Design, synthesis, and biological evaluation of curcumin analogues as multifunctional agents for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2011.07.033

A series of novel curcumin analogues were designed, synthesized, and evaluated as potential multifunctional agents for the treatment of AD. The in vitro studies showed that these compounds had better inhibitory properties against Aβ aggregation than curcu

Full text of DOI:10.1016/j.bmc.2011.07.033

Bioorganic & Medicinal Chemistry 19 (2011) 5596–5604
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of curcumin analogues
as multifunctional agents for the treatment of Alzheimer’s disease
⇑
Shang-Ying Chen, Yuan Chen, Yan-Ping Li, Shu-Han Chen, Jia-Heng Tan , Tian-Miao Ou, Lian-Quan Gu,
Zhi-Shu Huang
⇑
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel curcumin analogues were designed, synthesized, and evaluated as potential multifunc-
tional agents for the treatment of AD. The in vitro studies showed that these compounds had better
inhibitory properties against Ab aggregation than curcumin. Superior anti-oxidant properties (better than
the reference compound Trolox) of these compounds were observed by the oxygen radical absorbance
capacity (ORAC) method and a cell-based assay using DCFH-DA as a probe. In addition they were able
to chelate metals such as iron and copper and decrease metal-induced Ab aggregation. The structure–
activity relationships were discussed. The results suggested that our curcumin analogues could be
selected as multifunctional agents for further investigation of AD treatment.
Received 12 May 2011
Revised 16 July 2011
Accepted 19 July 2011
Available online 24 July 2011
Keywords:
Curcumin analogue
Anti-Alzheimer agent
Ab aggregation
Oxidative stress
Metal chelating
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
patients’ brian.11 Thus, therapeutic strategy that aimed at the
removal of free radicals or prevention of their formation might
Alzheimer’s disease (AD),
a
progressive neurodegenerative
be beneficial for AD.
brain disorder, is affecting more and more elderly all around the
world.1 Data have revealed that there are 17 million AD patients
until 2009 and the number would reach 70 million by 2050 if no
cure or preventive measure is found.2 The etiology of AD is still
enigmatic, and multiple factors have been suggested to contribute
to the development of AD. Amyloid-b (Ab) plaques, widely ac-
cepted as the key pathological feature of AD, are mainly consti-
tuted by aggregation of the Ab peptide, a 39- to 43-residue-long
protein, derived from the amyloid precursor protein (APP).3 Of
two most abundant forms of Ab, Ab1–42 has a higher propensity
to form fibrils than Ab1–40. Furthermore, Ab1–42 aggregates into
oligomers and fibrils in the brain and causes strong neuronal tox-
icity.4 Prevention of Ab aggregation in the brain is currently being
considered as potential therapies for AD. Several series of inhibi-
tors such as curcumin, rifampicin, benzofuran, and bis-styrylbenze
analogues or derivatives were developed, and these compounds
were found to interfere with fibrillization of Ab.5–8 Several studies
suggested that oxidative damage also plays an important role in
this chronic neurodegenerative disease.9,10 The direct evidences
of the oxidative stress hypothesis are increased lipid peroxidation
and the increased concentration of Fe, Cu, Al, and Hg in AD
Recently, abundant data has implicated the roles of biometals
such as iron, copper, and Zinc in the Ab aggregate deposition and
neurotoxicity including the formation of reactive oxygen species
(ROS).12,13 Firstly, abnormal enrichment of Cu, Fe, and Zn in post-
mortem AD brain has been confirmed.14 In vitro experiments re-
vealed that these metals are able to bind to Ab, thus promoting
its aggregation.15 On the other hand, redox-active metal ions like
Cu and Fe contribute to the production of ROS and widespread oxi-
dation damages observed in AD brains.16,17 Therefore, modulation
of such biometals in the brain provides a potential therapeutic
strategy for the treatment of AD. Small molecule chelating agents
have been proposed for this purpose. In particular, Cu chelating
agents have been widely explored to remove Cu from Cu-Ab spe-
cies and subsequently decrease metal-induced Ab deposits.18,19
The potential regulation of metal-induced Ab aggregation and
neurotoxicity through using traditional metal chelating agents,
such as desferrioxamine, clioquinol (CQ), and 8-hydroxyquinoline
derivative (PBT2) has been studied in clinical trials.20–22 However,
poor target specificity and consequential clinical safety of current
metal-complexing agents make them undesirable for wide applica-
tion. To circumvent this drawback, the new strategy of developing
bifunctional or multifunctional metal chelators has recently been
proposed.23–25 In addition to the metal chelating ability, these
agents are also designed to improve their uptake across the
blood–brain barrier, decrease Ab levels, inhibit cholinesterase
and increase anti-oxidant capabilities.25
⇑
Corresponding authors. Tel./fax: +86 20 39943056 (J.-H.T.).
