Novel class of quinone-bearing polyamines as multi-target-directed ligands to combat Alzheimer's disease

Article abstract of DOI:10.1021/jm070559a

One of the characteristics of Alzheimer's disease (AD) that hinders the discovery of effective disease-modifying therapies is the multifactorial nature of its etiopathology. To circumvent this drawback, the use of multi-target-directed ligands (MTDLs) has

Full text of DOI:10.1021/jm070559a

4882
J. Med. Chem. 2007, 50, 4882-4897
Novel Class of Quinone-Bearing Polyamines as Multi-Target-Directed Ligands To Combat
Alzheimer’s Disease
Maria Laura Bolognesi,*,† Rita Banzi,† Manuela Bartolini,† Andrea Cavalli,† Andrea Tarozzi,‡ Vincenza Andrisano,†
Anna Minarini,† Michela Rosini,† Vincenzo Tumiatti,† Christian Bergamini,§ Romana Fato,§ Giorgio Lenaz,§ Patrizia Hrelia,‡
Antonino Cattaneo,| Maurizio Recanatini,† and Carlo Melchiorre*,†
Department of Pharmaceutical Sciences, Alma Mater Studiorum, UniVersity of Bologna, Via Belmeloro 6, and Departments of Biochemistry
and Pharmacology, Alma Mater Studiorum, UniVersity of Bologna, Via Irnerio 48, 40126 Bologna, and Lay Line Genomics, Via di Castel
Romano 100, 00128 Rome, Italy
ReceiVed May 14, 2007
One of the characteristics of Alzheimer’s disease (AD) that hinders the discovery of effective disease-
modifying therapies is the multifactorial nature of its etiopathology. To circumvent this drawback, the use
of multi-target-directed ligands (MTDLs) has recently been proposed as a means of simultaneously hitting
several targets involved in the development of the AD syndrome. In this paper, a new class of MTDLs
based on a polyamine-quinone skeleton, whose lead (memoquin, 2) showed promising properties in
preclinical investigations (Cavalli et al. Angew. Chem., Int. Ed. 2007, 46, 3689-3692), is described. 3-29
were tested in vitro against a number of isolated AD-related targets, namely, AChE and BChE, and Aâ
aggregation (both AChE-mediated and self-induced). Furthermore, the ability of the compounds to counteract
the oxidative stress in a human neuronal-like cellular system (SH-SY5Y cells) was assayed, in both the
presence and absence of NQO1, an enzyme able to generate and maintain the reduced form of quinone.
Introduction
discovery paradigm from mono- to multi-target-directed ligands
(MTDLs), which are more adequate for addressing the com-
plexity of the disease. In this regard, recent results obtained
with MTDLs, i.e., single chemical entities able to hit distinct
molecular targets in the neurodegenerative cascade, have been
encouraging,7-10 convincing us that they might represent the
best pharmacological option for tackling the multifactorial nature
of AD and for stopping the disease progression.11,12
With these concepts in mind, we initiated a research program
aimed at identifying new MTDLs possessing different activities
toward AD-relevant targets, such as acetylcholinesterase (AChE),
Aâ processing and aggregation, and oxidative stress. Indeed,
oxidative damage is retained as one of the major pathogenic
mechanisms in AD, and since it occurs early in the pathogen-
esis,13 it represents an ideal target for intervention.
Although a few medicinal chemistry strategies have so far
been developed to create MTDLs by joining functionally distinct
pharmacophores,14 the design of MTDLs is a complex task.
Clearly, the success rate can be greatly increased by properly
selecting the starting scaffold: conceptually, it would appear
that the lack of tight molecular specificity for only one given
target may represent a prerequisite for the choice of a better
lead compound. In this respect, some years ago we postulated
that polyamines represent a universal template, able to interact
with all protein targets, whose affinity for a target can be
modulated by (a) inserting appropriate substituents on the
nitrogen atoms and (b) altering the chain length separating the
amine functions to fit the distance between the carboxylate or
aromatic residues of the biological counterpart.15,16
We therefore started from a set of polyamine derivatives that
already showed a dual action, being both AChEIs and musca-
rinic M2 receptor antagonists,17 and planned to modify their
structure by incorporating into their backbone an antioxidant
function. Among the possible carriers of radical scavenger
activity, attention was focused on the benzoquinone fragment
of coenzyme Q10 (CoQ), as this natural antioxidant offered
promise against AD both in vitro18 and in vivo.19 Moreover,
Treating Alzheimer’s disease (ADa) remains a challenge for
the pharmaceutical community. Despite intensive study, its
molecular etiology is still enigmatic, and although a variety of
drug treatments have been shown to benefit patients, none
represent a real cure. On the basis of the so-called cholinergic
hypothesis,1,2 a new generation of acetylcholinesterase inhibitors
(AChEIs) were developed and marketed in the 1990s to
specifically treat AD. These were joined in 2004 by memantine,
an NMDA receptor antagonist.3 AChEIs, although valuable in
restoring cholinergic dysfunction and in improving the patient’s
quality of life, have revealed themselves to be of limited effect,
suitable only for palliative use. In more recent times, mecha-
nism-based approaches directed toward two proteins, amyloid-â
(Aâ)4 and τ,5 have become a major focus of research. Aâ is
the major constituent of amyloid plaques, one of the key
pathological characteristics of AD, while phosphorylated τ is
the main component of neurofibrillary tangles, the second major
characterizing factor for AD.6 However, no new molecules
interfering with the biochemical pathways of either Aâ or τ
have so far reached the market. The reason for this drawback
appears to reside in the fact that, like many common diseases,
AD is multifactorial in origin, with both genetic and environ-
mental contributions likely to play an important role in the
pathogenesis. Accordingly, the quest for new and disease-
modifying agents could be addressed by shifting the drug
* To whom correspondence should be addressed. Phone +39 0512099700.
Fax: +39 0512099734. E-mail: marialaura.bolognesi@unibo.it (M.L.B.);
carlo.melchiorre@unibo.it (C.M.).
† Department of Pharmaceutical Sciences, University of Bologna.
‡ Department of Pharmacology, University of Bologna.
§ Department of Biochemistry, University of Bologna.
| Lay Line Genomics.
a Abbreviations: Aâ, amyloid-â; AChE, acetylcholinesterase; AChEI,
acetylcholinesterase inhibitor; AD, Alzheimer’s disease; APP, amyloid
precursor protein; BACE-1, â-secretase; BChE, butyrylcholinesterase; CoQ,
coenzyme Q10; MD, molecular dynamics; MTDL, multi-target-directed
ligand; NQO1, NAD(P)H:quinone oxidoreductase 1; PAS, peripheral anionic
site; ROS, reactive oxygen species; SAR, structure-activity relationship.
10.1021/jm070559a CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/13/2007

Quinone-Bearing Polyamines as MTDLs To Combat AD
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4883
Figure 1. Design strategy for compounds 2-29. The lead compound 1 (caproctamine) was modified to afford compounds 2-7 and 9-29 by
replacing (a) the inner octamethylene spacer with a 3,6-(un)substituted 2,5-diamino-1,4-benzoquinone to introduce a group with antioxidant properties
and reduced flexibility, (b) the hexamethylene spacer between inner and outer nitrogen atoms with different chain lengths to verify the importance,
if any, of the distance between the nitrogen atoms and the terminal aryl groups, (c) the 2-methoxybenzyl moiety with differently substituted benzyl
and phenethyl groups to assess the role of the aromatic ring and its substituents and of the distance of the ring from the outer nitrogen atoms as
well, and (d) the methyl group with different moieties to optimize the activity. Finally, the 1,4-benzoquinone moiety of 2 was replaced with a
phenyl ring, affording 8, to verify the importance of such a functionality to the biological profile. See Table 1 for the chemical structure of compounds
2-7 and 9-29.
CoQ and different benzoquinone derivatives have been shown
to modulate AD molecular targets, directly inhibiting Aâ
aggregation.20,21
effective recovery from the cholinergic deficit, τ hyperphos-
phorylation, Aâ deposition, and behavioral abnormalities in
AD11 anti-NGF mice, a comprehensive transgenic model of
AD.
As a first approach to convert these AChEIs into MTDLs,
the insertion of an antioxidant moiety into the polyamine
backbone was investigated. Detailed structure-activity relation-
ship (SAR) and docking studies have revealed that, in AChE,
the biophoric space around the octamethylene spacer separating
the two amide functions of 1 (caproctamine) is quite “tolerant”
and can accommodate a variety of cyclic moieties.22 We
therefore decided to introduce the benzoquinone nucleus to
replace the inner polymethylene chain (see Figure 1 for the
design strategy). Moreover, in the resulting prototypic structure
2,5-bis(diamino)-1,4-benzoquinone 2 (memoquin),23 the two
nitrogen atoms in positions 2 and 5 of the benzoquinone moiety
are amide-like in character, precisely mimicking the amide bonds
of 1. Due to a resonance effect, a hydrophobic and planar π
system is generated, which is able, in principle, to bind Aâ and
to perturb protein-protein interactions in the fibrillogenesis
process.24
On this basis, 2 was developed with the aim of obtaining an
MTDL effective on different biochemical cascades relevant for
AD pathology. As reported elsewhere,23 not only did this
compound show high AChE inhibitory activity, but it also
proved to be an inhibitor of the amyloid precursor protein (APP)
processing enzyme â-secretase (BACE-1). It was also able to
block in vitro Aâ aggregation mediated by AChE, together with
oxidative processes. In vivo, 2 showed the ability to rescue
scopolamine-induced amnesia in mice. Moreover, it caused an
However, systematic modifications of 2 to design more
effective ligands have not been reported so far. To clarify the
importance of the polyamine side chain and the quinone
functionality for the different activities relevant for AD, a
chemical modification of 2 was performed, affording compounds
3-29 according to the design strategy as outlined in Figure 1.
In the present paper, we delineate both the general SARs and
unique multiple mechanism of action of these novel MTDLs
as determined by a number of different in vitro assays
accounting for AChE inhibition, Aâ aggregation, and antioxidant
effects. The SAR for BACE-1 inhibition and a complete account
of the in vivo actions will be reported in separate papers.
Chemistry
The key intermediates for the synthesis of 2-13 and 16-29
were the N,N-disubstituted diamines 65-83 (Scheme 1). The
introduction of the aryl moiety on the terminal free amine
function of the commercially available N-Z-1,ω-diaminoalkanes
was performed through condensation with the appropriate aryl
aldehyde and subsequent reduction of the intermediate Schiff
bases with NaBH4 to give 30-46. Initially, N-ethylation of 30-
38 to afford 47-55 was achieved with diethyl sulfate. The
strategy depicted in Scheme 1 was suitable for our initial
purposes. However, in view of the need to produce a certain

4884 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Bolognesi et al.
Scheme 1. Synthesis of Diamines 65-83 and of
Quinone-Bearing Polyamines 2-7a
Scheme 2. Synthesis of Diamine 85a
a Reagents and conditions: BOC ) Me3COCO-; (a) DMF, KI, Et3N,
105 °C, 48 h; (b) CF3COOH, CHCl3, rt, 2 h.
Scheme 3. Synthesis of Amine Amide 87a
a Reagents and conditions: Z ) C6H5CH2OCO-; (a) EtOCOCl, Et3N,
dioxane, rt, 12 h; (b) H2, Pd/C, MeOH, rt,1 h.
corresponding salts or by silica gel chromatography, although
the latter technique is not realistic for large-scale purification.
Therefore, for compounds 39-46, a reductive amination
protocol with CH3COOH and NaBH4 in THF was used,25
leading to 56-63 with higher yields. This reaction does not
usually work on substrates carrying substituents in the 2 position
of the aromatic ring. Thus, in the case of 34,23 the standard
Borch reductive amination approach was used, which afforded
51 in 50% yield, employing washings as a purification step.
Eschweiler-Clarke methylation of 34 gave the methyl derivative
64. Removal of the protecting group of 47-64 in acidic medium
gave the desired synthons 65-83.
Due to the different reactivities and availabilities of the
starting materials, slightly modified procedures were followed
for the preparation of intermediates 85, 87, and 90 (Schemes
2-4). For 85, N-ethyl-N-(2-methoxy)phenethylamine26 was
alkylated with (6-chlorohexyl)carbamic acid tert-butyl ester27
to afford 84, which was treated with CF3COOH to cleave the
protective BOC group (Scheme 2). In the case of 87, N-Z-6-
aminocaproic acid was amidated with N-ethyl-N-(2-methoxy)-
benzylamine28 followed by the removal of the protecting group
with catalytic hydrogenation over 10% palladium on charcoal
(Scheme 3). The isopropyl derivative 90 was obtained by
alkylation with acetone in reductive conditions of N-Z-1,6-
diaminohexane, followed by the reaction of 88 with 2-meth-
oxybenzyl chloride to give 89 and subsequent deprotection to
the desired compound (Scheme 4).
The initial synthetic strategy to link the benzoquinone function
to the diamines relied on the straightforward reaction between
1,4-benzoquinone and the appropriate diamine. In this way, the
reaction between 1,4-benzoquinone and 69 gave 2 (Scheme 5).23
Similarly, compounds 6 and 7 were obtained through the
reaction of 69 with 2,5-dimethyl-1,4-benzoquinone and 2,5-
diphenyl-1,4-benzoquinone, whereas 3-5 were obtained through
the nucleophilic displacement of two halogen atoms respectively
from 2,3,5,6-tetrafluoro-, 2,3,5,6-tetrachloro-, or 2,3,5,6-tetra-
bromo-1,4-benzoquinone by 69 (Scheme 5).
a Reagents and conditions: Z ) C6H5CH2OCO-; (a) ArCHO, toluene,
Dean-Stark trap, reflux, 6 h, then NaBH4, EtOH, rt, overnight; (b) diethyl
sulfate, toluene, reflux, 6 h, then rt, overnight; (c) for 34 to give 51,
CH3CHO, NaBH3CN, MeOH, rt, overnight; for 39-46 to give 56-63,
AcOH, NaBH4, THF, 60 °C, 4 h; for 34 to give 64, HCOOH, HCHO, H2O,
reflux, 6 h; (d) 30% HBr, AcOH, rt, overnight.
amount of the final compounds, such as 2, for in vivo biological
testing and to create a library of compounds to expedite broad
SAR studies within this structural class of quinone-bearing
polyamines, the procedure had a number of undesirable
characteristics. In particular, a challenging step in Scheme 1
was the ethylation of the benzylic amino group of, for example,
34. When the reaction was performed on a 100 mg scale with
diethyl sulfate in toluene at 80 °C, the corresponding compound
51 was obtained in 75% yield. However, various byproducts,
such as quaternary ammonium cations, were produced when
the scale was increased to multigrams. As expected, these were
very difficult to separate from the desired 51 (yield estimated
to be less than 20%), even by repeated crystallization of the

