Tacripyrines, the first tacrine-dihydropyridine hybrids, as multitarget-directed ligands for the treatment of Alzheimer's disease

Article abstract of DOI:10.1021/jm801292b

Tacripyrines (1-14) have been designed by combining an AChE inhibitor (tacrine) with a calcium antagonist such as nimodipine and are targeted to develop a multitarget therapeutic strategy to confront AD. Tacripyrines are selective and potent AChE inhibito

Full text of DOI:10.1021/jm801292b

2724
J. Med. Chem. 2009, 52, 2724–2732
Tacripyrines, the First Tacrine-Dihydropyridine Hybrids, as Multitarget-Directed Ligands for
the Treatment of Alzheimer’s Disease
Jose´ Marco-Contelles,*,4 Rafael Leo´n,4,†,‡ Cristo´bal de los R´ıos,4,†,‡ Abdelouahid Samadi,4 Manuela Bartolini,§
Vincenza Andrisano,§ Oscar Huertas,| Xavier Barril, F. Javier Luque,| Mar´ıa I. Rodr´ıguez-Franco,# Beatriz Lo´pez,#
Manuela G. Lo´pez,†,‡ Antonio G. Garc´ıa,†,‡,3 Mar´ıa do Carmo Carreiras,O and Mercedes Villarroya†,‡
Laboratorio de Radicales Libres (IQOG, CSIC), C/Juan de la CierVa 3, 28006 Madrid, Spain, Laboratorio de Radicales Libres (IQOG, CSIC),
C/Juan de la CierVa 3, 28006-Madrid, Spain, Instituto Teo´filo Hernando, C/Arzobispo Morcillo 4, 28029 Madrid, Spain, Departamento de
Farmacolog´ıa y Terape´utica, Facultad de Medicina, UniVersidad Auto´noma de Madrid, C/Arzobispo Morcillo 4, 28029 Madrid, Spain,
Department of Pharmaceutical Sciences, Alma Mater Studiorum, UniVersity of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Department de
Fisicoqu´ımica and Institut de Biomedicina (IBUB), Facultat de Farma`cia, UniVersitat de Barcelona, AVenida Diagonal 643, 08028 Barcelona,
Spain, Institucio´ Catalana de Recerca i Estudis AVanc¸ats (ICREA), Instituto de Qu´ımica Me´dica (CSIC), C/Juan de la CierVa 3, 28006 Madrid,
Spain, SerVicio de Farmacolog´ıa Cl´ınica, Hospital UniVersitario de la Princesa, C/Diego de Leo´n 62, 28006 Madrid, Spain, iMEd.UL,
Research Institute for Medicines and Pharmaceutical Sciences, Faculdade de Farma´cia da UniVersidade de Lisboa, AV. Forças Armadas,
1600-083, Lisboa, Portugal
ReceiVed October 14, 2008
Tacripyrines (1-14) have been designed by combining an AChE inhibitor (tacrine) with a calcium antagonist
such as nimodipine and are targeted to develop a multitarget therapeutic strategy to confront AD. Tacripyrines
are selective and potent AChE inhibitors in the nanomolar range. The mixed type inhibition of hAChE
activity of compound 11 (IC50 105 ( 15 nM) is associated to a 30.7 ( 8.6% inhibition of the proaggregating
action of AChE on the Aꢀ and a moderate inhibition of Aꢀ self-aggregation (34.9 ( 5.4%). Molecular
modeling indicates that binding of compound 11 to the AChE PAS mainly involves the (R)-11 enantiomer,
which also agrees with the noncompetitive inhibition mechanism exhibited by p-methoxytacripyrine 11.
Tacripyrines are neuroprotective agents, show moderate Ca2+ channel blocking effect, and cross the
blood-brain barrier, emerging as lead candidates for treating AD.
Introduction
drugs (tacrine, donepezil, galantamine, and rivastigmine) that
improve AD symptoms by inhibiting acetylcholinesterase
(AChE), i.e., the enzyme responsible for the hydrolysis of ACh,
thereby raising the levels of ACh in the synaptic cleft.6 Recently,
a renewed interest for AChE inhibitors (AChEI) has been
stimulated by the potential role of AChE in accelerating the
formation of amyloid fibrils in the brain and forming stable
complexes with Aꢀ.7 This role involves the peripheral anionic
binding site (PAS) of AChE, as noted by the fact that propidium
iodide, which binds to the PAS, affects the Aꢀ aggregation in
vitro, whereas active-site inhibitors have no similar effect.8
Alzheimer’s disease (AD) is an age-related neurodegenerative
process characterized by progressive memory loss, decline in
language skills, and other cognitive impairments.1 Although the
etiology of AD is not well-known, several factors such as
amyloid-ꢀ (Aꢀ)2 deposits, τ-protein aggregation, oxidative
stress, or low levels of acetylcholine3 are thought to play
significant roles in the pathophysiology 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.a
The cholinergic theory1 suggests that the selective loss of
cholinergic neurons in AD results in a deficit of acetylcholine
(ACh) in specific regions of the brain that mediate learning and
memory functions.5 Consequently, there are four FDA-approved
The multifactorial nature of AD supports the most current
innovative therapeutic approach based on the “one molecule,
multiple targets” paradigm.9-13 The multitarget approach14
includes novel tacrine-melatonin hybrids,15 dual inhibitors of
AChE and monoamine oxidase16 or serotonin transporters,17
potent cholinesterase inhibitors with antioxidant and neuropro-
tective properties,18 gallamine-tacrine hybrids binding at cho-
linesterases and M2 muscarinic receptors,19 or NO-donor-tacrine
hybrids as hepatoprotective anti-Alzheimer drugs.20
It is known that Ca2+ overload is the main factor that triggers
the processes leading to cell death. Thus, it has been shown
that calcium dysfunction, involved in the pathogenesis of AD,
augments Aꢀ formation and τ hyperphosphorylation.21,22 More-
over, calcium entry through L-type Ca2+ channels (Cav 1.1-1.4)
causes both calcium overload and mitochondrial disruption,
which leads to the activation of the apoptotic cascade and cell
death.23 Hence, blocking the entrance of Ca2+ through this
specific subtype of Ca2+ channel could be valuable to prevent
cell death. In fact, nimodipine, a neuronal L-type Ca2+ channel
blocker, protects neurons from death evoked by focal cerebral
ischemia.24
* To whom correspondence should be addressed. Phone: +34-91-
5622900. Fax: +34-91-5644853. E-mail: iqoc21@iqog.csic.es.
4
Laboratorio de Radicales Libres (IQOG, CSIC), Madrid, Spain.
Instituto Teo´filo Hernando.
Departamento de Farmacolog´ıa y Terape´utica, Universidad Auto´noma
†
‡
de Madrid.
§
Department of Pharmaceutical Sciences, University of Bologna.
Department de Fisicoqu´ımica and Institut de Biomedicina (IBUB),
|
Universitat de Barcelona.
Institucio´ Catalana de Recerca i Estudis Avanc¸ats (ICREA), Universitat
de Barcelona.
#
Instituto de Qu´ımica Me´dica (CSIC).
Servicio de Farmacolog´ıa Cl´ınica, Hospital Universitario de la Princesa.
iMEd.UL, Research Institute for Medicines and Pharmaceutical
3
O
Sciences, Universidade de Lisboa.
a Abbreviations: AD, Alzheimer’s disease; ACh, acetylcholine; AChE,
acetylcholinesterase; Aꢀ, ꢀ-amyloid peptide; BuChE, butyrylcholinesterase;
AChEI, acetylcholinesterase inhibitors; hAChE, human acetylcholinesterase;
Ee, Electrophorus electricus; BBB, blood-brain barrier; LDH, lactic
dehydrogenase; VDCC, voltage-dependent Ca2+ channels; DHP, dihydro-
pyridines; PAS, peripheral anionic site; PAMPA, parallel artificial membrane
permeation assay.
