Multi-target strategy to address Alzheimer's disease: Design, synthesis and biological evaluation of new tacrine-based dimers

Article abstract of DOI:10.1016/j.ejmech.2011.07.004

The multifactorial nature of Alzheimer's disease (AD) offers us a textbook example where parental compounds, mostly marketed, are modified with the aim of improving and/or conferring two or even more biological activities to contrast or less frequently re

Full text of DOI:10.1016/j.ejmech.2011.07.004

European Journal of Medicinal Chemistry 46 (2011) 4336e4343
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Multi-target strategy to address Alzheimer’s disease: Design, synthesis
and biological evaluation of new tacrine-based dimers
Stefano Rizzo a, Alessandra Bisi b, Manuela Bartolini b, Francesca Mancini b, Federica Belluti b,
,
Silvia Gobbi b, Vincenza Andrisano b, Angela Rampa b
*
a Max-Planck-Institut für Molekulare Physiologie, Abteilung Chemische Biologie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany
b Department of Pharmaceutical Sciences, Alma Mater Studiorum-University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The multifactorial nature of Alzheimer’s disease (AD) offers us a textbook example where parental
compounds, mostly marketed, are modified with the aim of improving and/or conferring two or even
more biological activities to contrast or less frequently revert the disease’s symptoms. This is the case of
tacrine and its dimeric derivative bis(7)-tacrine which, for instance, paved the way for the development
of a broad collection of very interesting homo- and heterodimeric structures, conceived in light of the
emerging multi-target approach for AD-related drug discovery. As a contribution to the topic, we report
here the design, synthesis and biological evaluation of 12 compounds referable to bis(7)-tacrine. In
addition to the cholinesterase activity, some of the selected compounds (7e9 and 12) were capable of
inhibiting the non-enzymatic function of AChE and/or showed a remarkable activity against BACE1. Thus,
the present study outlines a series of newly synthesized molecules, structurally related to bis(7)-tacrine,
endowed with extended biological profile in agreement with the emerging multi-target paradigm.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 10 May 2011
Received in revised form
28 June 2011
Accepted 1 July 2011
Available online 8 July 2011
Keywords:
Alzheimer’s disease
bis(7)-tacrine analogs
Drug design
Heterodimers
Indenoquinoline
1. Introduction
that amyloid polymerization may play a critical role in the neuro-
degenerative process by disrupting the cell’s calcium homeostasis,
Alzheimer’s disease (AD) is an age-related neurodegenerative
disorder of the central nervous system (CNS) causing neuronal
death. As AD advances, a progressive loss of brain’s structure and
functions is observed and a series of symptoms arise which include
confusion, language breakdown, mood swings as well as loss of
memory and bodily functions [1]. The etiopathogenesis of this
multifactorial disease still remains unknown although, in the last
decades, several involved factors have been identified and found
consistent with its onset. Among them is the impairment of the
cholinergic system, as indicated by the presence of altered cholin-
ergic markers in AD patients, resulting in a pronounced acetyl-
choline (ACh) deficiency which translates into a generalized
withdrawal of the cholinergic tone [2,3]. Other involved factors
appear to be the presence of intracellular neurofibrillary tangles
[4,5], containing hyperphosphorylated tau protein, and extracel-
and inducing neuronal apoptosis [7]. The formation of senile pla-
ques involves a cascade of events triggered by the abnormal
catabolism of a transmembrane glycoprotein of undetermined
function known as the amyloid precursor protein (APP), which is
consecutively processed by two proteolytic enzymes, namely the
secretase or beta-site APP cleavage enzyme (BACE1), and the
b
g
-
-
secretase to generate soluble amyloid-beta peptides [8,9]. Mono-
meric A units can organize themselves, either spontaneously or in
b
a pathological chaperone-induced fashion, into aggregates of
increasing complexity, eventually forming neurotoxic senile
plaques.
Most of the currently available therapies on the market are
intended to treat AD by compensating for ACh deficiency so as to
enhance ACh-mediated transmission [10,11]. This strategy consists
in inhibiting cholinesterases (ChEs) [12], a family of enzymes that
catalyze the breakdown of ACh, such as acetylcholinesterase (AChE)
and butyrylcholinesterase (BuChE) [13]. Examples of this ChE
inhibitors are tacrine, donepezil, rivastigmine and galanthamine,
which lead to symptomatic cognitive, functional and behavioral
benefits although none of them has proven effective against the
progression of the disease. Aware of the multifactorial nature of AD,
researchers are nowadays focusing on the development of new
lular senile plaques [6], mostly composed of aggregated
b-amyloid
peptide (A ). Despite the lack of a firmly established causal link
b
between plaques formation and AD, increasing evidences suggest
* Corresponding author. Tel.: þ39 051 2099710; fax: þ39 051 2099734.
E-mail address: angela.rampa@unibo.it (A. Rampa).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.004

S. Rizzo et al. / European Journal of Medicinal Chemistry 46 (2011) 4336e4343
4337
therapeutic strategies that could arrest and revert its progress. This
is the case of the multi-target-directed ligand (MTDL) approach
[14,15], where drugs are designed to address selected activities
toward selected targets involved in the disease. To this regard, a lot
of interest has recently regained the so-called bis(7)-tacrine (Fig. 1),
an heptylene-tethered dimer of tacrine able to simultaneously
contact both the central anionic site and the peripheral anionic site
(PAS), and endowed with AD-related multi-target activity [16,17].
Indeed, besides its cholinesterase function, AChE can also act as
heterocyclic scaffolds, previously reported by our research group,
could allow us to evaluate the impact of structural diversification
upon the
p-p interactions with the PAS. As a first modification to
this second series of molecules, a chlorine atom was introduced at
position 6 of the THA resulting in the tacrine-indenoquinoline
heterodimer 8. This small structural change, as previously shown
[27,28], is thought to enhance the compound’s binding affinity
through favorable interactions with AChE. A further modification of
8 led to compound 9, where an additional chlorine was added at
position 7 of the indenoquinoline ring. In fact, the chloro-
substituted indenoquinoline ring loses the ability of penetrating
the enzymatic gorge of the hAChE, due to steric hindrance [25b],
therefore addressing the 6-chloro-THA moiety toward the central
anionic site of the enzyme. An indenoquinoline homodimer 12 was
prepared as well. In summary, 12 new derivatives were synthesized
and their structures are collected in Fig. 1 and Table 1. All
compounds were tested for their biological activities toward hAChE
and hBuChE. 5 compounds, properly selected, were also tested for
a promoter of Ab fibril formation. This activity is thought to take
place involving the PAS [18a,b] of the enzyme, located at the
entrance of its “catalytic gorge” and characterized by the presence
of the key aminoacid Trp286. Consequently, new dimeric families of
compounds, containing two identical or different structural units,
connected by a linker of suitable length, have been developed, with
tacrine being the active site acetylcholinesterase inhibitor (AChEI)
most extensively incorporated in their structure [19,20]. The
purpose of the homo- and heterodimers is to improve and enlarge
their biological profile beyond their ability to act as AChEI. Our
research group has been involved for many years in the develop-
ment of potential drugs for AD, including the design of dual binding
site AChEI [21]. In this regard, AP2238 was the first published
compound [21a] designed to bind to both anionic sites of the
human AChE (hAChE), for which the simultaneous inhibition of the
their ability to prevent the AChE-induced Ab aggregation, and were
evaluated for their ability to inhibit Ab42 production through the
inhibition of the aspartyl protease BACE1, which catalyzes the rate-
limiting step in the production of Ab.