Quinone-Bearing Polyamines as MTDLs To Combat AD
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4885
Scheme 6. Synthesis of Quinone-Bearing Polyamines 9-29a
Scheme 4. Synthesis of Diamine 90a
a Reagents and conditions: (a) EtOH, 60 °C, 5 h.
Scheme 7. Synthesis of the Aromatic Analogue 8a
a Reagents and conditions: Z ) C6H5CH2OCO-; (a) MeCOMe, NaBH4,
EtOH, 60 °C, 48 h; (b) 2-MeOC6H4CH2Cl, KI, Et3N, DMF, 105 °C, 96 h;
(c) 30% HBr, AcOH, 6 h.
Scheme 5. Synthesis of Quinone-Bearing Polyamines 2-7a
a Reagents and conditions: Z ) C6H5CH2OCO-; (a) EtOCOCl, Et3N,
dioxane, 12 h; (b) 30% HBr, AcOH/CF3COOH, 4 h; (c) 2-MeOC6H4CHO,
MeOH, reflux, 2 h, then NaBH4, EtOH, rt, overnight; (d) diethyl sulfate,
toluene, reflux, 6 h, then rt, overnight; (e) BH3·MeSMe, diglyme, 120 °C,
8 h, MeOH, gaseous HCl.
a Reagents and conditions: (a) for 2 see ref 21; for 3-5, ether, rt, 1 h;
for 6, MeOH, rt, 20 h; for 7, EtOH, 60 °C, 2 h.
potency is expressed as IC50, which represents the concentration
of inhibitor required to decrease the enzyme activity by 50%
(Table 1).
However, the synthetic procedure for 2 afforded very complex
mixtures, providing the desired 2,5-diaminoquinone in a yield
not higher than 20%. Therefore, a more efficient reaction was
developed, which involved substitution of the methoxy group
of 2,5-dimethoxy-1,4-benzoquinone with an appropriate diamine
(65-83). In this case, the yield increased to 60-100% and no
chromatography purification was required for the synthesis of
most of the final compounds (Scheme 6).
The use of inhibitors of Aâ oligomerization has emerged as
a promising pharmacotherapy for AD. Therefore, the ability of
selected quinone-bearing polyamines (5, 7, 8, 10, 15, 16, and
24) to inhibit the proaggregating action of AChE toward Aâ-
(1-40) was assessed through a thioflavin T-based fluorometric
assay (Table 1).29,30 Not all the compounds were assayed to
determine their ability to block AChE-mediated Aâ aggregation,
because this assay can be very expensive in light of the large
quantities of enzyme and peptide needed for screening. The
overall cost may be inaccessible for most of the academic
laboratories.31
Compounds 1 and 3-29 were also tested in vitro to assess
their potential to inhibit the self-induced aggregation of Aâ-
(1-42).32-34
Different studies on the antioxidant properties of 1,4-
benzoquinone derivatives, such as CoQ and idebenone, have
conclusively demonstrated that the antioxidant activity pertains
exclusively to their hydroquinone form, since the quinone, in
principle, cannot scavenge radicals.35 Furthermore, NAD(P)H:
quinone oxidoreductase 1 (DT-diaphorase, NQO1; EC 1.6.99.2)
Finally, the aromatic analogue 8 was synthesized, starting
from N-Z-6-aminocaproic acid and 1,4-phenylenediamine (Scheme
7). Treatment of 91 with HBr gave 92, which was treated with
2-methoxybenzaldehyde and NaBH4 to afford 93, which, in turn,
was ethylated (94) and finally reduced with borane to 8.
Biology
To determine their potential interest as MTDLs for the
treatment of AD, 3-29 were assayed for their human AChE
inhibitory activity. Furthermore, to establish the selectivity of
3-29, their butyrylcholinesterase (BChE) inhibitory activity was
also determined on BChE from human serum. The inhibitory

4886 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Bolognesi et al.
Table 1. Inhibition of AChE and BChE Activities and AChE-Mediated and Self-Induced Aâ Aggregation by Quinone-Bearing Polyamines
IC50b (nM)
inhibition of Aâ aggregation (%)
compd
no.a
Ar
X
R1
Y
n
R2
AChE
BChE
AChE-inducedc
self-inducedd
1e
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
170 ( 2
11600 ( 300
1440 ( 100g
1520 ( 90
800 ( 62
<5f
20.3 ( 1.9
66.8 ( 4.4
52.1 ( 11.7
68.2 ( 12.3
63.9 ( 9.7
68.0 ( 11.0
68.2 ( 2.1
56.5 ( 1.9
24.3 ( 3.8
32.4 ( 4.7
39.3 ( 1.8
50.5 ( 2.1
70.5 ( 0.5
71.4 ( 4.1
28.7 ( 10.2
69.9 ( 1.4
70.4 ( 4.2
58.5 ( 3.5
54.6 ( 1.7
68.8 ( 1.3
70.9 ( 1.4
46.9 ( 1.4
22.7 ( 2.4
46.7 ( 1.3
42.7 ( 1.4
43.8 ( 0.2
21.5 ( 1.5
13.1 ( 5.6
22.5 ( 1.5
<5
2-MeOC6H4
2-MeOC6H4
2-MeOC6H4
2-MeOC6H4
2-MeOC6H4
2-MeOC6H4
CH2
CH2
CH2
CH2
CH2
CH2
Et
Et
Et
Et
Et
Et
CH2
CH2
CH2
CH2
CH2
CH2
5
5
5
5
5
5
H
F
Cl
Br
Me
C6H5
1.55 ( 0.11g
5.17 ( 0.17
3.05 ( 0.29
2.66 ( 0.19
9.50 ( 0.23
123 ( 10
42.5 ( 1.9
61.5 ( 1.3
2.25 ( 0.21
13.6 ( 0.5
2.36 ( 0.13
7.79 ( 0.37
17.3 ( 1.4
11600 ( 300
123 ( 20
77.8 ( 4.3
12.9 ( 0.6
73.3 ( 3.2
51.1 ( 1.0
13.8 ( 1.7
71.9 ( 2.9
593 ( 51
2310 ( 130
144 ( 3
87.1 ( 1.7g
ndh
nd
84.2 ( 2.5
nd
696 ( 41
462 ( 2
2250 ( 190
505 ( 42
16800 ( 1100
9840 ( 340
12800 ( 800
3460 ( 180
1250 ( 250
518 ( 24
45.6 ( 0.8
72.4 ( 2.0
2-MeOC6H4
2-MeOC6H4
2-MeOC6H4
2-MeOC6H4
2-MeOC6H4
2-MeOC6H4
2-MeOC6H4
2-MeOC6H4
2-MeOC6H4
2-MeOC6H4
C6H5
4-MeOC6H4
3-MeOC6H4
2-MeC6H4
2-ClC6H4
2-NO2C6H4
2-furanyl
CH2
CH2
CH2
CH2
CH2
(CH2)2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
Et
Et
Et
Et
Et
Et
Et
H
Me
iPr
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
CH2
CH2
CH2
CH2
CH2
CH2
CO
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
1
2
3
4
6
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
nd
91.1 ( 0.3
nd
nd
nd
nd
<5
90.0 ( 5.4
nd
nd
nd
nd
nd
nd
nd
18500 ( 700
3230 ( 150
5110 ( 410
660 ( 46
645 ( 33
2390 ( 110
1690 ( 60
2080 ( 260
45100 ( 1000
67100 ( 4000
3410 ( 200
2790 ( 220
2470 ( 110
11900 ( 700
70100 ( 4700
45.8 ( 3.0
7420 ( 390
20700 ( 1500
30.1 ( 14
34.6 ( 4.1
nd
nd
nd
2-thienyl
2-pyridyl
3-pyridyl
4-pyridyl
60.4 ( 4.1
125 ( 6
133 ( 6
192 ( 10
424 ( 21
23.1 ( 4.8
2010 ( 150
3030 ( 210
32300
nd
nd
tacrine
7i
donepezil
galantamine
rivastigmine
propidium
Congo red
22i
<5
17.9 ( 0.1
<5
<5
17.8 ( 1.6
61.1 ( 4.6
78.8 ( 0.9
13200
nd
82.0 ( 2.5i
nd
nd
a 1, dihydrochloride; 2-29, free bases. b Human recombinant AChE and BChE 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. c Inhibition of
AChE-induced Aâ(1-40). The concentration of the tested inhibitor and Aâ(1-40) was 100 µM and 230 µM, respectively, whereas the Aâ(1-40):AChE
ratio was equal to 100:1. d Inhibition of self-induced Aâ(1-42) aggregation (50 µM) produced by the tested compound at 10 µM concentration. e See Figure
1 for structure. f Not significant. g Data from ref 21. h Not determined. i Data from ref 44.
Table 2. Ability of 2, 5, and 8 To Accept Electrons from the Enzyme
Table 3. Effects of 2, 5, and 8 on ROS Formation in SH-SY5Y Cellsa,b
NQO1
intracellular ROS increase, %
concn
Vmax
[(µmol/min)/
mg]
Vmax
[(µmol/min)/
mg]
compd
no.
Km
(µM)
compd
no.
Km
(µM)
(µM)
2
5
8
0
53.00 ( 6.55
52.67 ( 4.07
49.94 ( 6.12
40.25 ( 8.45c
30.50 ( 4.30d
53.00 ( 6.55
50.51 ( 4.05
52.35 ( 3.22
50.43 ( 4.35
42.12 ( 4.45c
53.00 ( 6.55
54.20 ( 5.45
50.45 ( 4.50
51.35 ( 6.50
49.54 ( 3.55
2
5
12.7 ( 1.3 3480 ( 20
8
not active
0.1
0.3
1.0
3.0
15.1 ( 0.1 2588 ( 68 menadione 1.20 ( 0.05 7286 ( 55
was shown to play a role in the regeneration and maintenance
of a CoQ-reduced antioxidant state. This inducible enzyme
catalyzes the two-electron reduction of quinones to hydroquino-
nes, bypassing the production of semiquinones and, conse-
quently, preventing their participation in redox-cycling and the
subsequent generation of reactive oxygen species (ROS).35 Thus,
compounds 2, 5, and 8 were tested with respect to their ability
to accept electrons from NAD(P)H via NQO1, in comparison
with menadione as a reference compound, by following the
absorbance change of NADH. The results are expressed in terms
of the apparent Vmax and Km (Table 2).
a The experiments were performed with human neuronal-like cells (SH-
SY5Y) expressing high levels of NQO1. b The results are expressed as the
percentage increase of intracellular ROS evoked by exposure to t-BuOOH.
The values are reported as the mean ( SD of three independent experiments
(treated vs untreated). c p < 0.01. d p < 0.001 at ANOVA with the Scheffe
post hoc test.
The antioxidant activity of 5 and 8 was also evaluated against
formation of ROS in human neuronal-like cells (SH-SY5Y) after
treatment with t-BuOOH, a compound used to induce oxidative
damage.36 Experiments were performed with SH-SY5Y cells

Quinone-Bearing Polyamines as MTDLs To Combat AD
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4887
treated with sulforaphane, a phase 2 enzyme inducer, to
overexpress cytosolic NQO1 (Table 3).37
In all the assays, 2 was used as a reference compound.
dependent on the type of substituent on the benzylic nitrogen
atoms, we included in this study compounds 16-18, in which
the N-ethyl group was replaced by a hydrogen atom, a methyl
group, or an isopropyl group. Furthermore, notwithstanding that
a 2-methoxybenzyl group seems to play a crucial role in the
affinity toward AChE as revealed by 1 analogues,20,32 we
decided to further investigate the role of different aromatic
substituents (19-29) in the hope of acquiring further information
about the structural requirements for targeting the biological
counterparts, other than AChE, investigated in the present study.
Docking, Cluster Analysis, and Molecular Dynamics
Simulations
Docking simulations were carried out using the GOLD v3.0.1
package of software.38 Compounds 2, 7, 8, and 10 were docked
at the gorge of the human AChE (PDB code 1B41).39 The
docking outcomes were rationalized using the software AClAP
v1.0.40,41 The binary complex between 2 and AChE was further
investigated by means of molecular dynamics (MD) simulations
in the nanosecond time scale carried out with the AMBER 8.0
package of software.42
AChE Inhibition. The inhibitory potency of compounds
3-29 on human recombinant AChE and BChE from human
serum was assessed through comparison with the prototypic
polyamines 1 and 2, the AChEIs donepezil, galantamine,
rivastigmine, and tacrine, marketed for the treatment of AD,
and propidium, a specific inhibitor of the AChE PAS (Table
1).
Results and Discussion
Recently, we reported that polyamines related to 1 could
represent a valuable lead for the design of compounds useful
for investigating AD because of (i) their improvement of
cholinergic transmission by inhibiting the catalytic site of AChE
and (ii) their interaction with the AChE peripheral anionic site
(PAS), which might prevent AChE-mediated Aâ aggrega-
tion.17,22,43
All the compounds 2-29 showed AChE inhibition activity
with IC50 values ranging from 1.55 to 11600 nM while
displaying a much lower affinity for BChE. These results are
particularly intriguing because, contrary to a previous view,44
BChE may have a role inverse to that of AChE in AD.45 Thus,
selectivity for AChE over BChE may be beneficial for AD
treatment. An analysis of the results reported in Table 1 reveals
that AChE, but not BChE, inhibition can be markedly affected
by the introduction of substituents on the benzoquinone moiety.
It turned out that the halogen-bearing analogues 3-5 were only
slightly less potent (2-3-fold) than 2, whereas 6 and 7 displayed
a significantly lower affinity toward AChE, being from 6- to
79-fold less potent. Interestingly, the replacement of the
benzoquinone moiety of 2 with a phenyl ring, as in 8, reduced
the inhibition of AChE 27-fold, revealing that the two oxygen
atoms of 2 may have, in addition to the other properties outlined
below, a role in the interaction process with the enzyme.
To obtain MTDLs useful for AD therapy, we inserted into
the polyamine backbone of 1 a 1,4-benzoquinone moiety that
could confer to the resulting molecules additional properties
beneficial in the treatment of AD. The significant effectiveness
of 2 in reverting neurodegeneration in vivo23 strongly supported
the feasibility of the MTDL approach to the treatment of AD.
It formed the basis for the development of a new class of small-
molecule-based MTDLs that can serve as powerful tools both
in understanding the role and function of the multiple emerging
biological targets and in further validating the design rationale.
A major concern regarding SAR studies of MTDLs is that
there is no a priori selection for small molecules to be
synthesized over others, because the properties of the selected
molecular targets usually differ. Nevertheless, it is worth noting
that, of the four selected targets, i.e., (a) cholinesterases (AChE
and BChE), (b) Aâ aggregation induced by human AChE, (c)
self-induced Aâ aggregation, and (d) oxidative stress, the first
two are supposedly related since AChEI binding at PAS may
interfere with AChE proaggregating action.29,30 Therefore, some
selection might be based on the inhibitor mechanism of action
and docking studies.
We circumvented the general lack of uniform structural
information by synthesizing a small unbiased library of deriva-
tives of 2, trying to analyze the contributions of the different
structural elements of the molecule to the different activities.
Starting from 2, the 2,3,5,6-tetrasubstituted 1,4-benzoquinones
3-7 were the first quinone-bearing polyamines to be synthe-
sized. Compound 8 was included in the present study because
its phenyl ring is planar, like the 1,4-benzoquinone moiety, and
can give information on the importance of the quinone
functionality on the biological profile of this new series of
MTDLs. The role of the distance between the inner and the
outer nitrogen atoms and between the outer nitrogen atoms and
the terminal phenyl ring was studied by synthesizing the
analogues 9-13 and 14, in which the hexamethylene chain and
the methylene group were respectively replaced by a spacer
composed of two to seven methylene units or by two methyl-
enes. Next, the importance of the terminal amine function of 2
on the biological profile was evaluated by synthesizing the amide
analogue 15. Since, in a previous study,43 we verified that, in
polyamines related to 1, AChE inhibition was significantly
The discovery that 10 and 12 (but not 9, 11, and 13), which
are endowed with a shorter distance between the inner and the
outer nitrogen atoms, showed an affinity for AChE that is only
slightly lower than that of 2 may indicate that the hexamethylene
spacer of 2 interacts with the enzyme with a (partially) folded
conformation. Interestingly, all the compounds with a shorter
distance (9-12) were weaker BChE inhibitors than 2. Length-
ening the distance between the outer nitrogen atoms and the
phenyl rings, as in 14, caused a significant decrease in the
affinity for AChE relative to that of 2, suggesting that the
terminal benzyl moieties are endowed with optimal properties
for interaction with the enzyme. The importance of the nature
of the terminal nitrogen atoms of 2 in the interaction with AChE
was evaluated by investigating compounds 15-18. The dramatic
decrease in affinity toward AChE observed for the amide
analogue 15 indicated that the terminal nitrogen atoms of this
series of quinone-bearing polyamines must be basic. In agree-
ment with previous results obtained with polyamines related to
1,43 compounds 16-18, bearing a hydrogen atom, a methyl
group, or an isopropyl group, respectively, inhibited AChE
activity with a lower affinity than that shown by 2, which bears
an ethyl group on the terminal nitrogen atoms. Finally, the
results observed for 19-29 clearly suggest that a 2-methoxy-
benzyl group on the terminal nitrogen atoms may be endowed
with the optimal electronic and lipophilic properties for interac-
tion with AChE (Table 1).
Computational Studies of the AChEI Binding Mode. To
investigate the binding mode of 2 and some of its derivatives
at the human AChE gorge, docking simulations and cluster
analyses were carried out. In addition, the binary complex

