Discovery of novel sesquistilbene indanone analogues as potent anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2016.02.021

To develop novel anti-inflammatory agents with improved pharmaceutical profiles, twenty-eight novel sesquistilbene indanone analogues were synthesized and evaluated for anti-inflammatory activity using RAW264.7 cells. Among these compounds, compound 11k was found to be one of the most potent analogues in inhibiting NO production in LPS-stimulated RAW264.7 cells. Furthermore, it could also significantly suppress LPS-induced iNOS and COX-2 expression and NO production through TLR4/JNK/NF-κB signaling pathway in a concentration dependent manner.

Full text of DOI:10.1016/j.ejmech.2016.02.021

Accepted Manuscript
Discovery of novel sesquistilbene indanone analogues as potent anti-inflammatory
agents
Mei-Lin Tang, Chen Zhong, Zheng-Yu Liu, Peng Peng, Xin-Hua Liu, Xun Sun
PII:
S0223-5234(16)30091-5
10.1016/j.ejmech.2016.02.021
EJMECH 8372
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 October 2015
Revised Date: 5 February 2016
Accepted Date: 7 February 2016
Please cite this article as: M.-L. Tang, C. Zhong, Z.-Y. Liu, P. Peng, X.-H. Liu, X. Sun, Discovery of novel
sesquistilbene indanone analogues as potent anti-inflammatory agents, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.02.021.
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ACCEPTED MANUSCRIPT
Graphical abstract
Sesquistilbene indanone analogue 11k was much more potent than resveratrol for
its anti-inflammatory activity. It could also significantly suppress LPS-induced iNOS
and COX-2 expression and NO production through TLR4/JNK/NF-κB signaling
pathway in a concentration dependent manner.

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Discovery of novel sesquistilbene indanone analogues as potent
anti-inflammatory agents
Mei-Lin Tanga, Chen Zhonga, Zheng-Yu Liua, Peng Penga, Xin-Hua Liub,*, Xun Suna,c,*
aDepartment of Natural Products Chemistry, School of Pharmacy, Fudan University, 826
Zhangheng Road, Shanghai 201203, China
bDepartment of Pharmacology, School of Pharmacy, Fudan University, 826 Zhangheng Road,
Shanghai 201203, China
cShanghai Key Laboratory of Clinical Geriatric Medicine, 221 West Yanan Road, Shanghai
200040, China
*To whom correspondence should be addressed. Tel.: +86-21-51980003; Fax:
+86-21-51980003.
E-mail
address:
sunxunf@shmu.edu.cn
(X.
Sun),
liuxinhua@fudan.edu.cn (X. Liu).

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Abstract
To develop novel anti-inflammatory agents with improved pharmaceutical profiles,
twenty-eight novel sesquistilbene indanone analogues were synthesized and evaluated
for anti-inflammatory activity using RAW264.7 cells. Among these compounds,
compound 11k was found to be one of the most potent analogues in inhibiting NO
production in LPS-stimulated RAW264.7 cells. Furthermore, it could also
significantly suppress LPS-induced iNOS and COX-2 expression and NO production
through TLR4/JNK/NF-κB signaling pathway in a concentration dependent manner.
Keywords: indanone, anti-inflammatory, TLR4/JNK/NF-κB

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1. Introduction
Resveratrol (3,5,4'-trihydroxy-trans-stilbene, 1, Fig. 1), a non-flavonoid, is a major
health-promoting compound in red wine and grape seeds [1]. It has been advocated as
the agent responsible for the “French Paradox” to protect the cardiovascular system
via a variety of mechanisms [2-6]. It demonstrated pharmacological outcomes, such
as coronary disease, diabetes, antitumor as well as neurodegeneration [7-11]. In
particular, resveratrol has been noted for its anti-inflammatory activity among these
beneficial actions [12]. Resveratrol can attenuate the activation of immune cells and
the subsequent synthesis and release of pro-inflammatory mediators through the
inhibition of transcriptional factors such as nuclear factor-κB (NF-κB) and activator
protein-1 (AP-1) [13]. Currently, resveratrol is under clinical trials as an
anti-inflammatory agent, though there is a challenge in translating pre-clinical
findings to patient outcomes. The data indicated that half of the studies found a
modest anti-inflammatory effect whereas the other half were unable to detect any
anti-inflammatory effect [12].
Inflammation is a fundamental protective response of the immune system to
pathogens or harmful irritants and the classical signs of inflammation are redness,
swelling, heat and pain [14, 15]. Paradoxically, the inflammatory process itself may
cause tissue damage and lead to a host of diseases and even cancer [15-17].
Nonsteroidal anti-inflammatory drugs (NSAIDs) are therapeutically important in the
treatment of rheumatic arthritis and various types of inflammatory conditions, but
found to be limited in clinical applications due to their gastrointestinal side effects
[18]. Therefore, the development of novel anti-inflammatory agents, possessing
improved pharmaceutical profiles and reduced adverse effects, is still urgently
needed.
Based on our previous studies as well as others [19, 20], it was revealed that
resveratrol oligomer families showed more potent anti-inflammatory activity than
resveratrol itself. We found out that sesquistilbene compound 2, which containing
1,2-diphenyl-2,3-dihydro-1H-indene core, exhibited much potent anti-inflammatory
activities than any other dimerized derivatives and resveratrol [19]. The mechanism of

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action (MOA) for 2 involved in its anti-inflammatory responses was through signaling
pathways of p38 MAPK and JNK [19]. During our previous course of work, it was
found that compounds bearing either indanol-type core or indanone-type core (e.g. 3)
had comparable anti-inflammatory activity [19]. However, compounds with
indanol-type core were chemically more unstable than indanone-type core when
subjected to light and heat. Considering this, efforts have been made to achieve
compounds with more potent and better pharmaceutical profile by focusing on
indanone-type structural modifications in our continuously structure-activity
relationship (SAR) study. Further, compared with indanol-type compounds, only two
enantiomers were produced owing to the fact that substituents at 2´- and 3´-position
were always posted as trans configurations in the synthesized analogues. Also, these
enantiomers could be easily separated by chiral HPLC. Thus, twenty-eight
indanone-type compounds were synthesized and evaluated for their biological activity
with lipopolysaccharide (LPS)-induced RAW264.7 cells as anti-inflammatory agents.
In addition, the most potent compound 11k, along with its optical pure compounds,
(+)-11k and (-)-11k, were tested for their nitric oxide (NO) production inhibitory
activity in RAW264.7 cells and acute toxicity studies in mice. The suppressive effects
on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) of 11k
were also examined in vitro and western blot experiments were performed in an
attempt to illustrate the preliminary mechanism of action.
2. Chemistry
Syntheses of analogues 7a-m have been achieved through our previous reported
methods with some modifications [21, 22]. As shown in Scheme 1, the synthetic
strategy involved a three step sequence via benzil formation, Wittig reaction and
Nazarov cyclization starting from commercially available 3,5-dimethoxybenzoic acid.
The acid 4 was rapidly dimerized to give diaryl ketone 5 in 44 % yield by the
treatment of Li-naphthalene reagent. By incorporating different substituents on R1 as
Wittig reagents, intermediates 6a-m were obtained as cis/trans mixtures in good
yields. It is noteworthy that such obtained mixtures could be used directly and did not
have influence on the next Nazarov cyclization step. Interestingly, when the mixtures

