Liberation of copper from amyloid plaques: Making a risk factor useful for alzheimers disease treatment

Article abstract of DOI:10.1021/jm3003813

Alzheimers disease (AD) is a complex multifactorial syndrome. Metal chelator and Aβ inhibitor are showing promise against AD. In this report, three small hybrid compounds (1, 2, and 3) have been designed and synthesized utilizing salicylaldehyde (SA) base

Full text of DOI:10.1021/jm3003813

Article
pubs.acs.org/jmc
Liberation of Copper from Amyloid Plaques: Making a Risk Factor
Useful for Alzheimer’s Disease Treatment
Jie Geng,†,‡,§ Meng Li,†,‡,§ Li Wu,†,‡ Jinsong Ren,† and Xiaogang Qu*,†
†Laboratory of Chemical Biology, Division of Biological Inorganic Chemistry, State Key Laboratory of Rare Earth Resource
Utilization, Changchun Institute of Applied Chemistry, Changchun, Jilin 130022, China
‡Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100039, P. R. China
S
* Supporting Information
ABSTRACT: Alzheimer’s disease (AD) is a complex multifactorial
syndrome. Metal chelator and Aβ inhibitor are showing promise against
AD. In this report, three small hybrid compounds (1, 2, and 3) have been
designed and synthesized utilizing salicylaldehyde (SA) based Schiff bases as
the chelators and benzothiazole (BT) as the recognition moiety for AD
treatment. These conjugates can capture Cu2+ from Aβ and become dimers
upon Cu2+ coordination and show high efficiency for both Cu2+ elimination
and Aβ assembly inhibition. Besides, the complexes have superoxide
dismutase (SOD) activity and significant antioxidant capacity and are
capable of decreasing intracellular reactive oxygen species (ROS) and
increasing cell viability. All these results indicate that the multifunctional
metal complexes which have Aβ specific recognition moiety and metal ion
chelating elements show the potential for AD treatment. Therefore, our work
will provide new insights into exploration of more potent amyloid inhibitors.
inhibitors. The hydrophobic core KLVFFA, positioned at the
β1 region in the structural model of Aβ1−40 fibrils, is crucial
for the self-assembly of Aβ into fibrils. Short peptides
containing the core sequence have been used as “binding
element” to design the inhibitors of fibrillization. A number of
low-molecular-mass Aβ aggregation inhibitors have also been
identified by screening of compound libraries as well as rational
design strategies. The resulting inhibitors include such
chemically diverse compounds as curcumin, (−)-epigallocha-
techingallate (EGCG), and rifampicin. However, a majority of
the above inhibitors show only moderate inhibition efficiency
attributed to their poor ability to interact over the large
interface of interaction of Aβ peptides during the aggregation
process or to interact simultaneously with the multiple binding
surfaces.11 Therefore, there is ever-increasing demand for
designing novel more potent AD inhibitors.
Evidences support the hypothesis that ligands exhibiting
greater affinity for the β-amyloid peptide are more effective at
preventing amyloid formation and inhibiting amyloid neuro-
toxicity.12 The use of multiple simultaneous interactions to
enhance target binding affinity and specificity is an important
concept and versatile in biology. Therefore, development of
new multimeric molecules used for AD therapy has received
much attention in recent years.13 These multimeric molecules
INTRODUCTION
■
Alzheimer’s disease (AD) is the most common form of
dementia, which affects more than 15 million people world-
wide.1 This figure is projected to double every 20 years as a
result of an increasing life span. AD is characterized by cerebral
extracellular amyloid plaques and intracellular neurofibrillary
tangles.2 The amyloid β peptide (Aβ) with 39−42 residues is
the major component of amyloid plaques found in brains of AD
patients. Although the molecular mechanisms of AD patho-
genesis have not been clearly understood due to its complexity,
recent advances have demonstrated that polymerization of Aβ
into amyloid fibrils is a critical step in the pathogenesis.2 The
origin of insoluble extracellular neurotoxic deposits is still not
clear, and multiple factors such as pH, temperature, protein
concentration, and ionic strength have been reported to trigger
their formation.3 Therefore, the inhibition of Aβ assembly has
been considered as the primary therapeutic strategy for the
neurodegenerative diseases.
Metal ions are involved in the assembly and neurotoxicity of
Aβ species.4 Copper, concentrated in amyloid plaques (≈0.4
mM), is involved in the pathogenesis of AD.5 Cu2+ is able to
accelerate the formation of Aβ aggregates and influence their
conformational transformation.6 In addition, the Aβ-bound
Cu2+ can contribute to the formation of neurotoxic reactive
oxygen species (ROS).7 Therefore, metal-ion chelation therapy
as a treatment for AD has been explored to prevent Aβ
deposition and ROS production.8
Special Issue: Alzheimer's Disease
Received: March 19, 2012
Peptide or peptide mimetics9 and small organic molecules
with aromatic groups10 are now the two main classes of Aβ
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm3003813 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. (A) Combination of the main features of ThT and CQ provides new ligands with potential intercalating and chelating properties. (B)
Synthesis of compounds 1−3. Compound 1, R = H; compound 2, R = Cl; compound 3, R = NO2.
show stronger ability to block fibril formation than the
corresponding monomers. Molecular self-assembly, which
combines the facile multimerization observed in the family of
antibodies, and the improved binding affinity and specificity of
synthetic organic molecules represent an intriguing hybrid
approach to targeting amyloid.13
Table 1. Physical Properties of XSABT Ligands Used in Our
Studies
a
calculation
1
2
3
rules
MW
254.05
1.80
3
288.75
2.52
3
299.30
1.55
6
≤450
≤5.0
≤10
≤5
clogP
HBA
HBD
log BB
Metal complexation has been used as scaffold to design
three- or four-helix bundles.14 Considering the high concen-
tration of Cu2+ in amyloid plaques, combining metal complex-
ation and ligand interaction may offer a novel method to
construct multivalent Aβ inhibitors. Benzothiazole is known to
possess binding affinity for β-amyloid plaques when appropri-
ately derivatized and has been used as essential part of some
imaging agents for β-amyloid plaques.15 Thus, we designed and
synthesized three small hybrid compounds, (E)-2-((benzo[d]-
thiazol-2-ylimino)methyl)phenol 1, (E)-2-((benzo[d]thiazol-2-
ylimino)methyl)-4-chlorophenol 2, and (E)-2-((benzo[d]-
thiazol-2-ylimino)methyl)-4-nitrophenol 3 (Figure 1) utilizing
salicylaldehyde based Schiff bases as the chelators and
benzothiazole as the recognition moiety. These conjugates are
designed to become dimers due to Cu2+ coordination, holding
great promise for Cu2+ elimination and Aβ assembly inhibition
abilities.
1
1
1
−0.26
−0.15
−0.97
≥-1.0
a
MW: molecular weight. clogP: calculated logarithm of the octanol−
water partition coefficient. HBA: hydrogen-bond acceptor atoms.
HBD: hydrogen-bond donor atoms. log BB = −0.0148 × PSA + 0.152
× clogP + 0.130.
group on the salicylaldehyde, the aqueous solubility, lip-
ophilicity, and BBB permeability can be modified. Defined by
the restrictive terms of Lipinski’s rules and calculated log BB for
potential applications in brains, compounds 1−3 fulfil drug-like
criteria and possible brain penetration (Table 1).
Chelating Properties of Compounds toward Cu2+. All
of the three compounds were studied by UV−vis spectroscopy
to determine their ability to act as Cu2+ chelators.18 To this end,
spectrophotometric titrations were used for the Cu2+ binding.
As an example, a series of UV−vis spectra of compound 1
titrated by Cu2+ were shown in Figure 2, while the spectra of
compound 2 and 3 were given in Figure S1 in Supporting
Information. In the absence of Cu2+, the UV−vis spectrum of
compound 1 showed absorption maximum at 375 nm and a
shoulder at 330 nm. When Cu2+ was added, a new band at 460
nm appeared which was associated with the copper complex.