4888 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Bolognesi et al.
Figure 2. Binding mode of some AChEIs as outcomes of docking simulations. H-bonds are dark-red dotted lines. Notably, an intramolecular
H-bond was always identified between one protonated nitrogen and one o-methoxyphenyl ring. (A) Binding mode of 2 (carbon atoms in green) at
the hAChE gorge. The conformation was selected within a stable MD simulation interval between 2 and 5 ns. The quinone moiety is able to tightly
anchor the molecule by means of H-bonds. (B) Binding mode of 8 (carbon atoms in magenta) at the hAChE gorge. The benzene ring can
hydrophobically interact with some aromatic residues (Tyr124 and Tyr341); however, these interactions are probably less tight than those observed
in the binding mode of 2. (C) Binding mode of 7 (carbon atoms in orange) at the hAChE gorge. The phenyl rings in positions 3 and 6 of the
quinone prevent the formation of the H-bonds that we could observe in the binding mode of 2. Moreover, one phenyl ring, pointing toward Asp74,
is probably responsible for an electrostatic repulsion between the π-system of 7 and the carboxylate group of the aspartic acid. (D) Binding mode
of 10 (carbon atoms in yellow) at the hAChE gorge. The aliphatic chain of three methylenes was long enough for a proper interaction between our
derivatives and both the Trp286 and Trp86 sites. The quinone moiety can establish H-bond interactions with residues located at the hAChE midgorge.
between 2 and AChE was then submitted to 7 ns of MD
simulations. The conformation of 2 reported in Figure 2A was
selected within a stable simulation interval (2-5 ns), and it
clearly shows that the benzylammonium ends of 2 were able to
contact Trp86 and Trp286 belonging to the catalytic and
peripheral sites of the enzyme, respectively. Both interactions
were quite stable during the MD simulations. This dynamic
behavior was quite different from that observed for the proto-
typic polyamine 1, which turned out to be dynamically less
stable during a few nanoseconds of MD simulations.17 In this
case, because of the highly flexible chain linking its benzylam-
monium moieties, we could not definitively conclude that 1 was
able to tightly interact with both sites of the AChE enzyme.17
Notwithstanding that 1 and 2 were able to simultaneously
contact both sites of AChE, only extensive MD simulations
could give meaningful information about their markedly dif-

Quinone-Bearing Polyamines as MTDLs To Combat AD
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4889
ferent AChE inhibitory profiles, providing a possible explanation
for the relatively low potency of 1. A pivotal role for the
different dynamical behavior of 2 when compared to 1 may be
related to the benzoquinone moiety, which could establish
favorable interactions with amino acids of the enzyme midg-
orge.46 Indeed, the carbonyl oxygen atoms can establish H-bonds
either with -OH of tyrosine side chains (Tyr341 and Tyr124)23
or with -NH of the Phe295 backbone (Figure 2A). One can
therefore conclude that the benzoquinone moiety of 2 is
fundamental for properly anchoring the molecule at the enzyme
gorge, thus allowing it to tightly interact with both the catalytic
and peripheral anionic sites of the enzyme. This structural feature
could be responsible for the experimentally observed capability
of 2 to inhibit the AChE-induced Aâ aggregation (see below).
To investigate more deeply the role of the benzoquinone
moiety in the inhibition of the AChE-induced Aâ aggregation,
docking simulations were also performed with 8, a compound
in which the benzoquinone moiety of 2 was substituted with a
phenyl ring. Notably, 8 was able to inhibit AChE-induced Aâ
aggregation to a lesser extent when compared to 2. However, 8
was a more potent AChEI than 1. Docking simulations (Figure
2B) clearly show that the central phenyl ring is responsible for
anchoring the molecule at the AChE gorge, by means of
hydrophobic and π-π stacking interactions. Obviously, the tight
H-bond network of the benzoquinone of 2 was not observed
for the phenyl ring of 8, which probably explains the slightly
lower inhibitory potency of 8 relative to 2 toward the AChE-
induced Aâ aggregation.
Docking simulations were also carried out with 7 and 10.
For 7 (Figure 2C), we could observe a detrimental role for two
phenyl rings in positions 3 and 6 of the benzoquinone ring. In
fact, such bulky substituents prevented a proper orientation of
7 within the gorge, which probably accounts for its reduced
inhibitory potencies, when compared to those of 2 (Table 1),
toward both AChE catalytic activity and AChE-induced Aâ
aggregation. Conversely, docking simulations carried out with
10 (Figure 2D) clearly showed that an aliphatic chain of three
methylenes was long enough for an optimal inhibition profile
toward both AChE and AChE-induced Aâ aggregation, as is
clearly demonstrated by its biological profile, reported in
Table 1.
Figure 3. Effect of the chain length n between the inner and outer
nitrogen atoms [N(CH2)nN] of 2 and 9-13 on the inhibition of the
self-induced Aâ(1-42) aggregation.
extent AChE-induced Aâ aggregation, whereas propidium
blocked the aggregation process by 82.0 ( 2.5%. Quinone-
bearing polyamines 2, 5, 10, and 16 at the same concentration
inhibited almost completely AChE-induced Aâ aggregation. The
amide analogue 15 was devoid of activity, whereas 7, 8, and
24 significantly blocked, although to a lesser extent than 2, Aâ
aggregation mediated by AChE. These results clearly suggest
that a basic nitrogen atom is essential for inhibiting Aâ
aggregation (compare 15 to 2), whereas a substituent on the
benzoquinone moiety may not favor the Aâ aggregation
inhibition process. Furthermore, the high antiaggregating activity
displayed by 16 and 8, despite their lower AChE inhibitory
potency, appears to validate our previous hypothesis that the
ability to simultaneously bind both the catalytic site and PAS
of AChE is not a sufficient condition for strongly inhibiting
Aâ aggregation mediated by the enzyme. In addition, a
comparison of 2 to 8 reveals that the benzoquinone moiety,
while important, may not be essential for inhibiting AChE-
induced Aâ aggregation.
Besides assessing the ability to inhibit AChE-induced Aâ-
(1-40) aggregation, which is likely to be relevant in the brain
of AD patients,49 we tested all the compounds (inhibitor:Aâ
ratio 1:5) to assess the structural elements responsible for the
in vitro inhibition of the self-assembly of Aâ(1-42), which is
the most amyloidogenic Aâ fragment found in the AD plaques.
The near-identical inhibition percentage provided by tetra-
substituted quinones 3-7 with respect to 2 points to the
conclusion that the presence of additional substituents on the
quinone core is irrelevant against the self-induced Aâ aggrega-
tion. Conversely, a major role seems to be played by the length
of the spacer, since the potency was increased by increasing
the length between the inner and outer nitrogen atoms from two
to six methylene units as graphically shown in Figure 3.
Interestingly, the potency remained almost unchanged on going
from six (2) to seven (13) methylenes. The conclusion that six/
seven methylenes may represent the optimal spacer length was
further confirmed by the observation that 14 was as potent as
2 and 13, which clearly suggests that the global length between
the benzoquinone moiety and the terminal phenyl rings may be
influential on the inhibition of the self-mediated Aâ aggregation.
Furthermore, the type of the terminal nitrogen atom also
seems relevant, since the amide derivative 15 was significantly
less potent than 2. In addition, the amine derivatives 16-18
were as potent as 2, whereas the N-i-Pr derivative 18 resulted
in a weaker inhibitor, owing to a possible steric hindrance of
the i-Pr over H/Me/Et groups. The results observed for 15-18
may allow us to hypothesize that the fibril-binding properties
of 2 are probably due to the presence of a symmetrical
protonated structure, with charges spaced across a hydrophobic
scaffold, which is also typical of classical azo-dye inhibitors of
amyloid aggregation, such as Congo red.50
Finally, MD simulations carried out on the binary complex
AChE-2 could also explain the 3 orders of magnitude difference
between AChE and BChE inhibitory potencies. Since the latter
enzyme lacks PAS, BChE cannot interact with 2 as tightly as
AChE does.
Inhibition of Aâ Aggregation. We have already demon-
strated that polyamines and quinone-bearing polyamines, ex-
emplified by 1 and 2, respectively, cause a mixed-type inhibition
of AChE, that is, inhibition of both the catalytic site and PAS
of the enzyme.22 The availability of a suitable test for the
determination of the Aâ(1-40) aggregation mediated by AChE
allowed us to verify whether such “dual acting” 47 AChEIs are
able to decrease or block AChE-induced Aâ aggregation. In
view of the high cost of the test, we selected a narrow number
of compounds to investigate some key aspects, namely, the
optimum spacer chain length (2 vs 10), the importance, if any,
of the benzoquinone fragment (2 vs 8) and of its substituents
(5 and 7), and the basicity of the terminal nitrogen atoms (2 vs
16 and 24 and 2 vs the amide analogue 15). The results were
compared with those of prototypes 1 and 2 and AChEIs tacrine,
donepezil, galantamine, rivastigmine, and propidium48 (Table
1). Of the reference compounds, 1, tacrine, donepezil, galan-
tamine, and rivastigmine were not able to inhibit to a significant

4890 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Bolognesi et al.
tion at a concentration of 1 µM (p < 0.01) and 3 µM (p <
0.001), while treatment with 5 produced a significant decrease
of ROS formation only with the highest concentration used (3
µM). In contrast, when treated with 8, the neuronal cells did
not show any difference on ROS formation. Taken together,
these results show that, via NQO1, compounds 2 and 5 (but
not 8) are able to protect neuronal cells against ROS formation
evoked by oxidative stress, with compound 2 being the most
active.
MTDLs for AD Treatment. The results of our efforts to
obtain new MTDLs endowed with an in vitro profile appropriate
for identifying novel lead candidates for a disease-modifying
treatment of AD show how, in the MTDL context, structural
variations can lead to different biological outcomes depending
on the target investigated. This highlights the difficulty of
designing proper analogue series targeted at several systems
simultaneously. Compounds 2, 5, and 8 are representative of
some modifications that we introduced into the polyamine
skeleton of 1 with the aim of “fine-tuning” the ligand’s structure
toward a suitable MTDL lead candidate. As stated above, given
the lack of a uniform structural guidance for the design of new
molecules, the adopted strategy involved the initial synthesis
of a small library of varied compounds followed by testing in
a first assay system (AChE/BChE inhibition) and then the
sequential exploration of other biological activities on the “most
interesting” compounds. Indeed, the parallel screening of all
the activities would have provided a complete set of results,
but we reasoned that, if the first test addressed an action deemed
crucial in the pathological context (in our case, AChE inhibi-
tion), it was reasonable to take a more conservative and
economic approach by selecting only the best performers for
subsequent steps of biological testing.
Figure 4. Inhibition of Aâ aggregation. When the AChE-mediated
aggregation was measured, the concentration of inhibitor and Aâ(1-
40) was 100 and 230 µM, respectively, whereas the Aâ(1-40):AChE
ratio was equal to 100:1. The inhibition of the self-induced Aâ(1-42)
aggregation (50 µM) was produced by the tested compound at a 10
µM concentration.
The main conclusions regarding the compounds modified in
the terminal aromatic moieties are that the methoxy derivatives
20 and 21 were as active as, if not slightly more potent than, 2
in inhibiting Aâ aggregation (it is worth noting that, unlike
AChE inhibitory potency, this potency was not influenced by
the position of the methoxy group). It is also clear that replacing
the phenyl moiety of 2 with different heteroaromatic rings
resulted in weaker inhibitors of the self-induced Aâ aggregation
(compare 25-29 to 2).
As shown in Figure 4, a comparison of the Aâ antiaggregating
properties of 2 to those of the four marketed AChEIs (tacrine,
donepezil, galantamine, and rivastigmine) highlights the much
better profile of 2 as far as both AChE- and self-induced Aâ
aggregation inhibition are concerned.
Antioxidant Activity. The antioxidant properties of 2, 5, and
8 were first verified by testing their ability to accept electrons
from NAD(P)H via NQO1 (Table 2). As one would expect, it
turned out that 2 and 5, but not 8, are good substrates for NQO1,
being not very different from menadione in terms of reduction
by the enzyme. To further confirm the NQO1-mediated mech-
anism of action, we showed that 2, 5, and menadione activities
were sensitive to the NQO1 competitive inhibitor dicoumarol.
In this situation, and parallel to its function in maintaining CoQ
in its reduced antioxidant state, NQO1 may extend the antioxi-
dant potential of this class of quinone derivatives through the
generation of the corresponding hydroquinones, which have been
suggested to have superior antioxidant properties.51 Furthermore,
the two-electron reduction of quinones by NQO1 should bypass
semiquinone production, preventing the generation of ROS that
would form by the interaction of the semiquinone with oxygen.
More interestingly, an increase in NQO1 activity and localized
changes have been shown in brain tissue from AD patients as
compared to controls, suggesting an NQO1 up-regulation in
response to the oxidative stress of the AD process.52 Quinone-
bearing compounds 2 and 5, being specifically reduced by
NQO1 into the corresponding hydroquinones, may exert their
antioxidant activity in those brain regions affected by AD, where
a close relationship exists between NQO1 activity and AD
pathology.
By examining the results of the present investigation, a kind
of “multivariate” SAR of the polyamine-quinones emerge,
showing how we can vary the biological profiles by changing
selected structural elements of the molecules. The resulting
information is the identification of the set of moieties that
provides the best overall action against the target biological
systems, leading to the optimization of the starting compounds.
In this regard, the data obtained confirm that 2 was indeed the
best molecule for addressing the different biological end points
relevant in the AD pathology.
As a general comment, the relevance of the results obtained
in in vitro models may suffer from the major drawback
represented by the different concentrations at which an MTDL
hits different targets. In this respect, only proof-of-concept by
means of in vivo experiments can provide a definite answer to
this issue, which, in our opinion, is the most relevant in the
development of efficacious MTDLs.
Conclusions
Nowadays the drug discovery approach is changing, because
the “one-molecule, one-target” paradigm cannot provide the
right answer to all the quests for new medicines, due to the
fact that many pathologies are multifactorial in nature. Hence,
a single drug, despite a high specificity for a given target, might
be devoid of practical utility for the treatment of complex
syndromes such as neurodegenerative diseases. It is emerging
that these latter disorders can be better approached with a drug
design strategy leading to MTDLs. Here, a critical feature of
the approach, i.e., the possibility of using small molecules to
target different functions of the AD pathogenic pathway, is
clearly demonstrated.
The antioxidant studies were extended to a cell-based system,
SH-SY5Y cells, in the presence or absence of an NQO1 inducer,
such as sulforaphane.
The intracellular antioxidant activity of 2, 5, and 8 against
the formation of ROS was assessed by using a range of
concentrations of tested compounds that did not affect neuronal
viability (0.1-3 µM). As shown in Table 3, SH-SY5Y cells
expressing high levels of NQO1 treated with 2 showed a
significant dose-dependent inhibitory effect on the ROS forma-

