Design, synthesis, and biological evaluation of dual binding site acetylcholinesterase inhibitors: New disease-modifying agents for Alzheimer's disease

Article abstract of DOI:10.1021/jm0503289

New dual binding site acetylcholinesterase (AChE) inhibitors have been designed and synthesized as new potent drugs that may simultaneously alleviate cognitive deficits and behave as disease-modifying agents by inhibiting the β-amyloid (Aβ) peptide aggreg

Full text of DOI:10.1021/jm0503289

J. Med. Chem. 2005, 48, 7223-7233
7223
Design, Synthesis, and Biological Evaluation of Dual Binding Site
Acetylcholinesterase Inhibitors: New Disease-Modifying Agents for Alzheimer’s
Disease
§
§
§
§
§
Pilar Mu n˜ oz-Ruiz, Laura Rubio, Esther Garc ´ı a-Palomero, Isabel Dorronsoro, Mar ´ı a del Monte-Mill a´ n,
§
§
§
‡
‡
#
Rita Valenzuela, Paola Us a´ n, Celia de Austria, Manuela Bartolini, Vincenza Andrisano, Axel Bidon-Chanal,
Modesto Orozco, F. Javier Luque, Miguel Medina, and Ana Mart ´ı nez*
†
#
§
,§
Neuropharma, S.A., Avda. de La Industria 52, 28760 Tres Cantos, Madrid, Spain, Department of Pharmaceutical Sciences,
University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Departamento de Fisicoqu ı´ mica, Facultad de Farmacia,
Universidad de Barcelona, Avda. Diagonal 643, 08028 Barcelona, Spain, and Departamento de Bioqu ´ı mica y Biolog ı´ a
Molecular, Facultad de Qu ı´ mica, Universidad de Barcelona, Mart ı´ i Franqu e´ s 1, 08028 Barcelona, Spain
Received April 11, 2005
New dual binding site acetylcholinesterase (AChE) inhibitors have been designed and
synthesized as new potent drugs that may simultaneously alleviate cognitive deficits and behave
as disease-modifying agents by inhibiting the â-amyloid (Aâ) peptide aggregation through
binding to both catalytic and peripheral sites of the enzyme. Particularly, compounds 5 and 6
emerged as the most potent heterodimers reported so far, displaying IC50 values for AChE
inhibition of 20 and 60 pM, respectively. More importantly, these dual AChE inhibitors inhibit
the AChE-induced Aâ peptide aggregation with IC50 values 1 order of magnitude lower than
that of propidium, thus being the most potent derivatives with this activity reported up to
date. We therefore conclude that these compounds are very promising disease-modifying agents
for the treatment of Alzheimer’s disease (AD).
Introduction
Alzheimer’s disease (AD), the most common form of
neurodegenerative senil dementia, is associated with
selective loss of cholinergic neurons and reduced levels
of acetylcholine neurotransmitter, and it is character-
ized by loss of memory and progressive impairment in
1
cognitive functions. Post-mortem brain studies, spe-
cially in neurocortex and hypocampus regions, have
revealed the presence of several distinct neuropatho-
logical hallmarks, including extracellular â-amyloid (Aâ)
containing plaques, intracellular neurofibrillary tangles
containing abnormally hyperphosphorylated τ protein,
and degeneration of cholinergic neurons of the basal
Figure 1. AChE inhibitors clinically used for the treatment
2
forebrain.
of AD.
In the past 2 decades, enormous research effort has
been spent to understand the molecular pathogenesis
role on the cognitive function, they might contribute to
slow the neurodegeneration in AD patients. In fact, it
is known that AChE exerts secondary noncholinergic
of AD, thus providing a robust framework to develop
effective pharmacological treatments.3 However, the
9
10,11
functions, related to its peripheral anionic site,
in
only therapeutic approach currently approved exploits4
cell adhesion1
2,13
and differentiation, and recent find-
14
the enhancement of the central cholinergic function,
ings also support its role in mediating the processing
which increasees the acetylcholine levels in the brain.
Thus, over the past decade various cholinergic drugs,
primarily inhibitors of the enzyme acetylcholinesterase
and deposition of Aâ peptide;1
5-18
AChE is one of the
proteins that colocalizes with Aâ peptide deposits in the
brain of AD patients15 and promotes Aâ fibrillogenesis
5
6
7
(
AChE) such as tacrine, donepezil, rivastigmine, and
16
8
by forming stable AChE-Aâ complexes. Additionally,
it has also been postulated that AChE binds through
its peripheral site to the Aâ nonamyloidogenic form and
acts as a pathological chaperone inducing a conforma-
more recently galantamine (Figure 1), have been
launched on the market for the symptomatic treatment
of AD.
The therapeutic potential of AChE inhibitors has been
strengthened by evidence showing that, besides their
1
7,18
tional transition to the amyloidogenic form.
On the
basis of this evidence, dual binding site AChE inhibitors
appear to be promising for the design of new antide-
mentia drugs, since they might simultaneously alleviate
the cognitive deficit in AD patients and act as disease-
*
To whom correspondence should be addressed. Phone: +34 91
8
061130. Fax: +34 91 8034660. E-mail: amartinez@neuropharma.es.
§
Neuropharma, S.A.
University of Bologna.
‡
#
Departamento de Fisicoqu ´ı mica, Universidad de Barcelona.
†
modifying agents delaying the amyloid plaque forma-
Departamento de Bioqu ´ı mica y Biolog ´ı a Molecular, Universidad
9,20
de Barcelona.
tion.1
Following this rationale, new AChE inhibitors
1
0.1021/jm0503289 CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/13/2005

7
224 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Mu n˜ oz-Ruiz et al.
The AChE and BChE inhibitory activity of each
compound was evaluated, and their selectivity ratio
(AChE/BChE) was also calculated. A molecular model-
ing study was performed to determine the binding mode
of these dual inhibitors to the AChE. Furthermore, for
those compounds showing very promising disease-
modifying profiles for the treatment of AD, their Aâ1-40
peptide aggregation inhibitory activity and their AChE-
induced Aâ1-40 peptide aggregation inhibitory activity
were studied to confirm their dual action. Finally,
cytotoxicity studies were performed in human neuro-
blastoma cells.
Chemistry
The synthetic methodology employed for the prepara-
tion of the new dual binding site heterodimers was
varied depending on the carbonylic bond type (amide
or carbamate) in the linker, which is inserted in the last
step of the synthetic pathway, as depicted in Schemes
1 or 2, respectively. Preparation of the new dual
heterodimers containing an amide bond, compounds
3-23 (Table 1), was carried out by coupling the required
Figure 2. Indole-tacrine heterodimers binding simulta-
neously to the catalytic and peripheral sites of the AChE.
have been designed to interact simultaneously with both
catalytic and peripheral sites.21-24
Continuing with our search for new dual-binding site
AChE inhibitors,25-27 we report here the design, syn-
9
-alkylaminotetrahydroacridines 1a-i to the corre-
thesis, and pharmacological evaluation of a new series
of potent compounds containing in their structures a
sponding indole carboxylic acid derivatives 2a-i, em-
ploying 1,1-carbonyldiimidazol (CDI) as coupling agent
1
,2,3,4-tetrahydroacridine fused ring (tacrine) as the
(
Scheme 1).36 The 9-alkylaminotetrahydroacridines 1a-i
catalytic anionic site binding unit, and an indole ring
as the peripheral site binding unit, connected to each
other by a linker of suitable length and nature (Figure
of different tether lengths were readily obtained follow-
ing the reported methodology.30 All the indole carboxylic
acid derivatives 2 employed for the preparation of the
new dual heterodimers 3-23 were commercially avail-
able except for 5-cyanoindole-3-propionic acid (2b),
which was synthesized according to a reported meth-
odology.37 Thus, activation of the indole carboxylic acid
derivatives 2 with CDI in THF for about 4 h followed
by treatment with the corresponding 9-alkylamino-
tetrahydroacridines 1a-i generated the desired dual
binding site inhibitors 3-23 in yields ranging from 8%
to 97%. In general, the yield of the reaction decreases
dramatically as the number of methylene units of the
2
8
2
). The tacrine unit was, in previous studies, incor-
porated in a series of bis-tacrines, designed by Pang et
1
9
al, that showed high potency and selectivity in AChE
inhibition, being up to 248-fold more selective and 149-
fold more potent than tacrine. Since then, tacrine has
been incorporated into several families of AChE dual
2
9-31
inhibitors.