2. Chemistry
catalytic and the amyloid-b pro-aggregating activities of AChE was
verified. As a follow-up of our studies, we turn here the attention to
bis(7)-tacrine designing dimeric compounds as potential MTDL
(Fig. 1). With this in mind, a 9-amino-1,2,3,4-tetrahydroacridine
(THA) core, for interaction with the central anionic site, and an
The synthesis of the studied compounds was accomplished as
shown in Scheme 1. 9-Chloro-1,2,3,4-tetrahydroacridine [29] was
heated with the selected morpholin-u-alkylamine [23] in phenol
to afford the final compounds 1e5. For the synthesis of derivative
6, 9-amino-1,2,3,4-tetrahydroacridine (THA) x [27] was reacted
with 13 using a phase transfer catalyst in a heterogeneous
medium. Similarly, when THA or 6-Cl-THA were reacted with
14e15 , and 16e17 (16 [26], for meta derivative 17 ) in DMSO,
using KOH as the base, compounds 7e9 and 10e11 were respec-
tively obtained. The intermediate 14 was converted to the desired
homodimer 12 upon bromine displacement with 11H-indeno [1,2-
b]quinolin-10-amine [25a].
amino group, for
p-cation interaction with the PAS, were linked
through alkyl tethers of diverse lengths. While a previous series of
derivatives carrying a diethylamino group was reported in litera-
ture [22] as AChE inhibitors, in the present paper the morpholino
group was chosen since its pKa was expected to improve the
pharmacokinetic properties of the compounds maintaining the
basicity of the inhibitors low enough to allow a sufficient rate of
penetration of the bloodebrain barrier [23]. This series of
compounds (1e5) was followed by a second one (6e12) in which
morpholine was replaced by different moieties, such as azax-
anthone [24], indenoquinoline [25], and benzofuran [26]. These
Table 1
Inhibitory activity on human AChE and BuChE, and IC50 Ratio of the studied
compounds.
1
NH(CH ) NH
R
2 n
N
NH(CH2)7 NH
N
N
NH(CH2)7 NH
N
N
NH(CH )
2 7 NH
N
R
N
12
bis-(7)-tacrine
12
1-11
Comp.
n
R
R1
IC50 (nM) ꢀ SEM
Ratio IC50
BuChE/AChE
R
N
R = H, Cl
n = 6-10
hAChE
hBuChE
101 ꢀ 4
1
2
3
4
5
6
7
8
6
7
8
9
10
7
7
7
7
7
7
e
e
e
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
e
e
e
A
A
A
A
A
B
C
C
D
E
68.9 ꢀ 3.8
44.5 ꢀ 3.1
16.5 ꢀ 0.8
34.0 ꢀ 1.6
28.4 ꢀ 1.8
283 ꢀ 6
1.46
2.03
2.59
1.12
0.52
0.35
1.78
60.7
137
23.5
0.16
0.06
0.20
6.99
O
N
O
90.5 ꢀ 4.0
42.8 ꢀ 1.5
38.1 ꢀ 0.9
14.7 ꢀ 1.5
101 ꢀ 6
NH
(CH2)n
A
1-11
O
F
O
O
1
O
R
13.9 ꢀ 2.1
1.05 ꢀ 0.08
12.1 ꢀ 1.6
310 ꢀ 13
258 ꢀ 6
24.8 ꢀ 0.8
63.7 ꢀ 3.5
1660 ꢀ 80
7280 ꢀ 550
41.4 ꢀ 3.8
28.6 ꢀ 1.5
50 ꢀ 2
N
O
O
E
B
9
NH
NH
10
11
12
THA
F
e
e
e
455 ꢀ 26
250 ꢀ 10
0.81 ꢀ 0.09
Cl
N
N
C
Bis(7)-THAa
5.66 ꢀ 0.15
D
a
Fig. 1. Bis-(7)-tacrine and design of target molecules.
from Ref. [40].

4338
S. Rizzo et al. / European Journal of Medicinal Chemistry 46 (2011) 4336e4343
Cl
N
O
N (CH2)nNH2
N
NH(CH2)n
1-5
N
O
a
O
O
N
HN(CH2)7NH
N
N
N
O(CH2)7NH
N
6
R1
R
7-9
O
b
c
O
13
O(CH2)7Br
N
HN(CH2)7Br
14-15
NH2
N
R = H
1
R1
H2N
N
R
= Cl
c
R
R = H
R = Cl
O(CH2)7Br
c
O
16-17
N
HN(CH2)7NH
N
O(CH2)7NH
N
O
12
10-11
Cl
Scheme 1. Synthesis of the studied compounds. Reagents and conditions: a) PhOH, 130 ꢁC; b) tetra-n-butyl ammonium hydrogen sulfate, 50% NaOH sol./DCM, r.t; c) KOH, DMSO, r.t.
3. Enzyme inhibition
In the second series (compounds 6e12), where morpholine was
replaced by different heterocyclic scaffolds to test the effect on pep
The inhibitory activities against AChE of the newly synthesized
compounds were investigated using the method described by Ell-
man [30]. The selectivity of the compounds was also tested by
determining their inhibitory activities against hBuChE. Some
compounds, rationally selected, were also tested for their abilities to
interactions with the PAS, the chain length was fixed to seven
methylene, as in bis(7)-tacrine. With regard to AChE inhibition,
switching the heterocycle to an azaxanthone (Fig. 1B), and to
a benzofuran (E-F), resulted in new molecules (compounds 6 and
10e11, respectively) with lower activity compared to the positive
reference compound THA, although compound 11 showed a satis-
factory BuChE inhibition (IC50 ¼ 41.4 nM). Much to our delight,
switching the heterocycle to an indenoquinoline (C) and to a 7-Cl-
indenoquinoline (D) afforded the most active heterodimers of the
prevent the hAChE-induced Ab aggregation by a ThT-based fluores-
cence assay. Moreover, in view of enlarging their spectrum of action,
some compounds were evaluated for their capabilities of inhibiting
BACE1, using a fluorescence resonance energy transfer (FRET) assay.
series (compounds 7e9). Remarkably, compound 8,
a THA-
4. Results and discussion
indenoquinoline heterodimer, showed an activity in the low nano-
molar range, comparable to that of the standard bis(7)-tacrine. It
could reasonablybeen assumed that four-ring heterocycles such as C
and D are best suited for stacking interactions, mostly with aromatic
residues at the PAS (i.e. Trp286 of hAChE). Furthermore, the 7-Cl-
indenoquinoline scaffold (D) exerted a noteworthy effect on the
selectivity of the inhibitor, as compound 9 showed a higher inhibi-
tory activity on AChE than on BuChE (ratio IC50 BuChE/AChE ¼ 137,
Table 1). Finally, the indenoquinoline homodimer 12 was a potent
BuChE inhibitorhaving IC50 ¼ 28.6 nM. Considering that in this latter
enzyme, Lys286 and Val288 line the acyl pocket, and that these ami-
noacid side chains are smaller, compared to the side chains of the
corresponding Phe residues of AChE, BuChE could better tolerate the
binding of larger compounds with respect to AChE.
The inhibitory activities against both human recombinant AChE
and BuChE from human serum of new derivatives, together with
those of tacrine and bis(7)-tacrine taken as references, are reported
in Table 1, and are expressed as IC50 values.
With regard to the first series (compounds 1e5), it appears that
variations of the chain length (n in the general formula) had
a stronger influence on BuChE with respect to AChE inhibition.
Indeed, the behavior of this series is rather uniform for AChE
inhibition and the most potent compound is
3
(n
¼
8;
IC50 ¼ 16.5 nM). On the contrary, for BuChE the inhibitory activity
increases with increasing chain length and the most potent
compound is 5 (n ¼ 10; IC50 ¼ 14.7 nM).

S. Rizzo et al. / European Journal of Medicinal Chemistry 46 (2011) 4336e4343
4339
Table 2
Concerning the potential therapeutic interest on BuChEI, it must
Inhibitory activity of AChE-mediated A
selected compounds.
b
aggregation and A
b
production of the
be mentioned that a possible role for BuChE inhibition in contrib-
uting to disease modification has been recently proposed [31].