E-mail addresses: tanjiah@mail.sysu.edu.cn (J.-H. Tan), ceshzs@mail.sysu.edu.cn
(Z.-S. Huang).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.033

S.-Y. Chen et al. / Bioorg. Med. Chem. 19 (2011) 5596–5604
5597
Indeed, the multifaceted condition of AD has encouraged active
research in the development of multifunctional drugs with two or
more complementary biological activities. Although a few note-
worthy advances in the area of multifunctional agents have been
achieved, the design of multifunctional drugs is still a tough
work.26,27 Clearly, the yield can be greatly increased by properly
selecting the lead compound. Inspired by this concept, curcumin
which already showed broad spectrum of biological activities re-
lated to AD was selected as the lead compound.28 Curcumin is a
yellowish polyphenol compound isolated from turmeric which
might be responsible for the low age-adjusted prevalence of AD
in India.28 Various experiments demonstrate that curcumin has
anti-oxidant, anti-inflammatory, anti-b-amyloid, and metal-
chelating properties.28,29 All these properties are critical in the
pathogenesis of AD. In this work, we rationally designed the syn-
thesized a novel series of curcumin analogues as multifunctional
agents for the treatment of AD. Their biological evaluation includes
inhibition of Ab aggregation, anti-oxidant capabilities in vitro and
in vivo, metal-chelating properties, and disassembly against
metal-induced Ab deposits.
peptide.27,32 On the other hand, since it has been pointed out that
the linkers are responsible for their activities.31,33 Thus, we varied
the flexibility of the linker as a key parameter for investigation. At
the same time, most of the designed compounds retained carbonyl
group which bears potential metal-chelating properties, along with
the styryl function for their anti-oxidant activities.
The facile synthetic pathway for curcumin analogues was illus-
trated in Scheme 1. Compounds A1–A5, A9–A10, and B4 were
synthesized by aldol reaction as reported.34,35 Compound A6
was obtained following the procedure of our previous work.36
A7 and A8 were synthesized in accordance with the Kyung Hyun
Lee method with little modification.37 The configuration of the
compounds was confirmed by the NOE analysis and coupling
constant displaying that all compounds had linear (E, E)
configuration.
2.2. Biological evaluation
2.2.1. Inhibition of self-mediated Ab1–42 aggregation
To investigate the self-mediated Ab1–42 aggregation, the Thiofla-
vin T (ThT) fluorescence assay was performed. Inhibition activities
of all substances against Ab1–42 aggregation were listed in Table 1
2. Results and discussion
as IC50 and inhibition ratio at a tested concentration of 50 lM.
Analysis of the data from ThT method revealed that all the tested
compounds inhibited the Ab aggregation while the inhibitory
activities of these compounds vary accordingly. Compounds
A1–A6 with a chain of aliphatic structure displayed excellent IC50
2.1. Design consideration and synthesis of curcumin analogues
In designing such curcumin analogues as multifunctional drugs,
we focused on improving their inhibition activities of Ab aggrega-
tion while retained the functional groups for anti-oxidation and
metal chelation. As shown in Figure 1, the structure of curcumin
is similar to that of the Congo Red and IMSB. They all have two aro-
matic end groups and a linker region in the middle.30 It was re-
ferred that the two aromatic groups and their polar substitution
are essential to their activities in terms of the efficient binding at
values (ranging from 2–22 lM), while reduced inhibitory activity
of A7–A10, with aromatous structure linkers in the middle was
observed. These findings suggested that the flexibility of the chain
between the two aromatic moieties might be an essential determi-
nant of the compound’s inhibitory activity as better inhibition
properties can be obtained from the aliphatic structure linkers
with less rigidity. Among A1–A6, compound A4 was recognized
the Ab peptide guided by
p-stacking and hydrogen bond interac-
tions.31 Therefore, we introduced an N-methylpiperazine on the
two aromatic ends (Scheme 1) expecting that the hydrogen bond-
ing substitutions and the additional electrostatic interactions
would significantly strengthen the binding affinity to the Ab
by the most potent activity with an IC50 value of 2.5
lM and 90%
inhibition at a concentration of 50 M. Substitution with a carbon
l
atom of the nitrogen atom or N-methylation in the linker of A4 (see
compound A3 and A5) induced a lessening of activity, suggesting
that piperidone structure fragment in the linker play a key role
in the significant inhibition activity of the compound. Hereafter,
compound B4 with a similar structure of A4 except a piperidine
pattern in the two aromatic ends was designed to investigate the
effect the terminal protonated nitrogen on Ab1–42 assembly.
Although B4 displayed less inhibitory potency against Ab1–42
aggregation, it still had a good inhibition of Ab1–42 aggregation
O
O
H3CO
HO
OCH3
OH
Curcumin
with its IC50 value of 17.2 lM. Therefore, it indicated that the
terminal protonated nitrogen in the ends may be less crucial than
the linker for the inhibition activity of the compound.
NH2
N
2.2.2. Effect on Ab b-sheet formation by A4
H2N
It was referred that Ab1–42 adopted a conformational mixture of
N
N
O
a-helix, b-sheet, and random coil in the aqueous solution and
S
N
O
underwent conformational change to form intramolecular
a
NaO
b-sheet structure in the fibrillation.38 In order to further investigate
the effect of the promising compound A4 on the structural transi-
tion of Ab1–42, the CD spectroscopy method was employed to
monitor the changes of the secondary structure of Ab1–42 during
the assembly stages (0–32 h). Figure 2 showed that the contents
of b-sheet structure (occurrence of a peak around 195 nm) and
O
S
O
ONa
Congo Red
I
a-helix structure (occurrence of a broad minimum around
217 nm) increased during the first 32 h incubation (Fig. 2). Notably,
the addition of compound A4 resulted in a significant decrease in
b-sheet structure at all tested time points during the first 32 h,
HO
HOOC
OH
COOH
but it had little effect on the content of
ing that A4 may reduce or retard b-sheet structure formation
through stabilizing the -helix structure of peptide.
