Targeting Alzheimer's disease: Novel indanone hybrids bearing a pharmacophoric fragment of AP2238

Article abstract of DOI:10.1016/j.bmc.2010.01.071

We report on a series of hybrid compounds structurally derived from donepezil and AP2238. This study was aimed at improving the activities of the reference compounds, donepezil and AP2238, and at broadening the range of activities of new derivatives as, due to the multifactorial nature of AD, molecules that modulate the activity of a single protein target are unable to significantly modify the progression of the disease. In particular, the indanone core from donepezil was linked to the phenyl-N-methylbenzylamino moiety from AP2238, through a double bond that was kept to evaluate the role of a lower flexibility in the biological activities. Moreover, SAR studies were performed to evaluate the role of different substituents in position 5 or 6 of the indanone ring in the interaction with the PAS, introducing also alkyl chains of different lengths carrying different amines at one end. Derivatives 21 and 22 proved to be the most active within the series and their potencies against AChE were in the same order of magnitude of the reference compounds. Compounds 15, 21-22, with a 5-carbon alkyl chain bearing an amino moiety at one end, better contacting the PAS, remarkably improved the inhibition of AChE-induced Aβ aggregation with respect to the reference compounds. They also showed activity against self-aggregation of Aβ₄₂ peptide, the most amyloidogenic form of amyloid produced in AD brains, while the reference compounds resulted completely ineffective.

Full text of DOI:10.1016/j.bmc.2010.01.071

Bioorganic & Medicinal Chemistry 18 (2010) 1749–1760
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Targeting Alzheimer’s disease: Novel indanone hybrids bearing
a pharmacophoric fragment of AP2238
Stefano Rizzo a, Manuela Bartolini a, Luisa Ceccarini a, Lorna Piazzi a, Silvia Gobbi a, Andrea Cavalli a,b
,
Maurizio Recanatini a, Vincenza Andrisano a, Angela Rampa a,
*
a Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
b Department of Drug Discovery and Development, Italian Institute of Technology, Via Morego 30, 16163 Genova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We report on a series of hybrid compounds structurally derived from donepezil and AP2238. This study
was aimed at improving the activities of the reference compounds, donepezil and AP2238, and at broad-
ening the range of activities of new derivatives as, due to the multifactorial nature of AD, molecules that
modulate the activity of a single protein target are unable to significantly modify the progression of the
disease. In particular, the indanone core from donepezil was linked to the phenyl-N-methylbenzylamino
moiety from AP2238, through a double bond that was kept to evaluate the role of a lower flexibility in the
biological activities. Moreover, SAR studies were performed to evaluate the role of different substituents
in position 5 or 6 of the indanone ring in the interaction with the PAS, introducing also alkyl chains of
different lengths carrying different amines at one end. Derivatives 21 and 22 proved to be the most active
within the series and their potencies against AChE were in the same order of magnitude of the reference
compounds. Compounds 15, 21–22, with a 5-carbon alkyl chain bearing an amino moiety at one end, bet-
ter contacting the PAS, remarkably improved the inhibition of AChE-induced Ab aggregation with respect
to the reference compounds. They also showed activity against self-aggregation of Ab42 peptide, the most
amyloidogenic form of amyloid produced in AD brains, while the reference compounds resulted com-
pletely ineffective.
Received 10 September 2009
Revised 22 January 2010
Accepted 29 January 2010
Available online 4 February 2010
Dedicated to Professor Piero Valenti on the
occasion of his 70th birthday
Keywords:
Alzheimer’s disease
AChE
Ab inhibition
Indanone
Hybrid molecules
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
block the amyloid polymerization process could be considered po-
tential drugs for AD. To date, no available treatment is known to
It is known that Alzheimer’s disease (AD) is a multifactorial dis-
order involving the malfunction of different, but related, biochem-
ical pathways in which certain proteins play a key role. A
significant alteration of cholinergic markers,1,2 together with extra-
neuronal deposition of b-amyloid (Ab) plaques, are the most signif-
icant hallmarks depicting the pathology.3,4 In particular, because of
an altered catabolism of the so called ‘amyloid precursor protein’
(APP), small insoluble peptides are formed that undergo aggrega-
tion into senile plaques, either spontaneously or through induction
by chaperone molecules such as acetylcholinesterase (AChE). In
fact, it has been shown that AChE, besides its catalytic function,
can also bind to Ab and act as a promoter of Ab fibril formation.5
This action was associated with the peripheral anionic site (PAS)
located at the lip of the enzyme’s gorge and several ligands that
bind to this site have been shown to retard aggregation.6,7 On
the other hand, growth of the fibrils occurs by assembly of the
Ab seeds into intermediate protofibrils, which in turn self-associate
to form mature fibers, and thus compounds that are able to slow or
stop the progression of AD, and currently approved drugs (includ-
ing donepezil, Fig. 1) only seem to act as palliative by temporary
ameliorating cognitive impairment.8 Moreover, it is widely ac-
cepted that targeting different biological entities, involved in the
same pathology, can be a better strategy to block the course of
multifactorial diseases rather than just reducing their symptoms.
For this reason, designing molecular entities able to address more
than one disease-related target has now become a challenging and
interesting approach in the drug design for multifactorial diseases
such as AD.
Our research group has been involved through the years in the
development of AChE inhibitors aimed at inhibiting ACh degrada-
tion, and recently focused the efforts on the design and synthesis
of dual-binding AChE inhibitors, that simultaneously inhibit AChE
and AChE-induced Ab aggregation.9–12 In particular, AP2238 was
the first published compound,9 designed to bind both anionic sites
of the human acetylcholinesterase (hAChE) for which the simulta-
neous inhibition of the catalytic and the amyloid-b pro-aggregating
activities of AChE was verified. The potency of AP2238 against
AChE is comparable to that of donepezil, while its ability to con-
trast Ab aggregation is higher. Docking studies of AP2238 at the
* Corresponding author. Fax: +39 051 2099734.
E-mail address: angela.rampa@unibo.it (A. Rampa).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.071

1750
S. Rizzo et al. / Bioorg. Med. Chem. 18 (2010) 1749–1760
O
H3CO
H3CO
O
O
H3CO
H3CO
CH3
N
N
AP2238
Donepezil
O
R
R
R
N
O
R
R
CH3
N
Figure 1. Design of target molecules.
hAChE gorge have shown that its benzyl group interacted by
means of a stacking with the indole ring of Trp86; the spacer
phenyl ring could establish both a interaction with the phenol
ring of Tyr341 and an OH- interaction with the hydroxyl group of
Tyr124; the protonated amino group was involved in a cation-
zylamine or N-ethylbenzylamine to afford amino derivatives 24
or 25, respectively. These latter were oxidized using the Ni/Raney
alloy to give aldehydes 26–27, which were then reacted with the
selected indanone or tetralone ring to give compounds 1–5 and
18–20, respectively, via the aldol condensation. The synthesis of
derivatives carrying substituents other than methoxy started by
the selected commercially available hydroxy indanone or tetra-
p–p
p–p
p
p
interaction with the phenol ring of Tyr337; the carbonyl group of
the coumarin moiety and eventually the endocyclic oxygen could
establish H-bond interactions with the backbone amide group of
Phe295; the aromatic moiety of the coumarin ring interacted with
lone, that underwent alkylation with 1-bromo-x-chloroalkanes
or -dibromoalkanes (according to commercial availability) in
x
Trp286, by means of a
p-
p
stacking.9 Indeed, AP2238 contacted the
the presence of K2CO3 affording aloalkyloxy derivatives 28a–c,
catalytic site of hAChE and apparently it reached the PAS of the en-
zyme (Trp286 and surrounding residues) better than donepezil.
Here, we report on a series of hybrid compounds structurally
derived from donepezil and AP2238. In particular, as depicted in
Figure 1, the indanone core from donepezil was linked to the phe-
nyl-N-methylbenzylamino moiety from AP2238, to maintain the
29a–f, and 30d, which were subsequently condensed with alde-
hyde 26 to give 31a–c, 32a–f and 33d. The
x-chloroalkyl deriva-
tives 31a–c, 32a–e and 33d were treated with NaI in refluxing
methylethyl ketone to give the corresponding iodo-derivatives
34a–c, 35a–e, 36d, and then subjected to nucleophilic substitution
reaction by the selected amine to give the desired compounds
6–17 and 21–22.
additional OH-p interaction with Tyr124, through a double bond
that was kept to evaluate the role of a lower flexibility in the bio-
logical activities. Moreover, SAR studies were performed to evalu-
ate the role of different substituents in positions 5 or 6 of the
indanone ring in the interaction with the PAS. In particular, since
6-substituted derivatives turned out to be the most promising
compounds, we further modified this position by introducing alkyl
chains of different lengths carrying different amines at one end,
with the purpose to mime the diethylmethylammoniumalkyl moi-
ety of propidium, a potent PAS ligand.7 A derivative in which the
phenyl-N-methylbenzylamino moiety from AP2238 was replaced
by a phenyl-N-ethylbenzylamino group was synthesized as well,
like AP2243.11 Furthermore, the indanone ring itself was replaced
by a tetralone scaffold. As a result, 22 new compounds were syn-
thesized and tested for their biological activities toward hAChE
and human butyrylcholinesterase (hBuChE). Among them, a group
of selected inhibitors was also tested for the inhibition of both
AChE-induced and self Ab aggregation.
3. Enzyme inhibition
The inhibitory activity against AChE of the newly synthesized
compounds was studied using the method described by Ellman13
to determine the rate of hydrolysis of acetylthiocholine in the pres-
ence of the inhibitor. The selectivity of the compounds was also
tested by determining their inhibitory activity against hBuChE.
Some compounds, rationally selected, were also tested for their
ability to prevent the hAChE-induced Ab aggregation by a ThT-
based fluorescence assay. Moreover, in view of enlarging their
spectrum of action, the same series of selected compounds were
in vitro evaluated for their capability of inhibiting b-amyloid self-
aggregation.