Therefore, the binding stoichiometry of compound 1 with
CuCl2 was determined by following the absorption changes at
460 nm. The presence of an isosbestic point revealed the
formation of a unique Cu2+−1 complex (Figure 2), and
titration analysis was consistent with a 1:2 Cu2+/ligand molar
ratio (Figure 2 inset). We have also made an attempt to
estimate the binding affinity of compound 1 to Cu2+ and the
log Kb value for the 2:1 complex was calculated to be 13. For
Cu2+, the binding affinity follows the order 1 > 2 > 3, which was
consistent with the general rule for a series of similar ligands
that the binding constants increased with increasing ligand
basicity.
RESULTS AND DISCUSSION
■
Prediction of BBB Penetration of compound 1, 2, and
3. A major impediment to the development of effective anti-Aβ
compounds for AD therapy is that essentially 100% of large-
molecule drugs and >98% of small-molecule drugs fail to cross
the blood−brain barrier (BBB).16 Filters to ensure that the
resultant molecules fulfill drug-like criteria that are most
commonly defined using Lipinski’s rules:17 molecular weight
(MW) less than 500, the calculated logarithm of the octanol−
water partition coefficient (clogP) less than 5, the number of
hydrogen bond donor atoms (HBD) less than 5, and the
number of hydrogen-bond acceptor atoms (HBA) less than 10.
Filter for prediction of BBB penetration involves calculation of
log BB by means of the equation shown in the footnote of
Table 1. This equation has shown good predictive ability and
comprises two simple variables that can be rapidly computed
for any structure: clogP and the topological polar surface area
(TPSA). The multifunctional small molecules were prepared
via Schiff base condensation between benzothiazole and
salicylaldehyde or its derivates (Figure 1). By changing the R
To explore the metal binding properties of compounds 1−3
with other biologically relevant metal ions, the optical
B
dx.doi.org/10.1021/jm3003813 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
precipitate formation,20 and atomic force microscopy (AFM),
which showed the morphology of Aβ aggregates.21
Synthetic human Aβ1−40 was dissolved in a buffered
aqueous solution (10 mM HEPES, pH 6.6, 150 mM NaCl, 2-
[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid
(HEPES)) to which Cu2+ was added. Addition of metal ion
caused significant Aβ aggregation, which was monitored by
recording the light scattering on a fluorescence spectropho-
tometry (Figure 3). Higher light scattering at 320 nm (LS320) of
Figure 2. UV−vis titration of compound 1 with Cu2+ in 50% (v/v)
DMSO/buffer (10 mM HEPES, 150 mM NaCl, pH 6.6) at room
temperature. The concentration of Cu2+ was varied from 0 to 100 μM.
Inset: change in absorbance at 460 nm with increased concentration of
Cu2+ derived from the UV−vis titration, a breakpoint was observed at
0.5:1 ratio. The concentration of compound 1 was 50 μM.
Figure 3. Inhibition and disaggregation of Cu2+-induced Aβ aggregates
by chelators. Left: Effect of chelators on the Cu2+-induced Aβ
aggregation was evaluated by the change of LS at 320 nm. Black,
inhibition experiments; red, disaggregation experiments. Right: The
morphology of Aβ aggregates after 24 h incubation without (A) or
with (B) compound 1, (C) 2, or (D) 3 was analyzed by AFM
(disaggregation experiments). Scale bars: 500 nm. Inhibition experi-
ments: compounds 1−3 was added before incubation of Aβ−Cu2+.
Disaggregation experiments: 1−3 was added after 24 h incubation of
Aβ−Cu2+. [Aβ] = 10 μM; [Cu2+] = 10 μM; [1], [2], or [3] = 20 μM.
Buffer: 10 mM HEPES, 150 mM NaCl, pH 6.6.
responses of compounds 1−3 in the presence of other metal
ions (Mg2+, Ca2+, Al3+, Fe3+, Co2+, or Ni2+) were examined19 as
depicted in Figure S2A,C,E in Supporting Information. Taking
compound 1 for example, less optical changes were observed
upon incubation of ligands with these metal ions. Then the
selectivity of compound 1 for Cu2+ in the presence of other
divalent metal ions was investigated by UV−vis (Figure S2B in
Supporting Information). The optical intensity at 460 nm
(absorption band for Cu2+-treated compound 1) was
monitored when the appropriate metal ions (25 μM) were
added to a 50 μM solution of compound 1 followed by the
subsequent treatment with 25 μM Cu2+. As shown in Figure
S2B in Supporting Information, binding of compound 1 (50
μM) to Cu2+ (25 μM) was observed in the presence of other
metal ions (25 μM), which indicated that compound 1 was
selective to Cu2+. Similar results were observed for compound 2
but not 3, which showed relatively poor Cu2+ selectivity.
Copper is concentrated in amyloid plaques and directly
coordinates Aβ at the histidine side chains in its amino
terminus.4 We next investigated Cu2+ transfer from Aβ to
compounds 1−3 by UV−vis spectra. Upon adding compounds
1−3 to Cu2+−Aβ, the UV−vis spectrum changed steadily
toward the typical spectrum of Cu2+−1, 2, and 3 (Figure S3 in
Supporting Information). The competitive binding of com-
pounds 1−3 for Cu2+ suggested that the chelators would
sequester Cu2+ from Aβ and therefore inhibited Cu2+ induced
Aβ aggregation.
the resultant solution indicated more aggregates existing. The
presence of 2 equiv of compounds 1−3 inhibited Cu2+-induced
aggregation significantly. Metal-associated aggregation could
also be attenuated upon subsequent addition of ligand.
Addition of metal-binding ligands to Aβ−Cu2+ aggregates
reduced the scattering by varying extents (Figure 3).
Morphological analysis of Aβ aggregates on the surface of
mica formed by interaction between Aβ40 and Cu2+ with or
without chelators was achieved by AFM. Sample without a
chelator showed a lot of Aβ40 deposits (Figure 3), while in the
samples with compounds 1−3, the large plaques of amorphous
Aβ40 aggregates became smaller and fewer (Figure 3B−D).
The Cu2+ trials showed that all the chelators significantly
reduced the amount of aggregated Aβ in the solution.
Interestingly, the relative disaggregation activities of the three
ligands were higher than that of CQ for the Cu2+ trials, despite
the large difference in metal binding affinity (Figure S4 in
Supporting Information). Aβ targeted metal chelator is known
to result in better modulation of Aβ aggregation.22 We
speculated that the increased disaggregation activity of
compounds 1−3 could be due to Aβ intercalating ability of
these benzothiazole compounds.
Inhibition of Aβ Self-Assembly. Molecular self-assembly,
which is the spontaneous association of molecules under
equilibrium conditions into stable, structurally well-defined
aggregates that are joined by noncovalent bonds, is ubiquitous
in biological systems and underlies the formation of a wide
variety of complex biological structures. An interesting goal of
molecular assembly is to obtain biomolecules to display
multivalent interactions, which are characterized by simulta-
neous binding of multiple ligands on one biological entity to
multiple receptors on another with high avidity.23 Various
Inhibition of Cu2+ Induced Aβ Aggregation. Cu2+ is
known to cause Aβ aggregation in solution, and the interaction
of Aβ peptide with Cu2+ is known to be pH dependent. It has
been reported that Cu2+-induced Aβ aggregation is more
significant at pH 6.6 than at pH 7.4.6a To investigate the
influence of ligands on Cu2+-induced Aβ aggregation, we
performed two individual studies in a weak acidic buffered
aqueous solution: inhibition (the prevention of forming metal-
induced Aβ aggregates) and disaggregation (the transformation
of metal−Aβ aggregates by chelators). The effect of ligands on
metal-induced Aβ1−40 aggregation was assessed, and clioqui-
nol (CQ) was used as a positive control because CQ is known
to attenuate metal ion−Aβ aggregation.8 The degree of Aβ
aggregation was probed mainly by light scattering, which
exhibited the change in solution turbidity as a result of
C
dx.doi.org/10.1021/jm3003813 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
approaches have been developed to engineer multivalency by
linking multiple ligands together.24 Considering the general
Cu2+ content in the AD brain is at least 1 order of magnitude
higher than that in the blood, we suggest that the compounds
1−3 can selectively assemble to dimer in the brain (Figure S5
in Supporting Information), which is able to inhibit Aβ self-
assembly effectively.
