Quinones bearing non-steroidal anti-inflammatory fragments as multitarget ligands for Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2013.09.091

The anti-amyloid properties shared by several quinones inspired the design of a new series of hybrids derived from the multi-target drug candidate memoquin (1). The hybrids consist of a central benzoquinone core and a fragment taken from non-steroidal anti-inflammatory drugs, connected through polyamine linkers. The new hybrids retain the potent anti-aggregating activity of the parent 1, while exhibiting micromolar AChE inhibitory activities. Remarkably, 2, 4, (R)-6 and (S)-6 were Aβ aggregation inhibitors even more potent than 1. The balanced amyloid/cholinesterase inhibitory profile is an added value that makes the present series of compounds promising leads against Alzheimer's disease.

Full text of DOI:10.1016/j.bmcl.2013.09.091

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6254–6258
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Quinones bearing non-steroidal anti-inflammatory fragments
as multitarget ligands for Alzheimer’s disease
Federica Prati a,b, Manuela Bartolini a, Elena Simoni a, Angela De Simone c, Antonella Pinto d,
Vincenza Andrisano c, Maria Laura Bolognesi a,
⇑
a Department of Pharmacy and Biotechnology, Alma Mater Studiorum University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
b Department of Drug Discovery and Development, Italian Institute of Technology, Via Morego 30, 16163 Genoa, Italy
c Department for Life Quality Studies, Alma Mater Studiorum University of Bologna, Corso D’Augusto 237, 47921 Rimini, Italy
d Department of Drug Sciences—Pharmacology, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2013
Revised 24 September 2013
Accepted 27 September 2013
Available online 5 October 2013
The anti-amyloid properties shared by several quinones inspired the design of a new series of hybrids
derived from the multi-target drug candidate memoquin (1). The hybrids consist of a central benzoqui-
none core and a fragment taken from non-steroidal anti-inflammatory drugs, connected through
polyamine linkers. The new hybrids retain the potent anti-aggregating activity of the parent 1, while
exhibiting micromolar AChE inhibitory activities. Remarkably, 2, 4, (R)-6 and (S)-6 were Ab aggregation
inhibitors even more potent than 1. The balanced amyloid/cholinesterase inhibitory profile is an added
value that makes the present series of compounds promising leads against Alzheimer’s disease.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Memoquin
Alzheimer’s disease
Non-steroidal anti-inflammatory drugs
Amyloid aggregation inhibitors
Acetylcholinesterase inhibitors
Alzheimer’s disease (AD) is an incurable neurodegenerative
malady of the central nervous system with a complex multifacto-
rial etiology.1,2 In response to such molecular complexity, a
polypharmacological drug discovery approach is envisaged to
provide therapeutic benefits where currently available single-
target drugs have failed. There are two main ways to achieve
polypharmacology: drug combinations and single chemical
entities that have multiple biological properties, that is the so
called multitarget drugs. It has been advanced that the use of
multitarget drugs could have general inherent advantages over
combination therapies. Administering one compound with
multiple biological actions guarantees the simultaneous presence
of the molecule in those districts of the body, where the active
principle needs to work and interact with its multiple targets.
Focusing on AD, two critical issues are addressed: (i) the risk of
possible drug–drug interactions in elderly patients would be
reduced and (ii) the therapeutic regimen greatly simplified, with
activity. In vivo, 1 acts as a cognitive enhancer in several AD mouse
models, strengthening the value of a multitarget strategy in AD.5,6
From a structural point of view, 1 is a hybrid molecule obtained
by integrating a benzoquinone core into a polyamine chain.7 The
resulting 2,5-diamino-benzoquinone scaffold of
1 has been
deemed to have a crucial role in conferring the multiple activities.