Because the insertion of chlorine into
position 6 of tacrine enhances the affinity toward
32
AChE, we have also combined the 6-chlorotacrine unit
with the indole ring unit in most of the synthesized
heterodimers. The indole ring moiety was chosen on the
basis of its pharmacological, structural, and electronic
properties. In this view, the indole ring linked to the
tacrine moiety by a tether of appropriate length could
reach the entrance of the catalytic gorge and interact
9
5
-alkylaminotetrahydroacridines 1 varies from 8 (7,
3%) to 10 (9, 19%). The presence of an electron-
withdrawing group at position 5 of the indole ring,
compounds 2b and 2f, seemed to decrease the activation
of the carboxylic group, since compounds 12 (22%) and
33,34
with the Trp279 by π-π stacking.
To our knowledge,
1
9 (54%) were obtained in much lower yields than their
the indole unit has been incorporated only once into a
dual binding site AChE inhibitor, in the case of
tryptamine in combination with tacrine, which resulted
unsubstituted analogues 5 (77%) and 18 (84%). Cou-
pling the indole-3-propenoic acid (2c) to the 9-alkyl-
aminotetrahydroacridines 1c resulted in low formation
of the heterodimer 13 (8%), probably because of the
conjugation of the double bond with the carboxylic acid
group that might reduce the nucleophilicity of the
hydroxyl group. Heterodimers 14-17, containing an
amide group directly attached to the indole fused-ring,
were also obtained in moderate yield as observed when
comparing 16 (51%) with 18 (80%) and 6 (79%), where
the carbonylic group is separated by one or two meth-
ylene groups from the indole ring, respectively. This
effect was even more remarkable at synthesizing the
heterodimers 22 (8%) and 23 (13%), which also contain
an amide directly connected to the N-Me indole and
indazole rings, respectively.
3
5
in 3-fold less potency than tacrine. Regarding the
linker, we first synthesized indole-tacrine inhibitors
containing a propionamide moiety linked to position 3
of the indole ring and to an alkylaminotacrine of
different methylene tether length. The influence of the
linker on the pharmacological activity was explored by
different chemical modifications: (i) conformational
restriction at the propionamide chain, (ii) inclusion of
an additional N-Me function in the central part of the
methylene chain linked to tacrine, (iii) variation of the
relative position of the carbonyl group and the hetero-
cyclic units, and (iv) replacement of the amide group
by a carbamate residue, which is present in other AChE
inhibitors such as rivastigmine. Finally, derivatives
containing other heterocycles such as 5-cyanoindole,
Regarding the synthesis of the new heterodimers
containing a carbamate functionality within the linker
(Scheme 2), we designed a synthetic approach in which
5-bromoindole, N-methylindole, and indazole derivatives
were also synthesized.

Acetylcholinesterase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7225
Scheme 1a
a
Reagents: (i) 1,1′-carbonyldiimidazole, THF.
Scheme 2a
The chemical structures of all new compounds syn-
thesized herein were fully characterized by analytical
and spectroscopic data reported in the Experimental
1
Section. Bidimensional spectroscopic experiments H-
1
1
13
1
13
H COSY and H- C HMQC, and H- C HMBC were
carried out for unequivocal assignment of the chemical
1
13
shifts observed in the H and C NMR spectra. The
molecular weights of all compounds synthesized were
confirmed by low-resolution mass spectrometry analysis
+
ESI (electrospray), and their degree of purity was also
determined by elemental and HPLC analyses.
Results and Discussion
AChE Inhibitory Activity. To determine the thera-
peutic potential of this new series of tacrine-indole
heterodimers (compounds 3-23 and 27-29) for the
treatment of AD, their AChE inhibitory potency was
assayed on AChE from bovine erythrocytes according
to Ellman et al.,39 using tacrine as a reference com-
pound. With only the exception of compound 14, the
tacrine-indole heterodimers were all clearly more active
as bovine AChE inhibitors than the parent compound
tacrine (see Table 1). In particular, compounds 5 and 6
were >8000- and >2500-fold, respectively, more potent
than tacrine. As previously reported in the literature,40
insertion of a chlorine atom at position 6 of tacrine leads
to a large enhancement in the binding affinity, 6-
chlorotacrine heterodimers 4 and 11 being 17- and 49-
fold more potent than their unsubstituted analogues 3
and 10, respectively.
With the aim of optimizing the interaction of the
heterodimers with both catalytic and peripheral binding
sites of AChE, both the length of the tether and the
position of the amide group within the linker were
varied. Thus, among the heterodimers bearing two
methylene groups between the indole ring and the
amide functionality, those in which the tether length
from the amide to the tacrine unit contains 6 or 7
methylenes (compounds 5 and 6) displayed the best
AChE inhibitory activity (see Table 1). This potency
a
Reagents: (i) N-methylmorpholine; (ii) DMAP, DMF.
preparation of the highly reactive intermediate phenyl-
oxycarbonyl derivative 26 was required before coupling
to the corresponding 9-alkylaminotetrahydroacridines
3
8
1
.
Thus, the carbonate intermediate 26 was prepared
in 32% yield by treatment of 2-(1H-indol-3-yl)ethanol
24) with p-nitrophenyl chloroformate (25) in N-meth-
(
ylmorpholine, followed by chromatographic purification.
Finally, treatment of the carbonate derivative 26 with
the corresponding 9-alkylaminotetrahydroacridines 1b-d
in the presence of DMAP resulted in the formation of
the carbamate derivatives 27-29 that were then puri-
fied by chromatography.

7
226 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Mu n˜ oz-Ruiz et al.
Table 1. Inhibition of AChE and BChE Activities and Selectivity Ratios Determined for the Indole-Tacrine Heterodimers
selectivity
AChE/ BChE
R1
R2
R3
X
Y
Z
IC50(AChE) (nM)
a
IC50(BChE) (nM)
a
compd
tacrine
167 (119-233)
70 (54-95)
24 (12-45)
6.9
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
7
8
9
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
(CH2)5
(CH2)5
(CH2)6
(CH2)7
(CH2)8
(CH2)9
(CH2)10
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CH2)2
(CHdCH)
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
1 (0.8-1.5)
70
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
CN
H
H
H
H
H
H
Br
H
H
H
H
H
H
H
4 (3-6)
16 (8-31)
0.25
0.007
0.6
0.09
0.4
0.4
4900
0.13
0.06
0.2
18.9
19
0.02 (0.01-0.03)
0.06 (0.04-0.10)
0.5 (0.3-1.0)
4.4 (2.7-6.8)
21.9 (16-29)
147 (100-202)
2.9 (2.2-3.8)
0.7 (0.3-1.4)
18 (12-26)
180 (95-350)
33 (20-53)
36 (18-70)
46 (23-83)
0.2 (0.11-0.29)
0.6 (0.4-1.0)
0.3 (0.2-0.5)
0.5 (0.2-0.9)
10.9 (6.6-17)
95 (53-170)
1.5 (1.0-2.3)
0.7 (0.4-1.0)
3.0 (2.0-4.4)
2.9 (1.1-7.5)
0.1 (0.06-0.13)
5.7 (3.7-8.7)
9.6 (3-25)
54 (30-97)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
(CH2)3NMe(CH2)3
(CH2)3NMe(CH2)3
(CH2)6
0.03 (0.01-0.06)
21.4 (12-37)
11.7 (5-23)
77 (50-120)
9.5 (7-12)
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
(CH2)6
(CH2)5
(CH2)6
1.7 (0.9-3)
19 (12-28)
(CH2)7
1.9
2.1
0.02
0.3
(CH2)8
22.4 (13-37)
11.7 (6.4-21)
1.7 (1.0-2.7)
3.2 (1.7-6.1)
5.6 (3.5-8.8)
206 (145-292)
79 (63-97)
(CH2)7
CH2
CH2
(CH2)3
(CH2)3
(CH2)7
(CH2)5
0.09
0.09
0.05
1.2
(CH2)6
(CH2)7
(CH2)7
(CH2)5
(CH2)2O
(CH2)2O
(CH2)2O
CH
CH
CH
136 (87-212)
60 (41-86)
59 (43-81)
0.1
(CH2)6
0.2
(CH2)7
0.05
a
95% confidence intervals are given in parentheses.