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of cis/trans 6a-m were subjected to the Nazarov cyclization condition (BF3·OEt2,
DCM), cyclized products 7a-m were achieved in good yields with the substituents on
2´- and 3´-position in trans configurations. A same synthetic route was adopted for
the syntheses of derivatives 11a-l starting from 4-methoxybenzoic acid (Scheme 2).
However, a higher temperature condition (60℃, toluene) was required for the
formation of 10a-l than the formation of analogues 6a-6m (room temperature,
toluene). Possibly, the methoxy group as an electron-donating group at para position
in compound 9 could decrease the reactivity of carbonyl group for the Wittig reaction.
With 10a-l in hand, our previous Nazarov cyclization condition (BF3·OEt2, DCM)
was executed for the syntheses of 11a-l. To our surprise, this condition was not
working here. Though many Lewis acid conditions were explored for this reaction
[e.g. AlCl3, FeCl3, TiCl4, SnCl4, ZnCl2, Cu(OTf)2, In(OTf)3, InCl3, and Bi(OTf)3],
there was no desired product formed (either no reaction or deposition). Gratifyingly,
analogues 11a-l could be obtained in the presence of trifluoroacetic acid (TFA) at
75°C in moderate yields (45-64%). The lower reactivity of compounds 6a-m as
regards compounds 10a-l may be due to the presence of the two methoxy groups in
compounds 6a-m. These groups help delocalize the positive charge on the
intermediate generated in the intramolecular Friedel-Crafts type cyclization, thus
speeding up the reaction. Thus, a total of twenty-five novel compounds (7a-m and
11a-l) were synthesized. These compounds along with our previous synthesized 7n-p
were evaluated for their anti-inflammatory activity.
3. Results and discussion
3.1. Inhibition of NO production in LPS-stimulated RAW264.7 cells
NO is a significant pro-inflammatory mediator. Excessive production of NO was
found to be associated with the pathogenesis of inflammation diseases. It is generally
accepted that NO inhibitors may offer potential opportunity to identify new
therapeutic method for the inflammatory diseases [21, 22]. To examine whether
resveratrol analogues inhibited LPS-mediated inflammatory response, NO production
was measured in LPS-stimulated RAW264.7 cells. Herein, resveratrol and the lead
compound 2 were chosen as positive controls. As depicted in Figure 1 and Table 1, all

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analogues displayed improved NO inhibitory activity compared to resveratrol. Among
them, analogues 7f, 7h, 7i, 7l, 11c-e and 11h-l exhibited similar or better NO
inhibitory activity than compound 2. In general, 4´-methoxy substituted analogues
showed better NO inhibitory activity than their corresponding 3´, 5´-dimethoxy
substituted analogues (11a, 11c-e, and 11h-l vs. 7b, 7c-e, and 7n-p, respectively).
Particularly, compound 11k exhibited the most potent inhibitory activity and it´s NO
inhibition rate was 87.7% at the concentration of 50 ꢀM. Interestingly, a benzyl
substituted analogue 7m exhibited a similar potent inhibitory activity to 11k. Also,
there was no significant biological activity effect observed whether electron-donating
or electron-withdrawing substituent was presented on A-phenyl ring. These results
could be very useful for our future SAR study.
3.2. Activity studies in RAW264.7 cells and acute toxicity studies in mice of 11k,
(+)-11k and (-)-11k
The structure of racemic 11k was confirmed by crystal X-ray structure
determination (Fig. 3). This racemic 11k was further separated by using chiral HPLC
to obtain optical pure (+)-11k and (-)-11k (Fig. 4). Initially, NO production inhibitory
activity of (+)-11k, (-)-11k and racemic 11k were measured in LPS-stimulated
RAW264.7 cells. As illustrated in Table 2, racemic 11k surprisingly exhibited similar
inhibitory activity as (+)-11k and (-)-11k in RAW264.7 cells, with IC50 value of 23.2,
23.7, and 23.5ꢀM, respectively. Furthermore, racemic-11k exhibited consistent acute
toxicity in mice with LD50 value of 275 mg/kg, and the result was the same as that of
(+)-11k and (-)-11k. For all three compounds, the acute toxicity study showed that the
mortality was dose-dependent for both male and female mice after oral administration
and the death was occurred after 7 days.
3.3. Compound 11k inhibited LPS-induced inflammatory mediators in RAW264.7
cells
The pro-inflammatory mediators (NO, PGE2, and TNF-α) play important roles in
inflammation-related diseases, which are also importantly related to the modulation of
iNOS and COX-2 expression [23]. Thus, the inhibitory effects of 11k on
LPS-mediated expression of iNOS and COX-2 were analyzed by Western blot. The

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cell viability experiment was performed at 12.5-50 ꢀM concentration and there was
no significantly cytotoxic of 11k at the concentration up to 50 ꢀM (Fig. 5A). As
shown in Figure 5B, the LPS (1 ꢀg/mL) stimulation for 24 h could markedly
augmented NO production. However, 11k concentration-dependently suppressed
LPS-induced NO generation. In accordance with the inhibitory effect on NO
production, 11k was also concentration-dependently reduced LPS-induced iNOS (130
kDa) expression (Fig. 5C). Meanwhile, 11k concentration-dependently suppressed
LPS-induced COX-2 (72 kDa) (Fig. 5D) expression. Taken together, our results
demonstrated that 11k prevented LPS-induced inflammatory response in
macrophages.
3.4. Compound 11k suppresses LPS-induced NF-κB activation in RAW264.7
cells
LPS signal transduction has been shown by other researchers to activate a variety
of transcription factors, including NF-κB, which controls the transcription of a
number of inflammatory cytokines. During NF-κB activation, IκB degradation frees
NF-κB p65 subunit from sequestration, allowing it to translocate to the nucleus, bind
to target promoters, and turn on transcriptions of inflammatory genes including iNOS
and COX-2 [18]. Herein, the effects of compound 11k on NF-κB p65 translocation
from cytoplasm to nuclei were analyzed by Western blot. As shown in Figure 6, LPS
increased NF-κB p65 nuclear translocation, whereas pretreatment with compound 11k
(12.5, 25, 50 ꢀM) concentration-dependently reversed LPS-induced NF-κB p65
nuclear translocation, suggesting that compound 11k inhibited NF-κB activation.
These results further confirmed the anti-inflammatory effect of 11k, at least in part,
through modulation of NF-κB activation.
3.5. Compound 11k inhibits LPS-induced JNK signaling activation in RAW264.7
cells
It has been reported that mitogen-activated protein kinases (MAPK, including
ERK, p38, and JNK) were quite significant in the regulation of inflammation because
of their crucial roles in the mediation of the production of NO, TNF-α, IL-6, and other
inflammatory mediators in activated macrophages [24-27]. Reports have shown that

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inhibiting the activation of MAPK brings down the expression of inflammatory
mediators and thus improve the outcome of experimental inflammatory diseases [28,
29]. In addition, TLR4 is traditionally accepted as the primary LPS receptor and has
been reported as critical for the inflammatory response to LPS [30]. Upon stimulation
with LPS, TLR4 initiates series of signaling cascades that result in the activation of
NF-κB and MAPK to induce the release of pro-inflammatory cytokines [28]. Recent
studies found that inhibition of TLR4 could decrease the expressions of NF-κB, IL-1β,
and TNF-α, which was consistent with the improvement of behaviors in inflammatory
diseases [31-33]. These findings imply the TLR4 signal pathway plays an important
role in inflammatory diseases and it may be a novel target in the treatment of
inflammatory disease. Thus, it was hypothesized that 11k might interfere with the
phosphorylation of ERK, p38, and JNK. Initially, we investigated whether 11k
inhibited the expression of TLR4 by Western blot. It was confirmed that TLR4
expression was strongly up-regulated in LPS-induced RAW264.7 cells, which was
concentration-dependently reversed by pretreated with 11k (Fig. 7A). LPS could
cause a significant phosphorylation of MAPK (p38, JNK, and ERK) after stimulated
for 45 min (Fig. 7B, 7C, and 7D). Compound 11k concentration-dependently
diminished LPS-induced phosphorylation of JNK (Fig. 7B), but had little effect on
phosphorylation of ERK or p38 (Fig. 7C, 7D). These results strongly suggested that
inhibition of TLR4 and JNK phosphorylation by 11k might be responsible for
achieving LPS-induced inflammatory response. It has been well established that JNK
is as a key transcriptional regulator of inflammatory cytokines and an upstream
co-regulator of NF-κB [34, 35]. Furthermore, 11k also concentration-dependently
(12.5, 25, and 50 ꢀM) inhibited LPS-induced JNK phosphorylation in RAW264.7
cells (Fig. 7B). These results suggested that the anti-inflammatory activity of 11k
might be associated with its negative effects on JNK/NF-κB activation.
4. Conclusions
In conclusion, a series of novel resveratrol-based sesquistilbene indanone
derivatives were designed, synthesized and their anti-inflammatory activities were
examined with LPS-induced RAW264.7 cells. Among these compounds, compound

