Novel donepezil-based inhibitors of acetyl- and butyrylcholinesterase and acetylcholinesterase-induced β-amyloid aggregation

Article abstract of DOI:10.1021/jm8001313

A novel series of donepezil-tacrine hybrids designed to simultaneously interact with the active, peripheral and midgorge binding sites of acetylcholinesterase (AChE) have been synthesized and tested for their ability to inhibit AChE, butyrylcholinesterase (BChE), and AChE-induced Aβ aggregation. These compounds consist of a unit of tacrine or 6-chlorotacrine, which occupies the same position as tacrine at the AChE active site, and the 5,6-dimethoxy-2-[(4-piperidinyl)methyl]-1-indanone moiety of donepezil (or the indane derivative thereof), whose position along the enzyme gorge and the peripheral site can be modulated by a suitable tether that connects tacrine and donepezil fragments. All of the new compounds are highly potent inhibitors of bovine and human AChE and BChE, exhibiting IC₅₀ values in the subnanomolar or low nanomolar range in most cases. Moreover, six out of the eight hybrids of the series, particularly those bearing an indane moiety, exhibit a significant Aβ antiaggregating activity, which makes them promising anti-Alzheimer drug candidates.

Full text of DOI:10.1021/jm8001313

3588
J. Med. Chem. 2008, 51, 3588–3598
Novel Donepezil-Based Inhibitors of Acetyl- and Butyrylcholinesterase and
Acetylcholinesterase-Induced ꢀ-Amyloid Aggregation
Pelayo Camps,*,† Xavier Formosa,† Carles Galdeano,† Ta`nia Go´mez,† Diego Mun˜oz-Torrero,*,† Michele Scarpellini,†
Elisabet Viayna,† Albert Badia,‡ M. Victo`ria Clos,‡ Antoni Camins,§ Merce` Palla`s,§ Manuela Bartolini,# Francesca Mancini,#
Vincenza Andrisano,# Joan Estelrich,|, Mo`nica Lizondo,|, Axel Bidon-Chanal,| and F. Javier Luque|
Laboratori de Qu´ımica Farmace`utica (Unitat Associada al CSIC), Facultat de Farma`cia, and Institut de Biomedicina (IBUB), UniVersitat de
Barcelona, AV. Diagonal 643, E-08028, Barcelona, Spain, Departament de Farmacologia, de Terape`utica i de Toxicologia, Facultat de
Medicina, UniVersitat Auto`noma de Barcelona, E-08193-Bellaterra, Barcelona, Spain, Unitat de Farmacologia i Farmacogno`sia, Facultat de
Farma`cia, UniVersitat de Barcelona, AV. Diagonal 643, E-08028, Barcelona, Spain, Department of Pharmaceutical Sciences, Alma Mater
Studiorum, Bologna UniVersity, Via Belmeloro 6, I-40126 Bologna, Italy, Departament de Fisicoqu´ımica, Facultat de Farma`cia, and Institut de
Biomedicina (IBUB), UniVersitat de Barcelona, AV. Diagonal 643, E-08028, Barcelona, Spain, and Institut de Nanocie`ncia i Nanotecnologia,
UniVersitat de Barcelona, Barcelona, Spain
ReceiVed February 7, 2008
A novel series of donepezil-tacrine hybrids designed to simultaneously interact with the active, peripheral
and midgorge binding sites of acetylcholinesterase (AChE) have been synthesized and tested for their ability
to inhibit AChE, butyrylcholinesterase (BChE), and AChE-induced Aꢀ aggregation. These compounds consist
of a unit of tacrine or 6-chlorotacrine, which occupies the same position as tacrine at the AChE active site,
and the 5,6-dimethoxy-2-[(4-piperidinyl)methyl]-1-indanone moiety of donepezil (or the indane derivative
thereof), whose position along the enzyme gorge and the peripheral site can be modulated by a suitable
tether that connects tacrine and donepezil fragments. All of the new compounds are highly potent inhibitors
of bovine and human AChE and BChE, exhibiting IC50 values in the subnanomolar or low nanomolar range
in most cases. Moreover, six out of the eight hybrids of the series, particularly those bearing an indane
moiety, exhibit a significant Aꢀ antiaggregating activity, which makes them promising anti-Alzheimer drug
candidates.
Chart 1. Structures of Tacrine, Donepezil, and the Known
Donepezil-Tacrine Hybrids 3 and 4
Introduction
Since bis(7)-tacrine, a heptamethylene-linked dimer of the
first marketed anti-Alzheimer drug tacrine (1, Chart 1), was
developed 1 decade ago,1–3 the search for inhibitors of acetyl-
cholinesterase (AChEa) able to simultaneously bind to its
catalytic and peripheral binding sites has become an area of
very active research. Several classes of dual binding site AChE
inhibitors have been developed by connecting through a suitable
linker the two interacting units, which are generally derived from
known AChE inhibitors either commercialized or under
development.2–8 The success of the dual binding site strategy
is evidenced by the large increase in AChE inhibitory potency
of these dimers or hybrids relative to the parent compounds
from which they have been designed. Further interest comes
from the fact that some of these dual binding site AChE
inhibitors have been shown to inhibit the aggregation of
ꢀ-amyloid peptide (Aꢀ),9–18 which is a key event in the
neurotoxic cascade of Alzheimer’s disease (AD).19 This effect,
* To whom correspondence should be addressed. For P.C.: phone, +34
+ 934024536; fax, + 34 + 934035941; e-mail, camps@ub.edu. For D.M.-
T.: phone, +34
+ 934024536; fax, + 34 + 934035941; e-mail,
dmunoztorrero@ub.edu.
†
Laboratori de Qu´ımica Farmace`utica and IBUB, Universitat de
Barcelona.
‡
§
#
|
which has been related to the blockade of the AChE peripheral
site20 by dual binding site AChE inhibitors, makes these
compounds very promising disease-modifying anti-Alzheimer
drug candidates.
To date, the sole dual binding site AChE inhibitor approved
for the treatment of AD is donepezil (2, Chart 1), though it was
not specifically designed as such.21 The X-ray crystallographic
structure of the complex between Torpedo californica AChE
(TcAChE) and donepezil (PDB entry 1EVE)22 shows that the
Universitat Auto`noma de Barcelona.
Unitat de Farmacologia i Farmacogno`sia, Universitat de Barcelona.
Bologna University.
Departament de Fisicoqu´ımica, Universitat de Barcelona.
Institut de Nanocie`ncia i Nanotecnologia, Universitat de Barcelona.
a Abbreviations: Aꢀ, ꢀ-amyloid peptide; AChE, acetylcholinesterase;
AChEI, acetylcholinesterase inhibitor; AD, Alzheimer’s disease; APP,
amyloid precursor protein; BChE, butyrylcholinesterase; DTNB, 5,5′-
dithiobis(2-nitrobenzoic) acid; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; MD,
molecular dynamics; PDB, protein data bank; PTFE, polytetrafluoroethylene;
rmsd, root-mean square deviation
10.1021/jm8001313 CCC: $40.75
2008 American Chemical Society
Published on Web 06/03/2008

Cholinesterases and ꢀ-Amyloid Aggregation Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3589
Scheme 1. Synthesis of the Donepezil-Tacrine Hybrids
14-17a,b
elongated structure of donepezil spans the entire length of the
enzyme active-site gorge forming a variety of interactions with
specific residues, such as aromatic stacking interactions between
the benzyl and indanone moieties and the indole rings of Trp84
and Trp279 (Trp86 and Trp286 in human AChE) at the catalytic
and peripheral sites, respectively, and the cation-π interaction
between the protonated piperidine nitrogen and the phenyl ring
of Phe330. As a result of its dual binding site character,
donepezil at 100 µM is able to inhibit by 22% the AChE-induced
aggregation of Aꢀ.23 As bis(7)-tacrine, most of the dual binding
site AChE inhibitors developed so far contain at least one unit
related to tacrine, probably because of its ease of synthesis and
itsaffinityforboththeactiveandperipheralsitesofAChE.10–12,17,24–34
Conversely, to the best of our knowledge, only two classes of
donepezil-based hybrids have been purposely designed as dual
binding site AChE inhibitors by combining different fragments
of donepezil with tacrine.30,31 Among these donepezil-tacrine
hybrids, compounds 3 and 4 (Chart 1), which retain the
N-benzylpiperidine and the 5,6-dimethoxy-1-indanone moieties
of donepezil, respectively, are the most potent, they being 37-
and 7-fold more potent than tacrine but nearly equipotent to
donepezil, while their Aꢀ-antiaggregating effect has not been
assessed.
