Synthesis and biological evaluation of novel neuroprotective agents for paraquat-induced apoptosis in human neuronal SH-SY5Y cells

Article abstract of DOI:10.1016/j.ejmech.2012.12.037

Three types of resveratrol analogues were designed, synthesized and evaluated in vitro against paraquat-induced apoptosis in SH-SY5Y cells. The results showed that some compounds, especially those containing an indene core, exhibit good activities. Analogue 3′a showed a potent neuroprotective effect at a low concentration (10 μM). Further investigation showed that compound 3′a could attenuate paraquat-induced nuclear morphological changes, significantly decrease paraquat-induced ROS (reactive oxygen species) generation in SH-SY5Y cells and elevate the expression of SOD (Superoxide Dismutase) and GPx (glutathione peroxidase) in a dose-dependent manner. Furthermore, analogue 3′a could decrease Bax protein levels in a concentration-dependent manner and increase Bcl-2 protein expression, which was accompanied by increasing chances of cell survival.

Full text of DOI:10.1016/j.ejmech.2012.12.037

European Journal of Medicinal Chemistry 62 (2013) 187e198
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel neuroprotective agents
for paraquat-induced apoptosis in human neuronal SH-SY5Y cells
,
,
,c,
Chen Zhong a 1, Xin-Hua Liu b 1, Xiao-Dong Hao a, Jun Chang a, Xun Sun a
*
a Department of Natural Products Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
b Department of Pharmacology, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
c Key Lab of Smart Drug Delivery, Ministry of Education & PLA, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three types of resveratrol analogues were designed, synthesized and evaluated in vitro against paraquat-
induced apoptosis in SH-SY5Y cells. The results showed that some compounds, especially those con-
taining an indene core, exhibit good activities. Analogue 30a showed a potent neuroprotective effect at
Received 27 July 2012
Received in revised form
28 November 2012
Accepted 23 December 2012
Available online 5 January 2013
a low concentration (10 m
M). Further investigation showed that compound 30a could attenuate paraquat-
induced nuclear morphological changes, significantly decrease paraquat-induced ROS (reactive oxygen
species) generation in SH-SY5Y cells and elevate the expression of SOD (Superoxide Dismutase) and GPx
(glutathione peroxidase) in a dose-dependent manner. Furthermore, analogue 30a could decrease Bax
protein levels in a concentration-dependent manner and increase Bcl-2 protein expression, which was
accompanied by increasing chances of cell survival.
Keywords:
Resveratrol dimers
Resveratrol analogues
Neuroprotection
SH-SY5Y cells
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
demonstrate more potent biological activities than resveratrol due
to their polyphenol motifs. For example, resveratrol trimer 5 was
Resveratrol (3,5,40-trihydroxy-trans-stilbene, 1) (Fig. 1) is a nat-
ural polyphenol stilbene found in grapes and certain plants used as
medicines. It is a phytoalexin which is produced in plants in
response to external stimuli, such as UV light, fungal or viral in-
fections and mechanical damage [1]. Resveratrol has been reported
to have a diverse range of pharmacological properties, including
antiinflammatory, antiplatelet and antioxidant activities [2e5]. It
has been shown to be an oestrogen receptor agonist [6] and exerts
effects on cell signalling pathways involved in tumor growth, cell
proliferation and survival [7e9]. Due to these biological activities,
resveratrol has been studied as a potential drug for the treatment of
maladies such as Parkinson’s disease [10] and Alzheimer disease
[11e13]. Resveratrol can be biotransformed by Botrytis cinerea,
a fungal grapevine pathogen, into resveratrol oligomers such as
pallidol (2a), quadrangularin A (E-3a), parthenocissin A (Z-3a),
approximately 3- to 4-fold more potent than resveratrol for inhi-
bition of the cyclooxygenase activity of prostaglandin H-2 synthase,
which was partially purified from sheep seminal vesicles [16].
During our course of work, we developed methods to be capable
of synthesizing resveratrol dimers including pallidol (2a), quad-
rangularin A (E-3a), isopaucifloralF (4a) [17]. It was known that most
polyphenolic compounds have antioxidant activities. And in our lab,
the preliminary HTS result showed that some resveratrol dimers
indeed have potential activity of antioxidant. Recently, Pan et al.
reported that another resveratrol dimer parthenocissin A showed
neuroprotective effects in focal cerebral ischaemia of rats [18] and
several research papers demonstrated the relationships between
antioxidation and neuroprotection [19,20]. So we were also looking
for new leads for our new project about neuroprotective agents, and
considered to include this type of compounds into our screening
pool. Thus, synthesis of these natural compounds and their de-
rivatives, and evaluation of their neuroprotective effects, maylead to
novel candidates for this treatment. It has been reported that hy-
droxyl groups on the phenyl rings of resveratrol analogues play an
important role in their neuroprotective activity [21]. Therefore, we
manipulated the numbers and substituent patterns of hydroxyl
groups, carbonyl groups of indenones, and olefinic bonds of indene
moieties to explore the structureeactivity relationships involved in
their neuroprotective effects. In our earlier work, pallidol (2a),
isopaucifloral F (4a) and (þ)-
a-viniferin (5) (Fig. 1) [14,15]. In recent
years, researchers have found that these oligomers also show
a
variety of biological activities. Some of these derivatives
* Corresponding author. Department of Natural Products Chemistry, School of
Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China. Tel./
fax: þ86 21 51980003.
E-mail address: sunxunf@shmu.edu.cn (X. Sun).
These authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.037

188
C. Zhong et al. / European Journal of Medicinal Chemistry 62 (2013) 187e198
HO
OH
OH
OH
OH
OH
H
H
HO
HO
OH
HO
OH
OH
HO
OH
OH
pallidol (2a)
quadrangularin A (E-3a)
resveratrol (1)
OH
HO
OH
OH
OH
OH
H
O
OH
OH
HO
H
H
H
O
HO
H
OH
O
H
HO
parthenocissin A (Z-3a)
O
OH
HO
isopaucifloral F (4a)
(+)-α-viniferin (5)
Fig. 1. Natural resveratrol oligomers.
quadrangularin A (E-3a), isopaucifloral F (4a) and 30a (endo-shifted
olefin isomer of parthenocissin A) (Fig. 1) were synthesized in
short sequences starting from commercially available 3,5-
dimethoxybenzoic acid (5) [17]. These natural compounds, along
with a series of derivatives, were evaluated for their neuroprotective
effects on the paraquat-induced apoptosis in human SH-SY5Y cells.
For ease of discussion, we divided these compounds into three types
of structures: indenone-type (e.g., isopaucifloral F), indene-type
(e.g., quadrangularin A) and octahydropentalene-type (e.g., pal-
lidol). Meanwhile, elucidation of the molecular mechanism involved
in such neuroprotective effects might provide novel insight into the
process of protection against neurodegenerative diseases caused by
oxidative stress and apoptosis.
obtained as the major product (43% yield) along with the minor
product 19 (23% yield). However, if the reaction was not performed
in the dark, 19 was obtained the as the sole product in 77% yield
(Scheme 4). Finally, the double bond of E-12a migrated into the ring
system to afford 20 in 99% yield in the presence of a Lewis acid
(BF3$Et2O) (Scheme 5). Thus, a total of twenty-four compounds
were synthesized and eight of them were novel.
2.2. In vitro protection activity
2.2.1. Inhibition of apoptosis in SH-SY5Y cells induced by paraquat
The protective activity of the twenty-four resveratrol dimers
against paraquat-induced apoptosis was evaluated in SH-SY5Y cells
and the results are shown in Table 1. For indenone-type compounds,
they only exhibited comparable inhibitory activity to that of
resveratrol for the apoptosis induced by paraquat in SH-SY5Y cells.
However, some indene-type and octahydropentalene-type com-
pounds, such as 2a, 2b, 30a and 30b, inhibited greater potency than
resveratrol. Like resveratrol, compounds with polyphenol struc-
tures, especially with resorcinol groups, such as 2a, 2b, 30a and 30b,
were more effective in the neuroprotective assay than their corre-
sponding methylated precursors (14a, 13b, E-12a and E-12b). Fur-
ther SAR study showed that the para-hydroxyl group on the indene-
type analogues was contributory for increasing the neuroprotective
activity (30a versus 30b). This may be due to the fact that the hydroxyl
group in para position in this scaffold could decrease the values of
HOMO (highest occupied molecular orbital), IP (ionization poten-
tial) and BDEOH (bond dissociation energies), and increase the value
2. Results and discussion
2.1. Chemistry
Concise total syntheses of pallidol (2a), quadrangularin A (E-3a),
isopaucifloral F (4a) have been achieved starting from commercially
available 3,5-dimethoxybenzoic acid via a sequential process [17].
