
by H2O2 in a statistically significant manner (see Table 7).
3. Conclusions
dine hybrids. Regarding AChE inhibition, compounds 1–5 have an
inhibitory potency similar to that of tacrine, but are highly selec-
tive with respect to BuChE. These new compounds present a
The present study reports the synthesis and biological evalua-
tion of tacripyrimedones, a series of new tacrine/1,4-dihydropyri-

7766
R. León et al. / Bioorg. Med. Chem. 16 (2008) 7759–7769
obtained on a Perkin-Elmer Spectrum One spectrophotometer. 1H
NMR spectra were recorded with a Varian VXR-200S spectrometer,
using tetramethylsilane as internal standard and 13C NMR spectra
were recorded with a Bruker WP-200-SY. All the assignments for
protons and carbons were in agreement with 2D COSY, gHSQC,
gHMBC, and 1D NOESY spectra. Elemental analyses were carried
out on a Carlo Erba EA 1108 apparatus.
Table 7
Decrease of H2O2-generated reactive oxygen species (ROS) in the presence of
compounds 1–5 (0.3
by 60 M H2O2
l
M) and catalase (50 U/mL) expressed as % fluorescence induced
l
X
O
NH2
4.2. General method for the Friedländer reaction
Aluminum chloride (1.2–1.7 equiv) was suspended in dry 1,2-
dichloroethane (10 mL) at rt under argon. The corresponding dihy-
dropyridine (1 equiv) and cyclohexanone (1.2–1.7 equiv) were
added. The reaction mixture was heated under reflux (10–24 h).
When the reaction was over (TLC analysis), a mixture of THF/H2O
(1:1) was added at rt. An aqueous solution of sodium hydroxide
(10%) was added dropwise to the mixture until the aqueous solu-
tion was basic. After stirring for 30 min, the mixture was extracted
three times with dichloromethane. The organic layer was washed
with brine, dried over anhydrous sodium sulfate, filtered, and the
solvent was evaporated. The resultant solid was purified by silica
gel flash chromatography using methanol/dichloromethane mix-
tures as eluent to give pure compounds.
N
H
N
1-5
X
Fluorescence (% H2O2)
Vehicle
Catalase
—
—
100
31 6.2***
87.9 7.7ns
95.7 5.6ns
79.9 8.1ns
87.8 3.2ns
82.9 3.1ns
1
2
3
4
5
C-H
C-F
C-Me
C-OMe
N
Data are expressed as means SEM of at least three different experiments in qua-
druplicate. ***p < 0.001; ns, not significant.
4.2.1. 11-Amino-3,3-dimethyl-3,4,5,7,8,9,10,12-octahydro-12-
phenyl-dibenzo[b,g][1,8] naphthyridine-1(2H)-one (1)
Following the procedures Section 4.1, the reaction of compound
615a (200 mg, 0.68 mmol) with AlCl3 (136 mg, 1.02 mmol), in
ClCH2CH2Cl (5 mL), and cyclohexanone (100 mg, 1.02 mmol), after
non-competitive type of AChE inhibition, suggesting that they bind
to the PAS of the enzyme, as confirmed by the results of docking
studies, which indicates that the inhibitory potency should be
mainly ascribed to the R-enantiomer. Moreover, the predicted
binding mode also allows us to explain the nearly 100-fold in-
crease in potency arising upon replacement of the oxygen atom
at C4 in 19–22 by the naphthyridine NH unit present in 1-5. Ki val-
ues show that tacripyrimedones present a high nanomolar affinity
for the PAS center of the enzyme.
3 h, gave product 1 (247 mg, 97%): mp 325–327 °C; IR (KBr)
m
3393,
3225, 2931, 2862, 1616, 1600, 1439, 1396, 1246 cmꢂ1
;
1H NMR
(see Table 8); 13C NMR (see Table 9); MS (API-ES+) m/z: [M+1]+
374.2; [M+Na]+ 396.1; [M+K]+ 412.2; [2M+Na]+ 769.5. Anal. Calcd
for C24H27N3O: C, 77.18; H, 7.29; N, 11.25. Found: C, 76.99; H,
7.30; N, 11.14.