Herein, we describe the synthesis, pharmacological evaluation
(AChE and butyrylcholinesterase (BChE) inhibition and Aꢀ-
antiaggregating effect), and molecular modeling of a novel class
of highly potent donepezil-tacrine hybrids. On the basis of the
binding modes of donepezil22 and tacrine35 within TcAChE,
these novel hybrids were designed by combining the 5,6-
dimethoxy-2-[(4-piperidinyl)methyl]-1-indanone moiety of done-
pezil with tacrine, which would thus replace the benzyl moiety
of donepezil. In contrast to the preceding approaches,30,31 the
strategy adopted here largely preserves the chemical skeleton
of donepezil, which should allow the new hybrid compounds
not only to mimic the interactions of tacrine at the catalytic
site and of the indanone ring at the peripheral one but also to
preserve the contacts of the piperidine moiety with the residues
that are lining the wall of the AChE gorge as a third binding
site within the enzyme.
Moreover, because the indane derivative 13 is readily avail-
able,44 the synthesis of a parallel series of achiral hybrids
16-17a,b was also carried out by following the same meth-
odology but using piperidine 13 in the last step instead of 12
(Scheme 1).
In a first approach, the amination of the chloroquinolines 5
and 6 was carried out on reaction with 3 equiv of 2-aminoethanol
(7a) or 3-amino-1-propanol (7b) in refluxing 1-pentanol45,46 for
18 h, followed by removal of the solvent and excess of
aminoalcohol by distillation under reduced pressure. Alterna-
tively, the change of this tedious microdistillation workup by a
standard acid-base extraction turned out to be much more
convenient for the isolation of the desired alcohols. In this way,
the new alcohols 8a and 9a,b and the known alcohol 8b40 were
obtained in excellent yields and used directly in the next reaction
without further purification. However, for characterization
purposes, the new alcohols 8a and 9a,b were transformed into
their hydrochlorides and recrystallized from MeOH/AcOEt
mixtures.
Chemistry
The structures of the novel donepezil-tacrine hybrids
14-15a,b are shown in Scheme 1. The linker was selected
taking into account the sizable increase in AChE inhibitory
potency observed in tacrine-based homo- and heterodimers upon
introduction in the tether of a protonatable amino group at a
distance equivalent to three methylene groups.28,29 Because this
protonatable amino group could be the piperidine nitrogen atom
of these donepezil-tacrine hybrids, a length of two to three
methylenes for the linker was considered. Moreover, introduc-
tion of a chlorine atom at position 6 of the tacrine skeleton
should lead to additional interactions within the active site of
the enzyme.36–38
The synthesis of the novel donepezil-tacrine hybrids
14-15a,b was carried out through a three-step sequence
involving amination of the known 9-chloro-1,2,3,4-tetrahy-
droacridines 5 or 639,40 with an ω-aminoalcohol 7, followed by
mesylation of the resulting alcohol 8 or 9 and subsequent
reaction with the known piperidine 1221,41,42 (Scheme 1). Taking
into account that both enantiomers of donepezil display similar
pharmacologic and pharmacokinetic profiles and span the entire
AChE gorge with a common pattern of interactions,43 com-
pounds 14-15a,b, which bear the stereocenter-containing
indanone unit of donepezil, were prepared in racemic form.
Mesylation of 8-9a,b proceeded almost quantitatively, af-
fording pure mesylates 10a,b and 11a and slightly impure 11b,
which were used directly in the following reaction.
Piperidine 12 was prepared in quantitative yield following a
described procedure42 based on the catalytic hydrogenation of
donepezil in MeOH at 1 atm and room temperature for 72 h,
while piperidine 13 was prepared in 82% yield by a new
procedure involving the catalytic hydrogenation of donepezil
hydrochloride in MeOH in the presence of 1 N HCl at 28 atm
and 65 °C for 6 days (Scheme 2). Alternatively, 13 was prepared
in better yield (92% overall) through a two-step procedure
involving aldol condensation of 5,6-dimethoxy-1-indanone with
pyridine-4-carboxaldehyde in the presence of p-TsOH·H2O in
refluxing toluene,41 followed by catalytic hydrogenation of the
resulting compound 18 under forcing conditions (Scheme 2).

3590 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Camps et al.
Table 1. Pharmacological Data of the Hydrochlorides of Tacrine,
6-Chlorotacrine, Donepezil, Compound 19, and the Dihydrochlorides of
the Donepezil-Tacrine Hybrids 14-15a,b and Their Indane Derivatives
16-17a,ba
Scheme 2. Synthesis of Piperidine 13 and the Indane Derivative
of Donepezil 19
IC50 (nM)
compd
bAChE
hAChE
hBChE
14a·2HCl
1.74 ( 0.02 4.04 ( 0.06
0.29 ( 0.03 0.88 ( 0.04
0.57 ( 0.05 0.67 ( 0.06
0.09 ( 0.01 0.27 ( 0.03
2.28 ( 0.06 5.13 ( 0.52
0.82 ( 0.06 2.16 ( 0.21
1.86 ( 0.07 2.60 ( 0.23
0.82 ( 0.08 1.06 ( 0.05
12.4 ( 0.6
12.3 ( 0.6
136 ( 9
14b·2HCl
15a·2HCl
15b·2HCl
66.3 ( 4.0
8.06 ( 0.32
7.25 ( 0.33
88.7 ( 0.2
72.7 ( 4.2
43.9 ( 1.7
916 ( 19
16a·2HCl
16b·2HCl
17a·2HCl
17b·2HCl
tacrine·HCl
6-chlorotacrine·HCl
donepezil·HCl
19·HCl
130 ( 10
205 ( 18
5.73 ( 0.44 8.32 ( 0.75
8.12 ( 0.26 11.6 ( 1.6
7273 ( 621
1451 ( 33
not determined 6401 ( 119
a Values are expressed as mean ( standard error of the mean of at least
four experiments. IC50 inhibitory concentration (nM) of AChE (from bovine
or human erythrocytes) or BChE (from human serum) activity.
Table 1 summarizes the data for the new compounds, as well
as for tacrine ·HCl, 6-chlorotacrine·HCl, and donepezil ·HCl as
reference compounds. Although the indane analogue of done-
pezil 19 is a known compound,44 its biological activity has not
been determined. In order to extend the comparison of activity
between indanone and indane derivatives planned for the novel
hybrids, the cholinesterase inhibitory activity of this indane
derivative of donepezil was also assessed. All of the new hybrids
are highly potent bAChE inhibitors, exhibiting IC50 values in
the subnanomolar range in most cases and being clearly more
potent than the parent compounds tacrine (57- to 1440-fold),
6-chlorotacrine (3- to 65-fold), donepezil (4- to 90-fold), and
19 (640- to 16100-fold). Hybrids 14-15, which bear the
indanone system of donepezil, are more potent bAChE inhibitors
(1.3- to 9-fold more potent) than their indane analogues 16-17,
although the difference is much more pronounced in the case
of donepezil and 19 (180-fold). The presence of a chlorine atom
in the tacrine moiety enhances the inhibitory potency by 3-fold
in indanone hybrids, while it has less effect in indane hybrids.
Moreover, the three-methylene tether in the hybrids of the b
series makes them 6- and 3-fold more potent than their indanone
and indane counterparts of the a series, respectively. Overall,
compound 15b is the most active compound with an IC50 of 90
pM.