a-helix structure, suggest-
IMSB
Figure 1. Structure of curcumin, Congo Red, and IMSB.
a

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S.-Y. Chen et al. / Bioorg. Med. Chem. 19 (2011) 5596–5604
O
O
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
A1
A5
O
O
O
O
O
N
N
N
N
A9
A2
path a
O
O
or
O
N
N
N
A3
A10
N
N
O
O
N
N
H
N
N
N
H
N
N
N
A4
B4
O
O
path b
path c
O
N
N
N
N
N
N
A6
N
N
N
N
N
N
N
N
N
A7
A8
Scheme 1. Synthesis of curcumin analogues. Reagents and conditions: (a) ketone, 95% EtOH, 10% NaOH, rt, 30 min; or CH3COOH, HCl, rt, 48 h; (b) acetylacetone, B2O3,
n-(BuO)3B, EtOAC, n-C4H9NH2, 70 °C 24 h; (c) dibromide, potassium t-butoxide, THF, rt, 12 h.
Table 1
Substantial changes in the morphology of the aggregates were
observed when Ab1–42 was incubated with A4 (Fig. 3). At point 0,
there was no aggregation for Ab1–42 with or without A4. However,
after 12 h incubation, the sample of Ab1–42 alone had mostly aggre-
gated into amyloid fibrils while less thin and short fibrils were
caught in the sample of Ab1–42 in presence of A4. After 32 h incu-
bation, numerous mature and bulky fibrils were observed in the
sample of Ab1–42 alone. Meanwhile, only a few short fibrils and
many breakage points were found in the Ab1–42 samples incubated
with A4. The EM results were well consistent with the results of
ThT and CD measurements, strongly proving that A4 can inhibit
and slow down the Ab1–42 fibrils formation.
Inhibition of Ab aggregation tested by ThT methods and ORAC values
a
Compounds
IC50
(lM)
Inhibition of Ab
ORACc
aggregationb (%)
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
B4
Curcumin
Trolox
21.5 3.7
12.6 3.9
6.0 4.1
2.5 1.2
17.6 2.7
9.2 5.2
>50
68.6 3.2
67.2 6.1
73.1 2.3
90.2 3.2
65.1 3. 5
77.8 2.2
42.1 2.0
24.0 5.2
52.8 2.7
35.8 1.9
64.5 3.3
71.3 1. 3
3.2 0.2
1.7 0.2
2.6 0.2
5.8 0.1
4.7 0.2
1.9 0.1
1.2 0.1
1.1 0.2
1.3 0.1
1.1 0.1
2.4 0.1
2.5 0.2
1.00
>50
37.8 4.1
>50
17.2 3.2
12.1 1.2
2.2.4. Anti-oxidant activity in vitro
The reduction of the oxidative stress is another crucial aspect of
designing the curcumin analogues. The oxygen radical absorbance
capacity (ORAC) method was implemented to determine the anti-
oxidant capacities of the synthesized compounds (Table 1). The
ability to scavenge radicals is expressed as Trolox equivalent (their
relative ability compared to the highly potent compound Trolox).
From Table 1, it is quite evident that all the tested analogues had
better anti-oxidant activities than Trolox. It’s surprising to see
several compounds (A1, A3–A5) even better than curcumin, since
they lack the phenolic groups such as methoxy or hydroxyl, which
are important for the anti-oxidant activities of curcumin. An
a
The Thioflavin-T fluorescence method was used. Values are expressed as the
mean SD from at least two independent measurements.
b
The maximum percent inhibition of aggregation in the ThT does dependence
studies, all of them were found at the inhibitors’ concentration of 50
l
M.
The mean SD of the three independent experiments and the measurements
were carried out in presence of 0.625, 1.25, 2.5 M compounds.
c
l
2.2.3. Effects on abundance of Ab fibrils by A4
To complement the ThT binding assay and CD assay, Ab1–42
aggregation was also monitored by electron microscopy (EM).

S.-Y. Chen et al. / Bioorg. Med. Chem. 19 (2011) 5596–5604
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Figure 2. CD spectroscopy of Ab1–42 alone or with compound A4 incubated at 0 h (A), 6 h (B), 12 h (C) and 32 h (D).
Figure 3. EM images of Ab1–42 (40 lM) in the presence and absence of 20 lM compound A4, after 0, 12, and 32 h of aggregation.

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S.-Y. Chen et al. / Bioorg. Med. Chem. 19 (2011) 5596–5604
explanation for this might be that the styryl function and steric or
electronic factors through the introduction of the piperazine
groups contribute to their anti-oxidant activities. Furthermore,
the trend of these compounds’ anti-oxidant activities is similar to
their inhibition activities of Ab aggregation: compounds A1–A6
exhibited better anti-oxidant activities than compounds A7–A10
and among all the tested compounds, A4 had the highest ORAC va-
lue (5.8), a much higher radical capture activity than Trolox (1.0)
and curcumin (2.5). Therefore, it is clear that A4 was more active
than curcumin and other synthesized compounds in inhibiting
Ab fibril formation and scavenging radical.