10.1021/jm801292b CCC: $40.75
2009 American Chemical Society
Published on Web 04/17/2009

Tacripyrines as Multitarget-Directed Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2725
Chart 1. Tacripyrines 1-14 Based on the Juxtaposition of an
AChEI (Tacrine) and a 1,4-DHP (Nimodipine)
dropyridine-3-carboxylic acids (31-44) were isolated from
moderate to good chemical yields. In this work, we have
described the synthesis of 3-oxo-2-(4′-biphenylmethylene)-
butanoic acid (22) for the first time, and only compound 31
has been previously described by Troschu¨tz.41 Next, Friedla¨nder
reaction42 between the ꢀ-enaminonitriles 31-44 and cyclohex-
anone, under standard conditions (AlCl3, 1,2-dichloroethane,
under reflux)43 provided the desired target molecules (Scheme
1).
Compounds 1-14 are racemic hexahydrobenzo[b][1,8]naph-
thyridines substituted at C-4 by an aromatic ring incorporating
different types of substituents, or by a 3′-(4′)pyridyl ring system.
These molecules have been conveniently characterized by their
analytical and NMR spectroscopic data (see Supporting Infor-
mation).
Biology. AChE/BuChE Inhibitory Activity. Tacripyrines
1-14 were evaluated as inhibitors of AChE from Electrophorus
electricus (Ee) following Rappaport’s method,44 as well as
inhibitors of AChE/BuChE from human serum according to
Ellman’s protocol.45 Most of the tacripyrines were more potent
inhibitors of EeAChE, at nanomolar level, than tacrine, which
was used as reference (Table 1). Compounds 3, 4, 6, and 14,
bearing strong electron-withdrawing groups at C2′ or C4′ (2′-
CF3, 2′-NO2, 4′-NO2, 4′-pyridyl) had the lowest EeAChE
inhibitory potency (IC50 values >200 nM). However, 4 and 6
were the only compounds able to inhibit hBuChE (IC50 values
of 4.8 and 34.4 µM, respectively). Conversely, compounds 10/
11 and 2/13, bearing electron-donating (3′-MeO, 4′-MeO) or
electron-withdrawing (4′-F, 3′-pyridyl) groups, respectively, had
the best EeAChE inhibitory potencies, besides high hBuChE/
EeAChE selectivity. Compound 5, which has a nitro group at
C3′, showed better EeAChE inhibition than its isomers 4 and
6, with the nitro group located at C2′ or C4′, respectively. In
general, compounds with a substituent at C2′ position were the
poorest EeAChE inhibitors. Regardless of the electronic effect
of the functional group, substituents at C4′ were well accepted
for the inhibitory activity, except for the nitro-substituted
compound 6.
Inhibition experiments using human serum AChE (hAChE)
provided results similar to those found with EeAChE. The
inhibition of hAChE by compounds 1-14 was generally
somewhat less potent than that found for EeAChE, but the
selectivity for AChE was preserved (Table 1). Compounds 10
and 12 were the most potent hAChE inhibitors, whereas the
inhibitory potency of 11 and 13 was slightly weakened relative
to EeAChE. Overall, compounds 8 and 10-12, whose inhibitory
activity varies only by a factor of 2, are among the most potent
inhibitors of hAChE (IC50 values ranging from 45 to 105 nM).
Finally, comparing with tacrine, the reference compound,
tacripyrines 8 and 10-12, proved more potent, regardless of
the AChE used, and selective for hBuChE.
Because 1,4-dihydropyridines (DHPs) are compounds that
selectively block L-type voltage-dependent Ca2+ channels
(VDCC), hybrid molecules that combine an AChEI and a DHP,
such as tacrine and nimodipine (Chart 1), might represent a
promising approach to the treatment of AD. Support to this
therapeutic strategy comes from the fact that bis(7)-tacrine
attenuates ꢀ-amyloid neuronal apoptosis by regulating L-type
calcium channels.25 Besides inhibition of AChE and blockade
of VDCC, compounds able to prevent the oxidative stress might
have increased therapeutic value because oxidative damage
precedes the appearance of other pathological hallmarks of
AD.26-28
On the basis of the preceding considerations, a preliminary
study on the synthesis and biological evaluation of tacripyrines
allowed us to conclude that these compounds are promising for
further development as new drugs for the treatment of AD.29
In this context, coupled to the simple synthetic schemes that
yield multigram quantities of tacripyrines, new compounds have
been synthesized in order to provide a broader structure-activity
(AChE/BuChE) relationship (SAR), and new studies are now
been incorporated describing the inhibition mechanism of AChE,
the inhibition of the AChE-induced Aꢀ aggregation, as well as
the inhibition of the self-aggregation of Aꢀ, molecular modeling
on the active centers, the effect of these compounds on the
regulation of the cytosolic calcium concentration, their neuro-
protective properties against different cytotoxic stimuli, and
finally studies directed to evaluate the ability of these compounds
to cross the blood-brain barrier (BBB).
To sum up, on going from tacrine to our new tacripyrines,
the AChE inhibitory activity is potentiated, while BuChE
inhibition dramatically drops, giving a new family of potent
and selective AChE inhibitors. Accordingly, they should
contribute to activate central cholinergic transmission, while it
is plausible that these compounds are devoid of the side effects
related to nonselective cholinesterase inhibitors.46
Results and Discussion
Chemistry. The synthesis of tacripyrines 1-14 [“ethyl
ester of 5-amino-4-(hetero)aryl-1,4,6,7,8,9-hexahydro-2-methyl-
benzo[b][1,8]naphthyridine-3-carboxylic acid”] was easily
achieved by reaction of the readily available ethyl esters of
3-oxo-2-(arylmethylene)-butanoic acids (15-28),30-38 with 3,3-
diaminoacrylonitrile (29),39 prepared in situ from ethyl cy-
anoacetimidate hydrochloride (30)40 (Scheme 1). The resulting
ethyl esters of (()-6-amino-4-aryl-5-cyano-2-methyl-1,4-dihy-
Kinetic Study of AChE Inhibition. The mechanism of
inhibition of hAChE was investigated using p-methoxytacripy-
rine 11, one of the most potent EeAChE inhibitors (see above).
The inhibitory constant, Ki, was determined from the analysis
of Lineweaver-Burk plots, which show both increasing slopes
(lower Vmax) and intercepts (higher Km) with increasing inhibitor

2726 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Scheme 1. Synthesis of Tacripyrines (1-14)
Marco-Contelles et al.
Table 1. Inhibition of AChE from Electrophorus electricus (EeAChE), Human AChE (hAChE), and Human BuChE (hBuChE) by Tacripyrines 1-14
IC50 (nM)a
EeAChEb
IC50 (nM)a
hBuChEc
IC50 (nM)a
hAChEc
Selectivity IC50
hBuChE/IC50 hAChE
X, Y, Z
X ) H, Y, Z ) CH
X ) 4′-F, Y ) C, Z ) CH
X ) 2′-CF3, Y, Z ) CH
Tacrine
1
2
3
4
5
6
7
8
9
10
11
12
13
14
180 ( 20
80 ( 2
52 ( 9
36 ( 4
147 ( 11
122 ( 21
193 ( 31
226 ( 27
338 ( 13
191 ( 29
309 ( 21
169 ( 15
71 ( 4
234 ( 32
58 ( 7
105 ( 15
45 ( 2
0.24
>100000
>100000
>100000
4800 ( 40
>100000
34400 ( 110
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>820
>518
>442
14.2
>524
111
>592
>1408
>427
>1724
>952
>2222
>633
>448
210 ( 3
304 ( 2
110 ( 2
600 ( 7
91 ( 4
X ) 2′-NO2, Y, Z ) CH
X ) 3′-NO2, Y, Z ) CH
X ) 4′-NO2, Y ) C, Z ) CH
X ) 4′-Me, Y ) C, Z ) CH
X ) 4′-C6H5, Y ) C, Z ) CH
X ) 2′-OMe, Y, Z ) CH
X ) 3′-OMe, Y, Z ) CH
X ) 4′-OMe, Y ) C, Z ) CH
X ) 3′, 4′-di-OMe, Y ) C, Z ) CH
X ) H, Y ) CH, Z ) N
90 ( 2
160 ( 6
61 ( 9
45 ( 5
103 ( 6
47 ( 2
158 ( 20
223 ( 43
X ) H, Y ) N, Z ) CH
220 ( 2
a IC50 values are the mean ( SEM of at least three independent measurements. b From Electrophorus electricus (Ee). c From human serum.
concentration, indicating mixed type inhibition. The graphical
analysis of steady-state inhibition data for 11 is shown in Figure
1. A Ki value of 109 ( 1 nM was estimated from the plots of
the slope versus the concentration of 11.
