In vitro antiamyloidogenic properties of 1,4-naphthoquinones

Article abstract of DOI:10.1016/j.bbrc.2010.08.038

The aim of this study is to find out whether several 1,4-naphthoquinones (1,4-NQ) can interact with the amyloidogenic pathway of the amyloid precursor protein processing, particularly targeting at β-secretase (BACE), as well as at β-amyloid peptide (Aβ) aggregation and disaggregating preformed Aβ fibrils. Compounds bearing hydroxyl groups at the quinoid (2) or benzenoid rings (5, 6) as well as some 2- and 3-aryl derivatives (11-15) showed BACE inhibitory activity, without effect on amyloid aggregation or disaggregation. The halogenated compounds 8 and 10 were selective for the inhibition of amyloid aggregation. On the other hand, 1,4-naphthoquinone (1), 6-hydroxy-1,4-naphthoquinone (4) and 2-(3,4-dichlorophenyl)-1,4-naphthoquinone (26) did not show any BACE inhibitory activity but were active on amyloid aggregation and disaggregation preformed Aβ fibrils. Juglone (5-hydroxy-1,4-naphthoquinone (3), and 3-(p-hydroxyphenyl)-5-methoxy-1,4-napththoquinone (19) were active on all the three targets. Therefore, we suggest that 1,4-NQ derivatives, specially 3 and 19, should be explored as possible drug candidates or lead compounds for the development of drugs to prevent amyloid aggregation and neurotoxicity in Alzheimer's disease.

Full text of DOI:10.1016/j.bbrc.2010.08.038

Biochemical and Biophysical Research Communications 400 (2010) 169–174
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
In vitro antiamyloidogenic properties of 1,4-naphthoquinones
a
a
a
Paloma Bermejo-Bescós a, , Sagrario Martín-Aragón , Karim L. Jiménez-Aliaga , Andrea Ortega ,
⇑
María Teresa Molina b, Eduardo Buxaderas c, Guillermo Orellana c, Aurelio G. Csákÿ c
a Departamento de Farmacología, Facultad de Farmacia, Universidad Complutense de Madrid, Spain
b Instituto de Química Médica (CSIC), Madrid, Spain
c Departamento de Química Orgánica, Facultad de Química, Universidad Complutense de Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 August 2010
Available online 13 August 2010
The aim of this study is to find out whether several 1,4-naphthoquinones (1,4-NQ) can interact with the
amyloidogenic pathway of the amyloid precursor protein processing, particularly targeting at b-secretase
(BACE), as well as at b-amyloid peptide (Ab) aggregation and disaggregating preformed Ab fibrils. Com-
pounds bearing hydroxyl groups at the quinoid (2) or benzenoid rings (5, 6) as well as some 2- and 3-aryl
derivatives (11–15) showed BACE inhibitory activity, without effect on amyloid aggregation or disaggre-
gation. The halogenated compounds 8 and 10 were selective for the inhibition of amyloid aggregation. On
the other hand, 1,4-naphthoquinone (1), 6-hydroxy-1,4-naphthoquinone (4) and 2-(3,4-dichlorophenyl)-1,4-
naphthoquinone (26) did not show any BACE inhibitory activity but were active on amyloid aggregation
and disaggregation preformed Ab fibrils. Juglone (5-hydroxy-1,4-naphthoquinone (3), and 3-(p-hydroxy-
phenyl)-5-methoxy-1,4-napththoquinone (19) were active on all the three targets. Therefore, we suggest
that 1,4-NQ derivatives, specially 3 and 19, should be explored as possible drug candidates or lead com-
pounds for the development of drugs to prevent amyloid aggregation and neurotoxicity in Alzheimer’s
disease.
Keywords:
1,4-Naphthoquinones
b-Secretase (BACE)
Amyloid aggregation
Ab fibrils
Alzheimer’s disease
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
insults based upon its ability to activate adaptive cellular stress re-
sponse pathways [10]. Some naphthoquinone derivatives from the
1,4-Naphthoquinones (1,4-NQ) are widely spread in nature and
their interest lies on their broad-range biological action though
mainly owing to naphthoquinones being redox active organic
cofactors and important species in biological systems [1]. Quinones
account for one of the largest families of antitumor agents [2]. The
basic knowledge on quinone studies has been used to design new
anticancer drugs, improving selectivity and providing a more
rational therapeutic application of them [3]. The 1,4-NQ scaffold
has been identified as a new class of Hsp90 inhibitors [4] which
could be useful for the treatment of cancer [5] and numerous
neurodegenerative disorders, including Alzheimer’s disease (AD)
and Parkinson’s disease, in which protein aggregation is a common
etiology [6,7]. Different derivatives of 2-hydroxy-1,4-naphthoqui-
none ring have shown to activate the nuclear receptor peroxisome
root of Lithospermum erythrorhizon [11] have been reported to
provide neuroprotection by reducing the release of various proin-
flammatory molecules from activated microglia. The 2,3,6-tri-
methyl-1,4-naphthoquinone, a MAO inhibitor which is present in
the tobacco plant and smoke, has been identified as neuroprotec-
tive in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
C57BL/6 mouse model of neurodegeneration [12]. MK-4, a naph-
thoquinone with a side chain of four isoprenoid units, which is
the predominant form of vitamin K in the brain [13], has shown
to prevent oxidative cell death induced by reduced glutathione
depletion in primary oligodendrocyte precursors and in primary
cortical neurons [14].