The synthetic strategy involved
a Nazarov cyclization [22],
RambergeBacklund olefination and FriedeleCrafts alkylation
(Scheme 1). Derivatives 2b, 4b and 8be13b were also synthesized by
adapting the same method simply by using 15b instead of 15a as the
WittigeHorner reagent. Intermediate Z-12a was also obtained as
a by-product in 7% yield during the conversion of 11a to E-12a. The
obtained Z-12a could be used as a potential precursor for the syn-
thesis of natural product parthenocissin A (Z-3a). To our surprise,
when Z-12a was subjected to the same demethylation conditions as
E-12a (BBr3, DCM), it was converted to 30a rather than to the
expected parthenocissin A (Z-3a) (Scheme 2). Similarly, E-3b could
not be synthesized via the demethylation of E-12b, and 30b turned
out to be the only product that could also be prepared from Z-12b.
Compounds 10a and 9a were utilized for the synthesis of several
indenone-type analogues. Although 10a was primarily converted to
11a in the presence of catalytic In(OTf)3, compound 17 was also
obtained as a minor product in 19% yield (Scheme 3). For the syn-
thesis of 18 and 19, two sets of conditions were explored. When the
olefination of 9a by Tebbe’s reagent was carried out in dark, 18 was
of DEiso (stabilization energies) [23,24]. All abovementioned factors
could tend to enhance the chance of compound being attacked by
the electrophilic agents, such as radicals or metal ions, which
resulted in antioxidant and cytoprotective effects. However, in the
case of indenone-type analogues, their in vitro neuroprotective ac-
tivities could not be improved even in the presence of one or more
polyphenol groups (e.g., 4a, 9a and 10a).
Interestingly, 30a showed more potent cytoprotective activity
than 3a. The only difference is that 30a has conjugated double bonds
rather than isolated double bonds in 3a. It was reasoned that this
conjugation between two resorcinol rings could stabilize the radi-
cal formed during oxidation via resonance effect, which may make

C. Zhong et al. / European Journal of Medicinal Chemistry 62 (2013) 187e198
189
Reagents and conditions: (a) LiN reagent, THF, 0 °C; (b) (CH3)3COK, Toluene; (c)
BF3·Et2O, 25 °C, 40 h; (d) BBr3, CH2Cl2, 0 to 25 °C, 8 h; (e) NaBH4, THF, CH3OH,
25 °C, 30 min; (f) p-CH3O-PhCH2SH, In(OTf)3, CH2Cl2, 25 °C, 1.5 h; (g) (i) mCPBA,
NaHCO3, CH2Cl2, 0 to 25 °C, 25 min; (ii) KOH, CCl4/t-BuOH/H2O=5/5/1, 80 °C, 12
h; (h) (i) BH3•THF, THF, 25 °C, 18 h; (ii) NaOH, 30%H2O2, Na2SO3, 25 to 0 °C; (i)
AlCl3, CH2Cl2, 25 °C, 2 h.
Scheme 1. Synthesis of resveratrol dimers and derivatives.
a contribution for the improvement of cytoprotective activity. Thus
the synergism of the existence of both the para-hydroxyl group and
conjugation between two resorcinol rings may be necessary for
achieving a good cytoprotective activity, which was also confirmed
by the activity of 30b.
Since compound 30a showed the highest neuroprotective ac-
tivity among the tested compounds, it was further conducted for
the study of mechanism of action.
2.2.2. The effects of 30a on paraquat-induced nuclear morphological
changes in SH-SY5Y cells
Reagents and conditions: (a) BBr3, CH2Cl2, 0 to 25 °C, 8 h
Apoptosis is characterized by various features such as shrinkage
of the cellular nucleus, chromatin condensation, intense
Scheme 2. Unexpected conversion of Z-12a and Z-12b into olefinic bond endo isomers
(30a & 30b).

190
C. Zhong et al. / European Journal of Medicinal Chemistry 62 (2013) 187e198
Reagents and conditions: (a) p-CH3O-PhCH2SH, In(OTf)3, CH2Cl2, 25 °C, 1 h.
Scheme 3. Synthesis of indenone derivative 17.
fluorescence and nuclear fragmentation. To further confirm the
protective function of 30a against paraquat-induced cell death in
SH-SY5Y cells, we performed Hoechst 33342 staining to identify
apoptotic cells on the basis of nuclear morphological features. Cells
that oxidative stress was induced by paraquat stimulation. In con-
trast, pretreatment with 30a significantly decreased paraquat-
induced ROS levels in a concentration dependent manner. The re-
sults suggest that the free radical scavenging properties of 30a may
contribute to its cytoprotective effects. The data also indicate that
the free radical scavenging activity of DSC may be responsible for
the attenuation of paraquat-induced apoptosis in SH-SY5Y cells.
were pretreated with various concentrations of 30a (0.1e10
mM),
followed by stimulation with paraquat (250
mM). Apoptosis was
then quantified by staining with Hoechst 33342. As shown in Fig. 2,
unstimulated cells did not show any signs of morphological nuclear
damage or chromatin condensation. However, the nuclei of cells
treated with paraquat appeared to be hypercondensed and dis-
played chromatin fragmentation (arrows). Treatment with 30a
attenuated paraquat-induced nuclear morphological changes in
a dose-dependent manner. The data confirmed that the novel
compound 30a inhibited paraquat-induced apoptosis in SH-SY5Y
cells.
2.2.4. The effects of 30a on SOD and GPx expression in paraquat-
stimulated SH-SY5Y cells
Expression of the anti-oxidative enzyme SOD indirectly reflects
the scavenging of intracellular ROS. Another enzyme, GPx, is used
to remove lipid peroxidation and plays an important role in pro-
tecting the structure and function of the cell membrane. Therefore,
we measured the level of GPx and SOD expression as markers of
oxidative stress. As shown in Fig. 4, levels of the anti-oxidative
enzymes GPx and SOD were analysed by immunoblot. Paraquat
did not hamper expression of GPx and SOD as compared with
2.2.3. The effects of 30a on paraquat-induced ROS production in SH-
SY5Y cells
Oxidative stress due to abnormal exposure to ROS plays a key
role in apoptosis. Paraquat is a potent ROS inducer and can increase
the production of intracellular ROS, such as superoxide anion,
singlet oxygen and hydroxyl and peroxyl radicals. Production of
ROS subsequently leads to lipid peroxidation that results in oxi-
dative damage to the cell. To determine whether paraquat induced
apoptosis in SH-SY5Y cells by stimulating oxidative stress, we
exposed SH-SY5Y cells to paraquat for 2 h and then loaded the cells
for 30 min with DHE, which upon reaction with ROS yields the
fluorescent product 2-hydroxyethidium. As shown in Fig. 3, treat-
ment with paraquat led to significant increases in intracellular ROS
levels compared with untreated cells. These results demonstrate
control cells. Intriguingly, pre-incubation with 30a (0.1e10
elevated expression of SOD and GPx in concentration-
mM)
a
dependent manner in paraquat-stimulated SH-SY5Y cells. This
result strongly indicates that treatment with 30a prior to exposure
to paraquat may be helpful for amelioration of oxidative damage.
This treatment may also decrease apoptosis through upregulation
of the antioxidative enzymes SOD and GPx.
2.2.5. The effects of 30a on Bax and Bcl-2 expression in paraquat-
stimulated SH-SY5Y cells
The Bcl-2 family of proteins plays a critical role in the decision of
cells to die or survive by affecting caspase activation [25]. To study
Reagents and conditions: (a) Tebbe reagent, Toluene, 52 °C, in dark, 48 h; (b) Tebbe
reagent, Toluene, 52 °C, 24 h.
Scheme 4. Synthesis of indenone derivatives 18 and 19.

C. Zhong et al. / European Journal of Medicinal Chemistry 62 (2013) 187e198
191
to a concentration-dependent attenuation of paraquat-induced
proteolytic cleavage of procaspase-9 and procaspase-3 (Fig. 6).
3. Conclusions
In conclusion, three-types of resveratrol dimersdincluding
natural products and their analoguesdwere synthesized and
evaluated
for
neuroprotective
activity.