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11k was found to be one of the most potent analogues to inhibit NO production in
LPS-stimulated RAW264.7 cells. Western blot experiments for analogue 11k
preliminarily suggested that the mechanism of action was through TLR4/JNK/NF-κB
signaling pathway (Fig. 8). In addition, studies revealed that compound 11k and its
two optical pure compounds [(+)-11k and (-)-11k] have no impacts on both activity in
RAW264.7 cells and acute toxicity in mice. Beside these, 11k also has a reasonable
water solubility (38.3 ꢀg/mL, 68.7 ꢀg/mL and 46.5ꢀg/mL at pH = 1.2, 6.8 and 7.4
respectively) and suitable log P (log P = 1.77). Thus 11k could be a promising lead
compound. Further lead optimization based on structure 11k is ongoing in our
laboratory and the results will be reported in due course.
5. Experimental section
5.1. Materials and physical measurements
Reagents were purchased from Aldrich and TCI Chemical companies. All
solvents were purified and dried in accordance with standard procedures unless
otherwise indicated. Reactions were monitored by TLC using Yantai (China) GF254
silica gel plates (5 × 10 cm). Silica gel column chromatography was performed on
silica gel (300-400 mesh) from Yantai (China). Melting points (mp) were determined
using an X-4 microscope melting point apparatus and were uncorrected. All 1H-NMR
13
spectra were recorded on Bruker DRX-400 (400 MHz) spectrometers and C-NMR
spectral data were recorded on a BrukerDRX-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 TeslaFTMS (MALDI)
or a Bruker Daltonics, Inc. APEXIII7.0 TESLA FMS (ESI).
5.2. Chemistry
5.2.1. 1. 2-Bis(3,5-dimethoxyphenyl)ethane-1,2-dione (5)
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 a solution of 3,5-dimethoxybenzoic acid (4, 15.0 g, 82.3 mmol) in

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anhydrous THF (300 mL) at 0ꢀ 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 5 as a yellow solid (6.0 g, 44%). mp:
171-172ꢀ; 1H-NMR(400 MHz, CDCl3) δ ppm: 3.83 (12H, s), 6.74 (2H, s), 7.07 (4H,
d, J = 1.7 Hz); 13C-NMR (100 MHz, CDCl3) δ ppm: 55.67 (4C), 107.38 (4C), 107.53
(2C),134.65 (4C), 161.10 (2C), 194.16 (2C); C18H18O6: HRMS: m/z calcd.
331.1181[M + H]+; found: 331.1178.
5.2.2. 2. 2-Bis(4-methoxyphenyl)ethane-1,2-dione (9)
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 8h. The resulting mixture was added
dropwise to a solution of 4-dimethoxybenzoic acid (13, 12.5 g, 82.3 mmol) in
anhydrous THF (300 mL) at 0ꢀ 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 from ethyl acetate to give pure 9 as a yellow solid (6.80 g, 50%). mp:
134-135ꢀ; 1H-NMR (400 MHz, CDCl3) δ ppm: 7.93 (4H, d, J = 8.40 Hz), 6.95 (4H,
d, J = 8.40 Hz), 3.89 (6H, s); 13C-NMR (100 MHz, CDCl3) δ ppm: 55.01 (2C), 113.66
(2C), 125.69 (2C), 131.75 (4C), 164.23 (2C), 192.87 (2C); C16H14O4: HRMS: m/z
calcd. 280.0892 [M + H]+; found: 271.0965.
5.2.3. General procedure for the synthesis of methoxylindanones (7a-7p)
To a solution of 5 (330 mg, 1mmol) and Wittig regent (l.5 mmol) in anhydrous
toluene (5 mL) was added NaHMDS (1.5 mmol, 2.0 M/L) under N2 at room
temperature. The reaction mixture was stirred for 1 h, then cooled to 0ꢀ and
quenched with water. The reaction mixture was concentrated and then diluted with

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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, filtered, and evaporated. The obtained residue was further
purified by silica gel chromatography (0-4% ethyl acetate/hexanes) to give 6a-6p as a
mixture of cis and trans yellow oil, which only measured by MS. Further the target
compounds 7a-7p were fully characterized by 1H-NMR, 13C-NMR, and HRMS.
To a solution of chalcones 6a-6p (1.0 mmol) in CH2Cl2 (10 mL) was dropwisely
added BF3·OEt2 (0.71 mL, 5 mmol) via syringe. After the reaction mixture was stirred
at room temperature for 48 h under N2, the reaction was quenched with H2O (10 mL).
The resulting solution was extracted with CH2Cl2 (3 × 10 mL) and the combined
organic phases were washed with brine and dried over Na2SO4. After evaporation the
solvent, the obtained residue was further purified by silica gel chromatography (0-5%
ethyl acetate/hexanes) to give 7a-7p as white solid.
5.2.3.1.2-(3,5-Dimethoxyphenyl)-4,6-dimethoxy-3-(naphthalen-2-yl)-2,3-dihydro-1H-
1
inden-1-one (7a). White solid in 92% yield. mp: 136-137ꢀ; H-NMR (400 MHz,
CDCl3) δ ppm: 7.80 (1H, m), 7.77-7.62 (2H, m), 7.48 (1H, s), 7.45-7.43 (2H , m),
7.14 (1H, dd), 6.95 (1H, d, J = 2.0 Hz), 6.71 (1H, d, J = 2.0 Hz,), 6.38 (1H, t), 6.26
(2H, d, J = 2.0 Hz), 4.72 (1H, d, J = 3.0 Hz), 3.90 (3H, s), 3.73 (6H, s), 3.70 (1H, d, J
= 3.0 Hz), 3.60 (3H, s). 13C-NMR (100 MHz, CDCl3) δ ppm: 51.57, 55.27 (2C), 55.53,
55.80, 65.12, 96.46, 98.98, 106.02 (2C), 106.53, 125.36, 125.43, 125.47, 125.97,
127.57, 127.57, 128.27, 127.66, 128.27, 132.31, 133.42, 138.02, 138.69, 140.65,
141.37, 157.78, 160.97 (2C), 162.03, 205.28. ESI-MS m/z: 455.2 [M + H]+; C29H26O5:
HRMS calcd. 454.1540 [M + H]+, found 455.1356.
5.2.3.2. 3-[(1,1'-Diphenyl)-4-yl]-2-(3,5-dimethoxyphenyl)-4,6-dimethoxy-2,3-dihydro-
1H-inden-1-one (7b). White solid in 74% yield. mp: 182-183ꢀ; 1H-NMR (400 MHz,
CDCl3) δ ppm: 7.58-7.57 (2H, m), 7.50-7.46 (2H, m), 7.44-7.4 (2H , m), 7.34-7.32
(1H, m), 7.10-7.08 (2H, m), 6.92 (1H, d, J = 2.0 Hz), 6.71 (1H, d, J = 2.0 Hz,), 6.37
(1H, t), 6.27 (2H, d, J = 2.0 Hz), 4.60 (1H, d, J = 2.74 Hz), 3.89 (3H, s), 3.75 (6H, s),
13
3.68 (4H, s). C-NMR (100 MHz, CDCl3) δ ppm: 51.58, 55.27 (2C), 55.58, 55.79,
65.08, 96.48, 98.89, 106.02 (2C), 106.53, 126.89 (2C), 127.12 (2C), 128.68 (2C),