The new hybrids result in less potent inhibitors of human
than bovine AChE (1.2- to 3-fold less potent), as it is the case
for the parent compounds (around 1.5-fold less potent). Nev-
ertheless, they are highly potent hAChE inhibitors, being more
active than tacrine (40- to 760-fold), 6-chlorotacrine (2-to 30-
fold), and donepezil (2- to 45-fold). As observed for bAChE,
the best substitution pattern involves the presence of a chlorine
atom at position 6 of the tacrine unit, the indanone ring of
donepezil, and a tether length of three methylenes. As a result,
15b is also the most active hAChE inhibitor (IC50 ) 0.27 nM).
Moreover, the new hybrids are potent inhibitors of hBChE,
though their activity is lower than that shown for hAChE. Thus,
these compounds are 1.6- to 246-fold more potent toward
hAChE than hBChE, as it is found for 6-chlorotacrine and
donepezil (110- and 627-fold more potent toward AChE) but
in contrast with tacrine (4.7-fold more potent toward BChE).
WhenhBChEinhibitoryactivityisconsidered,thestructure-activity
relationships in this novel class of compounds reveal trends
different from those observed for the AChE inhibitory activity.
Thus, the indane hybrids 16-17 are 1.5-fold more potent than
their analogues 14-15 bearing the indanone system of donepezil
Finally, alkylation of piperidines 12 and 13 with a stoichio-
metric amount of mesylates 10-11a,b in the presence of excess
of Et3N in DMSO at 85 °C for 2 days afforded the desired
donepezil-tacrine hybrids 14-15a,b and the indane derivatives
16-17a,b in low to moderate yields (Scheme 1) after a tedious
purification of the crude products by silica gel column
chromatography.
For comparison purposes, the indane derivative of donepezil,
19 (Scheme 2), was also prepared. This compound can be
prepared directly from donepezil by reduction with borane in
THF.44 However, in our hands this reaction failed. Other
attempts to prepare 19 involving Wolff-Kishner reduction of
donepezil or benzylation of piperidine 13 afforded intractable
mixtures containing the desired product together with starting
materials and some byproduct, as those arising from demethy-
lation of the 5-methoxy group, in the first case, or from N,N-
dibenzylation, in the latter. Finally, we prepared 19 in 53% yield
by NaBH3CN reductive alkylation of piperidine 13 with
benzaldehyde (Scheme 2), followed by purification by silica
gel column chromatography.
The novel hybrids 14-17a,b were fully characterized as
dihydrochlorides through their spectroscopic data and elemental
analyses (C, H, N, Cl), while the known indane analogue of
donepezil 19 was characterized by spectroscopic data and
HRMS analyses.
Pharmacology
AChE and BChE Inhibition. To determine the potential
interest of the new donepezil-tacrine hybrids 14-15a,b and
the indane derivatives 16-17a,b for the treatment of AD, their
AChE inhibitory activity was assayed by the method of Ellman
et al.47 on AChE from bovine (bAChE) and human (hAChE)
erythrocytes. Recent evidence has shown that in advanced AD
patients, AChE activity is greatly reduced in specific brain
regions while BChE activity increases, likely as compensation
for AChE decrease.48 The increasing role of BChE in the
hydrolysis of acetylcholine as the ratio AChE/BChE gradually
decreases in these patients suggests that inhibition of BChE
might be valuable in the search for anti-Alzheimer agents.48,49
Consequently, the inhibitory activity on human serum BChE
(hBChE) was also assayed by the same method.

Cholinesterases and ꢀ-Amyloid Aggregation Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3591
Table 2. Thioflavin T Competition Assay Results with the
(with the only exception of 17b, which is slightly less potent
than 15b), which agrees with the 1.1-fold increase in potency
of indane analogue 19 relative to donepezil. Moreover, a chlorine
atom at position 6 of the tacrine unit is detrimental for the
hBChE inhibitory activity, the unsubstituted hybrids 14 and 16
being 10-fold more potent than their 6-chloro derivatives 15
and 17, which agrees with the 21-fold increase in potency of
tacrine relative to 6-chlorotacrine and with the results reported
for other tacrine-based dual binding site AChE inhibitors.12,26
The effect of the tether length on the BChE inhibitory activity
is much lower than that observed when the AChE inhibitory
activity is considered, the hybrids of the b series being
equipotent or slightly more potent (up to 2-fold) than their
counterparts of the a series. Overall, the most active BChE
inhibitor is 16b (IC50 ) 7.25 nM), which is 6-, 125-, 1000-,
and 880-fold more potent than tacrine, 6-chlorotacrine, done-
pezil, and 19, respectively.
Although the higher AChE vs BChE inhibitory activity of
the first tacrine-based homo- and heterodimers was initially
ascribed to the lack of a peripheral binding site in BChE,1,46
recent studies have suggested that Phe278 would be responsible
for π-π interactions with aromatic moieties of tacrine-based
heterodimers.28 The higher BChE inhibitory activity of bis(7)-
tacrine and several tacrine-based heterodimers,28,29 as well as
that of some of the new donepezil-tacrine hybrids herein
described, relative to tacrine seems to validate this hypothesis.
At this point, the high inhibitory activity toward both AChE
and BChE of most of the donepezil-tacrine hybrids should not
be detrimental for an anti-Alzheimer agent because dual
inhibition of AChE and BChE could increase the efficacy of
treatment and broaden the indications.49
Dihydrochlorides of the Donepezil-Tacrine Hybrids 15a and 15b and
with the Hydrochlorides of Tacrine, 6-Chlorotacrine, and Donepezila
compd
reduction of fluorescence (%)
15a·2HCl
79.4 ( 3.5
57.0 ( 2.0
12.6 ( 3.8
12.4 ( 1.9
26.0 ( 3.5
15b·2HCl
tacrine·HCl
6-chlorotacrine·HCl
donepezil·HCl
a Percentage of thioflavin T fluorescence reduction by effect of several
AChE inhibitors at 100 µM concentration. Values are expressed as the mean
( standard error of the mean of three experiments.
Table 3. Inhibition of hAChE-Induced Aggregation of Aꢀ1-40 by
Donepezil-Tacrine Hybrids 15-17a,b and Tacrine, 6-Chlorotacrine,
Donepezil, and 19 as Reference Compoundsa
compd
inhibition at 100 µM ( SEM (%)
15a·2HCl
37.6 ( 8.8
46.1 ( 9.0
60.0 ( 3.2
65.9 ( 2.5
61.6 ( 1.8
63.5 ( 6.2
7b
15b·2HCl
16a·2HCl
16b·2HCl
17a·2HCl
17b·2HCl
tacrine·HCl
6-chlorotacrine·HCl
donepezil·HCl
19·HCl
8.5 ( 1.6
22b
17.5 ( 2.5
a Values are the mean of two independent measurements, each performed
in duplicate (SEM ) standard error of the mean). b Data from ref 23.
Since the excitation wavelength of propidium is very close to
the emission wavelength of thioflavin T, this compound was
not included in the assay. The active site inhibitors tacrine and
6-chlorotacrine reduce thioflavin T fluorescence, on average,
by 12%, an effect that might be ascribed to changes in the local
conformation at the peripheral site induced upon binding at the
AChE active site.56 The dual binding site AChE inhibitor
donepezil led to a 2-fold greater reduction in fluorescence (26%
reduction) relative to the preceding active site inhibitors, as
expected from the direct interaction of the indanone moiety with
the AChE peripheral site. Finally, the novel donepezil-tacrine
hybrids 15a and 15b produced the highest reductions in
thioflavin T fluorescence among all the tested compounds (79%
and 57% reduction, respectively), which could be ascribed to
the displacement of the fluorophore at the peripheral site of the
enzyme. Moreover, the 3- and 2-fold greater effect obtained
for 15a and 15b relative to donepezil suggests a larger
occupancy of the AChE peripheral site by the indanone unit of
the novel hybrids.