Overall,
Reagents and conditions: (a) BF3·Et2O, CH2Cl2, 25 °C, 48 h.
octahydropentalene-type and indene-type analogues were more
potent than indenone-type analogues. Among these, the natural
product 2a and some analogues (2b, 30a, 30b) were found to be
more effective than their methylated precursors. Further study
showed that compound 30a which contains conjugated double
bonds and para-hydroxyl group, could attenuate paraquat-induced
nuclear morphological changes, significantly decrease paraquat-
induced ROS levels in SH-SY5Y cells, and elevate expression of
SOD and GPx in a dose-dependent manner. Furthermore, pre-
Scheme 5. Synthesis of indene derivative 20.
the possible mechanisms of 30a inhibition of paraquat-induced
apoptosis, we investigated alterations in the expression of the
proapoptotic protein Bax and the antiapoptotic protein Bcl-2 in SH-
SY5Y cells treated with different concentrations of 30a. Exposure of
SH-SY5Y cells to paraquat leads to increased levels of Bax, which
heterodimerizes with Bcl-2 and inhibits Bcl-2 activity. This causes
a dramatic reduction in the level of Bcl-2, a potent cell death in-
hibitor, as shown in Fig. 5. However, pre-incubation with 30a (0.1e
incubation with 30a (0.1e10
and increase Bcl-2 protein expression in
mM) could decrease Bax protein levels
a
concentration-
dependent manner, accompanied by an increase in the chances of
cell survival. Further study demonstrated that compound 30a had
good protective effects against paraquat-induced apoptosis in SH-
SY5Y cells. This cytoprotective activity is associated with anti-
oxidant and antiapoptotic effects. Compound 30a could be
a potentially promising candidate for the treatment of Parkinson’s
and Alzheimer’s diseases. Because there are very few examples of
cell protection by these resveratrol dimers, our results provide
a theoretical basis for further detailed neuroprotective mechanistic
studies and lead optimization.
10 mM) could decrease Bax protein levels and increase Bcl-2 protein
expression in a concentration-dependent fashion, resulting in
increased chances of cell survival (Fig. 5).
2.2.6. The effects of 30a on caspase-3 and caspase-9 expression in
paraquat-stimulated SH-SY5Y cells
A change in the Bcl-2/Bax ratio is required for assembly of the
apoptosome and hence for the activation of the caspase cascade
during apoptosis. Therefore, the apoptotic protein markers caspase-
3 and caspase-9 were also examined by Western blot. Western blot
analysis showed that stimulation of cells with paraquat sig-
nificantly induced proteolytic cleavage of procaspase-9 and
procaspase-3. Conversely, treatment of SH-SY5Y cells with 30a led
4. Experimental section
4.1. Materials and physical measurements
Reagents were purchased from Aldrich and TCI Chemical com-
panies. Antibodies against Bax, caspase-3 and caspase-9 were
purchased from Cell Signaling Inc. (Beverly, MA, USA); antibodies
against SOD1, GPx, Bcl-2 and GAPDH were purchased from Santa
Cruz Biotechnology Inc. (Santa Cruz, CA, USA); Dihydroethidium
(DHE) was obtained from Molecular Probes (Eugene, OR, USA).
Paraquat and other chemicals were purchased from SigmaeAldrich
(St. Louis, MO). All solvents were purified and dried in accordance
with standard procedures unless otherwise indicated. Oxygen-free
and water-free operations were carried out under an argon atmo-
sphere in dried glassware unless otherwise noted. Reactions were
monitored by TLC using Yantai (China) GF254 silica gel plates
(5 ꢀ 10 cm). Silica gel column chromatography was performed on
silica gel (300e400 mesh) from Yantai (China). Melting points (mp)
were determined using an X-4 microscope melting point apparatus
and were uncorrected. All 1H NMR spectra were recorded on Varian
Mercury 300 (300 MHz) or Bruker DRX-400 (400 MHz) spec-
trometers and 13C NMR spectral data were recorded on a Bruker
DPX-400 (100 MHz) spectrometer. Low-resolution mass spectra
(ESI) were obtained on a Shimadzu LCMS-2010EV and high-
resolution mass spectra were obtained on an IonSpec 4.7 Tesla
FTMS (MALDI) or a Bruker Daltonics, Inc. APEXIII7.0 TESLA FMS
(ESI). Infrared spectra were obtained as thin films on KBr plates.
Table 1
The effects of novel compounds on paraquat-induced cell death in SH-SY5Y cells.
Compounds Control Paraquat
Compound
-treated
cells (%)
(%)
-stimulated
cells (%)
Indenone-type
derivatives
4a
4b
9a
9b
10a
10b
17
18
19
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
62.05 ꢁ 0.47a
58.5 ꢁ 3.45
62.05 ꢁ 0.47a 64.95 ꢁ 0.85
62.05 ꢁ 0.47a 59.86 ꢁ 3.74
62.05 ꢁ 0.47a 65.05 ꢁ 1.52
62.05 ꢁ 0.47a 59.02 ꢁ 4.37
62.05 ꢁ 0.47a
63.9 ꢁ 2.86
62.05 ꢁ 0.47a 58.63 ꢁ 4.95
62.05 ꢁ 0.47a 58.18 ꢁ 2.99
62.05 ꢁ 0.47a 55.77 ꢁ 3.56
62.05 ꢁ 0.47a 57.93 ꢁ 3.95
62.05 ꢁ 0.47a 89.56 ꢁ 4.13b
Indene-type
derivatives
E-3a
30a
30b
62.05 ꢁ 0.47a
82.9 ꢁ 1.95b
11a
11b
E-12a
Z-12a
E-12b
Z-12b
13a
13b
20
62.05 ꢁ 0.47a 65.15 ꢁ 3.07
62.05 ꢁ 0.47a 60.59 ꢁ 4.35
62.05 ꢁ 0.47a 62.38 ꢁ 4.31
62.05 ꢁ 0.47a 63.63 ꢁ 2.49
62.05 ꢁ 0.47a 64.03 ꢁ 3.08
62.05 ꢁ 0.47a 65.33 ꢁ 4.73
62.05 ꢁ 0.47a 69.75 ꢁ 5.39
62.05 ꢁ 0.47a 68.19 ꢁ 1.07
62.05 ꢁ 0.47a 61.35 ꢁ 5.50
62.05 ꢁ 0.47a 72.40 ꢁ 3.47
62.05 ꢁ 0.47a 77.23 ꢁ 2.13
62.05 ꢁ 0.47a 63.57 ꢁ 2.48
62.05 ꢁ 0.47a 66.33 ꢁ 2.01
Octahydropentalene 2a
-type
derivatives
Resveratrol
2b
14a
1
4.2. Chemistry
4.2.1. 1,2-Bis(3,5-dimethoxyphenyl)ethane-1,2-dione (7)
After preincubation with indicated compound (10
with paraquat (250
mM), cells were then stimulated
M) for 24 h. Cell viability was measured by the MTT assay. Data
To a solution of naphthalene (23.3 g, 181 mmol) in anhydrous
THF (100 mL) was added lithium (1.26 g, 181 mmol) under N2 at
room temperature. The mixture was continuously stirred at room
temperature for 8 h. The resulting mixture was added dropwise to
m
shown are means ꢁ SEM. aP < 0.05 compared with control cells. bP<0.05 compared
with paraquat-stimulated cells. Data were from at least three independent experi-
ments, each performed in duplicate.

192
C. Zhong et al. / European Journal of Medicinal Chemistry 62 (2013) 187e198
Fig. 2. The effects of 30a on paraquat-induced nuclear morphological changes in SH-SY5Y cells. Representative photomicrographs show nuclear morphological changes determined
by fluorescence microscope. The experiments were repeated in triplicate.
a solution of 3,5-dimethoxybenzoic acid (6, 15.0 g, 82.3 mmol) in
anhydrous THF (300 mL) at 0 ꢂC and quenched with water upon
completion of the addition. After removed the THF and diluted with
ethyl acetate (50 mL), the organic layer was separated and the
aqueous layer was extracted with ethyl acetate (3 ꢀ 30 mL). The
combined organic extracts were washed with brine (30 mL) and
dried over Na2SO4. After filtered, the solvent was evaporated to
obtain a solid which was further recrystallized in ethyl acetate to
give pure 7 as a yellow solid (12.0 g, 44%). mp: 171e172 ꢂC; 1H NMR
(20 mL) was added slowly at 0 ꢂC. The resulting mixture was
refluxed overnight and TLC indicated that the reaction was com-
pleted. After cooled to 0 ꢂC, 30% H2O2 (60 mL) was added dropwise
to the mixture at 0 ꢂC. Then the reaction was concentrated and
diluted with CH2Cl2 (50 mL). The organic layer was separated and
the aqueous layer was extracted with CH2Cl2 (3 ꢀ 60 mL). The
combined organic extracts were washed with brine (60 mL), dried
over Na2SO4, filtered, and evaporated. The obtained residue was
washed by hexane, ethyl acetate and ethanol to give 15a as a white
solid. Yield 81%; mp: 233e235 ꢂC; 1H NMR (300 MHz, CDCl3)
(400 MHz, CDCl3)
d ppm: 3.83 (12H, s), 6.74 (2H, s), 7.07 (4H, d,
J ¼ 1.7 Hz); 13C NMR (100 MHz, CDCl3)
d
ppm: 55.67, 107.38, 107.53,
d
ppm: 3.59 (2H, d, J ¼ 13.2 Hz), 3.74 (3H, s), 6.73 (2H, d, J ¼ 8.8 Hz),
134.65, 161.10, 194.16; C18H18O6: HRMS: m/z calcd. 331.1181
7.02 (2H, d, J ¼ 7.2 Hz), 7.43e7.71 (10H, m); 13C NMR (100 MHz,
[M þ H]þ; found: 331.1178.