We first compared the Aβ binding affinity of compounds 1−
3 and their copper complexes. Taking advantage of the Aβ
tyrosine fluorescence quenching effect of the compounds, the
apparent binding constants6d yielded by nonlinear least-squares
fitting (Figure S6 in Supporting Information) were shown in
Table 2. All the copper complexes exhibited stronger binding
AFM was used to determine the morphology of the Aβ40
assembly when maximal ThT binding was observed. Classical
amyloid fibrils were observed in samples of untreated Aβ40
(Figure 4A).21 The Aβ40 fibrils were nonbranched, helical
filaments with diameters of ∼10 nm and lengths of up to
several micrometers. The addition of compounds 1−3 (100 μM
concentration) could hardly or only slightly alter the assembly
of Aβ40 (date not shown). In contrast to the results with
compounds 1−3, stronger inhibition of fibril assembly was
observed in 4-, 5-, and 6-treated samples at the same
concentrations. In addition, numerous small, relatively
amorphous aggregates were observed (Figure 4B−D).
We next performed a variety of experiments in order to
determine whether these metal complexes could dissociate the
existing fibrils of Aβ. The addition of complexes after the
completion of the fibrillation reaction, as monitored by ThT,
led to a rapid, concentration dependent decrease in
fluorescence signal that was consistent with loss of fibrils,
whereas in controls with no complexes, the ThT signal
remained essentially constant (Figure 5). AFM experiments
further supported that the metal complexes 4−6 could
remarkably decrease fibril numbers (Figure 5) while increasing
the amorphous aggregates.
Table 2. Kb (Binding Constant) of Chelators or Their
Copper Complexes to Aβ
1
2
3
ligand
1.0 × 105 M−1
1.9 × 106 M−1
3.2 × 105 M−1
1.8 × 106 M−1
8.9 × 105 M−1
7.3 × 106 M−1
complex
affinity than their ligands with an enhancement of one order
due to the multivalent effect, which meant that these complexes
might have stronger Aβ aggregation inhibition ability.12
To directly compare the effects of compounds 1−3 and their
copper complexes 4−6 on Aβ aggregation, Aβ1−40 was
incubated for 7 days in the presence of chelators or their copper
complexes in different ratios. The ratios were expressed per
mole of ligand. Thioflavin T (ThT) analysis showed that 4−6
inhibited Aβ1−40 aggregation in a concentration dependent
manner; the strongest inhibition occurred at the highest
concentration used (Figure 4). Taking 6 for example, a 1:2 ratio
Figure 5. Disassembly of Aβ fibrils by 4−6. Left: Concentration-
dependent disassembly of Aβ fibrillogenesis assessed by ThT assay.
Right: The morphology of Aβ was analyzed by AFM in the absence
(A) or presence of 4 (B), 5 (C), or 6 (D). Aβ was fixed at 50 μM and
fibrillar Aβ40 was mixed with 4−6. Aliquots were kept at 37 °C for 24
h after being mixed. All the reactions were conducted without
agitation. Buffer: 10 mM HEPES, 150 mM NaCl, pH 7.3. Scale bars:
500 nm.
Figure 4. Inhibition of Aβ self-assembly by metal complex. Left:
Concentration-dependent inhibition of Aβ fibrillogenesis assessed by
ThT assay. Right: The morphology of Aβ aggregates was analyzed by
AFM images in the absence (A) or presence of 4 (B), 5 (C), or 6 (D).
Aβ was fixed at 50 μM and was mixed with 4−6. Aliquots were kept at
37 °C for 7 days after being mixed. All the reactions were conducted
without agitation. Buffer: 10 mM HEPES, 150 mM NaCl, pH 7.3.
Scale bars: 500 nm.
The 1:2 copper complexes of 1−3, having a high binding
constant, are expected to be stable in biological solutions,
without undergoing hydrolysis. They are desired to be more
stable than the ones formed by mixing the two reagents. Upon
Aβ binding, the metal-to-ligand charge-transfer (MLCT) bands
of the metal complexes did not change (Figure S8 in
Supporting Information), indicating that Aβ binding did not
destroy the complex structure.
The influence of copper ions on aggregation of Aβ has been
intensively studied over the past decade. Nevertheless, many
important aspects like the effect of metal ions on aggregation
kinetics and the effect of chelators on metal-induced
aggregation of Aβ are still elusive. In early studies, metal ions
have been reported to enhance the rate of Aβ fibrillization and
metal chelators acted in an opposite direction by reversing the
aggregation.6,7 Later on, evidence demonstrate that metal-
induced Aβ aggregates are predominantly nonfibrillar.25 Recent
results show that the aggregated state of Aβ peptide in vitro
depended on the concentration of Cu2+. Image study indicates
of Aβ/3 (in 6) inhibited the relative change of ThT
fluorescence by more than 75%, whereas the same ratio of 3
inhibited ThT by only 35% (Figure S8 in Supporting
Information). 6 was a stronger potent inhibitor of Aβ
aggregation than 3, which was consistent with our expectation.
As to compound 1 and 2, neither of them showed any
inhibition (Figure S7 in Supporting Information), while their
Cu2+ complexes exhibited significant ability to prevent Aβ
fibrillization (Figure 4).
D
dx.doi.org/10.1021/jm3003813 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
that at low concentrations of Cu2+, the aggregates are mature
fibrils, whereas at high Cu2+ concentrations, granular aggregates
are formed.26 Medium- and high-affinity metal chelators
including metallothioneins can initiate fast Aβ42 fibrillization.27
Consequently, the outcome of metal-chelation therapy might
vary between two opposite scenarios: removal of metal ions
especially of Cu2+ and reduction of their toxicity; solubilization
of metal induced Aβ aggregates and promotion of Aβ
fibrillization and amyloid formation under certain conditions,
which threw the chelator therapy into a dilemma. Thus,
alternative mechanisms should be applied to stop amyloido-
genesis and reduce amyloid load of AD patients. As
demonstrated above, compounds 1−3 can remove Cu2+ from
Aβ and reduce their toxicity, and the formed metal complexes
4−6, on the other hand, can inhibit Aβ fibrillization and
disassemble formed Aβ fibrils (Figure 6). Therefore, our
strategy described here may shed light on how to design more
potent amyloid inhibitors.
Figure 7. Production of H2O2 from reactions of Aβ, Cu2+, and
compound upon addition of ascorbate, as determined by a HRP/
luminol assay. Control: Aβ + Cu2+. [Aβ] = 10 μM, [Cu2+] = 20 μM,
[chelator] = 40 μM, [ascorbate] = 50 μM, [luminol] = 10 μM, [HRP]
= 3 U/mL.
β-amyloid-induced neuronal death and improve mitochondrial
respiratory function.34 Considering the fact that the active
centers of superoxide dismutase (SOD) are metal complexes,
we speculate that although the chelator is inert, when the
chelator captures Cu from Aβ−Cu complex, the formed
chelator−Cu complex may form an active artificial enzyme
center and show SOD activity.
The SOD activity of the chelator−Cu complex was
quantified using a modified NBT assay system.35 Superoxide
anion generation by the system was detected using spectros-
copy by following reduction of nitro blue tetrazelium (NBT) to
blue formazane (MF+) at 560 nm. To determine the activity of
the enzyme mimics, we measured the IC50 values of the
reactions, a generally used indicator for comparing the activity
of enzyme and enzyme mimics. The results indicated that all
copper complexes had SOD activity as expected while their
corresponding ligands showed little SOD activity (Figure S9A
in Supporting Information). IC50 values of the prepared
complexes were 1.31, 1.06, and 0.19 μM, respectively (Figure
8), and 6 was the most potent SOD mimic where the presence
of aromatic rings in the vicinity of metal center led to the
enhanced SOD activity. The results also indicated that copper
atom was essential for SOD activity and incorporation of
copper into the structure of antioxidant could tremendously
increase the mimic SOD activity. Besides, we adopted SOD
enzyme as reference to compare with these complexes (Figure
Figure 6. Schematic representation of the possible modes of
multifunctional effects of our chelators in the process of Aβ
aggregation. Aβ exists in equilibrium between monomers, oligomers,
and amyloid fibrils, while Cu2+ binds to Aβ monomer and prevents
amyloidogenesis by inducing the formation of nonfibrillar aggregates.