b-Amyloid peptide (Ab) is the major constituent of senile plaques
found in the brain of patients with AD, and the aggregation and
deposition of the Ab in neural tissue give rise to the neuronal atrophy
that is characteristic of the disease [15]. Decreasing Ab production or
aggregation are two therapeutic approaches for AD. The first one is
based primarily on the inhibition of two proteolytic enzymes,
proliferator-activated receptor gamma (PPARc) [8], strategy which
could be promising in the treatment of several neurological pathol-
ogies [9]. Exposure of neuroblastoma cells and primary cortical neu-
rons to 2-methyl-5-hydroxy-1,4-naphthoquinone (6) has shown to
provide protection against subsequent oxidative and metabolic
b- and c-secretases, that participate in the processing of the amyloid
precursor protein (APP) [16]. The second one lies on inhibiting Ab
aggregation and destabilizing preformed Ab fibrils [17].
Rifampicin and its derivatives, which possess a naphthohydro-
quinone or naphthoquinone structure, have demonstrated to
⇑
Corresponding author. Address: Departamento de Farmacología, Facultad de
Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal s/n, 28040
Madrid, Spain. Fax: +34 913941726.
E-mail address: bescos@farm.ucm.es (P. Bermejo-Bescós).
0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.08.038

170
P. Bermejo-Bescós et al. / Biochemical and Biophysical Research Communications 400 (2010) 169–174
inhibit Ab (1–40) aggregation and neurotoxicity in the PC12 cells.
Similarly, it has been shown that tetracyclines and doxycycline
not only inhibited the Ab (1–42) fibril formation but also disassem-
bled the preformed fibrils in vitro [18,19].
signal. The level of secretase enzymatic activity is proportional to
the fluorimetric reaction. BACE assay was carried out at 37 °C using
0.24 U of human recombinant BACE enzyme and 10
in 20 mM sodium acetate buffer (pH 4.5) in a final volume of
100 l. Fluorescence was measured using a FLx800 microplate
lM substrate
A wide array of anti-amyloid and neuroprotective therapeutic
approaches are under investigation on the basis of the hypothesis
that Ab peptide plays a pivotal role in disease onset and progression.
Development of b-secretase (BACE) inhibitors and targeting the
fibrillary aggregates of Ab peptide are strategies which are being ex-
plored and could promise for acute improvement in AD [20]. There-
fore, interventions with agents that reduce amyloid production or
limit aggregation, or increase removal might block the cascade of
events comprising AD pathogenesis. Based on some reported neuro-
protective effects of naphthoquinones, it was of our interest to find
out whether several 1,4-NQ can interact with the amyloidogenic
pathway of the APP processing, particularly targeting at BACE which
plays a crucial role in the rate-limiting step of the amyloid cascade,
as well as at Ab aggregation and destabilizing preformed Ab fibrils.
l
fluorescence reader (Bio-tek Instruments, Inc.). Wavelengths of
excitation and emission were 360 and 528 nm, respectively. The
enzyme activity assay was performed in the absence (control reac-
tion), and in the presence of the 1,4-NQ. Before the addition of the
substrate the human recombinant BACE enzyme and 1,4-NQ were
preincubated at 37 °C for 1 h. The inhibition ratio of BACE activity
was calculated from the percentage of control after 1 h of incuba-
tion once the substrate was added. Data were expressed as the 50%
inhibitory concentration (IC50).
2.5. Preparation of A (25–35) peptide
Ab (25–35) peptide was dissolved at 1 mM in PBS. For aggrega-
tion inhibition experiments, 10
with the 1,4-NQ and incubated at 37 °C for 4 days. For disaggregat-
ing experiments, 10 M Ab (25–35) peptide was incubated at 37 °C
lM Ab (25–35) peptide was mixed
2. Materials and methods
l
for 4 days to generate fibrils. Preformed fibrils were mixed with the
1,4-NQ for 4 additional days at 37 °C. The degree of Ab aggregation
was determined using both Thioflavin-T (Thio-T) fluorescence and
Electron microscopy (EM) analyses. The degree of Ab disaggrega-
tion was determined using Thio-T fluorescence analysis.
2.1. Reagents
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) and human recombinant b-secretase (BACE) were obtained
from Sigma–Aldrich Chemical Company (St. Louis, USA). Fluoro-
genic BACE (H-RE(EDANS) EVNLDAEFK(DABCYL)R-OH) substrate
was obtained from Calbiochem (La Jolla, CA). Ab (25–35) peptide
was purchased from GenScript (New Jersey, USA), 200-mesh cop-
per grids coated with Formvar/carbon film (TAAB Essentials for
Microscopy, UK).