CDCl3)
d ppm: 36.71, 37.26, 55.16, 113.67, 113.85, 113.88, 122.78,
122.86, 128.42, 128.54, 131.08, 131.11, 131.13, 131.20, 131.75, 131.78,
4.2.2. p-Methoxy-benzyl-diphenyl-phosphate oxygen (15a)
132.76, 158.48, 158.51; EI-MS m/z: 322 [M]þ.
1-(Chloromethyl)-4-methoxybenzene (12.01 g, 76.7 mmol) was
added dropwise to a stirred solution of magnesium (1.84 g,
76.7 mmol) in dry THF (100 mL) under N2. The reaction mixture was
heated to reflux for 5 h, then Ph2PCl (14.2 mL, 76.7 mmol) in THF
4.2.3. Benzyl-diphenyl-phosphate oxygen (15b)
Compound 15b was prepared in a manner identical to that
described for 15a as a white solid. Yield 71%; mp: 190e192 ꢂC; 1H
Fig. 3. The effects of 30a on paraquat-induced ROS production in SH-SY5Y cells. Cell morphology (A), intracellular ROS production (B), and cell fluorescence were detected by
fluorescence microscopy (200ꢀ) and the figure is representative of at least three experiments performed on different days.

C. Zhong et al. / European Journal of Medicinal Chemistry 62 (2013) 187e198
193
Fig. 4. The effects of 30a on SOD and GPx expression in paraquat-stimulated SH-SY5Y cells. Western blots for SOD (A) and GPx (B) protein expression. Bar graphs show quantitative
analysis of SOD and GPx protein levels, respectively. GAPDH was used as loading control. *P < 0.05 compared with unstimulated cells; &P < 0.05 compared with paraquat-stimulated
cells. Data are from at least three independent experiments, each performed in duplicate.
NMR (400 MHz, CDCl3)
d
ppm: 3.65 (2H, d, J ¼ 13.8 Hz), 7.11e7.73
(15H, m), 6.38e6.39 (1H, m), 6.56e6.59 (3H, m), 6.72e6.75 (2H, m),
7.09e7.23 (5H, m); 13C NMR (100 MHz, CDCl3)
ppm: 54.95, 55.03,
(15H, m); EI-MS m/z (%): 291 (Mþ, 43.38), 201 [(M ꢃ PhCH2)þ, 100].
d
55.16, 55.29, 55.32, 99.61, 100.06, 103.94, 104.36, 106.03, 107.11,
107.18, 107.26, 113.58, 113.77, 126.83, 127.74, 129.91, 130.19, 132.23,
138.05, 138.42, 138.69, 140.06, 140.37, 141.16, 159.28, 160.32, 160.77,
160.82, 161.04, 196.79, 198.77; MS m/z: 434 (M)þ; C26H26O6: HRMS
calcd. 434.1705 [M]þ, found 434.1681; IR (KBr) nmax ¼ 3090, 3004,
4.2.4. 1,2-Bis(3,5-dimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-
en-1-one (8a)
To a solution of 7 (1.00 g, 3.03 mmol) and 15a (0.98 g, 3.03 mmol)
in anhydrous THF (100 mL) was added potassium tert-butoxide
(0.56 g, 4.54 mmol) under N2 at room temperature. The reaction
mixture was stirred for 1 h, then cooled to 0 ꢂC and quenched with
water. The reaction mixture was concentrated and then diluted with
CH2Cl2 (50 mL). The organic layer was separated and the aqueous
layer was extracted with CH2Cl2 (3 ꢀ 20 mL). The combined organic
extracts were washed with brine (30 mL), dried over Na2SO4, fil-
tered, and evaporated. The obtained residue was further purified by
silica gel chromatography (ethyl acetate/hexane ¼ 1/10) to give 8a as
2901, 2939, 2839, 1666, 1591, 1512, 1457, 1426 cmꢃ1
.
4.2.5. 1,2-Bis(3,5-dimethoxyphenyl)-3-phenylprop-2-en-1-one (8b)
Compound 8b was prepared in a manner identical to that
described for 8a as yellow oil. Yield 71%; 1H NMR (400 MHz, CDCl3)
d
ppm: 3.69e3.81 (12H, m, multiple singlets overlapped), 6.42e
7.29 (12H, m); EI-MS m/z (%) 404 (Mþ, 62.24), 165 [(CH3O)2-m-
PhCOþ, 100]; IR (KBr): nmax ¼ 3086, 3003, 2960, 2939, 2839, 1667,
a yellow oil. Yield 74%; 1H NMR (300 MHz, CDCl3)
d
ppm: 3.72e3.77
1591, 1457, 1425, 1206, 1158, 1065 cmꢃ1
.
Fig. 5. The effects of 30a on Bax and Bcl-2 expression in paraquat-stimulated SH-SY5Y cells. Western blots for Bax (A) and Bcl-2 (B) protein expression. Bar graph shows quantitative
analysis of Bax and Bcl-2 protein levels, respectively. GAPDH was used as loading control. *P < 0.05 compared with unstimulated cells; &P < 0.05 compared with paraquat-
stimulated cells. Data are from at least three independent experiments, each performed in duplicate.

194
C. Zhong et al. / European Journal of Medicinal Chemistry 62 (2013) 187e198
Fig. 6. The effects of 30a on caspase-3 and caspase-9 expression in paraquat-stimulated SH-SY5Y cells. Western blots for caspase-3 (A) and caspase-9 (B) protein expression. Bar
graphs show quantitative analysis of caspase-3 and caspase-9 protein levels, respectively. GAPDH was used as loading control; *P < 0.05 compared with unstimulated cells;
&P < 0.05 compared with paraquat-stimulated cells. Data are from at least three independent experiments, each performed in duplicate.
4.2.6. Trans-2-(3,5-dimethoxyphenyl)-4,6-dimethoxy-3-(4-
solid. Yield 54%; mp: 159e161 ꢂC; 1H NMR (400 MHz, CDCl3)
d ppm:
methoxyphenyl)-2,3-dihydroinden-1-one (9a)
3.37 (1H, d, J ¼ 2 Hz), 4.39 (1H, d, J ¼ 2 Hz), 6.00 (2H, d, J ¼ 1.2 Hz),
To a solution of chalcones 8a (3.21 g, 7.40 mmol) in CH2Cl2 (70 mL)
was dropwisely added BF3$OEt2 (0.9 mL, 7.4 mmol) via syringe. After
the reaction mixture was stirred at room temperature for 40 h under
N2, the reaction was quenched with H2O (10 mL). The resulting so-
lution was extracted with CH2Cl2 (3 ꢀ 10 mL) and the combined
organic phases were washed with brine and dried over Na2SO4. After
evaporated the solvent, the resulting yellow residue was recrystal-
lized in ethyl acetate (3 mL)/petroleum ether (50 mL) to give 9a as
a white solid. Yield 85%; mp: 173e174 ꢂC; 1H NMR (300 MHz, CDCl3)
6.15 (1H, s), 6.61 (1H, d, J ¼ 1.6 Hz), 6.67e6.69 (3H, m), 6.82e6.84
(2H, m); 13C NMR (100 MHz, CDCl3)
d ppm: 52.40, 67.19, 102.26,
107.21, 111.03, 116.22, 129.01, 135.93, 136.73, 140.04, 143.33, 156.94,
157.21, 159.84, 160.75, 208.88; ESI-MS: 365.1 (M þ H)þ; C21H16O6:
HRMS (MALDI/DHB) calcd. 365.1030 [M þ H]þ, found 365.1020; IR
(KBr) nmax ¼ 3342, 1687, 1603, 1513, 1476, 1342, 1254, 1151 cmꢃ1
.