Quinone-Bearing Polyamines as MTDLs To Combat AD
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4891
(40). 40 was synthesized from (6-aminohexyl)carbamic acid benzyl
ester and 4-methoxybenzaldehyde: pale yellow oil; 78% yield.
[6-(3-Methoxybenzylamino)hexyl]carbamic Acid Benzyl Ester
(41). 41 was synthesized from (6-aminohexyl)carbamic acid benzyl
ester and 3-methoxybenzaldehyde: pale yellow oil; 76% yield.
{6-[(Furan-2-ylmethyl)amino]hexyl}carbamic Acid Benzyl
Ester (42). 42 was synthesized from (6-aminohexyl)carbamic acid
benzyl ester and furan-2-carboxaldehyde: brown solid; mp 132-
135 °C; 72% yield.
{6-[(Thiophen-2-ylmethyl)amino]hexyl}carbamic Acid Benzyl
Ester (43). 43 was synthesized from (6-aminohexyl)carbamic acid
benzyl ester and thiophene-2-carboxaldehyde: yellow oil; 90%
yield.
{6-[(Pyridin-2-ylmethyl)amino]hexyl}carbamic Acid Benzyl
Ester (44). 44 was synthesized from (6-aminohexyl)carbamic acid
benzyl ester and pyridine-2-carboxaldehyde: yellow oil; 55% yield.
{6-[(Pyridin-3-ylmethyl)amino]hexyl}carbamic Acid Benzyl
Ester (45). 45 was synthesized from (6-aminohexyl)carbamic acid
benzyl ester and pyridine-3-carboxaldehyde: yellow oil; 50% yield.
{6-[(Pyridin-4-ylmethyl)amino]hexyl}carbamic Acid Benzyl
Ester (46). 46 was synthesized from (6-aminohexyl)carbamic acid
benzyl ester and pyridine-4-carboxaldehyde: yellow oil; 60% yield.
General Procedure for the Synthesis of 47-50 and 52-55.
A mixture of compounds 30-33 or 35-38 and diethyl sulfate in
toluene (1:2 molar ratio) was refluxed for 6 h and then stirred
overnight at room temperature. After removal of toluene, the residue
was washed several times with petroleum ether. The crude material
was then dissolved in H2O, made basic with 2 N NaOH, and
extracted with CHCl3. The dried organic phases were evaporated
to afford an oil that was purified by flash chromatography.
{2-[Ethyl(2-methoxybenzyl)amino]ethyl}carbamic Acid Ben-
zyl Ester (47). 47 was obtained from 30: eluent CHCl3/toluene/
MeOH/aqueous 28% ammonia (8:2:0.3:0.02); yellow oil; 45% yield.
{3-[Ethyl(2-methoxybenzyl)amino]propyl}carbamic Acid Ben-
zyl Ester (48). 48 was obtained from 31: eluent petroleum ether/
CH2Cl2/MeOH/aqueous 28% ammonia (5.0:4.6:0.4:0.04); colorless
oil; 70% yield.
{4-[Ethyl(2-methoxybenzyl)amino]butyl}carbamic Acid Ben-
zyl Ester (49). 49 was obtained from 32: eluent CH2Cl2/petroleum
ether/EtOH/aqueous 28% ammonia (7:2.5:0.5:0.04); yellow oil;
51% yield.
{5-[Ethyl(2-methoxybenzyl)amino]pentyl}carbamic Acid Ben-
zyl Ester (50). 50 was obtained from 33: eluent CH2Cl2/MeOH
(9.25:0.75); yellow oil; 60% yield.
{7-[Ethyl(2-methoxybenzyl)amino]heptyl}carbamic Acid Ben-
zyl Ester (52). 52 was obtained from 35: eluent CHCl3/petroleum
ether/MeOH/aqueous 28% ammonia (8:2:0.5:0.02); yellow oil; 35%
yield.
{6-[Ethyl(2-methylbenzyl)amino]hexyl}carbamic Acid Benzyl
Ester (53). 53 was obtained from 36: eluent CH2Cl2/petroleum
ether/MeOH/aqueous 28% ammonia (8:2:0.3:0.015); yellow oil;
40% yield.
Experimental Section
Melting points were taken in glass capillary tubes on a Buchi
SMP-20 apparatus and are uncorrected. IR and direct infusion ESI-
MS spectra were recorded on Perkin-Elmer 297 and Waters ZQ
4000 apparatuses, respectively. 1H NMR spectra were recorded on
Varian VXR 200 and 300 instruments. Chemical shifts are reported
in parts per millions (ppm) relative to the peak for tetramethylsilane
(TMS), and spin multiplicities are given as s (singlet), br s (broad
singlet), d (doublet), t (triplet), br t (broad triplet), q (quartet), sept
(septet), or m (multiplet). Although IR spectral data are not included
(because of the lack of unusual features), they were obtained for
all compounds reported, and they were consistent with the assigned
structures. The elemental compositions of the compounds agreed
to within (0.4% of the calculated value. Where the elemental
analysis is not included, crude compounds were used in the next
step without further purification. Chromatographic separations were
performed on silica gel columns by flash (Kieselgel 40, 0.040-
0.063 mm, Merck) or gravity (Kieselgel 60, 0.063-0.200 mm,
Merck) column chromatography. Reactions were followed by thin-
layer chromatography (TLC) on Merck (0.25 mm) glass-packed
precoated silica gel plates (60 F254) and then visualized in an iodine
chamber or with a UV lamp. The term “dried” refers to the use of
anhydrous sodium sulfate. Compounds were named following
IUPAC rules as applied by Beilstein-Institute AutoNom (version
2.1), a PC-integrated software package for systematic names in
organic chemistry.
General Procedure for the Synthesis of 30-46. A solution of
the appropriate carbamic acid benzyl ester53 and the corresponding
substituted aldehyde in a 1:1.2 molar ratio in toluene was stirred at
the refluxing temperature in a Dean-Stark apparatus for 6 h. After
cooling, the solvent was evaporated, and the residue was dissolved
in EtOH, cooled in an ice bath, and treated with NaBH4 (1.2 equiv).
The reaction mixture was stirred overnight at room temperature
and then made acidic with 3 N HCl. The solution was concentrated,
and the residue was taken up with H2O and washed with ether.
The aqueous phase was made basic with 40% NaOH and extracted
with CHCl3. Removal of the dried solvents afforded the desired
compound.
[2-(2-Methoxybenzylamino)ethyl]carbamic Acid Benzyl Ester
(30). 30 was synthesized from (2-aminoethyl)carbamic acid benzyl
ester and 2-methoxybenzaldehyde: yellow oil; 80% yield.
[3-(2-Methoxybenzylamino)propyl]carbamic Acid Benzyl Es-
ter (31). 31 was synthesized from (3-aminopropyl)carbamic acid
benzyl ester and 2-methoxybenzaldehyde: colorless oil; 90% yield.
[4-(2-Methoxybenzylamino)butyl]carbamic Acid Benzyl Ester
(32). 32 was synthesized from (4-aminobutyl)carbamic acid benzyl
ester and 2-methoxybenzaldehyde: colorless oil; quantitative yield.
[5-(2-Methoxybenzylamino)pentyl]carbamic Acid Benzyl Es-
ter (33). 33 was synthesized from (5-aminopentyl)carbamic acid
benzyl ester and 2-methoxybenzaldehyde: colorless oil; 92% yield.
[6-(2-Methoxybenzylamino)hexyl]carbamic Acid Benzyl Ester
(34). 34 was synthesized as previously described.23
[7-(2-Methoxybenzylamino)heptyl]carbamic Acid Benzyl Es-
ter (35). 35 was synthesized from (7-aminoheptyl)carbamic acid
benzyl ester and 2-methoxybenzaldehyde: pale yellow oil; 90%
yield.
[6-(2-Methylbenzylamino)hexyl]carbamic Acid Benzyl Ester
(36). 36 was synthesized from (6-aminohexyl)carbamic acid benzyl
ester and 2-methylbenzaldehyde: yellow oil; 55% yield.
[6-(2-Chlorobenzylamino)hexyl]carbamic Acid Benzyl Ester
(37). 37 was synthesized from (6-aminohexyl)carbamic acid benzyl
ester and 2-chlorobenzaldehyde: pale yellow oil; 65% yield.
[6-(2-Nitrobenzylamino)hexyl]carbamic Acid Benzyl Ester
(38). 38 was synthesized from (6-aminohexyl)carbamic acid benzyl
ester and 2-nitrobenzaldehyde: yellow oil; 95% yield.
{6-[(2-Chlorobenzyl)ethylamino]hexyl}carbamic Acid Benzyl
Ester (54). 54 was obtained from 37: eluent petroleum ether/CH2-
Cl2/MeOH (7:2.5:0.5); colorless oil; 40% yield.
{6-[Ethyl(2-nitrobenzyl)amino]hexyl}carbamic Acid Benzyl
Ester (55). 55 was obtained from 38: eluent petroleum ether/
CHCl3/MeOH/aqueous 28% ammonia (8:2:0.4:0.02); green oil; 40%
yield.
{6-[Ethyl(2-methoxybenzyl)amino]hexyl}carbamic Acid Ben-
zyl Ester (51). Acetaldehyde (29 mL, 0.51 mol) was added to a
solution of KOH (4.2 g, 0.07 mol) and 3423 (hydrochloride salt;
104 g, 0.26 mol) in methanol (1.2 L) in a three-necked flask
equipped with a mechanical stirrer. After the resulting solution was
stirred at room temperature for 15 min, a solution of NaBH3CN
(6.3 g, 0.1 mol) in methanol (50 mL) was added dropwise, and the
resulting mixture was stirred overnight. Then KOH (15 g) was
added, and the obtained suspension was suction-filtered through
Celite and concentrated under vacuum at a temperature lower than
45 °C. A solid was obtained, which was partitioned between water
and CH2Cl2 (2 × 750 mL). The combined organic extracts were
(6-Benzylaminohexyl)carbamic Acid Benzyl Ester (39). 39 was
synthesized from (6-aminohexyl)carbamic acid benzyl ester and
benzaldehyde: colorless oil; quantitative yield.
[6-(4-Methoxybenzylamino)hexyl]carbamic Acid Benzyl Ester