4. Results and discussion
The inhibitory activities against both human recombinant AChE
and BuChE from human serum of new derivatives, together with
those of donepezil and AP2238 taken as references, are reported
in Tables 1 and 2, and are expressed as IC50 values.
It was previously proven that methoxy substituents could im-
prove the ability of the compounds to interact with the PAS, as
for both inhibitors donepezil14 and AP2238.9 With regard to the
2. Chemistry
The synthesis of the studied compounds was accomplished as
shown in Scheme 1. Benzylic bromination with N-bromosuccini-
mide (NBS) of the commercially available 4-methylbenzonitrile
afforded 23, which was subsequently reacted with N-methylben-

S. Rizzo et al. / Bioorg. Med. Chem. 18 (2010) 1749–1760
1751
NC
ii
i
R
N
NC
CH3
NC
CH2Br
23
24 R = Me
25 R = Et
OHC
R
N
iii
26 R = Me
27 R = Et
O
O
)
O
)
R2
R3
m = 1,2
iv
HO
( )
X (CH2)n
O
m
v
v
(
(
m
m
28a-c m = 1
29a-f m = 1
X = Cl
X = Cl, Br
X = Cl
30d
m = 2
O
O
R2
R3
R
N
X
(CH2)n
vi
O
CH3
( )m
( )m
N
X = Cl
X = I
34a-c
35a-e
36d
1-5
m=1
31a-c
32a-f
33d
n
18-20 m=2
a
b
c
d
e
f
2
3
4
5
6
7
R = CH3 or C2H5
R2,R3 = H or OCH3
see Table 1 and 2
vii
O
R1 (CH2)n
O
CH3
N
)
(
m
6-17 m = 1
21-22 m = 2
R1 see Table 1 and 2
Scheme 1. Synthesis of the studied compounds. Reagents and conditions: (i) NBS, CCl4, reflux; (ii) N-benzyl-N-methylamine or N-benzyl-N-ethylamine, toluene, reflux; (iii)
Ni/Raney, HCOOH, reflux; (iv) Br(CH2)nCl, K2CO3, acetone, reflux; (v) NaOH, EtOH, rt; (vi) NaI, EtCOMe, reflux; (vii) selected amine, toluene, reflux.
role of the methoxy substituents in this new series, it can be no-
ticed that, when placed in position 6 of the indanone ring, the
methoxy group conferred a good activity (3, Table 1), while when
it was moved to position 5, it led to a reduction in activity of
two order of magnitude. These results are in agreement with those
shown by the 5,6-dimethoxy derivative 1, which is two times less
active than compound 3. The role of the methoxy group in position
6 appeared to be crucial in the interaction with the enzyme, as
evicted by the lower activity (10 times less) of compound 5, whose
indanone ring does not carry any methoxy substituent, when com-
pared with 3. By analyzing Table 1, a detrimental effect on the bio-
logical activity can be noticed when the methyl substituent at the
nitrogen was replaced by the homologous ethyl residue (com-
pound 4). A completely reversed trend was observed within the
tetralone derivatives series, mono-methoxy substituted com-
pounds being 10 times less active than the dimethoxy substituted
compound 20 (Table 2).
To confirm the effective AChE binding mode of some representa-
tive novel AChE inhibitors here reported, 2, 3, 18, and 19 underwent
molecular modeling studies. Docking simulations were carried out
with the software GOLD15 and outcomes were rationalized by means
of the clustering algorithm AClAP.16,17 In Figure 2, the binding modes
of 2, 3, 18, and 19 are reported. It can be seen that all molecules could
favorably interact with both the catalytic pocket and the PAS of the
enzyme. In particular, the following major interactions, responsible
for the inhibiting profiles of the selected molecules, could be identi-
fied: (i) the benzyl group of all the inhibitors interacts by means of
and a T-shaped interaction with the side chain of Phe338; (iii) the
protonated amino group of all inhibitors is involved in a cation-
interaction with the phenol ring of Tyr337; the bicyclic aromatic
p
moiety (indanone of 2 and 3, and tetralone of 18 and 19) interacts
with Trp286 by means of a
p-p stacking; iv) the carbonyl group
(shared by all the docked inhibitors) can establish H-bond interac-
tions with the backbone amide groups of Phe295 and Arg296.
Furthermore, we investigated the role of the methoxy group, which
apparently did not specifically interact with AChE residues. Quan-
tum chemical calculations carried out at the density functional level
of theory (DFT) demonstrated that such a substituent could affect
the H-bond between the carbonyl group and Phe295 and Arg296
backbones by means of a mesomeric effect. In particular, DFT calcu-
lations showed that the H-bonds of 18 (a 6-methoxy derivative)
were about 0.5 kcal/mol more stable than H-bonds between 19
(the 7-methoxy derivative) and Phe295 and Arg296 backbones.
Notably, as shown in Figure 1, the two molecules presented a very
similar binding mode, and therefore the slight difference in biologi-
cal activities here observed (Table 2) could be due to an electronic
contribution to the carbonyl involved in the above mentioned H-
bond interactions. Furthermore, the methyl group of the methoxy
substituent was found to be at hydrophobic distance with the side
chain of Leu289, and therefore it contributes to the overall interac-
tion energy by means of a slight hydrophobic interaction. Such an
interaction was conversely not possible for 2 (Table 1) that points
the methoxy group in a rather different region when compared to
the others. This could help explaining the remarkably lower activity
of 2 when compared to 3, 18 and 19.
p
–
p
stacking with the indole ring of Trp86; (ii) the common spacer
phenyl ring can establish a interaction with the phenol of
Tyr341, an OH-
interaction18 with the hydroxyl group of Tyr124,
p–p
Methoxy substituents were also replaced by an alkyloxy chain
bearing a diethylamino moiety at one end, to evaluate the role of
p

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S. Rizzo et al. / Bioorg. Med. Chem. 18 (2010) 1749–1760
Table 1
Inhibitory activity on human AChE and BuChE, IC50 ratio of the studied compounds
O
O
7
7
6
6
1
1
H3CO
R1-(CH2)n
O
2
2
R
N
R
N
5
5
3
4
3
4
1-5
6-17
Compd
n
OCH3 or chain position
R
R1
IC50 hAChE (
l
M) SEMa
IC50 hBuChE (
l
M) SEMa
Ratio IC50 BuChE/AChE
1
2
3
4
5
6
7
8
—
—
—
—
—
2
3
4
2
3
5,6
5
6
6
—
5
5
5
6
6
CH3
CH3
CH3
C2H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
—
—
—
—
1.14 0.05
28.7 2.2
0.63 0.05
4.93 0.28
11.7 1.1
2.90 0.21
1.61 0.35
3.95 0.47
1.53 0.07
1.32 0.10
0.89 0.15
144 23
21.1 26
7.93 1.18
98.1 4.5
29.9 1.0
22.5 3.9
11.4 0.8
22.9 1.6
11.9 0.5
11.9 0.7
3.52 0.04
126
0.73
12.6
19.9
2.55
7.76
7.08
5.80
7.77
9.01
3.95
—
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
9
10
11
4
6
12
4
6
CH3
0.89 0.08
99.8 5.3
112
O
N
13
14
4
5
6
6
CH3
CH3
0.18 0.02
0.19 0.01
10.0 0.3
55.5
14.2
N
NEt2
2.70 0.15
15
5
6
CH3
0.20 0.02
7.70 0.27
38.5
N
16
17
Donepezil
AP2238
6
7
—
—
6
6
—
—
CH3
CH3
—
NEt2
NEt2
—
0.38 0.02
0.51 0.03
0.02 0.01
20.5 1.8
33.0 3.0
7.42 0.39
48.9 3.7
53.9
64.7
371
—
—
0.044 0.006
1111
a
Human recombinant AChE and BuChE from human serum were used. IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and
are the mean of two independent measurements, each performed in triplicate (SEM = standard error of the mean).
Table 2
Inhibitory activity on human AChE and BuChE, IC50 ratio of the studied compounds
O
O
8
5
8
5
7
6
7
6
1
1
CH3
N
CH3
N
2
2
R1 (CH2)5
O
H3CO
3
3
4
4
18-20
21-22
Compd
OCH3 or chain position
R1
IC50 hAChE (
l
M) SEMa
IC50 hBuChE (
l
M) SEMa
Ratio IC50 BuChE/AChE
18
19
20
21
6
7
6,7
6
—
—
—
NEt2
0.11 0.01
5.38 0.84
0.18 0.01
90.3 3.8
135 32
72.8 5.0
8.05 0.50
820
25.1
404
144
0.056 0.003
22
6
0.052 0.002
5.01 0.19
96.3
N
Donepezil
AP2238
—
—
—
—
0.02 0.01
0.044 0.006
7.42 0.39
48.9 3.7
371
1111
a
Human recombinant AChE and BuChE from human serum were used. IC50 values represent the concentration of inhibitor required to decrease enzyme activity by 50% and
are the mean of two independent measurements, each performed in triplicate (SEM = standard error of the mean).
its interaction with the PAS. Again, position 5 of the indanone ring
proved not to be optimal, independently of the chain’s length
(n = 2–4, compounds 6–8), while the substitution in position 6
(compounds 9–11, 14, 16–17) resulted in a general improvement
of the activity, along with the elongation of the chain, the optimum
being n = 5 (compound 14). The trend was inverted when n = 6–7
(compounds 16–17). We then changed the nature of the second
amino moiety by replacing the diethylamino group with morpho-
line and piperidine. While the effect of morpholine was negligible
(12), the introduction of piperidine with n = 4, (13) increased the
activity by 5 times compared to the diethylamino derivative (11).
Yet, when n = 5 no such increase in potency was noted for the
piperidine substituent (15) with respect to the diethylamino (14).