2.6. Time-resolved fluorescence spectroscopy
Aliquots from each incubated sample were treated with Thio-T
(final concentration 25 lM). Each test sample was shaken for 10 s
prior to each measurement. Fluorescence intensity decay was mea-
sured by using an apparatus FluoroMax-4Ò based on the time-cor-
related single-photon counting method, including a diode laser
(Horiba–Jobin–Yvon). The fluorescence spectroscopy technique
used in this work measures the time dependence of the emission
intensity. Lifetimes represent the average times during which the
fluorophore (Thio-T) remains in the first excited electronic state
prior to its return to the ground state. An examination of the life-
times will permit identification of 1,4-NQ as inhibitors of amyloid
aggregation and/or disaggregates of amyloid once the fibrils are
formed. Samples were excited at 463 nm and the fluorescence
intensity decay was registered at 482 nm. The pulses of laser exci-
tation on the sample result in a histogram in the multichannel ana-
lyzer, which represents the number of counts in each channel,
which in turn corresponds to the sample fluorescence decay pro-
file. The fluorescence was measured until a predetermined signal
count of 10,000 was reached. We conducted a control experiment
to rule out the possibility that the low values in the Thio-T fluores-
cence assay might result from a quenching effect by the inhibitor.
No significant quenching effect was observed (data not shown).
2.2. Compound synthesis
1,4-NQ 4 [21], 9 [22], 10 [23] and 26 [24] were synthesized fol-
lowing literature procedures. 1,4-NQ 11, 12 and 27, 28 were syn-
thesized from 1,4-naphthoquinone (1), 1,4-NQ 13–17, 20 and 21
from 5-hydroxy-1,4-naphthoquinone (juglone, 3) by direct aryla-
tion with boronic acids under dicationic Pd(II)-catalysis as recently
described [25]. 1,4-NQ 20 and 21 were synthesized, respectively,
by methylation or acetylation of 3 [25]. The rest of 1,4-NQ were
commercially available. All 1,4-NQ were dissolved in DMSO and
stored frozen at À20 °C.
2.3. Determination of cell viability
The mitochondrial-dependent reduction of MTT to formazan
was used to exclude a cytotoxic effect of the 1,4-NQ in SH-SY5Y
neuroblastoma cells. Cells were plated in 96-well polystyrene
plates with 40,000 cells per well and incubated at 37 °C for 24 h
to allow cells to attach. The plates were then incubated with the
different 1,4-NQ in a concentration range of 1–10
tional 24 h. Cell viability was determined by addition of 20
2 mg/ml MTT to each well. After incubation for 1 h at 37 °C, the
medium was removed and 100 l of DMSO was added to dissolve
Data were expressed as IC50
2.7. Electron microscopy (EM) analyses
A 3 l aliquot of each reaction mixture was spotted to 200-
.
l
M for an addi-
ll of
l
l
the formazan crystals. The absorbance was read at 550 nm using a
Digiscan Microplate Reader (Assys Hitech, Kornenburg, Austria).
The optical density of the formazan formed in the control cells (un-
treated) was taken as 100% viability.
mesh copper grids coated with Formvar/carbon film and incubated
for 30 s. Afterwards, the peptide was stained with 2% uranyl ace-
tate in water. This solution was wicked off and then the grid was
air-dried. Samples were examined using a JEOL JEM-4000 EX trans-
mission electron microscopy.
2.4. b-Secretase (BACE) enzyme activity assay
2.8. Data statistical analyses
The assay is based on the secretase-dependent cleavage of a
secretase-specific peptide conjugated to the fluorescent reporter
molecules and DABCYL, which results in the release of a fluorescent
All experiments were done at least in three independent exper-
iments and all assays were done in triplicates. Data were analyzed

P. Bermejo-Bescós et al. / Biochemical and Biophysical Research Communications 400 (2010) 169–174
171
Table 1
3.2. BACE inhibitory activity
b-Secretase (BACE) inhibitory activity of 1,4-naphthoquinones in a cell-free assay
system.
A series of 1,4-NQ were tested for their inhibition of BACE activ-
ity in a cell-free assay system (Table 1). Our results revealed that
the 1,4-NQ 2, 3, 5, 9, 11, 12 and 14 had BACE inhibitory activity
Compound
IC50 (lM)
1,4-Naphthoquinone (1)
–
2-Hydroxy-1,4-naphthoquinone (2)
5-Hydroxy-1,4-naphthoquinone (3)
6-Hydroxy-1,4-naphthoquinone (4)
5,8-Dihydroxy-1,4-naphthoquinone (5)
5.96
6.51
–
with IC50 values lower than 10 lM.
