Multitargeted drugs discovery: Balancing anti-amyloid and anticholinesterase capacity in a single chemical entity

Article abstract of DOI:10.1016/j.bmcl.2010.12.093

Memoquin (1) is a lead compound multitargeted against Alzheimer's disease (AD). It is an AChE inhibitor, free-radical scavenger, and inhibitor of amyloid-β (Aβ) aggregation. A new series of 1 derivatives was designed and synthesized by linking its 2,5-dia

Full text of DOI:10.1016/j.bmcl.2010.12.093

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2655–2658
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Multitargeted drugs discovery: Balancing anti-amyloid and anticholinesterase
capacity in a single chemical entity
Maria Laura Bolognesi a, , Manuela Bartolini a, Andrea Tarozzi b, Fabiana Morroni b, Federica Lizzi a,
⇑
Andrea Milelli a, Anna Minarini a, Michela Rosini a, Patrizia Hrelia b, Vincenza Andrisano a, Carlo Melchiorre a
a Department of Pharmaceutical Sciences—Alma Mater Studiorum-Bologna University, Via Belmeloro 6, 40126 Bologna, Italy
b Department of Pharmacology, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Memoquin (1) is a lead compound multitargeted against Alzheimer’s disease (AD). It is an AChE inhibitor,
free-radical scavenger, and inhibitor of amyloid-b (Ab) aggregation. A new series of 1 derivatives was
designed and synthesized by linking its 2,5-diamino-benzoquinone core with motifs that are present
in the structure of known amyloid binding agents like curcumin, the benzofuran derivative SKF64346,
or the benzothiazole bearing compounds KHG21834 and BTA-1. The weaker AChE inhibitory potencies
and the concomitant nearly equipotent anti-amyloid activities of the new compounds with respect to
1 resulted in a more balanced biological profile against both targets. Selected compounds turned out
to be effective Ab aggregation inhibitors in a cell-based assay. By properly combining two or more distinct
pharmacological properties in a molecule, we can achieve greater effectiveness compared to single-tar-
geted drugs for investigating AD.
Received 29 November 2010
Revised 17 December 2010
Accepted 20 December 2010
Available online 23 December 2010
Keywords:
Memoquin
Alzheimer’s disease
Protein–protein interactions
Ó 2010 Elsevier Ltd. All rights reserved.
In the fight against Alzheimer’s disease (AD), great gains in basic
science knowledge have not yet resulted in clinically effective
drugs. A contributing factor to this lack of translational progress
is the difficulty in defining an appropriate drug discovery strategy
that parallels the complexity of the AD pathology. The only
available drugs are acetylcholinesterase (AChE) inhibitors and
memantine, both of which target neurotransmission with modest
palliative clinical effects. Currently, AD drug development targets
the production or aggregation of amyloid-b peptide (Ab) into
oligomers, which are responsible for disrupting neuronal synaptic
plasticity and for the resulting early cognitive impairment associ-
ated with AD.1 However, no potential drug compound has success-
fully completed clinical trials. A careful re-examination of current
strategies is therefore warranted.
Since AD is a complex neurodegenerative disorder resulting
from multiple molecular abnormalities, and not from a single tar-
get/gene defect, we and others have proposed a move from the
‘one protein, one target, one drug’ strategy to a strategy of develop-
ing drugs that simultaneously affect multiple targets.2–5 This ap-
proach has led us to a new paradigm in medicinal chemistry, the
‘multitarget-directed ligand’ (MTDL) design strategy.6 In the past
decade, by following this strategy, we have developed several
ligands able to simultaneously hit multiple AD biological targets.7
Memoquin (1; Fig. 1) is a quinone-bearing polyamine currently in
preclinical investigation: it inhibits self- and AChE-induced Ab
aggregation or its formation, by inhibiting the beta secretase en-
zyme (BACE-1), and acts an antioxidant.8,9 Its development offered
the proof of concept of the effectiveness of the MTDL drug discov-
ery approach, with in vivo confirmation of its multitarget mecha-
nism of action.10
In the search for novel memoquin analogues, we focused on
MTDLs with a better and a more balanced anti-amyloid/anticholin-
esterase profile. The view that Ab is one of the (main) factors, and not
the only factor underlying AD pathogenesis, is more consistent with
current knowledge,11 and might account for the failure of purely
anti-amyloid strategies. Moreover, it further supports the rationale
that MTDLs could be more therapeutically effective if they have
other biological properties in addition to the anti-amyloid one. To
this end, we have rationally manipulated the structure of 1. Our
starting point was the assumption that its planar 2,5-diamino-
benzoquinone (BQ) scaffold could be a privileged motif for modulat-
ing protein–protein interactions (PPIs) and an optimal central core
for the design of bivalent ligands.9,12–15 The terminal 2-methoxy-
benzyl groups of 1 were replaced with several amyloid binding frag-
ments from known amyloidophilic agents, as depicted in Figure 1.
Curcumin (Curc) directly binds Ab, blocking aggregation and fibril
formation in vitro and in vivo.16 The vanillic (Van) ring seems to be
critically involved in the interaction.17 Several compounds contain-
ing a benzofuran (BFur) moiety (e.g., SKF64 346) have been identi-
fied as inhibitors of Ab fibril formation.18,19 Benzothiazole (BTh)
derivatives are extremely interesting molecules for neurodegenera-
tive drug development. As an example, the benzothiazole derivative
⇑
Corresponding author. Tel.: +39 051 2099718.
E-mail address: marialaura.bolognesi@unibo.it (M.L. Bolognesi).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.093