Compounds 1–5 decreased the rise in [Ca2+]c induced by K+ in
neuroblastoma cells. This effect correlates with their neuroprotec-
tive effect against Ca2+ overload, which was significant for all com-
pounds and independent of the type of substituent. Finally,
products 1–5 were also neuroprotective against free radical toxic-
ity with values of protection ranging from 17% to 40%, at the con-
4.2.2. 11-Amino-12-(40-fluorophenyl)-3,3-dimethyl-3,4,5,7,8,9,
10,12-octahydrodibenzo [b,g][1,8]naphthyridine-1(2H)-one (2)
Following the procedures in Section 4 and 1, the reaction of
compound 715a (200 mg, 0.64 mmol) with AlCl3 (128 mg,
0.96 mmol) and cyclohexanone (94 mg, 0.96 mmol), in ClCH2CH2Cl
(5 mL), after 4.5 h, gave molecule 2 (300 mg, 90%): mp 325–327 °C;
centration of 0.5 lM, the most active being compound 2, which
bears a 40-F group. This effect was not probably due to scavenging
of ROS, since none of the compounds reduced in a significant man-
ner the fluorescence due to H2O2, measured with the fluorescent
dye H2DCFDA.
IR (KBr)
m
;
3396, 3217, 2931, 2862, 1616, 1504, 1441, 1396,
1245 cmꢂ1
1H NMR (see Table 8); 13C NMR (see Table 9); MS
(API-ES+) m/z: [M+1]+ 392.3; [M+Na]+ 415.2; [2M+Na]+ 807.3. Anal.
Calcd for C24H26FN3O: C, 73.63; H, 6.69; N, 10.73. Found: C, 73.48;
H, 6.57; N, 10.48.
Based on these findings, tacripyrimedones 1–5 appear to be
promising compounds, and the present molecular modeling results
provide a relevant basis to pursue our efforts by exploring the
structure–activity relationships of enantiomerically pure R- and
S-tacripyrimedones.
4.2.3. 11-Amino-3,3-dimethyl-12-(40-methylphenyl)-3,4,5,7,8,9,
10,12-octahydrodibenzo[b,g][1,8]naphthyridine-1(2H)-one (3)
Following the procedures in Section 4.1, the reaction of com-
pound 815a (200 mg, 0.65 mmol) with AlCl3 (146 mg, 0.97 mmol)
and cyclohexanone (96 mg, 0.97 mmol), in ClCH2CH2Cl (5 mL),
after 4 h, afforded compound 3 (243 mg, 96%): mp 336–338 °C;
4. Experimental
4.1. General methods
IR (KBr) m ;
3394, 3217, 29.31, 2862, 1614, 1600, 1439, 1246 cmꢂ1
1H NMR (see Table 8); 13C NMR (see Table 9); MS (API-ES+) m/z:
Reactions were monitored by TLC using precoated silica gel alu-
minum plates containing a fluorescent indicator (Merck, 5539).
Detection was done by UV (254 nm) followed by charring with sul-
furic–acetic acid spray, 1% aqueous potassium permanganate solu-
tion, or 0.5% phosphomolybdic acid in 95% EtOH. Anhydrous
Na2SO4 was used to dry organic solutions during work-ups and
the removal of solvents was carried out under vacuum with a ro-
tary evaporator. Flash column chromatography was performed
using silica gel 60 (230–400 mesh, Merck). Melting points were
determined on a Kofler block and are uncorrected. IR spectra were
[M+1]+ 388.3; [M+Na]+ 410.2; [2M+Na]+ 797.5. Anal. Calcd for
C25H29N3O: C, 77.48; H, 7.54; N, 10.84. Found: C, 77.35; H, 7.66;
N, 10.57.