Concerning the tetralone series, replacing the methoxy substituent
in position 6 by a pentyl chain (compound 21) resulted in a
remarkable increase in activity, comparable with that of the corre-
sponding piperidine derivative (22). These two latter compounds
proved to be the most active within the series and their potency
against hAChE was in the same order of magnitude of the reference
compounds. These derivatives also showed a good inhibition of
hBuChE activity, with selectivity values lower than donepezil and
AP2238. Several new lines of evidence suggest that BuChE might
be a co-regulator of the activity of the neurotransmitter ACh, and
that it might be important to inhibit this enzyme in the treatment
of AD.19,20 The marketed drug Rivastigmine also inhibits BuChE,
providing dual AChE and BuChE inhibition.21 It has been suggested

S. Rizzo et al. / Bioorg. Med. Chem. 18 (2010) 1749–1760
1753
methylene chain at position 6. The obtained inhibitory activities,
expressed as% inhibition, are reported in Table 3. Compound 3,
bearing a methoxy substituent in position 6, was not able to inhibit
AChE-induced Ab aggregation, whereas 14–15 and 22, with a pen-
tyl chain bearing a second amino moiety, showed some activity. In
particular, 15 and 22, piperidine derivatives, proved to be more ac-
tive than the diethylamino derivative 14 (46.8% and 48.3% com-
pared with 29.7%). In order to highlight the specific residues
involved in the interaction, 14 and 15 underwent molecular mod-
eling studies (Fig. 4). The molecules were found to contact both
sites of the enzyme. However, the aliphatic chains of 14 and 15
can better contact Trp286 and partially protrude towards the bulk
of the solvent. These features can be responsible for the inhibition
of AChE-induced Ab aggregation experimentally observed for 14
and 15, but not for 3. We can argue that the positive charge at
the end of the aliphatic chains of 14 and 15 can be responsible
for an electrostatic repulsion with the Ab peptide, preventing its
interaction with the PAS, which is fundamental for the chaperon-
like effect of AChE towards Ab fibril formation.
Finally, in the search for new multipotent compounds, the se-
lected derivatives were evaluated against self-aggregation of Ab42
peptide, the most amyloidogenic form of amyloid produced in
AD brains. Previously selected lead compounds (donepezil and
AP2238) as well as 3 resulted completely ineffective in avoiding
amyloid spontaneous aggregation (Table 3). On the other hand,
14, 15, 21 and 22, when tested at 10 lM, showed a significant even
if low inhibitory activity (26–28%).
Figure 2. Docking of 2 (carbon atoms in yellow), 3 (carbon atoms in magenta), 18
(carbon atoms in green), and 19 (carbon atoms in cyan) into the gorge of the human
AChE enzyme. All molecules are able to properly contact both sites of the enzyme,
i.e. the catalytic pocket around Trp86 and the PAS in the region of Trp286. Further
interactions could be also identified with the side chains of the residues of the mid-
gorge27–29 (Tyr124, Tyr337, and Tyr341, for a detailed description see the text). The
interaction mode of the present inhibitors strictly resembles the binding of
donepezil at the Torpedo californica AChE gorge (PDB code: 1eve30).
5. Conclusions
We reported a new series of indanone hybrids bearing a phar-
macophoric fragment of AP2238. This study was aimed at improv-
ing the activities of the reference compounds, donepezil and
AP2238, and at broadening the range of activities of new deriva-
tives as, due to the multifactorial nature of AD, molecules that
that dual inhibition may afford several advantages, including
greater and broader symptomatic effects, particular behavioral
benefits and the absence of AChE upregulation. These benefits
are likely to increase with time, because, as AD progresses, AChE
activity decreases by up to 45%, while BuChE activity increases
by 40–90%. The gradual shift from AChE to BuChE in the enzyme
responsible for degrading ACh during AD progression could be
responsible for the inefficacy of AChE selective inhibitors.
Consequently, there is a rationale for switching from a selective
AChE inhibitor to an inhibitor of both AChE and BuChE.21
The in vitro determination of the inhibitory constant
(61.1 2.3 nM) of compound 22, the most active derivative of
these new series, confirmed the mixed type mechanism of action
and, therefore, the possibility for 22 of contacting both the active
and the peripheral binding sites of AChE. In fact, the pattern of
the overlaid Lineweaver–Burk plots (Fig. 3) showed both increas-
ing slopes and higher Km at increasing inhibitor concentrations.
Some compounds, properly selected on the basis of their inhib-
itory potencies, were also tested for their ability to prevent AChE-
induced Ab aggregation. In particular, compounds 3, 14–15 and 22
were screened at a single concentration and compared with refer-
ence compounds donepezil and AP2238. The limited selection of
compounds to undergo AChE-induced Ab aggregation assay is
due to the high costs of the recombinant proteins involved in this
assay. Considering that AChE inhibition and AChE-induced beta-
amyloid aggregation do not represent independent phenomena,
but both can be modulated by AChE PAS binding inhibitors, a ra-
tional selection was made on the basis of the lowest IC50 values ob-
tained in Ellman’s assay as well as of the dual binding mode of
action. On the other hand, compound 3 represents an internal ref-
erence of the indanone series with regards to the role of the amino
Figure 3. Steady-state inhibition by 22 of hydrolysis of acetylthiocholine (ATCh) by
human recombinant AChE. Reciprocal plot of initial velocity (v) and substrate
concentration at increasing inhibitor concentrations are reported. Reciprocal plot of
initial velocity in the absence of inhibitor gave an estimate of kapp for ATCh of
170 15
points.
lM. Lines were derived from a weighted least-squares analysis of the data

1754
S. Rizzo et al. / Bioorg. Med. Chem. 18 (2010) 1749–1760
Table 3
Inhibitory activity AChE-mediated Ab aggregation and against b-amyloid (1–42) self-aggregation of the selected compounds
O
O
6
5
R1-(CH2)n
O 7
6
O
R1 (CH2)n
B
A
Compd
Structure
n
Position
R1
Inhibition of AChE-induced Ab40 aggregationa SEM (%)
Inhibition of Ab42 self-aggregationb SEM (%)
3
14
A
A
—
5
6-OCH3
6
—
NEt2
<5
29.7 3.8
<5
27.7 3.6
15
21
A
B
5
5
6
6
46.8 2.0
42.9 0.8
27.9 4.0
26.4 1.1
N
N
NEt2
22
B
5
6
48.3 0.9
22c
35
26.9 3.4
Donepezil
AP2238
<5
<5
a
b
c
Determined at [inhibitor] = 100
[Ab42] = 50 M; [I] = 10
Data obtained from Ref. 22.
l
M, [Ab40] = 230 lM and [hAChE] = 2.3 lM.
l
lM. Values are the mean of two independent measurements, each performed in duplicate. (SEM = standard error of the mean).
6. Experimental section
6.1. Chemistry
6.1.1. General methods
All melting points were determined in open glass capillaries
using a Büchi apparatus and are uncorrected. 1H NMR and 13C
NMR spectra were recorded in CDCl3 solution on a Varian Gemini
300 MHz spectrometer with Me4Si as the internal standard. Chem-
ical shifts are reported in parts per million (ppm) relative to tetra-
methylsilane (TMS), and multiplicities are given as s (singlet), d
(doublet), t (triplet), m (multiplet) or br (broad). Mass spectra were
recorded on a Waters ZQ 4000 apparatus operating in electrospray
(ES) mode. Chromatographic separations were performed on silica
gel columns (Kieselgel 40, 0.040–0.063 mm; Merck) by flash chro-
matography. The purity of the tested compounds was determined
by HPLC analysis, performed on a Jasco LC 1500 PU-1587; the
column used was a Phenomenex Luna C18(2) 5
150 mm; elution conditions: mobile phase CH3CN/H2O 50:50; the
flow-rate was 0.8 mL/min and the injection volume was 5 L;
lm ꢀ 4.60 mm ꢀ
l
peaks were detected at 250 nm and results were >95% purity. Ele-
mental analyses are indicated with element symbols, analytical re-
sults obtained for those elements were within 0.4% of the
theoretical values. Compounds’ names were obtained using AUTO-
NOM, PC software for nomenclature in organic chemistry, Beil-
stein-Institute and Springer.
Figure 4. Docking of 14 (carbon atoms in orange), 15 (carbon atoms in light green),
and 3 (carbon atoms in magenta) into the gorge of the human AChE enzyme. The
molecules contact both sites of the enzyme. However, the aliphatic chain of 14 and
15 can better contact Trp286 and partially protrude towards the bulk of the solvent.
The poses reported in the Figure are docking configurations representative of
clusters statistically populated according to the Chaevenaut criterion, implemented
in the AClAP 1.0 code.
6.1.2. 4-Bromomethylbenzonitrile (23)
To a solution of 4-methylbenzonitrile (10.0 g, 0.088 mol) in CCl4
(100 mL), NBS (15.6 g, 0.088 mol) was added in the presence of a
catalytic amount of benzoyl peroxide. The mixture was refluxed
for 5 h and hot filtered to remove the succinimide. The filtrate
was evaporated under reduced pressure to give the crude product
23 (15.86 g, 92%) as yellow solid used for the next step without fur-
ther purification.
modulate the activity of a single protein target are unable to signif-
icantly modify the progression of the disease. Derivatives 21 and
22 proved to be the most active within the series and their potency
against AChE was in the same order of magnitude of the reference
compounds. Compounds 15, 21–22, with a 5-carbon alkyl chain
bearing an amino moiety at one end, better contacting the PAS,
remarkably improved the inhibition of AChE-induced Ab aggrega-
tion, with respect to the reference compounds. They also showed
activity against self-aggregation of Ab42 peptide, while the refer-
ence compounds resulted completely ineffective. On the basis of
the promising results obtained with the new compounds, 15, 21
and 22 could represent new templates for further optimization
studies.