Our chelators 1−3 can disassembly Cu2+ induced Aβ aggregates
through sequestration of Cu2+ from Aβ and the formed multivalent
complexes have strong inhibition and dissolution ability in Aβ
fibrillization.
Inhibition of H2O2 Production. The formation of ROS by
Cu−Aβ has been suggested as another proposed mechanism of
AD pathogenesis.7,28 This could cause an increase in oxidative
stress, which triggers damage of cellular components such as
DNA, lipids, and proteins.29 Under reducing conditions, the
Aβ−Cu complex reacts with O2 to generate H2O2. The effects
of the chelators on the generation of H2O2 by compounds 1−3
were examined in cell-free solutions by a widely used
horseradish peroxidase (HRP)/luminol chemiluminescence
assay. Samples containing Cu2+, Aβ, and compounds 1−3
showed less H2O2 at different levels (Figure 7), indicating that
all compounds 1−3 could reduce H2O2 produced by Cu−Aβ.
SOD (Superoxide Dismutase) Activity of Metal
Complexes. Partial deficiency of SOD-2, which plays an
important role in the clearance of ROS, has been shown to
accelerate plaque deposition30 and increase tau phosphorylation
in AD mice models.31 SOD-2 deficiency has also been shown to
accelerate the onset of a number of behavioral deficits in hAPP
mice.32 Overexpression of SOD-2 reduces hippocampal
superoxide and prevents memory deficits in a mouse model
of Alzheimer’s disease.33 Overexpression of Mn-SOD or
addition of MnTE-2-PyP5+ (SOD mimic) can protect against
Figure 8. Percentage inhibition of NBT oxidation by superoxide
radicals generated in riboflavin−NBT−light system in vitro assessed by
NBT+ absorption at 560 nm with CQ−Cu2+, 4, 5, or 6 ([ligand]:[
Cu2+] = 2:1).
E
dx.doi.org/10.1021/jm3003813 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
S9B in Supporting Information). The amounts of the
complexes equivalent to 1U of enzyme activity were 9, 7.5,
and 0.2 μM, respectively. Therefore, the prepared complexes of
low molecular weight and simple structures could be SOD
mimics as proposed.
Intracellular Determination of ROS. There is abundant
evidence of peroxidative attack in AD brain,28 which may
originate from Aβ−Cu electrochemistry.36 Exposure of the cells
to 5 μM Aβ−Cu caused 194% increase in content of ROS
relative to Aβ−Cu-untreated control cells (Figure 9). Pretreat-
Figure 10. Protection effects of ligands on Aβ40−Cu2+-induced
cytotoxicity of PC 12 cells. Cell viability was determined using MTT
method and data points shown are the mean values SEM from three
independent experiments.
in vitro Aβ inhibitors but also could decrease Aβ-mediated
cellular toxicity.
CONCLUSIONS
■
Alzheimer’s disease (AD) is a complex multifactorial syndrome
unlikely to arise from a single causal factor. Different number of
related biological alterations may contribute to AD patho-
genesis.39 None of the presently marketed drugs, although
valuable in improving cognitive, behavioral, and functional
impairments, can alter AD progression.40 Design of multifunc-
tional molecules may provide new insights into developing
more potent amyloid inhibitors. Here, we have shown that the
benzothiazole-containing compounds 1−3 can bind Cu2+ and
decrease Aβ-mediated cellular toxicity. More importantly, these
chelators can change copper from risk factor into useful species.
Compounds 1−3 can bind Cu2+ and assemble into metal
complexes, which are not only able to inhibit Aβ self-
aggregation but also to dissolve the formed fibrils due to
multivalent effect. In addition, the formed metal complexes
have significant SOD activity and therefore can act as a free-
radical scavenger. Our results strongly support the designed
mechanism of action that the compounds can compete
effectively for copper with the metal binding sites on Aβ
peptide, inhibit Aβ self-assembly, and function as antioxidants
and reduce Aβ cellular toxicity. This may open an avenue for
exploration of more potent amyloid inhibitors.
Figure 9. Cells were treated with aged Aβ40−Cu2+ at the
concentration of 5 μM in the absence or presence of ligands (10
μM), and 12 h later ROS generation inside the cells was measured
using DCF fluorescence. Samples (from left to right) are: control, Aβ−
Cu(II), Aβ−Cu(II) + 1, Aβ−Cu(II) + 2, Aβ−Cu(II) + 3. Data
represents mean SEM of at least three different experiments.
ment of the cells with compounds 1−3 at concentrations of 10
μM diminished the levels of ROS by 25%, 27%, and 41%,
respectively, compared to the cells exposed only to Aβ−Cu.
The intracellular level of ROS did not vary among the cells
treated solely with Aβ (5 μM). On the basis of these data, the
formed Cu complex could be a free-radical scavenger.
Radical scavengers and metal chelators have been scrutinized
in clinical studies of AD and positive results have been gained
in the past few years which have the ability to attenuate the
broad spectrum of oxidative stress associated neuropathology,
as well as APP translation, Aβ generation, and amyloid plaques
and NFT formation. It was proposed that if one compound
held radical-scavenging and metal−protein-attenuating proper-
ties simultaneously, it might be more potent than that with
single property, inspired by the preliminary successes of radical
scavengers and metal−protein-attenuating compound
(MPAC).37
Cell Viability Assay. The ability of compounds 1−3 to
inhibit Aβ assembly suggested that it might be useful in
blocking Aβ-mediated cellular toxicity. To address this
question, we used PC12 cells to perform MTT assays to
probe cellular metabolism.38 All assays were carried out in the
same manner, and the data were normalized using the results
from the cells not treated with Aβ−Cu2+ as a positive control.
Treatment of the cells with 5 μM Aβ−Cu2+ for 2 days reduced
cell viability to 58%. In the presence of compounds 1−3, the
survival of the cells increased to about 90% (Figure 10). The
survival rate was even higher than that of CQ (Figure 10). As
depicted in Figure S10 in Supporting Information, the
multifunctional compounds 1−3 themselves exhibited less
toxicity than the clinically available compound CQ. These
results showed that compounds 1−3 were not only effective as
EXPERIMENTAL SECTION
■
Materials. All of the solvents were reagent grade and used without
further purification unless otherwise specified. The commercial
compounds CuCl2·2H2O, salicylaldehyde, 5-chlorosalicylaldehyde, 5-
nitrosalicylaldehyde, 2-aminobenzothiazole, clioquinol, and thioflavin
T were purchased from Sigma-Aldrich. 1,1,1,3,3,3-Hexafluoro-2-
propanol (HFIP) and 4,4′-methylenedianiline were obtained from
Acros organics. Milli-Q water was used to prepare all of the buffer
solutions.
(E)-2-((Benzo[d]thiazol-2-ylimino)methyl)phenol (1). 1 was
prepared by Schiff base condensation of salicylaldehyde (5 mmol) with
2-aminobenzothiazole (5 mmol) in ethanol (40 mL), at reflux for 8 h.
After cooling, the Schiff base precipitated from the reaction mixture as
a pure yellow powder, washed with ethanol three times, and then dried
1
in vacuum (purity >95%). H NMR (400 MHz, CDCl3, 25 °C): δ =
12.23 (H, s), 9.28 (H, s), 7.99−7.97 (H, d), 7.86−7.84 (H, d), 7.53−
7.46 (3H, m), 7.41−7.39 (H, t), 7.08−7.05 (H, d), 7.02−7.00 (H, t).
MALDI-TOF-MS (CHCl3): m/z 255.1, [M + H]+. Elemental Anal.,
Calcd %: C, 66.12; H, 3.96; N, 11.02. Found %: C, 65.96; H, 3.92; N,
10.69.