4.2.4. 11-Amino-12-(40-methoxyphenyl)-3,3-dimethyl-3,4,5,7,8,
9,10,12-octahidrodibenzo[b,g][1,8]naphthyridine-1(2H)-one (4)
Following procedures in Section 4.1, the reaction of compound
915a (200 mg, 0.61 mmol) with AlCl3 (123 mg, 0.92 mmol) and
cyclohexanone (91 mg, 0.92 mmol), in ClCH2CH2Cl (5 mL), after
5 h, provided product 4 (203 mg, 83%): mp 318–320 °C; IR (KBr)

R. León et al. / Bioorg. Med. Chem. 16 (2008) 7759–7769
7767
Table 8
Selected 1H NMR (DMSO-d6, 300 MHz, d) for compounds 1–5
4´
X
3´
5´
6´
1´
O 2´
NH2
10
7
1 X= H
12a
11a
10a
9
8
2
1
2 X= C-F
3 X= C-Me
4 X= C-OMe
5 X= N
11
12
3
4a
6a
5a
N
H
N
4
(1-5)
NH
NH2
H12
H2
H4
H7
H10
H9,H8
(CH3)2C3
1
2
3
4
5
9.54 (s)
5,40 (s)
5.12 (s)
2.52–2.36
JAB = 16.1
2.23–1.96
JAB = 16.0
2.61 (s)
2.34–2.20 (m)
1.73 (s)
1.04, 0.87 (s)
0.97, 0.80 (s)
0.97, 0.87 (s)
0.97, 0.82 (s)
0.97, 0.82 (s)
9.50 (s)
9.44 (s)
9.42 (s)
9.59 (s)
5.36 (s)
5.25 (s)
5.25 (s)
5.47 (s)
5.09 (s)
4.99 (s)
4.98 (s)
5.15 (s)
2.44–2.27
JAB = 17.0
2.15–1.87
JAB = 16.0
2.53 (s)
2.52 (s)
2.53 (s)
2.53 (s)
2.30–1.92 (m)
2.33–2.13 (m)
2.29–2.18 (m)
2.20–2.18 (m)
1.66 (s)
1.65 (s)
1.66 (s)
1.66 (s)
2.49–2.34
JAB = 16.0
2.14–1.88
JAB = 16.0
2.48–2.27
JAB = 17.0
2.14–1.89
JAB = 16.0
2.6–2.8
2.16–1.91
JAB = 17.
JAB = 16.0
Table 9
Selected 13C NMR (DMSO-d6, 300 MHz, d) for compounds 1–5
C2
C3
C4
C4a
C5a
C6a
C7
C8
C9
C10
C10a
C11
C11a
C12
C12a
1
2
3
4
5
50.6
50.5
50.6
50.2
50.4
32.4
32.4
32.4
32.6
32.4
40.1
40.1
40.1
40.3
40.1
152.2
152.8
152.3
151.8
153.2
152.4
150.4
152.2
151.7
152.8
147.1
147.1
147.1
146.7
147.2
32.2
32.2
32.2
31.8
32.2
22.6
22.6
22.6
22.3
22.6
22.7
22.7
22.8
22.4
22.7
23.2
23.2
23.2
22.9
23.2
111.5
111.5
111.5
111.1
111.6
150.4
150.3
150.3
150.9
150.6
100.8
100.5
100.9
100.7
99.1
34.9
34.1
34.6
33.7
34.4
107.9
107.8
108.0
107.8
106.5
m
3390, 3210, 2934, 1609, 1601, 1507, 1439, 1249 cmꢂ1
;
1H NMR
as a function of enzyme activity. Inhibition curves were made by
incubating with the different compounds for 30 min; a sample
without any compound was always present to determine the
100% of enzyme activity. After the 30 min incubation, the loss of
yellow color by m-nitrophenol was evaluated by measuring absor-
bance at 405 nm in a spectrophometric plate reader (iEMS Reader
MF, Labsystems). The concentration of compound that produces
50% AChE activity inhibition (IC50) was calculated by transforming
the values of absorbance to Rappaport enzymatic activity units
extrapolating from a calibration curve previously obtained. Data
are means SEM of at least three different experiments in
triplicate.
(see Table 8); 13C NMR (see Table 9); MS (API-ES+) m/z: [M+1]+
404.2; [M+Na]+ 426.2; [2M+Na]+ 829.0. Anal. Calcd for
C
N, 10.37.