2.2.5. Anti-oxidant activity in SH-SY5Y cells
The ability of the compounds to counteract the formation of
ROS was assayed in human neuroblastoma cells (SH-SY5Y) after
the treatment with tert-butyl hydroperoxide (t-BuOOH), a com-
pound used to induce oxidative stress.39 The concentration of
Figure 5. UV spectrum of compound A4 (20 lM) alone or at the presence of 20 lM
ZnSO4, CuSO4, and FeSO4.
tested compounds did not affect the cell viability (2.5 lM)
(Fig. S1). As shown in Figure 4, the ROS levels of SH-SY5Y cells
incubated with tested compounds decreased compared to control
cells, particularly compounds A1–A4. Other anti-oxidants Trolox,
N-acetyl-L-cysteine (NAC) and curcumin were also tested at a
In order to determine the stoichiometry of the complex A4-me-
tal (II), the molar ration method was employed, by preparing the
solution of compound A4 with ascending amounts of CuSO4 or
FeSO4.40 For instance, the UV spectra were recorded by numerical
subtraction of CuSO4 and A4 at corresponding concentrations,
and the spectra peaked at 493 nm (data not shown). According to
Figure 6, it is implicit that the absorbance firstly increases at
493 nm and then tends to be stable versus the mole fraction of
Cu2+ to A4. Therefore, two straight lines were draw with the inter-
section point at a mole fraction of 1.04, revealing 1:1 stoichiometry
for metal(II)–ligand complexes. This is in accordance with other
similar structures reported elsewhere.29,40
higher concentration (20 lM). As can be seen in Figure 4, NAC
was unable to abolish ROS generation in SH-SY5Y cells treatment
with t-BuOOH, while both Trolox and curcumin decreased the
intensity of fluorescence. This result suggests that the action of
these compounds involved in lipid peroxidation and free radical
chain reaction might be close to that of Trolox.
2.2.6. Metal-chelating properties of A4
The chelation ability of compound A4 toward biometals such as
Cu, Fe and Zn was studied by UV–vis spectrometry.40 Electronic
spectral of compound A4 in ethanol changed in the presence of
Cu2+ and Fe2+ ions while remained unchanged after adding Zn2+
ions ( Fig. 5). Upon the addition of CuSO4 and FeSO4, the curve
had a red shift (after adding Cu2+, the peak at 267 nm shift to
274 nm whereas the peak at 427 nm shift to 434 nm) suggesting
the formation of complex A4-metal (II). However, after adding
ZnSO4, no significant difference was observed in the UV spectrum
which indicates that this compound may not complex with zinc.
Therefore, compound A4 had the same selectivity of metal chela-
tion as reported to curcumin (affinity for Cu2+ and Fe2+ rather than
Zn2+).29
2.2.7. Effects on metal-induced Ab1–42 aggregation by A4
To investigate the impact of A4 on metal-induced Ab1–42 aggrega-
tion, the ThT experiment was performed.41 The general Ab aggrega-
tion turbidity assay was not used because the absorbance of the A4
and its corresponding metal complexes (see Fig. 5) overlapped the
analysis window (405 nm) and significantly interfered with the re-
sult. ThT, however, showed a much higher fluorescent intensity than
the compound and its metal complexes. For the disassembly of the
metal fibril, all the samples containing Cu2+ and Fe2+ were buffered
at pH 6.6, whereas the ones with Zn2+ were buffered at pH 7.4. The
results were reported in Figure 7. Firstly, all three kinds of metal ions
accelerated the aggregation of Ab1–42 in 45 min, while Fe2+ showed a
Figure 4. ROS generation in SH-SY5Y cells incubated without or with compounds
measured using DCFH-DA. The results are expressed as the percentage of control
cells (untreated with compounds).
Figure 6. Determination of the stoichiometry of complex A4-Cu(II) by molar ration
method.

S.-Y. Chen et al. / Bioorg. Med. Chem. 19 (2011) 5596–5604
5601
water (35:65–80:20) containing 0.1% TFA at
a
flow rate of
0.5 mL/min. Melting points (mp) were determined using a SRS-
OptiMelt automated melting point instrument without correction.
4.2. Synthesis of curcumin analogues
4.2.1. (1E,4E)-1,5-Bis(4-(4-methylpiperazin-1-yl)phenyl)penta-
1,4-dien-3-one (A1)
To
a
mixture of 4-(4-methylpiperazin-1-yl)benzaldehyde
L, 0.5 mmol) in 1 mL 95% eth-
(204 mg, 1 mmol) and acetone (36
l
anol, 0.5 mL 10% NaOH aqueous solution was added slowly at room
temperature. The resulting reaction mixture was stirred for 30 min.
The precipitate was then separated from solvent by filtration and
washed with water to afford a yellow solid A1 (179 mg, 85%);
mp: 201–203 °C. 1H NMR (400 MHz, CDCl3) d 7.60 (d, J = 15.8 Hz,
2H), 7.46 (d, J = 8.6 Hz, 4H), 6.84 (dd, J = 12.0 Hz, 8.8, 6H), 3.26 (t,
J = 8.0 Hz, 8H), 2.52 (t, J = 8.0 Hz, 8H), 2.30 (s, 6H). 13C NMR
(100 MHz, CDCl3)
121.46, 113.85, 53.78, 46.73, 45.10. ESI-HRMS m/z: calcd for
27H34N4O [M+H]+ 431.2811, found 431.2805.
d 187.80, 151.44, 141.65, 128.90, 124.43,
Figure 7. Influence of A4 on metal-induced Ab1–42 aggregation. Values of ThT
fluorescence intensity depicted in the figure were obtained by subtraction from that
of the samples containing ThT solution and HEPEs buffer or the same concentra-
tions of A4.