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138.03, 138.57, 139.25, 140.72, 141.42, 142.32, 150.70, 160.96 (2C), 161.98, 205.24.
ESI-MS m/z: 481.2 [M + H]+; C31H28O5: HRMS calcd. 481.1931 [M + H]+, found
481.1656.
5.2.3.3.2-(3,5-Dimethoxyphenyl)-3-(3-fluorophenyl)-4,6-dimethoxy-2,3-dihydro-1H-
1
inden-1-one (7c). White solid in 65% yield. mp: 137-138ꢀ; H-NMR (400 MHz,
CDCl3) δ ppm: 7.24-7.19 (1H, m), 6.92-6.88 (2H, m), 6.82-6.80 (1H, dd, J = 2.30,
2.75 Hz), 6.73-6.70 (2H, m), 6.37 (1H, t), 6.23 (2H, d, J = 2.35 Hz), 4.53 (1H, d, J =
2.72 Hz), 3.88 (3H, s), 3.74 (6H, s), 3.66 (3H, s), 3.60 (1H, d, J = 2.72 Hz). 13C-NMR
(100 MHz, CDCl3) δ ppm: 51.19, 55.27 (2C), 55.53, 55.81, 64.88, 96.52, 98.93,
106.01 (2C), 106.53, 113.30, 113.50, 113.73, 113.94, 122.64, 129.84, 137.25, 138.57,
141.06, 145.79, 160.99 (2C), 161.65, 162.14, 204.77. ESI-MS m/z: 423.0 [M + H]+;
C25H23FO5: HRMS calcd. 481.1421 [M + H]+, found 481.1331.
5.2.3.4. 2-(3,5-Dimethoxyphenyl)-4,6-dimethoxy-3-(4-(trifluoromethoxy)phenyl)-2,3-
1
dihydro-1H-inden-1-one (7d).White solid in 74% yield. mp: 136-137ꢀ; H-NMR
(400 MHz, CDCl3) δ ppm: 7.11-7.03 (4H, m), 6.91 (1H, d, J = 2.0 Hz), 670 (1H, d, J
= 2.0 Hz), 6.23 (2H, d, J = 3.0 Hz), 4.55 (1H, d, J = 2.0 Hz), 3.88 (3H, s), 3.74 (6H, s),
13
3.65 (3H, s), 3.59 (1H, d, J = 2.0 Hz). C-NMR (100 MHz, CDCl3) δ ppm: 50.86,
55.27 (2C), 55.51, 55.82, 64.97, 96.55, 98.93, 106.63 (2C), 106.55, 120.96 (2C),
128.26 (2C), 137.37, 138.54, 141.04, 141.92, 147.77, 157.58, 161.03 (2C), 162.17,
204.75. ESI-MS m/z: 489.2 [M + H]+; C26H23F3O6: HRMS calcd. 488.1721 [M + H]+,
found 489.1631.
5.2.3.5. 2-(3,5-Dimethoxyphenyl)-4,6-dimethoxy-3-(4-(trifluoromethyl)phenyl)-2,3-
1
dihydro-1H-inden-1-one (7e). White solid in 90% yield. mp: 136-137ꢀ; H-NMR
(400 MHz, CDCl3) δ ppm: 7.51 (2H, d, J = 6.20 Hz), 7.13 (2H, d, J = 6.20 Hz), 6.92
(1H, d, J = 2.0 Hz), 6.81 (1H, d, J = 2.0 Hz), 6.37 (1H, t, J = 2.34 Hz), 6.22 (2H, d, J
= 2.34 Hz), 4.59 (1H, d, J = 2.74 Hz), 3.89 (3H, s), 3.74 (6H, s), 3.65 (3H, s), 3.58
(1H, d, J = 2.74 Hz). 13C-NMR (100 MHz, CDCl3) δ ppm: 51.29, 55.27 (2C), 55.52,
55.83, 64.81, 96.59, 98.98, 106.04 (2C), 106.52, 125.42, 125.46 (2C), 125.51, 136.94,
138.60, 140.86, 147.22, 157.52, 161.04, 162.24, 204.52. ESI-MS m/z: 473.0 [M + H]+;
C26H23F3O5: HRMS calcd. 472.1521 [M + H]+, found 473.1734.

ACCEPTED MANUSCRIPT
5.2.3.6.3-Benzyl-2-(3,5-dimethoxyphenyl)-4,6-dimethoxy-2,3-dihydro-1H-inden-
1
1-one (7f). White solid in 54% yield. mp: 159-160ꢀ; H-NMR (400 MHz, CDCl3) δ
ppm: 7.24-7.13 (5H, m), 6.81 (1H, d, J = 2.35 Hz), 6.74 (1H, d, J = 2.35 Hz), 6.23
(1H, t, J = 2.35 Hz), 5.89 (2H, d, J = 2.35 Hz), 3.89 (3H, s), 3.85 (6H, s), 3.65 (3H, s),
3.74-3.71 (1H, m), 3.63 (3H, s), 3.57-3.51 (2H, m), 2.73-2.68 (1H, m). 13C-NMR (100
MHz, CDCl3) δ ppm: 39.76, 47.51, 55.14 (2C), 55.52, 55.74, 59.25, 96.53, 98.67,
105.67 (2C), 106.04, 126.29, 128.30 (2C), 129.35, 138.62, 139.21, 139.48, 142.44,
157.71, 160.63 (2C), 161.52, 205.73. ESI-MS m/z: 419.2 [M + H]+; C26H26O5: HRMS
calcd. 418.1923 [M + H]+, found 419.1684.
5.2.3.7.2,3-Bis(3,5-dimethoxyphenyl)-4,6-dimethoxy-2,3-dihydro-1H-inden-1-one(7g).
1
White solid in 82% yield. mp: 146-147ꢀ; H-NMR (400 MHz, CDCl3) δ ppm: 6.89
(1H, d, J = 1.95 Hz), 6.69 (1H, d, J = 1.95 Hz), 6.35(1H, t, J = 1.95Hz), 6.31 (1H, t, J
= 1.95 Hz), 6.24 (2H, d, J = 2.34 Hz), 6.15 (2H, d, J = 2.34 Hz), 4.48 (1H, d, J = 2.74
Hz), 3.87 (3H, s), 3.74 (6H, s), 3.66 (3H, s), 3.71 (6H, s), 3.69 (3H, s), 3.62 (1H, d, J
= 2.74 Hz), 13C-NMR(100 MHz, CDCl3) δ ppm: 51.63, 55.21 (2C), 55.26 (2C), 55.60,
55.78, 64.88, 96.42, 98.13, 98.87, 105.14 (2C), 105.96 (2C), 106.48, 137.77, 138.63,
141.55, 145.85, 157.72, 160.73 (2C), 160.93 (2C), 161.96, 205.17. ESI-MS m/z:
465.2 [M + H]+; C27H28O7: HRMS calcd. 464.1376 [M + H]+, found 465.1255.
5.2.3.8.2-(3,5-Dimethoxyphenyl)-4,6-dimethoxy-3-(p-tolyl)-2,3-dihydro-1H-inden-
1-one (7h). White solid in 81% yield. mp: 124-125ꢀ; 1H-NMR (400 MHz, CDCl3) δ
ppm: 7.06-7.04 (2H, m), 6.92-6.90 (3H, m), 6.68 (1H, d, J = 1.95 Hz), 6.35 (1H, t, J =
2.35 Hz), 6.24 (2H, d, J = 2.73 Hz), 4.53 (1H, d, J = 2.73 Hz), 3.87 (3H, s), 3.73 (6H,
s), 3.66 (3H, s), 3.61 (1H, d, J = 2.73 Hz), 2.31 (3H, s). 13C-NMR (100 MHz, CDCl3)
δ ppm: 21.05, 51.18, 55.29, 55.61, 55.80, 65.25, 96.42, 98.86, 106.01 (2C), 106.54,
126.85 (2C), 129.15 (2C), 135.97, 138.41, 138.57, 140.27, 141.61, 157.75, 160.96
(2C), 161.90, 205.47. ESI-MS m/z: 419.2 [M + H]+; C26H26O5: HRMS calcd.
418.1876 [M + H]+, found 419.1276.
5.2.3.9. 2-(3,5-Dimethoxyphenyl)-3-(4-ethoxyphenyl)-4,6-dimethoxy-2,3-dihydro-1H-
1
inden-1-one (7i).White solid in 93% yield. mp: 145-146ꢀ; H-NMR (400 MHz,
CDCl3) δ ppm: 6.93-6.89 (3H, m), 6.78-6.76 (2H, m), 6.68 (1H, s), 6.35 (1H, s), 6.23