3.3. Amyloid aggregation process
9.08
2-Methyl-5-hydroxy-1,4-naphthoquinone (6)
2-Methyl-1,4-naphthoquinone (7)
2-Bromo-1,4-naphthoquinone (8)
2-Bromo-5-hydroxy-1,4-naphthoquinone (9)
2-Bromo-8-hydroxy-1,4-naphthoquinone (10)
2-Phenyl-1,4-naphthoquinone (11)
2-(p-Hydroxyphenyl)-1,4-naphthoquinone (12)
2-Phenyl-5-hydroxy-1,4-naphthoquinone (13)
3-Phenyl-5-hydroxy-1,4-naphthoquinone (14)
5-Hydroxy-2-(p-hydroxyphenyl)-1,4-naphthoquinone (15)
5-Hydroxy-2-(p-methoxyphenyl)-1,4-naphthoquinone (16)
5-Hydroxy-3-(p-methoxyphenyl)-1,4-naphthoquinone (17)
5-Methoxy-2-(p-methoxyphenyl)-1,4-naphthoquinone (18)
3-(p-Hydroxyphenyl)-5-methoxy-1,4-naphthoquinone (19)
5-Methoxy-1,4-naphthoquinone (20)
12.92
–
–
7.86
–
9.33
8.22
12.56
8.98
16.09
–
The effect of 1,4-NQ on b-amyloid aggregation was observed by
Thio-T-induced fluorescence assay and EM analysis. Fluorescence
decay analysis of the Thio-T in complexes with fibrillar amyloid re-
vealed that the fluorescence lifetime values were significantly
higher in comparison with those from amyloid plus 4, 8 and 24
(Table 2). Fig. 2 shows an analysis of time-resolved fluorescence
spectroscopy of 4 which has the lowest IC50 in inhibition of amy-
loid aggregation.
In order to detect the morphologies of Ab (25–35) aggregates,
we used EM to probe the aggregation process in the presence or
absence of the most active 1,4-NQ: 4, 8 and 24. When Ab (25–
35) was incubated alone for 4 days, there were robust fibrils ob-
served (Fig. 1A). But when Ab (25–35) was co-incubated with 4
–
11.21
10.31
–
–
–
–
–
–
–
5-Acetoxy-1,4-naphthoquinone (21)
2,3-Dibromo-1,4-naphthoquinone (22)
2,3-Dichloro-1,4-naphthoquinone (23)
5-Nitro-1,4-naphthoquinone (24)
2-(p-Bromophenyl)-1,4-naphthoquinone (25)
2-(3,4-Dichlorophenyl)-1,4-naphthoquinone (26)
(5
lM) (Fig. 1B), 8 (5 lM) (Fig. 1C) or 24 (1 lM) (Fig. 1D), the num-
ber of fibrils decreased.
To evaluate whether or not preformed fibrils were disaggregat-
ed, 1,4-NQ were incubated with Ab preformed fibrils. Compounds
19, 24 and 4 significantly decreased the fluorescence lifetime val-
ues in comparison with those from amyloid alone (Table 2).
using one-way analysis of variance (ANOVA) followed by a Student
test. The median inhibition concentration (IC50) was determined
using SPSS 11.0.1 statistical software (SPSS Inc., Chicago, USA).
4. Discussion
We observed that whereas 1,4-naphthoquinone (1) did not
show any inhibitory effect, the presence of several substituents al-
lowed for a significant increase of BACE inhibition. Whereas substi-
tution at C-2 by methyl (7) was ineffective, the presence of a
phenyl ring at C-2 (11) increased the inhibition of BACE. The inhi-
bition was further increased by the presence of a p-OH substituent
at the phenyl ring (12). However, other substitution patterns of the
phenyl ring such as p-bromo (25) or 3,4-dichloro (26) gave rise to a
3. Results
3.1. Cell viability
The treatment of neuroblastoma SH-SY5Y cells with the differ-
ent 1,4-NQ derivatives for 24 h did not show any significant toxic-
ity between 1 and 10 lM in comparison with untreated cells.
Table 2
Effect of 1,4-naphthoquinones on Ab aggregation by Thio-T-induced fluorescence assay.