C
4.2.2. (2E,5E)-2,5-Bis(4-(4-methylpiperazin-1-yl)benzylidene)-
cyclopentanone (A2)
weaker ability of promoting Ab1–42 aggregation than Cu2+ and Zn2+
.
Following the method described for production of A1, a yellow
solid A2 (152 mg, 68%) was obtained; mp: 189–191 °C. 1H NMR
(400 MHz, CDCl3) d 7.46 (d, J = 9.0 Hz, 6H), 6.87 (d, J = 8.8 Hz, 4H),
3.26 (t, J = 8.0 Hz, 8H), 3.00 (s, 4H), 2.50 (t, J = 8.0 Hz, 8H), 2.29 (s,
6H). 13C NMR (100 MHz, CDCl3) d 195.13, 150.35, 133.53, 132.27,
131.34, 125.71, 113.80, 53.81, 46.74, 45.12, 25.55. ESI-HRMS m/z:
calcd for C29H36N4O [M+H]+ 457.2967, found 457.2961.
Moreover, there was little aggregation of the Ab1–42 in the absence
of metal ions after such short incubation time. Secondly, compound
A4 clearly decreased fibrils buildup in the presence of Cu2+ and Fe2+
compared with a potent Cu chelator CQ. And it had less inhibition ef-
fect on the Zn2+-promoted fibrils, which was consistent with metal-
chelating properties of A4 (Fig. 5), selectively chelating to redox-ac-
tive metal Fe and Cu, but not Zn. However, since A4 also displayed
inhibition of Zn2+-promoted fibrils, suggesting that A4 reduced me-
tal-promoted fibril not only depending on its chelating ability.
4.2.3. (2E,6E)-2,6-Bis(4-(4-methylpiperazin-1-yl)benzylidene)-
cyclohexanone (A3)
Following the method described for production of A1, a yellow
solid A3 (146 mg, 61%) was obtained; mp: 170–172 °C. 1H NMR
(400 MHz, CDCl3) d 7.68–7.65 (m, 2H), 7.36 (d, J = 8.7 Hz, 4H),
6.84 (d, J = 8.8 Hz, 4H), 3.24 (t, J = 8.0 Hz, 8H), 2.85 (t, J = 5.3 Hz,
4H), 2.50 (t, J = 8.0 Hz, 8H), 2.29 (s, 6H). 13C NMR (100 MHz, CDCl3)
d 189.06, 149.95, 135.65, 132.52, 131.14, 125.86, 113.67, 53.87,
46.94, 45.11, 27.63, 22.09. ESI-HRMS m/z: calcd for C30H38N4O
[M+H]+ 471.3124, found 471.3119.
3. Conclusions
In summary, a series of curcumin analogues have been de-
signed, synthesized and evaluated as multifunctional anti-Alzhei-
mer agents. Majority of the synthesized analogues displayed
higher effective inhibitory potencies against Ab aggregation than
curcumin. Structure–activity relationships analysis for all synthe-
sized analogues was investigated and the analysis suggested that
the introduction of flexible moieties at the linker is crucial to the
inhibitory potencies of the compounds against Ab aggregation. Fur-
ther investigations of compound A4 by CD and EM experiments
confirmed that A4 can slow down or inhibit b-sheet aggregation
and fibril formation. Furthermore, A4 displayed better anti-oxidant
property than the Trolox and curcumin. It also possessed the pro-
spective property of acting as a metal chelator and inhibiting the
metal-induced Ab aggregation. These results strongly encourage
further structure optimization of A4 to develop more potent mul-
tifunctional anti-Alzheimer agents.
4.2.4. (3E,5E)-3,5-Bis(4-(4-methylpiperazin-1-yl)benzylidene)-
piperidin-4-one (A4)
Analog A4 was obtained using an acid promoted aldol reaction
(glacial acetic acid), following the procedure described by Adams.35
Glacial acetic acid (8 mL) was saturated with HCl gas at room tem-
perature, and then 4-piperidone hydrochloride monohydrate
(192 mg, 1.25 mmol) was suspended. To the resulting clear solu-
tion, 4-(4-methylpiperazin-1-yl)benzaldehyde (510 mg, 2.5 mmol)
was added and the reaction mixture was stirred for 3 days at room
temperature. Then 10% NaOH was added to adjust the pH to 7, the
forming yellow solids were filtered off, washed with water, and
dried under vacuum yielding A4 as yellow plates (315 mg, 67%);
mp: 190–193 °C. 1H NMR (400 MHz, CDCl3) d 7.67 (s, 2H), 7.27
(d, J = 8.8 Hz, 4H), 6.84 (d, J = 8.9 Hz, 4H), 4.09 (s, 4H), 3.25 (t,
J = 8.0 Hz, 8H), 2.50 (t, J = 8.0 Hz, 8H), 2.29 (s, 6H). 13C NMR
4. Experimental section
4.1. General
Commercially available reagents (chemicals) were used without
further purification, unless otherwise stated. NMR spectra were re-
corded using TMS as the internal standard in CDCl3 with a Bruker
AvanceIII 400 spectrometer. Proton coupling patterns are de-
scribed as single (s), double (d), triplet (t) and multiple (m). HRMS
was obtained with an Agilent Iron-TOF-LC/MC spectrometer. The
purities of synthesized compounds were confirmed by analytical
HPLC performed with a LC-20A system equipped with a Ultimate-
(100 MHz, CDCl3) d 186.66, 150.25, 134.73, 131.36, 131.27,
124.93, 113.64, 53.83, 47.27, 46.77, 45.12. ESI-HRMS m/z: calcd
for C29H37N5O [M+H]+ 472.3076, found 472.3072.