decreases as the number of methylene units is length-
Though the total length of the linker is identical in the
two compounds, compound 18 is around 10-fold less
potent than 5, which must be attributed to the fact that
in the former compound the amide group is separated
by only one methylene from the indole ring. Likewise,
when the amido group is separated by three methylenes
from the indole ring (compounds 20 and 21), the
inhibitory activity drops 10-fold with respect to 5 and
6, though the total length of tether is maintained. Thus,
the precise location of the amido group plays a decisive
role in modulating the inhibitory potency of the het-
erodimer.
Replacement of the methylene adjacent to the
carbonyl group in heterodimers 20 and 21 by an oxygen
(compounds 27 and 28) did not produce an important
variation in the inhibitory activity. Similarly, replace-
ment of the indole ring in 16 by an indazole ring (23)
or methylation of the indole nitrogen atom (22) has
modest effects on the inhibitory potency.
On the basis of the preceding results, heterodimers
5, 6, and 18-20, which were the most potent inhibitors
in bovine erythrocyte AChE, were also assayed in
human brain AChE. Within the uncertainty of the
experimental measurements, the results confirm the
trends mentioned above (see Table 2). Thus, compounds
5 and 6 have similar inhibitory activities, which are
close to the values determined for bovine AChE. More-
over, compounds 18-20 exhibit similar potencies, being
2- to 10-fold less active than compounds 5 and 6. These
results, therefore, confirm that these tacrine-indole
heterodimers possess very potent activities with IC50
values at subnanomolar levels.
ened from 8 to 10 (compounds 7-9) or decreased to 5
(
compound 4). Replacement of the central methylene
group of heterodimer 6 by a methylamine group de-
creases the AChE inhibitory activity of compound 11,
which is nearly 50-fold less potent. Moreover, confor-
mational restriction of heterodimer 5 by introduction
of unsaturation at the linker also substantially reduces
the AChE binding affinity, as noted by the fact that
compound 13 is about 900-fold less potent than 5. All
these findings suggest that along the gorge the tether
must satisfy very specific structural requirements to
allow the interaction of both tacrine and indole units
at both catalytic and peripheral binding sites. Finally,
the 5-cyanoindole heterodimer 12 was about 35-fold less
potent than the unsubstituted analogue 5.
Regarding the family of heterodimers where the
carbonyl group is directly attached to the indole ring,
the AChE inhibitory activity was similar for hetero-
dimers bearing six, seven, and eight methylene units
within the linker (compounds 15-17). However, short-
ening the length of the tether to five methylenes (14)
leads to a notable decrease in the inhibitory potency.
Furthermore, compounds 16 and 17, which possess the
same number of methylene units as 4 and 5 but differ
in the position of the amide group within the linker, are
clearly less potent, being particularly worse for com-
pound 17, which is around 2300-fold less potent than
5
. This result strongly suggests that the location of the
amide group within the linker is critical. Further
support of this conclusion comes from the comparison
of the potencies determined for heterodimers 18 and 5.

Acetylcholinesterase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7227
Table 2. Inhibition of Human AChE by Selected
Indole-Tacrine Heterodimers
selectivity
AChE/BChE
a
a
compd
IC50(AChE) (pM)
Ki(AChE) (pM)
5
6
70 (0.4-1.0)
20 (10-30)
120 (80-180)
230 (140-370)
130 (80-200)
14 (3-20)
9 (2-17)
15 (4-20)
20 (8-30)
1.7 (-0.7-4)
0.02
0.22
0.01
0.14
0.04
18
19
20
a
95% confidence intervals are given in parentheses.
Figure 4. Superposition of the backbone of AChE in the X-ray
crystallographic structure complexed with huprine X (PDB
entry 1E66; yellow) and in a representative structure of the
AChE complex with heterodimer 5 (blue). The inhibitor is
represented as a colored ball-and-stick model, and the side
chain of Trp279 is displayed using a stick model.
units of the inhibitor, thus explaining the notable
dependence of the inhibitory potency on the position of
the amido group within the tether (see above and Table
Figure 3. Representation of the heterodimer 5 docked into
the binding site of AChE highlighting the protein residues that
form the main interactions with the different structural units
of the inhibitor.
1
).
Finally, the indole ring is roughly stacked onto the
aromatic ring of Trp279. Moreover, after the first
nanosecond of the simulation, the NH group of the
indole ring forms a water-mediated interaction with the
hydroxyl oxygen of Tyr70 (average N‚‚‚O distance: 5.18
Å). In this orientation, position 5 of the indole unit lies
close to the backbone of Gly335 and to the side chain of
Ile287 (average distances from C5 to CR in Gly335 and
Câ in Ile287 ranging between 3.7 and 4.4 Å), which
justifies the decrease in inhibitory activity observed
upon inclusion of cyano and bromide substituents (see
above and Table 1). Remarkably, there is a notable
conformational rearrangement in the side chain of
Trp279 (see Figure 4). Since the peripheral site region
has been postulated to mediate the interaction of AChE
with the Aâ nonamyloidogenic form,17 it might be
hypothesized that binding of compound 5 might have a
significant effect in modulating the AChE-induced Aâ
peptide fibrillogenesis.
Molecular Modeling. To gain insight into the mo-
lecular determinants that modulate the inhibitory activ-
ity, a molecular modeling study was performed to
explore the binding of heterodimer 5 to the enzyme (see
Experimental Section for details).
The position of heterodimer 5 with respect to the key
residues in the binding site is shown in Figure 3. The
tacrine moiety is firmly bound to the catalytic site of
AChE, it being stacked against the aromatic rings of
Trp84 and Tyr330 (average distances between rings:
3
.52 (Trp84) and 3.56 (Tyr330) Å). The aromatic nitro-
gen of tacrine is hydrogen-bonded to the main-chain
carbonyl oxygen of His440 (average N‚‚‚O distance: 2.84
Å). In addition, a hydrogen-bond interaction is also
formed along the simulation between the tacrine amino
NH group and the main-chain carbonyl oxygen of Trp84
(
average N‚‚‚O distance: 3.27 Å). Finally, the chlorine
AChE/BChE Selectivity. Recent evidence has shown
that in AD patients with severe pathology, AChE is
greatly reduced in specific brain regions, while BChE,
which catalyzes the hydrolysis of a wide variety of
choline esters (butyrylcholine, succinylcholine, and
acetylcholine), noncholine esters, acyl amides, and
aromatic amines, increases.43 Therefore, to further
characterize the pharmacological profile of the het-
erodimers, their selectivity to inhibit AChE vs BChE
was determined.
The selectivity for BChE is generally small in the case
of the 6-chlorotacrine substituted heterodimers 14-17
and 23, it being much less pronounced than the effect
due to the detachment of the chlorine atom in com-
pounds 3 and 10, which reflect and even enhance the
larger binding affinity of tacrine for BChE. Particularly,
10 is around 700-fold more selective for BChE than
tacrine. Remarkably, the AChE/BChE selectivity of
compound 10 is reversed in its 6-chloro derivative
atom occupies a small hydrophobic pocket formed by
Trp432, Met436, and Ile439. This feature, which has
been identified in both modeling studies and the X-ray
41
4
2
crystallographic structure of the AChE-huprine Y
complex, justifies the higher potencies of heterodimers
having the 6-chlorotacrine unit compared to the unsub-
stituted compounds.