Compound
Inhibition of Ab fibrils, IC50
(lM) Disaggregation of Ab fibrils, IC50 (lM)
1,4-Naphthoquinone (1)
12.43
11.32
2-Hydroxy-1,4-naphthoquinone (2)
–
–
5-Hydroxy-1,4-naphthoquinone (3)
11.10
15.49
6-Hydroxy-1,4-naphthoquinone (4)
2.75
–
–
–
6.7
–
–
5,8-Dihydroxy-1,4-naphthoquinone (5)
2-Methyl-5-hydroxy-1,4-naphthoquinone (6)
2-Methyl-1,4-naphthoquinone (7)
–
2-Bromo-1,4-naphthoquinone (8)
6.44
–
2-Bromo-5-hydroxy-1,4-naphthoquinone (9)
2-Bromo-8-hydroxy-1,4-naphthoquinone (10)
2-Phenyl-1,4-naphthoquinone (11)
2-(p-Hydroxyphenyl)-1,4-naphthoquinone (12)
2-Phenyl-5-hydroxy-1,4-naphthoquinone (13)
3-Phenyl-5-hydroxy-1,4-naphthoquinone (14)
5-Hydroxy-2-(p-hydroxyphenyl)-1,4-naphthoquinone (15)
5-Hydroxy-2-(p-methoxyphenyl)-1,4-naphthoquinone (16)
5-Hydroxy-3-(p-methoxyphenyl)-1,4-naphthoquinone (17)
17.25
18.65
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
5-Methoxy-2-(p-methoxyphenyl)-1,4-naphthoquinone (18) 22.55
3-(p-Hydroxyphenyl)-5-methoxy-1,4-naphthoquinone (19) 16.59
–
5.05
–
–
–
–
6.28
–
5-Methoxy-1,4-naphthoquinone (20)
5-Acetoxy-1,4-naphthoquinone (21)
2,3-Dibromo-1,4-naphthoquinone (22)
2,3-Dichloro-1,4-naphthoquinone (23)
5-Nitro-1,4-naphthoquinone (24)
–
–
–
–
4.36
–
2-(p-Bromophenyl)-1,4-naphthoquinone (25)
2-(3,4-Dichlorophenyl)-1,4-naphthoquinone (26)
–
–

172
P. Bermejo-Bescós et al. / Biochemical and Biophysical Research Communications 400 (2010) 169–174
Fig. 1. A representative electron micrograph showing the formation of Ab fibrils after 4-day incubation as described in material and methods (A). A representative electron
micrograph showing the aggregation process in the presence of the most active 1,4-naphthoquinones: 4 (B) 8 (C) and 24 (D). Scale bar, 200 nm.
loss of the activity. Substitution at C-2 by bromo (8) did not pro-
mote any BACE inhibition, as well as simultaneous substitution
by halogen at C-2 and C-3 (22 and 24). Substitution of the parent
1,4-naphthoquinone ring by hydroxyl groups rendered interesting
compounds. Thus, substitution at C-2 by OH (2) afforded a notably
active compound. 5-Hydroxy-1,4-napthtoquinone (3) was also
found to produce BACE inhibition. The simultaneous presence of
an extra hydroxyl group at C-8 (5) gave rise to a slight loss of activ-
ity. Location of the OH group at C-6 (4) did not show any inhibition.
Modification of the 5-hydroxy-1,4-naphthoquinone scaffold by
the inclusion of substituents at C-2 was then tested. The presence
of Br at C-2 (9) gave rise to a slight decrease of the inhibition when
10000
1000
100
Prompt
10 uM amyloid (BA)
Q4-10 uM + BA
Q4-5 uM + BA
Q9-1 uM + BA
10
1600
1650
1700
1750
1800
Channels (nm)
Fig. 2. Inhibition of Ab aggregation by 6-hydroxy-1,4-naphthoquinone (4).

P. Bermejo-Bescós et al. / Biochemical and Biophysical Research Communications 400 (2010) 169–174
173
compared with the parent compound (3). On the other hand, the
presence of a methyl group gave rise to a stronger decrease of
the activity (6). This was also the case when substituting C-2 with
a phenyl group (13). Modification of the phenyl ring by a p-OH
group (15) further decreased the inhibition of BACE, and the pres-
ence of a p-methoxy group (16) gave a completely inactive
compound.
On the other hand, substitution with a phenyl group at C-3 (14)
gave a more active compound than substitution with phenyl at C-2
(13). Again, modification of the phenyl ring by a p-methoxy group
(17) led to a loss of the activity. No activity was found either for
bromo substitution at C-3 (10).
In the choice of candidate compounds as inhibitors of Ab aggre-
gation process we follow these criteria: (1) the compounds should
be sufficiently hydrophobic to interfere with key hydrophobic
interactions in Ab fibrils; (2) they should still be sufficiently
water-soluble to make their utilization straightforward in in vitro
assays. (3) Low molecular weight and (4) lack of neurotoxicity at
the concentrations required for effective inhibition Ab aggregation.
Therefore, the most interesting compounds would be the 1,4-NQ
derivatives with IC50 values lower than 10 lM.
5. Conclusion
The effect of the protection of the OH group of 5-hydroxy-1,4-
naphthoquinone was tested. Both 5-methoxy-1,4-naphthoquinone
(20) and 5-acetoxy-1,4-naphthoquinone (21) showed no BACE
inhibition at all. This was also the case when introducing aryl sub-
stituents either at C-2 or C-3 of the 5-methoxy-1,4-naphthoqui-
none scaffold (18 and 19). Replacement of the OH group at C-5
by a nitro group (24) afforded an inactive compound.
BACE inhibitors are under intensive study and a lot of pepti-
domimetic inhibitors have been reported and developed [26,27].
Considering the development of therapeutics for AD, chemical
compounds must cross the blood–brain barrier (BBB) and the plas-
ma membrane. The peptidomimetic inhibitors seem to have diffi-
cultly crossing the BBB. On the other hand, quinones in general
are very small compounds which are relatively advantageous for
crossing the BBB. Indeed, recent studies revealed that quinones like
plumbagin [10], thymoquinone [28] and coenzyme Q10 [29] are
able to traverse the BBB in vivo.
We have found several 1,4-NQ with activities on BACE inhibi-
tion, inhibition of Ab aggregation and disaggregation of Ab fibrils.