In particular, thanks to the planar and aromatic features and the
hydrogen bonding capability, it might be essential in modulating
protein–protein interactions involved in AD pathogenesis.8 In fact,
several other quinones (either benzo-, naphtho- or anthra-
quinones) have been shown to effectively inhibit the aggregation
of various amyloidogenic proteins.9–13
The anti-aggregating capability we could verify for several
hybrid molecules featuring a 2,5-diamino-1,4-benzoquinone core
connecting two aromatic appending moieties, lends further
support to this hypothesis.14–18 The selected aromatic moieties
where taken from known amyloidophilic agents, such as, among
others, curcumin, benzofurans and benzothiazoles. Intriguingly,
such molecules were shown to be effective inhibitors of Ab fibril
formation,14–16 but also of prion protein aggregation.17,18 For these
reasons, the 2,5-diamino-1,4-benzoquinone fragment can be
the prospect of enhanced patient compliance.3 Exploiting
a
multitarget drug discovery approach, memoquin (1; Fig. 1) was
developed as one of the first multitarget drug candidate against
AD.4 Compound 1 is a free-radical scavenger and an inhibitor of
amyloid-b (Ab) aggregation and acetylcholinesterase (AChE)
considered as
a truly privileged motif to interfere with
protein–protein interactions and a useful starting point for the
design of novel multitarget ligands against AD. Expanding this
basic idea, we developed herein a further series of quinone-based
hybrids. Looking for novel aromatic fragments to be appended to
⇑
Corresponding author.
E-mail address: marialaura.bolognesi@unibo.it (M.L. Bolognesi).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.09.091

F. Prati et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6254–6258
6255
Figure 1. Design strategy for compounds 2–9.
the benzoquinone core, we turned our attention to non-steroidal
anti-inflammatory drugs (NSAIDs), that is ibuprofen, sulindac,
indomethacin, diclofenac and flurbiprofen (Fig. 1). This choice
was motivated by the fact that this specific NSAID subset has been
demonstrated to directly inhibit Ab fibril formation and destabilize
preformed Ab fibrils in vitro.19 In addition, all of them have also
been shown to affect the production of Ab.20 As a further positive
evidence, epidemiological studies indicated that NSAIDs might
lower the risk of developing AD.21 Thus, hybrids 2–9 were designed
and synthesized by connecting the 2,5-diamino-benzoquinone
core with the NSAID-pharmacophoric elements through the poly-
amine linkers that were successfully exploited in a previous
endorsement.18 Considering that monomeric naphthoquinone ana-
logues of 1 retained the multitarget profile of the parent com-
pound,22 the monomeric hybrid 9 was also designed (Fig. 1).
The synthetic route used to access the target compounds is sum-
marized in Schemes 1 and 2.23 The presence in all the selected NSA-
IDs of a free carboxylic acid functionality provided a straightforward
attachment point to the amino group of the polyamine linker
through amide formation. Thus, the properly Boc-protected poly-
amines 10–1224 were coupled with the respective NSAID fragments
through standard coupling conditions, affording intermediates 13–
21. After a deprotection step, the primary amino groups of 22–30
were connected to the central benzoquinone core by exploiting an
efficient substitution reaction on 2,5-dimethoxy-benzoquinone.7
In the case of 9, the amino group of 26 was connected to the naph-
thoquinone moiety through a Michael addition reaction.
To characterize the in vitro anti-AD multitarget profile of our
derivatives, the ability to reduce Ab42 spontaneous aggregation
and secretion (through the inhibition of beta-secretase activity)
and the inhibitory activity on the cholinesterase enzymes that is,
human AChE and butyrylcholinesterase (BuChE), were investigated
in comparison to 1.
Even if the main triggering event leading to the AD pathology is
still under debate, Ab aggregation is well recognized to be con-
nected to AD pathogenesis and progression. Therefore, it is still fea-
sible that anti-amyloid agents might impact the disease
progression in a meaningful way.25,26 Thus, the in vitro anti-
amyloid potential of all synthesized quinone-NSAID hybrids was
determined using a Thioflavin T (ThT)-based fluorometric assay.8
Inhibition studies were carried out by incubating Ab42 (50
lM)
with and without a five fold lower concentration of tested com-
pound and by evaluating the decrease in ThT fluorescence intensity
at 490 nm (kexc = 446 nm). We were pleased to verify that all hy-
brids carrying a triamine linker (2–6) exhibited percentages of
inhibition ranging from 55% to 90% (Table 1). In agreement with
the anti-aggregating properties reported for some NSAIDs,19 the
presence of a flurbiprofen (6), ibuprofen (2), indomethacin (5), or
diclofenac (4) fragment leads to an inhibitory potency slightly
higher than that of the parent compound 1. On the basis of these
promising results, the correspondent IC50 values were determined.