4.2.5. (3E,5E)-1-Methyl-3,5-bis(4-(4-methylpiperazin-1-yl)ben-
zylidene)piperidin-4-one (A5)
Following the method described for production of A1, a yellow
solid A5 (89 mg, 37%) was obtained; mp: 193–196 °C. 1H NMR
(400 MHz, CDCl3) d 7.67 (s, 2H), 7.26 (d, J = 8.8 Hz, 4H), 6.84
QB-C18 column (4.6 ꢀ 250 mm, 5
lm) and eluted with methanol/

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S.-Y. Chen et al. / Bioorg. Med. Chem. 19 (2011) 5596–5604
(d, J = 8.9 Hz, 4H), 3.70 (s, 4H), 3.29 (t, J = 8.0 Hz, 8H), 2.51 (t,
J = 8.0 Hz, 8H), 2.39 (s, 3H), 2.27 (s, 6H). 13C NMR (100 MHz, CDCl3)
d 185.69, 150.19, 135.19, 131.26, 129.39, 125.01, 113.68, 56.26,
53.82, 46.76, 45.09, 44.81. ESI-HRMS m/z: calcd for C30H39N5O
[M+H]+ 486.3233, found 486.3225.
7.7 Hz, 2H), 7.73 (d, J = 12.8 Hz, 2H), 7.61–7.44 (m, 5H), 7.35 (d,
J = 15.5 Hz, 2H), 6.85 (d, J = 11.4 Hz, 4H), 3.28 (t, J = 8.0 Hz, 8H),
2.51 (t, 8H), 2.29 (s, 6H). 13C NMR (100 MHz, CDCl3) d 188.91,
151.78, 144.88, 138.12, 130.94, 129.39, 127.82, 127.02, 124.05,
116.89, 113.73, 53.74, 46.57, 45.07. ESI-HRMS m/z: calcd for
C
34H38N4O2 [M+H]+ 535.3073, found 535.3068.
4.2.6. (1E,6E)-1,7-Bis(4-(4-methylpiperazin-1-yl)phenyl)hepta-
1,6-diene-3,5-dione (A6)
4.2.10. (2E,20E)-1,10-(1,4-Phenylene)bis(3-(4-(4-methylpipera-
zin-1-yl)phenyl)prop-2-en-1-one) (A10)
Compound A6 was prepared according to our previous Letter.36
Boric anhydride (87 mg, 1.25 mmol) was suspended in 10 mL
EtOAc in the presence of acetylacetone (251 mg, 2.5 mmol), and
the mixture was stirred for 3 h at 70 °C. After evaporation of the
solvent, the residue was washed with hexane and then, 5 mL
EtOAc, 4-(4-methylpiperazin-1-yl) benzaldehyde (1.02 g, 5 mmol),
and tributylborate (1.15 g, 5 mmol) were added, and the mixture
was stirred for further 30 min. Butylamine (18 mg, 0.25 mmol) dis-
solved in EtOAc was added drop wise. The mixture acted at 70 °C
for 24 h. Then 1 N HCl was added to adjust the pH to 5, and the
solution was heated for 30 min at 60 °C. EtOAc was used to extract
the product from the water layer. The compound A6 was purified
by recrystalization from EtOAc to yield yellow crystals (595 mg,
50%); mp: 214–216 °C. 1H NMR (400 MHz, CDCl3) d 7.52 (d,
J = 15.8 Hz, 2H), 7.41 (d, J = 13.2 Hz, 4H), 6.83 (d, J = 8.0 Hz, 4H),
6.39 (d, J = 15.7 Hz, 2H), 5.69 (s, 1H), 3.26 (t, J = 8.0 Hz, 8H), 2.51
(t, J = 8.0 Hz, 8H), 2.30 (s, 6H). 13C NMR (100 MHz, CDCl3) d
182.30, 151.24, 139.22, 128.60, 124.69, 119.60, 113.94, 100.11,
53.83, 46.82, 45.12. ESI-HRMS m/z: calcd for C29H34N4O2 [M+H]+
473.2917, found 473.2911.
Following the method described for production of A1, a red so-
lid A10 (184 mg, 69%) was obtained; mp: 224–226 °C. 1H NMR
(400 MHz, CDCl3) d 8.01 (s, 4H), 7.72 (d, J = 15.6 Hz, 2H), 7.50 (d,
J = 8.8 Hz, 4H), 7.30 (d, J = 15.6 Hz, 2H), 6.84 (d, J = 8.9 Hz, 4H),
3.30 (t, J = 8.0 Hz, 8H), 2.53 (t, J = 8.0 Hz, 8H), 2.31 (s, 6H). 13C
NMR (100 MHz, CDCl3) d 189.23, 151.80, 145.00, 140.66, 129.36,
127.45, 124.08, 113.79, 53.72, 46.55, 45.02. ESI-HRMS m/z: calcd
for C34H38N4O2 [M+H]+ 535.3073, found 535.3071.