4892 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Bolognesi et al.
dried and evaporated to give the raw product (106 g), which was
purified by flash chromatography. Eluting first with CHCl3 and
then with CHCl3/EtOH (9.3:0.7) provided pure 51 in 48% yield.
Alternatively, the raw product could be purified by multiple
extractions with hot petroleum ether. The combined and dried
extracts gave 51 in 53% yield, which was sufficiently pure for most
purposes.
General Procedure for the Synthesis of 56-63. Glacial acetic
acid (2 mL) was added to a suspension of 39-46 (1.0 mmol) in
THF. NaBH4 (5.0 mmol) was then added in small portions, cooling
the solution in an ice bath. The mixture was heated at 60 °C under
nitrogen for 4 h. After cooling, H2O was added, and the solution
was made acidic with 3 N HCl and washed with ether. The aqueous
phase was then basified with NaOH pellets and extracted with CH2-
Cl2. The dried solvents were evaporated to afford the desired
compounds. 61-63 were purified by flash chromatography.
[6-(Benzylethylamino)hexyl]carbamic Acid Benzyl Ester (56).
56 was obtained from 39: colorless oil; 75% yield.
N1-Ethyl-N1-(2-nitrobenzyl)hexane-1,6-diamine (73). 73 was
obtained from 55: yellow oil; quantitative yield.
N1-(2-Methoxybenzyl)hexane-1,6-diamine (74). 74 was ob-
tained from 34:23 yellow oil; 65% yield.
N1-Benzyl-N1-ethylhexane-1,6-diamine (75). 75 was obtained
from 56: yellow oil; quantitative yield.
N1-Ethyl-N1-(4-methoxybenzyl)hexane-1,6-diamine (76). 76
was obtained from 57: yellow oil; 90% yield.
N1-Ethyl-N1-(3-methoxybenzyl)hexane-1,6-diamine (77). 77
was obtained from 58: yellow oil; quantitative yield.
N1-Ethyl-N1-(furan-2-ylmethyl)hexane-1,6-diamine (78). 78
was obtained from 59: colorless oil; 85% yield.
N1-Ethyl-N1-(thiophen-2-ylmethyl)hexane-1,6-diamine (79).
79 was obtained from 60: yellow oil; 80% yield.
N1-Ethyl-N1-(pyridin-2-ylmethyl)hexane-1,6-diamine (80). 80
was obtained from 61: colorless oil; 75% yield.
N1-Ethyl-N1-(pyridin-3-ylmethyl)hexane-1,6-diamine (81). 81
was obtained from 62: colorless oil; 75% yield.
N1-Ethyl-N1-(pyridin-4-ylmethyl)hexane-1,6-diamine (82). 82
was obtained from 63: colorless oil; 75% yield.
N1-(2-Methoxybenzyl)-N1-methylhexane-1,6-diamine (83). 83
was obtained from 64: yellow oil; quantitative yield.
{6-[Ethyl(4-methoxybenzyl)amino]hexyl}carbamic Acid Ben-
zyl Ester (57). 57 was obtained from 40: yellow oil; 71% yield.
{6-[Ethyl(3-methoxybenzyl)amino]hexyl}carbamic Acid Ben-
zyl Ester (58). 58 was obtained from 41: colorless oil; 60% yield.
{6-[Ethyl(furan-2-ylmethyl)amino]hexyl}carbamic Acid Ben-
zyl Ester (59). 59 was obtained from 42: yellow oil; 60% yield.
{6-[Ethyl(thiophen-2-ylmethyl)amino]hexyl}carbamic Acid
Benzyl Ester (60). 60 was obtained from 43: yellow oil; 70% yield.
{6-[Ethyl(pyridin-2-ylmethyl)amino]hexyl}carbamic Acid Ben-
zyl Ester (61). 61 was obtained from 44: eluent EtOAc/toluene/
EtOH/aqueous 28% ammonia (7:3:0.5:0.05); yellow oil; 40% yield.
{6-[Ethyl(pyridin-3-ylmethyl)amino]hexyl}carbamic Acid Ben-
zyl Ester (62). 62 was obtained from 45: eluent EtOAc/toluene/
EtOH/aqueous 28% ammonia (7:3:0.5:0.05); yellow oil; 40% yield
{6-[Ethyl(pyridin-4-ylmethyl)amino]hexyl}carbamic Acid Ben-
zyl Ester (63). 63 was obtained from 46: eluent EtOAc/CH2Cl2/
EtOH/aqueous 28% ammonia (6:4:0.5:0.05); yellow oil; 40% yield.
{6-[(2-Methoxyphenyl)methylamino]hexyl}carbamic Acid Ben-
zyl Ester (64). To a suspension of 34 (5.0 g, 13.5 mmol) in H2O
(25 mL) were added 95% HCOOH (1.6 mL, 40.5 mmol) and 37%
HCHO (3.1 mL, 40.5 mmol). The mixture was refluxed for 6 h
and then stirred at room temperature overnight. After partial
evaporation of the solvent, the resulting suspension was made basic
with 40% NaOH and extracted with CHCl3 (3 × 100 mL). The
dried extracts were evaporated to afford a residue which was
purified by flash chromatography. Elution with CHCl3/petroleum
ether/MeOH/aqueous 28% ammonia (8:2:0.7:0.05) afforded 64:
pale yellow oil; 75% yield.
General Procedure for the Synthesis of 65-83. A solution of
30% HBr in CH3COOH (6.5 mL) was added to a solution of 47-
64 or 34 (1.0 mmol) in CH3COOH, and the resulting mixture was
stirred overnight at room temperature. Ether was then added, and
the resulting oil was washed with ether and dissolved in H2O. The
solution was made basic with NaOH pellets and extracted with
CHCl3. Removal of the dried solvents afforded the desired
compounds.
N1-Ethyl-N1-(2-methoxybenzyl)ethane-1,2-diamine (65). 65
was obtained from 47: colorless oil; quantitative yield.
N1-Ethyl-N1-(2-methoxybenzyl)propane-1,3-diamine (66). 66
was obtained from 48: colorless oil; quantitative yield.
N1-Ethyl-N1-(2-methoxybenzyl)butane-1,4-diamine (67). 67
was obtained from 49: yellow oil; quantitative yield.
(6-{Ethyl[2-(2-methoxyphenyl)ethyl]amino}hexyl)carbamic
Acid tert-Butyl Ester (84). Some crystals of KI and a solution of
N-ethyl-N-(2-methoxy)phenethylamine26 (2.4 g, 1.2 mmol) in DMF
(5 mL) were added to a solution of (6-chlorohexyl)carbamic acid
tert-butyl ester27 (1.0 g, 5.6 mmol) and Et3N (1.3 mL, 11.2 mmol)
in DMF (10 mL). The resulting mixture was heated at 105 °C for
48 h, and then the solvent was removed to give a residue that was
partitioned between H2O and CHCl3. The dried organic fraction
was evaporated, affording an oil that was purified by flash
chromatography. Elution with CH2Cl2/petroleum ether/EtOH/aque-
ous 28% ammonia (9:1:0.4:0.07) afforded the desired compound:
yellow oil; 35% yield.
N1-Ethyl-N1-[2-(2-methoxyphenyl)ethyl]hexane-1,6-di-
amine (85). A solution of CF3COOH (4.5 mL) and 84 (0.75 g,
1.98 mmol) in CHCl3 (20 mL) was stirred at room temperature for
2 h. After removal of the solvent, the residue was dissolved in H2O,
washed with ether (2 × 50 mL), basified with 40% NaOH, and
extracted with CHCl3 (3 × 60 mL). Removal of the organic phase
afforded 85: yellow oil; 85% yield.
{5-[Ethyl(2-methoxybenzyl)carbamoyl]pentyl}carbamic Acid
Benzyl Ester (86). Ethyl chlorocarbonate (0.95 mL, 10.0 mmol)
in dry dioxane (3 mL) was added dropwise to a stirred and cooled
solution of N-Z-6-aminocaproic acid (2.65 g, 10.0 mmol) and Et3N
(1.4 mL, 10.0 mmol) in dioxane (60 mL), followed, after the
resulting solution was allowed to stand for 20 min, by the addition
of N-ethyl-N-(2-methoxy)benzylamine28 (1.65 g, 10.0 mmol) in
dioxane (10 mL). After being stirred at room temperature overnight,
the mixture was evaporated, affording a residue that was suspended
in water (150 mL). The precipitate was filtered and washed with 2
N KHSO4, aqueous NaHCO3 saturated solution, and water to give
86: 66% yield.
6-Aminohexanoic Acid Ethyl(2-methoxybenzyl)amide (87). A
solution of 86 (1.12 g, 14 mmol) in methanol (60 mL) was
hydrogenated over 10% Pd on charcoal (0.11 g) for 1 h at room
temperature and a pressure of 15 psi. Following catalyst removal,
the solvent was evaporated, affording 87: colorless oil; quantitative
yield.
[6-(Isopropylamino)hexyl]carbamic Acid Benzyl Ester (88).
Acetone (0.45 mL, 6.0 mmol) was added to a solution of
(6-aminohexyl)carbamic acid benzyl ester (0.50 g, 2.0 mmol) in
EtOH (10 mL). The resulting solution was stirred for 10 min
followed by the addition of NaBH4 (0.23 g, 6.0 mmol) and heating
at 60 °C for 48 h. After removal of the solvent, the residue was
purified by flash chromatography. Eluting with CHCl3/MeOH/
aqueous 28% ammonia (9:1:0.8) afforded the desired compound:
colorless oil; 78% yield.
N1-Ethyl-N1-(2-methoxybenzyl)pentane-1,5-diamine (68). 68
was obtained from 50: yellow oil; 70% yield.
N1-Ethyl-N1-(2-methoxybenzyl)hexane-1,6-diamine (69). 69
was obtained from 51: yellow oil; 90% yield.
N1-Ethyl-N1-(2-methoxybenzyl)heptane-1,7-diamine (70). 70
was obtained from 52: yellow oil; quantitative yield.
N1-Ethyl-N1-(2-methylbenzyl)hexane-1,6-diamine (71). 71
was obtained from 53: yellow oil; 90% yield.
{6-[Isopropyl(2-methoxybenzyl)amino]hexyl}carbamic Acid
Benzyl Ester (89). Et3N (0.45 mL), some crystals of KI, and,
dropwise, a solution of 2-methoxybenzyl chloride (0.28 mL, 2.06
N1-(2-Chlorobenzyl)-N1-ethylhexane-1,6-diamine (72). 72 was
obtained from 54: yellow oil; quantitative yield.

Quinone-Bearing Polyamines as MTDLs To Combat AD
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4893
mmol) in DMF (3 mL) were added to a solution of 88 (0.30 g,
1.03 mmol) in DMF (4 mL). The mixture was heated at 105 °C
for 4 days. The solvent was removed, and the residue was
partitioned between H2O and CHCl3. The dried organic fraction
was evaporated, affording an oil which was purified by flash
chromatography. Elution with CHCl3/EtOH (9.7:0.3) afforded 89:
brown oil; 50% yield.
N1-Isopropyl-N1-(2-methoxybenzyl)hexane-1,6-diamine (90).
90 was obtained from 89 following the procedure described for
65-83: yellow oil; 70% yield.
General Procedure for the Synthesis of 3-5. A suspension of
the proper 2,3,5,6-tetrahalogeno-p-benzoquinone (0.56 mmol) in
ether (2 mL) was added dropwise to a solution of 69 (1.23 g, 4.66
mmmol) in ether (3 mL). The reaction mixture turned a dark red
color almost immediately. After the reaction mixture was stirred
for 1 h at room temperature, the solvent was evaporated under
vacuum. The residue was then purified by flash chromatography.
Data for 2,5-bis{6-[ethyl(2-methoxybenzyl)amino]hexylamino}-
3,6-difluoro-[1,4]-benzoquinone (3): eluent CH2Cl2/MeOH/aque-
ous 28% ammonia (9:1:0.05); green oil; 90% yield; 1H NMR (free
base, CDCl3) δ 1.08 (t, 6H), 1.23-1.63 (m complex, 16H), 2.46-
2.64 (m, 8H), 3.51 (q, 4H), 3.64 (s, 4H), 3.84 (s, 6H), 6.17 (br s,
2H exchangeable with D2O), 6.87 (d, 2H), 6.95 (t, 2H), 7.24 (t,
2H), 7.43 (d, 2H); MS (ESI+) m/z ) 669 (M + H)+. Anal.
(C38H54F2N4O4) C, H, N.
Data for 2,5-bis{6-[ethyl(2-methoxybenzyl)amino]hexylamino}-
3,6-dichloro-[1,4]-benzoquinone (4): eluent CH2Cl2/MeOH/aque-
ous 28% ammonia (9:1:0.05); violet oil; 35% yield; 1H NMR (free
base, CDCl3) δ 1.08 (t, 6H), 1.28-1.78 (m complex, 16H), 2.42-
2.63 (m, 8H), 3.63 (s, 4H), 3.75-3.86 (m, 10H), 6.85 (d, 2H), 6.94
(t, 2H), 7.12 (br s, 2H exchangeable with D2O), 7.22 (t, 2H), 7.43
(d, 2H); MS (ESI+) m/z ) 701 (M + H)+. Anal. (C38H54Cl2N4O4)
C, H, N.
Data for 2,5-bis{6-[ethyl(2-methoxybenzyl)amino]hexylamino}-
3,6-dibromo-[1,4]-benzoquinone (5): eluent CH2Cl2/MeOH/aque-
ous 28% ammonia (9:1:0.05); violet oil; 25% yield; 1H NMR (free
base, CDCl3) δ 1.07 (t, 6H), 1.28-1.78 (m complex, 16H), 2.43-
2.60 (m, 8H), 3.60 (s, 4H), 3.84-3.94 (m, 10H), 6.87 (d, 2H), 6.95
(t, 2H), 7.19-7.28 (m, 2H + 2H exchangeable with D2O), 7.42
(d, 2H); MS (ESI+) m/z ) 791 (M + H)+. Anal. (C38H54Br2N4O4)
C, H, N.
2,5-Bis{6-[ethyl(2-methoxybenzyl)amino]hexylamino}-3,6-
dimethyl-[1,4]-benzoquinone (6) and 2,5-Bis{6-[ethyl(2-meth-
oxybenzyl)amino]hexylamino}-3,6-diphenyl-[1,4]-benzoquino-
ne (7). A solution of diamine 69 (2.1 mmol) was added dropwise
to a suspension of the appropriate 3,6-disubstituted 1,4-benzo-
quinone (1.0 mmol). The reaction solvent was MeOH for 6 and
EtOH for 7. The solution was stirred (room temperature and 60
°C, respectively), and the state of the reaction was continuously
monitored by TLC. After removal of the solvent, the residue was
purified by gravity chromatography.
lamino}-[1,4]-benzoquinone) as a red solid in quantitative yield,
which was identical to that obtained by a different synthetic
scheme.23
Compounds 9-29 were synthesized using the same procedure.
2,5-Bis{2-[ethyl(2-methoxybenzyl)amino]ethylamino}-[1,4]-
benzoquinone (9). 9 was obtained from 65: eluent petroleum ether/
EtOAc/MeOH/aqueous 28% ammonia (7:3:0.4:0.03); red-brown
1
solid; mp 127-130 °C; 60% yield; H NMR (free base, CD3OD)
δ 1.15 (t, 6H), 2.64 (q, 4H), 2.79 (t, 4H), 3.18-3.25 (m, 4H), 3.71
(s, 4H), 3.82 (s, 6H), 5.28 (s, 2H), 6.88-6.99 (m, 4H), 7.21-7.38
(m, 4H); MS (ESI+) m/z ) 521 (M + H)+. Anal. (C30H40N4O4) C,
H, N.
2,5-Bis{3-[ethyl(2-methoxybenzyl)amino]propylamino}-[1,4]-
benzoquinone (10). 10 was obtained from 66: eluent CH2Cl2/
MeOH/aqueous 28% ammonia (9.75:0.25:0.025); foam red solid;
85% yield; 1H NMR (free base, CDCl3) δ 1.11 (t, 6H), 1.72-1.85
(m, 4H), 2.56 (q, 8H), 3.18 (q, 4H), 3.62 (s, 4H), 3.82 (s, 6H),
5.26 (s, 2H), 6.83-6.95 (m, 4H), 7.19-7.43 (m, 4H + 2H
exchangeable with D2O); MS (ESI+) m/z ) 549 (M + H)+. Anal.
(C32H44N4O4) C, H, N.
2,5-Bis{4-[ethyl(2-methoxybenzyl)amino]butylamino}-[1,4]-
benzoquinone (11). 11 was obtained from 67: red solid; mp 97
°C (EtOH); 72% yield; 1H NMR (free base, CDCl3) δ 1.08 (t, 6H),
1.51-1.77 (m, 8H), 2.42-2.63 (m, 8H), 3.11 (q, 4H), 3.60 (s, 4H),
3.81 (s, 6H), 5.28 (s, 2H), 7.72 (br t, 2H exchangeable with D2O),
6.82-7.01 (m, 4H), 7.19-7.23 (m, 2H), 7.38-7.42 (m, 2H); MS
(ESI+) m/z ) 577 (M + H)+. Anal. (C34H48N4O4) C, H, N.
2,5-Bis{5-[ethyl(2-methoxybenzyl)amino]pentylamino}-[1,4]-
benzoquinone (12). 12 was obtained from 68: red solid (EtOH);
mp 88 °C; 68% yield; 1H NMR (free base, CDCl3) δ 1.18 (t, 6H),
1.28-1.72 (m, 12H), 2.43-2.61 (m, 8H), 3.15 (q, 4H), 3.62 (s,
4H), 3.84 (s, 6H), 5.33 (s, 2H), 6.61 (br t, 2H exchangeable with
D2O), 6.86-7.01 (m, 4H), 7.22-7.29 (m, 2H), 7.42-7.46 (m, 2H);
MS (ESI+) m/z ) 605 (M + H)+. Anal. (C36H52N4O4) C, H, N.
2,5-Bis{7-[ethyl(2-methoxybenzyl)amino]heptylamino}-[1,4]-
benzoquinone (13). 13 was obtained from 70: eluent CHCl3/
petroleum ether/MeOH/aqueous 28% ammonia (8:1:1:0.08); red oil;
62% yield; 1H NMR (free base, CDCl3) δ 1.08 (t, 6H), 1.28-1.42
(m, 12H), 1.45-1.60 (m, 4H), 1.62-1.75 (m, 4H), 2.48 (t, 4H),
2.58 (q, 4H), 3.18 (q, 4H), 3.62 (s, 4H), 3.83 (s, 6H), 5.35 (s, 2H),
6.62 (br t, 2H exchangeable with D2O), 6.85-6.90 (m, 2H), 6.97
(t, 2H), 7.22 (t, 2H), 7.43 (d, 2H); MS (ESI+) m/z ) 661 (M +
H)+. Anal. (C40H60N4O4) C, H, N.
2,5-Bis(6-{ethyl[2-(2-methoxyphenyl)ethyl]amino}hexylamino)-
[1,4]-benzoquinone (14). 14 was obtained from 85: foam red solid
1
(EtOH); 75% yield; H NMR (free base, CDCl3) δ 1.03 (t, 6H),
1.23-1.68 (m, 22H), 2.39-2.75 (m, 8H), 3.16 (q, 4H), 3.81 (s,
6H), 4.32 (q, 2H), 5.31 (s, 2H), 6.61 (t, 2H exchangeable with D2O),
6.83-7.01 (m, 4H), 7.17-7.29 (m, 2H), 7.39-7.48 (m, 2H); MS
(ESI+) m/z ) 661 (M + H)+. Anal. (C40H60N4O4) C, H, N.
6-(4-{5-[Ethyl(2-methoxybenzyl)carbamoyl]pentylamino}-3,6-
dioxocyclohexa-1,4-dienylamino)hexanoic Acid Ethyl(2-meth-
oxybenzyl)amide (15). 15 was obtained from 87: red solid (Et2O);
Data for 6: eluent CH2Cl2/MeOH/aqueous 28% ammonia (9.5:
1
0.5:0.04); purple oil; 10% yield; H NMR (free base, CDCl3) δ
1
1.08 (t, 6H), 1.29-1.63 (m, 16H), 2.07 (s, 6H), 2.48-2.60 (m,
8H), 3.53 (q, 4H), 3.64 (s, 4H), 3.85 (s, 6H), 6.73 (br t, 2H
exchangeable with D2O), 6.85-6.97 (m, 4H), 7.28 (t, 2H), 7.45
(d, 2H); MS (ESI+) m/z ) 661 (M + H)+. Anal. (C40H60N4O4) C,
H, N.
mp 134 °C; 65% yield; H NMR (free base, CDCl3) δ 1.11-1.21
(m, 6H), 1.28-1.76 (m complex, 12H), 2.34 (t, 2.4 H), 2.44 (t,
1.6H), 3.11-3.23 (m, 4H), 3.34 (q, 1.6H), 3.46 (q, 2.4H), 3.87 (s,
2.4H), 3.90 (s, 3.6), 4.53 (s, 2.4H), 4.64 (s, 1.6H), 5.30-5.33 (d,
2H), 6.59 (br s, 2H exchangeable with D2O), 6.82-7.32 (m, 8H);
MS (ESI+) m/z ) 683 (M + Na)+. Anal. (C38H52N4O6) C, H, N.
2,5-Bis[6-(2-methoxybenzylamino)hexylamino]-[1,4]-benzo-
quinone (16). 16 was obtained from 74: red solid (EtOH); mp
Data for 7: eluent CH2Cl2/MeOH/aqueous 28% ammonia (9.25:
1
0.75:0.075); pink solid; mp 104 °C (ether); 19% yield; H NMR
(free base, CDCl3) δ 1.01-1.83 (m complex, 22H), 2.36-2.69 (m
complex, 12H), 3.58 (s, 4H), 3.82 (s, 6H) 6.83-6.97 (m complex,
4H + 2H exchangeable with D2O), 7.19-7.40 (m complex, 14H);
MS (ESI+) m/z ) 786 (M + H)+. Anal. (C50H64N4O4) C, H, N.
General Procedure for the Synthesis of 2 and 9-29. A solution
of 69 (12.2 g, 46 mmol) in EtOH (150 mL) was added dropwise to
a suspension of 2,5-dimethoxyquinone (3.85 g; 23.0 mmol) in
boiling EtOH (450 mL). The reaction mixture became progressively
clear and red. It was heated at 60 °C for 5 h and, after cooling,
filtered through a paper filter. The filtrate was concentrated under
vacuum to give 2 (2,5-bis{6-[ethyl(2-methoxybenzyl)amino]hexy-
1
205 °C; quantitative yield; H NMR (free base, CDCl3) δ 1.28-
1.42 (m, 8H), 1.52-1.76 (m, 8H), 2.63 (t, 4H), 3.16 (q, 4H), 3.78-
3.85 (m, 10H + 2H exchangeable with D2O), 5.32 (s, 2H), 6.62
(br t, 2H exchangeable with D2O), 6.85-6.96 (m, 4H), 7.22-7.35
(m, 4H); MS (ESI+) m/z ) 577 (M + H)+. Anal. (C34H48N4O4) C,
H, N.
2,5-Bis{6-[(2-methoxybenzyl)methylamino]hexylamino}-[1,4]-
benzoquinone (17). 17 was obtained from 83: eluent CH2Cl2/
petroleum ether/EtOH/aqueous 28% ammonia (9:1:0.6:0.05); foam
1
red solid; 77% yield; H NMR (free base, CDCl3) δ 1.31-1.48