Effects on AChE-Induced Aꢀ40 Aggregation. The avail-
ability of a suitable test for the study of the AChE-mediated
Aꢀ40 aggregation allowed us to verify whether compound 11,
endowed with a mixed inhibition mechanism, could affect the
Aꢀ aggregation. The results confirmed that AChE inhibitor 11
was able to interfere with the pro-aggregating action of hAChE
with an inhibitory effect (%) of 30.7 ( 8.6. It is noteworthy
that the parent AChE inhibitor tacrine was unable to significantly
inhibit the AChE-induced Aꢀ40 aggregation.8 Moreover, p-
methoxytacripyrine 11 resulted in being more potent than
donepezil and as potent as 3-{4-[(benzylmethylamino)meth-
yl]phenyl}-6,7-dimethoxy-2H-2-chromenone (AP2238)47 in this

Tacripyrines as Multitarget-Directed Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2727
Figure 1. Steady-state inhibition of AChE hydrolysis of acetylthio-
choline (ATCh) by 11. Lineweaver-Burk reciprocal plots of initial
velocity and substrate concentrations (0.058-0.578 mM) are presented.
Lines were derived from a weighted least-squares analysis of the data
points.
action. For the sake of completeness, it has to be mentioned
that heterodimers of tacrine have been shown to be more potent
in inhibiting the AChE-induced of Aꢀ40 aggregation.8 It must
be noticed that the assay is performed using much higher
concentrations of hAChE and Aꢀ40 than those present in brain.
Also, the concentration of inhibitor used is much higher (100
µM) than necessary to inhibit hAChE. However, the high
hAChE concentration is necessary to accelerate the aggregation
process up to a reasonable extent for analytical purposes;
therefore to better estimate the obtained data, values should be
normalized. Thus, if the inhibitor/AChE concentration ratio in
both assays is taken into account, the resulting values are indeed
of the same magnitude and, therefore, it would seem reasonable
that similar amounts of a given inhibitor might afford both
inhibitory activities. Moreover, the proof-of-concept of the
therapeutic usefulness of the in vitro Aꢀ antiaggregating effect
of dual binding site AChEIs has been recently obtained in in
vivo studies with memoquin9
Effects on Aꢀ42 Self-Aggregation. Because Aꢀ42 is the most
amyloidogenic isoform of ꢀ-amyloid, the ability of compound
11 to inhibit Aꢀ42 self-aggregation was also investigated. When
tested in a 1:1 ratio with Aꢀ42 (50 µM), 11 gave rise to a 34.9
( 5.4% of inhibition, whereas no significant inhibition was
detected when the ratio [Aꢀ42]/[inhibitor 11] was 5/1. From these
data, it can be stated that 11 is a weak inhibitor of Aꢀ42 self-
aggregation. Its inhibitory action can be compared to that of
resveratrol (30.0 ( 8.7% if tested at 50 µM). Moreover, it has
to be noted that anti-Alzheimer drugs such as donepezil and
tacrine were found to be inactive.
Molecular Modeling. The AChE binding mode of compound
11 was explored by means of docking and molecular dynamics
(MD) studies (see Experimental Section). The reliability of the
docking protocol was verified by examining the best poses
predicted for tacrine and propidium by taking advantage of the
known X-ray crystallographic binding mode of these compounds
at the catalytic and peripheral sites, respectively, of AChE (data
not shown),48,49 thus giving confidence to the binding mode
predicted for inhibitor 11. Whereas compound 11 was not easily
accommodated in the catalytic site, it was successfully docked
in the PAS, which agrees with the noncompetitive inhibition
mechanism exhibited by p-methoxytacripyrine 11. According
to the putative binding mode of the (R)-11 enantiomer, the
inhibitor is stacked against the indole ring of Trp286, while the
Figure 2. Time dependence of the root-mean square deviation (Å) of
the backbone (gray) and all-heavy (black) atoms of the residues that
define the PAS for the AChE complexes with (top) (R)- and (bottom)
(S)-enantiomers of 11.
NH group of the 1,8-naphthyridine unit is hydrogen-bonded to
the hydroxyl group of Tyr72, which in turn is hydrogen-bonded
to Asp74. The amino group is oriented toward Ser293, and the
4′-methoxybenzene unit is pointing toward the solvent, adopting
a similar arrangement as the aliphatic chain containing the
quaternary ammonium group in propidium, thus avoiding steric
clashes with residues at the PAS. Compared to the enantiomer
(R)-11, the 1,8-naphthyridine unit of the enantiomer (S)-11 is
rotated by ca. 180° along the short axis of the cyclic ring, which
permits retention of the orientation of the 4′-methoxybenzene
unit in the PAS, though at the expense of weakening the stacking
with Trp286 because the ligand is located less deeply in the
binding site (see Supporting Information).
To examine the relative binding affinity of the two enanti-
omers, a series of 12 ns MD simulations were run to explore
the structural stability. Compared to the X-ray structure, the
root-mean square deviation (rmsd) determined for the backbone
and all-heavy atoms of the residues that define the PAS in
the complex with the enantiomer (R)-11 was around 1.1 and
1.5 Å, which indicates the structural stability of the complex
along the trajectory (Figure 2). However, notably larger values
were found for the rmsd of both the backbone (1.6 Å) and all-
heavy atoms (2.3 Å) in the complex with the enantiomer (S)-
11, suggesting that binding of the (S)-11 to the PAS is associated
with a large structural destabilization (Figure 2).
MM/PBSA computations were performed for the snapshots
collected along the last 5 ns of the trajectories in order to
examine the relative stabilities of the (R)-11-AChE and (S)-
11-AChE complexes (Table 2). The results indicate that
binding of 11 to the AChE PAS mainly inVolVes the (R)-
enantiomer. Thus, even though the electrostatic contribution due

2728 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Marco-Contelles et al.
Table 3. Effect of Compounds 1-14 on [Ca2+]c Increase Elicited by 70
mM K+ in SH-SY5Y Cells (% Inhibition with Respect to a Control
without Any Drug)a
% blockade in
X, Y, Z
SH-SY5Y cells
nimodipine
1
2
3
4
5
6
7
8
9
10
11
12
13
14
-
45.5 ( 6.1***d
36.64 ( 2.66*b
22.22 ( 3.00 nse
34.35 ( 2.12*b
32.65 ( 4.20**c
33.61 ( 5.10***d
33.65 ( 3.00***d
29.83 ( 3.98 ns
35.35 ( 4.94*b
46.50 ( 4.44***d
31.04 ( 4.10**c
32.75 ( 2.50**c
45.45 ( 5.70*b
48.78 ( 2.30***d
44.78 ( 5.60**c
X ) H, Y, Z ) CH
X ) 4′-F, Y ) C, Z ) CH
X ) 2′-CF3, Y, Z ) CH
X ) 2′-NO2, Y, Z ) CH
X ) 3′-NO2, Y, Z ) CH
X ) 4′-NO2, Y ) C, Z ) CH
X ) 4′-Me, Y ) C, Z ) CH
X ) 4′-C6H5, Y ) C, Z ) CH
X ) 2′-OMe, Y, Z ) CH
X ) 3′-OMe, Y, Z ) CH
X ) 4′-OMe, Y ) C, Z ) CH
X ) 3′, 4′-di-OMe, Y ) C, Z ) CH
X ) H, Y ) CH, Z ) N
X ) H, Y ) N, Z ) CH
Figure 3. Representation of the binding mode of (R)-11 in the PAS
of AChE. Relevant residues of the PAS are shown in stick. The red
contour denotes the presence of water at a density of two times that of
pure bulk water.
a Data are expressed as means ( SEM of at least three different cultures
in quadruplicate. b *p < 0.05. c **p < 0.01. d ***p < 0.001 with respect
to [Ca2+]c increase in control cells. All compounds were assayed at the
concentration of 0.3 µM. e ns ) not significant.