F
dx.doi.org/10.1021/jm3003813 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(E)-2-((Benzo[d]thiazol-2-ylimino)methyl)-4-chlorophenol
(2). 2 was prepared by Schiff base condensation of 5-chlorosalicy-
laldehyde (5 mmol) with 2-aminobenzothiazole (5 mmol) in ethanol
(40 mL) at reflux for 8 h. After cooling, the Schiff base precipitated
from the reaction mixture as a pure yellow powder, washed with
v) DMSO/buffer (10 mM HEPES, 150 mM NaCl, pH 6.6)), 25 or 50
μM of metal salt (MgCl2, CaCl2, AlCl3, FeCl3, CoCl2, NiCl2, or ZnCl2)
was added. The solution was incubated at room temperature for 5 min,
the spectrum was recorded, and then 25 or 50 μM CuCl2 was added.
The resulting solution was mixed well, and the spectrum was recorded
after additional 5 min incubation at room temperature. Selectivity was
quantified by comparing and normalizing the absorbance values of the
metal−ligand complex at λ = 460 nm to the absorbance of the solution
at this wavelength after the addition of CuCl2.
Aβ Sample Preparation. Aβ1−40 (lot no. U10012) was
purchased from American Peptide and prepared as previously
described.38 Briefly, the powered Aβ peptide was first dissolved in
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at the concentration of 1
mg/mL. The solution was shaken at 4 °C for 2 h in a sealed vial for
further dissolution and was then stored at −20 °C as a stock solution.
Before use, the solvent HFIP was removed by evaporation under a
gentle stream of nitrogen and peptide was dissolved in water. Cu2+
induced aggregation of Aβ 1−40 was accomplished by mixing an
aliquot of the peptides and CuCl2 at a molar ratio of 1:1 into 10 mM
HEPES (150 mM NaCl, pH 6.6) at 37 °C for 24 h. Aβ1−40 self-
aggregation was accomplished by mixing an aliquot of the peptides
from the stock solution into aggregation buffer (10 mM HEPES in 150
mM NaCl, pH 7.3) at 37 °C for 7 days. Due to the poor solubility of
compounds 1−3 in water or media, the final amount of DMSO used in
all biological experiment is 2% (v/v).
Light Scattering Measurements. Aβ aggregation were moni-
tored in a Jasco-FP6500 spectrofluorometer by following the increase
in light scattering using a 1.0 cm quartz cuvette lightening the samples
at 320 nm and collecting scattered light at 90° angles at 320 nm. Light
scattering by the buffer was used as baseline.
ThT Fluorescence Measurements. All of the measurements
were recorded in a fluorescence spectrophotometer (JASCO FP-6500)
at room temperature with excitation wavelength 444 nm. Fluorescence
data at 480 nm were collected after 5 min to ensure that thermal
equilibrium had been achieved. ThT was kept in excess at a final
concentration of 10 μM.
1
ethanol three times, and then dried in vacuum (purity >95%). H
NMR (400 MHz, CDCl3, 25 °C): δ = 12.21 (H, s), 9.22 (H, s), 8.00−
7.98 (H, d), 7.87−7.85 (H, d), 7.53−7.48 (2H, m), 7.42−7.38 (2H,
m), 7.03−7.01 (H, d). MALDI-TOF-MS (CHCl3): m/z 289.0, [M +
H]+. Elemental Anal., Calcd %: C, 58.23; H, 3.14; N, 9.70. Found %:
C, 58.47; H, 3.41, N, 9.64.
(E)-2-((Benzo[d]thiazol-2-ylimino)methyl)-4-nitrophenol (3).
3 was prepared by Schiff base condensation of 5-nitrosalicylaldehyde
(5 mmol) with 2-aminobenzothiazole (5 mmol) in ethanol (40 mL),
at reflux for 8 h. After cooling, the Schiff base precipitated from the
reaction mixture as a pure yellow powder, washed with ethanol three
times, and then dried in vacuum (purity >95%). 1H NMR (400 MHz,
CDCl3, 25 °C): δ = 13.10 (H, s), 9.40 (H, s), 8.53−8.52 (H, d), 8.37−
8.34 (H, d), 8.04−8.01 (H, d), 7.91−7.89 (H, d), 7.57−7.53 (H, t),
7.46−7.42 (H, t), 7.19−7.16 (H, d). MALDI-TOF-MS (CHCl3): m/z
300.0, [M + H]+. Elemental Anal., Calcd %: C, 56.18; H, 3.03; N,
14.04. Found %: C, 55.93; H, 2.99, N, 13.96.
(E)-2-((Benzo[d]thiazol-2-ylimino)methyl)phenol−CuCl2 (4).
CuCl2·2H2O (0.017 g, 0.1 mmol) in ethanol (1 mL) was added to
1 (0.2 mmol) in hot ethanol (25 mL). The resultant mixture was
stirred at 60 °C for ∼1 h, and the resultant solution was left to stand at
−20 °C for ∼12 h. The solid formed was collected by filtration,
washed with ethanol, and dried in vacuum (purity >95%). Elemental
Anal., Calcd %: C, 58.99; H, 3.18; N, 9.83. Found %: C, 58.74; H,
3.22; N, 9.67. ICP-MS, Calcd %: Cu, 11.15; S, 11.25. Found %: Cu,
11.18; S, 11.39.
(E)-2-((Benzo[d]thiazol-2-ylimino)methyl)-4-chlorophenol−
CuCl2 (5). CuCl2·2H2O (0.017 g, 0.1 mmol) in ethanol (1 mL) was
added to 2 (0.2 mmol) in hot ethanol (25 mL). The resultant mixture
was stirred at 60 °C for ∼1 h, and the resultant solution was left to
stand at −20 °C for ∼12 h. The solid formed was collected by
filtration, washed with ethanol, and dried in vacuum (purity >95%).
Elemental Anal., Calcd %: C, 52.63; H, 2.52; N, 8.77. Found %: C,
52.94; H,2.54; N, 8.61. ICP-MS, Calcd %: Cu, 9.94; S, 10.04. Found
%: Cu, 9.66; S, 10.08.
Atomic Force Microscopy Study. For the atomic force
microscopy (AFM) measurements, samples were diluted with
deionized H2O to yield a final concentration of 1 μM. Then the
sample (20 μL) was applied onto freshly cleaved muscovite mica and
allowed to dry. Data were acquired in the tapping mode on a
Nanoscope V multimode atomic force microscope (Veeco Instru-
ments, USA).
(E)-2-((Benzo[d]thiazol-2-ylimino)methyl)-4-nitrophenol−
CuCl2 (6). CuCl2·2H2O (0.017 g, 0.1 mmol) in ethanol (1 mL) was
added to 3 (0.2 mmol) in hot ethanol (25 mL). The resultant mixture
was stirred at 60 °C for ∼1 h, and the resultant solution was left to
stand at −20 °C for ∼12 h. The solid formed was collected by
filtration, washed with ethanol, and dried in vacuum (purity >95%).
Elemental Anal., Calcd %: C, 50.94; H, 2.44; N, 12.73. Found %: C,
50.82; H, 2.34; N, 12.51. ICP-MS, Calcd %: Cu, 9.63; S, 9.71. Found
%: Cu, 9.41; S, 9.87.
Light-Emission Measurements. Light emission was performed
by using a photon-counting spectrometer (ultra-weak luminescence
analyzer, BPCL-2-TGC) equipped with a cooled photomultiplier
detection system, connected to a computer. Before the samples
analyses, the background light was recorded and integrated. This
background was subtracted from the recorded integrated spectra of the
respective samples. Sample analyses were performed by adding the
sample solution (10 μL) to luminol (10 μM) and HRP (3 U/mL) in
buffer solution (150 μL; 150 mM Tris; pH 8.8) in a cuvette.
SOD Activity Determination. The SOD activity of the complexes
was assayed by measuring inhibition of the photoreduction of nitro
blue tetrazolium (NBT), by a method slightly modified from that
originally described by Beauchamps and Fridovich. The solutions
containing riboflavin (2 × 10−5 M), methionine (0.013 M), NBT (7.5
× 10−5 M), and complex of various concentrations were prepared with
50 mM phosphate buffer (pH 7.8). The mixtures were illuminated by a
lamp with a constant light intensity for 10 min at 25 °C. After
illumination, immediately the absorbance was measured at 560 nm.