1,4-NQ 2, 5, 6 and 11–15 were selective for BACE inhibition, with
no effect on Ab aggregation or disaggregation. 1,4-NQ 8 and 10
were selective for the inhibition of Ab aggregation. 1,4-NQ 1, 4
and 24 did not show any BACE inhibition but were active on Ab
aggregation and disaggregation. 1,4-NQ 3 and 19 were active on
all the three targets. Although these compounds are toxic at high
doses and some of them have been evaluated for their ability to
prevent the cell growth or kill cancer cells [35], investigations of
the possibility that subtoxic doses of them might activate adaptive
stress response pathways in neurons that may protect neurons
against more severe stress are currently performed and could be
promising for finding neuroprotective compounds [10,36]. 1,4-
NQ which inhibit BACE activity and/or inhibit Ab aggregation
and/or disaggregate Ab fibrils could be promising strategies for
the prevention and treatment of AD.
It could be speculated that those active 1,4-NQ, by inhibiting
the BACE activity, could make predominant the metabolism of
In conclusion, we propose that 1,4-NQ derivatives, specially 3
and 19, should be explored as possible drug candidates or lead
compounds for the development of drugs to prevent Ab aggrega-
tion and neurotoxicity in AD.
APP towards the
a-secretase pathway, thus increasing the release
of aAPPs [30]. In this regard, the
a-secretase activity increases with
enhanced membrane fluidity [31] and processing of APP by b-
secretase might be explained by alterations in cell membrane flu-
idity [32]. The activity of BACE is sensitive to oxidative stress [33].
There is convincing evidence that oxidative stress regulates the
BACE activity, resulting in Ab accumulation [34]. Therefore, BACE
inhibition by 1,4-NQ could contribute to an improvement of the
cell membrane fluidity.
Acknowledgments
Andrea Ortega is a Ph.D. fellow and is supported by the MECE-
SUP program from the Chile government (UCN0604). Projects
UCM-910815 and CTQ2009-14124-C02-01 are gratefully acknowl-
edged for financial support.
Some of the 1,4-NQ which had shown BACE inhibition were also
found to inhibit Ab aggregation and/or to disaggregate Ab fibrils
(Tables 1 and 2). Among the C-2 substituted 1,4-NQ, the 2-bromo-
derivative (8) inhibited Ab aggregation but had no effect on disag-
gregation. However, 2-methyl-1,4-naphthoquinone (7), the 2-aryl
derivatives (11, 12, 25, 26) as well as the dihalogenated derivatives
(22 and 23) were not active either on aggregation or disaggrega-
tion. On the other hand, 1,4-naphthoquinone (1), which had not
shown any significant BACE inhibition, was found to inhibit Ab
aggregation and also to disaggregate Ab fibrils.
Among the hydroxylated 1,4-NQ derivatives, 2-hydroxy-1,4-
naphthoquinone (2) was inactive upon both Ab aggregation or dis-
aggregation. However, 5-hydroxy-1,4-naphthoquinone (3) was
slightly active on both targets, and its 2-bromoderivative (9) inhib-
ited Ab aggregation but had no effect on disaggregation. The pres-
ence of an extra hydroxyl group (5) rendered an inactive
compound on both targets. On the other hand, 2-bromo-8-hydro-
xy-1,4-naphthoquinone (10) and 6-hydroxy-1,4-naphthoquinone
(4), which had not shown any BACE inhibition, were found to inhi-
bit Ab aggregation, and in addition the latter (4) was also found to
disaggregate Ab fibrils.
References
[1] D. Tauraite, V. Razumas, E. Butkus, Lipophilic 1,4-naphthoquinone derivatives:
synthesis and redox properties in solution and entrapped in the aqueous cubic
liquid-crystalline phase of monoolein, Chem. Phys. Lipids 159 (2009) 45–50.
[2] J.A. Valderrama, P. Colonelli, D. Vasquez, M.F. Gonzalez, J.A. Rodriguez, C.
Theoduloz, Studies on quinones. Part 44: novel angucyclinone N-heterocyclic
analogues endowed with antitumoral activity, Bioorg. Med. Chem. 16 (2008)
10172–10181.
[3] R.C. Montenegro, A.J. Araujo, M.T. Molina, J.D. Marinho Filho, D.D. Rocha, E.
Lopez-Montero, M.O. Goulart, E.S. Bento, A.P. Alves, C. Pessoa, M.O. de Moraes,
L.V. Costa-Lotufo, Cytotoxic activity of naphthoquinones with special emphasis
on juglone and its 5-O-methyl derivative, Chem. Biol. Interact. 184 (2010) 439–
448.
[4] M.K. Hadden, S.A. Hill, J. Davenport, R.L. Matts, B.S. Blagg, Synthesis and
evaluation of Hsp90 inhibitors that contain the 1,4-naphthoquinone scaffold,
Bioorg. Med. Chem. 17 (2009) 634–640.