With regard to the linker, which is aligned along the
gorge, the most relevant feature comes from the interac-
tions formed by the amido group. Thus, the NH group
is hydrogen-bonded to the hydroxyl oxygen of Try330
(average N‚‚‚O distance: 3.01 Å), which in turn remains
firmly stacked onto the tacrine unit. Indeed, the carbo-
nyl oxygen is hydrogen-bonded to the hydroxyl oxygen
of Tyr121 (average O‚‚‚O distance: 2.75 Å). Overall, the
presence of the amido group in the tether permits us to
define a network of interactions that couple several
residues of the enzyme gorge and different structural

7
228 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Mu n˜ oz-Ruiz et al.
of 5, 6, and 18-20 to inhibit the AChE-induced Aâ1-40
peptide fibrillogenesis was examined employing the
same thioflavin T-based fluorometric assay. Results are
also shown in Table 3, where propidium and some other
typical inhibitors of AChE have been used as reference
compounds.45 The most active compounds were 5 and
2
0, followed by 6, 18, and 19, the latter being around
-, 3-, and 10-fold less active than the former com-
2
pounds, respectively. With only the exception of het-
erodimer 19, all of them were more active than the
reference compound propidium, which is the most potent
AChE-induced Aâ1-40 peptide antiaggregating drug
reported so far (see Table 3 for comparison with com-
mercially available AChE inhibitors). Clearly, these
findings point out a potential disease-modifying action
for this new family of dual AChE inhibitors.
Cell Viability. The cytotoxic effect of this new family
of heterodimers was assayed in the human neuroblas-
toma cell line SH-SY5Y. For compounds 6, 7, and 11
only, toxicity was found at 10 µM (several orders of
magnitude higher than the active concentration). For
the rest of compounds we did not found any toxicity at
this concentration. Therefore, we state that these new
compounds possess a wide therapeutic safety range.
Figure 5. Representation of selected residues in the catalytic
binding site of the MD-averaged structure of the complex
between AChE (blue) and heterodimer 5 (yellow, but the
chlorine atom is shown in magenta), and the superposed
structure of BChE (PDB entry 1N5R; red). An atom-based color
code is used to distinguish between Met437 in BChE and
Ile439 in AChE. The numbering denotes the residues in BChE.
Table 3. Inhibition of Aâ1-40 Peptide Aggregation (with and
without Human AChE) by Some Heterodimers and Reference
Compounds
Conclusions
We have synthesized a series of indole-tacrine het-
erodimers as dual binding site AChE inhibitors. These
compounds display high inhibitory potency against
AChE (bovine and human) and, in general, are rather
selective for AChE regarding BChE. More importantly,
compounds 5, 6, and 18-20 inhibit the AChE-induced
Aâ peptide aggregation in vitro, showing lower IC50
values than propidium, thus rendering new potent
compounds acting as disease-modifying agents for the
treatment of AD. In fact, preliminary in vivo studies in
a transgenic mice overexpressing human APP (hAPP-
(751)) suggest that this class of tacrine-indole het-
erodimers leads to a net reduction of Αâ peptide plaque
load in the animals and an improvement in their
cognitive functions.46 This work is in progress and will
be published. Taken together, all the preceding results
suggest that the new dual binding AChE inhibitors have
potential impact for the future therapeutic treatment
of AD.
with AChE
without AChE
inhibition at
inhibition at
1
00 µM ( SEM IC50 ( SEM 100 µM ( SEM
compd
(%)
(µM)
(%)
5
6
1
2
1
98 ( 2
96 ( 0
84 ( 1
99 ( 4
82 ( 1
2 ( 1
6 ( 2
7 ( 4
2 ( 0
15 ( 3
13 ( 0
49 ( 7
65 ( 5
63 ( 10
63 ( 4
62 ( 14
46 ( 5
0
8
0
9
a
propidium
82
a
b
decamethonium
donepezil
tacrine
edrophonium
physostigmine
25
nd
a
b
22
nd
0
a
b
7
nd
0
nd
a
b
b
0
nd
a
b
b
30
nd
nd
a
SD within 3% (data from ref 43). b nd ) not determined.
(
compound 11), in agreement with the findings obtained
4
4
for homodimeric tacrine inhibitors. The influence of
the chlorine atom on the AChE/BChE selectivity can be
explained by the replacement of Ile439 in AChE by
Met437 in BChE, as noted in the superposition (Figure
Experimental Section
5
) of the MD-averaged structure of the AChE-5 complex
Chemistry. All reagents were of commercial quality. All
experiments involving water-sensitive compounds were carried
out under nitrogen and scrupulously dry conditions, using
anhydrous solvents purchased from Aldrich. Analytical thin-
layer chromatography (TLC) was performed on aluminum
sheets with precoated silica plates, 0.2 mm layer silica gel 60
to BChE (PDB entry 1N5R). Such a replacement elimi-
nates the hydrophobic pocket that anchors the chlorine
atom in AchE
39,40
and makes the terminal methyl group
of Met437 in BChE to be around 1.1 Å closer to the
chlorine atom, thus leading to steric hindrance with the
F254 (Merck). Preparative centrifugal circular thin-layer chro-
6
-chlorotacrine unit.
Effects on the Aâ Aggregation. The heterodimers
matography (CCTLC) was performed on 20 cm diameter
circular glass plates coated with a 1 mm layer of Kieselgel 60
PF254 (Merk), by using a Chromatotron, with the indicated
solvent as eluent. Flash column chromatography was per-
formed at medium pressure on silica gel 60 (particle size
showing best AChE inhibitory potency (5, 6, and 18-
2
0) were selected to assess their ability to inhibit Aâ1-40
peptide aggregation employing the thioflavin T fluores-
cence method and using propidium, a known inhibitor
acting at the AChE peripheral site, as reference com-
pound. Results showed that the heterodimers inhibited
the Aâ peptide aggregation at 100 µM in a range varying
from 49% to 63% (Table 3), they being at least as potent
as propidium, which caused 46% inhibition. To further
explore the dual action of these compounds, the capacity
1
0.040-0.063 mm, 230-240 mesh ASTM, Merck). H NMR and
1
3
C NMR spectra were recorded with Mercury Plus Varian
spectrometers operating at 400 and 50 MHz and using
tetramethylsilane (TMS) as reference. Chemical shifts are
reported in parts per million (ppm) relative to TMS. Elemental
analyses were obtained in a LECO CHNS-932 apparatus.
Analytical RP HPLC was performed on an Alliance 2605
(Waters) provided with a PDA2996 variable-wavelength detec-

Acetylcholinesterase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7229
+
tor and using an Xterra MS C18 column (4.6 mm × 150 mm),
a Symmetry C18 column (2.1 mm × 150 mm), or an Atlantis
DC18 3 µm (2.1 mm × 100 mm), depending on the case.
Mixtures of 0.1% of formic acid in water (solvent A) and 0.1%
of formic acid in CH
phases.
at 0:100 A/B). ESI-MS m/z 545 [M + H] . Anal. (C33
2
H
41ClN
4
O‚
H
O) C, H, N.
N-[10-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-
decyl]-3-(1H-indol-3-yl)propionamide (9). Indole-3-propi-
onic acid (2a) (47 mg, 0.25 mmol), anhydrous THF (4 mL), 1,1′-
carbonyldiimidazol (44 mg, 0.27 mmol), and 6-chloro-9-(10-
aminodecylamino)-1,2,3,4-tetrahydroacridine (1g) (105 mg,
0.27 mmol) were used to produce 9. Purification: DCM/MeOH
(10:1). Yellow foam (21 mg, 19%). RP HPLC (Atlantis DC18)
3
CN (solvent B) were used as mobile
General Procedure for the Synthesis of Indole-
Tacrine Analogues 3-23. To a solution of the corresponding
indole carboxylic acid 2a-i derivative in anhydrous THF was
R
t ) 9.18 (8 min gradient from 95:5 to 0:100 A/B and 2 min
2
added 1,1′-carbonyldiimidazol under N , and the resulting
+
isocratic at 0:100 A/B). ESI-MS m/z 559 [M + H] . Anal.