The entire reaction assembly was enclosed in a box lined with
aluminum foil. Identical tubes with the reaction mixture were kept in
the dark and served as blanks. Inhibition percentage was calculated
according to the following formula: % inhibition = [(A0 − A)/A0] ×
100, where A0 is the absorbance of the control and A is the absorbance
of the samples. The IC50 value, which obtained from linear regression
plots between percent SOD activities versus their concentrations,
represents the concentration of the SOD mimic that induces a 50%
Cu2+ Binding Stoichiometry and Binding Affinity. Cu2+
binding stoichiometry and binding affinity were determined by UV−
vis. Because the compounds are slightly soluble in water, 10 mM stock
solutions were prepared in DMSO. In each titration, the solvent
composition in the vessel was 50% (v/v) DMSO/buffer (10 mM
HEPES, 150 mM NaCl, pH 6.6). For each compound, the binding
constant was determined by titration of a 0.050 mM solution of the
compound with microadditions of 1 mM CuCl2 standard solution.
Spectra were recorded after 5 min incubation at room temperature.
Data were fitted to the theoretical equations by using the nonlinear
least-squares fitting procedure implemented with the Origin 7.5
program.
Metal Ion Selectivity Studies. The interaction of compounds 1−
3 with other metal ions was determined using UV−vis. To investigate
the metal binding ability of compounds 1−3, solutions of the ligands
(50 or 100 μM in 50% (v/v) DMSO/buffer (10 mM HEPES, 150 mM
NaCl, pH 6.6)) were prepared and treated with 0.5 equiv of MgCl2,
CaCl2, AlCl3, FeCl3, CoCl2, NiCl2, or ZnCl2. Spectra were recorded
after 5 min incubation at room temperature. The metal ion selectivity
of compounds 1−3 for Cu2+ over other metal ions was determined by
UV−vis. To solutions of compounds 1−3 (50 or 100 μM in 50% (v/
G
dx.doi.org/10.1021/jm3003813 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
inhibition of the reduction of NBT. The SOD activities of test
compounds were assayed in triplicate.
amyloid hypothesis of Alzheimer’s disease: progress and problems on
the road to therapeutics. Science 2002, 297, 353−356.
Intracellular Determination of ROS. The generation of reactive
oxygen radicals was monitored using 2′,7′-dichlorofluorescein diacetate
(DCFH-DA). This dye is a nonfluorescent compound which readily
diffuses into the cells and reacts with intracellular free radicals. The
product is dichlorofluorescein (DCF), which is a fluorophor. The DCF
fluorescence intensity correlates with the amount of intracellular
reactive oxygen radicals. To do the test, the DCFH-DA solution (20
μM) was added to the cells and the mixture was incubated at 37 °C for
1 h. The cells were then washed twice with PBS and, finally, the
fluorescence intensity was monitored on a fluorescence spectrofluor-
ometer (JASCO FP-6500) with excitation and emission wavelengths
of 485 and 530 nm, respectively.
Cell Toxicity Assays. PC12 cells (rat pheochromocytoma,
American Type Culture Collection) were cultured in IMDM (Gibco
BRL) medium supplemented with 5% FBS, 10% horse serum in a 5%
CO2 humidified environment at 37 °C. For the MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Al-
drich) assay, cells were plated at a density of 10000 cells per well
on 96-well plates, followed by introduction of Aβ (5 μM), CuCl2 (5
μM), and a chelator (10 or 50 μM) 24 h later. After 48 h, the cells
were treated with 10 μL MTT (5 mg/mL in PBS) for 4 h at 37 °C and
then were lysed in DMSO for 10 min at room temperature in the dark.
Absorbance values of formazan were determined at 570 nm with an
automatic plate reader.
(3) Bush, A. I. The metallobiology of Alzheimer’s disease. Trends
Neurosci. 2003, 26, 207−214.
(4) Lovell, M. A.; Robertson, J. D.; Teesdale, W. J.; Campbell, J. L.;
Markesbery, W. R. Copper, iron and zinc in Alzheimer’s disease senile
plaques. J. Neurol. Sci. 1998, 158, 47−52.
(5) Stine, W. B., Jr.; Dahlgren, K. N.; Krafft, G. A.; LaDu, M. J. In
vitro characterization of conditions for amyloid-beta peptide
oligomerization and fibrillogenesis. J. Biol. Chem. 2003, 278, 11612−
11622.
(6) (a) Sparks, D. L.; Schreurs, B. G. Trace amounts of copper in
water induce beta-amyloid plaques and learning deficits in a rabbit
model of Alzheimer’s disease. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
11065−11069. (b) Syme, C. D.; Nadal, R. C.; Rigby, S. E.; Viles, J. H.
Copper binding to the amyloid-beta (Abeta) peptide associated with
Alzheimer’s disease: folding, coordination geometry, pH dependence,
stoichiometry, and affinity of Abeta-(1−28): insights from a range of
complementary spectroscopic techniques. J. Biol. Chem. 2004, 279,
18169−18177. (c) Geng, J.; Yu, H. J.; Ren, J. S.; Qu, X. G. Rapid label-
free detection of metal-induced Alzheimer’s amyloid beta peptide
aggregation by electrochemical method. Electrochem. Commun. 2008,
10, 1797−1800. (d) Yu, H.; Ren, J.; Qu, X. Different hydration
changes accompanying copper and zinc binding to amyloid beta-
peptide: water contribution to metal binding. ChemBioChem 2008, 9,
879−882.
(7) (a) Huang, X.; Atwood, C. S.; Hartshorn, M. A.; Multhaup, G.;
Goldstein, L. E.; Scarpa, R. C.; Cuajungco, M. P.; Gray, D. N.; Lim, J.;
Moir, R. D.; Tanzi, R. E.; Bush, A. I. The A beta peptide of Alzheimer’s
disease directly produces hydrogen peroxide through metal ion
reduction. Biochemistry 1999, 38, 7609−7616. (b) Huang, X.;
Cuajungco, M. P.; Atwood, C. S.; Hartshorn, M. A.; Tyndall, J. D.;
Hanson, G. R.; Stokes, K. C.; Leopold, M.; Multhaup, G.; Goldstein, L.
E.; Scarpa, R. C.; Saunders, A. J.; Lim, J.; Moir, R. D.; Glabe, C.;
Bowden, E. F.; Masters, C. L.; Fairlie, D. P.; Tanzi, R. E.; Bush, A. I.
Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with
cell-free hydrogen peroxide production and metal reduction. J. Biol.
Chem. 1999, 274, 37111−37116.
(8) (a) Cherny, R. A.; Legg, J. T.; McLean, C. A.; Fairlie, D. P.;
Huang, X.; Atwood, C. S.; Beyreuther, K.; Tanzi, R. E.; Masters, C. L.;
Bush, A. I. Aqueous dissolution of Alzheimer’s disease Abeta amyloid
deposits by biometal depletion. J. Biol. Chem. 1999, 274, 23223−
23228. (b) Cherny, R. A.; Atwood, C. S.; Xilinas, M. E.; Gray, D. N.;
Jones, W. D.; McLean, C. A.; Barnham, K. J.; Volitakis, I.; Fraser, F.
W.; Kim, Y.; Huang, X.; Goldstein, L. E.; Moir, R. D.; Lim, J. T.;
Beyreuther, K.; Zheng, H.; Tanzi, R. E.; Masters, C. L.; Bush, A. I.
Treatment with a copper−zinc chelator markedly and rapidly inhibits
beta-amyloid accumulation in Alzheimer’s disease transgenic mice.
Neuron 2001, 30, 665−676.
ASSOCIATED CONTENT
* Supporting Information
■
S
UV−vis titration of compounds 2 and 3 with Cu2+; metal ion
selectivity studies; capture Cu2+ from Aβ by compounds 1−3
characterized by UV−vis spectrum; Cu2+ competitive binding
experiment; the model structures of complexes 4−6;
fluorescence titration of Aβ with compounds 1−3 and metal
complexes; effect of compounds 1−3 on Aβ self-assembly by
ThT fluorescence assay; absorption spectra of complexes 4−6
in the presence (red line) of Aβ40; SOD activity of compounds
1−3 and SOD enzyme. Effect of compounds 1−3 on PC12 cell
viability. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (+86) 431-85262656. Fax: (+86) 431-85262656. E-
mail: xqu@ciac.jl.cn.