Table 2. Relative Contributions to the Binding Affinity (kcal/mol)
between (R)- and (S)-Enantiomers of 11 to AChE Determined from
MM/PBSA Computations (Values Given Relative to the (R)-Enantiomer)
sol
sol
ε
∆GMM
∆G
∆G
∆∆Gbinding
ele
nonpolar
positive control such as nimodipine (0.3 µM), which caused
45.5% inhibition of K+-evoked Ca2+ uptake.
2
4
83.3
38.4
-80.9
-19.1
1.1
1.1
3.6
20.5
The calcium channel antagonist activities exhibited by
compounds 1-14 are shown in Table 3. Most of the tested
compounds promoted significant Ca2+ blockade, the most potent
being hybrid 13, whose activity (49% inhibition) was similar
to that obtained with nimodipine (i.e., the reference compound
used in this assays) at the same concentration. Tacripyrines 9,
12, and 14 also showed good blockade (>44% inhibition)
regardless of the electronic nature of the substituents or the
groups attached to the aromatic ring.
Neuroprotection. Prompted by the results obtained for
compounds 1-14 as [Ca2+]c blockers, their potential neuropro-
tective activity was investigated. For this purpose, we studied
the effects of 1-14 on SH-SY5Y cells exposed for 24 h to a
medium with a depolarizing concentration of KCl (70 mM),
which induced Ca2+ overload and cell death. Drugs at a
concentration of 0.3 µM were administered 24 h before
incubation of the cells with high K+ (70 mM; hypertonic) and
maintained during the entire experiment. Thereafter, release of
lactic dehydrogenase (LDH) was measured as a marker of cell
death.55
As shown in Table 4, all compounds afforded a higher degree
of neuroprotection compared to tacrine and within the same
range relative to nimodipine. Only 4 did not show a statistically
significant protection against Ca2+ overload. Tacripyrines 2, 6,
and 11 were the most effective compounds with protection
activities (%) of 48.8, 45.0, and 41.3, respectively. Again, it
has to be highlighted that the electronic nature of the substituents
in the aromatic ring does not seem to have a critical influence
on the observed activities.
to the solvent reaction field favors the interaction with the (S)-
enantiomer, this trend is compensated by the larger internal
destabilization, which agrees with the structural distortion
observed in the (S)-11-AChE complex (see above). In par-
ticular, the stacking with the indole ring of Trp286, which is a
common feature observed experimentally49-51 and from mo-
lecular modeling52,53 studies for a variety of inhibitors interacting
at the PAS, is lost in the binding mode of (S)-11.
The main features that mediate the binding of (R)-11 to the
PAS in the docking solution are preserved at the end of the
MD simulation (see Figure 3). Besides the stacking with Trp286,
it is worth noting the hydrogen-bond contact between the NH
group of the 1,8-naphthyridine unit and the hydroxyl group of
Tyr72 because this feature allows us to explain the 20-fold
increase in the inhibitory potency observed between compounds
11 and the analogue obtained upon replacement of the NH
moiety by O (IC50 of 870 nM in eeAChE).54 Furthermore, it is
also noteworthy the high density of water molecules found
between the NH2 group of the 1,8-naphthyridine unit and
Ser293, suggesting that they also contribute to the binding at
the PAS through water-mediated bridges. Finally, the lack of a
tryptophan residue mimicking Trp286 in the peripheral site of
hBuChE (replaced 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.
Ca2+ Channel Blockade Activity of Tacripyrines. To study
the multitarget activity of tacripyrines, we investigated the Ca2+
influx induced by K+-depolarization in SH-SY5Y neuroblastoma
cells, previously loaded with the fluorescent dye Fluo-4AM.
Fluo-4-loaded cells were incubated in the presence of
compounds 1-14 (0.3 µM) for 10 min and then stimulated with
a concentrated solution of KCl, so that the final K+ concentration
in the medium was 70 mM. In all experiments, we included a
Antioxidant Activity. The antioxidant activity of 1-14 was
evaluated at a concentration of 0.3 µM on SH-SY5Y neuro-
blastoma cells exposed to 60 µM H2O2 for 24 h.
The results shown in Table 5 indicate that tacripyrines protect
much more efficiently against free radicals than the parent
compounds, tacrine and nimodipine, which did not show any

Tacripyrines as Multitarget-Directed Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2729
Table 5. Effect of Tacripyrines 1-14 on SH-SY5Y Cells Viability
Table 4. Effect of Tacripyrines 1-14 on SH-SY5Y Cells Viability
a
Expressed as % LDH Released in the Presence of 70 mM K+ and %
Expressed as % LDH Released in the Presence of 60 µM H2O2 and %
a
Protection
Protection
LDH release
%
LDH release
%
X
(% of control)
protection
X, Y, Z
(% of control)
protection
18.93 ( 1.77***b 88.34 ( 2.80
tacrine
nimodipine
1
2
3
4
5
6
7
8
9
10
11
12
90.4 ( 4.1 nsc
13.4 ( 7.2
catalase
tacrine
nimodipine -
1
2
3
4
5
6
7
8
9
10
11
12
-
-
75.00 ( 2.24***b 35.93 ( 2.84
75.02 ( 2.56***b 35.92 ( 3.53
61.22 ( 2.10***b 48.79 ( 3.99
79.29 ( 1.64***b 29.79 ( 2.20
100.34 ( 3.37 nsc
0
75.42 ( 2.12***b 36.03 ( 2.82
76.61 ( 1.83***b 30.60 ( 1.96
64.17 ( 1.53***b 46.11 ( 2.02
71.72 ( 1.29***b 37.21 ( 1.27
71.91 ( 1.23***b 37.18 ( 1.61
77.19 ( 1.69***b 30.09 ( 2.18
X ) H, Y, Z ) CH
X ) 4′-F, Y ) C, Z ) CH
X ) 2′-CF3, Y, Z ) CH
X ) 2′-NO2, Y, Z ) CH
X ) 3′-NO2, Y, Z ) CH
X ) 4′-NO2, Y ) C, Z ) CH
X ) 4′-Me, Y ) C, Z ) CH
X ) H, Y, Z ) CH
X ) 4′-F, Y ) C, Z ) CH
X ) 2′-CF3, Y, Z ) CH
X ) 2′-NO2, Y, Z ) CH
X ) 3′-NO2, Y, Z ) CH
88.29 ( 2.99 ns
16.59 ( 4.18
77.87 ( 2.74***b 31.80 ( 3.81
63.91 ( 2.34***b 45.03 ( 4.02
72.43 ( 1.89***b 39.70 ( 2.47
X ) 4′-NO2, Y ) C, Z ) CH 72.43 ( 2.99***b 35.48 ( 3.78
X ) 4′-C6H5, Y ) C, Z ) CH 81.50 ( 4.61**b
26.51 ( 6.59
X ) 4′-Me, Y ) C, Z ) CH
59.60 ( 0.92***b 53.68 ( 1.62
X ) 2′-OMe, Y, Z ) CH
X ) 3′-OMe, Y, Z ) CH
77.61 ( 1.66***b 32.23 ( 2.21
68.96 ( 1.33***b 38.77 ( 3.43
X ) 4′-C6H5, Y ) C, Z ) CH 62.24 ( 3.65***b 47.82 ( 4.06
X ) 2′-OMe, Y, Z ) CH
X ) 3′-OMe, Y, Z ) CH
63.78 ( 1.35***b 48.23 ( 2.24
65.14 ( 1.23***b 44.80 ( 1.53
X ) 4′-OMe, Y ) C, Z ) CH 67.63 ( 2.24***b 41.30 ( 4.95
X ) 3′,4′-di-OMe,
73.66 ( 1.84***b 37.89 ( 2.37
X ) 4′-OMe, Y ) C, Z ) CH 56.55 ( 1.94***b 55.78 ( 2.32
Y ) C, Z ) CH
X ) 3′, 4′-di-OMe,
77.42 ( 2.22***b 29.41 ( 2.43
13
14
X ) H, Y ) CH, Z ) N
X ) H, Y ) N, Z ) CH
75.77 ( 2.18***b 32.82 ( 4.90
74.31 ( 1.48***b 37.11 ( 1.96
Y ) C, Z ) CH
13
14
X ) H, Y ) CH, Z ) N
X ) H, Y ) N, Z ) CH
57.51 ( 2.25***b 54.38 ( 2.03
a Data are expressed as the means ( SEM of at least three different
batches of cells in quadruplicate. LDH released was calculated for each
individual experiment, considering 100% the extracellular LDH released
in the presence of vehicle. To calculate % protection, LDH release was
normalized as follows: in each individual quadruplicate experiment, the
LDH release obtained in nontreated cells (basal) was subtracted from the
LDH released upon 70 mM K+ treatment and normalized to 100%, then
that value was subtracted from 100. All the compounds were assayed at a
concentration of 0.3 µM. b ***p < 0.001. c ns ) not significant.