[5] T.J. Turbyville, E.M. Wijeratne, M.X. Liu, A.M. Burns, C.J. Seliga, L.A. Luevano,
C.L. David, S.H. Faeth, L. Whitesell, A.A. Gunatilaka, Search for Hsp90 inhibitors
with potential anticancer activity: isolation and SAR studies of radicicol and
monocillin I from two plant-associated fungi of the Sonoran desert, J. Nat. Prod.
69 (2006) 178–184.
[6] F. Dou, W.J. Netzer, K. Tanemura, F. Li, F.U. Hartl, A. Takashima, G.K. Gouras, P.
Greengard, H. Xu, Chaperones increase association of tau protein with
microtubules, Proc. Natl. Acad. Sci. USA 100 (2003) 721–726.
[7] H.Y. Shen, J.C. He, Y. Wang, Q.Y. Huang, J.F. Chen, Geldanamycin induces heat
shock protein 70 and protects against MPTP-induced dopaminergic
neurotoxicity in mice, J. Biol. Chem. 280 (2005) 39962–39969.
[8] S. Sundriyal, B. Viswanad, P. Ramarao, A.K. Chakraborti, P.V. Bharatam, New
PPARgamma ligands based on barbituric acid: virtual screening, synthesis and
receptor binding studies, Bioorg. Med. Chem. Lett. 18 (2008) 4959–4962.
The 5-methoxyderivatives (18 and 19), which had not shown
BACE inhibition, were active only against Ab disaggregation, and
in a similar fashion, 5-nitro-1,4-naphthoquinone (24) was active
both on aggregation and disaggregation of Ab fibrils.

174
P. Bermejo-Bescós et al. / Biochemical and Biophysical Research Communications 400 (2010) 169–174
[9] X. Zhao, Z. Ou, J.C. Grotta, N. Waxham, J. Aronowski, Peroxisome-proliferator-
[25] M.T. Molina, C. Navarro, A. Moreno, A.G. Csaky, Arylation of benzo-fused 1,4-
quinones by the addition of boronic acids under dicationic Pd(II)-catalysis,
Org. Lett. 11 (2009) 4938–4941.
activated receptor-gamma (PPARgamma) activation protects neurons from
NMDA excitotoxicity, Brain Res. 1073–1074 (2006) 460–469.
[10] T.G. Son, S. Camandola, T.V. Arumugam, R.G. Cutler, R.S. Telljohann, M.R.
Mughal, T.A. Moore, W. Luo, Q.S. Yu, D.A. Johnson, J.A. Johnson, N.H. Greig, M.P.
Mattson, Plumbagin, a novel Nrf2/ARE activator, protects against cerebral
ischemia, J. Neurochem. 112 (2010) 1316–1326.
[11] K.N. Nam, M.S. Son, J.H. Park, E.H. Lee, Shikonins attenuate microglial
inflammatory responses by inhibition of ERK, Akt, and NF-kappaB:
neuroprotective implications, Neuropharmacology 55 (2008) 819–825.
[12] K.P. Castagnoli, S.J. Steyn, J.P. Petzer, C.J. Van der Schyf, N. Castagnoli Jr.,
[26] M.C. Maillard, R.K. Hom, T.E. Benson, J.B. Moon, S. Mamo, M. Bienkowski, A.G.
Tomasselli, D.D. Woods, D.B. Prince, D.J. Paddock, T.L. Emmons, J.A. Tucker,
M.S. Dappen, L. Brogley, E.D. Thorsett, N. Jewett, S. Sinha, V. John, Design,
synthesis, and crystal structure of hydroxyethyl secondary amine-based
peptidomimetic inhibitors of human beta-secretase, J. Med. Chem. 50 (2007)
776–781.
[27] R.K. Hom, A.F. Gailunas, S. Mamo, L.Y. Fang, J.S. Tung, D.E. Walker, D. Davis, E.D.
Thorsett, N.E. Jewett, J.B. Moon, V. John, Design and synthesis of
hydroxyethylene-based peptidomimetic inhibitors of human beta-secretase,
J. Med. Chem. 47 (2004) 158–164.
Neuroprotection in the MPTP Parkinsonian C57BL/6 mouse model by
compound isolated from tobacco, Chem. Res. Toxicol. 14 (2001) 523–527.
a
[13] H.H. Thijssen, M.J. Drittij-Reijnders, Vitamin K status in human tissues: tissue-
specific accumulation of phylloquinone and menaquinone-4, Br. J. Nutr. 75
(1996) 121–127.
[28] A.A. Al-Majed, F.A. Al-Omar, M.N. Nagi, Neuroprotective effects of
thymoquinone against transient forebrain ischemia in the rat hippocampus,
Eur. J. Pharmacol. 543 (2006) 40–47.
[14] J. Li, H. Wang, P.A. Rosenberg, Vitamin K prevents oxidative cell death by
inhibiting activation of 12-lipoxygenase in developing oligodendrocytes, J.
Neurosci. Res. 87 (2009) 1997–2005.
[29] X. Yang, G. Dai, G. Li, E.S. Yang, Coenzyme Q10 reduces beta-amyloid plaque in
an APP/PS1 transgenic mouse model of Alzheimer’s disease, J. Mol. Neurosci.