■
Author Contributions
§J.G. and M.L. contributed equally to this work.
(9) (a) Tjernberg, L. O.; Naslund, J.; Lindqvist, F.; Johansson, J.;
Karlstrom, A. R.; Thyberg, J.; Terenius, L.; Nordstedt, C. Arrest of
beta-amyloid fibril formation by a pentapeptide ligand. J. Biol. Chem.
1996, 271, 8545−8548. (b) Takahashi, T.; Mihara, H. Peptide and
protein mimetics inhibiting amyloid beta-peptide aggregation. Acc.
Chem. Res. 2008, 41, 1309−1318. (c) Frydman-Marom, A.; Rechter,
M.; Shefler, I.; Bram, Y.; Shalev, D. E.; Gazit, E. Cognitive-
performance recovery of Alzheimer’s disease model mice by
modulation of early soluble amyloidal assemblies. Angew. Chem., Int.
Ed. Engl. 2009, 48, 1981−1986.
(10) (a) Yang, F.; Lim, G. P.; Begum, A. N.; Ubeda, O. J.; Simmons,
M. R.; Ambegaokar, S. S.; Chen, P. P.; Kayed, R.; Glabe, C. G.;
Frautschy, S. A.; Cole, G. M. Curcumin inhibits formation of amyloid
beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo.
J. Biol. Chem. 2005, 280, 5892−5901. (b) Barnham, K. J.; Kenche, V.
B.; Ciccotosto, G. D.; Smith, D. P.; Tew, D. J.; Liu, X.; Perez, K.;
Cranston, G. A.; Johanssen, T. J.; Volitakis, I.; Bush, A. I.; Masters, C.
L.; White, A. R.; Smith, J. P.; Cherny, R. A.; Cappai, R. Platinum-based
inhibitors of amyloid-beta as therapeutic agents for Alzheimer’s
disease. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 6813−6818.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support by 973 Project (2011CB936004,
2012CB720602), NSFC (20831003, 90813001, 20833006,
90913007, 20903086), and Fund from CAS.
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; Aβ, amyloid β peptide; ROS, reactive
oxygen species; BBB, blood−brain barrier; ThT, thioflavin T;
CQ, clioquinol; SOD, superoxide dismutase
REFERENCES
■
(1) Blennow, K.; de Leon, M. J.; Zetterberg, H. Alzheimer’s disease.
Lancet 2006, 368, 387−403.
(2) (a) Hardy, J. Testing times for the “amyloid cascade hypothesis”.
Neurobiol. Aging 2002, 23, 1073−1074. (b) Hardy, J.; Selkoe, D. J. The
H
dx.doi.org/10.1021/jm3003813 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(c) Ehrnhoefer, D. E.; Bieschke, J.; Boeddrich, A.; Herbst, M.; Masino,
L.; Lurz, R.; Engemann, S.; Pastore, A.; Wanker, E. E. EGCG redirects
amyloidogenic polypeptides into unstructured, off-pathway oligomers.
Nature Struct. Mol. Biol. 2008, 15, 558−566.
(11) Ouberai, M.; Dumy, P.; Chierici, S.; Garcia, J. Synthesis and
Biological Evaluation of Clicked Curcumin and Clicked KLVFFA
Conjugates as Inhibitors beta-Amyloid Fibril Formation. Bioconjugate
Chem. 2009, 20, 2123−2132.
compounds with metal chelating properties for application in
Alzheimer’s disease. J. Am. Chem. Soc. 2009, 131, 1436−1451.
(23) M. Mammen, M.; Choi, S. K.; Whitesides, G. M. Polyvalent
Interactions in Biological Systems: Implications for Design and Use of
Multivalent Ligands and Inhibitors. Angew. Chem., Int. Ed. Engl. 1998,
37, 2755−2794.
(24) (a) Baldini, L.; Casnati, A.; Sansone, F.; Ungaro, R. Calixarene-
based multivalent ligands. Chem. Soc. Rev. 2007, 36, 254−266.
(b) Bowman, M. C.; Ballard, T. E.; Ackerson, C. J.; Feldheim, D. L.;
Margolis, D. M.; Melander, C. Inhibition of HIV fusion with
multivalent gold nanoparticles. J. Am. Chem. Soc. 2008, 130, 6896−
6897. (c) Rosenzweig, B. A.; Ross, N. T.; Tagore, D. M.;
Jayawickramarajah, J.; Saraogi, I.; Hamilton, A. D. Multivalent protein
binding and precipitation by self-assembling molecules on a DNA
pentaplex scaffold. J. Am. Chem. Soc. 2009, 131, 5020−5021.
(25) (a) Yoshiike, Y.; Tanemura, K.; Murayama, O.; Akagi, T.;
Murayama, M.; Sato, S.; Sun, X.; Tanaka, N.; Takashima, A. New
insights on how metals disrupt amyloid beta-aggregation and their
effects on amyloid-beta cytotoxicity. J. Biol. Chem. 2001, 276, 32293−
32299. (b) Zou, J.; Kajita, K.; Sugimoto, N. Cu(2+) Inhibits the
Aggregation of Amyloid beta-Peptide(1−42) in vitro. Angew. Chem.,
Int. Ed. Engl. 2001, 40, 2274−2277.
(26) Jun, S.; Saxena, S. The aggregated state of amyloid-beta peptide
in vitro depends on Cu2+ ion concentration. Angew. Chem., Int. Ed.
Engl. 2007, 46, 3959−3961.
(27) Tougu, V.; Karafin, A.; Zovo, K.; Chung, R. S.; Howells, C.;
West, A. K.; Palumaa, P. Zn(II)- and Cu(II)-induced non-fibrillar
aggregates of amyloid-beta (1−42) peptide are transformed to amyloid
fibrils, both spontaneously and under the influence of metal chelators.
J. Neurochem. 2009, 110, 1784−1795.
(28) Rauk, A. The chemistry of Alzheimer’s disease. Chem. Soc. Rev.
2009, 38, 2698−2715.
(29) Miranda, S.; Opazo, C.; Larrondo, L. F.; Munoz, F. J.; Ruiz, F.;
Leighton, F.; Inestrosa, N. C. The role of oxidative stress in the toxicity
induced by amyloid beta-peptide in Alzheimer’s disease. Prog.
Neurobiol. 2000, 62, 633−648.
(30) Li, F.; Calingasan, N. Y.; Yu, F.; Mauck, W. M.; Toidze, M.;
Almeida, C. G.; Takahashi, R. H.; Carlson, G. A.; Flint Beal, M.; Lin,
M. T.; Gouras, G. K. Increased plaque burden in brains of APP mutant
MnSOD heterozygous knockout mice. J. Neurochem. 2004, 89, 1308−
1312.
(31) Melov, S.; Adlard, P. A.; Morten, K.; Johnson, F.; Golden, T. R.;
Hinerfeld, D.; Schilling, B.; Mavros, C.; Masters, C. L.; Volitakis, I.; Li,
Q. X.; Laughton, K.; Hubbard, A.; Cherny, R. A.; Gibson, B.; Bush, A.
I. Mitochondrial oxidative stress causes hyperphosphorylation of tau.
PLoS One 2007, 2, e536.
(12) Cairo, C. W.; Strzelec, A.; Murphy, R. M.; Kiessling, L. L.
Affinity-based inhibition of beta-amyloid toxicity. Biochemistry 2002,
41, 8620−8629.
(13) (a) Zhang, G. B.; Leibowitz, M. J.; Sinko, P. J.; Stein, S.