6.1.3. 4-[(Benzylmethylamino)methyl]benzonitrile (24)
A
solution of 23 (18.91 g, 0.096 mol) and N-benzylmetil-
amine (24.9 mL, 0.19 mol) in toluene (250 mL) was refluxed
for 8 h. After cooling, the reaction mixture was washed with
H2O, dried (Na2SO4) and evaporated to dryness affording 24
(14.43 g, 64%). 1H NMR d 2.15 (s, 3H), 3.50 (s, 4H), 7.20–7.60
(m, 9H, Ar).

S. Rizzo et al. / Bioorg. Med. Chem. 18 (2010) 1749–1760
1755
6.1.4. 4-[(Benzylethylamino)methyl]benzonitrile (25)
Using the previous procedure and starting from 23 (19 g,
0.097 mol) and N-benzylethylamine (28.5 mL, 0.194 mol), 25
(18 g, 74%) was obtained as a yellow oil. 1H NMR d 1.00 (t, 3H),
2.35–2.55 (m, 2H), 3.50 (s, 4H), 7.00–7.75 (m, 9H, Ar).
Mp 62–63 °C. 1H NMR d 1.80–2.05 (m, 4H), 2.70 (t, 2H), 3.05 (t, 2H),
3.60 (t, 2H), 4.00 (t, 2H), 7.10–7.40 (m, 3H, Ar).
6.1.13. 6-(5-Chloropentyloxy)indan-1-one (29d)
Using the previous procedure and starting from 6-hydroxyin-
dan-1-one (1.2 g, 0.008 mol) and 1-bromo-5-chloropentane
(2.13 mL, 0.016 mol), 29d (1.69 g, 83%) was obtained as white so-
lid. Mp 59–60 °C. 1H NMR d 1.50–1.95 (m, 6H), 2.70 (t, 2H), 3.05
(t, 2H), 3.60 (t, 2H), 4.00 (t, 2H), 7.10–7.40 (m, 3H, Ar).
6.1.5. 4-[(Benzylmethylamino)methyl]benzaldehyde (26)
A mixture of 24 (6.85 g, 29 mmol) and Ni/Raney alloy (13.7 g) in
75% HCOOH (138 mL) was refluxed for 7 h, and then hot filtered.
The residue was diluted with 100–150 mL of H2O, basified by
K2CO3 and extracted three times with CH2Cl2. The combined organ-
ic extracts were dried (Na2SO4), filtered and concentrated under re-
duced pressure to afford 26 (6.0 g, 87%) as yellow oil (purified by
flash chromatography toluene/acetone 9:1 as eluent). 1H NMR d
2.20 (s, 3H), 3.50 (s, 4H), 7.20–7.80 (m, 9H, Ar), 10.00 (s, 1H).
6.1.14. 6-(6-Chlorohexyloxy)indan-1-one (29e)
Using the previous procedure and starting from 6-hydroxyin-
dan-1-one (1.0 g, 6.7 mmol) and 1-bromo-6-chlorohexane
(2.0 mL, 13.5 mmol), 29e (1.26 g, 71%) was obtained as white solid.
Mp 61–62 °C. 1H NMR d 1.35–1.55 (m, 6H), 1.70–1.90 (m, 2H), 2.70
(t, 2H), 3.05 (t, 2H), 3.50 (t, 2H), 4.00 (t, 2H), 7.10–7.40 (m, 3H, Ar).
6.1.6. 4-[(Benzylethylamino)methyl]benzaldehyde (27)
Using the previous procedure and starting from 25 (10 g,
0.097 mol), 27 (9.22 g, 78%) was obtained as yellow oil (purified
by flash chromatography with toluene/acetone 4.8:0.2). 1H NMR
d 1.00 (t, 3H), 2.35–2.55 (m, 2H), 3.50 (s, 4H), 7.00–7.75 (m, 9H,
Ar), 9.90 (s, 1H).
6.1.15. 6-(7-Bromoheptyloxy)indan-1-one (29f)
Using the previous procedure and starting from 6-hydroxyin-
dan-1-one (1.0 g, 6.7 mmol) and 1,7-dibromoheptane (2.76 mL,
3.4 mmol), 29f (0.9 g, 41%) was obtained as yellow solid. Mp 36–
37 °C. 1H NMR d 1.35–1.55 (m, 6H), 1.70–1.90 (m, 4H), 2.70 (t,
2H), 3.05 (t, 2H), 3.40 (t, 2H), 3.95 (t, 2H), 7.05–7.35 (m, 3H, Ar).
6.1.7. 5-(2-Chloroethoxy)indan-1-one (28a)
A stirred mixture of 5-hydroxyindan-1-one (3.0 g, 0.020 mol),
1-bromo-2-chloroethane (10 mL, 0.040 mol) and K2CO3 (6 g) in
acetone (250 mL) was refluxed for 24 h. The mixture was hot fil-
tered and the solvent was removed. After adding petroleum ether,
the residue was kept at ꢁ18 °C overnight, the white solid that
formed was filtered off affording 28a (3.83 g, 91%). Mp 81–82 °C.
1H NMR d 2.70 (t, 2H), 3.10 (t, 2H), 3.85 (t, 2H), 4.30 (t, 2H),
6.85–7.75 (m, 3H, Ar).
6.1.16. 6-(5-Chloropentyloxy)-3,4-dihydro-2H-naphthalen-1-
one (30d)
Using the previous procedure and starting from 6-hydroxy-
3,4-dihydro-2H-naphthalen-1-one (1.2 g, 7.4 mmol) and 1-bro-
mo-5-chloropentane (1.95 mL, 14.8 mmol), 30d (1.49 g, 76%) was
obtained. Mp 25–26 °C. 1H NMR d 1.60–1.90 (m, 6H), 2.00–2.20
(m, 2H), 2.60 (t, 2H), 2.90 (t, 2H), 3.60 (t, 2H), 4.00 (t, 2H), 6.70–
8.00 (m, 3H, Ar).
6.1.17. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-5-(2-
chloroethoxy)indan-1-one (31a)
6.1.8. 5-(3-Chloropropoxy)indan-1-one (28b)
Using the previous procedure and starting from 5-hydroxyin-
dan-1-one (3.0 g, 0.020 mol) and 1-bromo-3-chloropropane
(10.1 mL, 0.040 mol), 28b (3.69 g, 82%) was obtained as white so-
lid. Mp 90–91 °C. 1H NMR d 2.20–2.40 (m, 2H), 2.70 (t, 2H), 3.05
(t, 2H), 3.75 (t, 2H), 4.20 (t, 2H), 6.85–7.70 (m, 3H, Ar).
To a solution of NaOH (0.58 g) in EtOH (72 mL), 28a (1.5 g,
0.007 mol) was added and the reaction mixture was stirred for
1 h. Then, a solution of 26 (1.67 g, 0.007 mol) in a small amount
of EtOH was added dropwise and the resulting mixture was stirred
at room temperature overnight. The solid that formed was filtered
off affording 31a (1.30 g, 43%). Mp 100–101 °C. 1H NMR d 2.20 (s,
3H), 3.55 (s, 4H), 3.85 (t, 2H), 4.00 (s, 2H), 4.35 (t, 2H), 7.00–7.85
(m, 13H, Ar).
6.1.9. 5-(4-Chlorobutoxy)indan-1-one (28c)
Using the previous procedure and starting from 5-hydroxyin-
dan-1-one (4.0 g, 0.027 mol) and 1-bromo-4-chlorobutane
(13.8 mL, 0.054 mol), 28c (4.28 g, 67%) was obtained as white solid.
Mp 52–53 °C. 1H NMR d 1.90–2.05 (m, 4H), 2.70 (t, 2H), 3.10 (t, 2H),
3.65 (t, 2H), 4.10 (t, 2H), 6.85–7.75 (m, 3H, Ar).
6.1.18. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-5-(3-
chloropropoxy)indan-1-one (31b)
Using the previous procedure and starting from 28b (1.50 g,
6.7 mmol), 31b (1.45 g, 49%) was obtained as white solid. Mp
111–113 °C. 1H NMR d 2.20 (s, 3H), 2.20–2.35 (m, 2H), 3.55 (s,
4H), 3.75 (t, 2H), 4.00 (s, 2H), 4.25 (t, 2H), 7.00–7.85 (m, 13H, Ar).
6.1.10. 6-(2-Chloroethoxy)indan-1-one (29a)
Using the previous procedure and starting from 6-hydroxyin-
dan-1-one (1.5 g, di 0.010 mol) and 1-bromo-2-chloroethane
(1.66 mL, 0.020 mol), 29a (0.9 g, 43%) was obtained as white solid.
Mp 59–60 °C. 1H NMR d 2.70 (t, 2H), 3.10 (t, 2H), 3.85 (t, 2H), 4.25
(t, 2H,), 7.10–7.40 (m, 3H, Ar).
6.1.19. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-5-(4-
chlorobutoxy)indan-1-one (31c)
Using the previous procedure and starting from 28c (1.50 g,
6.3 mmol), 31c (1.32 g, 46%) was obtained as pale brown solid.
Mp 113–114 °C. 1H NMR d 1.90–2.05 (m, 4H), 2.20 (s, 3H), 3.55
(s, 4H), 3.65 (t, 2H), 4.00 (s, 2H), 4.10 (t, 2H), 6.85–7.85 (m, 13H,
Ar).
6.1.11. 6-(3-Chloropropoxy)indan-1-one (29b)
Using the previous procedure and starting from 6-hydroxyin-
dan-1-one (1.5 g, 0.010 mol) and 1-bromo-3-chloropropane
(2 mL, 0.020 mol), 29b (1.89 g, 84%) was obtained as white solid.
Mp 50–51 °C. 1H NMR d 2.05–2.45 (m, 2H), 2.70 (t, 2H), 3.10 (t,
2H), 3.75 (t, 2H), 4.10 (t, 2H), 7.10–7.40 (m, 3H, Ar).
