Design, synthesis, and evaluation of resveratrol derivatives as Ass1-42 aggregation inhibitors, antioxidants, and neuroprotective agents

Article abstract of DOI:10.1016/j.bmcl.2012.09.105

A series of novel resveratrol derivatives were designed, synthesised and evaluated as potential therapeutic agents for the treatment of Alzheimer's disease. Among these compounds, compound 7l, (E)-5-(4-(isopropylamino)styryl) benzene-1,3-diol, exhibited potent ss-amyloid aggregation inhibition activity, which was confirmed by a ThT fluorescence assay (71.65% at 20 μM) and transmission electron microscopy (TEM). Compound 7l also exhibited good antioxidant activity (4.12 Trolox equivalents in an oxygen radical absorbance capacity assay and a 37% reduction in reactive oxygen species in cells at 10 μM). The cytotoxicity analysis of compounds 7f, 7i, 7j and 7l indicated that these compounds have lower toxicities than resveratrol at 60 μM.

Full text of DOI:10.1016/j.bmcl.2012.09.105

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7683–7687
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and evaluation of resveratrol derivatives as Aß1–42
aggregation inhibitors, antioxidants, and neuroprotective agents
Chuanjun Lu a, Yueyan Guo a, Jianheng Li a, Meicun Yao a, Qiongfeng Liao b, Zhiyong Xie a, , Xingshu Li a,
⇑
⇑
a School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
b College of Chinese Traditional Medicine, Guangzhou University of Traditional Chinese Medicine, Guangzhou 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 July 2012
Revised 6 September 2012
Accepted 28 September 2012
Available online 13 October 2012
A series of novel resveratrol derivatives were designed, synthesised and evaluated as potential therapeu-
tic agents for the treatment of Alzheimer’s disease. Among these compounds, compound 7l, (E)-5-(4-(iso-
propylamino)styryl)benzene-1,3-diol, exhibited potent ß-amyloid aggregation inhibition activity, which
was confirmed by a ThT fluorescence assay (71.65% at 20
(TEM). Compound 7l also exhibited good antioxidant activity (4.12 Trolox equivalents in an oxygen rad-
ical absorbance capacity assay and a 37% reduction in reactive oxygen species in cells at 10 M). The cyto-
toxicity analysis of compounds 7f, 7i, 7j and 7l indicated that these compounds have lower toxicities than
resveratrol at 60 M.
lM) and transmission electron microscopy
l
Keywords:
Resveratrol derivatives
Alzheimer’s disease
Aß aggregation
Antioxidant
l
Ó 2012 Elsevier Ltd. All rights reserved.
Neuroprotective
Alzheimer’s disease (AD), characterised by the progressive loss
of memory and cognitive functions, is the most common fatal neu-
rodegenerative disorder in the world. Although much work has
been performed, there is currently no effective drug for the treat-
ment of this chronic debilitating illness.1–5 The etiology of AD is
still not completely known at present, but some major pathological
signs, such as ß-amyloid (Aß) plaques, tau neurofibrillary tangles
and synaptic loss, have been characterised.6–8 Among these signs,
the formation of Ab fibrils and their deposition into neurotoxic
amyloid plaques are recognised as the crucial cause of AD.9–11
These fibrils and plaques consist predominantly of Aß peptides,
primarily Aß40 and Aß42, a series of C- and N-terminally heteroge-
neous proteolytic fragments with 40 or 42 amino-acid residues.
Oxidative stress is an early feature of AD pathology, and during
the aging process, oxidative-induced apoptosis and the increased
of drugs for the treatment of AD. However, the rapid metabolism
of resveratrol’s three hydroxyl groups leads to the production of
sulfates and glucuronides in vivo (initial half-life of resveratrol:
8–14 min), and therefore, despite its efficient absorption after oral
administration, resveratrol has a low bioavailability.15,16 Therefore,
from both fundamental and practical standpoints, it is desirable to
develop resveratrol derivatives that have superior bioavailabilities.
It is interesting to note that many Aß binding agents possess
amino groups.17,18 For example, thioflavin T (ThT), a quaternary
ammonium salt with a substituted amino group, is widely used as
a probe for detecting Aß aggregates. To develop anti-AD agents with
better bioavailability based on resveratrol, we designed, synthes-
ised and evaluated a series of resveratrol derivatives with reduced
hydroxyl groups as antioxidants and inhibitors of b-amyloid
aggregation.
activity of ß- and
c
-secretases potentially contribute to Aß gener-
The synthetic route of resveratrol derivatives is shown in Scheme
1. Using commercially available 3,5-dimethoxybenzaldehyde as the
starting material, target compounds 7a to 7n were synthesised via
five steps. First, the reduction of 3,5-dimethoxybenzaldehyde with
sodium borohydride in methanol provided alcohol 2. Compound 2
was then reacted with phosphorus tribromide in the presence of
pyridine to give 3, which was converted into diethyl 3,5-
dimethoxybenzylphosphonate 4 in high yield. The substituted
amino aldehydes 5h–n were synthesised by reacting the corre-
sponding fluoroaldehydes with different amines. These reactions
were catalysed by tetrabutylammonium bromide in the presence
of potassium carbonate (Scheme 2). The Wittig olefination of
compound 4 with various substituted benzaldehydes was used to
ation. The early involvement of oxidative stress in the pathogenesis
and progression of AD is known.12
Resveratrol (Fig. 1), a powerful antioxidant that is present in
many plants, including grapes, peanuts and plums, protects against
environmental stresses. In recent years, resveratrol has been
extensively investigated as a cardioprotective, anti-inflammatory,
anticancer and anti-aging agent.13 A recent study also found that
resveratrol can inhibit Aß aggregation in vitro,14 indicating that
this compound could be used as a lead compound in the design
⇑
Corresponding author. Tel./fax: +86 20 39943050.
E-mail address: lixsh@mail.sysu.edu.cn (X. Li).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.105