O‚2H O) C, H, N.
N-(3-{[3-(1,2,3,4-Tetrahydroacridin-9-ylamino)propyl]-
mixture was stirred for 4 h at room temperature. A solution
of the corresponding 9-alkylaminotetrahydroacridine 1a-i in
THF was added, and the stirring was continued for a further
(
34
C H
43ClN
4
2
2
0 h. After evaporation of the solvent under reduced pressure,
methylamino}propyl)-3-(1H-indol-3-yl)propionamide (10).
Indole-3-propionic acid (2a) (56 mg, 0.29 mmol), anhydrous
THF (4 mL), 1,1′carbonyldiimidazole (50 mg, 0.31 mmol), and
N -[3-(1,2,3,4-tetrahydroacridin-9-ylamino)propyl]-N -methyl-
propane-1,3-diamine (1h) (100 mg, 0.31 mmol) were used to
water was added and the resulting mixture was extracted with
dichloromethane. The combined organic extracts were washed
1
1
with saturated NaCl solution and dried with Na
2
SO
4
. Evapo-
ration of the solvent under reduced pressure gave a residue
that was purified by silica gel flash-column chromatography
as indicated below for each case.
produce 10. Purification: DCM/MeOH (20:1 + 0.1% NH
+ 0.2% NH , 10:1 + 0.4% NH
RP HPLC (Symmetry C18) t ) 3.98 (8 min gradient from 95:5
to 0:100 A/B). ESI-MS: m/z 498 [M + H] . Anal. (C31
O) C, H, N.
3
, 10:1
3
3
). Yellow syrup (70 mg, 46%).
R
3
-(1H-Indol-3-yl)-N-[5-(1,2,3,4-tetrahydroacridin-9-yl-
amino)pentyl]propionamide (3). Indole-3-propionic acid
2a) (63 mg, 0.33 mmol), anhydrous THF (3 mL), 1,1′-
+
39 5
H N O‚
H
2
(
N-(3-{[3-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-
carbonyldiimidazol (57 mg, 0.35 mmol), and 9-(5-aminopent-
ylamino)-1,2,3,4-tetrahydroacridine (1a) (100 mg, 0.35 mmol)
were used to produce 3. Purification: DCM/MeOH (3:1). Yellow
propyl]methylamino}propyl)-3-(1H-indol-3-yl)propiona-
mide (11). Indole-3-propionic acid (2a) (56 mg, 0.29 mmol),
anhydrous THF (4 mL), 1,1′-carbonyldiimidazole (50 mg, 0.3
foam (147 mg, 97%). RP HPLC (Atlantis DC18) t
R
) 7.83 (8
1
1
mmol), and N -[3-(6-chloro-1,2,3,4-tetrahydroacridin-9-
min gradient from 95:5 to 0:100 A/B). ESI-MS m/z 455 [M +
1
+
ylamino)propyl]-N -methylpropane-1,3-diamine (1i) (100 mg,
.31 mmol) were used to produce 11. Purification: DCM/MeOH
20:1 + 0.1% NH , 10:1 + 0.2% NH3, 10:1 + 0.4% NH ). Yellow
syrup (70 mg, 46%). RP HPLC (Atlantis DC18) t ) 1.50 (8
min gradient from 95:5 to 0:100 A/B). ESI-MS: m/z 532
H] . Anal. (C29
34 4 2
H N O‚H O) C, H, N.
0
N-[5-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-
pentyl]-3-(1H-indol-3-yl)propionamide (4). Indole-3-pro-
pionic acid (2a) (57 mg, 0.3 mmol), anhydrous THF (3 mL),
(
3
3
R
1
,1′-carbonyldiimidazol (51 mg, 0.32 mmol), and 6-chloro-9-
5-aminopentylamino)-1,2,3,4-tetrahydroacridine (1b) (100 mg,
.32 mmol) were used to produce 4. Purification: DCM/MeOH
7:1). Yellow foam (121 mg, 83%). RP HPLC (Symmetry C18)
) 5.95 (10 min gradient from 100:0 to 0:100 A/B). ESI-MS
+
[
M + H] . Anal. (C31
N-[6-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-
hexyl]-3-(5-cyano-1H-indol-3-yl)propionamide (12). 5-Cy-
5
H38ClN O) C, H, N.
(
0
(
3
5
anoindole-3-propionic acid (2b) (111 mg, 0.52 mmol), anhy-
drous THF (10 mL), 1,1′-carbonyldiimidazol (84 mg, 0.52
mmol), and 6-chloro-9-(6-aminohexylamino)-1,2,3,4-tetra-
hydroacridine (1c) (164 mg, 0.49 mmol) were used to produce
t
R
+
4
m/z 489 [M + H] . Anal. (C29H33ClN O) C, H, N.
N-[5-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-
hexyl]-3-(1H-indol-3-yl)propionamide (5). Indole-3-propi-
onic acid (2a) (70 mg, 0.37 mmol), anhydrous THF (3 mL),
1
2
2. Purification: EtOAc/MeOH 50:1. Yellow syrup (60 mg
2%). RP HPLC (Symmetry C18) t ) 5.33 (8 min from 95:5
R
1
,1′carbonyldiimidazole (63 mg, 0.39 mmol), and 6-chloro-9-
6-aminohexylamino)-1,2,3,4-tetrahydroacridine (1c) (131 mg,
.39 mmol) were used to produce 5. Purification: DCM/MeOH
50:1, 25:1, 20:1). Yellow foam (143 mg, 77%). RP HPLC
Symmetry C18) t ) 5.43 (8 min from 95:5 to 0:100 A/B). ESI-
+
to 0:100 A/B). ESI-MS: m/z 528 [M + H] . Anal. (C31
H
34ClN
5
O‚
(
2
1.5H O) C, H, N.
0
(
(
N-[6-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-
hexyl]-3-(1H-indol-3-yl)acrylamide (13). Indole-3-acrylic
acid (2c) (88 mg, 0.47 mmol), anhydrous THF (6 mL), 1,1′-
carbonyldiimidazole (76 mg, 0.47 mmol), and 6-chloro-9-(6-
aminohexylamino)-1,2,3,4-tetrahydroacridine (1c) (150 mg,
R
+
4 2
MS m/z 503 [M + H] . Anal. (C30H35ClN O‚0.5H O) C, H, N.
N-[7-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-
heptyl]-3-(1H-indol-3-yl)propionamide (6). Indole-3-pro-
pionic acid (2a) (70 mg, 0.37 mmol), anhydrous THF (3 mL),
0
.45 mmol) were used to produce 13. Purification: EtOAc/
MeOH (100:1, 100:1 + 0.1% NH ). Yellow foam (20 mg, 8%).
) 5.46 (8 min from 95:5 to 0:100
3
1
,1′-carbonyldiimidazole (63 mg, 0.39 mmol), and 6-chloro-9-
7-aminoheptylamino)-1,2,3,4-tetrahydroacridine (1d) (135 mg,
.39 mmol) were used to produce 6. Purification: AcOEt/MeOH
50:1). Yellow foam (151 mg, 79%). RP HPLC (Symmetry C18)
) 5.60 (8 min from 95:5 to 0:100 A/B). ESI-MS m/z 517 [M
RP HPLC (Symmetry C18) t
R
(
+
A/B). ESI-MS: m/z 501 [M] . Anal. (C30
H
33ClN
4
O‚H
2
O) C, H,
0
(
N.