6.1.20. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(2-
chloroethoxy)indan-1-one (32a)
6.1.12. 6-(4-Chlorobutoxy)indan-1-one (29c)
Using the previous procedure and starting from 29a (0.9 g,
4.3 mmol), 32a (0.88 g, 48%) was obtained as pale brown solid.
Mp 92–93 °C. 1H NMR d 2.20 (s, 3H), 3.50 (s, 4H), 3.85 (t, 2H),
4.00 (s, 2H), 4.30 (t, 2H), 7.20–7.70 (m, 13H, Ar).
Using the previous procedure and starting from 6-hydroxyin-
dan-1-one (1.5 g, 0.010 mol) and 1-bromo-4-chlorobutane
(2.31 mL, 0.020 mol), 29c (2.17 g, 91%) was obtained as white solid.

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S. Rizzo et al. / Bioorg. Med. Chem. 18 (2010) 1749–1760
6.1.21. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(3-
chloropropoxy)indan-1-one (32b)
6.1.30. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(2-
iodoethoxy)indan-1-one (35a)
Using the previous procedure and starting from 29b (1.21 g,
5.4 mmol), 32b (1.06 g, 37%) was obtained as pale brown solid.
Mp 120–121 °C d. 1H NMR d 1.80–2.05 (m, 2H), 2.20 (s, 3H), 3.25
(t, 2H), 3.55 (s, 4H), 3.60 (t, 2H), 3.90 (s, 2H), 4.05 (t, 2H), 7.10–
7.60 (m, 13H, Ar).
Using the previous procedure and starting from 32a (0.40 g,
0.93 mmol), 35a (0.31 g, 64%) was obtained as yellow oil. 1H
NMR d 2.20 (s, 3H), 3.55 (s, 4H), 3.80 (t, 2H), 3.95 (s, 2H), 4.30 (t,
2H), 7.20–7.60 (m, 13H, Ar).
6.1.31. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(3-
iodopropoxy)indan-1-one (35b)
Using the previous procedure and starting from 32b (0.80 g,
1.8 mmol), 35b (1.22 g, 72%) was obtained as yellow solid. Mp
110–112 °C. 1H NMR d 2.20 (s, 3H), 2.25–2.40 (m, 2H), 3.40 (t,
2H), 3.55 (s, 4H), 4.00 (s, 2H), 4.25 (t, 2H), 7.00–7.85 (m, 13H, Ar).
6.1.22. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(4-
chlorobutoxy)indan-1-one (32c)
Using the previous procedure and starting from 29c (1.44 g,
6 mmol), 32c (1.66 g, 60%) was obtained as pale brown solid. Mp
90–91 °C. 1H NMR d 1.90–2.10 (m, 4H), 2.20 (s, 3H), 3.55 (s, 4H),
3.60 (t, 2H), 4.00 (t, 2H), 4.10 (t, 2H), 7.10–7.65 (m, 13H, Ar).
6.1.32. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(4-
iodobutoxy)indan-1-one (35c)
Using the previous procedure and starting from 32c (0.80 g,
1.8 mmol), 35c (0.89 g, 95%) was obtained as yellow oil. 1H NMR
d 1.80–2.05 (m, 4H), 2.20 (s, 3H), 3.25 (t, 2H), 3.55 (s, 4H), 3.60
(t, 2H), 3.90 (s, 2H), 4.05 (t, 2H), 7.10–7.60 (m, 13H, Ar).
6.1.23. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(5-
chloropentoxy)indan-1-one (32d)
Using the previous procedure and starting from 29d (1.69 g,
6.7 mmol), 32d (1.72 g, 55%) was obtained as solid. Mp 71–72 °C.
1H NMR d 1.55–1.95 (m, 6H), 2.20 (s, 3H), 3.55 (s, 4H), 3.60 (t,
2H), 3.95 (t, 2H), 4.10 (t, 2H), 7.20–7.75 (m, 13H, Ar).
6.1.33. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(5-
iodopentyloxy)indan-1-one (35d)
Using the previous procedure and starting from 32d (0.30 g,
0.63 mmol), 35d (0.22 g, 69%) was obtained as yellow oil. 1H
NMR d 1.40–1.90 (m, 6H), 2.20 (s, 3H), 3.20 (t, 2H), 3.55 (s, 4H),
3.60 (t, 2H), 3.80–4.10 (m, 4H), 7.00–7.60 (m, 13H, Ar).
6.1.24. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(6-
chlorohexiloxy)indan-1-one (32e)
Using the previous procedure and starting from 29e (1.00 g,
3.8 mmol), 32e (1.30 g, 70%) was obtained as white solid. Mp 94–
95 °C. 1H NMR d 1.45–1.65 (m, 8H), 2.20 (s, 3H), 3.55 (s, 4H),
3.70 (t, 2H), 4.00 (t, 2H), 4.10 (t, 2H), 7.10–7.65 (m, 13H, Ar).
6.1.34. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(6-
iodohexiloxy)indan-1-one (35e)
6.1.25. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(7-
bromoheptyloxy)indan-1-one (32f)
Using the previous procedure and starting from 32e (1.30 g,
2.7 mmol), 35e (1.50 g, 97%) was obtained as yellow solid. Mp
90–91 °C. 1H NMR d 1.40–1.60 (m, 4H), 1.70–1.90 (m, 4H), 2.20
(s, 3H), 3.20 (t, 2H), 3.55 (s, 4H), 3.90–4.10 (m, 4H), 7.10–7.70
(m, 13H, Ar).
Using the previous procedure and starting from 29f (1.30 g,
4 mmol), 32f (1.20 g, 55%) was obtained as white solid. Mp 90–
91 °C. 1H NMR d 1.35–1.60 (m, 6H), 1.75–1.95 (m, 4H), 2.20 (s,
3H), 3.40 (t, 2H), 3.55 (s, 4H), 3.80–4.10 (m, 4H), 7.20–7.65 (m,
13H, Ar).
6.1.35. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(5-
iodopentyloxy)-3,4-dihydro-2H-naphthalen-1-one (36d)
Using the previous procedure and starting from 33d (0.39 g,
0.8 mmol), 36d (0.32 g, 69%) was obtained as yellow oil. 1H NMR
d 1.50–2.00 (m, 6H), 2.20 (s, 3H), 2.90 (t, 2H), 3.10 (t, 2H), 3.20 (t,
2H), 3.55 (s, 4H), 4.00 (t, 2H), 6.65–8.10 (m, 13H, Ar).
6.1.26. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(5-
chloropentyloxy)-3,4-dihydro-2H-naphthalen-1-one (33d)
Using the previous procedure and starting from 30d (1.50 g,
5.6 mmol), 33d (0.42 g, 15%) was obtained as yellow oil (purified
by flash chromatography with toluene/acetone 4.8:0.2). 1H NMR
d 1.55–1.70 (m, 2H), 1.75–1.90 (m, 4H), 2.20 (s, 3H), 2.90 (t, 2H),
3.10 (t, 2H), 3.50–3.55 (m, 6H), 4.00 (t, 2H), 6.65–8.10 (m, 13H, Ar).
6.1.36. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-5,6-
dimethoxyindan-1-one (1)
6.1.27. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-5-(2-
iodoethoxy)indan-1-one (34a)
To a solution of pellets NaOH (0.21 g) in EtOH (30 mL), 5,6-dim-
ethoxyindan-1-one (0.5 g, 2.6 mmol) was added and the reaction
mixture was stirred for 1 h. A solution of 26 (0.62 g, 2.6 mmol) in
a small amount of EtOH was added dropwise and the resulting
mixture was stirred at room temperature overnight. The solid that
formed was filtered off affording 1 (0.87 g, 81%) as yellow solid. Mp
147–148 °C (ligroine), HCl mp 249–250 °C (EtOH/Et2O). 1H NMR d
2.20 (s, 3H), 3.55 (s, 4H), 3.95–4.10 (m, 8H), 7.00–7.75 (m, 12H,
Ar). 13C NMR d 31.83, 42.31, 56.12, 56.64, 61.42, 61.91, 105.75,
118.66, 118.82, 123.84, 126.87, 127.03, 128.26, 133.74, 134.13,
135.09, 139.29, 141.46, 150.95, 153.37, 145.23, 197.99. MS (ES)
m/z: 414 (M+H+). Anal. C27H27NO3 (C, H, N).
A mixture of 31a (1.20 g, 2.8 mmol) and NaI (0.42 g, 2.8 mmol)
in methylethylketone (20 mL) was refluxed for 4 h. After cooling,
the separated solid was collected by filtration, affording 34a
(0.9 g, 61%). Mp 94–96 °C. 1H NMR d 2.20 (s, 3H), 3.55 (s, 4H),
3.85 (t, 2H), 4.00 (s, 2H), 4.35 (t, 2H), 7.00–7.85 (m, 13H, Ar).
6.1.28. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-5-(3-
iodopropoxy)indan-1-one (34b)
Using the previous procedure and starting from 31b (1.60 g,
3.6 mmol), 34b (2.44 g, 72%) was obtained as yellow solid. Mp
112–114 °C. 1H NMR d 2.20 (s, 3H), 2.25–2.40 (m, 2H), 3.40 (t,
2H), 3.55 (s, 4H), 4.00 (s, 2H), 4.25 (t, 2H), 7.00–7.85 (m, 13H, Ar).
6.1.37. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-5-
methoxyindan-1-one (2)
6.1.29. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-5-(4-
iodobutoxy)indan-1-one (34c)
Using the previous procedure and starting from 31c (1.20 g,
2.6 mmol), 34c (1.10 g, 77%) was obtained as solid. Mp 87–88 °C.
1H NMR d 1.90–2.10 (m, 4H), 2.20 (s, 3H), 3.30 (t, 2H), 3.55 (s,
4H), 4.00 (s, 2H), 4.10 (t, 2H), 6.90–7.85 (m, 13H, Ar).