4.2.11. (3E,5E)-3,5-Bis(4-(4-piperidin-1-yl)benzylidene)piper-
idin-4-one (B4)
Following the method described for production of A4, a red so-
lid B4 (174 mg, 32%) was obtained; mp: 208–210 °C. 1H NMR
(400 MHz, CDCl3) d 7.67 (s, 2H), 7.25 (d, J = 8.8 Hz, 4H), 6.83 (d,
J = 8.8 Hz, 4H), 4.10 (s, 4H), 3.26–3.17 (t, J = 8.0 Hz, 8H), 1.69–
1.50 (m, 12H). 13C NMR (100 MHz, CDCl3) d 186.47, 150.81,
135.03, 131.48, 130.55, 124.05, 113.65, 48.10, 47.22, 24.47, 23.32.
ESI-HRMS m/z: calcd for C29H35N3O [M+H]+ 442.2858, found
442.2864.
4.2.7. 1,3-Bis(4-(4-methylpiperazin-1-yl)styryl)benzene (A7)
The mixture of m-xylylene dibromide (625 mg, 2.5 mmol) and
triethyl phosphate (1 mL, 5 mmol) was refluxed at 120 °C for 2 h.
The reaction mixture was cooled to room temperature and then
eluted with THF (50 mL), adding 570 mg (5 mmol) potassium
tert-butoxide at 0 °C. After stirring for 20 min, 4-(4-methylpipera-
zin-1-yl)benzaldehyde (1.02 g, 5 mmol) was added and the reac-
tion mixture was stirred for 30 min at room temperature. After
slow addition of chilled water and stirred at room temperature
for 30 min, the mixture was extracted with 30 mL ethylacetate
twice. The combined organic layer was washed with saturated
NaHCO3 solution and brine, dried over MgSO4, and concentrated
to dryness to give a light yellow solid (199 mg, 17%); mp: 218–
4.3. Biological assay
4.3.1. ThT assay
Experiments were performed by incubating the peptides in
10 mM phosphate buffer (pH 7.4) at 37 °C for 48 h (final Ab1–42
20
centrations (2, 5, 10, 20, 50
were diluted to a final volume of 180
buffer (pH 8.5) containing 5 M Thioflavin T. Fluorescence signal
l
M) with and without the tested compounds at different con-
M). After incubation, the samples
L with 50 mM glycine-NaOH
l
l
l
was measured (excitation wavelength 450 nm, emission wave-
length 485 nm and slit widths set to 5 nm) on a monochromators
based multimode microplate reader (INFINITE M1000), adapted
for 96-well microtiter plates. The fluorescence intensities were re-
corded, and the percentage of inhibition on aggregation was calcu-
lated by the following expression: (1ꢁIFi/IFc) ꢀ 100% in which IFi
and IFc were the fluorescence intensities obtained for absorbance
in the presence and absence of inhibitors, respectively, after sub-
220 °C. 1H NMR (400 MHz, CDCl3)
d 7.51 (s, 1H), 7.37 (d,
J = 8.7 Hz, 4H), 7.30–7.20 (m, 3H), 7.01 (d, J = 16.3 Hz, 2H), 6.90
(d, J = 16.3 Hz, 2H), 6.85 (d, J = 8.8 Hz, 4H), 3.23 (t, J = 8.0 Hz, 8H),
2.59 (t, J = 8.0 Hz, 8H), 2.34 (s, 6H). 13C NMR (100 MHz, CDCl3) d
149.61, 137.13, 127.84, 127.75, 127.54, 126.48, 124.80, 123.86,
123.09, 114.80, 53.94, 47.60, 45.03. ESI-HRMS m/z: calcd for
tracting the background fluorescence of the 5
solution.
lM Thioflavin T
C
32H38N4 [M+H]+ 479.3175, found 479.3183.
4.3.2. CD assay
4.2.8. 2,6-Bis(4-(4-methylpiperazin-1-yl)styryl)pyridine (A8)
Following the method described for production of A7, a light
yellow solid A8 (102 mg, 8%) was obtained; mp: 212–214 °C. 1H
NMR (400 MHz, CDCl3) d 7.64–7.55 (m, 3H), 7.52 (d, J = 8.7 Hz,
4H), 7.20 (d, J = 16.1 Hz, 2H), 7.06 (d, J = 16.1 Hz, 2H), 6.92 (d, J =
8.8 Hz, 4H), 3.29 (t, J = 8.0 Hz, 8H), 2.60 (t, J = 8.0 Hz, 8H), 2.37 (s,
6H). 13C NMR (100 MHz, CDCl3) d 155.90, 151.02, 136.64, 132.45,
128.19, 128.06, 125.51, 119.30, 115.52, 54.98, 50.76, 48.44, 46.09.
Ab1–42 (20 lM) was mixed with and without 10 lM A4 in
10 mM sodium phosphate buffer (pH 7.4). All solutions were incu-
bated at 37 °C. CD spectra were obtained using a Jasco-810-150S
spectropolarimeter (Jasco, Japan). A quartz cell with 1 mm optical
path was used. Spectra were recorded at 25 °C between 190 and
260 nm with a bandwidth of 0.5 nm, a 3 s response time, and scan
speed of 10 nm/min. Background spectra and when applicable,
spectra of A4 were subtracted.
ESI-HRMS m/z: calcd for
C
31H37N5 [M+H]+ 480.3127, found
480.3121.