Furthermore, BuChE activity progressively increases as the severity
of dementia advances, while AChE activity declines. Therefore, the
inhibition of BuChE may become an increasingly important thera-
peutic target over time [13,32].
The ability of new cholinesterase inhibitors to interact with the
AChE peripheral binding site has gained interest in the last two
decades in view of developing a new disease-modifying drug able
to interfere with the pro-aggregating action of AChE on amyloid-
Comp.
Inhibition of
BACE1 inhibitionb
(%)ꢀSEM
BACE1
IC50mM ꢀ SEM
AChE-induced Ab40
aggregationa (%)ꢀSEM
3
7
8
9
27.7 ꢀ 1.5
44.7 ꢀ 7.4
46.1 ꢀ 0.3
30.5 ꢀ 0.3
30.2 ꢀ 5.7
<5c
9.3 ꢀ 2.6
36.1 ꢀ 3.8
78.2 ꢀ 1.4
4.7 ꢀ 1.3
92.8 ꢀ 1.7
n.a.
1.0 ꢀ 0.1
12
THA
Bis-(7)-THA
0.4 ꢀ 0.1
e
7.5 ꢀ 0.4e
68.0 ꢀ 3.5d
e
peptides, likely exerted through the interaction of A
b with PAS
SEM ¼ standard error of the mean; n.a. ¼ not active.
[18a,b]. Therefore, the mechanism of action of the most active
compound in the series was investigated. As shown in Fig. 2,
binding of 8 to hAChE changed both vmax and Km values, a trend
that is generally ascribed to mixed-type inhibition. In particular,
a
Determined at [inhibitor]c ¼ 100
m
mM.
M, [Ab40] ¼ 230
mM and [hAChE] ¼ 2.3
mM.
b
c
Determined at [inhibitor] ¼ 5
From Ref. [38].
d
e
From Ref. [40].
From Ref. [17].
a
decreased vmax at increasing inhibitor concentrations and
increasing intercepts (higher Km) with higher inhibitor concentra-
tion were observed. Ki value for compound
1.01 ꢀ 0.01 nM.
8
resulted
means of hydrophobic interactions with some residues within the
enzyme gorge [27,28], and this is confirmed by the finding that 8
showed AChE inhibiting activity 10 fold higher than 7.
On the basis of these promising results, compound 8 and other
properly selected tacrine-derivatives were also tested for their
ability to prevent AChE-induced Ab aggregation. A rational selec-
tion was made on the basis of the lowest IC50 values obtained in
Ellman’s assay since AChE inhibition remains one of the desired
feature of the new MTDLs. Furthermore, it must be considered that
With regard to BACE1 inhibition, compound 3, the most potent
AChE inhibitor of the first series, was shown to be ineffective, as
well as compound 9, a THA-7-Cl-indenoquinoline dimer.
All the other compounds tested (7e8, 12) were found more
active than the standard bis(7)-tacrine, which displayed a 27%
AChE inhibition and AChE-induced b-amyloid aggregation do not
inhibition when tested at 10
mM. This figure is consistent with the
represent independent phenomena, but can both be modulated by
inhibitor binding to PAS. The ability of interfering with the trig-
gering activity of AChE on amyloid aggregation, expressed as %
inhibition, is reported in Table 2. Compound 3, carrying a morpho-
lino group, showed to act as a weak but significant inhibitor of
previously published IC50 value of 7.5
m
M [17]. The most potent
compounds were 8 and 12, which inhibited enzyme activity by 78
and 93%, respectively, while the indenoquinoline fragment alone
was inactive (data not shown).
It is noteworthy that the presence of a chlorine atom on the
indenoquinoline nucleus leads to a complete loss in activity
(compare compounds 8, which has no chlorine, and 9, which
carries a Cl atom at position 7, Table 2). For 8 and 12, which dis-
AChE-induced A
b
aggregation (% inhibition ¼ 27.7). With the
introduction of indenoquinoline (C, compounds 7e8) or 7-Cl-
indenoquinoline (D, compound 9), the compounds showed
a significant increase of the ability to inhibit the Ab aggregation
played at 5
corresponding IC50 values were also calculated. 12 exhibited
a considerable activity with a submicromolar IC50 value of 0.4
and it was found to be more potent than 8 (IC50 value of 1.0
mM a percentage of inhibition higher than 50%, the
induced by AChE. The most potent agents are 7 and 8, heterodimers
of a THA or a 6-Cl-THA nucleus with indenoquinoline. As expected,
the compounds showed the same ability to block the AChE-induced
mM
mM).
A
b aggregation, as they only differ for a chlorine atom on the
Thus, the indenoquinoline fragment was found to be the most
suitable substituent to obtain a submicromolar BACE1 inhibition.
It has to be noted that these compounds show a lower potency
toward BACE1 compared to AChE and BuChE inhibition. Neverthe-
less, it is known that BACE1 is a more challenging target and our
compounds are in line with non peptidic inhibitors reported in
literature showing activity in the micromolar range, among these the
non competitive inhibitor bis(7)-tacrine [17], myricetin [33] or other
tacrine-based dual binding site acetylcholinesterase inhibitors [34].
Interestingly, recent studies have determined that BACE1 levels
and activity are approximately twofold elevated in AD brain, sug-
gesting the possibility that BACE1 increase might initiate or accel-
erate AD pathogenesis [35]. Moreover, it has recently been
nucleus of tacrine. Indeed, the chlorine atom in position 6 of the
THA core is thought to enhance affinity toward AChE, possibly by
demonstrated that bis(7)-tacrine may reduce the generation of A
b
by inhibiting BACE1 and simultaneously activating -secretase
a
activity, the enzyme responsible for the physiological cleavage of
APP, through the direct activation of protein kinase C [36]. From the
results obtained for BACE1 inhibition and due to the structural
similarity with bis(7)-tacrine, a comparable antiamyloidogenic
profile could also be envisaged for derivatives 8 and 12.
5. Conclusions
Fig. 2. Steady-state inhibition by
8 of the hydrolysis of acetylthiocholine (ATCh)
catalyzed by hAChE. LineweavereBurk reciprocal plots of initial velocity vs substrate
concentrations in the presence and absence of 8 (three concentrations) are presented.
Lines were derived from a weighted least-squares analysis of the data points.
In conclusion, we report the design, synthesis and biological
evaluation of 12 novel homo- and heterodimer molecules referable

4340
S. Rizzo et al. / European Journal of Medicinal Chemistry 46 (2011) 4336e4343
to bis(7)-tacrine-derivatives. The potential of these analogs as
gateway molecules’ for the development of multi-target drugs was
demonstrated by evaluating their biological profile toward a series
6.1.2. (7-Morpholin-4-yl-heptyl)-(1,2,3,4-tetrahydroacridin-9-yl)
amine (2)
Starting from 7-morpholin-4-yl-heptylamine and following the
of AD-related targets such as cholinesterases, A
b
aggregation and
previous procedure, 2 was obtained as a brownish oil. 1H NMR
d :
BACE1. In agreement with the trend observed for bis(7)-tacrine, the
majority of the newly synthesized derivatives were found to be
active in the nanomolar range against AChE. In addition to the
cholinesterase activity, some of the selected compounds (7e9, and
12) were capable of inhibiting the non-enzymatic function of AChE
and/or showed a remarkable activity against BACE1, with a slightly
(compound 7) or significantly (compounds 8 and 12) higher
potency compared to bis(7)-tacrine. Thus, our study outlines
a series of newly synthesized molecules, structurally related to
bis(7)-tacrine, endowed with extended biological profile. Particu-
larly interesting proved to be compound 8, which resulted the most
potent AChE inhibitor of the series, equaling the activity of bis(7)-
tacrine, and showed a good activity against BuChE, useful in
1.20e1.50 (m, 8H), 1.60e1.78 (m, 2H), 1.85e1.99 (m, 4H), 2.30 (t, 2H,
J ¼ 18 Hz), 2.39e2.48 (m, 4H), 2.65e2.75 (m, 2H), 3.01e3.17 (m, 2H),
3.50(t, 2H, J¼ 18Hz), 3.65e3.79(m, 4H), 7.30e7.40(m,1H),7.58(t,1H,
J ¼ 18 Hz), 7.88e7.97 (m, 2H). 13C NMR
d : 21.4, 22.6, 24.0, 26.0, 26.7,
27.1, 29.0, 31.2, 32.8, 49.1, 53.7, 58.9, 66.5, 115.1, 120.1, 122.7, 123.8,
127.9,129.1,139.8,151.5,156.9. MS (ES) m/z: 382 (M þ Hþ). Anal. calcd
for C24H35N3O: C 75.55, H 9.25, N 11.01, found:C 75.48, H 9.23, N 11.08.