Using the previous procedure and starting from 5-methoxyin-
dan-1-one (1.00 g, 6.0 mmol) and 26 (1.47 g, 6.0 mmol), 2 (0.84 g,
31%) was obtained as white solid. Mp 130–131 °C (toluene). 1H
NMR d 2.20 (s, 3H), 3.55 (s, 4H), 3.90 (s, 3H), 4.00 (s, 2H), 7.00–
7.90 (m, 13H, Ar). 13C NMR d 31.83, 42.31, 55.63, 61.42, 61.91,
105.75, 118.72, 115.08, 123.84, 126.87, 127.03, 128.26, 129.35,

S. Rizzo et al. / Bioorg. Med. Chem. 18 (2010) 1749–1760
1757
130.74, 134.13, 135.09, 139.29, 141.46, 150.95, 167.56, 197.99. MS
135.95, 145.69, 157.57, 186.78. MS (ES) m/z: 398 (M+H+). Anal.
C27H27NO2 (C, H, N).
(ES) m/z: 384 (M+H+). Anal. C26H25NO2 (C, H, N).
6.1.38. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-
methoxyindan-1-one (3)
6.1.43. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6,7-
dimethoxy-3,4-dihydro-2H-naphthalen-1-one (20)
Using the previous procedure and starting from 6-methoxyin-
dan-1-one (1.47 g, 9.0 mmol) and 26 (2.17 g, 9.0 mmol), 3 (0.30 g,
10%, purified by flash chromatography with toluene/acetone
4.5:0.5) was obtained as white solid. Mp 119–120 °C. 1H NMR d
2.20 (s, 3H), 3.55 (s, 4H), 3.80 (s, 3H), 3.90 (s, 2H), 7.10–7.65 (m,
13H, Ar). 13C NMR d 31.83, 42.31, 55.63, 61.42, 61.91, 105.75,
123.84, 126.87, 127.03, 128.26, 129.35, 130.72, 133.74, 134.13,
135.09, 139.04, 139.29, 141.46, 142.42, 150.95, 159.57, 197.99.
MS (ES) m/z: 384 (M+H+). Anal. C26H25NO2 (C, H, N).
Using the previous procedure and starting from 6,7-methoxy-
3,4-dihydro-2H-naphthalen-1-one (0.62 g, 3 mmol) and 26
(1.15 g, 4.8 mmol), 20 was obtained as yellow oil and purified by
flash chromatography with toluene/acetone 96:4. 1H NMR d 2.20
(s, 3H), 2.90 (t, 2H), 3.10 (t, 2H), 3.55 (s, 4H), 3.85 (s, 6H), 6.70–
8.10 (m, 12H, Ar). 13C NMR d 27.26, 29.28, 42.27, 55.43, 55.84,
61.47, 61.88, 113.29, 127.03, 127.10, 128.26, 128.91, 129.84,
130.74, 135.23, 135.95, 145.69, 147.34, 155.58, 186.78. MS (ES)
m/z: 428 (M+H+). Anal. C28H29NO2 (C, H, N)
6.1.39. 2-{4-[(Benzylethylamino)methyl]benzylidene}-6-
methoxyindan-1-one (4)
6.1.44. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-5-(2-
diethylaminoethoxy)indan-1-one (6)
Using the previous procedure and starting from 6-methoxyin-
dan-1-one (0.50 g, 3.1 mmol) and 27 (0.78 g, 3.1 mmol), 4 was ob-
tained as oil, purified by flash chromatography with toluene/
acetone 4.7:0.3. The purified compound was converted into the
hydrochloride salt affording 4ꢂHCl (0.63 g, 20%): mp 218–219 °C
(EtOH/Et2O). 1H NMR d 1.05 (t, 3H), 2.50–2.55 (m, 2H), 3.55 (s,
4H), 3.85 (s, 3H), 3.95 (s, 2H), 7.10–7.65 (m, 13H, Ar). 13C NMR d
10.78, 31.83, 48.93, 55.63, 59.02, 59.48, 105.75, 126.01, 126.87,
127.03, 128.26, 129.35, 130.72, 133.74, 134.13, 135.09, 139.29,
141.46, 141.84, 150.95, 197.99. MS (ES) m/z: 398 (M+H+). Anal.
C27H27NO2 (C, H, N).
A solution of 34a (0.35 g, 0.67 mmol) and diethylamine
(0.21 mL, 2 mmol) in toluene (50 mL) was refluxed for 20 h. The
mixture was washed with water (3 ꢀ 25 mL) and the organic layer
was dried over Na2SO4. The solvent was removed under reduced
pressure and the residue was purified by flash chromatography
(toluene/acetone 3:2), affording 6 as a yellowish solid (0.05 g,
16%). 1H NMR d 1.00 (t, 6H), 2.20 (s, 3H), 2.50–2.65 (m, 6H), 3.55
(s, 4H), 3.95 (s, 2H), 4.10 (t, 2H), 6.90–7.80 (m, 13H, Ar). 13C NMR
d 9.82, 10.74, 31.83, 42.33, 46.66, 52.27, 61.42, 61.92, 66.13,
106.47, 124.16, 127.00, 128.24, 128.84, 129.33, 130.70, 133.70,
134.10, 135.09, 139.09, 141.52, 142.29, 158.95, 197.97. MS (ES)
m/z: 469 (M+H+). Anal. C31H36N2O2 (C, H, N).
6.1.40. 2-{4-[(Benzylmethylamino)methyl]benzylidene}indan-
1-one (5)
6.1.45. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-5-(3-
diethylaminopropoxy)indan-1-one (7)
Using the previous procedure and starting from indan-1-one
(0.28 g, 2.0 mmol) and 26 (0.50 g, 2.0 mmol), 5 was obtained as
oil, purified by flash chromatography with toluene/acetone
4.8:0.2. The purified compound was converted in the hydrochlo-
ride salt affording 5ꢂHCl (0.10 g, 14%): mp 246–247 °C (EtOH/
Et2O).1H NMR d 2.20 (s, 3H), 3.55 (s, 4H), 4.00 (s, 2H), 7.10–7.90
(m, 14H, Ar). 13C NMR d 31.83, 42.31, 61.42, 61.91, 105.75,
123.84, 126.87, 127.03, 128.26, 129.35, 130.72, 133.74, 134.13,
135.09, 139.04, 139.29, 141.46, 142.42, 150.95, 159.57, 197.99.
MS (ES) m/z: 354 (M+H+). Anal. C25H23NO (C, H, N).
Using the previous procedure and starting from 34b (0.40 g,
0.75 mmol), 7 (purified by flash chromatography with toluene/ace-
tone 3:2) was obtained as yellow solid (0.24 g, 66%), mp 65–67 °C.
1H NMR d 1.00 (t, 6H), 1.85–2.00 (m, 2H), 2.20 (s, 3H), 2.55–2.65
(m, 6H), 3.55 (s, 4H), 3.95 (s, 2H), 4.10 (t, 2H), 6.90–7.80 (m, 13H,
Ar). 13C NMR d 9.82, 10.74, 29.73, 31.81, 42.33, 46.66, 52.27,
61.42, 61.92, 66.13, 106.47, 124.16, 127.00, 128.24, 128.84,
129.33, 130.70, 133.70, 134.10, 135.09, 139.09, 141.52, 142.29,
158.95, 197.97. MS (ES) m/z: 483 (M+H+). Anal. C32H38N2O2 (C, H,
N).
6.1.41. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-
methoxy-3,4-dihydro-2H-naphthalen-1-one (18)
6.1.46. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-5-(4-
diethylaminobutoxy)indan-1-one (8)
Using the previous procedure and starting from 6-methoxy-3,4-
dihydro-2H-naphthalen-1-one (1.60 g, 9 mmol) and 26 (2.17 g,
9 mmol), 18 was obtained and purified by flash chromatography
with toluene/acetone 4.7:0.3. The purified compound was obtained
as white solid (0.50 g, 14%) mp 91–93 °C. 1H NMR d 2.20 (s, 3H),
2.85 (t, 2H), 3.10 (t, 2H), 3.55 (s, 4H), 3.85 (s, 3H), 6.70–8.10 (m,
13H, Ar). 13C NMR d 27.26, 29.28, 42.27, 55.43, 61.47, 61.88,
112.24, 113.29, 127.03, 127.10, 128.26, 128.91, 129.84, 130.74,
135.23, 135.95, 145.69, 163.53, 186.78. MS (ES) m/z: 398 (M+H+).
Anal. C27H27NO2 (C, H, N).
Using the previous procedure and starting from 34c (0.10 g,
0.75 mmol), 8 (purified by flash chromatography with toluene/ace-
tone 3:2) was obtained as yellow oil (0.03 g, 33%). 1H NMR d 1.00 (t,
6H), 1.60–1.90 (m, 4H), 2.20 (s, 3H), 2.40–2.55 (m, 6H), 3.55 (s, 4H),
4.00 (s, 2H), 4.10 (t, 2H), 6.90–7.80 (m, 13H, Ar). 13C NMR d 9.82,
10.74, 25.70, 28.93, 31.81, 42.33, 46.66, 50.07, 61.42, 61.92,
67.85, 106.47, 124.16, 127.00, 128.24, 128.84, 129.33, 130.70,
133.70, 134.10, 135.09, 139.09, 141.52, 142.29, 158.95, 197.97.
MS (ES) m/z: 497 (M+H+). Anal. C33H40N2O2 (C, H, N).