ACCEPTED MANUSCRIPT
(2H, s), 4.50 (1H, s), 3.99 (2H, q, J = 7.05 Hz), 3.87 (3H, s), 3.73 (6H, s), 3.65 (3H, s),
13
3.60 (1H, s), 1.39 (3H, t, J = 7.05 Hz). C-NMR (100 MHz, CDCl3) δ ppm: 50.86,
55.29 (2C), 55.60, 55.80, 83.32, 65.34, 96.38, 98.86, 106.01 (2C), 106.57, 114.31,
127.93, 135.26, 138.50, 141.55, 157.52, 157.74, 160.95 (2C), 161.88, 205.54.
ESI-MS m/z: 449.2 [M + H]+; C27H28O6: HRMS calcd. 448.1976 [M + H]+, found
419.1975.
5.2.3.10. 3-(4-(Benzyloxy)phenyl)-2-(3,5-dimethoxyphenyl)-4,6-dimethoxy-2,3-
1
dihydro-1H-inden-1-one (7j). White solid in 59% yield. mp: 101-102ꢀ; H-NMR
(400 MHz, CDCl3) δ ppm: 7.34 (5H, m), 6.96 (1H, s), 6.72 (1H, s), 6.60 (2H, d, J =
2.30 Hz), 6.43 (2H, d, J = 2.30 Hz), 6.17 (1H, s), 5.90 (2H, d, J = 2.30 Hz), 4.92 (2H,
s), 4.26 (1H, d, J = 2.75 Hz), 3.90 (3H, s), 3.72 (3H, s), 3.55 (4H, s). 13H-NMR (400
MHz, CDCl3) δ ppm: 47.39, 55.15 (2C), 55.72, 55.87, 61.99, 69.87, 96.17, 99.41,
106.26, 108.41 (2C), 114.03 (2C), 127.39 (2C), 127.88, 128.54, 129.37, 132.79,
136.69, 137.06, 138.37, 139.06, 157.32, 157.67, 160.04, 161.75, 205.35. ESI-MS m/z:
551.2 [M + H]+; C32H30O6: HRMS calcd. 550.2176 [M + H]+, found 551.1765.
5.2.3.11. 2-(3,5-Dimethoxyphenyl)-3-(4-hydroxyphenyl)-4,6-dimethoxy-2,3-dihydro-
1H-inden-1-one (7k). White solid in 83% yield. mp: 192-193ꢀ; 1H-NMR (400 MHz,
CDCl3) δ ppm: 6.96 (1H, s), 6.72 (1H, s), 6.44 (4H, s), 6.17 (1H, s), 5.91 (2H, d, J =
2.30 Hz), 4.90 (1H, d, J = 2.75 Hz), 4.26 (1H, d, J = 2.75 Hz), 3.88 (3H, s), 3.70 (3H,
s), 3.57 (6H, s). 13C-NMR (100 MHz, CDCl3) δ ppm: 47.37, 55.16 (2C), 55.70, 55.86,
61.90, 96.17, 99.30, 104.56, 106.29, 108.46 (2C), 114.53, 129.54 (2C), 130.34,
132.67, 136.69, 138.38, 139.05, 153.91, 157.65, 160.06, 161.77, 205.39. ESI-MS m/z:
421.2 [M + H]+; C25H24O6: HRMS calcd. 420.1682 [M + H]+, found 421.1335.
5.2.3.12. 3-Cyclohexyl-2-(3,5-dimethoxyphenyl)-4,6-dimethoxy-2,3-dihydro-1H-
inden-1-one (7l).White solid in 79% yield. mp: 90-91ꢀ; 1H-NMR (400 MHz, CDCl3)
δ ppm: 6.78 (1H, d, J = 1.96 Hz), 6.68 (1H, d, J = 1.96 Hz), 6.30 (1H, t, J = 2.35 Hz),
6.25 (2H, d, J = 2.73 Hz), 3.86 (3H, s), 3.83 (3H, s), 3.73 (6H, s), 3.61 (1H, s), 3.50
(1H, s), 2.21 (1H, m), 1.75-0.83 (10H, m). 13C-NMR (100 MHz, CDCl3) δ ppm: 26.25,
26.45, 26.67, 26.75, 32.50, 39.93, 51.33, 55.22 (2C), 55.43, 55.67, 56.62, 96.42, 98.19,
105.86, 105.96 (2C), 138.59, 139.02, 143.10, 157.69, 168.80 (2C), 161.20, 205.54.

ACCEPTED MANUSCRIPT
ESI-MS m/z: 411.0 [M + H]+; C25H30O5: HRMS calcd. 410.2112 [M + H]+, found
411.2136.
5.2.3.13. Ethyl 2-(3,5-dimethoxyphenyl)-5,7-dimethoxy-3-oxo-2,3-dihydro-
1
1H-indene-1-carboxylate (7m). White solid in 79% yield. mp: 142-143ꢀ; H-NMR
(400 MHz, CDCl3) δ ppm: 6.91 (1H, d, J = 2.00 Hz), 4.56 (1H, d, J = 8.80 Hz), 4.16
(2H, m), 3.88 (1H, d, J = 8.80 Hz), 3.86 (3H, s), 3.84 (3H, s), 3.74 (6H, s), 1.24 (3H,
t). 13C-NMR (100 MHz, CDCl3) δ ppm: 47.67, 54.71, 55.08, 55.13, 55.25, 56.40,
58.32, 60.17, 96.04, 96.37, 105.43 (2C), 107.36 (2C), 132.62, 137.01, 139.43, 156.72,
159.90, 169.83, 202.60, 203.06. ESI-MS m/z: 401.0 [M + H]+; C22H24O7: HRMS
calcd. 400.1522 [M + H]+, found 401.1599.
5.2.3.14.2-(3,5-Dimethoxyphenyl)-3-(4-fluorophenyl)-4,6-dimethoxy-2,3-dihydro-
1H-inden-1-one (7n). White solid in 67% yield. mp: 148-149ꢀ; 1H-NMR (400 MHz,
CDCl3) δ ppm: 6.90-7.00 (5H, m), 6.69 (1H, d, J = 2.1 Hz), 6.36 (1H, t), 6.22 (2H, d,
J = 2.2 Hz), 4.52 (1H, d, J = 2.9 Hz), 3.87 (3H, s), 3.74 (6H, s), 3.65 (3H, s), 3.58 (1H,
d, J = 2.9 Hz). 13C-NMR (100 MHz, CDCl3) δ ppm: 50.85, 55.28, 55.53, 55.81, 65.19,
96.44, 98.88, 106.60, 106.55, 115.44, 115.35, 128.34, 128.41, 137.76, 138.50, 138.89,
141.14, 157.60, 160.98, 162.04, 205.04. ESI-MS m/z: 423.1 [M + H]+; C25H23FO5:
HRMS calcd. 481.1421 [M + H]+, found 481.1441.
5.2.3.15.2-(3,5-Dimethoxyphenyl)-4,6-dimethoxy-3-phenyl-2,3-dihydro-1H-inden-
1-one (7o). White solid in 66% yield. mp: 144-145ꢀ; 1H-NMR (400 MHz, CDCl3) δ
ppm: 7.02-7.28 (5H, m), 6.92 (1H, d, J = 2.1 Hz), 6.70 (1H, d, J = 2.1 Hz), 6.40-6.24
(3H, m), 4.56 (1H, d, J = 2.4 Hz), 3.88 (3H, s), 3.74 (6H, s), 3.65 (3H, s), 3.64 (1H, d,
J = 2.4 Hz). 13C-NMR (100 MHz, CDCl3) δ ppm: 51.66, 55.3, 55.8, 65.2, 96.5, 99.0,
106.1, 106.6, 126.0, 127.0, 128.5, 138.2, 138.7, 141.5, 143.3, 157.8, 161.0, 205.3.
ESI-MS m/z: 405.2 [M + H]+; C25H24O5: HRMS calcd. 404.1673 [M + H]+, found
405.1684.
5.2.3.16. 2-(3,5-Dimethoxyphenyl)-4,6-dimethoxy-3-(4-methoxyphenyl)-2,3-dihydro-
1
1H-inden-1-one (7p).White solid in 72% yield. mp: 165-166ꢀ; H NMR (400 MHz,
CDCl3) δ ppm: 6.96-6.23 (m, 9H), 4.52 (1H, d, J = 2.60 Hz), 3.87 (3H, s), 3.78 (3H,
s), 3.73 (6H, s). 13C-NMR (100 MHz, CDCl3) δ ppm: 50.84, 55.17, 55.29, 55.60,