6.1.3. (8-Morpholin-4-yloctyl)-(1,2,3,4-tetrahydroacridin-9-yl)
amine (3)
Starting from 8-morpholin-4-yloctylamine and following the
previous procedure, 3 was obtained as a brownish oil. 1H NMR
d :
1.20e1.58 (m, 10H), 1.60e1.75 (m, 2H), 1.80e1.99 (m, 4H), 2.35 (t,
2H, J ¼ 18 Hz), 2.39e2.48 (m, 4H), 2.65e2.78 (m, 2H), 3.05e3.17 (m,
2H), 3.52 (t, 2H, J ¼ 18 Hz), 3.68e3.79 (m, 4H), 7.35 (t, 1H, J ¼ 18 Hz),
advanced stages of the disease. Even if only a slight reduction of A
b
aggregation induced by AChE was observed with 8, it also proved to
be remarkably more potent than the reference compound as BACE1
inhibitor, which may represent the most direct approach in anti-
amyloid therapy.
Moreover, our selected compounds 7e9 and 12 could serve as
a platform for further studies aimed at fine-tuning their multifac-
eted activities for the identification of new molecular entities, in
which the activities on the different targets would be appropriately
balanced, endowed with broader biological profile in awareness of
the emerging multi-target paradigm for drug discovery.
7.58 (t, 1H, J ¼ 18 Hz), 7.80e7.88 (m, 2H). 13C NMR
d: 22.1, 22.8, 24.4,
26.3, 26.7, 27.5, 29.1, 29.3, 31.3, 32.7, 49.2, 53.6, 58.9, 66.4, 115.0,
118.5, 121.9, 123.8, 128.1, 129.1, 139.3, 151.6, 157.3. MS (ES) m/z: 396
(M þ Hþ). Anal. calcd for C25H37N3O: C 75.91, H 9.43, N 10.62, found:
C 75.88, H 9.40, N 10.68.
6.1.4. (9-Morpholin-4-ylnonyl)-(1,2,3,4-tetrahydroacridin-9-yl)
amine (4)
Starting from 9-morpholin-4-ylnonylamine and following the
previous procedure, 4 was obtained as a brownish oil. 1H NMR
d:
1.20e1.52 (m, 12H), 1.60e1.75 (m, 2H), 1.84e1.98 (m, 4H), 2.30 (t,
2H, J ¼ 19 Hz), 2.39e2.48 (m, 4H), 2.65e2.78 (m, 2H), 3.00e3.11 (m,
2H), 3.48 (t, 2H, J ¼ 19 Hz), 3.65e3.79 (m, 4H), 7.35 (t, 1H, J ¼ 18 Hz),
6. Experimental section
6.1. Chemistry. General methods
7.55 (t, 1H, J ¼ 18 Hz), 7.91e7.99 (m, 2H). 13C NMR
d : 21.9, 22.6, 24.8,
26.3, 26.7, 27.1, 28.8, 29.1, 29.3, 31.6, 32.6, 49.5, 53.3, 58.9, 66.5,
115.0, 119.5, 122.6, 123.8, 127.9, 128.9, 139.4, 151.5, 157.2. MS (ES) m/
z: 410 (M þ Hþ). Anal. calcd for C26H39N3O: C 76.24, H 9.60, N 10.26,
found: C 76.29, H 9.59, N 10.28.
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/
400 MHz spectrometer. Chemical shifts are reported in parts per
million (ppm) relative to tetramethylsilane (TMS), and multiplici-
ties 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,
0.040e0.063 mm; Merck) by flash chromatography. Compounds’
names were obtained using AUTONOM, PC software for nomen-
clature in organic chemistry, Beilstein-Institut and Springer.
6.1.5. (10-Morpholin-4-yldecyl)-(1,2,3,4-tetrahydroacridin-9-yl)
amine (5)
Starting from 10-morpholin-4-yldecylamine and following the
previous procedure, 5 was obtained as a brownish oil. 1H NMR
d:
1.20e1.58 (m, 14H), 1.60e1.75 (m, 2H), 1.85e1.99 (m, 4H), 2.30 (t,
2H, J ¼ 18 Hz), 2.39e2.45 (m, 4H), 2.68e2.75 (m, 2H), 3.02e3.12 (m,
2H), 3.50 (t, 2H, J ¼ 18 Hz), 3.65e3.79 (m, 4H), 7.35 (t, 1H, J ¼ 18 Hz),
7.58 (t, 1H, J ¼ 18 Hz), 7.90e8.05 (m, 2H). 13C NMR
d: 20.7, 21.9, 23.7,
25.9, 26.6, 27.3, 28.6, 29.1, 29.3, 29.3, 31.1, 48.6, 53.4, 58.9, 66.4,
110.9, 116.0, 121.3, 124.2, 125.0, 132.0, 139.3, 151.7, 155.2. MS (ES) m/
z: 424 (M þ Hþ). Anal. calcd for C27H41N3O: C 76.55, H 9.76, N 9.92,
found: C 76.49, H 9.79, N 9.94.
6.1.1. (6-Morpholin-4-ylhexyl)-(1,2,3,4-tetrahydroacridin-9-yl)
amine (1)
A stirred suspension of 9-chloro-1,2,3,4-tetrahydroacridine [29]
(0.35 g, 2.76 mmol) and phenol (1.50 mL) was heated at 85e90 ꢁC
until a solution was obtained. 6-Morpholin-4-ylhexylamine [23]
(0.6 g, 5.53 mmol) was then added and the reaction mixture
was heated to 130 ꢁC for 4 h. The crude was washed with NaOH
2 N solution and extracted with EtOAc, dried over Na2SO4 and
6.1.6. 7-[7-(1,2,3,4-Tetrahydroacridin-9-ylamino)heptyloxy]-9-oxa-
1-azaanthracen-10-one (6)
A
mixture of 9-amino-1,2,3,4-tetrahydroacridine (0.078 g,
0.39 mmol), 13 (0.31 g, 0.79 mmol) and a catalytic amount of tetra-
n-butyl ammonium hydrogensulphate (0.0195 g) was stirred at
room temperature for 4 h in a biphasic mixture composed of DCM/
NaOH 50% aq. sol. (7.5 mL/5 mL). The organic layer was separated
and washed with water, then dried and evaporated. The crude was
purified by flash chromatography (DCM/MeOH 97:3) affording 6 as
evaporated.