4.2.9. 2-(3,5-Dihydroxyphenyl)-4,6-dihydroxy-3-phenyl-2,3-
dihydroinden-1-one (4b)
d
ppm: 3.61 (1H, d, J ¼ 2.6 Hz), 3.66 (3H, s), 3.73 (6H, s), 3.78 (3H, s),
Compound 4b was prepared in a manner identical to that
3.87 (3H, s), 4.52 (1H, d, J ¼ 2.6 Hz), 6.23e6.96 (9H, m); 13C NMR
described for 4a as a light brown solid. Yield 39%; 1H NMR
(100 MHz, CDCl3)
d
ppm: 50.84, 55.17, 55.29, 55.60, 55.80, 65.33,
(400 MHz, CDCl3)
(1H, s), 6.63 (1H, s), 6.71 (1H, s), 7.00e7.25 (5H, m); 13C NMR
(100 MHz, CDCl3) ppm: 53.05, 66.91, 100.58, 102.31, 107.21, 110.97,
d ppm: 3.41 (1H, s), 4.48 (1H, s), 6.02 (2H, s), 6.17
96.38, 98.85, 106.00, 106.56, 113.77, 127.94, 135.40, 138.49, 141.53,
157.72, 158.11, 160.95, 161.89, 205.50; EI-MS m/z: 434 (M, 100)þ;
C26H26O6: HRMS calcd. 434.1721 [M]þ, found 434.1713; IR (KBr)
d
127.46, 128.03, 129.50, 136.29, 140.08, 143.14, 144.95, 157.21, 159.83,
160.85, 208.56; ESI-MS: 349.1 [M þ H]þ; C21H16O5: HRMS calcd.
349.1031 [M þ H]þ, found 349.1046.
nmax ¼ 3009, 2937, 2840, 1711, 1610, 1514, 1463 cmꢃ1
.
4.2.7. (2S,3S)-2-(3,5-Dimethoxyphenyl)-4,6-dimethoxy-3-phenyl-
2,3-dihydroinden-1-one (9b)
4.2.10. Trans-2-(3,5-dimethoxyphenyl)-4,6-dimethoxy-3-(4-
Compound 9b was prepared in a manner identical to that descri-
methoxyphenyl)-2,3-dihydro-1H-inden-1-ol (10a)
bed for 9a as a white solid. Yield 94.4%; mp: 103e105 ꢂC; 1H NMR
To a solution of 9a (2.00 g, 4.60 mmol) in THF (50 mL) was added
NaBH4 (1.74 g, 46 mmol) at room temperature. Then the reaction
mixture was added methanol (50 mL) and continuously stirred at
room temperature for 30 min. Upon completion, the reaction
mixture was concentrated. After separated the organic layer, the
aqueous layer was extracted with CH2Cl2 (3 ꢀ 15 mL). The com-
bined organic extracts were washed with brine (30 mL), dried over
Na2SO4, filtered, and evaporated in vacuo to give 10a as a white
solid which will be used directly for the next step. Yield 99%; mp:
62e63 ꢂC; ESI-MS m/z: 895.5 [2M þ Na]þ; IR (KBr) nmax ¼ 3416,
(400 MHz, CDCl3)
d
ppm: 3.64 (1H, d, J ¼ 2.4 Hz), 3.65 (3H, s), 3.74 (6H,
brs), 3.88 (3H, s), 4.55 (1H, d, J ¼ 2.4 Hz), 6.24e6.40 (3H, m), 6.70 (1H,
d, J ¼ 2.1 Hz), 6.92 (1H, d, J ¼ 2.1 Hz), 7.02e7.28 (5H, m); 13C NMR
(100 MHz, CDCl3) d ppm: 51.6, 55.3, 55.6, 55.8, 65.2, 96.5, 99.0, 106.1,
106.6, 126.5, 127.0, 128.5, 138.2, 138.7, 141.5, 143.3, 157.8, 161.0, 162.0,
205.3;EI-MS m/z (%) 404 ([M]þ,100); IR (KBr) nmax ¼ 3008, 2930, 2841,
1714, 1594, 1455, 1431, 1207, 1161, 1149, 1066, 1057, 1030 cmꢃ1
.
4.2.8. (ꢁ)Isopaucifloral F (4a)
To a solution of 9a (0.20 g, 0.46 mmol) in anhydrous CH2Cl2
(15 mL) was added BBr3 (0.66 mL, 6.91 mmol) at 0 ꢂC under N2. The
resulting mixture was stirred at room temperature for 8 h. Upon
completion, the reaction mixture was quenched with water and
extracted with ethyl acetate (3 ꢀ 5 mL). The combined organic
layers were then washed with brine, dried over MgSO4, filtered, and
evaporated. The residue was purified by Rp silica gel column
chromatography (MeOH:H2O ¼ 1:2) to give 4a as a light brown
2999, 2937, 2837, 1595, 1511, 1463, 1203, 1149 cmꢃ1
.
4.2.11. Trans-2-(3,5-dimethoxyphenyl)-4,6-dimethoxy-3-phenyl-
2,3-dihydro-1H-inden-1-ol (10b)
Compound 10b was prepared in a manner identical to that
described for 10a as a white solid. Yield 99%; ESI-MS m/z: 389.2
[M ꢃ H2O]þ; IR (KBr) nmax ¼ 3441, 2935, 2836, 1596, 1511, 1455,
1203, 1148 cmꢃ1
.

C. Zhong et al. / European Journal of Medicinal Chemistry 62 (2013) 187e198
195
4.2.12. (4-Methoxybenzyl)(2-(3,5-dimethoxyphenyl)-4,6-
dimethoxy-3-(4-methoxyphenyl)-2,3-dihydro-1H-inden-1-yl)
sulfane (11a)
solution was then stirred for 12 h at 80 ꢂC. Upon completion, the
reaction mixture was quenched with saturated aqueous NH4Cl
(10 mL), poured into water (20 mL), and extracted with CH2Cl2
(3 ꢀ 15 mL). The combined organic layers were washed with water,
brine, and dried over MgSO4. After filtered, the solution was con-
centrated in vacuo and further purified by silica gel column chro-
matography (ethyl acetate/hexanes ¼ 1/5) to give both the desired
E-alkene E-12a as a yellow oil along with a small and separable
portion of its Z-alkene Z-12a as a light yellow oil. Yield 61%; mp:
To a solution of 10a (0.44 g, 1.00 mmol) and In(OTf)3 (0.56 g,
1.00 mmol) at 25 ꢂC was added benzyl mercaptans (2.7 mmol) in
CH2Cl2. The resulting viscous red mixture was protected from light
and stirred for 60 min at 25 ꢂC. Upon completion, the reaction
mixture was quenched with saturated aqueous NaHCO3 (15 mL),
filtered through Celite, and extracted with ethyl acetate (3 ꢀ 10 mL).
The combined organic layers were washed with brine and dried
over MgSO4. After filtered, the solution was concentrated in vacuo
and further purified by silica gel column chromatography (ethyl
acetate/hexanes ¼ 1/10) to give sulfide 11a as a yellow solid and by-
product 17 as a white solid. Yield 75%; mp: 46e47 ꢂC; 1H NMR
64e66 ꢂC; 1H NMR (400 MHz, CDCl3)
d ppm: 3.61 (3H, s), 3.73e3.74
(12H, m), 3.92 (3H, s), 4.31 (2H, m), 6.31e6.32 (2H, m), 6.43e6.44
(2H, m), 6.73e6.77 (4H, m), 6.84 (1H, m), 7.03 (2H, d, J ¼ 8.4 Hz),
7.11 (1H, s), 7.22 (2H, d, J ¼ 8.8 Hz); 13C NMR (100 MHz, CDCl3)
d
ppm: 55.14, 55.18, 55.21, 55.55, 56.78, 59.22, 94.84, 97.69, 99.16,
(400 MHz, CDCl3)
d
ppm: 3.54 (3H, s), 3.75e3.78 (15H, m), 3.83 (3H,
105.33, 113.58, 113.72, 122.44, 126.87, 127.84, 129.70, 130.06, 137.94,
142.28, 145.28, 147.70, 157.43, 157.88, 158.47, 160.98, 161.43; ESI-MS
m/z :539.3 [M þ H]þ, IR (KBr) nmax ¼ 2955, 2916, 2849, 2868, 1462,
s), 4.26 (1H, d, J ¼ 6.7 Hz), 4.30 (1H, d, J ¼ 7.0 Hz), 6.27 (2H, d,
J ¼ 2.0 Hz), 6.37 (2H, m), 6.56 (1H, m), 6.75 (4H, m), 6.93 (2H, d,
J ¼ 8.6 Hz), 7.04 (2H, d, J ¼ 8.6 Hz); 13C NMR (100 MHz, CDCl3)
1377, 1260, 1021, 801, 739 cmꢃ1
.