ACCEPTED MANUSCRIPT
55.80, 65.33, 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. ESI-MS m/z: 435.2 [M + H]+; C26H26O6:
HRMS calcd. 434.1721 [M + H]+, found 435.1713.
5.2.4. General procedure for the synthesis of methoxyl-indanones (11a-11l)
To a solution of 10 (330 mg, 1mmol) and Wittig regent (1.5 mmol) in anhydrous
0
toluene (5 mL) was added NaHMDS (l.5 mmol, 2.0 M/L) under N2 at 60 C. The
reaction mixture was stirred for 1 h, then cooled to 0ꢀ 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,
filtered, and evaporated. The obtained residue was further purified by silica gel
chromatography (0-4% ethyl acetate/hexanes) to give 11a-11l as a mixture of cis and
trans yellow oil, which only measured by MS. Further the target compounds 11a-11l
were fully characterized by 1H-NMR, 13C-NMR, and HRMS.
To a solution of chalcones 11a-11c (1.0 mmol) was dropwisely added TFA (2 mL,
50 mmol) via syringe. After the reaction mixture was stirred at 75°C for 48 h under N2,
then cooled to 0ꢀ, the reaction was quenched with H2O (10 mL). The resulting
solution was extracted with CH2Cl2 (3 × 10 mL) and the combined organic phases
were washed with brine and dried over Na2SO4. After evaporation the solvent, the
obtained residue was further purified by silica gel chromatography (0-4% ethyl
acetate/hexanes) to give 11a-11l as a white solid.
5.2.4.1.5-Methoxy-2-(4-methoxyphenyl)-3-(naphthalen-2-yl)-2,3-dihydro-1H-
1
inden-1-one (11a). White solid in 53% yield. mp: 99-100ꢀ; H-NMR (400 MHz,
CDCl3) δ ppm: 7.87-7.77 (4H, m), 7.60 (1H, s), 7.49-7.47 (2H, m), 7.17-7.15 (1H, m),
7.04-6.87 (3H, m), 6.86-6.84 (2H, m), 6.68 (1H, m), 4.60 (1H, d, J = 4.70 Hz), 3.84
(1H, d, J = 4.70 Hz), 3.79 (3H, s), 3.78 (3H, s). 13C-NMR (100 MHz, CDCl3) δ ppm:
55.25, 55.40, 55.73, 63.99, 109.47, 114.28 (2C), 116.65, 125.64, 125.81, 125.91,
126.35, 126.92, 127.69 (2C), 128.97, 129.36 (2C), 129.59, 130.95, 132.56, 133.40,
139.75, 158.68, 159.10, 165.93, 203.74. ESI-MS m/z: 395.2 [M + H]+; C27H22O3:
HRMS calcd. 394.1612 [M + H]+, found 395.1536.

ACCEPTED MANUSCRIPT
5.2.4.2.3-([1,1'-Biphenyl]-4-yl)-5-methoxy-2-(4-methoxyphenyl)-2,3-dihydro-1H-
1
inden-1-one (11b). White solid in 47% yield. mp: 144-145ꢀ; H-NMR (400 MHz,
CDCl3) δ ppm: 7.85-7.83 (1H, d, J = 8.22 Hz), 7.60-7.54 (4H, s), 7.46-7.42 (2H, m),
7.37-7.33 (1H, m), 7.18-7.15 (2H, m), 7.06-7.01 (3H, m), 6.68-6.85 (2H, m), 6.74 (1H,
s), 4.49 (1H, d, J = 4.70 Hz), 3.83 (3H, s), 3.80 (3H, s), 3.77 (1H, d, J = 4.70 Hz).
13C-NMR (100 MHz, CDCl3) δ ppm: 54.73, 55.22, 55.73, 64.06, 109.54, 114.24 (2C),
116.42, 125.77, 126.94 (2C), 127.29, 127.58 (2C), 128.27 (2C), 128.75 (2C), 129.30
(2C), 129.50, 130.92, 139.95, 140.54, 141.52, 158.65, 158.94, 165.83, 203.68.
ESI-MS m/z: 421.2 [M + H]+; C29H24O3: HRMS calcd. 420.1765 [M + H]+, found
421.1437.
5.2.4.3.3-(3-Fluorophenyl)-5-methoxy-2-(4-methoxyphenyl)-2,3-dihydro-1H-
1
inden-1-one (11c). White solid in 61% yield. mp: 122-123ꢀ; H-NMR (400 MHz,
CDCl3) δ ppm: 7.84-7.81 (1H, m), 7.31-7.27 (1H, m), 7.04-6.97 (4H, m), 6.89-6.84
(3H, m), 6.81-6.77 (1H, m), 6.68 (1H, s), 4.44 (1H, d, J = 4.70 Hz), 3.83 (3H, s), 3.79
(3H, s), 3.70 (1H, d, J = 4.70 Hz). 13C-NMR (100 MHz, CDCl3) δ ppm: 54.69, 55.22,
55.73, 63.90, 109.52, 113.98, 114.19 (2C), 114.60, 114.82, 116.43, 123.58, 125.86,
129.43 (2C), 130.42, 144.92, 158.17, 158.71, 161.81, 164.26, 165.86, 203.19.
ESI-MS m/z: 363.2 [M + H]+; C23H19FO3: HRMS calcd. 362.1391 [M + H]+, found
363.1737.
5.2.4.4.5-Methoxy-2-(4-methoxyphenyl)-3-(4-(trifluoromethoxy)phenyl)-2,3-dihydro-
1H-inden-1-one (11d). White solid in 63% yield. mp: 126-127ꢀ; 1H-NMR (400 MHz,
CDCl3) δ ppm: 7.84-7.82 (1H, m), 7.18-7.10 (4H, m), 7.04-7.00 (3H, m), 6.86 (2H, d,
J = 7.02 Hz), 6.68 (1H, s), 4.46 (1H, d, J = 4.68 Hz), 3.83 (3H, s), 3.79 (3H, s), 3.67
(1H, d, J = 4.68 Hz). 13C-NMR (100 MHz, CDCl3) δ ppm: 54.35, 55.24, 55.79, 64.11,
109.61, 114.34 (2C), 116.48, 121.42 (2C), 125.93 (2C), 129.23 (2C), 129.29 (2C),
129.47, 130.57, 141.23, 148.23, 158.30, 158.77, 165.95, 203.21. ESI-MS m/z: 429.0
[M + H]+; C24H19F3O4: HRMS calcd. 428.1283 [M + H]+, found 429.1233.
5.2.4.5.5-Methoxy-2-(4-methoxyphenyl)-3-(4-(trifluoromethyl)phenyl)-2,3-dihydro-
1H-inden-1-one (11e). White solid in 59% yield. mp: 126-127ꢀ; 1H-NMR (400 MHz,
CDCl3) δ ppm: 7.84 (1H, d, J = 8.60 Hz), 7.58 (1H, d, J = 8.60 Hz), 7.21 (2H, d), 7.04