Flash
chromatography
(DCM/MeOH/NH4OH
70:30:0.7) afforded 1 as a brownish oil (0.26 g, 26%). 1H NMR
d:
1.20e1.58 (m, 6H), 1.60e1.75 (m, 2H), 1.80e1.99 (m, 4H), 2.30 (t,
2H, J ¼ 18 Hz), 2.39e2.45 (m, 4H), 2.60e2.68 (m, 2H), 2.98e3.17
(m, 2H), 3.50 (t, 2H, J ¼ 18 Hz), 3.65e3.79 (m, 4H), 7.35 (t, 1H,
J ¼ 18 Hz), 7.55 (t, 1H, J ¼ 18 Hz), 7.85e7.90 (m, 2H). 13C NMR
d
:
a brownish oil (0.015 g, 7.6%). 1H NMR
d:1.42e1.65 (m, 6H),
22.4, 22.8, 24.5, 26.3, 26.7, 27.1, 31.6, 32.9, 49.2, 53.7, 58.9, 66.9,
114.9, 119.4, 122.5, 123.8, 127.9, 128.9, 139.3, 151.4, 157.1. MS (ES) m/
z: 368 (M þ Hþ). Anal. calcd for C23H33N3O: C 75.16, H 9.05, N
11.43, found: C 75.28, H 9.03, N 11.48.
1.77e2.05 (m, 8H), 2.60 (t, 2H, J ¼ 16 Hz), 3.30 (t, 2H, J ¼ 16 Hz), 3.90
(t, 2H, J ¼ 16 Hz), 4.15 (t, 2H, J ¼ 18 Hz), 5.80 (br, 1H), 7.05e7.71 (m,
8H), 8.00e8.17 (m, 2H). 13C NMR
d: 23.1, 26.6, 27.7, 29.9, 30.6, 31.8,
31.9, 34.7, 52.3, 72.3, 106.9, 110.2, 115.7 (2C), 117.4, 118.1, 119.3, 121.1,

S. Rizzo et al. / European Journal of Medicinal Chemistry 46 (2011) 4336e4343
4341
124.2, 127.7, 128.4, 131.1, 140.7, 146.7, 147.6, 151.5, 155.5, 155.9, 163.7,
164.8. ES-MS m/z: 508 (M þ Hþ). Anal. calcd for C32H33N3O3: C
75.71, H 6.55, N 8.28, found: C 75.69, H 6.52, N 8.24.
CeliteÒ and the filtrate diluted with water (300 mL), extracted with
EtOAc (3 25 mL), anhydrified over Na2SO4 and concentrated to
dryness giving 0.26 g of crude. Flash chromatography (DCM/MeOH
98:2) afforded 10 as a yellowish oil (0.18 g, 51%). 1H NMR
d:
6.1.7. N-(11H-Indeno[1,2-b]quinolin-10-yl)-N’-(1,2,3,4-
tetrahydroacridin-9-yl)heptane-1,7-diamine (7)
Following the previous procedure and starting from 14 (0.1 g,
0.24 mmol) and 9-amino-1,2,3,4-tetrahydroacridine (0.048 g,
0.24 mmol), 7 was obtained as a pale brown oil (0.03 g, 24%). 1H
1.28e1.52 (m, 6H), 1.54e1.92 (m, 8H), 2.52e2.68 (m, 2H), 2.92e3.12
(m, 2H), 3.41 (t, 2H, J ¼ 18 Hz), 3.92 (t, 2H, J ¼ 18 Hz), 5.24 (s, 1H),
6.82 (s, 1H), 6.90 (d, 2H, J 8.8 Hz), 7.18e7.25 (m, 3H), 7.45e7.49 (m,
2H), 7.73 (d, 2H, J 8.8 Hz,), 7.86 (d, 2H, J 10.6 Hz). 13C NMR (CDCl3)
d:
22.5, 22.7, 24.4, 25.8, 26.7, 28.9, 31.5, 33.9, 40.8, 49.3, 67.7, 99.4,
110.8, 114.6, 115.5, 118.2, 120.4, 122.7, 122.9, 123.5, 123.9, 124.5,
126.2, 127.4, 129.3, 133.7, 148.0, 150.6, 150.8, 154.5, 155.9, 159.3. ES-
MS m/z: 539 (M þ Hþ). Anal. calcd for C34H35ClN2O2: C 75.75, H
6.54, N 5.20, found: C 75.79, H 6.56, N 5.18.
NMR d: 1.20e1.60 (m, 8H), 1.62e1.97 (m, 6H), 2.59e2.67 (m, 2H),
3.02e3.18 (m, 2H), 3.60 (t, 2H, J ¼ 12 Hz), 3.80 (t, 2H, J ¼ 12 Hz), 4.08
(s, 2H), 5.60 (br, 1H), 7.20e7.60 (m, 7H), 7.85e8.17 (m, 4H),
8.32e8.41 (m, 1H). 13C NMR
d: 21.4, 21.7, 23.8, 26.1, 28.1, 27.9, 29.9,
30.1, 31.3, 35.8, 44.1, 53.7, 61.1, 111.6, 111.9, 118.0, 119.0, 120.2, 120.8,
125.3, 127.8, 128.1, 128.9, 129.5, 130.2, 130.3, 132.1, 138.9, 145.8,
150.9, 156.2, 158.1, 161.8. ES-MS m/z: 527 (M þ Hþ). Anal. calcd for
C36H38N4: C 82.09, H 7.27, N 10.64, found: C 82.19, H 7.28, N 10.61.
6.1.12. [7-(3-Benzofuran-2-yl-phenoxy)heptyl]-(6-chloro-1,2,3,4-
tetrahydroacridin-9-yl)amine (11)
Following the previous procedure and starting from 6-chloro-9-
amino-1,2,3,4-tetrahydroacridine (0.15 g, 0.65 mmol) and 17 (0.3 g,
0.78 mmol),11 was obtained as a yellowish oil (0.05 g,14%). 1H NMR
6.1.8. N-(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)-N’-(11H-indeno
[1,2-b]quinolin-10-yl)heptane-1,7-diamine (8)
d: 1.38e1.58 (m, 6H), 1.60e1.93 (m, 8H), 2.60e2.65 (m, 2H),
Following the previous procedure and starting from 14 (0.1 g,
2.98e3.10 (m, 2H), 3.48 (t, 2H, J ¼ 18 Hz), 4.03 (t, 2H, J ¼ 18 Hz), 5.28
0.24 mmol)
(0.055 g, 0.24 mmol), 8 was obtained as a pale brown oil (0.07 g,
51%). 1H NMR
: 1.27e1.58 (m, 6H), 1.59e1.80 (m, 4H), 1.81e1.96 (m,
and
6-chloro-9-amino-1,2,3,4-tetrahydroacridine
(s, 1H), 6.81e6.92 (m, 1H), 7.00 (s, 1H), 7.21e7.59 (m, 8H), 7.87e7.91
(m, 2H). 13C NMR (CDCl3)
d: 22.4, 22.8, 24.4, 25.9, 26.8, 29.0, 29.1,
d
29.6, 31.6, 33.6, 49.4, 67.7,101.6,110.8,113.9, 114.8, 114.9,115.3,117.4,
120.9,122.9,124.3,124.6,124.7,126.9,129.1,129.8,131.7,134.3,151.0,
154.8, 155.7, 158.9, 159.4. ES-MS m/z: 539 (M Hþ). Anal. calcd for
C34H35ClN2O2: C 75.75, H 6.54, N 5.20, found: C 75.77, H 6.53, N 5.19.
4H), 2.57e2.65 (m, 2H), 3.00e3.11 (m, 2H), 3.55 (t, 2H, J ¼ 17 Hz),
3.70 (t, 2H, J ¼ 17 Hz), 3.98 (s, 2H), 7.20e7.60 (m, 6H), 7.85e8.17 (m,
4H), 8.32e8.41 (m, 1H). 13C NMR
d: 21.4, 21.7, 23.7, 26.1, 28.1, 27.9,
29.9, 30.1, 31.3, 35.8, 44.1, 53.7, 61.1, 111.6, 111.8, 118.0, 120.2, 120.5,
120.8,126.1,128.0,128.9,129.5,130.2,130.3,132.1,134.1,138.9,146.7,
150.9, 156.2, 158.1, 162.4. ES-MS m/z: 561 (M þ Hþ). Anal. calcd for
C36H37ClN4: C 77.05, H 6.65, N 9.98, found: C 77.09, H 6.67, N 9.96.