25H29N3O2: C, 74.41; H, 7.24; N, 10.41. Found: C, 74.33; H, 7.15;
4.2.5. 11-Amino-3,3-dimethyl-3,4,5,7,8,9,10,12-octahydro-12-
(40-pyridyl)-dibenzo[b,g][1,8]naphthyridine-1(2H)-one (5)
Following the procedures in Section 4.1, the reaction of com-
pound 1015b (200 mg, 0.68 mmol) with AlCl3 (136 mg, 1.02 mmol)
and cyclohexanone (100 mg, 1.02 mmol), in ClCH2CH2Cl (5 mL),
after 4 h, furnished compound 10 (221 mg, 88%): mp 315–318 °C;
IR (KBr)
m
;
3392, 3227, 2932, 2869, 1618, 1601, 1499, 1441,
1245 cmꢂ1
1H NMR (see Table 8); 13C NMR (see Table 9); MS
4.3.2. Measurement of AChE activity in hAChE
(API-ES+) m/z: [M+1]+ 375.2; [M+Na]+ 397.2; [2M+Na]+ 771.5. Anal.
Calcd for C23H26N4O: C, 73.77; H, 7.00; N, 14.96. Found: C, 74.02; H,
6.98; N, 14.87.
The inhibitory activity of the compounds toward hAChE was
determined following the method of Ellman18 using AChE from hu-
man erythrocytes and acetylthiocholine chloride (0.53 mM) as a
substrate. The reaction took place in a final volume of 3 mL of a
phosphate-buffered solution at pH 8, containing 0.09 U of hAChE
4.3. Pharmacological studies
and 333 l
M of 5,50-dithiobis-2-nitrobenzoic acid (DTNB), which
4.3.1. Measurement of AChE activity in Electrophorus electricus
To assess the inhibitory activity of the compounds toward AChE,
we followed the spectrophotometric method of Rappaport17 using
purified AChE from Electric eel ( E. electricus) and acetylcholine
chloride (29.5 mM) as a substrate. The reaction took place in a final
volume of 2.5 mL of an aqueous solution containing 0.78 U AChE
and 1.9 mM m-nitrophenol to produce a yellow color which is lost
produces the yellow anion 5-thio-2-nitrobenzoic acid. Inhibition
curves were made by incubating with the different compounds
for 15 min; a sample without any compound was always present
to determine the 100% of enzymatic activity. After the 15 min incu-
bation period, the production of color, as an indication of enzymatic
activity, was evaluated by measuring absorbance at 412 nm in a
spectrophotometer plate reader (iEMS Reader MF, Labsystems).

7768
R. León et al. / Bioorg. Med. Chem. 16 (2008) 7759–7769
KM
KM
4.3.3. Measurement of BuChE activity
q
¼
þ
½Iꢃ
ð4Þ
Vmax Vmax ꢁ Ki
The inhibitory activity of the compounds toward BuChE was
determined following the method of Ellman18 using BuChE from
human serum and butyrylthiocholine chloride (5 mM) as a sub-
strate. The reaction took place in a final volume of 1 mL of a phos-
phate-buffered solution at pH 7.2, containing 0.035 U of BuChE and
0.25 mM 5,50-dithiobis-2-nitrobenzoic acid (DTNB), which pro-
duces the yellow anion 5-thio-2-nitrobenzoic acid. Inhibition
curves were made by incubating with the different compounds
for 15 min; a sample without any compound was always present
to determine the 100% of enzymatic activity. After the 15 min incu-
bation period, the production of color, as an indication of enzy-
matic activity, was evaluated by measuring absorbance at
412 nm in a spectrophotometric plate reader (iEMS Reader MF,
Labsystems).
Thus, representing slopes versus concentration inhibitor, we can cal-
culate KM/Vmax from y axis intercept (parameter a) and the Ki as a/b.
All rate measurements were performed in quadruplicate.