The novel compounds reported in this study compare quite
favorably with the previously known donepezil-tacrine
hybrids.30,31 Thus, hybrids 14-17a,b are more potent AChE
inhibitors than compounds 3 (IC50 ) 6.0 nM, using rat cortex
AChE; IC50 ) 223 and 5.7 nM21 for tacrine and donepezil,
respectively, with the same enzyme source) and 4 (IC50 ) 25
nM, using bovine erythrocyte AChE; IC50 ) 167 and 19 nM
for tacrine and donepezil, respectively, in the same assay).
Regarding the BChE inhibitory activity, compounds 14a,b and
16a,b are more potent than 3 (IC50 ) 76 nM, using rat serum
BChE; IC50 ) 92 and 7138 nM21 for tacrine and donepezil,
respectively, with the same enzyme source), though none of
them is as potent as 4 (IC50 ) 0.6 nM, using hBChE; IC50
24 and 930 nM for tacrine and donepezil, respectively).
)
Thioflavin T Competition Assay. Inestrosa et al. reported
that AChE binds, through its peripheral site, to Aꢀ, thereby
inducing amyloid fibril formation,20,50,51 thus making blockade
of the AChE peripheral site an interesting pharmacological target
to develop disease-modifying anti-Alzheimer drug candidates.
Since propidium, a selective inhibitor of the AChE peripheral
site, exhibits an increase in fluorescence upon binding to AChE,
it has been used as a probe for competitive ligand binding to
the enzyme.31,52,53 Thioflavin T is another fluorescent probe
widely used to detect amyloid structures,54 though it can also
bind to other proteins.55,56 In particular, thioflavin T binds to
the AChE peripheral site, and the fluorescence enhancement
reported for thioflavin T upon binding to AChE is much greater
than that observed with propidium.56 Therefore, we chose
thioflavin T to study the interaction of selected donepezil-tacrine
hybrids (compounds 15a and 15b) at the AChE peripheral site.
Table 2 shows the reduction of thioflavin T fluorescence
arising from the competition with 15a and 15b, as well as
tacrine, 6-chlorotacrine, and donepezil as reference compounds.
Inhibiton of AChE-Induced Aꢀ Aggregation. The results
of the thioflavin T competition assay provide indirect evidence
of the ability of these donepezil-tacrine hybrids to interfere
with the AChE-induced Aꢀ aggregation. A direct measure
involving AChE and Aꢀ was therefore required to assess the
potential disease-modifying role of these hybrids. Thus, the
inhibitory activity of hybrids 15-17a,b on the AChE-induced
aggregation of Aꢀ1-40 was determined by a thioflavin T
fluorescent method.23 Also, the Aꢀ-antiaggregating effect of
6-chlorotacrine and the indane derivative of donepezil 19 was
determined, while those of tacrine and donepezil were already
described.23
Table 3 summarizes the Aꢀ-antiaggregating activity of the
novel hybrids and reference compounds. The six tested
donepezil-tacrine hybrids exhibit, at a 100 µM concentration,
a significant Aꢀ-antiaggregating effect with percentages of
inhibition ranging from 38% to 66%, being 5.4- to 9.4-fold more
potent than tacrine, 4.4- to 7.8-fold more potent than 6-chlo-
rotacrine, 1.7- to 3-fold more potent than donepezil, and 2.1-

3592 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Camps et al.
Figure 1. Time dependence of the potential energy (×105, kcal/mol) and the positional root-mean-squared deviation (rmsd, Å) determined for the
backbone and heavy atoms in the mobile part of the simulation system for the hAChE complexes with 15a (gray) and 15b (black). The profiles
were smoothed in 50 ps windows for the sake of clarity.
to 3.8-fold more potent than 19. Indane derivatives 16-17a,b
are clearly more potent than indanone derivatives 15a,b (about
1.5-fold), in contrast with the similar effect determined for
donepezil and its indane derivative 19. The length of the linker
and the substitution at the active site interacting unit, i.e., the
presence or absence of the chlorine atom at position 6 of the
tacrine unit, have little effect on the Aꢀ-antiaggregating activity
of the hybrids, observing only a slightly increased effect in the
hybrids bearing the trimethylene linker. The Aꢀ-antiaggregating
effects exhibited by these donepezil-tacrine hybrids are in the
same range as those of known dual binding site AChE inhibitors
such as lipocrine,11 xanthostigmine derivatives,13 and bis-(7)-
tacrine derivatives,17 while other families with lower9,14 or
higher10,12,15,16 potencies have been developed.
and memory in 15-month-old AD11 mice, a kind of transgenic
mice that display a full set of hallmarks of AD.15 Also,
Neuropharma’s NP-61 (formerly NP-0361), a dual binding site
AChEI that in vitro inhibited the AChE-induced Aꢀ aggregation
with an IC50 value 1 order of magnitude lower than that of
propidium, reduced the number, surface area, and size of
amyloid plaques in the cortex and hippocampus in human
amyloid precursor protein (hAPP) transgenic mice (Swedish and
London mutation) after oral administration during 3 months,
and the reduced brain amyloid burden resulted in a significantly
increased cognition (spatial learning and memory in the Morris
water maze test).57 In the first half of 2007, NP-61 entered phase
I clinical trials for Alzheimer’s disease in the U.K.,58 which
constitutes clear evidence of the promising role of these
compounds as anti-Alzheimer disease-modifying agents.
The ability of dual binding site AChE inhibitors to block the
AChE-induced Aꢀ aggregation constitutes their most interesting
property because of the derived potential to interfere upstream
in the neurotoxic cascade of AD. However, some concerns exist
about the biological relevance of the results of the in vitro Aꢀ
antiaggregating effects of these compounds. Indeed, these assays
are performed using much higher concentrations of AChE and
Aꢀ than those present in brain, but they are necessary to
accelerate the aggregation process up to a reasonable extent for
analytical purposes. Also, the concentration of inhibitor used
in these assays is much higher (usually 100 µM) than those
necessary to inhibit AChE (in the nanomolar or picomolar
range). However, as pointed out elsewhere,23 these high
concentration values should be normalized in order to compare
the inhibition data. Thus, if the inhibitor/AChE concentration
ratio in both the Ellman’s assay for determination of the AChE
inhibitory activity and in the thioflavin T-based fluorometric
assay for determination of the Aꢀ antiaggregating effect is taken
into account, the resulting values are indeed of the same
magnitude. Thus, 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. Thus,
memoquin, a dual binding site AChEI that in vitro blocked by
87% the AChE-induced Aꢀ aggregation at 100 µM,15,16 has
been shown to delay Aꢀ expression to significantly reduce Aꢀ
deposits and to rescue behavioral deficits linked to attention
Molecular Modeling Studies
To gain insight into the molecular determinants that modulate
the inhibitory activity of the novel donepezil-tacrine hybrids,
the binding modes of 15a and 15b were explored by means of
10 ns molecular dynamics (MD) simulations performed for their
complexes to hAChE. In the two cases the potential energy
dropped smoothly during the first nanosecond, but it remained
nearly constant for the rest of the trajectory (Figure 1). Indeed,
the stability of the trajectories is supported by the small
positional root-mean-squared deviations determined for the
backbone and heavy atoms, which amount to around 0.9 and
1.4 Å, respectively (Figure 1).
The position of 15a with respect to selected key residues in
the binding site is shown in Figure 2. The tacrine moiety is
firmly bound to the catalytic site of hAChE, it being stacked
against the aromatic rings of Trp86 and Tyr337 (average
distances from the central ring of tacrine of 3.73 and 4.35 Å,
respectively, and numbering of residues corresponding to
hAChE). The aromatic nitrogen of tacrine, protonated at
physiological pH, is hydrogen-bonded to the main chain
carbonyl oxygen of His447 (average N· · ·O distance of 2.81
Å). Finally, the chlorine atom occupies a small hydrophobic
pocket formed by Trp439, Met443, and Pro446, which parallels
the interaction observed between the chlorine atom at position
3 of huprine X, a hybrid compound whose structure contains a

Cholinesterases and ꢀ-Amyloid Aggregation Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3593
Figure 2. Representation of the heterodimers 15a (left) and 15b (right) in the binding site of hAChE highlighting selected residues that form the
main interactions with tacrine, piperidine, and indanone units of the inhibitors. Most hydrogen atoms are omitted for the sake of clarity.