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O
O
H
N
N
N
N
H
O
O
PPIs privileged motif
1
O
O
O
O
O
NH
S
N
NH
curcumin
HO
OH
O
O
KHG21834
Van
BTh
S
N
O
NH
BTA-1
N
PBTh
O
O
SKF64346
BFur
O
O
H
H
N
R
N
n
N
H
N
H
R
n
2: R = Van; n= 1
3: R = Van; n= 4
4: R = Bfur; n= 1
5: R = Bfur; n= 4
6: R = BTh; n= 1
7: R = BTh; n= 4
8: R = PBTh; n=1
9: R = PBTh; n= 4
Figure 1. Design strategy for compounds 2–9.
KHG21834 is neuroprotective against the Ab-induced degeneration
of neuronal cells in vitro and in vivo.20,21 Furthermore, starting from
the 2-phenylbenzothiazole (PBTh) dye thioflavin T (ThT), several
amyloid imaging agents were developed. It was reported that the
uncharged ThT analogue BTA-122 shows very good brain entry and
a specific binding to Ab deposits in AD brain while the 11C-labelled
N-methyl 6-hydroxy derivative of BTA-1 ([11C]PIB), also known as
Pittsburgh compound B, is the most commonly used agent for the
positron emission tomography (PET) imaging of Ab plaques.23 The
selected amyloid recognition motifs were linked to the BQ nucleus
through proper spacers, that is, a diaminopropane or a diaminohex-
ane chain, which conferred a better MTDL profile in previous SAR
studies on 1 derivatives.9
From the synthetic standpoint, a retrosynthetic analysis revealed
that, as with 1, the target compounds can be obtained from substitu-
tion reaction of 2,5-dimethoxy-1,4-benzoquinone with the appro-
priate diamines 10–17 (Scheme 1).9,24 Compounds 12–17, in turn,
were synthesized by reductive amination of an aldehyde with
N-Z-diaminohexane or N-Z-diaminopropane (20–25), followed by
cleavage of the N-protecting group with HBr. A milder reductive
amination protocol was used for vanillin-derived diamines 10 and
11. The reaction using trimethylorthoformate as solvent required
only 1 equiv of the respective protected diamine, and corresponding
equivalents of sodium cyanoborohydride together with 5% glacial
acetic acid. This gave high yields of the expected product without
protection of the phenolic group. In this case, removal of the Z group
was achieved by hydrogenolysis.
1, which inhibits AChE in the nanomolar range, 2–9 were weaker
inhibitors with IC50 values in the micromolar range.
Previous results demonstrated that quinone-bearing polyamines
can be potent AChE inhibitors only when the terminal protonable
nitrogen atom bears an ethyl substituent, with the des-ethyl-mem-
oquin nearly 100 times less active than 1.9 This could also account
for the decreased activity observed for 2–9. However, 2 and 3, car-
rying the vanillic moiety, showed IC50 values in the submicromolar
range on hAChE (0.198 and 0.102 lM, respectively). A submicrom-
olar inhibitory potency was also observed for compound 9. These
levels of inhibition are relatively modest in comparison to 1, yet
it is important to note that inhibition of acetylcholine cleavage
by current drugs, such as rivastigmine25 and donepezil,26 is not
dissimilar.
The activity of 2–9 towards the inhibition of Ab1–42 self-induced
aggregation was then investigated using a ThT-based fluorometric
assay to quantify Ab fibril formation in the presence and absence of
inhibitor (Table 1).27 The similar inhibition percentages provided
by 2–9 with respect to 1 point to the conclusion that the presence
of different amyloid binding motifs is not so relevant against Ab
self-induced aggregation. However, the fact that all the derivatives
at 10 lM concentration inhibited amyloid aggregation at a similar
extent to the known anti-aggregator curcumin (curc) reinforce the
rationale that the BQ is a privileged motif for bivalent ligands tar-
geting PPI. Furthermore, the length of the spacer appears to play a
role, since the potency of all tested compounds (except 2 and 3)
was always higher for the three methylene-spaced compounds
than for the corresponding six methylene ones. Compound 3, bear-
ing the vanillic fragment and a 6-methylene chain, maintained an
inhibitory potency in the same range as the reference compound 1,
and was the most potent inhibitor of the series.
First, we tested the ability of 2–9 to inhibit AChE and butyrylch-
olinesterase (BuChE) catalytic activity in comparison with 1. As
reported in Table 1, the nature of the R residue strongly influenced
the ability to bind and inhibit cholinesterase activity. In contrast to