6.1.42. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-7-
methoxy-3,4-dihydro-2H-naphthalen-1-one (19)
6.1.47. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(2-
diethylaminoethoxy)indan-1-one (9)
Using the previous procedure and starting from 7-methoxy-3,4-
dihydro-2H-naphthalen-1-one (0.50 g, 2.8 mmol) and 26 (0.68 g,
2.8 mmol), 19 was obtained as oil and purified by flash chromatog-
raphy with toluene/acetone 4.9:0.1. The purified compound was
converted in the hydrochloride salt affording 20ꢂHCl (0.10 g,
13%): mp 220–221 °C (EtOH/Et2O). 1H NMR d 2.20 (s, 3H), 2.85 (t,
2H), 3.10 (t, 2H), 3.55 (s, 4H), 3.85 (s, 3H), 7.00–7.85 (m, 13H,
Ar). 13C NMR d 27.26, 29.28, 42.27, 55.43, 61.47, 61.88, 113.29,
114.24, 127.03, 127.10, 128.26, 128.91, 129.84, 130.74, 135.23,
Using the previous procedure and starting from 35a (0.31 g,
0.6 mmol), 9 (purified by flash chromatography with toluene/ace-
tone 4.5:0.5) was obtained as orange solid (0.06 g, 21%), mp 58–
59 °C. 1H NMR d 1.00 (t, 6H), 2.20 (s, 3H), 2.50–2.65 (m, 4H), 2.95
(t, 2H), 3.55 (s, 4H), 4.00 (s, 2H), 4.15 (t, 2H), 7.10–7.70 (m, 13H,
Ar). 13C NMR d 9.82, 10.74, 31.83, 42.33, 46.66, 52.27, 61.42,
61.92, 66.13, 106.47, 124.16, 127.00, 128.24, 128.84, 129.33,
130.70, 133.70, 134.10, 135.09, 139.09, 141.52, 142.29, 158.95,
197.97. MS (ES) m/z: 469 (M+H+). Anal. C31H36N2O2 (C, H, N).

1758
S. Rizzo et al. / Bioorg. Med. Chem. 18 (2010) 1749–1760
6.1.48. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(3-
diethylaminopropoxy)indan-1-one (10)
6.1.53. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(5-
piperidin-1-yl-pentyloxy)indan-1-one (15)
Using the previous procedure and starting from 35b (0.50 g,
0.9 mmol), 10 (purified by flash chromatography with toluene/ace-
tone 90:10) was obtained as yellow solid (0.16 g, 37%), mp 80–
81 °C. 1H NMR d 1.00 (t, 6H), 1.90–2.05 (m, 2H), 2.20 (s, 3H),
2.50–2.65 (m, 6H), 3.55 (s, 4H), 4.00 (s, 2H), 4.10 (t, 2H), 7.10–
7.70 (m, 13H, Ar). 13C NMR d 9.82, 10.74, 29.73, 31.81, 42.33,
46.66, 52.27, 61.42, 61.92, 66.13, 106.47, 124.16, 127.00, 128.24,
128.84, 129.33, 130.70, 133.70, 134.10, 135.09, 139.09, 141.52,
142.29, 158.95, 197.97. MS (ES) m/z: 483 (M+H+). Anal.
C32H38N2O2 (C, H, N).
Using the previous procedure and starting from 35d (0.25 g,
0.44 mmol) and piperidine (0.1 mL, 0.88 mmol), 15 (purified by
flash chromatography with toluene/acetone 4:1) was obtained as
yellow oil (0.08 g, 38%). 1H NMR d 1.30–1.65 (m, 8H), 1.75–2.00
(m, 4H), 2.20 (s, 3H), 2.25–2.45 (m, 6H), 3.75 (s, 4H), 3.95 (s, 2H),
4.05 (t, 2H), 7.10–7.70 (m, 13H, Ar). 13C NMR d 23.47, 24.47,
25.99, 27.31, 27.33, 31.81, 42.33, 54.61, 59.04, 61.45, 61.95,
68.02, 106.46, 124.22, 126.82, 126.99, 128.24, 128.84, 129.31,
130.69, 133.63, 134.14, 135.14, 139.13, 139.24, 141.50, 142.23,
159.01, 197.96. MS (ES) m/z: 523 (M+H+). Anal. C35H42N2O2 (C, H,
N).
6.1.49. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(4-
diethylaminobutoxy)indan-1-one (11)
6.1.54. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(6-
diethylaminohexyloxy)indan-1-one (16)
Using the previous procedure and starting from 35c (0.89 g,
1.6 mmol), 11 (purified by flash chromatography with toluene/ace-
tone 4:1) was obtained as orange oil (0.09 g, 11%). 1H NMR d 1.00
(t, 6H), 1.55–1.70 (m, 2H), 1.75–1.85 (m, 2H), 2.20 (s, 3H), 2.45–
2.65 (m, 6H), 3.55 (s, 4H), 4.00 (s, 2H), 4.05 (t, 2H), 7.10–7.70 (m,
13H, Ar). 13C NMR d 9.82, 10.74, 25.70, 28.93, 31.81, 42.33, 46.66,
50.07, 61.42, 61.92, 67.85, 106.47, 124.16, 127.00, 128.24, 128.84,
129.33, 130.70, 133.70, 134.10, 135.09, 139.09, 141.52, 142.29,
158.95, 197.97. MS (ES) m/z: 497 (M+H+). Anal. C33H40N2O2 (C, H,
N).
Using the previous procedure and starting from 35e (0.30 g,
0.5 mmol), 16 (purified by flash chromatography with toluene/ace-
tone 4:1) was obtained as yellow solid (0.10 g, 38%), mp 63–64 °C.
1H NMR d 1.00 (t, 6H), 1.40–1.90 (m, 8H), 2.20 (s, 3H), 2.40–2.60
(m, 6H), 3.55 (s, 4H), 3.95–4.05 (m, 4H), 7.10–7.75 (m, 13H, Ar).
13C NMR d 9.82, 10.74, 24.01, 25.70, 28.93, 29.26, 31.81, 42.33,
46.66, 52.26, 61.42, 61.92, 68.14, 106.47, 124.16, 127.00, 128.24,
128.84, 129.33, 130.70, 133.70, 134.10, 135.09, 139.09, 141.52,
142.29, 158.95, 197.97. MS (ES) m/z: 525 (M+H+). Anal.
C35H44N2O2 (C, H, N).
6.1.50. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(4-
morpholin-4-yl-butoxy)indan-1-one (12)
6.1.55. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(7-
diethylaminoheptyloxy)indan-1-one (17)
Using the previous procedure and starting from 35c (0.25 g,
0.45 mmol) and morpholine (0.08 mL, 0.91 mmol), 12 (purified
by flash chromatography with toluene/acetone 4:1) was obtained
as yellow solid (0.08 g, 35%), mp 93–94 °C. 1H NMR d 1.60–1.70
(m, 2H), 1.75–1.90 (m, 2H), 2.20 (s, 3H), 2.35–2.50 (m, 6H), 3.55
(s, 4H), 3.70 (t, 4H), 3.95 (s, 2H), 4.05 (t, 2H), 7.10–7.65 (m, 13H,
Ar). 13C NMR d 25.89, 27.32, 31.81, 42.33, 54.61, 61.45, 61.95,
63.36, 68.20, 106.46, 124.22, 126.82, 126.99, 128.24, 128.84,
129.31, 130.69, 133.63, 134.14, 135.14, 139.13, 139.24, 141.50,
142.23, 159.01, 197.96. MS (ES) m/z: 511 (M+H+). Anal.
C33H38N2O3 (C, H, N).
Using the previous procedure and starting from 32f (0.30 g,
0.55 mmol), 17 (purified by flash chromatography with toluene/
acetone 4:1) was obtained as yellow solid (0.07 g, 24%), mp
72 °C. 1H NMR d 1.00 (t, 6H), 1.20–1.60 (m, 8H), 1.70–1.90 (m,
2H), 2.20 (s, 3H), 2.35–2.60 (m, 6H), 3.55 (s, 4H), 3.90–4.10 (m,
4H), 7.10–7.65 (m, 13H, Ar). 13C NMR d 9.82, 10.74, 25.01, 25.70,
28.93, 29.26, 29.82, 31.81, 42.33, 46.66, 52.28, 61.42, 61.92,
68.14, 106.47, 124.16, 127.00, 128.24, 128.84, 129.33, 130.70,
133.70, 134.10, 135.09, 139.09, 141.52, 142.29, 158.95, 197.97.
MS (ES) m/z: 539 (M+H+). Anal. C36H46N2O2 (C, H, N).
6.1.51. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(4-
piperidin-1-yl-butoxy)indan-1-one (13)
6.1.56. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(5-
diethylaminopentyloxy)-3,4-dihydro-2H-naphthalen-1-one
(21)
Using the previous procedure and starting from 35c (0.25 g,
0.45 mmol) and piperidine (0.09 mL, 0.91 mmol), 13 (purified by
flash chromatography with toluene/acetone 4:1) was obtained as
yellow solid (0.08 g, 35%), mp 76–77 °C. 1H NMR d 1.30–1.90 (m,
10H), 2.20 (s, 3H), 2.25–2.45 (m, 6H), 3.55 (s, 4H), 3.95 (s, 2H),
4.05 (t, 2H), 7.10–7.70 (m, 13H, Ar). 13C NMR d 23.47, 24.47,
25.99, 27.31, 31.81, 42.33, 54.61, 59.04, 61.45, 61.95, 68.20,
106.46, 124.22, 126.82, 126.99, 128.24, 128.84, 129.31, 130.69,
133.63, 134.14, 135.14, 139.13, 139.24, 141.50, 142.23, 159.01,
197.96. MS (ES) m/z: 509 (M+H+). Anal. C34H40N2O2 (C, H, N).
Using the previous procedure and starting from 36d (0.32 g,
0.55 mmol), 21 (purified by flash chromatography with toluene/
acetone 4:1) was obtained as yellow oil (0.14 g, 49%). 1H NMR d
1.00 (t, 6H), 1.40–1.60 (m, 4H), 1.75–1.85 (m, 2H), 2.20 (s, 3H),
2.40 (t, 2H), 2.40–2.55 (m, 4H), 2.90 (t, 2H), 3.10 (t, 2H), 3.55 (s,
4H), 4.00 (t, 2H), 6.70–8.10 (m, 13H, Ar). 13C NMR d 9.82, 10.74,
24.01, 25.70, 28.93, 29.28, 27.25, 42.33, 46.66, 52.27, 61.42,
61.92, 68.13, 124.16, 127.00, 128.24, 128.84, 129.33, 130.70,
133.70, 134.10, 135.09, 139.09, 141.52, 142.29, 162.45, 187.97.