60.32 ( 1.50***b 52.61 ( 2.01
a Data expressed as the means ( SEM of at least three different cultures
in quadruplicate. LDH released was calculated for each individual experi-
ment, considering 100% the extracellular LDH released in the presence of
vehicle. To calculate % protection, LDH release was normalized as follows:
in each individual quadruplicate experiment, the LDH release obtained in
nontreated cells (basal) was subtracted from the LDH released upon 70
mM K+ treatment and normalized to 100%, then that value was subtracted
from 100. All the compounds were assayed at a concentration of 0.3 µM.
b ***p < 0.001. c ns ) not significant.
neuroprotective effect. Compounds 7, 11, 13, and 14 exhibited
neuroprotection values higher than 50%, whereas 2 and 8-10
elicited neuroprotection activities ranging from 44% to 48%.
Thus, three of the most potent neuroprotectors in this assay (2,
7, and 11) also proved to be among the most efficient in the
previous test (see Table 3).
Table 6. Permeability (Pe 10-6 cm s-1) in the PAMPA-BBB Assay of
18 Commercial Drugs and Tacripyrines 1-14a (Predictive Penetration in
the CNS)
commercial
drugs
Pe
CNS
prediction
c
biblb
expc
compd (10-6 cm s-1
)
testosterone
verapamil
17.0 23.1 ( 0.1
16.0 12.6 ( 0.2
1
2
3
4
5
6
7
8
12.6 ( 0.2
9.6 ( 0.1
1.4 ( 0.1
8.6 ( 0.2
7.0 ( 0.1
5.8 ( 0.1
1.8 ( 0.1
ndd
6.1 ( 0.1
5.5 ( 0.1
12.8 ( 0.2
9.4 ( 0.1
7.2 ( 0.4
6.2 ( 0.1
CNS+
CNS+
CNS(
CNS+
CNS+
CNS+
CNS(
In Vitro Blood-Brain Barrier (BBB) Permeation Assay. To
evaluate the brain penetration of tacripyrines, we used a parallel
artificial membrane permeation assay for blood-brain barrier
(PAMPA-BBB), following in part the method described by Di
et al.56 that was successfully applied by us to different
compounds57,58 The in vitro permeabilities (Pe) of tacripyrines
1-14 and 18 commercial drugs through a lipid extract of porcine
brain were determined (see Table 6). Assay validation was made
by comparing the experimental permeability with the reported
values of the commercial drugs, which showed a good correla-
tion, Pe (exp) ) 0.99 Pe (bibl) - 1.32 (r ) 0.90). From this
equation and taking into account the limits established by Di et
al. for BBB permeation,56 we established the ranges of perme-
ability as (i) compounds of high BBB permeation (CNS+): Pe
(10-6 cm s-1) > 2.7, (ii) compounds of low BBB permeation
(CNS-): Pe (10-6 cm s-1) < 0.7, and (iii) compounds of
uncertain BBB permeation (CNS(): 2.7 > Pe (10-6 cm s-1) >
0.7. As it can be seen in Table 6, the assay showed that almost
all tacripyrines could cross the BBB and reach their biological
targets in the central nervous system. Only two compounds, 3
and 7, showed uncertain brain permeation (CNS ().
imipramine
desipramine
astemizole
progesterone
promazine
chlorpromazine
clonidine
corticosterone
piroxicam
hydrocortisone
caffeine
aldosterone
lomefloxacin
enoxacin
atenolol
ofloxacin
13.0
7.3 ( 0.2
12.0 13.9 ( 0.1
11.0
9.3
8.8
6.5
5.3
5.1
2.5
1.9
1.3
1.2
1.1
0.9
6.1 ( 0.2
6.6 ( 0.5
7.7 ( 0.2
1.4 ( 0.2
4.2 ( 0.3
3.6 ( 0.3
0.4 ( 0.1
1.2 ( 0.2
1.0 ( 0.1
0.6 ( 0.1
0.5 ( 0.1
0.6 ( 0.2
9
CNS+
CNS+
CNS+
CNS+
CNS+
CNS+
10
11
12
13
14
0.8 0.03 ( 0.01
0.8 0.3 ( 0.01
a Commercial drugs and tacripyrines were tested using a mixture of PBS:
EtOH (80:20). b Taken from ref 56. c Data are expressed as the means (
standard deviation of three different experiments in quadruplicate. nd: not
determined. d Permeability was not determined due to the poor solubility
in PBS:EtOH (80:20).
A variety of substituted compounds have been easily prepared
by simple, high-yielding protocols from readily available
precursors. From the AChE inhibition results, we conclude that
tacripyrines are potent AChE inhibitors regardless of the
electronic nature of the substitution at C4, the best AChEI
tacripyrine being compounds 8 and 10-12 on both EeAChE
and hAChE. These hybrids were also devoid of hBuChE
Conclusions
We have synthesized and evaluated a series of tacrine-DHP
hybrids, named tacripyrines, which are potent and selective
inhibitors of AChE and show potent neuroprotection activity.

2730 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Marco-Contelles et al.
mmol) and cyclohexanone (61.7 mg, 0.63 mmol), in ClCH2CH2Cl
(5 mL), after 7 h, gave product 8 (131.0 mg, 71%); mp 195-197
°C. IR (KBr) ν 3414, 3021, 2931, 2862, 1630, 1573, 1483, 1449,
inhibition activity, showing, therefore, an extremely high
selectivity. Moreover, the mixed type inhibition of hAChE
activity of compound 11, one of the most potent derivatives,
was associated to a 30.7 ( 8.6% inhibition of the proaggregating
action of AChE on the ꢀ-amyloid peptide. Compound 11 was
also a moderate inhibitor of ꢀ-amyloid self-aggregation (34.9
( 5.4%). Molecular modeling results point out that AChE-
inhibitor 11 is not easily accommodated in the catalytic site,
but it can be successfully docked in the PAS, which agrees with
the noncompetitive inhibition mechanism exhibited by 11. In
addition, the inhibitor (R)-11 is stacked against the indole ring
of Trp286, while 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 Asp74. The amino group is oriented
toward Ser293, and the 4′-methoxybenzene unit is pointing
toward the solvent, adopting a similar arrangement as the
aliphatic chain containing the quaternary ammonium group in
propidium, thus avoiding steric clashes with residues at the PAS.
Compared with the enantiomer (S)-11, the binding of this
inhibitor in the AChE PAS mainly involves the (R)-11 enan-
tiomer.59
1
1243 cm-1. H NMR (see Table 9, Supporting Information). 13C
NMR (see Table 10, Supporting Information). MS (API-ES+) m/z:
[M + 1]+ 440.2; [2M + Na]+ 901.5. Anal. (C28H29N3O2) C, H, N.
Ethyl Ester of 5-Amino-4-(3′,4′-dimethoxyphenyl)-1,4,6,7,8,9-
hexahydro-2-methyl-benzo[b][1,8]naphthyridine-3-carboxylic (12).
Following the general method for the Friedla¨nder synthesis, reaction
of compound 42 (200 mg, 0.58 mmol) with AlCl3 (105.8 mg, 0.87
mmol) and cyclohexanone (85.62 mg, 0.87 mmol), in ClCH2CH2Cl
(5 mL), after 4 h, gave product 12 (233.1 mg, 94%); mp 112-115
°C. IR (KBr) ν 3392, 2932, 1627, 1511, 1448, 1264, 1230 cm-1
.