d
ppm: 34.78, 36.07, 52.15, 53.38, 53.44, 55.04, 55.11, 55.14, 55.18,
55.23, 55.44, 55.51, 55.95, 56.03, 61.63, 66.33, 98.35, 98.48, 98.87,
100.22, 100.68, 105.71, 107.26, 113.17, 113.37, 113.61, 113.68, 123.95,
124.43, 128.08, 128.17, 130.00, 130.15, 130.19, 135.78, 136.71, 143.15,
145.06, 145.85, 156.80, 156.89, 157.66, 157.74, 158.37, 158.42, 160.19,
160.65, 161.04, 161.33; ESI-MS m/z :595.3 [M þ Na]þ; IR (KBr)
4.2.16. Z-Permethylated quadrangularin A (Z-12a)
Compound Z-12a was separated as by-product when preparing
E-12a as light yellow oil. Yield 7%; mp: 128e129 ꢂC; 1H NMR
(400 MHz, CDCl3) d ppm: 3.55 (3H, s), 3.58 (3H, s), 3.74 (6H, s), 3.78
(3H, s), 3.82 (3H, s), 3.92 (1H, m), 4.31 (1H, d, J ¼ 2.7 Hz), 6.29 (1H, d,
J ¼ 2.1 Hz), 6.33e6.35 (3H, m), 6.39 (1H, s), 6.51 (1H, d, J ¼ 2.1 Hz),
6.80 (2H, d, J ¼ 8.7 Hz), 6.88 (2H, d, J ¼ 8.7 Hz), 7.06 (2H, d,
J ¼ 8.7 Hz), 7.35 (2H, d, J ¼ 8.7 Hz); 13C NMR (100 MHz, CDCl3)
nmax ¼ 2997, 2935, 2834, 1606, 1591, 1462, 1246, 1149, 829 cmꢃ1
.
4.2.13. 2-(3,5-Dimethoxyphenyl)-5,7-dimethoxy-1-(4-
methoxyphenyl)-1H-indene (17)
d
ppm: 54.52, 55.17, 55.23, 55.27, 63.17, 97.82, 99.55, 99.70, 105.78,
Compound 17 was separated as by-product when preparing 11a
113.53, 113.55, 113.69, 124.87, 127.84, 127.98, 129.68, 129.96, 130.06,
130.21, 137.72, 141.51, 144.91, 148.44, 157.07, 157.83, 158.61, 160.19,
160.71, 160.97; ESI-MS m/z: 539.3 [M þ H]þ, IR (KBr) nmax ¼ 2958,
as a white solid. Yield 19%; 1H NMR (400 MHz, CDCl3)
d ppm: 3.63
(3H, s), 3.72e3.73 (9H, m), 3.83 (3H, s), 4.93 (1H, s), 6.26 (1H, d,
J ¼ 2.0 Hz), 6.29e6.30 (1H, m), 6.62 (1H, d, J ¼ 2.0 Hz) 6.64 (2H, m),
6.69e6.72 (2H, m), 7.05e7.09 (2H, m), 7.18 (1H, m); ESI-MS m/z
:419.2 [M þ H]þ; IR (KBr) nmax ¼ 2957, 2939, 2835, 1592, 1511, 1457,
2923, 2850, 1593, 1509, 1541, 1246, 1150, 1033, 804, 737 cmꢃ1
.
4.2.17. (E)-1-(4-Methoxybenzylidene)-2-(3,5-dimethoxyphenyl)-
4,6-dimethoxy-3-phenyl-2,3-dihydro-1H-indene (E-12b)
1425, 1250, 1206, 1155, 820 cmꢃ1
.
Compound E-12b was prepared as by-product in a manner
identical to that described for E-12a as light brown oil. Yield 52%;
4.2.14. (4-Methoxybenzyl)(2-(3,5-dimethoxyphenyl)-4,6-
dimethoxy-3-phenyl-2,3-dihydro-1H-inden-1-yl)sulfane (11b)
Compound 11b was prepared in a manner identical to that
described for 11a as a yellow solid. Yield 65%; 1H NMR (400 MHz,
1H NMR (400 MHz, CDCl3)
d ppm: 3.57 (3H, s), 3.66 (6H, s), 3.68 (3H,
s), 3.88 (3H, s), 4.22 (2H, d, J ¼ 5.9 Hz), 6.29e6.38 (4H, m), 6.71 (2H,
d, J ¼ 8.6 Hz), 6.93e7.21 (9H, m); 13C NMR (100 MHz, CDCl3)
d ppm:
CDCl3)
d
ppm: 3.40 (1H, m), 3.52 (3H, s), 3.59 (2H, m), 3.73 (6H, s),
55.62, 56.07, 59.11, 60.77, 96.14, 98.92, 99.97, 106.17, 114.66, 123.62,
127.17, 127.52, 127.90, 129.39, 130.96, 131.14, 143.48, 147.00, 147.16,
148.92, 158.86, 160.11, 162.63, 163.35; ESI-MS m/z: 509.2 [M þ H]þ;
C33H32O5: HRMS calcd. 509.2283 [M þ H]þ, found 509.2310; IR
3.76 (3H, s), 3.83 (3H, s), 4.27 (1H, d, J ¼ 7.1 Hz), 4.33 (1H, d,
J ¼ 7.1 Hz), 6.26 (2H, d, J ¼ 2.0 Hz), 6.36 (2H, d, J ¼ 1.6 Hz), 6.56 (1H,
s), 6.74 (2H, d, J ¼ 8.6 Hz), 6.99e7.21 (7H, m); 13C NMR (100 MHz,
CDCl3)
d
ppm: 35.07, 55.45, 55.51, 55.85, 56.45, 57.00, 66.50, 98.89,
(KBr) nmax ¼ 2926, 2816, 1593, 1512, 1447, 1243, 1150 cmꢃ1
.
99.19, 100.57, 106.04, 113.93, 124.05, 126.14, 127.58, 128.14, 130.31,
130.47, 144.74, 145.48, 146.01, 157.18, 158.69, 160.96, 161.71; ESI-MS
m/z: 543.2 [M þ H]þ; C33H34O5S: HRMS calcd. 565.2019 [M þ Na]þ,
found 565.2037; IR (KBr) nmax ¼ 2926, 2822, 1594, 1510, 1455, 1247,
4.2.18. (Z)-1-(4-Methoxybenzylidene)-2-(3,5-dimethoxyphenyl)-
4,6-dimethoxy-3-phenyl-2,3-dihydro-1H-indene (Z-12b)
Compound Z-12b was separated as by-product when preparing
1204, 1145, 822 cmꢃ1
.
E-12b as light brown oil. Yield 11%; 1H NMR (400 MHz, CDCl3)
d
ppm: 3.54 (3H, s), 3.76 (6H, s), 3.77 (3H, s), 3.83 (3H, s), 4.41 (1H, d,
4.2.15. Permethylated quadrangularin A (E-12a)
J ¼ 5.1 Hz), 4.72 (1H, d, J ¼ 5.9 Hz), 6.31e6.46 (4H, m), 6.76 (2H, d,
To a solution of sulfide 11a (2.10 g, 3.67 mmol) in CH2Cl2 (50 mL)
were added NaHCO3 (1.55 g, 18.4 mmol) and mCPBA (85%, 2.53 g,
14.7 mmol) at 0 ꢂC to give a milk-coloured slurry. After slowly
warmed to 25 ꢂC, the reaction mixture was stirred for 25 min, then
quenched with saturated aqueous NaHCO3 (10 mL). The mixture
was poured into water (20 mL) and extracted with CH2Cl2
(3 ꢀ 10 mL). The combined organic layers were washed with water,
brine, and dried over MgSO4. After filtered, the solution was con-
centrated in vacuo and further purified by silica gel column chro-
matography (ethyl acetate/hexanes ¼ 1/5) to give the desired
sulfone intermediate as yellow pink oil. The resulting oil was dis-
solved in a mixture of CCl4/t-BuOH/H2O (45 mL, 5/5/1) and KOH
(3.93 g, 70.20 mmol) was added at 25 ꢂC. The resulting suspended
J ¼ 8.6 Hz), 6.86 (2H, d, J ¼ 8.6 Hz), 6.94 (1H, s), 7.06e7.26 (6H, m);
13C NMR (100 MHz, CDCl3)
d ppm: 55.21, 55.28, 55.41, 55.70, 56.88,
57.75, 58.12, 99.03, 100.73, 101.62, 105.56, 114.02, 118.64, 126.27,
126.39, 127.32, 128.19, 132.16, 135.95, 143.81, 145.46, 156.92, 159.86,
161.38, 161.63; ESI-MS m/z: 509.2 [M þ H]þ; C33H32O5: HRMS calcd.