M. L. Bolognesi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2655–2658
2657
O
O
= Van
= BFur
= BTh
HO
H2N
NHZ
R
H
+
n
n=1 or 4
O
S
for 18 and 19
a
N
S
N
for 20-25
= PBTh
b
for 10 and 11
c
R
N
H
R
N
NHZ
NH2
n
n
H
for 12-17
10: R = Van; n = 1
11: R = Van; n = 4
18: R = Van; n = 1
19: R = Van; n = 4
20: R = BFur; n = 1
21: R = BFur; n = 4
22: R = BTh; n = 1
23: R = BTh; n = 4
24: R = PBTh; n = 1
25: R = PBTh; n = 4
d
12: R = BFur; n = 1
13: R = BFur; n = 4
14: R = BTh; n = 1
15: R = BTh; n = 4
16: R = PBTh; n = 1
17: R = PBTh; n = 4
O
O
O
O
2-9
Scheme 1. Reagents and conditions: (a) 1 equiv RCHO, trymethylorthoformate, rt, 1 h, then NaBH3CN, 5% CH3COOH, rt, overnight; (b) 1.1 equiv RCHO, toluene, Dean–Stark
trap, reflux, 3 h, then EtOH, NaBH4, rt, overnight; (c) H2, 10% Pd/C (10% w/w), MeOH, rt,2 h; (d) 30% HBr, AcOH, rt, overnight; (e) 0.5 equiv 2,5-dimethoxybenzoquinone, EtOH,
50 °C for 3 h, then rt, overnight.
Table 1
The neuroprotective effects of selected compounds were also
Inhibitory activity of human AChE and BuChE and amyloid self-aggregation by 2–9
and reference compounds 1 and curcumine (Curc)
determined against the Ab1–42 induced toxicity in human neuro-
nal-like SH-SY5Y cells using a colorimetric MTT assay.19 The
Compd
IC50 hAChE
M)a SEM
IC50 hBuChE
M)a SEM
Ab self-aggregation
3-methylene-spaced compounds were tested because of their
greater compliance with Lipinski’s rules (MW and lipophilicity)
and also because preliminary results showed that they were gener-
ally less cytotoxic than the higher homologues (data not shown).
Thus, intrinsic cell toxicity of newly synthesized compounds was
(l
(l
(%)b SEM
1c
2
3
4
5
6
7
8
9
0.00155 0.00011c
0.198 0.008
0.102 0.014
21.8 0.1
>>10
1.44 0.10c
8.24 0.43
1.60 0.01
2.60 0.19
84.2 0.2
>>10
66.8 4.4
22.0 1.6
50.4 0.6
27.2 2.0
18.4 1.8
16.2 1.4
18.5 1.0
41.4 6.8
25.7 1.1
34.4 1.1
first evaluated. Treating SH-SY5Y cells with 2 and 6 (1–50 lM)
>>10
did not lead to modified neuronal viability, whereas, 24 h treat-
ment of SH-SY5Y cells with 1 and 4 decreased neuronal viability
with IC50 (concentration of compound resulting in 50% inhibition
22.0 2.5
31.4 0.2
0.305 0.009
ndd
>>10
35.3 2.5
27.3 0.4
ndd
of neuronal viability) values of 19.4
Notably, curcumin exhibited a similar toxicity profile (IC50
12.8
M).30 We then assessed the protective effects of 1, 2, 4, and
lM and 14.8 lM, respectively.
Curc
=
a
Human recombinant AChE and BuChE from human serum were used. IC50
l
values represent the concentration of inhibitor required to decrease enzyme
activity by 50% and are the mean of two independent measurements, each per-
formed in duplicate; IC50 values were determined by following Ellman’s method;
6 against neurotoxicity induced by Ab1–42 oligomers in SH-SY5Y
cells. We used a range of concentrations of tested compounds
SEM = standard error of the mean.
(0.1–1
ronal viability. As shown in Figure 2, compounds 2, 4, and 6 at
M partially inhibited the Ab1–42 oligomer-induced neurotoxicity
(25–36%), while 1 exerted a stronger inhibition (70%) at the same
concentration. However, 2, at higher concentration (10 M),
produced a neuroprotective effect similar to that of 1 (ꢀ 80%).