4.3.5. Culture of SH-SY5Y cells
SH-SY5Y cells, at passages between 3 and 16 after de-freezing,
were maintained in
(DMEM) containing 15 non-essential amino-acids (NEAAs) and
supplemented with 10% fetal calf serum (FCS), 1 mM glutamine,
50 U/mL penicillin, and 50 lg/mL streptomycin (reagents from
GIBCO, Madrid, Spain). Cultures were seeded into flasks containing
supplemented medium and maintained at 37 °C in 5% CO2/humid-
ified air. Stock cultures were passaged 1:4 twice weekly. For as-
says, SH-SY5Y cells were sub-cultured in 48-well plates at a
seeding density of 2 ꢄ 105 cells per well, or in 96-well plates at a
seeding density of 8 ꢄ 104 cells per well. For the cytotoxicity
experiments, cells were treated with drugs before confluence, in
DMEM free of serum.
a Dulbecco’s modified Eagle’s medium
4.3.4. Kinetic characterization of AChE inhibition
Acetylcholinesterase (Electric eel), DTNB, and acetylthiocholine
were purchased from Sigma–Aldrich chemical company. Inhibi-
tors were solubilized in DMSO at a stock concentration of 10ꢂ2
M and maintained at +4 °C. AChE inhibition was assayed spectro-
photometrically at 30 °C according to the method of Ellman.18 As-
say buffer was phosphate-buffered solution (PBS) 0.1 M, pH 8.0. A
stock solution of AChE (270 U/mL) in assay buffer was kept at
ꢂ80 °C, and a 1:70 dilution was prepared immediately before
starting the measurement. ATCh (10 mM) and DTNB (1 mM) were
dissolved in assay buffer and kept at ꢂ80 °C. Tacrine–HCl was dis-
solved in water and stock solutions of compounds 1–5 were pre-
pared in DMSO. For inhibitors, at least five concentrations were
used.
4.3.6. Measurement of cytosolic Ca2+ concentrations
For these experiments, SH-SY5Y neuroblastoma cells were
grown at confluence in 96-well black dishes. Cells were loaded
with 4 lM fluo 4/AM for 1 h at 37 °C in DMEM. Then, cells were
washed twice with Krebs–Hepes solution and kept at room tem-
perature for 30 min before the beginning of the experiment. Fluo-
rescence was measured in
(FLUOstar Optima, BMG, Germany). Wavelengths of excitation
and emission were 485 and 520 nm, respectively.
a fluorescence microplate reader
Experiments were performed in a transparent 48-well plate
containing each well 350
umes of buffer solution between 604 and 504
inhibitor solution to give desired final concentration, and five dif-
ferent concentrations of ATCh (0.1, 0.25, 0.5, 0.75, and 1 mM).
The mixture was thoroughly mixed. The reaction was initiated by
l
L of the DTNB solution, variable vol-
L, 1 L DMSO or
4.3.7. Measurement of lactic dehydrogenase (LDH) activity
Extracellular and intracellular LDH activity was spectrophoto-
metrically measured using a Cytotoxicity Cell Death kit (Roche-
Boehringer. Mannheim, Germany) according to the manufac-
turer’s indications. Total LDH activity was defined as the sum
of intracellular and extracellular LDH activity; released LDH
was defined as the percentage of extracellular compared to total
LDH activity.
l
l
adding 45 lL of an AChE solution (0.18 U/mL) at 30 °C to give a fi-
nal volume of 1 mL. Uninhibited enzyme activity was determined
by adding DMSO instead of the inhibitor solution. Determination
of Michaelis constant for the substrate ATCh was done at 6 differ-
ent concentrations (0.1–1.5 mM) to give
Km = 650 40
M, and Vmax = 1.48 0.05 minꢂ1
AChE-catalyzed ATCh hydrolysis were corrected by those of the
non-enzymatic hydrolysis of ATCh as determined by using 45
a
value of
4.3.8. Measurement of reactive oxygen species (ROS)
l
. The rates of
To measure cellular ROS, we have used the molecular probe
H2DCFDA.29 SH-SY5Y cells were loaded with 10
lM/L H2DCFDA,
lL
which diffuses through the cell membrane and is hydrolyzed by
intracellular esterases to the non-fluorescent form dichlorofluores-
cein (DCFH). DCFH reacts with intracellular H2O2 to form dichloro-
fluorescein (DFC), a green fluorescent dye. Fluorescence was
measured in a fluorescence microplate reader (FLUOstar Optima,
Biogen). Wavelengths of excitation and emission were 485 and
520 nm, respectively.