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C. Lu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7683–7687
provide compounds 6a–n. Finally, the target compounds 7a–n were
obtained by the demethylation of 6a–n with boron tribromide at
ꢀ20 °C. The hydrogenation of compound 7f gave compound 7o,
which is a diphenylethane derivative (Scheme 3). The purities of
all synthesised compounds were determined by HPLC.
Using a thioflavin T (ThT) fluorescence assay, the inhibitory
activities of all resveratrol derivatives at 20 lM against self-medi-
ated Aß1–42 aggregation were evaluated, and the results are shown
in Table 1. Curcumin, a known amyloid aggregation inhibitor, was
used as a reference compound. The results in Table 1 indicate that
most compounds with reduced hydroxyl groups had similar or bet-
ter inhibition aggregation activities (59.40–71.65%) relative to res-
veratrol (69.73%). The simple analysis of the relationship between
structure and activity indicated that two hydroxyl groups are
necessary. Compound 6f, which possesses two methoxy groups
at the 3 and 5 positions on one of the benzene rings, had almost
Figure 1. Design strategy for resveratrol derivatives.
Scheme 1. Synthesis of resveratrol derivatives. Reagents and conditions: (a) NaBH4, MeOH; (b) PBr3, pyridine, 0 °C; (c) triethyl phosphate, 120 °C; (d) 5aꢁn, CH3ONa, 0 °C, 1 h,
rt, 12 h; (e) BBr3, CH2Cl2, ꢀ20 °C, 2 h; rt, 4 h.
Scheme 2. Synthesis of different substituted benzaldehydes. Reagents and conditions: (f) Different amines, tetrabutylammonium bromide, K2CO3, DMSO, 95 °C, 3d.

C. Lu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7683–7687
7685
groups, pyridyl groups and bromine), the dimethylamino group
gave the best result (7f, 65.20%; 7a–c). Altering the position of
the dimethylamino group resulted in compounds 7d–e, in which
the amino group is at the 20 or 30 position of the benzene ring.
These compounds exhibited reduced activities. Finally, exploratory
structure–activity relationship (SAR) revealed that the replace-
ment of the dimethylamino group with other mono- or bi-substi-
tuted amino groups did not change the inhibitory activity
significantly (7f–n, 63.70–71.65%, among them, compound 7l
exhibited the most potent Aß aggregation inhibitive activity,
Scheme 3. Synthesis of 7o. Reagents and conditions: (g) Pd/C, H2, rt, 12 h.
no inhibitory activity, whereas the related compound 7f exhibited
65.20% inhibition at 20 lM. These results indicate that hydrogen
bonds are crucial in the interactions between polyphenols and pro-
teins, as many previous studies have indicated.19 The stilbene frag-
ment in the structure of resveratrol derivatives may play a key role
in the inhibition of Aß1–42 aggregation. Compound 7o, in which the
carbon–carbon double bound was reduced relative to the structure
of compound 7f, exhibited a lower level of activity. The influence of
the different R groups on the inhibitory activity was also investi-
gated, and among the four groups (substituted amino groups, alkyl
71.65% at 20 lM).
To further investigate the Aß aggregation inhibitory activities of
the resveratrol derivatives, compound 7l and resveratrol were cho-
sen for study by transmission electron microscopy (TEM), and the
results are shown inFigure 2. The aggregation of Aß1–42 led to well-
defined Aß fibrils after 24 h of incubation at 37 °C (Fig. 2C). In con-
trast, few Aß fibrils were observed in presence of compound 7l
(Fig. 2A) or resveratrol (Fig. 2B) under the same experiment
Table 1
The inhibition of Aß aggregation by the ThT method and ORAC values
R2
R3
HO
HO
HO
R1
R1
N
N
R4
HO
7a
7o
Compd
R1
R2
H
R3
R4
Inhibition of Aß aggregationa (%)
ORACb
6f
OCH3
H
7.02 3.01
3.46 0.48
N
7a
7b
—
OH
—
H
—
H
—
Br
13.56 2.12
25.23 4.57
1.71 0.06
2.33 0.07
7c
7d
7e
7f
OH
OH
OH
OH
OH
H
H
H
43.56 3.40
59.40 3.30
61.55 2.18
65.20 4.24
64.85 3.04
1.28 0.35
2.05 0.11
3.93 0.41
4.70 0.45
3.31 0.25
H
H
N
H
H
H
N
H
H
N
N
7g
7h
OH
H
H
69.00 1.41
2.37 0.11
N
H
N
7i
OH
OH
OH
H
H
H
H
H
H
63.70 3.82
68.60 0.57
66.70 0.42
5.54 0.14
5.19 0.21
4.33 0.03
H
7j
N
H
N
7k
H
7l
OH
OH
OH
H
H
H
H
H
H
71.65 3.32
68.70 5.08
65.50 0.71
4.12 0.17
3.37 0.36
2.25 0.04
N
H
N
7m
H
N
7n
7o
—
—
—
—
—
—
—
—
—
—
—
—
48.54 3.54
69.73 2.35
52.77 1.08
5.21 0.23
5.92 0.32
n.t.c
Resveratrol
Curcumin
a
b
c
The Thioflavin-T fluorescence method was used. Values are expressed as the mean SD from at least three independent measurements. Tested compounds are 20 lM.
Data are expressed as
lmol of trolox equivalent/lmol of tested compound (means SD) of three independent experiments.
n.t. means not tested.