1
H-Indole-3-carboxylic Acid [5-(6-Chloro-1,2,3,4-tet-
t
R
rahydroacridin-9-ylamino)pentyl]amide (14). Indole-3-
carboxylic acid (2d) (151 mg, 0.94 mmol), anhydrous THF (4
mL), 1,1′-carbonyldiimidazole (153 mg, 0.94 mmol), and 6-chloro-
9-(5-aminopentylamino)-1,2,3,4-tetrahydroacridine (1b) (276
mg, 0.90 mmol) were used to produce 14. Purification: EtOAc/
MeOH 50:1. Yellow foam (198 mg, 52%). RP HPLC (Symmetry
+
+
H] . Anal. (C31
4 2
H37ClN O‚0.5H O) C, H, N.
N-[8-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-
octyl]-3-(1H-indol-3-yl)propionamide (7). Indole-3-propi-
onic acid (2a) (70 mg, 0.37 mmol), anhydrous THF (3 mL), 1,1′-
carbonyldiimidazole (63 mg, 0.39 mmol), and 6-chloro-9-(8-
aminooctylamino)-1,2,3,4-tetrahydroacridine (1e) (140 mg, 0.39
mmol) were used to produce 7. Purification: AcOEt/MeOH
C18) t
R
) 5.12 (8 min from 95:5 to 0:100 A/B). ESI-MS: m/z
+
461.07 [M] . Anal. (C27
H
4 2
29ClN O‚H O) C, H, N.
(
50:1). Yellow syrup (104 mg, 53%). RP HPLC (Symmetry C18)
1H-Indole-3-carboxylic Acid [6-(6-Chloro-1,2,3,4-tet-
rahydroacridin-9-ylamino)hexyl]amide (15). Indole-3-car-
boxylic acid (2d) (153 mg, 0.95 mmol), anhydrous THF (10 mL),
1,1′-carbonyldiimidazole (154 mg, 0.95 mmol), and 6-chloro-
9-(6-aminohexylamino)-1,2,3,4-tetrahydroacridine (1c) (300
mg, 0.90 mmol) were used to produce 15. Purification: EtOAc/
MeOH 50:1. Clear yellow foam (120 mg, 28%). RP HPLC
t
R
) 5.77 (8 min from 95:5 to 0:100 A/B). ESI-MS m/z 531
+
[
M + H] . Anal. (C32
4 2
H39ClN O‚0.5H O) C, H, N.
N-[9-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-
nonyl]-3-(1H-indol-3-yl)propionamide (8). Indole-3-propi-
onic acid (2a) (28 mg, 0.15 mmol), anhydrous THF (3 mL), 1,1′-
carbonyldiimidazole (25 mg, 0.15 mmol), and 6-chloro-9-(9-
aminononylamino)-1,2,3,4-tetrahydroacridine (1f) (57 mg, 0.15
mmol) were used to produce 8. Purification: DCM/MeOH (7:
(Symmetry C18) t
R
) 5.32 (8 min from 95:5 to 0:100 A/B). ESI-
+
MS: m/z 475 [M + H] . Anal. (C28
H
4
31ClN O) C, H, N.
1
).Yellow foam (10 mg, 14%). RP HPLC (Atlantis DC18) t
R
)
1H-Indole-3-carboxylic Acid [7-(6-Chloro-1,2,3,4-tet-
rahydroacridin-9-ylamino)heptyl]amide (16). Indole-3-
8
.90 (8 min gradient from 95:5 to 0:100 A/B and 2 min isocratic

7
230 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Mu n˜ oz-Ruiz et al.
carboxylic acid (2d) (147 mg, 0.91 mmol), anhydrous THF (10
mL), 1,1′-carbonyldiimidazole (147 mg, 0.91 mmol), and 6-chloro-
Synthesis of Intermediate Carbonic Acid 2-(1H-Indol-
3-yl)ethyl Ester 4-Nitrophenyl Ester (26). To a solution of
2-(1H-indol-3-yl)ethanol (24) (1600 mg, 9.92 mmol) in N-
methylmorpholine (2000 mg, 19.84 mmol) was added p-
nitrophenyl chloroformate (25) (4000 mg, 19.84 mmol), and
the mixture was stirred for 24 h at room temperature. Water
was added, and the mixture was extracted with dichlo-
romethane. Evaporation of the solvent gave a residue that was
purified by silica gel column chromatography using a mixture
of DCM/hexane (3:1) as eluent to produce the title compound
26 (1034 mg, 32%) as a yellow solid. ESI-MS 327 m/z
9
-(7-aminoheptylamino)-1,2,3,4-tetrahydroacridine (1d) (300
mg, 0.87 mmol). Purification: EtOAc/MeOH 50:1. Yellow foam
226 mg, 51%). RP HPLC (Symmetry C18) t ) 5.49 (8 min
from 95:5 to 0:100 A/B). ESI-MS: m/z 489 [M + H] . Anal.
O‚H O) C, H, N.
H-Indole-3-carboxylic Acid [8-(6-Chloro-1,2,3,4-tet-
(
R
+
(
29
C H
33ClN
4
2
1
rahydroacridin-9-ylamino)octyl]amide (17). Indole-3-car-
boxylic acid (2d) (92 mg, 0.57 mmol), anhydrous THF (3 mL),
1
,1′-carbonyldiimidazol (92 mg, 0.57 mmol), and 6-chloro-9-
+
(
8-aminooctylamino)-1,2,3,4-tetrahydroacridine (1e) (196 mg,
[M + H] .
0
.54 mmol) were used to produce 17. Purification: hexane/
General Synthesis of Carbamate Derivatives 27-29.
To a solution of the carbonic acid 2-(1H-indol-3-yl)ethyl ester
4-nitrophenyl ester (26) in MeOH was added a solution of the
corresponding alkylaminotetrahydroacridine in DMF, in the
presence of DMAP, and the resulting mixture was stirred for
24 h at room temperature. After evaporation of the solvent
under reduced pressure, water was added and the resulting
mixture was extracted with dichloromethane. The combined
organic extracts were washed with saturated NaCl solution
and filtered, and the solvent was evaporated to give a residue
that was purified by silica gel column chromatography as
indicated below for each case.
EtOAc (1:2 + 0.1% NH
mg, 40%). RP HPLC (Symmetry C18) t
3
, 1:3 + 0.2% NH
3
). Yellow foam (110
R
) 5.67 (8 min from
+
9
5:5 to 0:100 A/B). ESI-MS: m/z 503 [M + H] . Anal. (C30
H
35
-
4
ClN O) C, H, N.
N-[7-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-
heptyl]-2-(1H-indol-3-yl)acetamide (18). Indole-3-acetic acid
(
2e) (1.10 g, 6.3 mmol), anhydrous THF (50 mL), 1,1′-
carbonyldiimidazole (1.07 g, 6.6 mmol), and 6-chloro-9-(7-
aminoheptylamino)-1,2,3,4-tetrahydroacridine (1d) (2.29 g, 6.6
mmol) were used to produce 18. Purification: EtOAc/MeOH
5
0:1. Clear yellow foam (2.48 g, 80%). RP HPLC (Symmetry
C18) t ) 5.52 (8 min from 95:5 to 0:100 A/B). ESI-MS: m/z
03 [M + H] . Anal. (C30
-(5-Bromo-1H-indol-3-yl)-N-[7-(6-chloro-1,2,3,4-tetra-
R
[5-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-
+
5
H
35ClN
4
O‚H
2
O) C, H, N.
pentyl]carbamic Acid 2-(1H-Indol-3-yl)ethyl Ester (27).
1
2
N -(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)pentane-1,5-di-
hydroacridin-9-ylamino)heptyl]acetamide (19). 5-Bro-
moindole-3-acetic acid (2f) (155 mg, 0.61 mmol), anhydrous
THF (10 mL), 1,1′-carbonyldiimidazol (99 mg, 0.61 mmol), and
amine (1b) (500 mg, 1.58 mmol), carbonic acid 2-(1H-indol-3-
yl)ethyl ester 4-nitrophenyl ester (26) (260 mg, 0.79 mmol),
and DMAP (1930 mg, 1.58 mmol) were used to produce
27. Purification: DCM/MeOH (7:0.5). Yellow syrup (126
6
-chloro-9-(7-aminoheptylamino)-1,2,3,4-tetrahydroacridine (1d)
200 mg, 0.58 mmol) were used to produce 19. Purification:
EtOAc/MeOH 50:1. Clear yellow foam (185 mg, 54%). RP
HPLC (Symmetry C18) t ) 5.76 (8 min from 95:5 to 0:100
A/B). ESI-MS: m/z 581 [M + 1, Br] , 583 [M + 1, Br] .