6-chlorotacrine moiety, and TcAChE,38,59 though in this latter
case Pro446 is replaced by Ile439. The occupancy of such
hydrophobic pocket by the chlorine atom contributes to the
higher potency of heterodimers having the 6-chlorotacrine unit
relativetotheirunsubstitutedcounterparts.Asnotedpreviously,12,28
in hBChE, Met437 replaces Pro446 of hAChE and Ile439 of
TcAChE, which makes the terminal methyl group of Met437
to be around 1.2 Å closer to the chlorine atom. The larger steric
hindrance due to the greater proximity of the chlorine atom to
Met437 could account for the detrimental influence of this
substituent on the hBChE inhibitory activity.
Regarding the linker, which is aligned along the gorge, the
most relevant features come from the interactions formed by
the piperidine ring. This unit occupies a position slightly shifted
from that found in the X-ray crystallographic structure of the
TcAChE-donepezil complex (PDB entry 1EVE), although it still
retains the electrostatic interaction with the carboxylate group
of Asp74 (average N(piperidine) · · ·O(carboxylate) distance of
4.72 Å). In turn, this latter residue is hydrogen-bonded to the
hydroxyl group of Tyr72 (average O · · ·O distance of 2.87 Å).
Moreover, the protonated piperidine nitrogen is hydrogen-
bondedtothehydroxylgroupofTyr337(averageN(piperidine) · · ·O
distance of 3.20 Å). Thus, a network of interactions that couple
several residues in the gorge and the catalytic binding site is
formed. Finally, the indanone ring is stacked onto the aromatic
ring of Trp286 (average distance between the centers of
indanone and indole rings of 4.38 Å), whose orientation
resembles that found in the TcAChE-donepezil complex.
Moreover, the carbonyl group of the indanone unit forms water-
mediated contacts with the backbone N-H groups of Phe295
and Arg296 and the CdO groups of Pro290 and Ser293, which
should contribute to the higher AChE inhibitory activity of
indanone derivatives relative to the indane analogues.
15a are lost in the complex with 15b. Thus, Asp74 forms a
hydrogen-bond interaction with the backbone N-H unit of
Leu76 and the hydroxyl group of Tyr337 establishes a hydrogen-
bond contact with Tyr124. Second, though at the beginning of
the simulation the indanone ring was stacked onto the indole
ring of Trp286, its orientation changed along the first nanosec-
ond of MD simulation and adopted an arrangement roughly
normal to the indole ring (Figure 3). This change was ac-
companied by a conformational change in the indole ring of
Trp286, whose position differed from that found in the X-ray
structure by a rotation around the CR-Cꢀ-C3indole-C3aindole
from -83° (in 1EVE) to +32°. This finding, therefore, supports
the conformational plasticity of this residue in the peripheral
site of the enzyme already noted by other studies.12,60–62
Comparison of the binding mode of compounds 15a and 15b
with that of tacrine and donepezil (taken from X-ray structures
1ACJ and 1EVE, respectively) shows that the tacrine unit in
the dual binding site inhibitors closely matches the position
occupied by tacrine in the catalytic site (Figure 4). There are,
however, notable differences in the relative arrangement of the
indanone units of 15a and 15b. Thus, in 15a this unit is shifted
compared to the position occupied by the corresponding moiety
of donepezil, leading to a more efficient π-π stacking interac-
tion between the indanone ring of 15a and the indole ring of
Trp286 (Figure 4). In 15b the strong electrostatic interaction
formed between the protonated piperidine and Asp74 explains
the distinct orientation of the indanone unit relative to donepezil
and the conformational change adopted by the indole ring of
Trp286, which impedes the formation of a π-π stacking
interaction (Figure 4).
Conclusion
The binding mode of 15b at the catalytic site of hAChE
closely mimics the interactions noted above for 15a (Figure 2).
Thus, the tacrine ring is stacked against Trp86 and Tyr337
(average distances between rings of 3.63 and 4.44 Å, respec-
tively), the protonated quinoline nitrogen is hydrogen-bonded
to the main chain carbonyl oxygen of His447 (average N · · ·O
distance of 2.82 Å), and the chlorine atom fills the hydrophobic
pocket formed by Trp439, Met443, and Pro446. The enlarge-
ment of the tether length, however, leads to differences in the
interactions found along the gorge and at the peripheral site.
First, the protonated piperidine N-H group points directly to
the carboxylate group of Asp74 (average N · · ·O distance of 2.73
Å), which would strengthen the electrostatic interaction. The
hydrogen bonds between Asp74 and Tyr72 and between the
piperidine N-H unit and Tyr337 observed in the binding of
We have synthesized a new series of donepezil-tacrine
hybrids, designed to simultaneously interact with the active,
peripheral, and mid-gorge binding sites of AChE. In contrast
to previous approaches, the conjunctive strategy adopted here
largely preserves the chemical skeleton of donepezil while its
benzyl moiety is replaced by a tacrine unit. The length of the
tether that connects the two constituting fragments of the novel
hybrids, i.e., the 5,6-dimethoxy-2-[(4-piperidinyl)methyl]-1-
indanone moiety of donepezil (or its corresponding indane
derivative) and the tacrine (or 6-chlorotacrine) unit, has a
relevant effect on the arrangement of the hybrid along the gorge,
leading to different orientations of the piperidine ring and the
indanone system within the enzyme gorge and at the peripheral
site, respectively, while their tacrine unit closely matches the
position occupied by tacrine within the AChE active site. Thus,

3594 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Camps et al.
Figure 3. Time dependence of the relative orientation of the indanone ring of heterodimers 15a (black) and 15b (gray) and the indole ring of
Trp286 at the peripheral site. The relative orientation was measured from the cosine function of the angle formed by the vectors normal to the
indanone and indole rings. Accordingly, a perfect stacking would correspond to cos θ ) 1, while a perpendicular arrangement is denoted by cos θ
) 0.
those bearing an unsubstituted tacrine unit irrespective of the
presence of an indanone or indane system and the tether length,
are also more potent hBChE inhibitors than the parent com-
pounds. Following some interesting results arising from a
thioflavin T competition assay to assess their interaction with
the AChE peripheral site, six out of the eight hybrids of the
series were tested for their ability to block the AChE-induced
Aꢀ-aggregation. All the tested hybrids exhibited a significant
Aꢀ antiaggregating activity, being more potent than the parent
compounds, including the dual binding site inhibitor donepezil.
Overall, these results suggest that the novel donepezil-tacrine
hybrids herein reported may have a potential disease-modifying
role in the treatment of AD.
Experimental Section
Chemistry. General Methods. Melting points were determined
in open capillary tubes with a MFB 595010 M Gallenkamp melting
1
point apparatus. The 300 MHz H/75.4 MHz 13C NMR spectra,
400 MHz 1H/100.6 MHz 13C NMR spectra, and 500 MHz 1H NMR
spectra were recorded on Varian Gemini 300, Varian Mercury 400,
and Varian Inova 500 spectrometers, respectively. The chemical
shifts are reported in ppm (δ scale) relative to internal tetrameth-
ylsilane, and coupling constants are reported in hertz (Hz).
Assignments given for the NMR spectra of the new compounds
have been carried out by comparison with the NMR data of 15a,
16a, 17b, and 6-chlorotacrine, as model compounds, which in turn
were assigned on the basis of DEPT, COSY 1H/1H (standard
procedures), NOESY 1H/1H, and COSY 1H/13C (gHSQC and
gHMBC sequences) experiments. IR spectra were run on a Perkin-
Elmer Spectrum RX I spectrophotometer. Absorption values are
expressed as wavenumbers (cm-1); only significant absorption
bands are given. Column chromatography was performed on silica
gel 60 AC.C (35-70 mesh, SDS, no. 2000027). Thin-layer
chromatography was performed with aluminum-backed sheets with
silica gel 60 F254 (Merck, no. 1.05554), and spots were visualized
with UV light and 1% aqueous solution of KMnO4. Analytical grade
solvents were used for crystallization, while pure for synthesis solvents
were used in the reactions, extractions, and column chromatography.