In addition, the high inhibitory potency (% inhibition >90%) shown
by 6 as racemic mixture prompted us to investigate the activity of
the single enantiomers ((R)-6 and (S)-6). The effect of the
CF3
b, c
a
Boc
H2N
N
H
NH2
O
N
H
N
H
NH2
H2N
N
Boc
N
H
10
Scheme 1. Reagents and conditions: (a) EtOCOCF3, MeOH, À50 °C; (b) (Boc)2O; (c); NaOH/H2O.

6256
F. Prati et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6254–6258
O
a
Boc
Boc
RCOOH
+
H2N
X
N
H
R
N
H
X
N
H
carboxylic acid
10: X= -(CH2)2N(Boc)(CH2)2-
11: X= -(CH2)4-
13: R= Ibuprofen; X= -(CH2)2N(Boc)(CH2)2-
14: R= Sulindac; X= -(CH2)2N(Boc)(CH2)2-
: R= Diclofenac; X=-(CH2)2N(Boc)(CH2)2-
: R= Indomethacin; X= -(CH2)2N(Boc)(CH2)2-
17: R= Flurbiprofen; X= -(CH2)2N(Boc)(CH2)2-
18: R= (R)-Flurbiprofen; X= -(CH2)2N(Boc)(CH2)2-
19: R= (S)-Flurbiprofen; X= -(CH2)2N(Boc)(CH2)2-
: R= Flurbiprofen; X= -(CH2)4-
: R= Flurbiprofen; X= -CH2-
12
: X= -CH2-
Ibuprofen:
Indomethacin:
15
16
COOH
O
O
COOH
COOH
N
Sulindac:
F
20
21
Cl
(
)/( )/( )-Flurbiprofen:
S RS
R
O
b
S
F
O
COOH
Diclofenac:
R
N
H
X
NH2
Cl
NH Cl
22: R= Ibuprofen; X= -(CH2)2NH(CH2)2-
23: R= Sulindac; X= -(CH2)2NH(CH2)2-
24: R= Diclofenac; X=-(CH2)2NH(CH2)2-
HOOC
25
26
: R= Indomethacin; X= -(CH2)2NH(CH2)2-
: R= Flurbiprofen; X= -(CH2)2NH(CH2)2-
27: R= (R)-Flurbiprofen; X= -(CH2)2NH(CH2)2-
28: R= (S)-Flurbiprofen; X= -(CH2)2NH(CH2)2-
29: R= Flurbiprofen; X= -(CH2)4-
O
O
30
: R= Flurbiprofen; X= -CH2-
O
O
O
c
d
O
O
H
N
H
N
O
R
X
O
O
N
H
X
N
R
H
O
H
N
H
N
X
R
2
: R= Ibuprofen; X= -(CH2)2NH(CH2)2-
: R= Sulindac; X= -(CH2)2NH(CH2)2-
O
3
4: R= Diclofenac; X=-(CH2)2NH(CH2)2-
O
5: R= Indomethacin; X= -(CH2)2NH(CH2)2-
6: R= Flurbiprofen; X= -(CH2)2NH(CH2)2-
9
: R= Flurbiprofen ; X= - (CH2)2NH(CH2)2-
6
(R)- : R= (R)-Flurbiprofen; X= -(CH2)2NH(CH2)2-
6
(S)- : R= (S)-Flurbiprofen; X= -(CH2)2NH(CH2)2-
7: R= Flurbiprofen; X= -(CH2)4-
8: R= Flurbiprofen; X= -CH2-
Scheme 2. Reagents and conditions: (a) THF, EDC, Et3N; (b) TFA; (c) 0.5 equiv 2,5-dimethoxybenzoquinone, EtOH, 50–80 °C for 3–5 h; (d) 1 equiv naphthoquinone, MeOH, rt,
overnight.