1H NMR (see Table 9, Supporting Information). 13C NMR (see
Table 10, Supporting Information)). MS (API-ES+) m/z: [M + 1]+
424.2. Anal. (C24H29N3O4) C, H, N.
Ethyl Ester of 5-Amino-1,4,6,7,8,9-hexahydro-2-methyl-4-(3′-
pyridyl)benzo[b][1,8]naphthyridine-3-carboxylic Acid (13). Fol-
lowing the general method for the Friedla¨nder synthesis, reaction
of compound 43 (250 mg, 0.88 mmol) with AlCl3 (175.56 mg, 1.32
mmol) and cyclohexanone (129.4 mg, 1.32 mmol), in ClCH2CH2Cl
(5 mL), after 6 h, gave product 13 (259 mg, 71%); mp 132-134
°C. IR (KBr) ν 3379, 3217, 3079, 2971, 2933, 2172, 1627, 1576,
Most of the compounds blocked [K+]-induced [Ca2+
]
1501, 1447, 1383 cm-1 1H NMR (see Table 9, Supporting
.
c
Information). 13C NMR (see Table 10, Supporting Information).
MS (API-ES+) m/z: [M + 1]+ 365.2; [2M + Na]+ 751.3. Anal.
(C23H27N3O3) C, H, N.
increase in a statistically significant manner and induced a
remarkable neuroprotective effect against both Ca2+ overload
and oxidative stress. The fact that these compounds provide
protection against two stimuli with different mechanisms of
action (i.e., calcium overload and free radical generation)
indicates that they could exert their effects on the apoptotic
cell death cascade beyond the particular mechanism of each
toxic agent. In particular, they might induce the expression
of proteins implicated in cell survival, as demonstrated
previously for a tacrine analogue.54 These findings, in
conjunction with the predicted ability to enter the central
nervous system, indicate that tacripyrines can be considered
as interesting new chemical entities with potential therapeutic
application for AD patients.
Ethyl Ester of 5-Amino-1,4,6,7,8,9-hexahydro-2-methyl-4-(4′-
pyridyl)benzo[b][1,8]naphthyridine-3-carboxylic Acid (14). Fol-
lowing the general method for the Friedla¨nder synthesis, reaction
of compound 44 (200 mg, 0.70 mmol) with AlCl3 (139.65 mg, 1.05
mmol) and cyclohexanone (103.5 mg, 1.05 mmol), in ClCH2CH2Cl
(5 mL), after 15 h, gave product 14 (232 mg, 90%); mp 194-7
°C. IR (KBr) ν 3406, 2928, 2855, 1631, 1602, 1448, 1270 cm-1
.
1H NMR (see Table 9, Supporting Information). 13C NMR (see
Table 10, Supporting Information). MS (API-ES+) m/z: [M + 1]+
365.2; [M + Na]+ 387.1; [2M + Na]+ 751.5. Anal. (C21H24N4O2)
C, H, N.
Acknowledgment. J.M.C. thanks Dr. Ma. Luz de la Puente
(Analytical Technologies Department, Lilly SA), and Dr. Ma.
Angeles Mart´ınez-Grau (Lilly SA) for the resolution of com-
pound 11. J.M.C. and also R.L. thank MEC for a fellowship
(AP20020576) and B.L. thanks CSIC for a I3P Training
Contract. The present work has been supported by Fundacio´n
Teo´filo Hernando, MEC grants BFI2003-02722; SAF2006-
08764-C02-01, SAF-2006-08540, SAF2006-1249, and CTQ2005-
09365, CAM (S/SAL-0275-2006), ISCIII [Red RENEVAS
(RD06/0026/1002)], CSIC-GRICES project (2007PT-13), and
Fundacio´n La Caixa (Barcelona, Spain).
Experimental Section
General Method for the Friedla¨nder Reaction. Aluminum
chloride (1.2-1.7 equiv) was suspended in dry 1,2-dichloroethane
(10 mL) at rt under argon. The corresponding 4H-benzopyran (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 solution 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, and filtered, and the solvent was
evaporated. The resultant solid was purified by silica gel flash
chromatography using methanol/dichloromethane mixtures as eluent
to give pure compounds.
Supporting Information Available: Experimental details for
chemistry, spectroscopy [NMR data for intermediate (32-44) and
target compounds (1, 2, 4-6, 7, 9-11)], elemental analyses for
new intermediate (32-44) and target molecules (1-14), biology,
modeling, including the best poses for the (R)- and (S)-
enantiomers of 11 predicted from docking studies, and references
cited therein. Chromatograms and experimental conditions for
the chiral HPLC-mediated resolution of racemic p-methoxytac-
Ethyl Ester of 5-Amino-1,4,6,7,8,9-hexahydro-2-methyl-4-(2′-
trifluoromethylphenyl)-drobenzo[b][1,8]naphthyridine-3-carboxy-
lic Acid (3). Following the general method for the Friedla¨nder
synthesis, reaction of compound 33 (200 mg, 0.57 mmol) with AlCl3
(113.05 mg, 0.85 mmol) and cyclohexanone (83.68 mg, 0.85 mmol),
in ClCH2CH2Cl (5 mL), after 6 h, gave product 3 (194 mg, 79%);
mp 124-126 °C. IR (KBr) ν 3421,2934, 2862, 1688,1630, 1557,
1
ripyrine (11), H NMR spectra, quiroptical properties, and the
inhibition of AChE/BuChE, of the separated enantiomers (A)-
11 and (B)-11. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
1502, 1448, 1310, 1233 cm-1. H NMR (see Table 9, Supporting
Information). 13C NMR (see Table 10, Supporting Information).
MS (API-ES+) m/z: [M + 1]+ 432.1; [2M + Na]+ 885.1. Anal.
(C23H24F3N3O2) C, H, N.
References
(1) Goedert, M.; Spillantini, M. G. A century of Alzheimer’s disease.
Science 2006, 314, 777–781.
(2) Castro, A.; Mart´ınez, A. Targeting beta-amyloid pathogenesis through
acetylcholinesterase inhibitors. Curr. Pharm. Des. 2006, 12, 4377–
4387.
Ethyl Ester of 5-Amino-4-(4′-biphenyl)-1,4,6,7,8,9-hexahydro-
2-methyl-benzo[b][1,8]naphthyridine-3-carboxylic Acid (8). Fol-
lowing the general method for the Friedla¨nder synthesis, reaction
of compound 38 (150 mg, 0.42 mmol) with AlCl3 (125.7 mg, 0.63

Tacripyrines as Multitarget-Directed Ligands
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2731
(3) Cummings, J. L. Treatment of Alzheimer’s disease: current and future
therapeutic approaches. ReV. Neurol. Dis. 2004, 1, 60–69.
(4) Scarpini, E.; Scheltens, P.; Feldman, H. Treatment of Alzheimer’s
disease: current status and new perspectives. Lancet Neurol. 2003, 2,
539–547.
(5) Talesa, V. N. Acetylcholinesterase in Alzheimer’s disease. Mech.
Ageing DeV. 2001, 122, 1961–1969.
(24) Sobrado, M.; Lo´pez, M. G.; Carceller, F.; Garc´ıa, A. G.; Roda, J. M.
Combined nimodipine and citicoline reduce infarct size, attenuate
apoptosis, and increase bcl-2 expression after focal cerebral ischemia.
Neuroscience 2003, 118, 107–113.
(25) Fu, H.; Li, W.; Lao, Y.; Luo, J.; Lee, N. T.; Kan, K. K.; Tsang,
H. W.; Tsim, K. W.; Pang, Y.; Li, Z.; Chang, D. C.; Li, M.; Han,
Y. Bis(7)-tacrine attenuates beta amyloid-induced neuronal apop-
tosis by regulating L-type calcium channels. J. Neurochem. 2006,
98, 1400–1410.
(6) Racchi, M.; Mazzucchelli, M.; Porrello, E.; Lanni, C.; Govoni, S.
Acetylcholinesterase inhibitors: novel activities of old molecules.
Pharmacol. Res. 2004, 50, 441–451.
(26) Perry, G.; Cash, A. D.; Smith, M. A. Alzheimer’ s disease and oxidative
stress. J. Biomed. Biotechnol. 2002, 2, 120–123.