4.3.3. EM study
Ab1–42 peptide (Anaspec Inc.) was dissolved in 10 mM phos-
4.2.9. (2E,20E)-1,10-(1,3-Phenylene)bis(3-(4-(4-methylpiperazin-
1-yl)phenyl)prop-2-en-1-one) (A9)
Following the method described for production of A1, a yellow
solid A9 (154 mg, 57%) was obtained; mp: 216–218 °C. 1H NMR
(400 MHz, CDCl3) d 8.62–8.46 (m, 1H), 8.10 (dd, J = 15.6 Hz,
phate buffer (pH 7.4) at 4 °C to give an 80
was incubated in the presence and absence of A4 in 37 °C. The fi-
nal concentrations of and A4 were 40 M and 20 M, respec-
tively. At specified time points, aliquots of 10 L samples were
placed on carbon-coated copper/rhodium grid. After 1 min, the
lM solution. Ab1–42
l
l
l

S.-Y. Chen et al. / Bioorg. Med. Chem. 19 (2011) 5596–5604
5603
grid was washed with water and negatively stained with 2% ura-
nyl acetate solution for 1 min. After draining off the excess of
staining solution by means of a filter paper, the specimen was
transferred for examination in a transmission electron microscope
(JEOL JEM-1400).
4.3.6. Metal-chelating study
The chelating studies were made in ethanol using a UV–vis
spectrophotometer (SHIMADZC UV-2450PC). The absorption spec-
tral of compound A4, alone or in the presence of CuSO4, FeSO4, and
ZnCl2, was recorded at room temperature in a 1 cm quartz cell.
4.3.4. Anti-oxidant activity in vitro-ORAC-FL assay
4.3.7. Effects of A4 on metal-induced Ab1–42 aggregation by ThT
method
Two 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid
(HEPES) buffer solutions (20 lM) containing 150 lM NaCl were
Compounds were directly dissolved in DMSO, and diluted with
75 mM potassium phosphate buffer (pH 7.4) for analysis. AAPH
(0.414 g) was completely dissolved in 10 mL of 75 mM phosphate
buffer (pH 7.4) to a final concentration of 40 mM. The unused
AAPH solution was discarded within 8 h. Fluorescein stock solution
(3.5 mM/L) was made in 75 mM phosphate buffer (pH 7.4) and was
kept at 4 °C in dark condition. The fluorescein stock solution at
such condition can last several months. The 140 nM fresh fluores-
cein working solution was made daily by further diluting the stock
solution in 75 mM phosphate buffer (pH 7.4). Trolox standard was
prepared as following: 0.25 g of Trolox was dissolved in 50 mL
DMSO to give a 10 mM stock solution. The compounds and the
Trolox stock solution were diluted with the same phosphate buffer
prepared with distilled, deionized water at pH values of 6.6 and
7.4. Solutions of Fe2+ and Cu2+ were prepared from standards to fi-
nal concentrations of 200 lM using the HEPES buffer at pH 6.6, and
the Zn2+ solution was prepared to the same concentration with
HEPES buffer at pH 7.4. Solutions of A4 and CQ were prepared in
DMSO in 10 mM for store, and diluted with HEPES buffer before
use. To study effects of A4 on the metal-induced Ab1–42 aggrega-
tion, Ab1–42 (20
in HEPES buffer at pH 6.6 and 7.4, respectively, without or with
compound A4 (40 M) or a potent metal chelator, CQ (40 M).
The incubation was performed at 37 °C for 45 min. After incuba-
tion, the samples were diluted to a final volume of 180 L with
50 mM glycine–NaOH buffer (pH 8.5) containing 5 M Thioflavin
T. Fluorescence was measured at 450 nm (kex) and 485 nm (kem
lM) was co-incubated 40 l
M Cu2+, Fe2+, and Zn2+
l
l
to 25, 12.5, and 6.25
lM working solutions, and measured at final
concentrations of, 2.5, 1.25, and 0.625
lM. All reaction mixtures
l
were prepared fourfold and at least four independent runs were
performed for each sample. Fluorescence measurements were nor-
malized to the curve of the blank (without anti-oxidant). From the
normalized curves, the area under the fluorescence decay curve
(AUC) was calculated as:
l
)
using a monochromators based multimode microplate reader
(INFINITE M1000).
AUC ¼ 1 þ i¼90 fi=f0
Acknowledgments
X
(1)
i¼1
We thank the Natural Science Foundation of China (Grants
U0832005, 90813011, 81001400), The International S&T Coopera-
tion Program of China (2010DFA34630), The Specialized Research
Fund for the Doctoral Program of Higher Education (2009017111
0050), and The Science Foundation of Guangzhou (2009A1-E011-
6) for financial support of this study.
where f0 is the initial fluorescence at 0 min and fi is the
fluorescence at time i. The net AUC for a sample was calcu-
lated as follows:
(2) Net AUC = AUCanti-oxidantꢁAUCblank. The ORAC-FL values were
calculated.
(3) [(aucsample ꢁAUCblank)/(AUCTrolox ꢁAUCblank)] ꢀ [(concentra-
tion of Trolox/concentration of sample)] and expressed as
Trolox equivalents by using the standard curve calculated
Supplementary data
for each assay. Final results were in
equivalent/ M of pure compound.
lM of Trolox
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.07.033.
l
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