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C. Lu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7683–7687
Figure 2. TEM images ([Aß1–42] = 40 lM, [7l] = 20 lM, [Resveratrol] = 20 lM, 37 °C, 24 h). Samples: (A) Aß1–42 + 7l at 24 h; (B) Aß1–42 + Resveratrol at 24 h; (C) Aß1–42 at 24 h.
conditions. The results clearly indicate that both compound 7l and
resveratrol exhibited good inhibitory effect on Aß fibrillisation.
The ability of the resveratrol derivatives to scavenge oxygen
radicals was determined by the oxygen radical absorbance capacity
(ORAC) assay,20 and the results are listed in Table 1. Trolox (6-
hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), a vita-
min E analogue, was used as a standard, and the results are
expressed as Trolox equivalents (the antioxidant activity of the
tested compounds was compared with the activity of Trolox at
1 lM). The results showed that amino groups play an important
role in the antioxidant activities of the tested compounds. Further-
more, secondary amines gave better antioxidant activity than ter-
tiary amines. Among all of the resveratrol derivatives, 7i showed
the best antioxidant activity, which was close to that of resveratrol
(5.54 for 7i, 5.92 for resveratrol). Additionally, the most potent Aß
fibrillisation inhibitor, 7l, also exhibited good activity (4.12). Final-
ly, this assay also indicated that increasing the length of the carbon
linker or using bulkier cycloalkane on the amino group resulted in
decreased antioxidant activity.
The cytotoxicity effects of the resveratrol derivatives were
quantified using the colorimetric MTT [3-(4,5-dimethyl-2-thiazol-
yl)-2,5-diphenyl-2H-tetrazolium bromide] assay with the human
neuronal-like cell line SH-SY5Y. As indicated in Figure 3, all of
the tested compounds had lower toxicities than resveratrol at
Figure 4. Percentage increase in the intracellular ROS induced by exposure to t-
BuOOH determined using DCFH-DA. The results are reported as the mean of three
independent experiments performed in quadruplicate.
The antioxidant activity of the resveratrol derivatives in SH-
SY5Y21 cells was evaluated using a cellular antioxidant assay based
on dichlorofluorescein diacetate (DCFH-DA), a method that has
been widely used recently.22,23 Reactive oxygen species (ROS) were
detected after SH-SY5Y cells were treated with tert-butylhydroper-
oxide. The concentrations of the tested compounds did not affect
60
did not affect the viability of SH-SY5Y cells after incubation for
48 h. In contrast, 7j exhibited slight toxicity at 25 M.
lM. Among the tested compounds, 7f, 7i and 7l (12.5–25 lM)
l
the cell viability (2.5–10
tested at 20 M. The results are expressed as the percentage in-
crease in the level of intracellular ROS (Fig. 4.). Surprisingly, the
treatment of cells with 7i at 5 M resulted in a significant decrease
lM). Trolox, a known antioxidant, was
l
l
in ROS formation. Biological data indicated that the amino moiety
is important for this compound’s antioxidant activity. The antioxi-
dant effects of the tested compounds were concentration depen-
dent. Although 7l showed low activity at 5
lM, it had a moderate
level of activity at 10 M. The low toxicity of this compound indi-
l
cates that it has a wider therapeutic range.
In conclusion, we synthesised a series of resveratrol derivatives
that exhibited potent ß-amyloid aggregation inhibition activities
and antioxidant activities. Among the synthesised compounds, 7l
gave the best results and is a potential agent for the treatment of
Alzheimer’s disease. Further studies of this compound’s druggabil-
ity are in progress in our lab.
Acknowledgements
We thank the Natural Science Foundation of China (20972198
and 81173564) for financial support of this study.
Figure 3. Effects of compounds on the viability of SH-SY5Y cells. The results are
reported as the mean of three independent experiments performed in triplicate.

C. Lu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7683–7687
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.09.105.
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