´
(7) Inestrosa, N. C.; Alvarez, A.; Pe´rez, C. A.; Moreno, R. D.; Vicente,
M.; Linker, C.; Casanueva, O. I.; Soto, C.; Garrido, J. Acetylcho-
linesterase accelerates assembly of amyloid-beta-peptides into Alzhe-
imer’s fibrils: possible role of the peripheral site of the enzyme. Neuron
1996, 16, 881–891.
(27) Tan, D. X.; Manchester, L. C.; Sainz, R.; Mayo, J. C.; Alvares, F. L.;
Reiter, R. J. Antioxidant strategies in protection against neurodegen-
erative disorders. Expert Opin. Ther. Pat. 2003, 13, 1513–1543.
(28) Klatte, E. T.; Scharre, D. W.; Nagaraja, H. N.; Davis, R. A.;
Beversdorf, D. Q. Combination therapy of donepezil and vitamin E
in Alzheimer’s disease. Alzheimer Dis. Assoc. Disord. 2003, 17, 113–
116.
(8) Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V. beta-Amyloid
aggregation induced by human acetylcholinesterase: inhibition studies.
Biochem. Pharmacol. 2003, 65, 407–416.
(9) Cavalli, A.; Bolognesi, M. L.; Capsoni, S.; Andrisano, V.; Bartolini,
M.; Margotti, E.; Cattaneo, A.; Recanatini, M.; Melchiorre, C. A small
molecule targeting the multifactorial nature of Alzheimer’s disease.
Angew. Chem., Int. Ed. 2007, 46, 3689–3692.
(29) Marco-Contelles, J.; Leo´n, R.; de Los R´ıos, C.; Guglietta, A.; Terencio,
J.; Lo´pez, M. G.; Garc´ıa, A. G.; Villarroya, M. Novel multipotent
tacrine-dihydropyridine hybrids with improved acetylcholinesterase
inhibitory and neuroprotective activities as potential drugs for the
treatment of Alzheimer’s disease. J. Med. Chem. 2006, 49, 7607–
7610.
(10) Mun˜oz-Torrero, D.; Camps, P. Dimeric and hybrid anti-Alzheimer
drug candidates. Curr. Med. Chem. 2006, 13, 399–422.
(11) Savini, L.; Gaeta, A.; Fattorusso, C.; Catalanotti, B.; Campiani, G.;
Chiasserini, L.; Pellerano, C.; Novellino, E.; McKissic, D.; Saxena,
A. Specific targeting of acetylcholinesterase and butyrylcholinesterase
recognition sites. Rational design of novel, selective, and highly potent
cholinesterase inhibitors. J. Med. Chem. 2003, 46, 1–4.
(12) Decker, M. Homobivalent quinazolinimines as novel nanomolar
inhibitors of cholinesterases with dirigible selectivity toward butyryl-
cholinesterase. J. Med. Chem. 2006, 49, 5411–5413.
(13) Carlier, P. R.; Chow, E. S.; Han, Y.; Liu, J.; El Yazal, J.; Pang, Y. P.
Heterodimeric tacrine-based acetylcholinesterase inhibitors: investigat-
ing ligand-peripheral site interactions. J. Med. Chem. 1999, 42, 4225–
4231.
(14) Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.;
Recanatini, M.; Melchiorre, C. Multi-target-directed ligands to combat
neurodegenerative diseases. J. Med. Chem. 2008, 51, 347–372.
(15) Rodr´ıguez-Franco, M. I.; Ferna´ndez-Bachiller, M. I.; Pe´rez, C.;
Herna´ndez-Ledesma, B.; Bartolome´, B. Novel tacrine-melatonin
hybrids as dual-acting drugs for Alzheimer’s disease with improved
acetylcholinesterase inhibitory and antioxidant properties. J. Med.
Chem. 2006, 49, 459–462.
(16) Sterling, J.; Herzig, Y.; Goren, T.; Finkelstein, N.; Lerner, D.;
Goldenberg, W.; Miskolczi, I.; Molnar, S.; Rantal, F.; Tamas, T.; Toth,
G.; Zagyva, A.; Zekany, A.; Finberg, J.; Lavian, G.; Gross, A.;
Friedman, R.; Razin, M.; Huang, W.; Krais, B.; Chorev, M.; Youdim,
M. B.; Weinstock, M. Novel dual inhibitors of AChE and MAO
derived from hydroxy aminoindan and phenethylamine as potential
treatment for Alzheimer’s disease. J. Med. Chem. 2002, 45, 5260–
5279.
(17) Toda, N.; Tago, K.; Marumoto, S.; Takami, K.; Ori, M.; Yamada, N.;
Koyama, K.; Naruto, S.; Abe, K.; Yamazaki, R.; Hara, T.; Aoyagi,
A.; Abe, Y.; Kaneko, T.; Kogen, H. A conformational restriction
approach to the development of dual inhibitors of acetylcholinesterase
and serotonin transporter as potential agents for Alzheimer’s disease.
Bioorg. Med. Chem. 2003, 11, 4389–4415.
(30) Schwyzer, A.; Cruikshanks, G. S. Reaction products of ethyl benza-
lacetoacetate an cyclic ketones in the presence of secondary bases or
sodium etylate. J. Prakt. Chem. 1914, 89, 189–193.
(31) Fey, P.; Angerbauer, R.; Huebsch, W.; Bischoff, H.; Petzinna, D.;
Schmidt, D.; Thomas, G. Preparation of pyrimidinyldihydroxyala-
kanoates as antihypercholesterolemics. Patent EP 330057, 1989.
(32) Meyer, H.; Bossert, F.; Vater, W.; Stoepel, K. Dialkyl 4-aryl-2,6-
dimethyl-1,4-dihydro-3,5-pyridinecarboxylates. Patent DE 2117572,
1972.
(33) Loudon, J. D.; Tennant, G. Substituent interactions in ortho-substituted
nitrobenzenes. J. Chem. Soc. 1962, 3092–3097.
(34) Edwards, J. D.; Pianka, M. Fungitoxicity. IV. Fungitocixity of certain
ethylenic compounds. J. Sci. Food Agric. 1992, 14, 55–58.
(35) Heller, G.; Lauth, H.; Buchwaldt, A. Reactivity of the nitrobenzal-
dehydes. Chem. Ber. 1922, 55B, 483–489.
(36) Silver, R. F.; Kerr, K.; Frandsen, P. D.; Kelley, S. J.; Holmes, H. L.
Synthesis and chemical reactions of some conjugated heteroenoid
compounds. Can. J. Chem. 1967, 9, 45.
(37) Baltzly, R. The preparation and properties of a platinized charcoal
catalyst with some observations on the behaviour of inhibitors. J. Am.
Chem. Soc. 1952, 74, 4586–4589.
(38) Marvel, C. S.; Stille, J. K. Preparation of the pyridalacetones and the
inductive effect of nitrogen on the degradation of the intermediate
aldols. J. Org. Chem. 1957, 22, 1451–1457.
(39) Troschu¨tz, R.; Dennstedt, T. Synthese von substituierten 2-Aminoni-
cotinonitrilen. Arch. Pharm. 1994, 327, 33–40.
(40) McElvain, S. M.; Schoeder, J. P. Orthoesters and related compounds
from malono- and succinonitriles. J. Am. Chem. Soc. 1949, 71, 40–
46.
(41) Troschu¨tz, R. Synthesis of pyrido[2,3-d]pyrimidines with a trimetho-
prom partial structure. Arch. Pharm. 1989, 322, 285–290.
(42) Cheng, C. C.; Yan, S. J. The Friedla¨nder synthesis of quinolines. Org.
React. 1982, 28, 37–201.
(43) Leo´n, R.; Marco-Contelles, J.; Garc´ıa, A. G.; Villarroya, M. Synthesis,
acetylcholinesterase inhibition and neuroprotective activity of new
tacrine analogues. Bioorg. Med. Chem. 2005, 13, 1167–1175.
(44) Rappaport, F.; Fischl, J.; Pinto, N. An improved method for the
estimation of cholinesterase activity in serum. Clin. Chim. Acta 1959,
4, 227–230.
(45) Ellman, G. L.; Courtney, K. D.; Andres, B. J.; Featherstone, R. M. A
new and rapid colorimetric determination of acetylcholinesterase
activity. Biochem. Pharmacol. 1961, 7, 88–95.