For characterization purposes, the new donepezil-tacrine hybrids
were transformed into the corresponding dihydrochlorides and
recrystallized. Worthy of note, as previously reported for some
tacrine-related dimeric compounds,63 the new donepezil-tacrine
Figure 4. Superposition of the heterodimers 15a (left) and 15b (right)
with tacrine (taken from the 1ACJ X-ray structure of the
TcAChE-tacrine complex, blue) and donepezil (from the 1EVE X-ray
structure of the TcAChE-donepezil complex, orange), showing the
relative positions of Trp86 (Trp84 in 1ACJ) and Trp286 (Trp279 in
1EVE) at the catalytic and peripheral sites, respectively. Compounds
15a and 15b are colored by atom.
enlargement of the linker from two to three methylenes permits
the piperidine ring to form a direct electrostatic interaction with
Asp74, though at the expense of a less efficient π-π interaction
between the indanone unit and the indole ring of Trp286. In
spite of these structural differences, all of the new hybrids are
highly potent bovine and human AChE inhibitors, exhibiting
IC50 values in the subnanomolar range in most cases and being
clearly more potent than the parent compounds from which they
were designed and the previously described donepezil-tacrine
hybrids. The most potent AChE inhibitors are those bearing an
indanone system, a chlorine atom at the tacrine unit and a tether
length of three methylenes. Though all of the new hybrids are
less potent toward hBChE, the most active compounds, i.e.,

Cholinesterases and ꢀ-Amyloid Aggregation Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3595
hybrids have the ability to retain molecules of water, which cannot
be removed after drying the analytical samples at 80 °C/30 Torr
for 2 days. Thus, the elemental analyses of these compounds showed
the presence of variable amounts of water. NMR spectra of all of
the new compounds were performed at the Serveis Cient´ıfico-
Te`cnics of the University of Barcelona, while elemental analyses
and high resolution mass spectra were carried out at the My-
croanalysis Service of the IIQAB (CSIC, Barcelona, Spain) with a
Carlo Erba model 1106 analyzer and at the Mass Spectrometry
Laboratory of the University of Santiago de Compostela (Spain)
with a Micromass Autospec spectrometer, respectively.
9-[(2-Methanesulfonyloxyethyl)amino]-1,2,3,4-tetrahydroacri-
dine (10a). From alcohol 8a(3.52 g, 14.5 mmol), mesylate 10a (4.41
g, 95% yield) was obtained: Rf ) 0.67 (CH2Cl2/MeOH/25% aqueous
NH4OH, 9:1:0.1); IR (NaCl) ν 3391 and 3339 (N-H st), 1638,
1586, and 1524 (ar-C-C and ar-C-N st), 1352 (SO2 st as), 1178
(SO2 st s) cm-1 1H NMR (300 MHz, CDCl3) δ 1.88-1.98
;
(complex signal, 4H, 2-H2 and 3-H2), 2.76 (m, 2H, 1-H2), 2.97 (s,
3H, OSO2CH3), 3.08 (m, 2H, 4-H2), 3.81 (m, 2H, 1′-H2), 4.34 (t,
J ) 4.9 Hz, 2H, 2′-H2), 4.54 (broad s, 1H, NH), 7.39 (ddd, J ) 8.4
Hz, J′ ) 6.8 Hz, J′′ ) 1.5 Hz, 1H, 7-H), 7.57 (ddd, J ) 8.4 Hz, J′
) 6.8 Hz, J′′ ) 1.5 Hz, 1H, 6-H), 7.91-7.95 (complex signal, 2H,
5-H and 8-H); 13C NMR (75.4 MHz, CDCl3) δ 22.4 (CH2) and
22.7 (CH2) (C2 and C3), 24.7 (CH2, C1), 33.3 (CH2, C4), 37.3
(CH3, OSO2CH3), 47.4 (CH2, C1′), 69.0 (CH2, C2′), 117.7 (C, C9a),
120.7 (C, C8a), 122.2 (CH), 124.3 (CH), 127.8 (CH), 128.7 (CH)
(C5, C6, C7, and C8), 146.2 (C, C10a), 149.6 (C, C4a), 158.2 (C,
C9). HRMS calcd for (C16H20N2O3S + H+) 321.127, found
321.128.
General Procedure for the Coupling of Mesylates 10 and
11 with 5,6-Dimethoxy-2-[(4-piperidinyl)methyl]indan-1-one
(12) or 5,6-Dimethoxy-2-[(4-piperidinyl)methyl]indane (13). A
solution of the mesylate 10 or 11 (1 mmol), the piperidine 12 or
13 (1 mmol), and anhydrous Et3N (2.5 mmol) in DMSO (8 mL)
was heated at 85 °C for 48 h. The resulting solution was allowed
to cool to room temperature and was concentrated in vacuo. The
brown oily residue was treated with aqueous 2 N NaOH (25 mL)
and extracted with CH2Cl2 (3 × 35 mL). The combined organic
extracts were washed with water (6 × 40 mL) and brine (4 × 30
mL), dried with anhydrous Na2SO4, filtered, and evaporated under
reduced pressure to give an oily residue, which was submitted to
column chromatography (35-70 µm silica gel, CH2Cl2/MeOH/25%
aqueous NH4OH mixtures as eluent).
General Procedure for the Reaction of 9-Chloro-1,2,3,4-
tetrahydroacridine, 5, or 6,9-Dichloro-1,2,3,4-tetrahydroacri-
dine, 6, with ω-Aminoalcohols. A mixture of 5 or 6 (1 mmol)
and an excess of the aminoalcohol 7 (3 mmol) in 1-pentanol (1
mL) was heated under reflux with magnetic stirring for 18 h. The
resulting mixture was cooled to room temperature, diluted with
AcOEt (5 mL), and extracted with 1 N HCl (4 × 3 mL). The
combined aqueous extracts were washed with AcOEt (3 × 3 mL),
alkalinized with NaOH pellets (until pH 12), and extracted with
CH2Cl2 (3 × 8 mL). The combined organic extracts were dried
with anhydrous Na2SO4 and concentrated in vacuo to give alcohol
8 or 9 as a pale-brown solid or oily residue, which was used in the
next step without further purification.
For characterization purposes, analytical samples of the hydro-
chlorides of 8 and 9 were prepared as follows: The alcohol 8 or 9
(1 mmol) was dissolved in MeOH (10 mL). The solution was
filtered through a 0.45 µm polytetrafluoroethylene (PTFE) filter and
treated with an excess of a methanolic solution of HCl (3 mmol),
and the resulting solution was concentrated in vacuo to dryness.
The solid was recrystallized from a mixture MeOH/AcOEt in a
ratio of 1:4 (5 mL) and dried at 80 °C/30 Torr for 48 h to give the
9-(ω-hydroxyalkylamino)tetrahydroacridine hydrochloride 8·HCl
or 9·HCl as a light-brown solid.
The isolated donepezil-tacrine hybrids 14-17 were transformed
into the corresponding dihydrochlorides as follows: A solution of
the free base (1 mmol) in MeOH (40 mL) was filtered through a
0.45 µm PTFE filter and treated with excess of a methanolic solution
of HCl (5 mmol). The solution was concentrated in vacuo to
dryness, and the solid residue was recrystallized from MeOH (20
mL) and dried at 80 °C/30 Torr for 48 h.