4894 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Bolognesi et al.
(m, 16H), 2.22 (s, 6H), 2.41 (t, 4H), 3.15 (q, 4H), 3.52 (s, 4H),
3.83 (s, 6H), 5.32 (s, 2H), 6.62 (br t, 2H exchangeable with D2O),
6.83-6.99 (m, 4H), 7.19-7.38 (m, 4H); MS (ESI+) m/z ) 605
(M + H)+. Anal. (C36H52N4O4) C, H, N.
(t, 6H), 1.35-1.75 (m, 16H), 2.41-2.62 (m, 8H), 3.18 (q, 4H),
3.81 (s, 4H), 5.38 (s, 2H), 6.62 (br s, 2H exchangeable with D2O),
6.88-7.00 (m, 4H), 7.21-7.25 (m, 2H); MS (ESI+) m/z ) 585
(M + H)+. Anal. (C32H48N4O2S2) C, H, N.
2,5-Bis{6-[isopropyl(2-methoxybenzyl)amino]hexylamino}-
[1,4]-benzoquinone (18). 18 was obtained from 90: red solid
2,5-Bis{6-[ethyl(pyridin-2-ylmethyl)amino]hexylamino}-[1,4]-
benzoquinone (27). 27 was obtained from 80: foam red solid (H2O/
1
1
(EtOH); mp 116 °C; 80% yield; H NMR (free base, CDCl3) δ
EtOH); 60% yield; H NMR (free base, CDCl3) δ 1.02 (t, 6H),
1.05 (d, 12H), 1.34-1.48 (m, 12H), 1.59-1.70 (m, 4H), 2.46 (t,
4H), 2.97 (sept, 2H), 3.14 (q, 4H), 3.60 (s, 4H), 3.85 (s, 6H), 5.32
(s, 2H), 6.60 (br t, 2H exchangeable with D2O), 6.83-6.89 (m,
2H), 6.87 (t, 2H), 7.23 (t, 2H), 7.58 (d, 2H); MS (ESI+) m/z ) 660
(M + H)+. Anal. (C40H60N4O4) C, H, N.
2,5-Bis[6-(benzylethylamino)hexylamino]-[1,4]-benzoquino-
ne (19). 19 was obtained from 75: eluent CH2Cl2/EtOH/aqueous
28% ammonia (9.5:0.5:0.07); foam red solid; 82% yield; 1H NMR
(free base, CDCl3) δ 1.05 (t, 6H), 1.14-1.39 (m, 8H), 1.43-1.53
(m, 4H), 1.58-1.65 (m, 4H), 2.41-2.56 (m, 8H), 3.10 (q, 4H),
3.71 (s, 4H), 5.28 (s, 2H), 6.44 (br t, 2H exchangeable with D2O),
7.10-7.24 (m, 10H); MS (ESI+) m/z ) 573 (M + H)+. Anal.
(C36H52N4O2) C, H, N.
1.18-1.68 (m, 16H), 2.38-2.60 (m, 8H), 3.12 (q, 4H), 3.64 (s,
4H), 5.22 (s, 2H), 6.60 (br s, 2H exchangeable with D2O), 7.08-
7.17 (m, 2H), 7.40-7.48 (m, 2H), 7.57-7.68 (m, 2H), 8.42-8.48
(m, 2H); MS (ESI+) m/z ) 575 (M + H)+. Anal. (C34H50N6O2) C,
H, N.
2,5-Bis{6-[ethyl(pyridin-3-ylmethyl)amino]hexylamino}-[1,4]-
benzoquinone (28). 28 was obtained from 81: foam red solid (H2O/
1
EtOH); 50% yield; H NMR (free base, CDCl3) δ 1.02 (t, 6H),
1.22-1.66 (m, 16H), 2.33-2.56 (m, 8H), 3.12 (q, 4H), 3.53 (s,
4H), 5.25 (s, 2H), 6.62 (br s, 2H exchangeable with D2O), 7.19-
7.24 (m, 2H), 7.62-7.68 (m, 2H), 7.57-7.68 (m, 2H), 8.41-8.55
(m, 2H); MS (ESI+) m/z ) 575 (M + H)+. Anal. (C34H50N6O2) C,
H, N.
2,5-Bis{6-[ethyl(4-methoxybenzyl)amino]hexylamino}-[1,4]-
benzoquinone (20). 20 was obtained from 76: eluent CH2Cl2/
petroleum ether/EtOH/aqueous 28% ammonia (8:1:1:0.05); foam
2,5-Bis{6-[ethyl(pyridin-4-ylmethyl)amino]hexylamino}-[1,4]-
benzoquinone (29). 29 was obtained from 82: eluent CH2Cl2/
EtOH/aqueous 28% ammonia (9.5:1:0.06); foam red solid; 67%
1
1
red solid; 79% yield; H NMR (free base, CDCl3) δ 1.04 (t, 6H),
yield; H NMR (free base, CDCl3) δ 0.98 (t, 6H), 1.22-1.75 (m,
1.22-1.56 (m, 12H), 1.58-1.72 (m, 4H), 2.38-2.56 (m, 8H), 3.12
(q, 4H), 3.47 (s, 4H), 3.79 (s, 6H), 5.32 (s, 2H), 6.59 (br t, 2H
exchangeable with D2O), 6.82-6.93 (m, 4H), 7.21-7.30 (m, 4H);
MS (ESI+) m/z ) 633 (M + H)+. Anal. (C38H56N4O4) C, H, N.
2,5-Bis{6-[ethyl(3-methoxybenzyl)amino]hexylamino}-[1,4]-
benzoquinone (21). 21 was obtained from 77: eluent CH2Cl2/
petroleum ether/EtOH/aqueous 28% ammonia (8:1:1:0.05); foam
16H), 2.35-2.58 (m, 8H), 3.12 (q, 4H), 3.52 (s, 4H), 5.25 (s, 2H),
6.63 (br s, 2H exchangeable with D2O), 7.21-7.28 (m, 4H), 8.42-
8.48 (m, 4H); MS (ESI+) m/z ) 575 (M + H)+. Anal. (C34H50N6O2)
C, H, N.
{5-[4-(6-Benzyloxycarbonylaminohexanoylamino)-
phenylcarbamoyl]pentyl}carbamic Acid Benzyl Ester (91). 91
was obtained from ethyl chlorocarbonate (0.72 mL, 7.24 mmol),
N-Z-6-aminocaproic acid (1.0 mL, 7.24 mmol), Et3N (2.0 g, 7.24
mmol), and 1,4-phenylenediamine (0.41 g, 3.77 mmol) following
the procedure described for 86. After being stirred at room
temperature overnight, the mixture was poured into water, affording
a white solid which was collected by filtration and washed with 1
N HCl, 1 N NaOH, and water to give 91: 70% yield; mp 195 °C
dec.
6-Aminohexanoic Acid [4-(6-Aminohexanoylamino)phenyl]-
amide (92). 92 was obtained from 91 (1.53 g, 2.55 mmol) following
the procedure described for 65-83: white solid; mp 160-165 °C;
85% yield.
6-(2-Methoxybenzylamino)hexanoic Acid {4-[6-(2-Methoxy-
benzylamino)hexanoylamino]phenyl}amide (93). 93 was obtained
from 92 (0.70 g, 2.09 mmol) and 2-methoxybenzaldehyde (0.63 g,
4.6 mmol) following the procedure described for 30-46: oil; 75%
yield.
1
red solid; 75% yield; H NMR (free base, CDCl3) δ 1.04 (t, 6H),
1.24-1.57 (m, 12H), 1.60-1.75 (m, 4H), 2.39-2.60 (m, 8H), 3.09
(q, 4H), 3.57 (s, 4H), 3.81 (s, 6H), 5.32 (s, 2H), 6.63 (br t, 2H
exchangeable with D2O), 6.76-6.95 (m, 6H), 7.18-7.28 (t, 2H);
MS (ESI+) m/z ) 633 (M + H)+. Anal. (C38H56N4O4) C, H, N.
2,5-Bis{6-[ethyl(2-methylbenzyl)amino]hexylamino}-[1,4]-
benzoquinone (22). 22 was obtained from 71: eluent CH2Cl2/
petroleum ether/EtOH/aqueous 28% ammonia (7:3:0.3:0.035); foam
1
red solid; 78% yield; H NMR (free base, CDCl3) δ 1.03 (t, 6H),
1.22-1.73 (m, 16H), 2.28-2.58 (m, 14H), 3.12 (q, 4H), 3.55 (s,
4H), 5.32 (s, 2H), 6.61 (br t, 2H exchangeable with D2O), 7.12-
7.21 (m, 6H), 7.25-7.40 (m, 2H); MS (ESI+) m/z ) 601 (M +
H)+. Anal. (C38H56N4O2) C, H, N.
2,5-Bis{6-[(2-chlorobenzyl)ethylamino]hexylamino}-[1,4]-ben-
zoquinone (23). 23 was obtained from 72: red solid (EtOH); mp
70 °C; 64% yield; 1H NMR (free base, CDCl3) δ 1.04 (t, 6H), 1.10-
1.35 (m, 8H), 1.43-1.53 (m, 4H), 1.57-1.65 (m, 4H), 2.41-2.56
(m, 8H), 3.08 (q, 4H), 3.46 (s, 4H), 5.31 (s, 2H), 6.59 (br t, 2H
exchangeable with D2O), 7.05-7.23 (m, 6H), 7.45 (d, 2H); MS
(ESI+) m/z ) 641 (M + H)+. Anal. (C36H50Cl2N4O2) C, H, N.
2,5-Bis{6-[ethyl(2-nitrobenzyl)amino]hexylamino}-[1,4]-ben-
zoquinone (24). 24 was obtained from 73: eluent CH2Cl2/petroleum
ether/EtOH/aqueous 28% ammonia (8:2:0.5:0.035); foam red solid;
61% yield; 1H NMR (free base, CDCl3) δ 0.98 (t, 6H), 1.19-1.50
(m, 12H), 1.52-1.72 (m, 4H), 2.35-2.57 (m, 8H), 3.13 (q, 4H),
3.83 (s, 4H), 5.30 (s, 2H), 6.58-6.66 (br t, 2H exchangeable with
D2O), 7.32-7.41 (m, 2H), 7.48-7.58 (m, 2H), 7.62-7.70 (m, 2H),
7.78-7.82 (m, 2H); MS (ESI+) m/z ) 663 (M + H)+. Anal.
(C36H50N6O6) C, H, N.
6-[Ethyl(2-methoxybenzyl)amino]hexanoic Acid (4-{6-[Ethyl-
(2-methoxybenzyl)amino]hexanoylamino}phenyl)amide (94). 94
was obtained from 93 (0.86 g, 1.50 mmol) following the procedure
described for 47-55 and purified by gravity chromatography.
Eluting with CHCl3/MeOH/aqueous 28% ammonia (9:1:0.05) gave
the desired compound: yellow oil; 32% yield.
N,N′-Bis{6-[ethyl(2-methoxybenzyl)amino]hexyl}benzene-1,4-
diamine (8). A solution of 10 M BH3·CH3SCH3 (0.15 mL) in dry
diglyme (10 mL) was added dropwise at room temperature to a
solution of 94 (0.30 g, 0.47 mmol) in dry diglyme (20 mL) with
stirring under a stream of dry nitrogen. When the addition was
completed, the reaction mixture was heated at 120 °C for 8 h. After
the reaction mixture was cooled at 0 °C, excess borane was
destroyed by cautious dropwise addition of MeOH (10 mL). The
resulting mixture was left to stand overnight at room temperature,
cooled at 0 °C, treated with HCl gas for 30 min, and then heated
at 120 °C for 5 h. After cooling, ether was added and the resulting
solid collected by filtration and dissolved in water. The aqueous
phase was washed with ether, made basic with 3 N NaOH, and
extracted with CHCl3. Removal of the dried solvent gave a residue
that was purified by gravity column chromatography. Elution with
CH2Cl2/EtOH/aqueous 28% ammonia (9:1:0.02) afforded 8: dark
2,5-Bis{6-[ethyl(furan-2-ylmethyl)amino]hexylamino}-[1,4]-
benzoquinone (25). 25 was obtained from 78: eluent CH2Cl2/
EtOH/aqueous 28% ammonia (9.5:0.5:0.07); foam red solid; 80%
1
yield; H NMR (free base, CDCl3) δ 1.09 (t, 6H), 1.22-1.58 (m,
12H), 1.60-1.73 (m, 4H), 2.43 (t, 4H), 2.57 (q, 4H), 3.18 (q, 4H),
3.63 (s, 4H), 5.32 (s, 2H), 6.18-6.22 (m, 2H), 6.32-6.39 (m, 2H),
6.62 (br t, 2H exchangeable with D2O), 7.38-7.41 (m, 2H); MS
(ESI+) m/z ) 553 (M + H)+. Anal. (C32H48N4O4) C, H, N.
2,5-Bis{6-[ethyl(thiophen-2-ylmethyl)amino]hexylamino}-
[1,4]-benzoquinone (26). 26 was obtained from 79: red solid; mp
55 °C (H2O/EtOH); 85% yield; 1H NMR (free base, CDCl3) δ 1.08
1
red oil; 50% yield; H NMR (free base, CDCl3) δ 1.12 (t, 6H),
1.28-1.48 (m, 8H + 2H exchangeable with D2O), 1.50-1.68 (m,