41 (2010) 110–113.
[15] K. Blennow, M.J. de Leon, H. Zetterberg, Alzheimer’s disease, Lancet 368 (2006)
387–403.
[16] B. De Strooper, L. Umans, F. Van Leuven, H. Van Den Berghe, Study of the
synthesis and secretion of normal and artificial mutants of murine amyloid
precursor protein (APP): cleavage of APP occurs in a late compartment of the
default secretion pathway, J. Cell Biol. 121 (1993) 295–304.
[30] Z. Mei, B. Situ, X. Tan, S. Zheng, F. Zhang, P. Yan, and P. Liu, Cryptotanshinone
upregulates alpha-secretase by activation PI3K pathway in cortical neurons.
Brain Res. June 2 (2010) [Epub ahead of print].
[31] E. Kojro, G. Gimpl, S. Lammich, W. Marz, F. Fahrenholz, Low cholesterol
stimulates the nonamyloidogenic pathway by its effect on the alpha-secretase
ADAM 10, Proc. Natl. Acad. Sci. USA 98 (2001) 5815–5820.
[17] F. Yang, G.P. Lim, A.N. Begum, O.J. Ubeda, M.R. Simmons, S.S. Ambegaokar, P.P.
Chen, R. Kayed, C.G. Glabe, S.A. Frautschy, G.M. Cole, Curcumin inhibits
formation of amyloid beta oligomers and fibrils, binds plaques, and reduces
amyloid in vivo, J. Biol. Chem. 280 (2005) 5892–5901.
[32] C.A. von Arnim, B. von Einem, P. Weber, M. Wagner, D. Schwanzar, R. Spoelgen,
W.L. Strauss, H. Schneckenburger, Impact of cholesterol level upon APP and
BACE proximity and APP cleavage, Biochem. Biophys. Res. Commun. 370
(2008) 207–212.
[18] G. Forloni, L. Colombo, L. Girola, F. Tagliavini, M. Salmona, Anti-amyloidogenic
activity of tetracyclines: studies in vitro, FEBS Lett. 487 (2001) 404–407.
[19] H. Kim, B.S. Park, K.G. Lee, C.Y. Choi, S.S. Jang, Y.H. Kim, S.E. Lee, Effects of
naturally occurring compounds on fibril formation and oxidative stress of
beta-amyloid, J. Agric. Food Chem. 53 (2005) 8537–8541.
[20] S. Salloway, J. Mintzer, M.F. Weiner, J.L. Cummings, Disease-modifying
therapies in Alzheimer’s disease, Alzheimers Demen. 4 (2008) 65–79.
[21] T.R. Kelly, N.D. Parekh, E.N. Trachtenberg, Regiochemical control in the Diels-
Alder reaction of substituted naphthoquinones. The directing effects of C-6
oxygen substitutents, J. Organic Chem. 47 (1982) 5009–5013.
[22] T.N. Van, N. De Kimpe, Synthesis of 6H-naphtho[2,3-c]chromene-7,12-diones
via palladium-catalyzed intramolecular cyclization of 2-bromo-3-
aryloxymethyl-1,4-naphthoquinones, Tetrahedron 59 (2003) 5941–5946.
[23] L.F. Tietze, R.R. Singidi, K.M. Gericke, Total synthesis of the proposed structure
of the anthrapyran metabolite delta-indomycinone, Chem.-Eur. J. 13 (2007)
9939–9947.
[33] E. Tamagno, P. Bardini, A. Obbili, A. Vitali, R. Borghi, D. Zaccheo, M.A. Pronzato,
O. Danni, M.A. Smith, G. Perry, M. Tabaton, Oxidative stress increases
expression and activity of BACE in NT2 neurons, Neurobiol. Dis. 10 (2002)
279–288.
[34] J. Apelt, M. Bigl, P. Wunderlich, R. Schliebs, Aging-related increase in oxidative
stress correlates with developmental pattern of beta-secretase activity and
beta-amyloid plaque formation in transgenic Tg2576 mice with Alzheimer-
like pathology, Int. J. Dev. Neurosci. 22 (2004) 475–484.
[35] S. Sugie, K. Okamoto, K.M. Rahman, T. Tanaka, K. Kawai, J. Yamahara, H. Mori,
Inhibitory effects of plumbagin and juglone on azoxymethane-induced
intestinal carcinogenesis in rats, Cancer Lett. 127 (1998) 177–183.
[36] V. Calabrese, C. Cornelius, C. Mancuso, G. Pennisi, S. Calafato, F. Bellia, T.E.
Bates, A.M. Giuffrida Stella, T. Schapira, A.T. Dinkova Kostova, E. Rizzarelli,
Cellular stress response: a novel target for chemoprevention and nutritional
neuroprotection in aging, neurodegenerative disorders and longevity,
Neurochem. Res. 33 (2008) 2444–2471.
[24] N. Maugel, B.B. Snider, Efficient synthesis of the tetracyclic aminoquinone
moiety of marmycin A, Org. Lett. 11 (2009) 4926–4929.