Table 1
Inhibitory activity on amyloid self-aggregation and human AChE and BuChE activities by 2À9 and reference compound 1a
Inhibition of Ab42 aggregation
Inhibition of cholinesterase activity
hAChE hBuChE
b
R
X
[I] = 10
l
Ma (%)
IC50
(l
M)
c
c
IC50
(
lM)
IC50 (lM)
1
2
3
4
5
6
(R)-6
(S)-6
66.8 4.4
82.6 4.5
55.0 3.0
74.9 0.2
80.1 3.0
>90
5.93
4.83
8.18
3.15
6.11
—
4.77
4.43
ndd
nd
(1.55 0.11)10-3
0.60 0.02
9.54 0.29
15.4 1.1
10.3 3.7
—
27.8 1.1
2.89 0.12
>20
1.44 0.10
22.5 1.5
14.7 0.2
0.36 0.02
18.7 1.0
—
9.98 0.60
7.77 0.21
>20
Ibuprofen
Sulindac
Diclofenac
–(CH2)2NH–(CH2)2–
–(CH2)2NH–(CH2)2–
–(CH2)2NH–(CH2)2–
–(CH2)2NH–(CH2)2–
–(CH2)2NH–(CH2)2–
––(CH2)2NH–(CH2)2–
–(CH2)2NH–(CH2)2–
–(CH2)4–
Indomethacin
Flurbiprofen
(R)-Flurbiprofen
(S)-Flurbiprofen
Flurbiprofen
Flurbiprofen
See Figure 1 for structure
Tacrine
87.2 1.6
>90
7
8
9
ndd
–CH2–
12.7 3.8
76.4 2.5
nae
>20
116
2
6.88
—
107
7
16.4 0.2
0.058 0.005
0.383 0.02
a
b
c
Percent inhibition of 50
IC50 represents the concentration of inhibitor required to decrease the ThT fluorescence intensity at 490 nm by 50%.
Human recombinant AChE and BuChE from human serum were used. IC50 values, determined by using Ellman’s method,29 represent the concentration of inhibitor
lM Ab42 aggregation by 10 lM compound. The Ab42/inhibitor ratio was equal to 5:1.
required to decrease enzyme activity by 50%.
d
nd = not determined. Not determined because not soluble in the assay conditions.
% Inhibition <5% at 50 lM. na = not active.
e

F. Prati et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6254–6258
6257
polyamine linker was also evaluated, by synthesizing the
1,3-hexane- (7) and 1,6-propane-diamino (8) congeners of 6.
All the tested hybrids showed low micromolar activities that
make them potent anti-aggregating agents. Differences in the
inhibitory potencies are quite little within the series, being the
difference between the less and the most active within two
folds.
inhibition obtained at the screening concentration, IC50 values
were not determined.
To assess safety profile, the in vitro cytotoxicity of the most
interesting derivatives, namely compounds 4, (R)- and (S)-6 and
9, was evaluated using HEK 293 cell line. Results showed that all
the tested compounds, but (S)-6, did not significantly reduce cell
viability up to 10 lM (Fig. 2).
No enantioselectivity was found for 6, as (R)-6 and (S)-6 display
very similar inhibitory activities on amyloid aggregation.
Conversely, the depletion in the polyamine linker of 6 of the
protonable nitrogen atom not only influenced the physical–chem-
ical properties, but also modified its ability of interacting with
amyloid oligomers and fibrils. In fact, 7 could be not tested because
not soluble in the assay conditions, whereas 8 was a very weak
inhibitor with a percentage of inhibition of only 12%.