Quinone-Bearing Polyamines as MTDLs To Combat AD
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4895
8H), 2.52 (t, 4H), 2.59 (q, 4H), 3.06 (t, 4H), 3.63 (s, 4H), 3.82 (s,
6H), 6.58 (s, 4H), 6.88-6.90 (m, 2H), 6.93-7.01 (m, 2H), 7.22-
7.32 (m, 2H), 7.43-7.48 (m, 2H); MS (ESI+) m/z ) 603 (M +
H)+. Anal. (C38H58N4O2) C, H, N.
(2′,7′-dichlorofluorescein diacetate, DCFH-DA), by measuring the
intracellular ROS formation evoked by exposure of SH-SY5Y cells
to tert-butyl hydroperoxide (t-BuOOH), a compound used to induce
oxidative stress.36 After 24 h of treatment with the compounds, the
SH-SY5Y cells were washed with PBS and then incubated with 5
µM DCFH-DA in PBS at 37 °C in 5% CO2 for 30 min. After
removal of DCFH-DA and further washing, the cells were incubated
with 0.1 mM t-BuOOH in PBS for 30 min. At the end of incubation,
Determination of the Inhibitory Effect on AChE and BChE
Activities. The method of Ellman et al.54 was followed. Prototypes
1 and 2 and the AChEIs tacrine, donepezil, galantamine, and
rivastigmine were used as reference compounds. Five different
concentrations of each compound were used to obtain inhibition
of AChE or BChE activity comprised between 20% and 80%. The
assay solution consisted of a 0.1 M phosphate buffer, pH 8.0, with
the addition of 340 µM 5,5′-dithiobis(2-nitrobenzoic acid), 0.02
unit/mL human recombinant AChE or human serum BChE (Sigma
Chemical), and 550 µM substrate (acetylthiocholine iodide or
butyrylthiocholine iodide). 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 done with a
blank containing all components except AChE or BChE to account
for nonenzymatic reaction. The reaction rates were compared, and
the percentage of inhibition due to the presence of test compounds
was calculated. Each concentration was analyzed in triplicate, and
IC50 values were determined graphically from log concentration-
inhibition curves.
Determination of the Inhibitory Effect on Aâ Aggregation
Induced by AChE. Aliquots of 2 µL of Aâ(1-40) (Bachem AG,
Switzerland), lyophilized from 2 mg/mL HFIP and dissolved in
DMSO at a final concentration of 230 µM, were incubated for 24
h at room temperature in 0.215 M sodium phosphate buffer (pH
8.0). For coincubation experiments, aliquots of AChE (2.30 µM,
ratio 100:1) and AChE in the presence of the tested compound (100
µM) were added. Blanks containing Aâ, AChE, Aâ plus the tested
compound, and AChE plus the tested compound in 0.215 M sodium
phosphate buffer (pH 8.0) were prepared. The final volume of each
vial was 20 µL. To quantify amyloid fibril formation, the thioflavin
T fluorescence method was used.32,33,55 Thioflavin T binds to
amyloid fibrils, giving rise to an intense specific emission band at
490 nm in its fluorescent emission spectrum. Therefore, after
incubation, the samples were diluted to a final volume of 2 mL
with 50 mM glycine-NaOH buffer (pH 8.5) containing 1.5 µM
thioflavin T. A 300 s time scan of fluorescence intensity was carried
out (λexc ) 446 nm, λem ) 490 nm), and values at the plateau were
averaged after subtraction of the background fluorescence of the
1.5 µM thioflavin T solution.
Determination of the Inhibitory Effect on the Self-Mediated
Aâ(1-42) Aggregation. To investigate the self-mediated Aâ(1-
42) aggregation, a thioflavin T based fluorometric assay was
performed. HFIP-pretreated Aâ(1-42) samples (Bachem AG) were
resolubilized with a CH3CN/Na2CO3/NaOH (48.4:48.4:3.2) to have
a stable stock solution ([Aâ] ) 500 µM). Experiments were
performed by incubating the peptide in 10 mM phosphate buffer
(pH 8.0) containing 10 mM NaCl at 30 °C for 24 h (final Aâ
concentration 50 µM) with and without the tested compound at 10
µM. To quantify amyloid fibril formation, the thioflavin T
fluorescence method was used.32,33,55 After incubation, the samples
were diluted to a final volume of 2.0 mL with 50 mM glycine-
NaOH buffer (pH 8.5) containing 1.5 µM thioflavin T. A 300 s
time scan of fluorescence intensity was carried out (λexc ) 446
nm, λem ) 490 nm), and values at the plateau were averaged after
subtraction of the background fluorescence of the 1.5 µM thioflavin
T solution.
Determination of Antioxidant Activity in SH-SY5Y Cells.
Human neuronal-like cells, SH-SY5Y, were routinely grown at 37
°C in a humidified incubator with 5% CO2 in Dulbecco’s modified
Eagle’s medium supplemented with 10% fetal calf serum (FCS), 2
mM glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin.
To evaluate the antioxidant activity of the compounds, SH-SY5Y
cells were seeded in 96-well plates at 3 × 104 cells/well.
Experiments were performed after 24 h of incubation at 37 °C in
5% CO2 with 4-methylsulfinylbutyl isothiocyanate (2.5 µM), a
potent inducer of cytosolic NQO1. The antioxidant activity of
compounds 2, 5, and 8 was evaluated using a fluorescent probe
the fluorescence of the cells from each well was measured (λexc
)
485 nm, λem ) 535 nm) with a spectrofluorometer (Wallac Victor
multilabel counter, Perkin-Elmer Inc., Boston, MA). The results
are expressed as the percentage increase of intracellular ROS evoked
by exposure to t-BuOOH and calculated by the formula [(Ft - Fnt)/
Fnt × 100], where Ft ) fluorescence of treated neurones and Fnt )
fluorescence of untreated neurones.
Data are reported as the mean ( SD of at least three independent
experiments. Statistical analysis was performed using ANOVA (the
Scheffe post hoc test was used), and the differences were considered
significant at p < 0.05. Analyses were performed using STATIS-
TICA 4.5 software on a Windows platform.
Substrate Specificity for NQO1. 2, 5, and 8 were tested with
respect to their ability to accept electrons from NADH via human
NQO1 (Sigma), by following the absorbance change of NADH.
Menadione was used as a reference compound. Briefly, each
reaction consisted of NADH, NQO1, and the tested compounds in
a final volume of Tris-HCl buffer containing bovine serum albumin
(0.07%). Reactions were started by the addition of NADH. The
time course of the reaction was followed by monitoring the
absorbance decrease of NADH at 340 nm, using an extinction
coefficient of 6.22 mM-1 cm-1 in a Jasco 7850 double-beam
spectrophotometer. The extent of nonenzymatic quinone reduction
by NADH was determined in all cases either in the absence of the
enzyme or in the presence of 20 µM dicoumarol. The results are
expressed in terms of the apparent Vmax and Km for three
independent titrations.
Computational Studies. The 3D models of compounds 2, 7, 8,
and 10 were built using Sybyl 7.1.1 (Tripos Associates, Inc., St.
Louis, MO, 2001) and geometry optimized at the density functional
level of theory (B3LYP/6-31G*) by means of the software
Gaussian03.56 Docking simulations were carried out by means of
the GOLD software38 (v. 3.0.1) and using the crystal structure of
human AChE in complex with fasciculin (PDB code 1B41).39
Fasciculin was removed, and the ligand binding site was defined
at 10 Å from the oxygen of the Tyr124 side chain. As suggested
by the GOLD authors,38 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, migrate was 10, mutate was 95, and crossover
was 95. A total of 100 poses were generated by GOLD and then
clusterized by means of AClAP (v. 1.0).40,41 Briefly, AClAP is a
newly developed clustering protocol implemented in a MATLAB
metalanguage program, which combines a hierarchical agglomera-
tive cluster analysis with a clusterability assessment method and a
user-independent cutting rule.41 In particular, when applied to
docking outcomes, we demonstrated that the combination of the
average linkage rule with the cutting function developed by Sutcliffe
and co-workers57 turned out to be an approach that meets all the
criteria required for a robust clustering protocol.40 The pose reported
in Figure 2 for compounds 7, 8, and 10 is a low-energy conforma-
tion representative of the most populated cluster that, according to
the Chauvenet criterion,40,41 was statistically populated. Concerning
2, the pose representative of the most populated cluster was used
as the starting conformation for subsequent molecular dynamics
simulations.
The binary complex between AChE and 2 was investigated by
means of MD simulations carried out with the AMBER 8.0 package
software.42 Seven nanosecond MD simulations on the 2-hAChE
binary complex were carried out in explicit solvent and periodic
boundary conditions. Six Na+ counterions were added to the solvent
bulk of the protein-water complexes to maintain neutrality in the
systems. First, water shells and counterions were minimized using