Anal. (C30 O‚H O) C, H, N.
(
R
mg, 32%). RP HPLC (Atlantis DC18) t ) 8.60 (8 min gradient
from 95:5 to 0:100 A/B and 2 min isocratic at 0:100 A/B).
+
R
4 2 2
ESI-MS: m/z 505.1 [M + H] . Anal. (C29H33ClN O ‚H O) C,
7
9
+
81
+
H, N.
H
35ClN
4
2
[6-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)hexyl]-
1
N-[5-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-
pentyl]-4-(1H-indol-3-yl)butyramide (20). Indole-3-butyric
acid (2g) (134 mg, 0.66 mmol), anhydrous THF (10 mL), 1,1′-
carbonyldiimidazole (107 mg, 0.66 mmol), and 6-chloro-9-(5-
aminopentylamino)-1,2,3,4-tetrahydroacridine (1b) (200 mg,
carbamic Acid 2-(1H-Indol-3-yl)ethyl Ester (28). N -(6-
Chloro-1,2,3,4-tetrahydroacridin-9-yl)hexane-1,6-diamine (1c)
(610 mg, 1.84 mmol), carbonic acid 2-(1H-indol-3-yl)ethyl ester
4-nitrophenyl ester (26) (300 mg, 0.92 mmol), and DMAP (225
mg, 1.84 mmol) were used to produce 28. Purification: DCM/
MeOH (7:0.5). Amber syrup (100 mg, 21%). RP HPLC (Atlantis
0
.63 mmol) were used to produce 20. Purification: EtOAc/
MeOH 100:1. Yellow foam (220 mg, 44%). RP HPLC (Sym-
metry C18) t ) 5.43 (8 min from 95:5 to 0:100 A/B). ESI-MS:
m/z 503 [M + H] . Anal. (C30
DC18) t
min isocratic at 0:100 A/B). ESI-MS: m/z 519.2 [M + H] . Anal.
‚0.5H O) C, H, N.
R
) 8.70 (8 min gradient from 95:5 to 0:100 A/B and 2
+
R
+
H
35ClN
4
O‚H
2
O) C, H, N.
(C30H
35ClN
4
O
2
2
N-[6-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-
hexyl]-4-(1H-indol-3-yl)butyramide (21). Indole-3-butyric
acid (2g) (134 mg, 0.66 mmol), anhydrous THF (8 mL), 1,1′-
carbonyldiimidazol (107 mg, 0.66 mmol), and 6-chloro-9-(6-
aminohexylamino)-1,2,3,4-tetrahydroacridine (1c) (200 mg,
[7-(6-Chloro-1,2,3,4-tetrahydroacridin-9-ylamino)-
heptyl]carbamic Acid 2-(1H-Indol-3-yl)ethyl Ester (29).
1
N -(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)heptane-1,7-di-
amine (1d) (344 mg, 1.0 mmol), carbonic acid 2-(1H-indol-3-
yl)ethyl ester 4-nitrophenyl ester (26) (166 mg, 0.5 mmol), and
DMAP (122 mg, 1.0 mmol) were used to produce 29. Purifica-
tion: DCM/MeOH (7:0.5). Amber syrup (70 mg, 40%). RP
0
.63 mmol) were used to produce 21. Purification: silica gel
column chromatography using DCM/MeOH (20:1, 20:1 +
.01% NH ). Yellow syrup (163 mg, 48%). RP HPLC (Sym-
metry C18) t
0
3
HPLC (Atlantis DC18) t ) 8.90 (8 min gradient from 95:5 to
R
R
+
) 5.32 (8 min from 95:5 to 0:100 A/B). ESI-MS:
0:100 A/B and 2 min isocratic at 0:100 A/B). ESI-MS: m/z 533.1
+
m/z [M + H] 517. Anal. (C31
H
37ClN
4
O‚H
2
O) C, H, N.
[M + H] . Anal. (C H ClN O ‚0.5H O) C, H, N.
3
1
37
4
2
2
1H-Methylindole-3-carboxylic Acid [7-(6-Chloro-1,2,3,4-
Biochemical Methods. In Vitro AChE Inhibition As-
tetrahydroacridin-9-ylamino)heptyl]amide (22). 1-Meth-
ylindole-3-carboxylic acid (2h) (214 mg, 1.22 mmol), anhydrous
THF (20 mL), 1,1′-carbonyldiimidazole (197 mg, 1.22 mmol),
and 6-chloro-9-(7-aminoheptylamino)-1,2,3,4-tetrahydroacri-
dine (1d) (400 mg, 1.16 mmol) were used to produce 22.
Purification: EtOAc/MeOH 50:1. Yellow foam (141 mg, 23%).
say. Inhibitory activity against AChE was evaluated at 30 °C
by the colorimetric method reported by Ellman et al.39 The
assay solution consisted of 0.1 M phosphate buffer, pH 8, 0.3
mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s re-
agent), 0.02 units of AChE (Sigma Chemical Co. from bovine
erythrocytes or human brain), and 0.5 mM acetylthiocholine
iodide as the substrate of the enzymatic reaction. Compounds
tested were added to the assay solution and preincubated with
the enzyme for 10 min at 30 °C. After that period, the substrate
was added. The absorbance changes at 405 nm were recorded
for 5 min with a microplate reader Digiscan 340T, the reaction
rates were compared, and the percent inhibition due to the
presence of the test compounds was calculated. The compound
concentration producing 50% of AChE inhibition (IC50) was
determined for each compound. The sigmoidal dose response
curves have been fit by nonlinear regression (GraphPad Prism
4 software). The experimental uncertainties were calculated
as 95% confidence interval.
R
RP HPLC (Symmetry C18) t ) 5.70 (8 min from 95:5 to 0:100
+
4 2
A/B). ESI-MS: m/z 503 [M + H] . Anal. (C30H35ClN O‚H O)
C, H, N.
1H-Indazole-3-carboxylic Acid [7-(6-Chloro-1,2,3,4-tet-
rahydroacridin-9-ylamino)heptyl]amide (23). Indazole-3-
carboxylic acid (2h) (187 mg, 1.15 mmol), anhydrous THF (5
mL), 1,1′-carbonyldiimidazol (162 mg, 1.20 mmol), and 6-chloro-
9
-(7-aminoheptylamino)-1,2,3,4-tetrahydroacridine (1d) (345
mg, 1.15 mmol) were used to produce 23. Purification: EtOAc/
MeOH 50:1. Yellow syrup (79 mg, 13%). RP HPLC (Symmetry
C18) t
R
) 5.58 (8 min from 95:5 to 0:100 A/B). ESI-MS: m/z
+
490 [M + H] . Anal. (C28H32ClN O) C, H, N.