Building on the low in vitro cytotoxicity shown by 9 in HEK cell
line (Fig. 2) and the good cell toxicity profile on primary neurons
shown by similar naphtoquinone derivatives,22 the effect of 9 on
APP processing was evaluated in a cellular context. The study
was carried out in embryonic chicken telencephalon neurons to as-
sess the effect on Ab secretion.22 All data were corrected with
mean neurons viability evaluated in the MTT assay. A mild reduc-
tion on Ab38, Ab40, and Ab42 secretion was observed at 10
because of some detrimental toxicity displayed at 25 M and 50
M, a clear concentration-dependent decrease in Ab secretion
could not be observed (Fig. 3).
lM, but,
Notably, 4 showed a remarkable IC50 of 3.15
lM, which makes it
l
the most active of the current series, but also the top-ranked
l
among all 1’s derivatives tested so far.7,15,16,22
As a general comment, the significant anti-aggregating activity
shown by 2–8, which all share a common bivalent structure,
reinforces the intriguing hypothesis that such palindromic
compounds could cross-link two amyloid molecules and
consequently effectively perturb the fibrillogenesis process.27
The anti-fibrillogenic activities showed by naphthoquinone 9 is
also intriguing. Indeed, despite the lack of a bivalent structure, 9
shows an IC50 value which is slightly higher than that of 1 (6.33
From the clinical use it is well known that AChE inhibitors, al-
beit palliative, are effective in improving activities of daily living,
behavior, and slowing cognitive decline in moderate to severe AD
patients. Moreover, APP processing is under the control of several
major neurotransmitters such as acetylcholine (ACh), therefore
an ACh level increase by AChE inhibitors can also lower amyloid
production.28 Thus, the anticholinesterase activity can still be con-
sidered an important component in the overall multitarget profile
of an AD drug candidate. On this basis, the anticholinesterase activ-
ities of 2–9 were also evaluated (Table 1) in comparison with 1. An
analysis of the results revealed that the new hybrids still keep
inhibitory potencies against human AChE (hAChE) and human
BuChE (hBuChE), in the micromolar range. However, as expected
on the basis of previous results,7 they are three fold less potent
than 1, which is a nanomolar AChE inhibitor. It is highly feasible
that the lack of a protonable nitrogen atom (interacting with the
AChE anionic site) in the case of 7 and 8, or of the N-ethyl
lM vs 5.88 lM, respectively). This result is in line with the
outstanding anti-amyloid profile shown by the 1,4-naphthoqui-
non-2-yl-L-tryptophan developed by Gazit and coworkers.13
To expand the anti-amyloid profile of 2–9, their ability to inhibit
the amyloidogenic activity of human recombinant beta-secretase-
1 (BACE-1) enzyme was also investigated. When screened at a
concentration of 3 lM using M-2420 (Bachem) as assay substrate,
these compounds showed to be weak BACE-1 inhibitors with per-
centages of inhibition ranging from 20% to 30%. Due to the low % of
Figure 2. MTT assay to measure cell viability in HEK 293 cell line after treatment with 4 (a), (R)-6 (b), (S)-6 (c), and 9 (d) at concentrations ranging from 0.3125 to 20
control. Statistical analysis was performed with Dunnett’s multiple comparison test with⁄p <0.05 ⁄⁄p <0.01 versus untreated cells.
lM. Ctrl:

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F. Prati et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6254–6258
Figure 3. Secretion of Ab38, Ab40, and Ab42 after treatment with 9. Values represent the mean and the SEM of Ab38, Ab40, and Ab42 secretion in percent from one experiment
performed in 24-well plates (n = 3 per experiment) for each inhibitor concentration and for controls. Since cells were cultured in two 24-well plates, each plate contained a
separate vehicle control (VC) group. VC1B indicates the vehicle control on plate 1 and VC2B on plate 2. Vehicle controls were set as 100%.
substituent for 2–6 can account for that.7 Among the homogenous
triamine series 2–6, the most active is the ibuprofen derivative 2
4. Cavalli, A.; Bolognesi, M. L.; Capsoni, S.; Andrisano, V.; Bartolini, M.; Margotti,
E.; Cattaneo, A.; Recanatini, M.; Melchiorre, C. Angew. Chem., Int. Ed. 2007, 46,
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(IC50 = 0.60
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M), which turned out more potent than the marketed
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PLoS One 2010, 5, e11101.