lM for 1, 4, and 6; 0.1–10 lM for 2) that did not affect neu-
b
% inhibition of 50 lM Ab(1–42) self-induced aggregation by 10 lM compound.
The Ab(1–42)/inhibitor ratio was equal to 5/1. Values are the mean of two inde-
1
l
pendent experiments, each performed in duplicate.
c
From Ref. 9.
nd = not determined.
d
l
It should be also mentioned that PBTh derivatives may displace
ThT from amyloid fibrils.28 This may translate in an overestimation
of the inhibitory activity in the ThT-based assay. Therefore, to en-
sure that the fluorescence decrease observed for compounds 8 and
9 was exclusively due to the inhibition of fibril formation, control
In conclusion, an appropriate decoration of the BQ nucleus of 1
allowed us to identify novel multitargeted hit compounds.
Although the cholinesterase activities decreased significantly, the
new compounds were endowed with more balanced biological
profiles. In principle, novel MTDLs active in the micromolar range,
such as 2 and 4, represent interesting starting points for further
development. In fact, where connections exist between two or
more targets, as seems to be the case for AChE and Ab, MTDLs with
only moderate activities are expected to produce superior in vivo
effects compared to higher-affinity single-targeted compounds. In
this context, because most links in cellular networks are weak,
low-affinity MTDLs such as those developed herein, might well
experiments were performed adding compounds 8 and 9 (10 lM)
to aggregated Ab samples after dilution with ThT. Samples in
which compounds 8 and 9 were added to aggregated Ab samples
diluted with the ThT solution showed the same fluorescence out-
put as those containing only aggregated Ab and ThT, providing con-
vincing evidence that no displacement of ThT occurred in the used
experimental conditions. Therefore, any observed reduction in
fluorescence intensity can be exclusively attributed to the inhibi-
tion of Ab fibril formation by the tested compounds.
accomplish
cascade.
a significant modification of the AD neurotoxic

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100
80
60
40
20
0
100
a
b
80
60
40
20
0
1
2
4
6
0
0.1
1
10
Compound (1 µM)
Compound 2
Figure 2. (a) Effects of compounds (1 lM) on neurotoxicity induced by Ab1–42 oligomers in SH-SY5Y cells. (b) Effects of various concentrations of 2 (0.1–10 lM) on
neurotoxicity induced by Ab1–42 oligomers in SH-SY5Y cells. The neuronal viability in SH-SY5Y cells was determined by the MTT assay, after 3 h of incubation with Ab1–42
aggregated to oligomers in presence of compounds.29 The results are expressed as a percentage of inhibition of neurotoxicity induced by Ab1–42 oligomers and shown as
mean SD of at least two independent experiments.
18. Howlett, D. R.; Perry, A. E.; Godfrey, F.; Swatton, J. E.; Jennings, K. H.;
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This work was supported by grants from MIUR (FIRB RBNE03F-
H5Y) and the University of Bologna. The present work was also
founded by an European FP7 grant (Bio-Inspired Self-assembled
Nano-Enabled Surfaces–BISNES).
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