(18) Rosini, M.; Antonello, A.; Cavalli, A.; Bolognesi, M. L.; Minarini,
A.; Marucci, G.; Poggesi, E.; Leonardi, A.; Melchiorre, C. Prazosin-
related compounds. Effect of transforming the piperazinylquinazoline
moiety into an aminomethyltetrahydroacridine system on the affinity
for alpha1-adrenoreceptors. J. Med. Chem. 2003, 46, 4895–4903.
(19) Elsinghorst, P. W.; Cieslik, J. S.; Mohr, K.; Tra¨nkle, C.; Gu¨tschow,
M. First gallamine-tacrine hybrid: design and characterization at
cholinesterases and the M2 muscarinic receptor. J. Med. Chem. 2007,
50, 5685–5695.
(20) Fang, L.; Appenroth, D.; Decker, M.; Kiehntopf, M.; Roegler, C.;
Deufel, T.; Fleck, C.; Peng, S.; Zhang, Y.; Lehmann, J. Synthesis and
biological evaluation of NO-donor-tacrine hybrids as hepatoprotective
anti-Alzheimer drug candidates. J. Med. Chem. 2008, 51, 713–716.
(21) Kruman, I.; Guo, Q.; Mattson, M. P. Calcium and reactive oxygen
species mediate staurosporine-induced mitochondrial dysfunction and
apoptosis in PC12 cells. J. Neurosci. Res. 1998, 51, 293–308.
(22) Mattson, M. P.; Cheng, B.; Davis, D.; Bryant, K.; Lieberburg, I.; Rydel,
R. E. beta-Amyloid peptides destabilize calcium homeostasis and
render human cortical neurons vulnerable to excitotoxicity. J. Neurosci.
1992, 12, 376–389.
(46) Cousins, M. S.; Carriero, D. L.; Salamone, J. D. Tremulous jaw
movements induced by the acetylcholinesterase inhibitor tacrine:
effects of antiparkinsonian drugs. Eur. J. Pharmacol. 1997, 322, 137–
145.
(47) Piazzi, L.; Rampa, A.; Bisi, A.; Gobbi, S.; Belluti, F.; Cavalli, A.;
Bartolini, M.; Andrisano, V.; Valenti, P.; Recanatini, M. 3-(4-
{[Benzyl(methyl)amino]methyl}phenyl)-6,7-dimethoxy-2H-2-chrome-
none (AP2238) Inhibits Both Acetylcholinesterase and Acetylcho-
linesterase-Induced ꢀ-Amyloid Aggregation: A Dual Function Lead
for Alzheimer’s Disease Therapy. J. Med. Chem. 2003, 46, 2279–
2282.
(23) Cano-Abad, M. F.; Villarroya, M.; Garc´ıa, A. G.; Gabila´n, N. H.;
Lo´pez, M. G. Calcium entry through L-type calcium channels causes
mitochondrial disruption and chromaffin cell death. J. Biol. Chem.
2001, 276, 39695–39704.
(48) Harel, M.; Schalk, I.; Ehret-Sabatier, L.; Bouet, F.; Goeldner, M.; Hirth,
C.; Axelsen, P. H.; Silman, I.; Sussman, J. L. Quaternary ligand binding
to aromatic residues in the active-site gorge of acetylcholinesterase.
Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 9031–9035.

2732 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Marco-Contelles et al.
(49) Bourne, Y.; Taylor, P.; Radic, Z.; Marchot, P. Structural insights into
ligand interactions at the acetylcholinesterase peripheral anionic site.
EMBO J. 2003, 22, 1–12.
(56) Di, L.; Kerns, E. H.; Fan, K.; McConnell, O. J.; Carter, G. T. High
throughput artificial membrane permeability assay for blood-brain
barrier. Eur. J. Med. Chem. 2003, 38, 223–232.
(50) Bourne, Y.; Kolb, H. C.; Radic, Z.; Sharpless, B.; Taylor, P.; Marchit,
P. Freeze-frame inhibitor captures acetylcholinesterase in a unique
conformation. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1449–1454.
(51) Harel, M.; Sonoda, L. K.; Silman, I.; Sussman, J. L.; Rosenberry, T.
Crystal structure of thioflavin T bound to the peripheral site of Torpedo
californica acetylcholinesterase reveals how thioflavin T acts as a
sensitive fluorescent reporter of ligand binding to the acylation site.
J. Am. Chem. Soc. 2008, 130, 7856–7861.
(52) Senapati, S.; Bui, J. M.; McCammon, J. A. Induced fit in mouse
acetylcholinesterase upon binding a femtomolar inhibitor: A molecular
dynamics study. J. Med. Chem. 2005, 48, 8155–8162.
(53) Mu˜oz-Ruiz, P.; Rubio, L.; Garc’a-Palomero, E.; Dorronsoro, I.; del
Monte-Milln´, M.; Valenzuela, R.; Usn´, P.; de Austria, C.; Bartolini,
M.; Andrisano, V.; Bidon-Chanal, A.; Orozco, M.; Luque, F. J.;
Medina, M.; Mart´ınez, A. Design, synthesis, and biological evaluation
of dual binding site acetylcholinesterase inhibitors: New disease-
modifying agents for Alzheimer’s disease. J. Med. Chem. 2005, 48,
7223–7233.
(54) Marco, J. L.; de los R´ıos, C.; Garc´ıa, A. C.; Villarroya, M.; Carreiras,
M. C.; Martins, C.; Eleute´rio, A.; Morreale, A.; Orozco, M.; Luque,
F. J. Synthesis, biological evaluation and molecular modelling of
diversely functionalized heterocyclic derivatives as inhibitors of
acetylcholinesterase/butyrylcholinesterase and modulators of Ca2+
channels and nicotinic receptors. Bioorg. Med. Chem. 2004, 12, 2199–
2218.
(55) Maroto, R.; De la Fuente, M. T.; Artalejo, A. R.; Abad, F.; Lo´pez,
M. G.; Garc´ıa-Sancho, J.; Garc´ıa, A. G. Effects of Ca2+ channel
antagonists on chromaffin cell death and cytosolic Ca2+ oscillations
induced by veratridine. Eur. J. Pharmacol. 1994, 270, 331–339.
(57) Reviriego, F.; Rodr´ıguez-Franco, M. I.; Navarro, P.; Garc´ıa-
Espan˜a, E.; Liu-Gonza´lez, M.; Verdejo, B.; Dome`nech, A. The
sodium salt of diethyl 1H-pyrazole-3,5-dicarboxylate as an efficient
amphiphilic receptor for dopamine and amphetamines. Crystal
structure and solution studies. J. Am. Chem. Soc. 2006, 128, 16458–
16459.
(58) Pavo´n, F. J.; Herna´ndez-Folgado, L.; Bilbao, A.; Cippitelli, A.;
Jagerovic, N.; Abella´n, G.; Rodr´ıguez-Franco, M. I.; Serrano, A.;
Mac´ıas, M.; Navarro, M.; Goya, P.; Rodr´ıguez de Fonseca, F.
Antiobesity effects of the novel in vivo neutral cannabinoid receptor
antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-
1,2,4-triazole-LH 21. Neuropharmacology 2006, 51, 358–366.
(59) Racemic p-methoxytacripyrine 11 has been resolved by chiral
25
HPLC-semipreparative chromatography. Enantiomer (A)-11 {R]D
+179 (c 0.5, CHCl3)} and enantiomer (B)-11 {[R]D25-180 (c 0.62,
CHCl3)} showed AChE/BuChE inhibition values [(A)-11: (IC50
EeAChE ) 80 ( 12 nM; IC50 hAChE ) 378 ( 40 nM; IC50
hBuChE ) 11000 ( 2000 µM); (B)-11: (IC50 EeAChE ) 9 ( 1
nM; IC50 hAChE ) 36 ( 3 nM; IC50 hBuChE ) >100000)]
(Supporting Information), in good agreement with the predictions
of the molecular modeling. The assignment of the absolute
configuration at the stereogenic center, the X-ray analysis of one
or both enantiomers of compound 11 with AChE, as well as some
biological tests in vivo on each enantiomer, are now in progress
and will be reported here in due course.
JM801292B