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Bartolini, M.; Andrisano, V.; Melchiorre, C. J. Med. Chem. 2009, 52, 7883.
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23. Spectra data for compound 4: 1H NMR (200 MHz, CDCl3) d 1.68–1.80 (m, 8H),
2.61–2.73 (m, 8H), 3.26–3.30 (m, 4H), 3.42–3.50 (m, 4H), 3.51 (s, 2H
exchangeable with D2O), 3.85 (s, 4H), 5.35 (s, 2H), 6.51 (d, J = 8.0, 2H), 6.83–
7.18 (m complex, 8H), 7.18–7.37 (m, 4H+2H exchangeable with D2O), 7.76 (br
s, 2H exchangeable with D2O), 8.48 (br s, 2H exchangeable with D2O); ESI–MS
(m/z): 923 (M+H+), 945 (M+Na+).
l
M)7 and rivastigmine (3.03 M).7
l
A slightly lower activity is observed for (S)-6, whereas the enantio-
mer (R)-6 is ten times less effective. Compound 9, carrying the flur-
biprofen fragment, but devoid of a bivalent structure, resulted less
potent than (R)-6 and (S)-6. Moreover, the type of NSAID fragment
seems to influence the selectivity for one of the two ChEs. Indeed
while 2 (bearing the ibuprofen fragment) is 37.5 times more potent
on AChE, the derivative 4 (bearing the diclofenac fragment) is 42.8
more potent on BuChE. To confirm the importance of a proper
integration of single fragments into a new chemical entity, an equi-
molar mixture of the three fragments composing 2 (bis-(3-amino-
propyl)amine, 1,4-benzoquinone and ibuprofen) was assayed. At
67
lM, inhibition resulted 26.3% 1.8% and <10% on hAChE and
hBuChE, respectively whereas, at the same concentration, com-
pound 2 gave a complete inhibition of hAChE activity and ꢀ70%
inhibition of the hBuChE’s hydrolyzing activity. These results fur-
ther confirm that the suitable incorporation of the different struc-
tural elements into a new single chemical entity enables the
achievement of higher inhibitory potency and the selectivity.
Taken together, the results presented here indicate that targeting
Ab by a quinone structure is a promising approach for the inhibition
of amyloid fibrillogenesis. Because Ab misfolding and aggregation
are still considered key early pathogenic events in AD, it is of great
interest for a potential disease-modifying treatment.
Additionally, 2–9 have been found to display another pharma-
cological effect, namely a cholinesterase inhibitory activity, which
should nicely complement the anti-amyloid one.
In conclusion, thanks to a balanced micromolar Ab/cholinester-
ase profile, the here presented quinone–NSAID hybrids could be a
promising starting point in the search for new multitarget ligands
against AD.
Acknowledgements
This work was supported by grants from MIUR ((PRIN
2009ESXPT2_001, PRIN 2009Z8YTYC_003) and the University of
Bologna. Thanks are also due to UniRimini S.p.A. for financial
support.
24. Bolognesi, M. L.; Calonghi, N.; Mangano, C.; Masotti, L.; Melchiorre, C. J. Med.
Chem. 2008, 51, 5463.
25. Bartolini, M.; Andrisano, V. ChemBioChem 2010, 11, 1018.
26. Belluti, F.; Rampa, A.; Gobbi, S.; Bisi, A. Expert Opin. Ther. Pat. 2013, 23, 581.
27. Kolstoe, S. E.; Mangione, P. P.; Bellotti, V.; Taylor, G. W.; Tennent, G. A.; Deroo,
S.; Morrison, A. J.; Cobb, A. J.; Coyne, A.; McCammon, M. G.; Warner, T. D.;
Mitchell, J.; Gill, R.; Smith, M. D.; Ley, S. V.; Robinson, C. V.; Wood, S. P.; Pepys,
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