6.1.13. 7-(7-Bromoheptyloxy)-9-oxa-1-azaanthracen-10-one (13)
A stirred suspension of 7-hydroxy-9-azaanthracen-10-one [24]
(0.8 g, 3.7 mmol), 1,7-dibromoheptane (1.2 mL, 7.5 mmol) and
K2CO3 (0.97 mg) in acetone (30 mL) was refluxed for 20 h. The reac-
tion mixture was hot filtered and the filtrate evaporated. The residue
was purified by flash chromatography (toluene/acetone 90:10),
6.1.9. N-(7-Chloro-11H-indeno[1,2-b]quinolin-10-yl)-N’-(3-chloro-
1,2,3,4-tetrahydroacridin-9-yl)heptane-1,7-diamine (9)
Following the previous procedure and starting from 15 (0.15 g,
affording 13 as an oil (0.65 g, 45%). 1H NMR (CDCl3):
d 1.35e1.57 (m,
0.34 mmol)
(0.078 g, 0.34 mmol), 9 was obtained as a pale brown oil (0.03 g,
15%). 1H NMR
: 1.27e1.58 (m, 6H), 1.59e1.80 (m, 4H), 1.81e1.96 (m,
and
6-chloro-9-amino-1,2,3,4-tetrahydroacridine
6H), 1.79e1.97 (m, 4H), 3.43 (t, 2H), 4.10 (t, 2H), 6.91e7.03 (m, 2H),
7.39e7.45 (m, 1H), 8.16e8.23 (m, 1H), 8.60e8.75 (m, 2H).
d
4H), 2.57e2.65 (m, 2H), 3.00e3.11 (m, 2H), 3.55 (t, 2H, J ¼ 18 Hz),
6.1.14. (7-Bromoheptyl)-(11H-indeno [1,2-b]quinolin-10-yl)amine
(14)
3.70 (t, 2H, J ¼ 18 Hz), 3.98 (s, 2H), 7.20e7.60 (m, 6H), 7.85e8.17 (m,
4H). 13C NMR
d: 21.4, 21.7, 23.8, 26.1, 28.1, 28.0, 29.9, 30.1, 31.3, 35.8,
A suspension of 11H-indeno [1,2-b]quinolin-10-ylamine [25a]
(0.65 g, 2.8 mmol), 1,7-dibromoheptane (0.57 mL, 3.4 mmol) and
KOH (0.25 g, 4.5 mmol) in dry DMSO (10 mL) was stirred for 16 at
room temperature under nitrogen in the presence of freshly acti-
vated 4 Å molecular sieves (100 mg). The mixture was filtered
through CeliteÒ and the filtrate diluted with water (250 mL),
extracted with EtOAc (3 x 30 mL), dried over Na2SO4 and the
solvent was evaporated. Flash chromatography (DCM/MeOH 98:2)
afforded 0.11 g of 14 as a yellowish oil (10%). 1H NMR (CDCl3):
44.1, 53.7, 61.1, 111.6, 111.9, 118.7, 119.9120.5, 120.8, 127.9, 129.1,
129.9, 130.2, 130.3, 132.1, 132.3, 134.1, 138.1, 146.7, 150.9, 156.2, 158.1,
162.4. ES-MS m/z: 595 (M þ Hþ). Anal. calcd for C36H36Cl2N4: C
72.60, H 6.09, N 9.41, found: C 72.59, H 6.08, N 9.41.
6.1.10. N,N’-Bis-(11H-indeno[1,2-b]quinolin-10-yl)heptane-1,7-
diamine (12)
Following the previous procedure and starting from 14 (0.1 g,
0.24 mmol) and 11H-indeno [1,2-b]quinolin-10-ylamine (0.056 g,
0.24 mmol), 12 was obtained as a pale brown oil (0.05 g, 37%). 1H
d
1.38e1.60 (m, 6H), 1.62e1.83 (m, 4H), 3.60e3.78 (m, 4H), 4.12 (s,
2H), 7.40e7.61 (m, 4H), 7.75 (t, 1H), 8.16e8.29 (m, 3H).
NMR
4.11 (s, 2H), 4.80 (br, 2H), 7.05e7.71 (m, 12H), 8.00e8.17 (m, 2H),
8.20e8.32 (m, 2H). 13C NMR
: 26.1, 28.1, 30.1, 35.8, 44.1, 53.7, 61.1,
d: 1.35e1.59 (m, 6H), 1.60e1.77 (m, 4H), 3.59e3.72 (m, 4H),
6.1.15. (7-Bromoheptyl)-(7-chloro-11H-indeno [1,2-b]quinolin-10-
yl)amine (15)
d
111.9,119.0,120.8,123.9,128.1,129.5,130.3,138.9,145.8,147.4,150.9,
158.7, 159.8. ES-MS m/z: 561 (M þ Hþ). Anal. calcd for C39H36N4: C
83.54, H 6.47, N 9.99, found: C 83.59, H 6.48, N 10.01.
Following the previous procedure and starting from 7-chloro-
11H-indeno [1,2-b]quinolin-10-ylamine [25b] (0.5 g, 1.88 mmol), 15
was obtained as a brownish oil (0.18 g, 22%). 1H NMR (CDCl3):
d
1.37e1.59 (m, 6H), 1.63e1.85 (m, 4H), 3.58e3.76 (m, 4H), 4.11 (s,
6.1.11. [7-(4-Benzofuran-2-yl-phenoxy)heptyl]-(6-chloro-1,2,3,4-
2H), 7.42e7.63 (m, 4H), 7.77 (t, 1H), 8.15e8.32 (m, 2H).
tetrahydroacridin-9-yl)amine (10)
A
solution of 6-chloro-9-amino-1,2,3,4-tetrahydroacridine
6.1.16. Synthesis of 3-Benzofuran-2-yl-phenol: 2-(3-
Methoxyphenyl)benzofuran
(0.15 g, 0.65 mmol), 16 [25] (0.3 g, 0.78 mmol) and KOH (0.06 g,
1.04 mmol) in dry DMSO (8 mL) was stirred for 16 at room
temperature under nitrogen in the presence of freshly activated 4 Å
molecular sieves (100 mg). The mixture was filtered through
A
stirred suspension of 3-methoxybenzoylchloride (5 g,
29 mmol), 2-hydroxybenzyltriphenyl phosphonium bromide (12 g,
27 mmol) and Et3N (11.1 mL, 80 mmol) in toluene (125 mL) was

4342
S. Rizzo et al. / European Journal of Medicinal Chemistry 46 (2011) 4336e4343
refluxed for 10 . The reaction mixture was filtered under vacuum
and evaporated to dryness. EtOH (150 mL) was added to the oily
residue and the flask kept in the freezer overnight. A yellowish solid
formed which was filtered off and purified by flash chromatog-
raphy on silica gel (petroleum ether/ethyl acetate 95:5), affording
temperature in 0.215 M sodium phosphate buffer (pH 8.0) at a final
concentration of 230 M. For co-incubation experiments aliquots
(16 L) of human recombinant AChE (final concentration 2.30 M,
/AChE molar ratio 100:1) and AChE in the presence of 2 L of
tested inhibitor (final concentration ¼ 100 M) were added. Blanks
containing A , AChE, and A plus inhibitors, in 0.215 M sodium
phosphate buffer (pH 8.0) were prepared. The final volume of each
vial was 20 L. Each assay was run in duplicate. To quantify amyloid
m
m
m
Ab
m
m
the compound as a colorless oil (3.58 g, 55%). 1H NMR (CDCl3)
d:
b
b
3.87(s,3H), 6.82e6.95 (m, 1H), 7.00 (s, 1H), 7.18e7.60 (m, 6H). 3-
Benzofuran-2-yl-phenol. To
a
cold (0 ꢁC) solution of 2-(3-
m
methoxyphenyl)benzofuran (3.0 g, 13 mmol) in anhydrous DCM
(300 mL), BBr3 (18 mL of 1 M solution in DCM) was added slowly.