4896 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Bolognesi et al.
(12) Bolognesi, M. L.; Minarini, A.; Tumiatti, V.; Melchiorre, C. Lipoic
acid, a lead structure for multi-target-directed drugs for neurodegen-
eration. Mini ReV. Med. Chem. 2006, 6, 1269-1274.
steepest descent and conjugate gradient algorithms. Then a mini-
mization of the entire ensemble was performed setting a conver-
gence criterion on the gradient of 0.001 kcal mol-1 Å-1. Then
equilibration runs were carried out by heating the system to 300 K
in 100 ps. This was followed by 7 ns MD simulations in the NPT
ensemble (constant temperature and pressure). The parm99 version58
of the all-atom Amber force field59 was used for the protein and
the counterions, whereas the TIP3P model60 was employed to
explicitly represent water molecules. van der Waals and short-range
electrostatic interactions were estimated within a 10 Å cutoff,
whereas the long-range electrostatic interactions were assessed by
using the particle mesh Ewald (PME) method,61 with a ∼1 Å charge
grid spacing interpolated by a fourth-order B-spline, and by setting
the direct sum tolerance to 10-5. Bonds involving hydrogen atoms
were constrained by using the SHAKE algorithm62 with a relative
geometric tolerance for coordinate resetting of 0.00001 Å. Ber-
endsen’s coupling algorithms63 were employed to maintain constant
temperature and pressure with the same scaling factor for both
solvent and solutes and with the time constant for heat bath coupling
maintained at 1.5 ps. The pressure for the isothermal-isobaric
ensemble was regulated by using a pressure relaxation time of 1
ps in Berendsen’s algorithm. The simulations of the solvated protein
models were performed using a constant pressure of 1 atm and a
constant temperature of 300 K.
(13) Nunomura, A.; Perry, G.; Aliev, G.; Hirai, K.; Takeda, A.; Balraj,
E. K.; Jones, P. K.; Ghanbari, H.; Wataya, T.; Shimohama, S.; Chiba,
S.; Atwood, C. S.; Petersen, R. B.; Smith, M. A. Oxidative damage
is the earliest event in Alzheimer disease. J. Neuropathol. Exp.
Neurol. 2001, 60, 759-767.
(14) Hopkins, A. L.; Mason, J. S.; Overington, J. P. Can we rationally
design promiscuous drugs? Curr. Opin. Struct. Biol. 2006, 16, 127-
136.
(15) Melchiorre, C.; Angeli, P.; Brasili, L.; Giardina`, D.; Pigini, M.;
Quaglia, W. Polyamines: a possible “passe-partout” for receptor
characterization. Actual. Chim. The´r., 15e Se´r. 1988, 149-168.
(16) Bolognesi, M. L.; Minarini, A.; Budriesi, R.; Cacciaguerra, S.;
Chiarini, A.; Spampinato, S.; Tumiatti, V.; Melchiorre, C. Universal
template approach to drug design: polyamines as selective muscarinic
receptor antagonists. J. Med. Chem. 1998, 41, 4150-4160.
(17) Melchiorre, C.; Andrisano, V.; Bolognesi, M. L.; Budriesi, R.; Cavalli,
A.; Cavrini, V.; Rosini, M.; Tumiatti, V.; Recanatini, M. Acetyl-
cholinesterase noncovalent inhibitors based on a polyamine backbone
for potential use against Alzheimer’s disease. J. Med. Chem. 1998,
41, 4186-4189.
(18) Beal, M. F. Mitochondrial dysfunction and oxidative damage in
Alzheimer’s and Parkinson’s diseases and coenzyme Q10 as a
potential treatment. J. Bioenerg. Biomembr. 2004, 36, 381-386.
(19) Bragin, V.; Chemodanova, M.; Dzhafarova, N.; Bragin, I.; Czerni-
awski, J. L.; Aliev, G. Integrated treatment approach improves
cognitive function in demented and clinically depressed patients. Am.
J. Alzheimer’s Dis. Other Dementias 2005, 20, 21-26.
(20) Ono, K.; Hasegawa, K.; Naiki, H.; Yamada, M. Preformed beta-
amyloid fibrils are destabilized by coenzyme Q10 in vitro. Biochem.
Biophys. Res. Commun. 2005, 330, 111-116.
(21) Tomiyama, T.; Shoji, A.; Kataoka, K.; Suwa, Y.; Asano, S.; Kaneko,
H.; Endo, N. Inhibition of amyloid beta protein aggregation and
neurotoxicity by rifampicin. Its possible function as a hydroxyl radical
scavenger. J. Biol. Chem. 1996, 271, 6839-6844.
(22) Tumiatti, V.; Andrisano, V.; Banzi, R.; Bartolini, M.; Minarini, A.;
Rosini, M.; Melchiorre, C. Structure-activity relationships of ace-
tylcholinesterase noncovalent inhibitors based on a polyamine
backbone. 3. Effect of replacing the inner polymethylene chain with
cyclic moieties. J. Med. Chem. 2004, 47, 6490-6498.
All calculations were performed on a Linux cluster employing
an openMosix architecture.
Acknowledgment. This research was supported by a grant
from MIUR (FIRB RBNE03FH5Y), the University of Bologna,
and Lay Line Genomics.
Supporting Information Available: Elemental analysis results
for target compounds, chemical structures of reference compounds,
and NMR data for intermediate compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
(23) Cavalli, A.; Bolognesi, M. L.; Capsoni, S.; Andrisano, V.; Bartolini,
M.; Margotti, E.; Cattaneo, A.; Recanatini, M.; Melchiorre, C. A
small molecule targeting the multifactorial nature of Alzheimer’s
disease. Angew. Chem., Int. Ed. 2007, 46, 3689-3692.
(24) Nesterov, E. E.; Skoch, J.; Hyman, B. T.; Klunk, W. E.; Bacskai, B.
J.; Swager, T. M. In vivo optical imaging of amyloid aggregates in
brain: design of fluorescent markers. Angew. Chem., Int. Ed. 2005,
44, 5452-5456.
(25) Gribble, G. W.; Jasinski, J. M.; Pellicone, J. T.; Panetta, J. A.
N-Alkylation of aliphatic secondary amines with carboxylic acids.
Synthesis 1998, 766-768.
(26) Dubowchik, G. M.; Michne, J. A.; Zuev, D. An efficient sequence
for the preparation of small secondary amine hydrochloride salts for
focused library generation without need for distillation or chromato-
graphic purification. Bioorg. Med. Chem. Lett. 2004, 14, 3147-3149.
(27) Bolognesi, M. L.; Bixel, M. G.; Marucci, G.; Bartolini, M.; Krauss,
M.; Angeli, P.; Antonello, A.; Rosini, M.; Tumiatti, V.; Hucho, F.;
Melchiorre, C. Structure-activity relationships of methoctramine-
related polyamines as muscular nicotinic receptor noncompetitive
antagonists. 3. Effect of inserting the tetraamine backbone into a
macrocyclic structure. J. Med. Chem. 2002, 45, 3286-3295.
(28) Piazzi, L.; Belluti, F.; Bisi, A.; Gobbi, S.; Rizzo, S.; Bartolini, M.;
Andrisano, V.; Recanatini, M.; Rampa, A. Cholinesterase inhibi-
tors: SAR and enzyme inhibitory activity of 3-[omega-(benzylm-
ethylamino)alkoxy]xanthen-9-ones. Bioorg. Med. Chem. 2007, 15,
575-585.
(29) Inestrosa, N. C.; Alvarez, A.; Calderon, F. Acetylcholinesterase is a
senile plaque component that promotes assembly of amyloid beta-
peptide into Alzheimer’s filaments. Mol. Psychiatry 1996, 1, 359-
361.
(30) Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V. beta-Amyloid
aggregation induced by human acetylcholinesterase: inhibition
studies. Biochem. Pharmacol. 2003, 65, 407-416.
References
(1) Bartus, R. T.; Dean, R. L., III; Beer, B.; Lippa, A. S. The cholinergic
hypothesis of geriatric memory dysfunction. Science 1982, 217, 408-
414.
(2) Terry, A. V., Jr.; Buccafusco, J. J. The cholinergic hypothesis of
age and Alzheimer’s disease-related cognitive deficits: recent
challenges and their implications for novel drug development. J.
Pharmacol. Exp. Ther. 2003, 306, 821-827.
(3) Cummings, J. L. Treatment of Alzheimer’s disease: current and future
therapeutic approaches. ReV. Neurol. Dis. 2004, 1, 60-69.
(4) McLaurin, J.; Kierstead, M. E.; Brown, M. E.; Hawkes, C. A.;
Lambermon, M. H.; Phinney, A. L.; Darabie, A. A.; Cousins, J. E.;
French, J. E.; Lan, M. F.; Chen, F.; Wong, S. S.; Mount, H. T.; Fraser,
P. E.; Westaway, D.; St George-Hyslop, P. Cyclohexanehexol
inhibitors of Abeta aggregation prevent and reverse Alzheimer
phenotype in a mouse model. Nat. Med. 2006, 12, 801-808.
(5) Khlistunova, I.; Biernat, J.; Wang, Y.; Pickhardt, M.; von Bergen,
M.; Gazova, Z.; Mandelkow, E.; Mandelkow, E. M. Inducible
expression of Tau repeat domain in cell models of tauopathy:
aggregation is toxic to cells but can be reversed by inhibitor drugs.
J. Biol. Chem. 2006, 281, 1205-1214.
(6) Hardy, J.; Duff, K.; Hardy, K. G.; Perez-Tur, J.; Hutton, M. Genetic
dissection of Alzheimer’s disease and related dementias: amyloid
and its relationship to tau. Nat. Neurosci. 1998, 1, 355-358.
(7) Youdim, M. B.; Buccafusco, J. J. Multi-functional drugs for various
CNS targets in the treatment of neurodegenerative disorders. Trends
Pharmacol. Sci. 2005, 26, 27-35.
(8) Youdim, M. B.; Buccafusco, J. J. CNS Targets for multi-functional
drugs in the treatment of Alzheimer’s and Parkinson’s diseases. J.
Neural Transm. 2005, 112, 519-537.
(9) Csermely, P.; Agoston, V.; Pongor, S. The efficiency of multi-target
drugs: the network approach might help drug design. Trends
Pharmacol. Sci. 2005, 26, 178-182.
(31) Dickerson, T. J.; Beuscher, A. E. t.; Rogers, C. J.; Hixon, M. S.;
Yamamoto, N.; Xu, Y.; Olson, A. J.; Janda, K. D. Discovery of
acetylcholinesterase peripheral anionic site ligands through compu-
tational refinement of a directed library. Biochemistry 2005, 44,
14845-14853.
(32) Naiki, H.; Higuchi, K.; Nakakuki, K.; Takeda, T. Kinetic analysis
of amyloid fibril polymerization in vitro. Lab. InVest. 1991, 65, 104-
110.
(10) Espinoza-Fonseca, L. M. The benefits of the multi-target approach
in drug design and discovery. Bioorg. Med. Chem. 2006, 14, 896-
897.
(11) Rosini, M.; Andrisano, V.; Bartolini, M.; Bolognesi, M. L.; Hrelia,
P.; Minarini, A.; Tarozzi, A.; Melchiorre, C. Rational approach to
discover multipotent anti-Alzheimer drugs. J. Med. Chem. 2005, 48,
360-363.

Quinone-Bearing Polyamines as MTDLs To Combat AD
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 4897
(33) LeVine, H., 3rd Thioflavine T interaction with synthetic Alzheimer’s
disease beta-amyloid peptides: detection of amyloid aggregation in
solution. Protein Sci. 1993, 2, 404-410.
(34) Datki, Z.; Papp, R.; Zadori, D.; Soos, K.; Fulop, L.; Juhasz, A.;
Laskay, G.; Hetenyi, C.; Mihalik, E.; Zarandi, M.; Penke, B. In vitro
model of neurotoxicity of Abeta 1-42 and neuroprotection by a
pentapeptide: irreversible events during the first hour. Neurobiol.
Dis. 2004, 17, 507-515.
(35) Mordente, A.; Martorana, G. E.; Minotti, G.; Giardina, B. Antioxidant
properties of 2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-ben-
zoquinone (idebenone). Chem. Res. Toxicol. 1998, 11, 54-63.
(36) Wang, H.; Joseph, J. A. Quantifying cellular oxidative stress by
dichlorofluorescein assay using microplate reader. Free Radical Biol.
Med. 1999, 27, 612-616.
(37) Fahey, J. W.; Zhang, Y.; Talalay, P. Broccoli sprouts: an exception-
ally rich source of inducers of enzymes that protect against chemical
carcinogens. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10367-10372.
(38) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
Development and validation of a genetic algorithm for flexible
docking. J. Mol. Biol. 1997, 267, 727-748.
(39) Kryger, G.; Harel, M.; Giles, K.; Toker, L.; Velan, B.; Lazar, A.;
Kronman, C.; Barak, D.; Ariel, N.; Shafferman, A.; Silman, I.;
Sussman, J. L. Structures of recombinant native and E202Q mutant
human acetylcholinesterase complexed with the snake-venom toxin
fasciculin-II. Acta Crystallogr., D: Biol. Crystallogr. 2000, 56,
1385-1394.
(40) Bottegoni, G.; Cavalli, A.; Recanatini, M. A comparative study on
the application of hierarchical-agglomerative clustering approaches
to organize outputs of reiterated docking runs. J. Chem. Inf. Model
2006, 46, 852-862.
(41) Bottegoni, G.; Rocchia, W.; Recanatini, M.; Cavalli, A. AClAP,
Autonomous hierarchical agglomerative Cluster Analysis based
protocol to partition conformational datasets. Bioinformatics 2006,
22, e58-65.
(42) Case, D. A.; Darden, T. E.; Cheatham, T. E. I.; Simmerling, C. L.;
Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Wang, B.; Pearlman,
D. A.; Crowley, M.; Brozell, S.; Tsui, V.; Gohlke, H.; Mongan, J.;
Hornak, V.; Cui, G.; Beroza, P.; Schafmeister, C.; Caldwell, J. W.;
Ross, W. S.; Kollman, P. A. AMBER 8; University of California:
San Francisco.
(49) Rees, T.; Hammond, P. I.; Soreq, H.; Younkin, S.; Brimijoin, S.
Acetylcholinesterase promotes beta-amyloid plaques in cerebral
cortex. Neurobiol. Aging 2003, 24, 777-787.
(50) Abe, K.; Kato, M.; Saito, H. Congo red reverses amyloid beta protein-
induced cellular stress in astrocytes. Neurosci. Res. 1997, 29, 129-
134.
(51) Beyer, R. E.; Segura-Aguilar, J.; Di Bernardo, S.; Cavazzoni, M.;
Fato, R.; Fiorentini, D.; Galli, M. C.; Setti, M.; Landi, L.; Lenaz, G.
The role of DT-diaphorase in the maintenance of the reduced
antioxidant form of coenzyme Q in membrane systems. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 2528-2532.
(52) SantaCruz, K. S.; Yazlovitskaya, E.; Collins, J.; Johnson, J.; DeCarli,
C. Regional NAD(P)H:quinone oxidoreductase activity in Alzhe-
imer’s disease. Neurobiol. Aging 2004, 25, 63-69.
(53) Atwell, G. J.; Denny, W. A. Monoprotection of R,ω-alkanediamines
with the N-benzyloxycarbonyl group. Synthesis 1984, 1032-1033.
(54) Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Feather-Stone, R.
M. A new and rapid colorimetric determination of acetylcholinest-
erase activity. Biochem. Pharmacol. 1961, 7, 88-95.
(55) Inestrosa, N. C.; Alvarez, A.; Perez, C. A.; Moreno, R. D.; Vicente,
M.; Linker, C.; Casanueva, O. I.; Soto, C.; Garrido, J. Acetylcho-
linesterase accelerates assembly of amyloid-beta-peptides into Alzhe-
imer’s fibrils: possible role of the peripheral site of the enzyme.
Neuron 1996, 16, 881-891.
(56) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A.,
Jr.; Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.;
Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.;
Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.;
Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P.
Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.;
Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.;
Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M.
A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.;
Gill, P. M. W.; Johnson, B. G.; Chen, W.; Wong, M. W.; Andres, J.
L.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 03,
Revision C.02; Gaussian, Inc.: Pittsburgh, PA.
(57) Kelley, L. A.; Gardner, S. P.; Sutcliffe, M. J. An automated approach
for defining core atoms and domains in an ensemble of NMR-derived
protein structures. Protein Eng. 1997, 10, 737-741.
(58) Wang, J.; Cieplak, P.; Kollman, P. A. How well does a restrained
electrostatic potential (RESP) model perform in calculating confor-
mational energies of organic and biological molecules? J. Comput.
Chem. 2000, 21, 1049-1074.
(43) Tumiatti, V.; Rosini, M.; Bartolini, M.; Cavalli, A.; Marucci, G.;
Andrisano, V.; Angeli, P.; Banzi, R.; Minarini, A.; Recanatini, M.;
Melchiorre, C. Structure-activity relationships of acetylcholinesterase
noncovalent inhibitors based on a polyamine backbone. 2. Role of
the substituents on the phenyl ring and nitrogen atoms of caproc-
tamine. J. Med. Chem. 2003, 46, 954-966.
(44) Giacobini, E.; Spiegel, R.; Enz, A.; Veroff, A. E.; Cutler, N. R.
Inhibition of acetyl- and butyryl-cholinesterase in the cerebrospinal
fluid of patients with Alzheimer’s disease by rivastigmine: correlation
with cognitive benefit. J. Neural Transm. 2002, 109, 1053-1065.
(45) Diamant, S.; Podoly, E.; Friedler, A.; Ligumsky, H.; Livnah, O.;
Soreq, H. Butyrylcholinesterase attenuates amyloid fibril formation
in vitro. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 8628-8633.
(46) Campiani, G.; Fattorusso, C.; Butini, S.; Gaeta, A.; Agnusdei, M.;
Gemma, S.; Persico, M.; Catalanotti, B.; Savini, L.; Nacci, V.;
Novellino, E.; Holloway, H. W.; Greig, N. H.; Belinskaya, T.;
Fedorko, J. M.; Saxena, A. Development of molecular probes for
the identification of extra interaction sites in the mid-gorge and
peripheral sites of butyrylcholinesterase (BuChE). Rational design
of novel, selective, and highly potent BuChE inhibitors. J. Med.
Chem. 2005, 48, 1919-1929.
(59) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M.;
Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.;
Kollman, P. A. A second generation force field for the simulation of
proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc.
1995, 117, 5179-5197.
(60) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.;
Klein, L. M. Comparison of simple potential functions for simulating
liquid water. J. Chem. Phys. 1983, 79, 926-935.
(61) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.;
Pedersen, L. G. A smooth particle mesh Ewald method. J. Chem.
Phys. 1995, 103, 8577-8593.
(62) Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C. Numerical integration
of the cartesian equations of motion of a system with constraints:
molecular dynamics of n-alkanes. J. Comput. Phys. 1977, 23, 327-
341.
(47) Recanatini, M.; Valenti, P. Acetylcholinesterase inhibitors as a starting
point towards improved Alzheimer’s disease therapeutics. Curr.
Pharm. Des. 2004, 10, 3157-3166.
(63) Berendsen, H. J. C.; Postma, J. P. M.; Van, Gunsteren, W. F.; Di,
Nola, A.; Haak, J. R. Molecular dynamics with coupling to an external
bath. J. Chem. Phys. 1984, 81, 3684-3690.
(48) Bolognesi, M. L.; Andrisano, V.; Bartolini, M.; Banzi, R.; Melchiorre,
C. Propidium-based polyamine ligands as potent inhibitors of
acetylcholinesterase and acetylcholinesterase-induced amyloid-beta
aggregation. J. Med. Chem. 2005, 48, 24-27.
JM070559A