Multiple-peptide conjugates for binding beta-amyloid plaques of
Alzheimer’s disease. Bioconjugate Chem. 2003, 14, 86−92. (b) Chafekar,
S. M.; Malda, H.; Merkx, M.; Meijer, E. W.; Viertl, D.; Lashuel, H. A.;
Baas, F.; Scheper, W. Branched KLVFF tetramers strongly potentiate
inhibition of beta-amyloid aggregation. ChemBioChem 2007, 8, 1857−
1864. (c) Dolphin, G. T.; Chierici, S.; Ouberai, M.; Dumy, P.; Garcia,
J. A multimeric quinacrine conjugate as a potential inhibitor of
Alzheimer’s beta-amyloid fibril formation. ChemBioChem 2008, 9,
952−963.
(14) (a) Ghadiri, M. R.; Soares, C.; Choi, C. Design of an Artificial 4-
Helix Bundle Metalloprotein Via a Novel Ruthenium(II)-Assisted Self-
Assembly Process. J. Am. Chem. Soc. 1992, 114, 4000−4002. (b) M.
Lieberman, M.; Sasaki, T. Iron(II) organizes a synthetic peptide into
three-helix bundles. J. Am. Chem. Soc. 1991, 113, 1470−1471.
(15) (a) Wu, C.; Pike, V. W.; Wang, Y. Amyloid imaging: from
benchtop to bedside. Curr. Top. Dev. Biol. 2005, 70, 171−213.
(b) Ono, M.; Hayashi, S.; Kimura, H.; Kawashima, H.; Nakayama, M.;
Saji, H. Push−pull benzothiazole derivatives as probes for detecting
beta-amyloid plaques in Alzheimer’s brains. Bioorgan. Med. Chem.
2009, 17, 7002−7007.
(16) Pardridge, W. M. Alzheimer’s disease drug development and the
problem of the blood−brain barrier. Alzheimer's Dementia 2009, 5,
427−432.
(17) Clark, D. E.; Pickett, S. D. Computational methods for the
prediction of 'drug-likeness. Drug Discovery Today 2000, 5, 49−58.
(18) Ferrada, E.; Arancibia, V.; Loeb, B.; Norambuena, E.; Olea-Azar,
C.; Huidobro-Toro, J. P. Stoichiometry and conditional stability
constants of Cu(II) or Zn(II) clioquinol complexes; implications for
Alzheimer’s and Huntington’s disease therapy. Neurotoxicology 2007,
28, 445−449.
(19) Choi, J. S.; Braymer, J. J.; Nanga, R. P.; Ramamoorthy, A.; Lim,
M. H. Design of small molecules that target metal-A{beta} species and
regulate metal-induced A{beta} aggregation and neurotoxicity. Proc.
Natl. Acad. Sci. U.S.A. 2010, 107, 21990−21995.
(20) (a) Cordeiro, Y.; Machado, F.; Juliano, L.; Juliano, M. A.;
Brentani, R. R.; Foguel, D.; Silva, J. L. DNA converts cellular prion
protein into the beta-sheet conformation and inhibits prion peptide
aggregation. J. Biol. Chem. 2001, 276, 49400−49409. (b) Ferrao-
Gonzales, A. D.; Robbs, B. K.; Moreau, V. H.; Ferreira, A.; Juliano, L.;
Valente, A. P.; Almeida, F. C.; Silva, J. L.; Foguel, D. Controlling
{beta}-amyloid oligomerization by the use of naphthalene sulfonates:
trapping low molecular weight oligomeric species. J. Biol. Chem. 2005,
280, 34747−34754.
(21) (a) Stine, W. B., Jr.; Snyder, S. W.; Ladror, U. S.; Wade, W. S.;
Miller, M. F.; Perun, T. J.; Holzman, T. F.; Krafft, G. A. The
nanometer-scale structure of amyloid-beta visualized by atomic force
microscopy. J. Protein Chem. 1996, 15, 193−203. (b) Arimon, M.;
Diez-Perez, I.; Kogan, M. J.; Durany, N.; Giralt, E.; Sanz, F.;
Fernandez-Busquets, X. Fine structure study of Abeta1−42 fibrillo-
genesis with atomic force microscopy. FASEB J. 2005, 19, 1344−1346.
(22) (a) Hindo, S. S.; Mancino, A. M.; Braymer, J. J.; Liu, Y.;
Vivekanandan, S.; Ramamoorthy, A.; Lim, M. H. Small molecule
modulators of copper-induced Abeta aggregation. J. Am. Chem. Soc.
2009, 131, 16663−16665. (b) Rodriguez-Rodriguez, C.; Sanchez de
Groot, N.; Rimola, A.; Alvarez-Larena, A.; Lloveras, V.; Vidal-Gancedo,
J.; Ventura, S.; Vendrell, J.; Sodupe, M.; Gonzalez-Duarte, P. Design,
selection, and characterization of thioflavin-based intercalation
(32) Esposito, L.; Raber, J.; Kekonius, L.; Yan, F.; Yu, G. Q.; Bien-Ly,
N.; Puolivali, J.; Scearce-Levie, K.; Masliah, E.; Mucke, L. Reduction in
mitochondrial superoxide dismutase modulates Alzheimer’s disease-
like pathology and accelerates the onset of behavioral changes in
human amyloid precursor protein transgenic mice. J. Neurosci. 2006,
26, 5167−5179.
(33) Massaad, C. A.; Washington, T. M.; Pautler, R. G.; Klann, E.
Overexpression of SOD-2 reduces hippocampal superoxide and
prevents memory deficits in a mouse model of Alzheimer’s disease.
Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 13576−13581.
(34) Sompol, P.; Ittarat, W.; Tangpong, J.; Chen, Y.; Doubinskaia, I.;
Batinic-Haberle, I.; Abdul, H. M.; Butterfield, D. A.; St. Clair, D. K. A
neuronal model of Alzheimer’s disease: an insight into the mechanisms
of oxidative stress-mediated mitochondrial injury. Neuroscience 2008,
153, 120−130.
(35) Wu, Y.; Wang, D. A new class of natural glycopeptides with
sugar moiety-dependent antioxidant activities derived from Ganoder-
ma lucidum fruiting bodies. J. Proteome Res. 2009, 8, 436−442.
(36) (a) Guilloreau, L.; Combalbert, S.; Sournia-Saquet, A.;
Mazarguil, H.; Faller, P. Redox chemistry of copper-amyloid-beta:
the generation of hydroxyl radical in the presence of ascorbate is linked
to redox-potentials and aggregation state. ChemBioChem 2007, 8,
1317−1325. (b) Jiang, D.; Men, L.; Wang, J.; Zhang, Y.; Chickenyen,
I
dx.doi.org/10.1021/jm3003813 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
S.; Wang, Y.; Zhou, F. Redox reactions of copper complexes formed
with different beta-amyloid peptides and their neuropathological
relevance. Biochemistry 2007, 46, 9270−9282.
(37) Ji, H. F.; Zhang, H. Y. A new strategy to combat Alzheimer’s
disease. Combining radical-scavenging potential with metal-protein-
attenuating ability in one molecule. Bioorg. Med. Chem. Lett. 2005, 15,
21−24.
(38) (a) Geng, J.; Zhao, C.; Ren, J.; Qu, X. Alzheimer’s disease
amyloid beta converting left-handed Z-DNA back to right-handed B-
form. Chem. Commun. 2010, 46, 7187−7189. (b) Yu, H.; Ren, J.; Qu,
X. Time-dependent DNA condensation induced by amyloid beta-
peptide. Biophys. J. 2007, 92, 185−191. (c) Geng, J.; Li, M.; Ren, J.;
Wang, E.; Qu, X. Polyoxometalates as inhibitors of the aggregation of
amyloid beta peptides associated with Alzheimer’s disease. Angew.
Chem., Int. Ed. Engl. 2011, 50, 4184−4188.
(39) Goedert, M.; Spillantini, M. G. A century of Alzheimer’s disease.
Science 2006, 314, 777−781.
(40) Klafki, H. W.; Staufenbiel, M.; Kornhuber, J.; Wiltfang, J.
Therapeutic approaches to Alzheimer’s disease. Brain 2006, 129,
2840−2855.
J
dx.doi.org/10.1021/jm3003813 | J. Med. Chem. XXXX, XXX, XXX−XXX