509.2283 [M þ H]þ, found 509.2229; IR (KBr) nmax ¼ 2934, 2836,
1607, 1507, 1494, 1463, 1206, 1148 cmꢃ1
.
4.2.19. (ꢁ)Quadrangularin A (E-3a)
Compound E-3a was prepared from E-12a in a manner identical
to that described for 4a as a light brown solid. Yield 39%; mp: 178e
180 ꢂC; 1H NMR (400 MHz, MeOD)
d
ppm: 4.02 (1H, brs), 4.16 (1H,
brs), 6.09 (1H, m), 6.17 (1H, d, J ¼ 2.0 Hz), 6.22 (2H, d, J ¼ 2.0 Hz),

196
C. Zhong et al. / European Journal of Medicinal Chemistry 62 (2013) 187e198
6.59e6.63 (4H, m), 6.70 (1H, d, J ¼ 2.0 Hz), 6.88 (2H, d, J ¼ 8.4 Hz),
6.98 (1H, s), 7.12 (2H, d, J ¼ 8.8 Hz); 13C NMR (100 MHz, CDCl3)
d ppm: 55.17, 61.43, 102.96, 103.81, 116.44, 124.27, 129.68, 138.91,
151.30, 156.02, 156.77, 159.78; ESI-MS m/z: 455.2 [M þ H]þ;
C28H22O6: HRMS calcd. 455.1489 [M þ H]þ, found 455.1496. IR (KBr)
nmax ¼ 3380, 1610, 1511, 1465, 1339, 1242, 1172, 1129, 1039,
d
ppm: 58.11, 61.17, 98.39, 101.52, 103.74, 106.56, 116.01, 123.11,
125.40, 128.92, 130.24, 131.21, 138.51, 143.33, 147.71, 149.75, 156.16,
156.47, 157.44, 159.69; ESI-MS m/z: 455.2 [M þ H]þ; C28H22O6:
HRMS calcd. 455.1489 [M þ H]þ, found 455.1502; IR (KBr)
836 cmꢃ1
.
nmax ¼ 3333, 2920, 1603, 1510, 1243, 1149 cmꢃ1
.
4.2.25. Dehydroxyl pallidol derivative (2b)
Compound 2b was prepared from 13b in a manner identical to
4.2.20. 1-(4-Hydroxybenzyl)-2-(3,5-dihydroxyphenyl)-3-(4-
hydroxyphenyl)-3H-indene-4,6-diol (30a)
that described for 4a as a light brown solid. Yield 44%; 1H NMR
(400 MHz, MeOD)
(2H, s), 6.54 (2H, s), 6.65 (2H, d, J ¼ 8.2 Hz), 6.92 (2H, d, J ¼ 8.6 Hz),
7.08e7.10 (5H, m); 13C NMR (100 MHz, MeOD)
ppm: 54.70, 55.53,
d
ppm: 3.74 (2H, s), 4.51 (2H, d, J ¼ 12.3 Hz), 6.11
Compound 30a was prepared from Z-12a in a manner identical to
that described for 4a as a light brown solid. Yield 43%; mp: 232e
d
235 ꢂC; 1H NMR (400 MHz, MeOD)
d
ppm: 3.87 (2H, m), 4.83 (1H,
60.81, 61.03, 102.47, 102.54, 103.31, 115.96, 123.34, 123.78, 126.78,
128.32, 129.18, 129.25, 138.38, 147.42, 150.65, 150.95, 155.58, 155.63,
156.31, 159.37, 159.46; ESI-MS m/z: 439.1 [M þ H]þ; C28H22O5:
HRMS calcd. 439.1540 [M þ H]þ, found 439.1540.
s), 6.06 (1H, d, J ¼ 2.0 Hz), 6.09 (1H, d, J ¼ 1.6 Hz), 6.17 (1H, d,
J ¼ 2.0 Hz), 6.21 (2H, m), 6.54 (2H, d, J ¼ 8.4 Hz), 6.70 (2H, d,
J ¼ 8.8 Hz), 6.84 (2H, d, J ¼ 8.8 Hz), 7.09 (2H, d, J ¼ 8.0 Hz); 13C NMR
(100 MHz, CDCl3) d ppm: 32.22, 56.18, 101.07, 101.27, 102.41, 108.83,
115.73, 116.26, 125.87, 130.21, 130.32, 131.82, 132.00, 137.46, 139.66,
149.36, 150.98, 153.97, 156.33, 156.56, 158.85, 158.98; ESI-MS m/z:
455.2 [M þ H]þ C28H22O6: HRMS calcd. 455.1489 [M þ H]þ, found
455.1499; IR (KBr) nmax ¼ 3424, 2919, 2531, 1615, 1511, 1443,
4.2.26. Permethylated pallidol (14a)
To a solution of AlCl3 (0.48 g, 3.6 mmol) in anhydrous CH2Cl2
(5 mL) was added compound 13a (0.20 g, 0.36 mmol) in anhydrous
CH2Cl2 (10 mL) at 0 ꢂC under N2. After stirred at room temperature
for 2 h, the resulting mixture was quenched with water. The organic
layer was separated and the aqueous layer was extracted with
CH2Cl2 (3 ꢀ 20 mL). The combined organic layers were washed with
brine and dried over MgSO4. After filtered, the solution was con-
centrated in vacuo and further purified by silica gel column chro-
matography (ethyl acetate/hexanes ¼ 1/5) to give 14a as a light red
solid (0.13 g, 68%). mp: 173e175 ꢂC; 1H NMR (400 MHz, CDCl3)
1384 cmꢃ1
.
4.2.21. 1-(4-Hydroxybenzyl)-2-(3,5-dihydroxyphenyl)-3-phenyl-
3H-indene-4,6-diol (30b)
Compound 30b was prepared from E-12b or Z-12b in a manner
identical to that described for 4a as a light brown solid. Yield 42%;
1H NMR (400 MHz, MeOD)
d ppm: 3.90 (2H, m), 4.85 (1H, s), 6.07e
6.24 (5H, m), 6.72 (2H, d, J ¼ 8.2 Hz), 7.02e7.12 (7H, m); 13C NMR
d ppm: 3.62 (6H, s), 3.78 (6H, s), 3.87 (6H, s), 4.00 (2H, s), 4.62 (2H,
(100 MHz, CDCl3)
d
ppm: 32.24, 56.83,101.04,101.24,102.40, 108.71,
s), 6.27 (2H, d, J ¼ 2 Hz), 6.70 (2H, d, J ¼ 2 Hz), 6.80 (4H, d,
116.26, 125.68, 126.90, 128.83, 129.33, 130.31, 131.91, 137.89, 139.42,
141.23, 149.46, 150.66, 154.06, 156.57, 158.96, 159.01; ESI-MS m/z:
439.1 [M þ H]þ; C28H22O5: HRMS calcd. 439.1540 [M þ H]þ, found
439.1541.
J ¼ 8.4 Hz), 7.07 (4H, d, J ¼ 8.7 Hz); 13C NMR (100 MHz, CDCl3)
d
ppm: 53.39, 55.13, 55.15, 55.47, 59.64, 97.65,100.16,113.65,124.82,
128.07, 137.88, 148.56, 156.95, 157.76, 161.19; ESI-MS m/z: 539.2
[M þ H]þ.
4.2.22. (2-(3,5-Dimethoxyphenyl)-4,6-dimethoxy-3-(4-
4.2.27. 2-(3,5-Dimethoxyphenyl)-4,6-dimethoxy-3-(4-
methoxyphenyl)-2,3-dihydro-1H-inden-1-yl)(4-methoxyphenyl)-
methanol (13a)
methoxyphenyl)-1-methylene-2,3-dihyd-o-1H-indene (18)
To a solution of 9a (0.20 g, 0.46 mmol) in anhydrous toluene
(10 mL) was added Tebbe reagent (0.51 mL, 5.40 mmol) at room
temperature under N2 The resulting mixture was stirred in dark at
room temperature at 52 ꢂC for 48 h. Upon completion, the reaction
mixture was quenched with hexane and extracted with ethyl ace-
tate (3 ꢀ 5 mL). The combined organic layers were then washed
with brine and dried over MgSO4. After filtered, the solution was
concentrated in vacuo and further purified by silica gel column
chromatography (ethyl acetate/hexanes ¼ 1/8) to give 18 as a white
To a solution of E-12a (054 g, 1.00 mmol) in anhydrous THF
(15 mL) was added BH3$THF (10 mmol) at room temperature under
N2 and the resulting mixture was stirred for 18 h. After subse-
quently added aqueous NaOH (0.24 mmol/mL, 10 mL, stirred for
5 min), 30% H2O2 (0.6 mL, stirred for 15 min), and saturated
aqueous Na2SO3 (3 mL, stirred for 5 min), the reaction went to
completion. The organic layer was separated and the aqueous layer
was extracted with ethyl acetate (3 ꢀ 30 mL). The combined organic
layers were then washed with brine and dried over MgSO4. After
filtered, the solution was concentrated in vacuo and further purified
by silica gel column chromatography (ethyl acetate/hexanes ¼ 1/3)
to give 13a as white foam-like solid which will be used directly for
the next step. Yield 81%; mp: 85e86 ꢂC; ESI-MS m/z: 539.2
[M ꢃ OH]þ.