ACCEPTED MANUSCRIPT
(1H, dd, J = 1.96 Hz, J = 8.60 Hz), 7.00 (2H, d, J = 8.60 Hz), 6.85 (2H, d, J = 8.60
Hz), 6.65 (1H, s), 4.50 (1H, d, J = 4.70 Hz), 3.86 (3H, s), 3.79 (3H, s), 3.69 (1H, d, J
= 4.70 Hz). 13C-NMR (100 MHz, CDCl3) δ ppm: 54.83, 55.27, 55.79, 64.00, 109.54,
114.38, 116.60, 125.90, 125.93, 126.02, 128.27 (2C), 129.29 (2C), 130.42, 146.58,
157.91, 158.84, 165.98, 202.95. ESI-MS m/z: 413.2 [M + H]+; C24H19F3O3: HRMS
calcd. 412.1412 [M + H]+, found 413.1536.
5.2.4.6.3-(3,5-Dimethoxyphenyl)-5-methoxy-2-(4-methoxyphenyl)-2,3-dihydro-1H-
1
inden-1-one (11f). White solid in 59% yield. mp: 140-141ꢀ; H-NMR (400 MHz,
CDCl3) δ ppm: 7.81-7.79 (1H, m), 7.03-7.01 (3H, m), 6.86-6.64 (2H, m), 6.72 (1H, d,
J = 1.56 Hz), 6.37 (1H, t, J = 2.35 Hz), 6.22 (2H, d, J = 2.35 Hz), 4.36 (1H, d, J =
4.70 Hz), 3.83 (3H, s), 3.78 (3H, s), 3.75 (1H, d, J = 4.70 Hz), 3.73 (6H, s). 13C-NMR
(100 MHz, CDCl3) δ ppm: 54.27, 54.25 (2C), 54.31, 54.75, 62.66, 97.63, 105.21 (2C),
108.52, 113.23 (2C), 115.52, 124.73, 128.28 (2C), 128.47, 130.14, 143.93, 157.63,
160.07 (2C), 164.83, 202.74. ESI-MS m/z: 405.0 [M + H]+; C25H24O5 : HRMS calcd.
404.1613 [M + H]+, found 405.1367.
5.2.4.7.5-Methoxy-2-(4-methoxyphenyl)-3-(p-tolyl)-2,3-dihydro-1H-inden-1-one
(11g).White solid in 53% yield. mp: 148-149ꢀ; 1H-NMR (400 MHz, CDCl3) δ ppm:
7.82-7.80 (1H, m), 7.12 (2H, d, J = 8.82 Hz), 7.02-6.97 (5H, m), 6.84 (2H, d, J = 8.82
Hz), 6.69 (1H, s), 4.40 (1H, d, J = 4.70 Hz), 3.81 (3H, s), 3.78 (3H, s), 3.70 (1H, d, J
13
= 4.70 Hz), 2.34 (3H, s). C-NMR (100 MHz, CDCl3) δ ppm: 21.07, 54.75, 55.25,
55.72, 64.16, 109.47, 114.23 (2C), 116.36, 125.70, 127.77 (2C), 129.30 (2C), 129.56
(2C), 131.09 (2C), 136.72, 139.51, 158.63, 159.28, 165.81, 203.86. ESI-MS m/z:
359.2 [M + H]+; C24H22O3 :HRMS calcd. 358.1639 [M + H]+, found 359.1226.
5.2.4.8.3-(4-Ethoxyphenyl)-5-methoxy-2-(4-methoxyphenyl)-2,3-dihydro-1H-inden-1-
one (11h).White solid in 45% yield. mp: 162-163ꢀ;1H-NMR (400 MHz, CDCl3) δ
ppm: 7.80 (1H, d, J = 8.60 Hz), 7.02-6.98 (5H, m), 6.86-6.83 (4H, m), 6.68 (1H, s),
4.38 (1H, d, J = 5.08 Hz), 4.01 (2H, m), 3.81 (3H, s), 3.78 (3H, s), 3.69 (1H, d, J =
4.70 Hz), 1.4 (3H, t) . 13C-NMR (100 MHz, CDCl3) δ ppm: 14.83, 54.39, 55.21, 55.69,
63.38, 64.26, 109.41, 114.19 (2C), 114.71, 116.31, 125.64, 128.87 (2C), 129.29 (2C),
131.01, 134.33, 157.93, 158.58, 159.38, 165.79, 203.90. ESI-MS m/z: 389.0 [M + H]+;

ACCEPTED MANUSCRIPT
C25H24O4: HRMS calcd. 388.1756 [ M+ H]+, found 389.1796.
5.2.4.9.3-Cyclohexyl-5-methoxy-2-(4-methoxyphenyl)-2,3-dihydro-1H-inden-1-one
1
(11i).White solid in 64% yield. mp: 134-135ꢀ; H-NMR (400 MHz, CDCl3) δ ppm:
7.72-7.70 (1H, m), 7.03 (2H, d, J = 8.60 Hz), 7.01-6.93 (2H, m), 6.81 (2H, d, J = 8.60
Hz), 3.92 (3H, s), 3.76 (3H, s), 3.60 (2H, d, J = 2.74 Hz), 3.36 (1H, t, J = 3.13 Hz),
1.91-1.90 (1H, m), 1.65-1.76 (5H, m), 1.34-1.06 (5H, m). 13C-NMR (100 MHz,
CDCl3) δ ppm: 26.34 (2C), 26.61, 27.84, 31.63, 42.67, 53.80, 55.18, 55.66, 56.15,
109.42, 114.12 (2C), 115.25, 125.96, 128.72 (2C), 129.78, 133.12, 158.32, 159.59,
165.41, 204.59. ESI-MS m/z: 351.8 [M + H]+; C25H24O4: HRMS calcd. 350.1936 [M
+ H]+, found 351.1756.
5.2.4.10. 3-(4-Fluorophenyl)-5-methoxy-2-(4-methoxyphenyl)-2,3-dihydro-1H-inden-
1-one (11j).White solid in 54% yield. mp: 104-105ꢀ; 1H-NMR (400 MHz, CDCl3) δ
ppm: 7.83-7.81 (1H, m), 7.08-6.99 (7H, m), 6.85 (2H, d, J = 8.60 Hz), 6.67 (1H, s),
4.43 (1H, d, J = 4.70 Hz), 3.82 (3H, s), 3.78 (3H, s), 3.67 (1H, d, J = 4.70 Hz).
13C-NMR (100 MHz, CDCl3) δ ppm: 54.32, 55.20, 55.70, 64.24, 109.48, 114.27 (2C),
115.64, 115.85, 116.37, 125.77, 129.26 (2C), 129.33, 129.41, 129.44, 130.66, 138.20,
158.64, 160.66, 163.10, 165.85, 203.30. ESI-MS m/z: 363.2 [M + H]+; C23H19FO3:
HRMS calcd. 362.1374 [M + H]+, found 363.1673.
5.2.4.11.5-Methoxy-2-(4-methoxyphenyl)-3-phenyl-2,3-dihydro-1H-inden-1-one (11k).
1
White solid in 61% yield. mp: 150-152ꢀ; H-NMR (400 MHz, CDCl3) δ ppm: 7.82
(1H, d, J = 8.60 Hz), 7.34-7.30 (3H, m), 7.10-7.08 (3H, m), 7.02-6.99 (3H, m), 6.85
(2H, d, J = 8.60 Hz), 6.69 (1H, s), 4.44 (1H, d, J = 4.70 Hz), 3.82 (3H, s), 3.79 (3H, s),
13
3.73 (1H, d, J = 4.70 Hz). C-NMR (100 MHz, CDCl3) δ ppm: 55.06, 55.20, 55.70,
64.07, 109.51, 114.21 (2C), 116.54, 125.72, 127.06, 127.86 (2C), 128.86 (2C), 129.48
(2C), 130.93, 142.48, 158.61, 159.03, 203.74. ESI-MS m/z: 345.2 [M + H]+;
C23H20O3: HRMS calcd. 344.1412 [M + H]+, found 345.1736.
5.2.4.12. 5-Methoxy-2,3-bis(4-methoxyphenyl)-2,3-dihydro-1H-inden-1-one (11l).
1
White solid in 56% yield. mp: 160-162ꢀ; H-NMR (400 MHz, CDCl3) δ ppm:
7.82-7.80 (1H, m), 7.02-6.98 (5H, m), 6.86-6.84 (2H, m), 6.84 (2H, d, J = 1.60 Hz),
4.39 (1H, d, J = 4.70 Hz), 3.81 (3H, s), 3.80 (3H, s), 3.78 (3H, s), 3.69 (1H, d, J =