The resulting mixture was stirred overnight at room temperature.
The reaction was quenched with ice/water and stirred for 30 min.
The organic layer was washed with water (3 x 50 mL), then with
brine (3 x 25 mL) and dried over Na2SO4. The solvent was removed
affording the compound (2.65 g, 97%) as a white solid, mp 123 ꢁC 1H
fibril formation, the thioflavin T (ThT)-based fluorescence method
was applied [37e39].
After incubation, the samples containing A
plus AChE in the presence of inhibitors were diluted with
50 mM glycine-NaOH buffer (pH 8.5) containing 1.5 M thioflavin T
b, or Ab plus AChE, or
Ab
m
to a final volume of 2.0 mL. A 300 s-time scan of the emitted
fluorescence (lexc ¼ 446 nm, lem ¼ 490 nm) was performed and the
intensity values at the plateau were averaged after subtracting the
NMR (CDCl3) d: 4.92 (br, 1H), 6.83e6.97 (m, 3H), 7.18e7.28 (m, 2H),
7.43e7.58 (m, 2H), 7.70e7.79 (m, 2H).
background fluorescence of 1.5 mM thioflavin T and AChE.
The fluorescence intensities in the presence and in the
absence of inhibitor were compared and the percentage of inhi-
bition was calculated by comparing the fluorescence intensities
6.1.17. 2-[3-(7-Bromoheptyloxy)phenyl]benzofuran (17)
A
stirred mixture of 3-benzofuran-2-yl-phenol (1.00 g,
4.7 mmol), 1,7-dibromoheptane (0.57 mL, 3.3 mmol) and K2CO3
(1.2 g) was refluxed in acetone (100 mL) for 20 . The suspension was
hot filtered and the solvent was removed. After adding petroleum
ether, the residue was kept in the freezer overnight and the white
solid that formed was filtered off, affording 17, mp 145 ꢁC 1H NMR
obtained for A
inhibitor [37].
b plus AChE in the presence and in the absence of
6.2.4. Inhibition of BACE1
Purified Baculovirus-expressed BACE1 (b-secretase) in 50 mM
(CDCl3)
d
: 1.23e1.58 (m, 6H), 1.69e1.98 (m, 4H), 3.40 (t, 2H), 4.02 (t,
Tris (pH 7.5), 10% glycerol and rhodamine derivative substrate
(Panvera peptide) were purchased from Invitrogen (Milan, Italy).
Sodium acetate and DMSO were obtained from SigmaeAldrich
(Milan, Italy). Purified water from Milli-RX system (Millipore,
Milford, USA) was used to prepare buffers and standard solutions.
Spectrofluorometric analyses were carried out on a Fluoroskan
Ascent multiwell spectrofluorometer (excitation: 544 nm; emis-
sion: 590 nm) using black microwell (96 wells) Corning plates
(SigmaeAldrich, Milan, Italy). Stock solutions of the test
compounds were prepared in DMSO and diluted with 50 mM
2H), 6.89 (s, 1H), 6.87e6.95 (m, 2H), 7.24e7.31 (m, 2H), 7.50e7.56
(m, 2H), 7.717.83 (m, 2H).
6.2. Biology
6.2.1. Inhibition of AChE and BuChE
The method of Ellman et al. was followed [30]. Five different
concentrations 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
nitrobenzoic acid), 0.02 unit/mL of human recombinant AChE or
BuChE derived from human serum (Sigma Chemical), and 550
of substrate (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
account for non-enzymatic reaction. The reaction rates were
compared, 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,
GraphPad Prism 4.0, GraphPad Software Inc).
sodium acetate buffer (pH 4.5). Specifically, 20
enzyme (11.7 nM, final concentration) were incubated with 20
of test compound for 60 min. To start the reaction, 20 L of Pan-
vera peptide (0.25 M, final concentration) were added to each
well. The mixture was incubated at 37 ꢁC for 60 min. To stop the
reaction, 20 L of BACE1 stop solution (sodium acetate, 2.5 M)
mL of BACE1
m
M 5,50-dithio-bis(2-
mL
m
mM
m
m
were added to each well. The spectrofluorometric assay was
subsequently performed by reading the fluorescence signal at
590 nm. The DMSO concentration in the final mixture was main-
tained below 5% (v/v) to guarantee no significant loss of enzyme
activity. The fluorescence intensities with and without inhibitor
were compared, and the percent inhibition due to the presence of
test compounds was calculated. The background signal was
measured in control wells containing all the reagents except
BACE1 and subtracted. The inhibition (%) due to the presence of
five or six increasing concentrations of test compound was
calculated by the following expression: 100-(IFi/IFo ꢃ 100) where
IFi and IFo are the fluorescence intensities obtained for BACE1 in
the presence and in the absence of inhibitor, respectively. Inhibi-
tion curves were obtained by plotting the percent inhibition
versus the logarithm of inhibitor concentration in the assay
sample, when possible. The linear regression parameters were
determined and the IC50 value extrapolated (GraphPad Prism 4.0,
GraphPad Software Inc.). To demonstrate inhibition of BACE1
6.2.2. Determination of steady-state inhibition constant
To obtain graphical estimates of the mechanism of inhibition,
reciprocal plots of 1/V versus 1/[S] were constructed in the pres-
ence of 3 different concentrations of 8 ranging from 0.143 to
0.937 nM and increasing concentration of substrate (below
0.56 mM). The plots were assessed by a weighted least square
analysis that assumed the variance of v to be a constant percentage
of v for the entire data set. Data analysis was performed with
GraphPad Prism 4.03 software (GraphPad Software Inc.).
activity, a peptidomimetic inhibitor (
biochem, Merck; Nottingham, UK) was serially diluted into the
M).
b-secretase inhibitor IV, Cal-
6.2.3. Inhibition of AChE-induced amyloid-
b
peptide aggregation
reaction wells (IC50 ¼ 0.013
m
Aliquots of 2
mL Ab40 peptide (Bachem AG, Germany), lyophi-
Additional measurements were performed for bis(7)-tacrine, 8
and 12 in the presence of a detergent (CHAPS, 0.1% w/v) to check for
non specific effects.
lized from a 2 mg mLꢂ1 HFIP (1,1,1,3,3,3-hexafluoro-2-propanol)
solution and dissolved in DMSO, were incubated for 24 h at room

S. Rizzo et al. / European Journal of Medicinal Chemistry 46 (2011) 4336e4343
4343
M. Recanatini, V. Andrisano, A. Rampa, Bioorg. Med. Chem. 18 (2010)
1749e1760.
References
[22] P.R. Carlier, E.S.H. Chow, Y. Han, J. Liu, J. El Yazal, Y.-P. Pang, J. Med. Chem. 42
(1999) 4225e4231.
[1] M. Goedert, M.G. Spillantini, Science 314 (2006) 777e781.
[2] A.V. Terry Jr., J.J. Buccafusco, J. Pharmacol. Exp. Ther. 306 (2003) 821e827.
[3] R.T. Bartus, Exp. Neurol. 163 (2000) 495e529.
[4] L. Buée, T. Bussière, V. Buée-Scherrer, A. Delacourte, P.R. Hof, Brain Res. Rev. 33
(2000) 95e130.