solid. Yield 43%; 1H NMR (300 MHz, CDCl3)
d ppm: 3.54 (6H, s), 3.65
(3H, s), 3.67 (3H, s), 3.90 (3H, s), 4.42e4.44 (1H, m), 4.56e4.59 (1H,
d, J ¼ 8.1 Hz), 4.81 (1H, d, J ¼ 2.1 Hz), 5.59 (1H, d, J ¼ 2.1 Hz), 5.94
(1H, d, J ¼ 2.1 Hz), 6.17e6.6 (8H, m); 13C NMR (100 MHz, CDCl3)
d
ppm: 51.16, 55.06, 55.08, 55.43, 55.56, 58.14, 95.53, 98.89, 99.79,
106.07, 108.32, 112.85, 126.89, 129.26, 133.80, 141.74, 143.42, 151.87,
157.17, 157.79, 159.70,161.15; C27H28O5: HRMS calcd. 432.1937 [M]þ,
found 432.1911. IR (KBr) nmax ¼ 3080, 3000, 2980, 2909, 2803, 1605,
4.2.23. (2-(3,5-Dimethoxyphenyl)-4,6-dimethoxy-3-phenyl-2,3-
dihydro-1H-inden-1-yl)(4-methoxyphenyl)-methanol (13b)
Compound 13b was prepared in a manner identical to that
described for 13a as a white foam-like solid. Yield 62%; ESI-MS m/z:
509.2 [M ꢃ OH]þ.
1593, 1512, 1466, 1206, 1155 cmꢃ1
.
4.2.28. 2-(3,5-Dimethoxyphenyl)-5,7-dimethoxy-1-(4-
methoxyphenyl)-3-methyl-1H-indene (19)
Adopted the same procedure described as for 18, 19 was pre-
4.2.24. (ꢁ)Pallidol (2a)
pared as a white solid. Yield 77%; 1H NMR (300 MHz, CDCl3)
d ppm:
Compound 2a was prepared from 13a in a manner identical to
that described for 4a as a light brown solid. Yield 55%; mp: 213e
2.26 (3H, m), 3.63 (3H, s), 3.70e3.71 (9H, m), 3.87 (3H, s) 4.83 (1H,
m), 6.29e6.59 (5H, m), 6.65 (2H, d, J ¼ 8.8 Hz), 6.93 (2H, d,
214 ꢂC; 1H NMR (400 MHz, MeOD)
d
ppm: 3.73 (2H, s), 4.47 (2H,
J ¼ 8.8 Hz); 13C NMR (100 MHz, CDCl3)
d ppm: 29.70, 54.91, 55.00,
55.18, 55.57, 55.59, 96.60, 96.92, 98.68, 107.31, 113.32, 127.23,
129.06, 131.27, 134.79, 138.27, 147.12, 148.34, 155.63, 157.68, 160.16,
s), 6.11 (2H, d, J ¼ 2.2 Hz), 6.53 (2H, d, J ¼ 1.8 Hz), 6.66 (4H, d,
J ¼ 8.8 Hz), 6.93 (4H, d, J ¼ 8.4 Hz); 13C NMR (100 MHz, MeOD)

C. Zhong et al. / European Journal of Medicinal Chemistry 62 (2013) 187e198
197
161.08; C27H28O5: HRMS calcd. 432.1937 [M]þ, found 432.1933. IR
were pretreated with 30a, then loaded with DHE (10
mM) for 30 min,
(KBr) nmax ¼ 3002, 2963, 2937, 2838, 1606, 1592, 1509, 1328, 1206,
washed to remove excess probe, and stimulated with paraquat
(250 M) for 2 h. Intracellular ROS generation was detected by
1158, 1048, 827 cmꢃ1
.
m
a fluorescence microscope (Carl Zeiss) at excitation and emission
wavelengths of 300 nm and 610 nm, respectively. Photographic
images were taken from four random fields.
4.2.29. (R)-3-(4-Methoxybenzyl)-2-(3,5-dimethoxyphenyl)-5,7-
dimethoxy-1-(4-methoxyphenyl)-1H-indene (20)
To a solution of E-12a (0.36 g, 0.67 mmol) in CH2Cl2 (30 mL) was
added dropwise BF3$Et2O (0.9 mL, 7.4 mmol) via syringe. After the
reaction mixture was stirred at room temperature for 24 h under
N2, the reaction was quenched by the addition of H2O (10 mL). The
resulting mixture was extracted with CH2Cl2 (3 ꢀ 10 mL). The
combined organic layers were washed with brine and dried over
MgSO4. After filtered, the solution was concentrated in vacuo and
further purified by silica gel column chromatography (ethyl ace-
tate/hexanes ¼ 1/10) to give 20 as a yellow oil (0.36 g, 99%). 1H NMR
4.3.5. Western blot analysis
Western blot analysis was performed on proteins isolated from
whole cell lysates as described previously [28]. Equal amounts of
cell lysate protein were separated with SDS-PAGE and transferred
to a nitrocellulose membrane (GE Healthcare). The membrane was
probed with primary antibody overnight at 4 ꢂC. Immunocom-
plexes were detected by enhanced chemiluminescence (Pierce)
with horseradish peroxidase-conjugated anti-mouse or anti-rabbit
immunoglobulin G as the secondary antibody (ICL Lab, Newberg,
OR). Proteins were visualized by enhanced chemiluminescence
(Pierce, Inc.) using a camera-based imaging system (Alpha Inno-
tech, Santa Clara, CA, USA). The densities of the signals were
quantified with AlphaEase software.
(400 MHz, CDCl3) d: 3.61 (6H, s), 3.63 (3H, s), 3.72 (3H, s), 3.73 (3H,
s), 3.79 (3H, s) 4.02 (2H, q, J ¼ 7.8 Hz), 4.95 (1H, s), 6.28e6.41 (5H,
m), 6.69 (2H, d, J ¼ 8.2 Hz), 6.85 (2H, d, J ¼ 8.2 Hz), 7.00 (2H, d,
J ¼ 8.2 Hz), 7.22 (2H, d, J ¼ 8.2 Hz); ESI-MS m/z :539.3 [M þ H]þ.
4.3. Pharmacological method
4.3.6. Statistical analysis
4.3.1. Neuroprotection activity on SH-SY5Y
Data are reported as the mean ꢁ SEM. All data analysis was
performed with the use of SPSS 13.0 statistical software. Differ-
ences between mean values of multiple groups were analysed by
one-way analysis of variance with Dunnett’s test for post hoc
comparisons. Statistical significance was considered at P < 0.05.
Potential neuroprotective activity was evaluated in human
neuroblastoma SH-SY5Y cells treated with paraquat. The com-
pounds were pre-dissolved in DMSO and diluted in medium to at
least five required concentrations. The content of DMSO in the final
concentrations did not exceed 0.1%. At this concentration, DMSO
was found to be nontoxic to the cells tested. All cells were cultured
at 37 ꢂC in DMEM/F12 medium supplemented with 10% FBS in
a humidified atmosphere of 5% CO2. The cells were seeded at
a density of 0.4e1.0 ꢀ 104 cells per well in 96-well microplates.
After 24 h, cells were preincubated with the indicated compounds
Acknowledgements
Financial support by the National Natural Science Foundation of
China (No: 30973612), Shanghai Municipal Committee of Science
and Technology (No: 10431903100) and National Basic Research
Program of China (973 Program, No: 2010CB912603) are
acknowledged.
(10
mM) for 2 h (viability of compound). Cells were then stimulated
with paraquat (250
m
M) for 24 h (viability of paraquat). Cell viability
was measured using a CCK assay. The optical density (OD) was read
at 450 nm.
Appendix A. Supplementary data
4.3.2. Cell culture and treatment
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.12.037.
Human neuroblastoma cells (SH-SY5Y) were grown in DMEM/F-
12 medium (Gibco) containing 10% FBS (Gibco) and maintained at
37 ꢂC with 95% humidified air and 5% CO2. In some experiments, the
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