of assay buffer, instead of the enzyme solution. Progress curves
were monitored at 412 nm over 2 min in a fluorescence plate-read-
er 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 fit-
ted according to Lineweaver–Burk replots using Origin software
(version 7.0). Double reciprocal plots were generated using the
same statistical package. The relative Ki’s and types of inhibition
were determined by plotting the slopes of the double reciprocal
plots versus concentration of inhibitor as described elsewhere.28
Briefly, from the initial Michaelis–Menten equation for Linewe-
aver–Burk plot that one for a mixed non-competitive inhibition
is developed
4.3.9. Molecular modeling
Docking was performed with the program rDock, which is an
extension of the program RiboDock30 using an empirical scoring
function calibrated based on protein–ligand complexes.31 This pro-
gram uses an empirical scoring function for attractive and repul-
sive polar interactions in combination with a Lennard–Jones
potential to account for the van der Waals term. Ligand internal
energies are accounted for using the same terms as the intermolec-
ular potential, plus a dihedral potential derived from the Tripos
force field.32 A full search can be performed using a genetic algo-
rithm where ligand docking poses are represented using a conven-
tional chromosome representation of translation, rotation, and
rotatable bond dihedral angles, while the receptor site was kept
fixed.
ð1=V0 ¼ 1=Vmax þ ðKM=VmaxÞ=½SꢃÞ
ð1Þ
ð2Þ
ꢀ
ꢁ
ꢀ
ꢁ
1
½Iꢃ
1
KM
½Iꢃ
1
¼
1 þ
þ
1 þ
;
Vo
KI Vmax Vmax
Ki ½Sꢃ
where the slope for each inhibitor concentration is
¼ ðKM=VmaxÞð1 þ ½Iꢃ=KiÞ
or written as a ‘‘y = a + bx” equation
q
ð3Þ

R. León et al. / Bioorg. Med. Chem. 16 (2008) 7759–7769
7769
11. Tan, D. X.; Manchester, L. C.; Sainz, R.; Mayo, J. C.; Alvares, F. L., et al Expert
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The reliability of rDock was assessed by docking tacrine at the
catalytic site of the Torpedo californica AChE and propidium at
the peripheral site of the mouse AChE, taking advantage of the X-
ray crystallographic structures of the two complexes (PDB entries
1ACJ and 1N5R).33,34 Docking of compound 4 was performed using
a structural model of the human enzyme used in our previous
studies.35,36 Superposition of the X-ray crystallographic structures
confirmed unambiguously the structural similarity of the ligand-
binding sites. Water molecules were removed from the coordi-
nates, and the docking volume was defined as the space within
10 Å of the ligands for both catalytic and peripheral binding sites.
Before docking, the structure of the ligands was built up and en-
ergy minimized at the MP2/6-31G* level using Gaussian03.37 Each
compound was subjected to 100 docking runs, and the output
docking modes were analyzed by visual inspection in conjunction
with the docking scores.
19. (a) Marco, J. L.; de los Ríos, C.; García, A. G.; Villarroya, M.; Carreiras, M. C.;
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20. Marco-Contelles, J.; León, R.; López, M. G.; García, A. G.; Villarroya, M. Eur. J.
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R.L. thanks the Ministerio de Educación y Ciencia (MEC) for a
fellowship (AP20020576). This work was supported by Fundación
Teófilo Hernando, FMUAM Red CIEN (Instituto de Salud Carlos
III), MEC (Grants Nos. BFI2003-02722; SAF2006-08764-C02-01;
SAF2006-08540; CTQ2005-08797), Instituto de Salud Carlos III [Re-
tic ‘‘RENEVAS” (RD06-2006-1002)], Comunidad de Madrid S/SAL-
0275-2006), CSIC-GRICES project (2007PT-13), AECI (A/7492/07).
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