[5] M. Tolnay, A. Probst, Neuropathol. Appl. Neurobiol. 25 (1999) 171e187.
[6] J. Hardy, D.J. Selkoe, Science 297 (2002) 353e356.
[7] P.J. Harrison, J.R. Soc, Med 79 (1986) 347e352.
[23] M.A. Alisi, M. Brufani, L. Filocamo, G. Gostoli, E. Licandro, M.C. Cesta, S. Lappa,
D. Marcherini, P. Pagella, Bioorg. Med. Chem. Lett. 5 (1995) 2077e2080.
[24] A. Rampa, A. Bisi, P. Valenti, M. Recanatini, A. Cavalli, V. Andrisano, V. Cavrini,
L. Fin, A. Buriani, P. Giusti, J. Med, Chem 41 (1998) 3976e3986.
[25] a) P. Valenti, A. Rampa, A. Bisi, V. Andrisano, V. Cavrini, L. Fin, A. Buriani,
P. Giusti, Bioorg. Med. Chem. Lett. 7 (1997) 2599e2602;
b) A. Rampa, A. Bisi, F. Belluti, S. Gobbi, P. Valenti, V. Andrisano, V. Cavrini,
A. Cavalli, M. Recanatini, Bioorg. Med. Chem. 8 (2008) 497e506.
[26] S. Rizzo, C. Riviere, L. Piazzi, A. Bisi, S. Gobbi, M. Bartolini, V. Andrisano,
F. Morroni, A. Tarozzi, P. J-Monti, A. Rampa, J. Med. Chem. 51 (2008)
2883e2886.
[27] V.E. Gregor, M.R. Emmerling, C. Lee, C.J. Moore, Bioorg. Med. Chem. Lett. 2
(1992) 861e864.
[28] M. Recanatini, A. Cavalli, F. Belluti, L. Piazzi, A. Rampa, A. Bisi, S. Gobbi,
P. Valenti, V. Andrisano, M. Bartolini, V. Cavrini, J. Med, Chem 43 (2000)
2007e2018.
[29] a) L.J. Sargent, L. Small, J. Org. Chem. 11 (1946) 359e362;
b) M.K. Hu, C.F. Lu, Tetrahedron. Lett. 41 (2000) 1815e1818.
[30] G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Featherstone, Biochem. Phar-
macol. 7 (1961) 88e95.
[31] M. Shanks, M. Kivipelto, R. Bullock, R. Lane, Curr. Med. Res. Opin. 25 (2009)
2439e2446.
[32] R.M. Lane, S.G. Potkin, A. Enz, Intern. J. Neuropsychopharmcol. 9 (2006) 101e124.
[33] Y. Shimmyo, T. Kihara, A. Akaike, T. Niidome, H. Sugimoto, Biochim. Biophys.
Acta. 1780 (2008) 819e825.
[34] P. Camps, X. Formosa, C. Galdeano, T. Gómez, D. Muñoz-Torrero, L. Ramírez,
E. Viayna, E. Gómez, N. Isambert, R. Lavilla, A. Badia, M.V. Clos, M. Bartolini,
F. Mancini, V. Andrisano, A. Bidon-Chanal, O. Huertas, T. Dafni, F.J. Luque,
Chem. Biol. Interact. 187 (2010) 411e415.
[8] J. Varghese, J.P. Beck, M.J. Bienkowski, S. Sinha, R.L. Heinrikson, J. Med. Chem.
46 (2003) 4625e4630.
[9] D.J. Selkoe, Annu. Rev. Neurosci. 17 (1994) 489e517.
[10] E. Giacobini, Int. J. Geriatr. Psychiatry. 18 (2003) S1eS5.
[11] I. Silman, J.L. Sussman, Curr. Opin. Pharmacol. 5 (2005) 293e302.
[12] E. Giacobini, Pharmacol. Res. 50 (2004) 433e440.
[13] a) S. Darvesh, D.A. Hopkins, C. Geula, Nat. Rev. Neurosci. 4 (2003) 131e138;
b) N.H. Greig, T. Utsuki, D.K. Ingram, Y. Wang, G. Pepeu, C. Scali, Q.S. Yu,
J. Mamczarz, H.W. Holloway, T. Giordano, D. Chen, K. Furukawa,
K. Sambamurti, A. Brossi, D.K. Lahiri, Proc. Natl. Acad. Sci. USA 102 (2005)
17213e17218;
c) N.H. Greig, T. Utsuki, Q.S. Yu, X. Zhu, H.W. Holloway, T. Perry, B. Lee,
D.K. Ingram, D.K. Lahiri, Curr. Med. Res. Opin. 17 (2001) 159e165.
[14] R. Morphy, Z. Rankovic, J. Med, Chem 48 (2005) 6523e6543.
[15] A. Cavalli, M.L. Bolognesi, A. Minarini, M. Rosini, V. Tumiatti, M. Recanatini,
C. Melchiorre, J. Med, Chem 51 (2008) 347e372.
[16] Y.P. Pang, P. Quiram, T. Jelacic, F. Hong, S. Brimijoin, J. Biol, Chem 271 (1996)
23646e23649.
[17] H. Fu, W. Li, J. Luo, N.T.K. Lee, M. Li, K.W.K. Tsim, Y. Pang, M.B.H. Youdim,
Y. Han, Biochem. Biophys. Res. Commun. 366 (2008) 631e636.
[18] a) G.V. De Ferrari, M.A. Canales, I. Shin, L.M. Weiner, I. Silman, N.C. Inestrosa,
Biochemistry 40 (2001) 10447e10457;
b) N.C. Inestrosa, A. Alvarez, C.A. Perez, R.D. Moreno, M. Vicente, C. Linker,
O.I. Casanueva, C. Soto, J. Garrido, Neuron 16 (1996) 881e891.
[19] V. Tumiatti, A. Minarini, M.L. Bolognesi, A. Milelli, M. Rosini, C. Melchiorre,
Curr. Med. Chem. 17 (2010) 1825e1838.
[20] A. Rampa, F. Belluti, S. Gobbi, A. Bisi, Curr. Top. Med. Chem., in press.
[21] a) L. Piazzi, A. Rampa, A. Bisi, S. Gobbi, F. Belluti, A. Cavalli, M. Bartolini,
V. Andrisano, P. Valenti, M. Recanatini, J. Med. Chem. 46 (2003) 2279e2282;
b) F. Belluti, A. Rampa, L. Piazzi, A. Bisi, S. Gobbi, M. Bartolini, V. Andrisano,
A. Cavalli, M. Recanatini, P. Valenti, J. Med. Chem. 48 (2005) 4444e4456;
c) S. Rizzo, M. Bartolini, L. Ceccarini, L. Piazzi, S. Gobbi, A. Cavalli,
[35] R. Vassar, D.M. Kovacs, R. Yan, P.C. Wong, J. Neurosci. 29 (2009) 12787e12794.
[36] H. Fu, J. Dou, W. Li, W. Cui, S. Mak, Q. Hu, J. Luo, C.S.C. Lam, Y. Pang,
M.B.H. Youdim, Eur. J. Pharmacol. 623 (2009) 14e21.
[37] M. Bartolini, C. Bertucci, V. Cavrini, V. Andrisano, Biochem. Pharmacol. 65
(2003) 407e416.
[38] H. Naiki, K. Higuchi, K. Nakakuki, T. Takeda, Lab. Invest. 65 (1991) 104e110.
[39] H. LeVine 3rd., Protein. Sci. 2 (1993) 404e410.
[40] M.L. Bolognesi, A. Cavalli, L. Valgimigli, M. Bartolini, M. Rosini, V. Andrisano,
M. Recanatini, C. Melchiorre, J. Med. Chem. 50 (2007) 6446e6449.