MS (ES) m/z: 525 (M+H+). Anal. C35H44N2O2 (C, H, N).
6.1.52. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(5-
diethylaminopentyloxy)indan-1-one (14)
6.1.57. 2-{4-[(Benzylmethylamino)methyl]benzylidene}-6-(5-
piperidin-1-yl-pentyloxy)-3,4-dihydro-2H-naphthalen-1-one
(22)
Using the previous procedure and starting from 35d (0.30 g,
0.53 mmol), 14 (purified by flash chromatography with toluene/
acetone 4:1) was obtained as yellow oil (0.03 g, 11%). 1H NMR d
1.00 (t, 6H), 1.40–1.90 (m, 6H), 2.20 (s, 3H), 2.40–2.60 (m, 6H),
3.55 (s, 4H), 3.95–4.05 (m, 4H), 7.10–7.65 (m, 13H, Ar). 13C NMR
d 9.82, 10.74, 24.01, 25.70, 28.93, 31.81, 42.33, 46.66, 52.27,
61.42, 61.92, 68.13, 106.47, 124.16, 127.00, 128.24, 128.84,
129.33, 130.70, 133.70, 134.10, 135.09, 139.09, 141.52, 142.29,
158.95, 197.97. MS (ES) m/z: 511 (M+H+). Anal. C34H42N2O2 (C, H,
N).
Using the previous procedure and starting from 36d (0.2 g,
0.35 mmol), 23 (purified by flash chromatography with toluene/
acetone 4:1) was obtained as yellow oil (0.14 g, 77%). 1H NMR d
1.25–1.65 (m, 8H), 1.75–1.95 (m, 4H), 2.20 (s, 3H), 2.25–2.40 (m,
6H), 2.95 (t, 2H), 3.05 (t, 2H), 3.75 (s, 4H), 4.00 (t, 2H), 6.70–8.10
(m, 13H, Ar). 13C NMR d 23.47, 24.47, 25.99, 27.31, 27.33, 29.29,
27.27, 42.33, 54.61, 59.04, 61.45, 61.95, 68.02, 124.22, 126.82,
126.99, 128.24, 128.84, 129.31, 130.69, 133.63, 134.14, 135.14,

S. Rizzo et al. / Bioorg. Med. Chem. 18 (2010) 1749–1760
1759
139.13, 139.24, 141.50, 142.23, 162.88, 186.79. MS (ES) m/z: 537
6.5. Inhibition of A42 self-aggregation
(M+H+). Anal. C36H44N2O2 (C, H, N).
The spontaneous fibrilization of peptide Ab42 was investigated
by a ThT-based fluorometric assay. HFIP (1,1,1,3,3,3-hexafluoro-
2-propanol) pretreated Ab42 samples (Bachem AG, Switzerland)
were resolubilized with a CH3CN/Na2CO3/NaOH (48.4:48.4:3.2)
6.2. Inhibition of AChE and BuChE
The method of Ellman et al. was followed.13 Five different con-
centrations of each compound were selected in order to obtain
inhibition of AChE or BuChE activity comprised between 20% and
80%. The assay solution consisted of a 0.1 M potassium phosphate
buffer pH 8.0, with the addition of 340
benzoic acid), 0.02 unit/mL of human recombinant AChE or BuChE
derived from human serum (Sigma Chemical), and 550 M of sub-
strate (acetylthiocholine iodide or butyrylthiocholine iodide,
respectively). Test compounds were added to the assay solution
and preincubated at 37 °C with the enzyme for 20 min followed
by the addition of substrate. Assays were carried out with a blank
containing all components except AChE or BuChE in order to ac-
count for non-enzymatic reaction. The reaction rates were com-
pared, and the percent inhibition due to the presence of test
compounds was calculated. Each concentration was analyzed in
triplicate, and IC50 values were determined graphically from inhi-
bition curves (log inhibitor concentration vs percent inhibition).
mixture to have a stable stock solution ([Ab] = 500 l
M).25 Experi-
ments were performed by incubating Ab42 in 10 mM phosphate
buffer (pH 8.0) containing 10 mM NaCl, at 30 °C for 24 h (final
l
M 5,50-dithio-bis(2-nitro-
Ab42 concentration = 50
pound at 10
lM) with and without the tested com-
l
M. To quantify amyloid fibril formation, the ThT fluo-
l
rescence method was used.23,24 After incubation, samples were
diluted to a final volume of 2.0 mL with 50 mM glycine–NaOH buf-
fer (pH 8.5) containing 1.5 lM thioflavin T. A 300-seconds-time
scan of fluorescence intensity was carried out (kexc = 446 nm;
kem = 490 nm), and values at plateau were averaged after subtract-
ing the background fluorescence of 1.5 lM thioflavin T solution.%
inhibition was calculated from the ratio between florescence inten-
sities obtained in the presence and in the absence of inhibitor.
6.6. Computational studies
The molecular structure of human AChE was obtained from
the Protein Data Bank (pdb accession code: 1b41).26 The investi-
gated compounds (2, 3, 18, and 19) were build using the SYBYL 7.3
molecular modeling suite (Tripos Inc., St. Louis, MO), and each
structure was optimized in the density functional theory (DFT)
framework. All the DFT computations described in the text were
6.3. Inhibition of AChE-induced b-amyloid aggregation
Aliquots of 2 lL Ab40 peptide (Bachem AG, Germany), lyophilized
from a 2 mg mLꢁ1 HFIP (1,1,1,3,3,3-hexafluoro-2-propanol) solution
and dissolved in DMSO, were incubated for 24 h at room tempera-
ture in 0.215 M sodium phosphate buffer (pH 8.0) at a final concen-
*
carried out at the same level of theory (B3LYP/6-31G ) using
tration of 230
human recombinant AChE (final concentration 2.30
molar ratio 100:1) and AChE in the presence of 2 L of tested inhib-
itor (final concentration = 100 M) were added. Blanks containing
lM. For co-incubation experiments aliquots (16
lL) of
the gussian03 software (Gaussian, Inc. Wallingford, CT). For
the target, fasciculin and all water molecules were removed and
the hydrogen atoms were added using the Biopolymer module
of SYBYL. The ligand binding site was defined as 13 Å from the oxy-
gen of Tyr124 side chain. Docking simulations were carried out
using GOLD 3.0.1.15 As suggested by the GOLD authors,15 genetic
algorithm default parameters were set: the population size was
100, the selection pressure was 1.1, the number of operations
was 105, the number of islands was 5, the niche size was 2, mi-
grate was 10, mutate was 95, and crossover was 95. For each
compound, 100 poses were generated by GOLD and then cluster-
ized by means of AClAP (v. 1.0).16,17 Briefly, AClAP is a newly
developed clustering protocol implemented in a MATLAB meta-
language program, which combines a hierarchical agglomerative
cluster analysis with a clusterability assessment method and a
user independent cutting rule. Several mathematical rules for
both hierarchical tree construction and cutting are implemented
in the algorithm. However, we have demonstrated that, when ap-
plied to docking outcomes, the combination of the average link-
age rule with the cutting function developed by Sutcliffe and
co-workers27 turned out to be an approach that meets all of the
criteria required for a robust clustering protocol.16,17 The poses
reported in Figure 1 are docking configurations representative
of clusters statistically populated according to the Chaevenaut cri-
terion, implemented in the code.16,17 Notably, such poses were
also low energy docking poses according to the GOLD scoring
function.
lM, Ab/AChE
l
l
Ab, AChE, and Ab plus inhibitors, in 0.215 M sodium phosphate
buffer (pH 8.0) were prepared. The final volume of each vial was
20 lL. Each assay was run in duplicate. To quantify amyloid fibril
formation, the thioflavin T (ThT) fluorescence method was then
applied.22–24
Analyses were performed with a Jasco Spectrofluorometer FP-
6200 using a 3 mL quartz cell. After incubation, the samples con-
taining Ab, or Ab plus AChE, or Ab plus AChE in the presence of
inhibitors were diluted with 50 mM glycine-NaOH buffer (pH
8.5) containing 1.5 lM thioflavin T to a final volume of 2.0 mL. A
300s-time scan of the emitted fluorescence (kexc = 446 nm,
kem = 490 nm) was performed and the intensity values at the pla-
teau were averaged after subtracting the background fluorescence
of 1.5 lM thioflavin T and AChE.
The fluorescence intensities in the presence and in the absence
of inhibitor were compared and the percentage of inhibition was
calculated by the following expression: 100 ꢁ (IFi/IFo ꢀ 100) where
IFi and IFo are the fluorescence intensities obtained for Ab plus
AChE in the presence and in the absence of inhibitor,
respectively.22
6.4. Determination of steady state inhibition constant
To obtain estimates of the competitive inhibition constant Ki,
reciprocal plots of 1/v versus 1/[ATCh] were constructed at rela-
tively low concentration of substrate (0.60–0.06 mM) and increas-
ing inhibitor concentrations (range 0–50 nM). The plots were
assessed by a weighted least square analysis that assumed the var-
iance of v to be a constant percentage of v for the entire data set.
Slopes of these reciprocal plots were then plotted against the con-
centration of 22 in a weighted analysis and Ki was determined as
the intersect on the negative x-axis. Data analysis was performed
with GraphPad Prism 4.03 software (GraphPad Software Inc.).
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