9-[(2-Hydroxyethyl)amino]-1,2,3,4-tetrahydroacridine Hy-
drochloride (8a ·HCl). From 5 (3.34 g, 15.4 mmol) and 2-ami-
noethanol 7a (2.48 mL, 2.83 g, 46.4 mmol), alcohol 8a (3.52 g,
95% yield, free base) was obtained: Rf ) 0.38 (CH2Cl2/MeOH/
25% aqueous NH4OH, 9:1:0.1). 8a·HCl: mp 184-185 °C (MeOH/
AcOEt, 1:4); IR (KBr) ν 3700-2400 (max at 3358, 3142, 3058,
3016, 2938, 2872, O-H, N-H, +N-H, and C-H st), 1633, 1592,
9-[(2-{4-[(5,6-Dimethoxy-1-oxoindan-2-yl)methyl]piperidin-1-
yl}ethyl)amino]-1,2,3,4-tetrahydroacridine Dihydrochloride (14a ·
2HCl). From mesylate 10a (276 mg, 0.86 mmol) and piperidine
12 (249 mg, 0.86 mmol), compound 14a (87 mg, 20% yield) was
obtained as a pale-brown solid on elution with a mixture of CH2Cl2/
MeOH/25% aqueous NH4OH, 99.5:0.5:0.4: Rf ) 0.91 (CH2Cl2/
MeOH/25% aqueous NH4OH, 90:10:0.1). 14a·2HCl: mp 190-191
°C (MeOH); IR (KBr) ν 3700-2400 (max at 3401, 2928, 2871,
2718, N-H, +N-H, and C-H st), 1685, 1672 (CdO st), 1636,
1
1577, and 1524 (ar-C-C and ar-C-N st) cm-1; H NMR (300
MHz, CD3OD) δ 1.90-2.04 (complex signal, 4H, 2-H2 and 3-H2),
2.74 (m, 2H, 1-H2), 3.01 (m, 2H, 4-H2), 3.86 (t, J ) 5.4 Hz, 2H,
2′-H2), 4.01 (t, J ) 5.4 Hz, 2H, 1′-H2), 4.87 (s, OH, NH and +NH),
7.54 (ddd, J ) 8.4 Hz, J′ ) 6.0 Hz, J′′ ) 2.4 Hz, 1H, 7-H),
7.76-7.84 (complex signal, 2H, 5-H and 6-H), 8.40 (d, J ) 8.4
Hz, 1H, 8-H); 13C NMR (75.4 MHz, CD3OD) δ 21.1 (CH2, C3),
23.1 (CH2, C2), 24.8 (CH2, C1), 30.1 (CH2, C4), 51.2 (CH2, C1′),
61.7 (CH2, C2′), 113.7 (C, C9a), 117.7 (C, C8a), 121.2 (CH, C5),
126.0 (CH, C8), 126.1 (CH, C7), 133.3 (CH, C6), 140.9 (C, C10a),
152.8 (C, C4a), 157.4 (C, C9). HRMS calcd for (C15H18N2O +
H+) 243.1497, found 243.1494.
1
1588, 1523, and 1500 (ar-C–C and ar-C–N st) cm-1; H NMR
(500 MHz, CD3OD) δ 1.45 (m, 1H, indanone-2-CHa), 1.62-1.73
(broad signal, 2H, piperidine 3-Hax and 5-Hax), 1.86 (m, 1H,
indanone-2-CHb), 1.90-2.02 (broad signal, 1H, piperidine 4-H),
superimposed 1.99 (complex signal, 4H, acridine 2-H2 and 3-H2),
2.02 (broad d, J ) 14.5 Hz, 1H) and 2.14 (broad d, J ) 14.0 Hz,
1H) (piperidine 3-Heq and 5-Heq), 2.74-2.80 (complex signal, 2H,
indanone 2-H and 3-Ha), 2.84 (m, 2H, acridine 1-H2), 3.08 (m, 2H,
acridine 4-H2), 3.12 (broad signal, 2H, piperidine 2-Hax and 6-Hax),
superimposed in part 3.34 (dd, J ) 18.0 Hz, J′ ) 8.5 Hz, 1H,
indanone 3-Hb), 3.60 (broad signal, 2H, NHCH2CH2N), 3.72 (broad
signal, 2H, piperidine 2-Heq and 6-Heq), 3.85 (s, 3H, 6-OCH3), 3.94
(s, 3H, 5-OCH3), 4.42 (t, J ) 7.7 Hz, 2H, NHCH2CH2N), 4.85 (s,
NH and +NH), 7.07 (s, 1H, indanone 4-H), 7.15 (s, 1H, indanone
7-H), 7.68 (ddd, J ) 8.5 Hz, J′ ) 7.0 Hz, J′′ ) 1.0 Hz, 1H, acridine
7-H), 7.83 (dd, J ) 8.5 Hz, J′ ) 1.0 Hz, 1H, acridine 5-H), 7.91
(ddd, J ) 8.5 Hz, J′ ) 7.0 Hz, J′′ ) 1.0 Hz, 1H, acridine 6-H),
8.43 (d, J ) 8.5 Hz, 1H, acridine 8-H); 13C NMR (100.6 MHz,
CD3OD) δ 21.7 (CH2, acridine C3), 23.0 (CH2, acridine C2), 25.7
(CH2, acridine C1), 29.5 (CH2, acridine C4), 30.3 (CH2) and 31.1
(CH2) (piperidine C3 and C5), 33.0 (CH, piperidine C4), 34.2 (CH2,
indanone C3), 39.0 (CH2, indanone-2-CH2), 43.1 (CH2,
NHCH2CH2N), 46.1 (CH, indanone C2), 54.1 (2 CH2, piperidine
General Procedure for the Mesylation of Alcohols 8 and 9.
To a cold solution (-10 °C, ice-salt bath) of the alcohol 8 or 9 (1
mmol) and anhydrous Et3N (1.7 mmol) in anhydrous CH2Cl2 (6
mL), methanesulfonyl chloride (1.5 mmol) was added dropwise,
and the reaction mixture was stirred for 30 min at this temperature.
The mixture was concentrated in vacuo, the residue was taken in
CH2Cl2 (4 mL), and the resulting organic solution was washed with
2 N NaOH (3 × 3 mL) until the aqueous phase remained basic
(pH > 10), dried with anhydrous Na2SO4, and concentrated in vacuo
to give the corresponding mesylate 10 or 11 as a brown oily residue,
which was used in the next step without further purification.
For characterization purposes, analytical samples of the mesylates
were obtained by extraction of the oily crude product with hot
AcOEt except in the case of 11a, whose analytical sample was
prepared by recrystallization of the corresponding hydrochloride,
obtained in a similar way to that described for the hydrochlorides
of alcohols 8 and 9.

3596 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Camps et al.
C2 and C6), 56.5 (CH3, 6-OCH3), 56.7 (CH3, 5-OCH3), 57.4 (CH2,
NHCH2CH2N), 105.3 (CH, indanone C7), 109.0 (CH, indanone C4),
114.4 (C, acridine C9a), 117.6 (C, acridine C8a), 120.3 (CH,
acridine C5), 125.9 (CH, acridine C8), 127.2 (CH, acridine C7), 129.8
(C, indanone C7a), 134.3 (CH, acridine C6), 139.4 (C, acridine C10a),
151.1 (C, indanone C6), 151.3 (C, indanone C3a), 153.1 (C, acridine
C4a), 157.8 (C) and 158.0 (C) (indanone C5 and acridine C9), 209.7
(C, indanone C1). Anal. (C32H39N3O3 ·2HCl·1.5H2O) C, H, N, Cl.
Biochemical Studies. AChE inhibitory activity was evaluated
spectrophotometrically at 25 °C by the method of Ellman,47 using
AChE from bovine or human erythrocytes and acetylthiocholine
iodide (0.53 and 0.13 mM for bovine and human AChE, respec-
tively) as substrate. The reaction took place in a final volume of 3
mL of 0.1 M phosphate-buffered solution, pH 8.0, containing 0.025
or 0.04 unit of bovine or human AChE, respectively, and 333 µM
5,5′-dithiobis(2-nitrobenzoic) acid (DTNB) solution used to produce
the yellow anion of 5-thio-2-nitrobenzoic acid. Inhibition curves
were performed in triplicate by incubating at least 12 concentrations
of inhibitor for 15 min. One triplicate sample without inhibitor was
always present to yield 100% of AChE activity. The reaction was
stopped by the addition of 100 µL of 1 mM eserine, and the color
production was measured at 414 nm. BChE inhibitory activity
determinations were carried out similarly, using 0.035 unit of human
serum BChE and 0.56 mM butyrylthiocholine, instead of AChE
and acetylthiocholine, in a final volume of 1 mL.