5

Acetylcholinesterase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7231
In Vitro BChE Inhibition Assay. BChE inhibitory activ-
presence and in the absence of inhibitor, respectively, after
subtracting the fluorescence of respective blanks. Inhibition
curves were obtained for each compound by plotting the
percent inhibition versus the logarithm of inhibitor concentra-
tion in the assay sample. The linear regression parameters
were then determined and the IC50 was extrapolated, when
possible (Microcal Origin, version 3.5, Microcal Software Inc.).
ity was evaluated at 30 °C by the colorimetric method reported
3
9
by Ellman et al. The assay solution consisted of 0.01 units
of BChE (Sigma Chemical Co. from human serum), 0.1 M
sodium phosphate buffer, pH 8, 0.3 mM 5,5’-dithiobis(2-
nitrobenzoic acid) (DTNB, Ellman’s reagent), and 0.5 mM
butyrylthiocholine iodide as the substrate of the enzymatic
reaction. Enzyme activity was determined by measuring the
absorbance at 405 nm during 5 min with a microplate reader
Digiscan 340T. The tested compounds were preincubated with
the enzyme for 10 min at 30 °C before starting the reaction
by adding the substrate. The reaction rate was calculated with
at least triplicate measurements. The IC50 is defined as the
concentration of compounds that reduces by 50% the enzymatic
activity with respect to that without inhibitors. The sigmoidal
dose response curves have been fit by nonlinear regression
Cytotoxicity. The cytotoxicity effect of the synthesized
compounds was studied in the human neuroblastoma cell line
SH-SY5Y. These cells were cultured in 96-well plates in
minimum essential medium and Ham’s F12 medium, supple-
mented with 10% fetal bovine serum, 1% glutamine, and 1%
2
penicillin/streptomycin, and grown in a 5% CO humidified
4
incubator at 37 °C. Cells were plated at 10 cells for each well
at least 48 h before the toxicity measurements. Cells were
exposed for 24 h to the compound at different concentrations,
and quantitative assessment of cell death was made by
measurement of the intracellular enzyme lactate dehydroge-
nase (LDH) (cytotoxicity detection kit, Roche). The quantity
of LDH was evaluated in a microplate reader, Anthos 2010,
at 492 nm (λ excitation) and 620 nm (λ emission). Controls
were taken as having 100% viability.
(
GraphPad Prism 4 software). The experimental uncertainties
were calculated as 95% confidence interval.
Inhibition of â-Amyloid Peptide Aggregation Assay.
Thioflavin T-Based Fluorometric Assay. Aâ peptide (1-
0), lyophilized from HFIP solution (rPeptide, GA), was
4
dissolved in DMSO to obtain a 2.3 mM solution. Aliquots of
Aâ in DMSO were then incubated in constant rotation for 24
h at room temperature in 0.215 M sodium phosphate buffer
Molecular modeling methods. Setup of the Model. The
simulation system was defined following the protocol used in
(
pH 8.0) at a final Aâ concentration of 10 µM in the presence
41,50
our previous studies on AChE-ligand complexes,
which
or absence of compounds or propidium at 100 µM, used as
reference. To quantify amyloid fibril formation, the thioflavin
T fluorescence method was used. Analyses were performed
with a Fluostar Optima plate reader (BMG). Fluorescence was
measured at 450 nm (λ excitation) and 485 nm (λ emission).
To determine amyloid fibril formation, after incubation, the
solutions containing Aâ or Aâ plus AChE inhibitors were added
to 50 mM glycine-NaOH buffer, pH 8.5, containing 3 µM
thioflavin T in a final volume of 150 µL. Each assay was run
in triplicate. The fluorescence intensities were recorded, and
the percent aggregation was calculated by the following
is briefly summarized here. The simulation system was based
on the X-ray crystallographic structures of the AChE com-
plexes with tacrine (PDB entry 1ACJ), huprine Y (1E66),
51
42
52
and donepezil (1EVE), which were used as templates to
position the 6-chlorotacrine unit in the catalytic binding site,
the linker along the gorge, and the indole unit in the peripheral
binding site. The enzyme was modeled in its physiologically
active form with neutral His440 and deprotonated Glu327,
which form together with Ser200 the catalytic triad. The
standard ionization state at neutral pH was considered for the
rest of the ionizable residues with the exception of Asp392 and
Glu443, which were neutral, and His471, which was proto-
expression: 100 - (IF
i
/IF
o
× 100) where IF
i o
and IF are the
fluorescence intensities obtained for Aâ in the presence and
in the absence of inhibitor, respectively, after subtracting the
fluorescence of respective blanks (method adapted from ref 45).
53
nated, according to previous numerical titration studies.
Finally, Phe330 was replaced by Tyr to reflect the binding site
sequence in bovine AChE. According to the basicity of the
Inhibition of AChE-Induced â-Amyloid Peptide Ag-
gregation Assay. Thioflavin T-Based Fluorometric As-
say. Aliquots of 2 µL Aâ(1-40) peptide (Bachem AG, Buben-
quinoline ring,54 the heterodimer was protonated at the tacrine
ring. The orientation of the methylene side chain with regard
to tacrine was determined by means of geometry optimizations
-
1
55
dorf, Switzerland), lyophilized from 2 mg mL 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 human recombinant AChE (final concentra-
tion of 2.30 µM, Aâ/AChE molar ratio of 100:1) and AChE in
the presence of 2 µL of the tested inhibitors were added. Blanks
containing Aâ, AChE, and Aâ plus inhibitors at various
concentrations 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. Inhibitor stock solutions were
prepared (c ) 1.0 or 0.5 mM) and diluted in methanol. To
quantify amyloid fibril formation, the thioflavin T fluorescence
performed at the MP2/6-31G(d) level using Gaussian 03.
With regard to the indole moiety, two different starting
orientations were initially chosen. In both cases the indole ring
was stacked against Trp279, though they differed by a 180°
rotation around the bond linking the indole unit to the
methylene side chain of the linker. The system was hydrated
by centering a sphere of 40 Å of TIP3P56 water molecules at
the inhibitor, paying attention to filling the position of the
crystallographic waters inside the binding cavity. The parm98
57
file of the AMBER force field was used to describe the
enzyme. For the inhibitor, the charge distribution of tacrine,
linker, and indole units was determined from fitting to the
HF/6-31G(d) electrostatic potential using the RESP proce-
4
7-49
method is then applied.
58
dure, and the van der Waals parameters were taken from
Analyses were performed with a Jasco spectrofluorimeter
FP-750 using a 3 mL quartz cell. After incubation, the samples
containing Aâ, Aâ plus AChE, or Aâ plus AChE in the presence
of inhibitors were diluted with 50 mM glycine-NaOH buffer
those defined for related atoms in the AMBER force field. The
final model system was partitioned into a mobile region and
a rigid region. The former included the inhibitor, all the protein
residues containing at least one atom within 15 Å from the
inhibitor, and all the water molecules, while the rest of atoms
defined the rigid part.
(
pH 8.5) containing 1.5 µM thioflavin T to a final volume of
.0 mL. Fluorescence was monitored with excitation at 446
2
nm and emission at 490 nm. A time scan of fluorescence is
performed, and the intensity values reached at the plateau
Molecular Dynamics Simulation. Starting from the two
models of the inhibitor bound to the enzyme (differing in the
orientation of the indole ring), the system was energy-
(
around 300 s) are averaged after subtracting the background
fluorescence from 1.5 µM thioflavin T and AChE.
59
minimized and equilibrated using the AMBER program.
The fluorescence intensities were compared, and the per-
centage of inhibition due to the presence of the tested
compounds was calculated. The percent inhibition of the
AChE-induced aggregation due to the presence of increasing
concentrations of the inhibitor was calculated by the following
First, all hydrogen atoms were minimized for 2000 steps of
steepest descent. Next, the positions of water molecules were
relaxed for 5000 steps of steepest descent plus 3000 steps of
conjugate gradient. At this point, the rigid part of the system
was kept frozen and the thermalization of the mobile part was
started by running four 10 ps molecular dynamics (MD)
simulations to increase the temperature to 298 K. Subse-
expression: 100 - (IF
i
/IF
o
× 100) where IF
i o
and IF are the
fluorescence intensities obtained for Aâ plus AChE in the

7
232 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Mu n˜ oz-Ruiz et al.
quently, a 6 ns MD simulation was carried out. Only one of
the two simulations provided a stable trajectory, as noted in
a small positional root-mean-square deviation (around 0.7 Å
for the backbone atoms in the mobile region with regard to
the crystallographic structure) and favorable contacts with the
enzyme (see text). The characterization of the structural
features that mediate the binding of compound 5 to the enzyme
was determined by averaging the geometrical parameters for
the snapshots (saved every picosecond) sampled along the last
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1
13
data for 3-23 and 27-29 and H and C NMR data for 3-23
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Internet at http://pubs.acs.org.
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