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4.70 Hz). 13C-NMR (100 MHz, CDCl3) δ ppm: 54.36, 55.21 (2C), 55.68, 64.25,
109.40, 114.17 (2C), 114.18 (2C), 114.19, 116.31, 125.64, 128.88 (2C), 129.28 (2C),
129.43, 130.97, 134.94, 158.58, 159.32, 165.78, 203.87. ESI-MS m/z: 375.2 [M + H]+;
C24H22O4: HRMS calcd. 374.1564 [M + H]+, found 375.1435.
5.3
ꢁBiological assays and experimental procedures
5.3.1. Cell culture
Murine monocyte-macrophage RAW264.7 cells maintained in Dulbecco’s
modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with
5% fetal bovine serum (Invitrogen), 100 units/mL penicillin, and 100 mg/mL
streptomycin and incubated at 37ꢀin a humidified atmosphere containing 5% CO2.
5.3.2. Cell viability assay
Cell cytotoxicity was evaluated by MTT assay as previous reported [19]. The
medium was changed before the assay. MTT dissolved in phosphate buffered saline
(PBS) and was added to the culture medium to reach a final concentration of
0.5 mg/ml. After incubation at 37°C for 4 h, the culture media containing MTT were
removed, and then DMSO was added into each well and the absorbance at 570 nm
was measured by a microplate reader (TECAN M1000, Austria GmbH, Austria).The
IC50 values were determined by GraphPad Prism 5.
5.3.3. Assay for NO production
NO production was quantified by nitrite accumulation in the culture medium using
the Griess reaction. Briefly, RAW264.7 cells were pretreated with compounds for 4 h,
and then stimulated with or without LPS (1 ꢀg/ml) for 24 h. The isolated supernatants
were mixed with an equal volume of Griess reagent (Beyotime Biotechnology, China).
NaNO2 was used to generate a standard curve, and nitrite production was determined
by measuring the optical density at 540 nm by a microplate reader (M1000, TECAN,
Austria GmbH, Austria).The IC50 values were determined by GraphPad Prism 5.
5.3.4. Western blot analysis
RAW264.7 cells were serum starved overnight before exposing to the treatment
conditions. For whole cell extraction, cells were lyzed in RIPA buffer with protease
and phosphatase inhibitors. After centrifugation (4°C, 10 min, 10, 000 g), samples

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were prepared for Western blot analysis. For preparation of nuclear fraction, nuclear
protein was extracted using the nuclear and cytoplasmic extraction kit (Pierce, Inc)
according to manufacturer's instructions. Western blot was performed as previously
described [19]. Equal amounts of protein extracts (30 ꢀg) were subject to immunoblot
analysis using p-JNK1/2 (Thr183/Tyr185), p-p38 (Thr180/Tyr182), p-ERK1/2
(Thr202/Tyr204), JNK1/2, ERK1/2, p38, NF-κB p65 (all in 1:1000 dilution), COX-2
(1:10000 dilution), iNOS, TLR4 (all in 1:500 dilution), β-actin (1:2000 dilution) and
Lamin (1:2000 dilution) antibodies followed by horseradish peroxidase-conjugated
secondary antibodies (1:5000 dilution, ICL Lab, Newberg, OR, USA), and developed
by enhanced chemiluminescence reaction (Pierce, Inc). Signal intensity was
quantified using the software Alpha Imager (Alpha Innotech Corp, San Leandro, CA)
in each group with β-actin as internal control. The results were expressed as fold
changes by normalizing the data to the control values.
5.4. Acute toxicity study in mice
Healthy Kunming mice of both sexes, weighing 18-22 g, were divided into 7
groups of 10 animals matched for weight and size. Compounds11k, (+)-11k and
(-)-11k were oral administration at the dose of 92.0, 115.0, 143.0, 179.0, 224.0, 280.0,
350.0 mg/kg body weight. Behavior and changes in the weight of mice were
monitored and the death was recorded within 14 days. The LD50 values were
calculated using Bliss method.
Acknowledgementꢂ
This work was supported by the National Natural Science Foundation of China (No:
81373276), Shanghai Municipal Committee of Science and Technology (No:
14XD1400300, 13JC1401200).
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Figure captions:
1. Figure 1. Design strategy for sesquistilbene indanone analogues;
2. Figure 2. Synthetic indanone derivatives inhibited LPS-induced NO production in
RAW 264.7 macrophages;
3. Figure 3. X-ray crystal structure of racemic-11k;
4. Figure 4.Separation of racemic11k using chiral HPLC;
5. Figure 5. Compound 11k inhibited LPS-induced inflammatory response in RAW
264.7 cells;
6. Figure 6.Compound 11k suppressed LPS-induced activation of NF-κB signaling
pathway in RAW 264.7 cells;
7. Figure 7. Compound 11k suppressed LPS-induced activation of MAPK signaling
pathway in RAW 264.7 cells;
8. Figure 8.Proposed anti-inflammatory mechanism of 11k via TLR4/JNK/ NF-κB
signaling pathway;
9. Scheme 1. General synthesis of compound 7a-7p;
10. Scheme 2. General synthesis of compound 11a-11l;
11. Table 1: Inhibitory effects of the synthesized compounds on NO production in
LPS-stimulatedRAW264.7 cells;
12. Table 2: Inhibitory effects of racemic-11k, (+)-11k and (-)-11k on NO production
in LPS-stimulated RAW264.7 cells and acute toxicity studies in mice.

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Figure 1. Design strategy for sesquistilbene indanone analogues.
Figure 2. Synthetic indanone derivatives inhibited LPS-induced NO production in
RAW 264.7 macrophages. RAW 264.7 cells were pretreated with indanones (50ꢀM)
for 4 h, and then stimulated with or without LPS (1ꢀg/mL) for 24h. Resveratrol and
compound 2 were chosen as positive controls, and NO production was measured
&
using nitrite and nitrate assay. Data shown are means ± SEM, P < 0.05 compared
*
with unstimulated cells, P < 0.05 compared with LPS-stimulated cells; Data were
from at least three independent experiments, each performed in duplicate.

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Figure 3. X-ray crystal structure of racemic-11k.
Figure 4. Separation of racemic-11k using chiral HPLC.

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Figure 5. Compound 11k inhibited LPS-induced inflammatory response in RAW
264.7 cells. After pretreatment with 11k (12.5-50 ꢀM), RAW 264.7 cells were
stimulated with LPS (1ꢀg/mL) for 24 h. Cell viability was evaluated using the MTT
assay. NO production was measured using nitrite and nitrate assay. iNOS and COX-2
expression were detected by Western blot analysis. (A) Cell viability assay; (B)
Quantitative analysis of NO production; (C) Quantitative analysis of iNOS expression,
β-actin was used as loading control; (D) Quantitative analysis of COX-2 expression,
ꢀ
#
β-actin was used as loading control. P < 0.05 compared with unstimulated cells, P
< 0.05 compared with LPS-stimulated cells; Data were obtained by at least three
independent experiments, and each was performed in duplicate.

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Figure 6. Compound 11k suppressed LPS-induced activation of NF-κB in RAW
264.7 cells. After pretreatment with 11k (12.5-50ꢀM), RAW 264.7 cells were
stimulated with LPS (1 ꢀg/mL) for 2 h. The nuclear and cytoplasmic levels of NF-κB
p65 were detected by Western blot, β-actin and lamin were used as loading controls
for cytoplasmic and nuclear extracts, respectively. CytꢁcytoplasmaꢂNuc, nuclei; #P <
ꢀ
0.05 compared with unstimulated cells; P < 0.05 compared with LPS stimulated
cells; Data were by at least three independent experiments, and each was performed in
duplicate.