Data from concentration-inhibition experiments of the inhibitors
were calculated by nonlinear regression analysis, using the Graph-
Pad Prism program package (GraphPad Software; San Diego, CA),
which gave estimates of the IC50 (concentration of drug producing
50% of enzyme activity inhibition). Results are expressed as the
mean ( SEM of at least four experiments performed in triplicate.
DTNB, acetylthiocholine, butyrylthiocholine, and the enzymes were
purchased from Sigma, and eserine was purchased from Fluka.
Thioflavin T Competition Assay. Fluorescence measurements
were carried out at room temperature in a SFM-25 spectrofluo-
rimeter (Biotek, Italy) using a 1 mL quartz cell. Fluorescence was
monitored at 448 and 488 nm for excitation and emission,
respectively. An aqueous solution of hAChE at 2.3 µM was stirred
at room temperature for 24 h. After incubation, an amount of 50
µL of the enzyme solution was mixed with 750 µL of 50 mM
glycine-NaOH buffer (pH 8.5) containing 15 µM thioflavin T. The
resulting solution was stirred for 1 h, and the fluorescence of the
sample was recorded (F1). To check the ability of reducing the
fluorescence arising from the interaction of AChE and thioflavin
T, the enzyme, at a final concentration of 2.3 µM, was mixed with
the inhibitor (final concentration of 100 µM), and the solution was
stirred for 24 h. After incubation, an amount of 50 µL of the protein
solution was mixed with 750 µL of fluorophore solution, as
indicated above, and the corresponding fluorescence was recorded
(F2). Finally, the fluorescence of a solution formed by 750 µL of
the buffer containing the dye and 50 µL of water was recorded
(F0). The percentage of reduction of ThT fluorescence was
determined as
AChE molar ratio of 100:1) and AChE in the presence of 2 µL of
the tested inhibitor (final inhibitor concentration 100 µM) in 0.215
M sodium phosphate buffer, pH 8.0, solution were added. Blanks
containing Aꢀ1-40 alone, human recombinant AChE alone, and
Aꢀ1-40 plus tested inhibitors in 0.215 M sodium phosphate buffer
(pH 8.0) were prepared. The final volume of each vial was 20 µL.
Each assay was run in duplicate. To quantify amyloid fibril
formation, the thioflavin T fluorescence method was then applied.23
The fluorescence intensities due to ꢀ-sheet conformation were
monitored for 300 s at λem ) 490 nm (λexc ) 446 nm). The percent
inhibition of the AChE-induced aggregation due to the presence
of the tested compound was calculated by the following expression:
100 - [(IFi/IFo) × 100] where IFi and IFo are the fluorescence
intensities obtained for Aꢀ plus AChE in the presence and in the
absence of inhibitor, respectively, minus the fluorescence intensities
due to the respective blanks.
Molecular Modeling Methods. The binding modes of com-
pounds 15a and 15b were explored by means of 10 ns molecular
dynamics simulations performed for their complexes to human
acetylcholinesterase (hAChE). To this end, models of the bonded
ligands were built up using the X-ray crystallographic structure of
the hAChE-fasciculin complex (PDB code 1B41).64 Fasciculin was
removed from the structure, truncated residues were reconstructed,
and missing residues were modeled using InsightII graphics
package.65 The starting pose of the ligands was determined by
means of docking computations with GOLD66 (using GoldScore
scoring function), which was successful for predicting the docking
of donepezil in the enzyme (see Supporting Information), and was
later refined by inspection of the X-ray crystallographic structures
of the TcAChE complexes with tacrine (1ACJ),35 huprine X
(1E66),38 and donepezil (1EVE)22 and the final structures of
previous heterodimer studies.12,31 The system was hydrated by
centering a sphere of 50 Å of TIP3P67 water molecules at the
inhibitor, paying attention to filling the position of crystallographic
waters inside the binding cavity. Finally, six Na+ cations were
added to neutralize the negative charge of the system with the xleap
module of AMBER8.68
Molecular dynamics simulations were run using the sander
module of AMBER8 and the parm98 parameters for the protein.
The charge distribution of the inhibitor was determined from a fit
to the HF/6s-31G(d) electrostatic potential obtained with Gaussi-
an’0369 using the RESP procedure,70 and the van der Waals
parameters were taken from those defined for related atoms in the
AMBER force field. The system was partitioned into a mobile
region, which included the ligand, all the protein residues containing
at least one atom within 20 Å from the ligand, and all the water
molecules and Na+ cations. The geometry of the system was
minimized in four steps. First, the position of hydrogen atoms was
optimized using 3000 steps of steepest descent algorithm. Then
water molecules were refined through 2000 steps of steepest
descent followed by 3000 steps of conjugate gradient. Next, the
ligand, water molecules, and counterions were optimized with
2000 steps of steepest descent and 4000 steps of conjugate
gradient, and finally the whole system was optimized with 3000
steps of steepest descent and 7000 steps of conjugate gradient.
Thermalization of the mobile part of the system was performed
in five steps of 20 ps, incrementing the temperature up to 298
K. At this point, a 10 ns molecular dynamics simulation was
carried out using a time step of 1 fs. SHAKE was used for those
bonds containing hydrogen atoms, and a cutoff of 11 Å was
used for nonbonded interactions.
F2 - F0
F1 - F0
% ) 1 -
(1)
Inhibition of AChE-Induced Aꢀ40 Aggregation Assay. Thiofla-
vin T (Basic Yellow 1), human recombinant AChE lyophilized
powder, and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were pur-
chased from Sigma Chemicals. Buffers and other chemicals were
of analytical grade. Absolute DMSO over molecular sieves was
from Fluka. Water was deionized and doubly distilled. ꢀ-Amyloid
1-40 (Aꢀ40), supplied as trifluoroacetate salt, was purchased from
Bachem AG (Bubendorf, Switzerland). Aꢀ40 (2 mg mL-1) was
dissolved in HFIP and lyophilized. The 1 mM solutions of tested
inhibitors were prepared by dissolution in MeOH.
The analysis of the structural features that mediate the binding
mode to the enzyme was determined by averaging the parameters
for the snapshots (saved every picosecond) sampled along the last
5 ns of the molecular dynamics simulations.
Aliquots of 2 µL of Aꢀ40 peptide, lyophilized from 2 mg mL-1
HFIP solution and dissolved in DMSO, were incubated for 24 h at
room temperature in 0.215 M sodium phosphate buffer (pH 8.0) at
a final concentration of 230 µM. For co-incubation experiments,
aliquots (16 µL) of hAChE (final concentration of 2.30 µM, Aꢀ/
Acknowledgment. Financial support from Direccio´n General
de Investigacio´n of Ministerio de Ciencia y Tecnolog´ıa and
FEDER (Projects CTQ2005-02192/BQU, SAF2006-04339,
CTQ2005-09365, and SAF2005-01604) and Comissionat per a

Cholinesterases and ꢀ-Amyloid Aggregation Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3597
Target-Directed Ligands To Combat Alzheimer’s Disease. J. Med.
Chem. 2007, 50, 4882–4897.
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Directed Drug Design Strategy: From a Dual Binding Site Acetyl-
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Universitats i Recerca of the Generalitat de Catalunya (Projects
2005-SGR00180, 2001-SGR00216, and 2005-SGR00893) are
gratefully acknowledged. We also thank the Serveis Cient´ıfico-
Te`cnics of the University of Barcelona for NMR facilities, and
we thank P. Dome`nech from the IIQAB (CSIC, Barcelona,
Spain) for carrying out the elemental analyses.
Supporting Information Available: Experimental procedures,
spectral and analytical characterization data of all of the new
compounds (except for 8a, 10a, and 14a) and the known compounds
13 and 19, and the predicted pose of donepezil obtained by using
GOLD. This material is available free of charge via the Internet at
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